University of Southern California Dissertations and Theses

Non-viral and viral hematopoietic progenitor cell gene therapy

(USC Thesis Other)

Non-viral and viral hematopoietic progenitor cell gene therapy

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content
NON-VIRAL AND VIRAL
HEMATOPOIETIC PROGENITOR CELL GENE THERAPY

by

Teiko Sumiyoshi

A Dissertation Presented to the
FACULTY OF THE GRADUATED SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR MICROLOGY AND IMMUNOLOGY)

August 2009

Copyright 2009 Teiko Sumiyoshi
ii

Dedication

For My Family
&
Dr. Ingrid Bahner

iii

Acknowledgements

I would like to thank my PI, Dr. Donald B. Kohn, for giving me the opportunity to work
in his lab. I am extremely grateful for him always pushing me to be a better scientist and
to be the best I can be. I thank him for being very tough, patient, and supportive of me
throughout the years. I hope I have made him proud.

I would also like to thank my mentor, Dr. Ingrid Bahner, for teaching me everything in
the lab. I thank her for spending countless hours of her precious time to help me practice
my talks or designing and troubleshooting experiments. It was an honor to work with
her. I don’t know how, but she always has an answer to anything I ask! I thank her for
believing in me and being there for me when I most needed it. She has inspired me in
many aspects of my life. I am forever grateful to have her as my teacher.

I would also like to thank Nathalia Holts for her generous help to my in vivo work. Her
dedication to science is remarkable. It was an honor to be able to work with such a
talented scientist. Another person who deserves thanks is Shundi Ge, who helped me
with my secondary transplant. Without Nat and Shundi’s help, I couldn’t have completed
my in vivo work.

iv

I would like to thank Dr. Roger Hollis for his guidance and help in my Sleeping Beauty
project. I thank him for teaching me everything I need to know about cloning and for
being extremely helpful and resourceful. I will miss his witty jokes and clever ideas. It
has been a great pleasure working with him.

I would like to thank my committee members: Dr. Minnie McMillan, Dr. Paula Cannon,
and Dr. James Ou. I thank them for their patience and constant support and for always
providing valuable feedback on my projects. I was very fortunate to have them guiding
me through my graduate career.

Many thanks to the graduate students in the lab: Denise Carbonaro, Eszter Pais, and Chris
Choi. Having them around just made my graduate work so much more fun and less
frustrating when things did not work. I want to thank everyone in the division of
Immunology and Bone Marrow Transplant at Childrens Hospital, Los Angeles. I thank
them for always making me feel like a part of the BMT family.

Last but not least, I would like to thank my family for their unconditional love and
support. I thank them for giving me strength and encouragement. Pursuing my Ph.D. has
truly been a humbling experience. This is a life lesson that I will never forget and will
forever benefit from. I have gained an appreciation for all the obstacles and challenges I
had to overcome, as well as all the wonderful scientists I met along the way.

v

Table of Contents

Dedication ii
Acknowledgements iii

List of Tables vii

List of Figures viii

Abstract xi

Chapter One – Introduction
1.1 Gene Therapy 1
1.2 Hematopoietic Stem Cells 1
1.3 Hematopoietic Stem Cell Identification and Isolation 2
1.4 Hematopoietic Stem Cell (HSC) Gene Therapy 4
1.5 Gene Transfer to HSC 5
1.5.1 Viral Vectors 6
1.5.1.1 Retroviral Vectors 6
1.5.1.2 Lentiviral Vectors 9
1.5.1.3 Non-Integrating Lentiviral (NIL) Vectors 11
1.5.2 Non-Viral Vectors 12
1.5.2.1 Delivery of Non-Viral Vectors to Cells 13
1.5.2.2 Integration of Non-Viral Vectors 14
1.5.3 Hybrid Gene Transfer System 16
1.6 Host Conditioning in HSC Gene Therapy 17
1.6.1 Myeloablative conditioning 18
1.6.2 Non-myeloablative conditioning

19
Chapter Two – Optimization of the Sleeping Beauty Transposon
System to Achieve Stable Transgene Expression in Human CD34+
Hematopoietic Stem/Progenitor Cells

2.1 Introduction 21
2.2 Materials and Methods 24
2.3 Results 31
2.4 Discussion

57
vi

Chapter Three – Hybrid of Sleeping Beauty Transposon System and
Non-Integrating Lentiviral Vectors for Efficient Gene Transfer and
Stable Transgene Expression

3.1 Introduction 66
3.2 Materials and Methods 69
3.3 Results 72
3.4 Discussion

87
Chapter Four – Low-Dose Busulfan and Fludarabine Conditioning
for Non-Myeloablative Hematopoietic Stem Cell Transplant in Infant
Non-human Primates

4.1 Introduction 90
4.2 Materials and Methods 96
4.3 Results 101
4.4 Discussion

112
Chapter Five – Concluding Remarks and Future Directions

115
Bibliography

119

vii

List of Tables

Table 2-1. Summary of HSB- vs. SB-mediated gene transfer in K562,
Jurkat, primary human CD34+.

35
Table 2-2. Comparison of the stable transgene expression achieved using
HSB transposon expressed by MNDU3, CMV, or EF1-α promoter in
K562, Jurkat, and primary human CD34+.

43
Table 2-3. Summary of engraftment levels of human CD34+ progenitor
cells in NSG mice.

49
Table 2-4. Engraftment level and transgene expression in secondary
transplanted NSG mice.

56
Table 4-8C. Table summary of the AUC and percent gene marking in
PBMC and CD34+ cells of the animals in the study.

108

viii

List of Figures

Figure 1-1. The development of hematopoietic hierarchy.

2
Figure 1-2. HSC phenotypic profile.

4
Figure 1-3. Model of retroviral vector gene transfer.

7
Figure 1-4A. Schematic diagram of the SB transposase.

16
Figure 1-4B. Cut and paste mechanism of SB-mediated gene transfer.

16
Figure 2-1. Vectors for Sleeping Beauty-mediated gene transfer.

25
Figure 2-2. Stable long-term HSB- and SB-mediated gene transfer.

33
Figure 2-3. Optimization of transposon-mediated gene transfer.

39
Figure 2-4. Optimization of promoter transposase and transposon reporter
expression.

45
Figure 2-5. Overall SB-transposon system optimization in primary human
CD34+ hematopoietic progenitor/stem cells in vitro.

46
Figure 2-6. Schematic diagram of the experimental timeline for in vivo
analysis of SB-mediated gene transfer to human CD34+ cells by
NOD/SCID/γC neonatal transplantation.

47
Figure 2-7. Comparison of engraftment levels of human CD34+
progenitor cells in NSG mice.

50
Figure 2-8. Representative FACS analysis of eGFP expression detected in
multi-organ and multi-lineage differentiated cells from engrafted HSB-
modified human hematopoietic CD34+ cells at 5 months post-
transplantation.

52
Figure 2-9. Summary of multi-lineage eGFP expression levels determined
in peripheral blood, bone marrow, thymus, and spleen harvested from the
NSGs after 5 months neonatal transplantation of HSB-modified human
CD34+ cells.

54
Figure 2-10. Summary of eGFP gene marking in total cells isolated from
BM and spleen of the NSGs 5 moths post-transplantation.

55
ix

Figure 3-1. Schematic demonstration of SB delivery using non-
integrating lentiviral (NIL) vectors.

67
Figure 3-2. NILting beauty vectors constructed in this study.

71
Figure 3-3. Comparison of stable gene transfer efficacy between NILting
beauty viral delivery and electroporation delivery of SB components in
K562.

74
Figure 3-4. Validation of the cis NILting beauty vector construct.

76
Figure 3-5. NILting beauty rescue study.

78
Figure 3-6. Dissecting the NILting beauty vector system.

79
Figure 3-7. The trans NILting beauty delivery system in K562.

80
Figure 3-8. NIL-HSB16 supernatant dose escalation study.

82
Figure 3-9. Optimization of NIL-eGFP for the trans NILting beauty
system in K562 cells.

82
Figure 3-10. Stable eGFP transgene expression achieved by the trans
NILting beauty system in K562 cells.

84
Figure 3-11. Role of defective integrase in trans NILting beauty system.

85
Figure 3-12. The preliminary study of trans NILting beauty double
transduction in human CD34+ cells.

86
Figure 3-13. Linear v.s. circular HSB transposon for HSB-mediated gene
transfer in K562 cells.

89
Figure 4-1. Study design and outline to achieve immune reconstitution
and tolerance towards transgene product using busulfan and fludarabine
conditioning for non-myeloablative hematopoietic stem cell transplant in
infant nonhuman primates.

93
Figure 4-2. Proviral maps of NoN and eGFP SIV-based lentiviral vectors.

97
Figure 4-3. Experimental setup for HSCT in infant rhesus macaques with
non-myeloablative conditioning.

98
Figure 4-4. Flow diagram of rhesus CD34+ transduction for each monkey
subject in this study.

100
x

Figure 4-5. The preliminary study of trans NILting beauty double
transduction in human CD34+ cells.

102
Figure 4-6. Absolute neutrophil count nadir v.s. busulfan AUC by 2
months post-HSCT.

103
Figure 4-7. Effect of busulfan on total white blood cell count, neutrophil
count, and lymphocyte count.

105
Figure 4-8. Average NoN and eGFP gene marking in peripheral blood
mononuclear cells (PBMCs) at 6 months post-bone marrow transplant.

108
Figure 4-9. Average % eGFP gene marking in PBMC v.s. humoral
immune response.

112

xi

Abstract

The pluripotent characteristic of hematopoietic stem cells (HSCs) makes them a
good candidate for gene therapy. The safety drawbacks of the commonly used viral gene
transfer system have made the search for alternative gene transfer methods such as non-
viral or hybrid gene transfer systems became increasingly appealing in the field. One
such system is the Sleeping Beauty (SB) transposon-mediated gene transfer system.
Using a non-viral approach to delivery SB plasmids we were able to significantly
increase the efficiency of stable gene up to 20-fold higher than previously published data
by incrementally optimizing each element of the SB transposon system. In vivo studies
demonstrated that SB-modified human CD34+ cells were engrafted in
NOD/SCID/γC(null) (NSG) mice and differentiated into multi-lineage cell types with
stable transgene expression. Transgene expression remained persistent in the secondary
transplanted NSG mice indicating a long-term stable integration achieved by HSB-
transposon system. Non-integrating lentiviral (NIL) vectors were also investigated as
another method for SB plasmid delivery. Combining the stable integration of the SB
transposon system with the delivery efficiency of NIL, termed NILting beauty, could
produce a hybrid vector system that synergizes the advantages of both viral and non-viral
vector systems and provide a more effective and safer approach to genetically modify
HSCs. The feasibility and potential of utilizing NILting beauty to achieve stable
transgene integration was evaluated using K562 and human HSCs. Up to 7% stable
transgene expression was achieved in K562 cells and around 1% for human CD34+ cells
when transduced with NILting beauty vectors. The other approach to increase long-term
xii

transgene expression with relatively minimal adverse effects in clinical HSC gene
therapy is using non-myeloablative conditioning regimen. The feasibility of combining
busulfan with fludarabine as an alternative and potentially more effective conditioning
regimen was explored to achieve long-term stable gene marking in HSC gene therapy.
We hypothesized that the addition of the immunosuppressive chemotherapeutic agent
fludarabine may contribute to better HSC engraftment and long term transgene
expression by reducing host immunological responses to the foreign transgene product.
To evaluate this hypothesis, a clinically relevant infant rhesus monkey bone marrow
transplant (BMT) model was used. Preliminary data showed a strong correlation between
the busulfan dose and the busulfan area-under-the curve (AUC). Transient neutropenia
was noted whereas lymphopenia was not observed. While monkeys with high levels of
eGFP gene marking also showed detectable levels of anti-eGFP antibodies when no
fludarabine was given, they lacked humoral immune responses to eGFP if they received
fludarabine. These data suggest that the immune responses against the transgene may
play a significant role in the successful outcome of HSC gene therapy and that
fludarabine may be able to modulate these responses. Since significant lymphodepletion
was not achieved by the fludarabine treatment, higher doses of fludarabine may need to
be evaluated for an effect on engraftment and long-term transgene expression. Although
further improvements and optimization are required for the NILting beauty hybrid system
and the host conditioning regimen, studies described in this thesis demonstrated that the
application of the optimized HSB transposon system holds great promise for further
advancement of SB-transposon based gene therapy using hematopoietic stem cells.
1

Chapter 1 – Introduction

1.1 Gene Therapy
Gene therapy has provided a novel approach and holds a great promise for the
treatment of inherited genetic or acquired diseases such as severe combined immune
deficiency (SCID),(Aiuti, 2004; Qasim et al., 2004; Kohn, 2008; Aiuti et al., 2009)
Lesch-Nyhan syndrome,(Lowenstein et al., 1998; Glorioso et al., 2003) cancer,(Bachtarzi
et al., 2008; Johnson et al., 2009) or human immunodeficiency virus (HIV)
infection.(2009; Mitsuyasu et al., 2009; Tsygankov, 2009) The general idea of gene
therapy is to permanently introduce a therapeutic gene in replacing the absent or faulty
gene in patients. The transgenes used in gene therapy usually are either designed to
introduce new functions or to correct the defective genes to correct its phenotype and
ultimately eliminate the root cause of the disease.

1.2 Hematopoietic Stem Cells
Hematopoietic stem cells (HSC) represent a major focus for efforts at gene
therapy for genetic childhood immunological diseases, hemoglobinopathies, lysosomal
and metabolic disorders.(Kohn, 2008) HSCs are defined by their ability to self-renew
and differentiate into various lineages of hematopoietic cells, including all cells of the
myeloid and lymphoid lineages, essentially for a life time.(Bellantuono, 2004) These
unique qualities enable gene modified HSCs to achieve long-term expression of the
therapeutic transgene in the mature blood cells.

2

1.3 Hematopoietic Stem Cell Identification and Isolation
Hematopoietic stem cells are small, quiescent, and lack lineage (lin-) cell surface
markers. Murine HSCs were first identified by in vivo competitive repopulation assays
followed by secondary and tertiary transplant to assess the long-term engraftment of
HSCs.(Spangrude et al., 1988a) A panel of cell surface antigens has been identified as
markers for each distinct developmental stages of hematopoiesis that begins with the
long-term (LT) functional HSC.(Weissman et al., 1989) LT-HSCs produce less potent
short-term (ST) HSCs that still possess self-renew capability. ST-HSCs then differentiate
into multi-potent progenitors (MPP) which can give rise to terminally differentiated cells
through a variety of committed progenitor cells such as common lymphoid progenitors
(CLP) for T-cells, B-cells, and NK cells, and common myeloid progenitor (CMP) for
myeloid cell lineages such as dendritic cells, red blood cells, granulocytes, and platelets
(Fig. 1-1).(Bellantuono, 2004)

Figure 1-1. The development of hematopoietic hierarchy. Hematopoietic stem cells
(HSCs) can either be long-term repopulating cells (LT-HSC) or short-term repopulating
cells (ST-HSC). STRC differentiate into multipotent progenitors (MPP) which can then
give rise to the common lymphoid progenitor (CLP) and common myeloid progenitor
(CMP).
3

While human and murine HSCs share similar developmental hierarchies, the
markers used to identify human HSCs are different from the ones used for murine HSCs
(Fig. 1-2). One of the most prominent cell surface markers for human HSCs is the
glycoprotein CD34 which is expressed on 1 to 4% of human bone marrow cells and is
found on early progenitor cells but not on their mature counterparts.(Andrews et al.,
1986; Berenson et al., 1988; Civin et al., 1990) The self-renew property of CD34+ cells
was first demonstrated in a non-human primate transplantation model.(Berenson et al.,
1988) CD34+ cells were shown to restore hematopoiesis in lethally irradiated baboons
after autologous transplantation. In a clinical phase I/II trial, autologous transplantation
of positively selected human peripheral blood CD34+ cells showed rapid and stable
engraftment and reconstitution of hematopoiesis in patients with advanced
malignancies.(Brugger et al., 1994) Although the specific function of CD34 still remains
unknown, it has been commonly used as a marker to quantify and purify HSCs for
research and for clinical HSC-based gene therapy.
Other surface markers such as CD38, CD33, thy-1, CD10, and CD7 have also
been used in conjunction with CD34 to identify more primitive populations of
HSCs.(Spangrude et al., 1988b; Spangrude et al., 1989) Human CD34+ cells that
express high levels of CD34 and absence of CD38 (CD34+CD38-) have been found to be
highly enriched with LT-HSCs. Co-expression of CD10 or CD7 on CD34+ cells defines
the earliest human CLP. However, expressions of one or more of these surface markers
can also be found in more mature progenitors. Despite the growing knowledge and
rigorous isolation protocols that have been developed to date, it remains necessary for
further optimizations in the identification and isolation of highly enriched human HSCs.
4

Figure 1-2. HSC phenotypic profile. Cells at key stages of the murine and human
hematopoietic hierarchy are listed including LT-HSCs, ST-HSCs, MMPs, and MBCs.
MBC stands for mature blood cell populations. Figures adapted and modified from
Ivanova et al., 2002.(Ivanova et al., 2002)

At present, human HSCs can be assessed in vitro by colony forming unit (CFU)
assay to examine their ability to generate colonies that contain cells of different lineages.
Human HSCs can also be identified in vivo by transplanting human cells into immune-
deficient animals such as NOD/SCID (non-obese diabetic/severe combined
immunodeficient) mouse model and evaluating the engraftment levels of human cells in
the host.(Greiner et al., 1998) For a more stringent assay to assess the self-renew and
multi-potent abilities of human HSCs, a secondary transplantation is usually performed.

1.4 Hematopoietic Stem Cell (HSC) Gene Therapy
The pluripotent characteristic of HSCs makes them a good candidate for gene
therapy. Human HSCs can be obtained from cord blood, bone marrow, or cytokine-
mobilized peripheral blood, modified ex vivo, and re-transplanted into a patient to provide
a long-lasting source of blood cells engineered to improve or correct the defected
function. Clinical trials using genetically modified HSC for genes involved in the
5

pathogenesis of congenital immune deficiencies (ADA-deficient and X-linked forms of
SCID and Chronic Granulomatous Disease [CGD]) have led to clear-cut clinical benefits,
with the sustained production of genetically-corrected lymphocytes and neutrophils,
respectively, and restoration of protective immunity in a majority of treated
patients.(Cavazzana-Calvo et al., 2000; Horwitz et al., 2001; Aiuti et al., 2002; Gaspar et
al., 2006; Ott et al., 2006) In addition to the clinical trials on congenital immune
deficiencies, HSC-based gene therapy has also been actively explored as alternative
treatments for HIV infection and various cancers such as melanoma, leukemia, breast
cancer. Furthermore, gene modification of HSCs can also be used as an investigational
tool to understand the regulation and development of hematopoiesis.
To enable HSC gene therapy transitioning from the bench to clinical trials in
patients, two key requirements need to be met simultaneously: (1) expression of
therapeutic gene(s) in target cells/tissue must result in a sustained gene expression
duration to produce a clinically relevant therapeutic response; (2) expression of
transferred genes and their products must not cause serious adverse side effects such as
cancer or graft verse host diseases (GVHD). There has been significant progress in the
methods for gene modification of human HSCs and the optimization of HSC
transplantation protocols that result in a select number of promising clinical trials.(Liu
and Visner, 2007; Kohn and Candotti, 2009)
1.5 Gene Transfer to HSCs
Vehicles used to transfer a foreign gene into target cells are known as vectors.
The purpose of a vector is to protect the transgene(s), promote entry, and express the
transgene(s) in the target cells. Two major types of vectors used for HSC-based gene
6

therapy are viral and non-viral vectors. Currently, viral vectors have been widely used
for gene transfer to HSCs. However, viral-base gene transfer does raise potential safety
issues. These drawbacks of the viral gene transfer system have made the search for
alternative gene transfer methods such as non-viral or hybrid gene transfer systems
became increasingly appealing in the field. The advantages and limitations of all three
gene transfer systems are discussed in detail below.

1.5.1 Viral Vectors
Gene transfer vectors used in current HSC gene therapy clinical trials are mostly
derived from retroviruses, lentiviruses, and adenovirus.(Dunbar et al., 1995; Cavazzana-
Calvo et al., 2000; Thomas et al., 2003) The recombinant viruses are formed by
removing the viral genes and replacing them with therapeutic genes, and are replication
defective. The most effective methods to modified HSC has been using integrating
retroviral and lentiviral vectors due their ability to stably integrate into chromosomal
DNA and remain for long-term transgene expression as the cells proliferate.(Logan et al.,
2002; Sinn et al., 2005) These viral vectors can be used to transduce relatively high
percentages of HSC with minimal acute cytotoxicity.

1.5.1.1 Retroviral Vectors
Retroviral vectors can effectively integrate transgenes into the target cells based
on the fact that their viral genome encode an integrase protein that specifically recognizes
sequences in the viral long terminal repeats (LTR att sequences) and catalyzes their
covalent insertion into host chromosomes. Once the viral vector enters the target cell, the
viral RNA carrying transgene then undergoes reverse transcription and produces a blunt-
7

ended double stranded linear viral DNA that serves as the substrate for viral integration.
The viral integrase binds to the LTR region located on both ends of the double stranded
DNA and mediates transgene integration in to the target cells (Fig. 1-3). The integrated
viral genome is also known as provirus.

Figure 1-3. Model of retroviral vector gene transfer showing the formation of the
three different episomal DNA molecules and interationg of the proviral genome. The
LTR circles are formed by non-homologous end joining and homologous recombination.
Diagram is take from Philpott and Thrasher, 2007.(Philpott and Thrasher, 2007)

8

Despite their efficiency, one of the major limitations of the retroviral vector gene
transfer system is its incapability to transfer genes to quiescent HSCs. Retroviral vector
mediated gene transfer is limited only to dividing cells. In addition, retroviral-based gene
transfer requires HSCs undergo cell division via in vitro cytokine stimulation which is
frequently associated with loss of pluripotency and/or long-term engraftment capabilities.
Transgene silencing has also been an issue observed in HSCs and embryonic stem cells
(ESCs) transduced with retroviral vectors.(Teich et al., 1977; Feuer et al., 1989) This
restricted expression of retroviral vectors is due to restriction factors that recognize a
conserved sequence carried by retroviral vectors and/or de novo cytosine
methylation.(Jahner et al., 1982; Stewart et al., 1982; Linney et al., 1984) Studies have
shown that the transcriptional silencing is due to the repressor binding site (RBS) located
downstream of the 5’ LTR of the retroviral vectors.(Petersen et al., 1991; Haas et al.,
2003) Several host restriction factors such as TRIM28 and ZFP809 have been identified
to be responsible for blocking retroviral replication.(Wolf and Goff, 2007; Wolf et al.,
2008a; Wolf and Goff, 2008; Wolf et al., 2008b; Wolf and Goff, 2009) De novo
methylation of CpG dinucleotides also contributes to the integrated retroviral vector
silencing by inducing histone deacetylation and chromatin condensation.(Jaenisch et al.,
1982; Barklis et al., 1986)
Lastly, safety is still the major concern with using retroviral vector as the
transgene delivery vehicle in HSC therapy. As a safety precaution, self-inactivating
(SIN) lentiviral vectors are designed where the enhancer/promoter sequences from the U3
region of a retroviral vector are deleted to prevent unwanted LTR activity.(Yu et al.,
1986) SIN vectors are developed to improve the safety of viral-based gene transfer
9

system by eliminating the viral LTR enhancer/promoter activity.(Logan et al., 2004a)
However, the requirement for an internal promoter to drive transgene expression of the
viral vector does not completely abolish the potential for unwanted trans-activation
events.(Weber and Cannon, 2007)
Unfortunately, despite the promising potential of HSC gene therapy as an
effective treatment for SCID patients, five out of 26 treated children (16 showed clinical
benefit) who received retroviral-based HSC gene therapy developed T cell leukemia-like
complication due insertional oncogenesis 2.5 - 5 years post-treatment.(2003; Hacein-Bey-
Abina et al., 2003; Kaiser, 2003; Kohn, 2008) The retroviral gene construct was found to
integrate nearby the LMO2 gene and trans-activated the oncogene, which led to the
development of leukemia in these patients. Mapping of retroviral vector insertion sites
shows that approximately 17% of its integration occurred preferentially in and around
transcriptional start regions and another 17% of integrations occurred within 1 Kb of
CpG islands.(Wu et al., 2003) This integration preference near promoters may increase
the probability of a retroviral vector integrating near a cellular proto-oncogene with trans-
activation, which could lead to cellular transformation. Due to these reasons, retroviral
vectors might not be the safest gene transfer system to achieve the most optimal
therapeutic outcome for HSC gene therapy.

1.5.1.2 Lentiviral Vectors
As a result, a new generation of viral vectors derived from HIV-1 lentiviruses has
been developed.(Naldini et al., 1996; Miyoshi et al., 1999) In addition to all the
advantages of retroviral vectors, lentiviral vectors possess several unique characteristics
10

that are superior to retroviral vector systems for HSC gene therapy. First, lentiviral
vectors have the ability to integrate a transgene into both dividing and non-dividing cells.
This allows stable transgene integration into more primitive HSC populations to achieve
substantial long-term transgene expression. For the transduction of HSCs, it is ideal to
use a vector system that can stably integrate the transgene into non-dividing quiescent
cells that possess highest level of pluripotency and long-term engraftment capabilities.
Since lentiviral vectors can transduce quiescent HSCs, the pre-stimulation step that is
normally required during retroviral transduction is eliminated in a lentiviral vector
system. Shorter ex vivo transduction protocols might also better maintain the
pluripotency of HSCs and their engraftment capability.
Second, lentiviral vectors are more resistant to silencing than retroviral vectors.
Lentiviral genomes contain fewer CpG dinucleotides and consequently reduce the
incidences of transcription silencing due to de novo cytosine methylation.(Pfeifer et al.,
2002) Third, lentiviral vectors may be less prone to cause insertional mutagenesis in
comparison to retroviral vectors. Unlike the integration pattern of retroviral vectors, the
integration pattern of lentiviral vectors shows no preference for regions near the
transcription start site of genes. They integrate across the entire transcribed region of
genes. The chances of lentiviral vectors activating nearby proto-oncogene could be
relatively lower than retroviral vectors.(Mantovani et al., 2009) Several safety
modifications such as SIN vectors, chromosomal insulators, and tissue-specific promoters
have also been implemented and validated to demonstrate insertional oncogensis in
current lentiviral vector system.(Logan et al., 2002)
11

Despite major advances in retro- and lentiviral vector designs for gene
modification of HSCs, the potential risk of insertional oncogenesis is still the major
drawback for the viral vectors. The other drawback of viral-based gene therapy is the
possible formation of replication-competent virus generated by either recombination
between virus vector and packaging functions, or endogenous defective proviruses.
Finally, the production of viral vectors is a complex and expensive process that may be
challenging to develop into cost-efficient large-scale processes. Therefore, exploring
other alternatives for a safer and more effective approach to gene transfer system is
imperative for HSC gene therapy.

1.5.1.3 Non-Integrating Lentiviral (NIL) Vectors
As mentioned above, nonspecific integration can be problematic because of
possible gene silencing, formation of replication-competent viruses and, most
importantly, insertional mutagenesis, which can lead to undesirable effects such as
malignant transformation. One way to avoid non-specific integrations during viral
transduction is to use a gene transfer system that maintains the advantages of viral-based
vectors (efficient and non-cytotoxic) in the absence of integration to provide an additional
safety measure. Non-integrating lentiviral (NIL) vectors are designed to disable vector
integration but reserve their normal viral entry function, DNA synthesis, and
accumulation of double stranded DNA circles in the cell nucleus.(Engelman, 1999;
Nightingale et al., 2006) NIL vectors generally are created by introducing specific
mutations in the gene encoding integrase and/or in the recognition or attachment
sequences (att) found at the terminal ends of the non-integrated linear viral
12

DNA.(Nightingale et al., 2006; Yanez-Munoz et al., 2006) The double stranded DNA
circles found in NIL transduced cells are normally the by-products of integration during
wild-type HIV and retrovirus infection.(Pang et al., 1990; Pauza et al., 1990; Robinson
and Zinkus, 1990) These circles have been shown to be functional templates for
transcription by host machinery. (Stevenson et al., 1990; Engelman et al., 1995;
Wiskerchen and Muesing, 1995; Nakajima et al., 2001; Wu, 2004) This enables NIL
vectors to be used as a gene transfer system for efficient and transient transgene
expression in cycling cells.(Nightingale et al., 2006; Yanez-Munoz et al., 2006;
Lombardo et al., 2007) NIL vectors have been shown to achieve effective gene delivery
and mediate stable transduction in various cell types, including human primary HSCs and
ES cells.(Nightingale et al., 2006; Yanez-Munoz et al., 2006; Lombardo et al., 2007)

1.5.2 Non-Viral Vectors
As an alternative to viral vectors, delivery of non-viral vectors (plasmids) to cells
may have several advantages. Plasmids may carry larger gene expression units than viral
vectors allowing more sophisticated patterns of gene expression rather than the
constitutive, ubiquitous expression that is often achieved using viral vectors and strong
enhancer/promoters. Genomic elements, such as extended upstream, down-stream and
intronic sequences affecting transcription, locus-control regions, insulators, etc. can be
included more readily in plasmids than in viral vectors. For safety concerns, non-viral
vectors are much less immunogenic and toxic compared to viral vectors.(Lehrman, 1999;
Lozier et al., 1999) In addition, the risks of forming replication-competent virus are not
13

existent in non-viral vector systems.(Kay et al., 2001) Lastly, the purification and quality
control processes of non-viral vectors are much easier, because plasmids are more stable
than viral vectors, and therefore are more amenable to pharmaceutical formulation.

1.5.2.1 Delivery of Non-Viral Vectors to Cells
Two major hurdles for non-viral gene transfer systems are the ability to deliver
plasmid DNA into the target cell and to achieve stable integration after the DNA
delivery. Delivering naked DNA to the nucleus of the target cells has been one common
challenge all non-viral DNA-based therapeutic approaches face. Non-viral vectors
cannot penetrate cellular membranes effectively without assistance. Both plasma and
nuclear membranes of the target cells serve as natural barriers preventing plasmid DNA
from entering the nucleus where the gene integration occurs. As a result, a delivery
carrier/method is required to actively deliver non-viral vectors to the target cells. Current
delivery methods that have been used to deliver non-viral vectors to the target cells and
achieve detectable transgene expression include hydrodynamic injection, liposomes, and
cationic polymer polyethyleneimine (PEI).(Boussif et al., 1995; Yant et al., 2000) More
recently, lipofection and electroporation-based techniques has been developed to deliver
plasmid DNA into more primary human cells such as primary human T cells, HSCs
(CD34+), and ESCs.(Wu et al., 2001b; Lundqvist et al., 2002; Oldak et al., 2002;
Weissinger et al., 2003; Cao and Zou, 2004; Gresch et al., 2004; Hollis et al., 2006;
Huang et al., 2006)

14

1.5.2.2 Integration of Non-Viral Vectors
The primary limitation of non-viral vector systems is the relatively short duration
of gene expression. Plasmids by themselves would not persist in HSC once the HSC
begin their massive proliferation for blood cell production. Several approaches may be
used to make the results of plasmid-mediated gene delivery have long-lasting
effects.(Porteus and Baltimore, 2003; Dorigo et al., 2004; Porteus and Carroll, 2005;
Hollis et al., 2006) Approaches including the Sleeping Beauty (SB),(Ivics et al., 2004)
piggyBac,(Ding et al., 2005) and Tol2(Balciunas et al., 2006) transposon systems and the
φC31(Thyagarajan et al., 2001; Olivares et al., 2002) and φBT1(Chen and Woo, 2005)
phage integrase systems all have shown great promise for non-viral based HSC gene
therapy.
The SB transposon system is the first non-viral vector used in gene therapy
experiments. The SB transposable element is a nonviral-based gene transfer system with
the ability to achieve stably integrated genes.(Izsvak and Ivics, 2004; Essner et al., 2005;
Hackett et al., 2005) SB transposon belongs to the Tc1/mariner family of transposable
elements and was reconstructed from the fish (salmonid) genome.(Ivics et al., 1997) In
its natural configuration, SB transposase consists of a DNA-binding domain at the N-
terminus, a nuclear localization signal (NLS), and a catalytic core domain at the C-
terminus of the transposase (Fig. 1-4A).(Yant et al., 2004) There are two components in
SB-mediated gene integration system: a transposon that carries the therapeutic gene and
a transposase that catalyzes the mobilization of the transposon. SB-mediated gene
integration is based on a cut-and-paste mechanism that integrates the transgene into the
chromosomal target site (Fig. 4B).(Walisko and Ivics, 2006) SB first binds to the
15

inverted repeats (IR) on both ends of transposon and excises the transposon from the
donor site. The excised transposon is then integrated into TA dinucleotide base pairs of
the host chromosomal DNA. The integration of SB transposon is found to be reversible
and occurs in a fairly random manner with respect to integration sites.(Vigdal et al.,
2002; Yant et al., 2005)
SB-mediated integration has been shown to achieve long-term transgene
expression and is capable of integrating into a wide range of host cells.(Izsvak et al.,
2000) Animal studies of SB transposon system have shown that SB transposon-mediated
gene therapy can effectively treat diseases such as hemophilia A (both factor VIII and
factor IX deficiencies), tyrosinemia type I, diabetes, and cardiovascular diseases.(Yant et
al., 2000; Montini et al., 2002; He et al., 2004; Liu et al., 2004; Ohlfest et al., 2005)
Furthermore, SB also has been used as the gene transfer system for an adaptive
immunotherapy clinical trial for B-lymphoid malignancies.(Williams, 2008) The
application and advances of SB transposon system as a strategy to introduce a stable and
permanent transgene to primary human HSCs both in vitro and in vivo is discussed in
detail in Chapter 2.

16

1.5.3 Hybrid Gene Transfer System
The hybrid systems are designed to combine the strengths such as the high
intracellular efficiency of the viral vectors and the high systemic potential and safety of
the non-viral vectors. Recent studies have reported that merging of viral and non-viral
vector gene transfer systems could provide a promising strategy for an alternative method
of gene delivery. A hybrid system with gene-deleted recombinant adenovirus (as a viral
component) and the SB transposon system (as a non-viral component) has been shown to
achieve a stable transgene expression in a NOD/SCID model.(Yant et al., 2002) The
non-viral component can also be modified with targeting ligands to facilitate the delivery
efficiency of the viral component and showed synergistic effect.(Diebold et al., 1999;
Boeckle and Wagner, 2006) When the viral component, such as adenoviral vector, was
coupled with a non-viral transferring-polylysine/DNA complexes, this hybrid system
greatly enhanced the receptor-mediated gene delivery and expression of transfected
Transposon
The transposon before
jumping
The transposase binds to the inverted repeats
Synaptic complex
The excised transposon
integrates into the target DNA
Excised transposon
Integrated transposon at a new
genomic site
Figure 1-4. (A) Schematic diagram of the
SB transposase. Figure was taken and
modified from Yant et al., 2004.
97
(B) Cut
and paste mechanism of SB-mediated gene
transfer. Figure was modified from
Walisko et al., 2006.
98

A.
B.
17

gene.(Wagner et al., 1992) A combination of baculovirus and galactosylated
polyethylenimine/DNA complexes also showed an enhanced transduction efficiency and
cell-type specificity which could be utilized as a potential hepatocyte-targeting gene
delivery system.(Kim et al., 2009)
NIL vectors have been shown to achieve gene delivery and transient gene
expression in various cell types, including human primary HPCs and ES cells.(Yanez-
Munoz et al., 2006; Lombardo et al., 2007) In combination with the non-viral SB
transposon system, the NIL/transponson hybrid gene transfer system could potentially
provide not only the benefit of viral transduction (high-level transient gene expression)
but also SB-mediated permanent transgene integration. The feasibility and potential of
utilizing NIL/SB transposon hybrid system as an alternative approach to achieve stable
transgene expression in the context of HSC gene therapy will be explored and evaluated
in Chapter 3.

1.6 Host Conditioning in HSC Gene Therapy
In addition to improving the gene transfer system to HSCs, the other strategy to
increase HSC engraftment efficiency in HSC gene therapy is by conditioning patients
with immune ablation such as irradiation or chemotherapy to remove their pre-existing
immunity. Evidence has shown that the host immunologic clearance of cells carrying
foreign or new genes influences the outcome of gene therapy. Specific cytotoxic T
lymphocytes (CTLs) against the fusion gene products of the herpersvirus thymidine
kinase (HSV-TK)(Bordignon et al., 1995a) and the hygromycin phosphotransferase
(Hy)(Riddell et al., 1996) or neo(Heim et al., 2000; Berger et al., 2001) gene led to the
18

drastic reduction of the autologous transduced T cells in humans. T cell-mediated
cytotoxicity against cellular expression of eGFP in vivo has been observed in murine
models.(Stripecke et al., 1999; Gambotto et al., 2000) Similar effects by host immune
response on HSC engraftment was also demonstrated in the canine model.(Lutzko et al.,
1999) In a more clinically relevant non-human primate model, induction of CTL and
antibody responses against the eGFP transgene product was observed and determined to
be responsible for the disappearance of eGFP-expressing cells in vivo.(Rosenzweig et al.,
2001; Morris et al., 2004) Based on the success of the recent HSC SCID gene therapy
trials, the absence of a functional immune system in the patients is believed to be a major
reason contributing to the clinical therapeutic outcome.(Cavazzana-Calvo et al., 2000;
Aiuti et al., 2002) The absent immune response against the genetically corrected HSCs
prevented the elimination of the gene modified HSCs and ultimately improved the
efficiency of the HSC engraftment and expansion in X-SCID patients. However, in
diseases where the host immunity remains competent, the gene modified HSCs are likely
to provoke host immune response towards the transgene product and subsequently result
in an inadequate engraftment (low transgene expression) and ultimately lead to absent or
limited clinical benefit. Therefore, eliminating host immune responses against the
transgene is crucial for stable transgene expression in HSC gene therapy.

1.6.1 Myeloablative Conditioning
There are several ways to achieve elimination or reduction of host immune
responses against transgene products and subsequently induce immune tolerance to a
therapeutic transgene. In a non-human primate model, long-term expression of a foreign
19

gene is achieved by delivery of the transgene with a lentiviral vector in combination with
myeloablative total body irradiation (TBI).(Heim et al., 2000; An et al., 2001; Kung et
al., 2003) The level of gene engraftment reached 10% or higher. Other methods to
induce immune tolerance have also been explored. Blockage of the co-stimulatory
signals at the initial time of encounter with the transplanted antigen is implicated to
induce anergy of potentially reactive T cells.(Wekerle et al., 2002; Vincenti et al., 2005)
In addition, modifications of the host immune system by creating mixed pools of donor-
host hematopoietic chimerism also show improved gene marking.(Forman, 2005; Koporc
et al., 2006)
High intensity myeloablation such as high dose TBI (1,000 – 1,300 cGY) and
chemotherapy such as high dosage of busulfan is used to “make space” in the bone
marrow for HSC engraftment. Immunosuppressants are often added to prevent
immunologic rejection. However, the adverse effects associated with such intensive
cytoreductive regimens are usually high and this regimen-related toxicity often leads to
even higher risk of morbidity and mortality.(Vassal et al., 1990; Vassal et al., 1996)
Tremendous efforts have been devoted into establishing non-myeloablative conditioning
that could achieve similar levels of clinical effect with relatively moderate adverse
effects.(Rosenzweig et al., 1999; Aiuti et al., 2002; Andersson et al., 2003; Kahl et al.,
2006; Kang et al., 2006)
1.6.2 Non-Myeloablative Conditioning
Non-myeloablative conditioning is defined as using low dose of TBI (200 – 400
cGY) or busulfan to create space in the bone marrow micro-environment and thereby
increasing the efficacy of HSCT. In an ADA-SCID clinical gene therapy trial, a
20

combination of gene therapy with non-myeloablative conditioning has demonstrated an
increased level (5% - 15%) of gene engraftment.(Aiuti et al., 2002) In contrast to the X-
SCID(Bordignon et al., 1993; Bordignon et al., 1995b; Kohn et al., 1995; Cavazzana-
Calvo et al., 2000) and other earlier ADA-SCID gene therapy trials(Bordignon et al.,
1993; Bordignon et al., 1995b; Kohn et al., 1995; Cavazzana-Calvo et al., 2000) where
no conditioning regime protocol was used, low dosage of busulfan (4 mg/kg) used as
non-myeloablative conditioning has improved the host environment for efficient
engraftment of the gene modified HSCs. The success of this ADA-SCID gene therapy
trial has demonstrated the feasibility of using a lower dose of busulfan as the non-
myeloablative conditioning for gene therapy.
Although non-myeloablative TBI has also been demonstrated to induce immune
tolerance and increase gene engraftment in nonhuman primate models, the level of
engraftment was still too low to achieve any clinical benefit for gene
therapy.(Rosenzweig et al., 1999; Kang et al., 2001) More importantly, the sensitivity to
irradiation follows a steep dose-response curve.(Giri et al., 2001) Therefore, providing
an alternative method for non-myeloablative conditioning, such as using a low-dose
chemotherapeutic agent in a clinical-relevant nonhuman primate model, could be
beneficial. A non-myeloablative conditioning regimen with a combination of
chemotherapeutic agent busulfan and fludarabine is discussed in Chapter 4.

21

Chapter 2 - Optimization of the Sleeping Beauty Transposon System to Achieve
Stable Transgene Expression in Human CD34+ Hematopoietic Stem/Progenitor
Cells

2.1 Introduction
The Sleeping Beauty (SB) transposon system, as one of the plasmid-mediated
gene delivery approaches, is a recombinase-based gene integration system with the ability
to stably integrate genes in various target host cells.(Izsvak et al., 2000; Izsvak and Ivics,
2004; Essner et al., 2005; Hackett et al., 2005) SB transposon belongs to the
Tc1/mariner family of transposable elements and was reconstructed from the fish
(salmonid) genome.(Ivics et al., 1997) In its natural configuration, SB transposase
consists of a DNA-binding domain at the N-terminus, a nuclear localization signal (NLS),
and a catalytic core domain at the C-terminus of the transposase.(Yant et al., 2004)
There are two components in the SB-mediated gene integration system: a transposon that
carries the therapeutic gene and a transposase that catalysis the mobilization of the
transposon. The components are encoded by two separate plasmids that are co-delivered
to the target cells. In this two-plasmid system,(Walisko et al., 2007) one plasmid
carrying an eGFP reporter cassette (or a therapeutic gene) flanked by the SB
inverted/direct repeats (IR/DR) sequences for permanent integration is co-electroporated
with a second plasmid carrying the SB transposase expression cassette for transient
production of the SB transposase enzyme. SB-mediated gene integration is based on a
cut-and-paste mechanism that integrates the transgene into the chromosomal target
22

site.(Walisko and Ivics, 2006) SB first binds to the IR/DR sequences on both ends of
transposon and excises the transposon from the donor site. The excised transposon is
then integrated into TA dinucleotide base pairs of the host chromosomal DNA.
The major hindrance to using non-viral gene modifications in HSC has been the
great difficulty getting them into HSC, compared to the more efficient gene delivery by
viral vectors. Methods such as transfection, lipofection, or electroporation, suffer from
low efficiency and/or high cytotoxicity. More recently, stable gene transfer and
expression in primary human T cells, neurons, embryonic cells, and HSC (CD34+) by the
SB transposon system in vitro have been reported by utilizing the Amaxa nucleoporator
(an electroporation-based device; AMAXA Inc., Walkersville, MD) as the SB carrier in
SB-mediated gene therapy.(Hollis et al., 2006; Huang et al., 2006; Wilber et al., 2007;
Zeitelhofer et al., 2007; Hohenstein et al., 2008; Zeitelhofer et al., 2009) Amaxa
nucleoporation has been developed as a more efficient and gentle approach, delivering
SB plasmid components via electroporation.
Despite the success of SB gene transfer, the efficacy of gene transfer by SB-
mediated system to primary cell populations for HPC gene therapy still remains to be
optimized. Based on the data reported by Hollis et al., suboptimal stable gene expression
was achieved. The level of eGFP expression in CD34+ cells (enriched for HSCs) by SB-
transposon system drastically decreased from 30-70% to 1-6% by two weeks post-
electroporation.(Hollis et al., 2006) The gene transfer efficiency of CD34+ cells
nucleoporated with the SB plasmids was also evaluated in vivo. The non-obese diabetic
NOD/SCID/B2m(null) mice were transplanted with electroporated human CD34+ cells to
demonstrate human cell engraftment and reporter gene transduction. Unfortunately, the
23

results were inconclusive because of significant impairment of cell survival and
engraftment due to cytotoxicity in electroporated cells. Therefore, further optimization
remains necessary to verify and improve the efficacy of the SB transposon gene transfer
system for HSC gene therapy.
In this chapter, we optimized the SB transposon-mediated gene transfer system to
achieve higher stable transgene expression in K562 human erythroleukemia cells, Jurkat
human T-lymphoid cells, and primary human CD34+ hematopoietic stem/progenitor
cells. A hyperactive mutant of SB, HSB,(Baus et al., 2005) with higher transposition
efficiency than the original SB transposase was used to increase the transposition efficacy
in the target cells. In addition, the expression of the SB transposase and the transgene
cassette carried by the transposon were also optimized analyzing three different viral and
cellular promoters: the modified MPSV long terminal repeat enhancer-promoter
(MNDU3), the human cytomegalovirus (CMV)

immediate-early region enhancer-
promoter, and the human elongation factor 1 (EF1α) promoter. The stable gene transfer
efficiency of the optimized SB-mediated gene delivery system was also evaluated in vivo
in a NOD/SCID/γC(null) (NSG) neonatal transplant model. The optimized SB
transposon system in primary human CD34+ hematopoietic progenitors reported here has
improved the stable gene transfer efficiency by ~30-fold, compared to our prior published
data.(Hollis et al., 2006)
Furthermore, this study is demonstrating that SB-modified human CD34+
stem/progenitor cells can be engrafted and differentiated into multi-lineage cell types in
vivo. More importantly, long-term repopulating capacity of the SB-modified human
CD34+ cells is conserved following secondary transplantation. This suggests that SB
24

transposon can achieve stable transgene integration in primitive Long term HSCs (LT-
HSCs) which shows promise for further advancement of non-viral based gene therapy
using hematopoietic stem cells.

2.2 Materials and Methods
Plasmid construction
The transposon plasmid, pT-MNDU3-eGFP-BGH(Hollis et al., 2006), used in
this study contains the Sleeping Beauty (SB) inverted repeat (IR) sequences flanking the
eGFP expression cassette. The pT-MNDU3-eGFP-BGH plasmid was modified from
plasmid pT-MC containing the IRs from SB flanking a multi-cloning site (kind gift from
Dr. Mark Kay, Stanford University) as described perviously(Hollis et al., 2006). The
transient expression plasmids pCMV-SB and pCMV-mSB containing regular and
defective versions of the SB transposase respectively were also obtained from Mark
Kay(Yant et al., 2000). The plasmid pCMV-HSB16, containing the “hyper-active”
version of the SB transposase, HSB16, was obtained from Dr. Bradley Fletcher
(University of Florida)(Baus et al., 2005).
The plasmid pT-EF1α-eGFP-BGH-attB was created from pT-MNDU3-eGFP-
BGH-attB(Hollis et al., 2006) and pCCL-EF1α-GFP(Uetsuki et al., 1989; Mizushima
and Nagata, 1990; Nakai et al., 1998; Ramezani et al., 2000; Haas et al., 2003; Nakai et
al., 2003). The MNDU3 fragment was removed from pT-MNDU3-eGFP-BGH-attB with
BgIII. The human EF1-α promoter was removed from pCCL-EF1α -GFP with PvuI and
BamHI, blunted-ended and inserted into the BgIII site of pT-X-eGFP-BGH-attB to create
pT-EF1α-eGFP-BGH-attB. The human EF1-α promoter fragment generated from PvuI
25

and BamHI was also used to create pEF1α-HSB16. The CMV promoter was removed
from the plasmid pCMV-HSB16(Baus et al., 2005) with SpeI and BamHI to create pX-
HSB16. The EF1-α promoter was then inserted into the blunted SpeI site to obtain
plasmid pEF1α-HSB16. The plasmid pMNDU3-HSB16 was created by inserting
MNDU3 promoter into pX-HSB16. The MNDU3 promoter was obtained from pT-
MNDU3-eGFP-BGH-attB using BgIII and SpeI, blunted-ended and ligated into blunted
BamHI site of pX-HSB16. The plasmids used in this study are illustrated in Fig. 2-1.

Figure 2-1. Vectors for Sleeping Beauty-mediated gene transfer. The pTransposon
transposon-plasmids include the SB inverted repeat (IR/DR) sequences flanking the
expression cassette, which consists of the promoter, the eGFP coding sequences (eGFP),
and a bovine growth hormone polyadenylation site (pA). The pSB transposase transient
expression plasmids contain the Sleeping Beauty (SB) or hyperactive Sleeping Beauty
(HSB16) transposase (or the mutant inactive SB, not shown) and the BGH polyA
sequence driven by various viral and cellular promoters including human CMV, MNDU3
retroviral LTR, and human EF1-α.

26

Cells
Human K562 myeloid leukemia and Jurkat T cells were both obtained from
ATCC (Manassas VA) and cultured in RPMI 1640, 10% fetal calf serum, with 2 mM
glutamine and penicillin/streptomycin (100 U/mL). CD34+ cells were isolated from
human cord blood (CB) obtained from normal deliveries using Ficoll-paque-PLUS
(Amersham Pharmacia Biotech, Piscataway, NJ) density gradient centrifugation followed
by Miltenyi MidiMACs separation columns (Miltenyi Biotech, Sunnyvale, CA). Use of
the CB was approved by the Committee on Clinical Investigations at Childrens Hospital
Los Angeles, CA(Hao et al., 1995).

Electroporation
Cells were electroporated using the Amaxa nucleoporator according to the
manufacturers’ instructions. Amaxa nucleoporation Cell Line Kit with program T-16
was used to electroporate 1 x 10
6
cells for K562 cells, and program C-16 was used to
electroporate 2 x 10
6
cells for Jurkat cells. For human CD34+ cells, 2 x 10
6
were used
with AMAXA program U-08. Prior to electroporation, the freshly isolated CD34+ cells
were stimulated overnight in X-vivo 15 medium (Cambrex, East Rutherford, NJ)
containing 2 mM L-glutamine with 50 ng/mL Stem Cell Factor (SCF; R&D system
[R&D]. Minneapolis, MN), 50 ng/mL flt-3 ligand (R&D), and 50 ng/mL thrombopoietin
(R&D).
Following electroporation, CD34+ cells for in vitro analyses were cultured for
four weeks under conditions inducing myeloid differentiation in retronectin-coated plates
(Takara Mirus Bio, Madison, WI) in Iscove’s Modified Dulbecco’s Medium with 20%
27

fetal calf serum, 0.5% bovine serum albumin (BSA), 2 mM glutamine, and
penicillin/streptomycin (100 U/mL) plus 5 ng/mL human interleukin IL-3 (Biosource
International, Camarillo, CA), 10 ng/mL IL-6 (Biosource International), and 25 ng/mL
SCF (R&D).
Human CD34+ cell transplantation into neonatal immune-deficient mice
The NOD/SCID/γC(null) (NSG) mice (NOD.Cg-Prkdc
scid
Il2rg
tm1Wjl
/SzJ , Stock#
005557; Jackson Labs, Bar Harbor, ME) were housed in accordance with the guidelines
of the Institutional Animal Care and Use Committee (Saban Research Institution at
Childrens Hospital Los Angeles, CA) and the National Institutes of Health. All animals
were handled in laminar flow hoods and housed in micro-insulator cages in a pathogen-
free colony.
On the day of transplantation, CD34+ cells were electroporated after 5 - 8 hours
of pre-stimulation in X-vivo 15 medium (Cambrex, East Rutherford, NJ) containing 2
mM L-glutamine with 50 ng/mL Stem Cell Factor (SCF; R&D system [R&D].
Minneapolis, MN), 50 ng/mL flt-3 ligand (R&D), and 50 ng/mL thrombopoietin (R&D).
Electroporated cells were with trypan blue exclusion to determine numbers of live cells
and resuspended in PBS with heparin (10 U/mL) for transplantation at a concentration of
1 x 10
6
live cells per 50 μl within 1-2 hours post-electropration. Prior to transplantation,
NSG mice received 150 cGy sub-lethal total body irradiation from a
137
Cesium source
with attenuator. Neonatal NSG mice (1 to 2 day-old) received approximately 1 x 10
6

modified human CD34
+
cells by facial vein injection using a 28-gauge needle.
Transplanted pups were housed with nursing mothers and weaned to separate cages at
three weeks. Blood samples were collected via retro-orbital venous plexus under general
28

anesthesia with isoflurane at 6 and 10 weeks post-transplant to evaluate the engraftment
efficiency. Bone marrow, thymus, spleen and blood were harvested from each animal at
5 months post-transplant for FACS and quantitative PCR analyses.
Secondary bone marrow transplantation
Adult (6 to 10-week-old) NSG mice were used as recipient for the secondary
transplants of marrow from the mice that had received primary transplants of human
CD34+ cells as neonates five months earlier. At 5 months after transplantation, the
primary recipient mice were euthanized under CO
2
narcosis. Femurs and tibias were
harvested and washed in phosphate-buffered saline (PBS) with 5% fetal bovine serum
(FBS; Omega Scientific, Inc., Tarzana, CA). Marrow was flushed using a 23-G needle
and 1-mL syringe filled with PBS with 5% FBS. Cells were counted with trypan blue
exclusion to determine numbers of live cells and were resuspended in 100 μl PBS with
5% FBS for transplantation. Recipient mice received 270 cGy of sub-lethal irradiation
from a
137
Cesium source with attenuator one hour pre-transplant. Approximately 2-7 x
10
7
cells bone marrow cells were transplanted into each recipient mouse via retro-orbital
venous plexus under general anesthesia with isoflurane. Bone marrow cells of the
secondary recipients were analyzed by FACS for human CD45 and eGFP expression 2
months post-transplant.
Flow cytometry
In vitro transgene expression analysis: For in vitro studies, the electroporated
cells were analyzed by flow cytometry performed on a FACSCalibur (Becton-Dickinson
Immunocytometry Systems, San Jose, CA) using CellQuest software for eGFP transgene
expression weekly for up to 4 weeks. Initial eGFP expression and regimen-related
29

toxicity in the electroporated cultures were analyzed by fluorescent-activated cell sorting
(FACS) on day 3 post-electroporation. Toxicity was determined using propidium iodide
(Sigma-Aldrich, St. Louis, MO) staining followed by FACS analysis. Levels of eGFP
expression obtained after week 4 of culture were defined as an indication of stable long-
term transgene integration in this study.
In vivo hematopoietic chimerism analysis: To detect human cells in NSG mice,
multicolor cytometric analysis was performed using a FACSCalibur. Peripheral blood
(PB) was taken from the retro-orbital venous plexus at 6 and 10 weeks after
transplantation under general anesthesia to assess the level of engraftment. Blood was
collected through heparinized calibrated pipettes (Drummond Scientific, Broomall, PA)
and transferred to 50 μl of FBS for FACS analysis. At 5 months post transplantation, the
mice were euthanized under CO
2
narcosis. The PB, femurs, thymus, and spleens were
harvested from each animal. Cell suspensions from each organ were made by pressing
tissue through a 70-μm sterile nylon cell strainer (BD Biosciences, San Jose, CA) and
were subjected to FACS analysis and quantitative PCR. Samples were depleted of
erythrocytes with BD FACS Lysing Solution (BD PharMingen, San Diego, CA) and
approximately 3 x 10
6
cells from each sample were incubated in 100% FBS for 30
minutes at 4°C to block non-specific staining. The remaining cells were used for PCR
analysis (see below). The appropriate volumes of indicated antibodies (BD Pharmingen)
were added to the blocked samples for 30 minutes in the dark at room temperature.
Human T cells were examined by triple staining with peridinin-chlorophyll-protein
(perCP) conjugated anti-human CD45 antibody, allophycocyanin (APC)-conjugated anti-
human CD4 antibody, and phycoerythrin (PE)-conjugated anti-human CD8 antibody. To
30

detect human B and NK cells in each sample, 3-color cytometric analysis with perCP-
conjugated anti-human CD45 antibody, APC-conjugated anti-human CD19 antibody, and
PE-conjugated anti-human CD56 antibody was also performed. Lastly, human
granulocytes were detected by double staining with perCP-conjugated anti-human CD45
antibody and PE-conjugated anti-human CD14 antibody.
Hematopoietic repopulation analysis: Bone marrow was harvested from adult
NSG mice at 2 months post-secondary transplantation. To detect repopulating human
eGFP
+
cells in the recipients, bone marrow cells were stained with perCP-conjugated
anti-human CD45 antibody (BD PharMingen) and analyzed by FACS for human CD45
+
and eGFP
+
expression.
Quantitative PCR analysis
eGFP DNA copy numbers were determined by real-time quantitative PCR
(qPCR). DNA from in vitro experiments was extracted using the Qiagen DNeasy Tissue
Kit (Qiagen, Valencia, CA). DNA from tissues and cell populations purified from organs
were extracted using phenol/chloroform and resuspended in Tris-
ethylenediaminetetraacetate (TE). All DNA was quantified using fluorimetry and a
DNA-specific dye (Hoescht dye; Sigma, St. Louis, MO). qPCR was performed with
primers and probe designed to amplify integrated eGFP sequence: sense primer (ctg ctg
ccc gac aac ca), antisense primer (gaa ctc cag cag gac cat gtg), and the TAMRA probe
sequence (ccc tga gca aag acc cca acg aga). The primer concentrations (sense and
antisense) were 400 nM and the probe concentration was 50 nM in all reactions. All
reactions utilized Universal Master Mix (Applied Biosystems, Inc. (ABI), Fullerton, CA)
and were run under default conditions in a 7900HT Fast Real-Time PCR System (ABI).
31

Each of the wells contained 350 ng of template DNA and were compared to a standard
curve made by diluting DNA from a cell line containing one copy of an integrated eGFP
gene diluted into DNA of non-transduced cells, yielding a detection sensitivity of
1:100,000 vector-containing cells.
Statistical analysis
The Student t test was used to determine statistical significance, and p < 0.05 was
considered significant.

2.3 Results
HSB is a more efficient enzyme in comparison to the original SB
Baus et al derived a hyperactive mutant version of the SB transposase (HSB)
which they reported to increase SB-mediated transposition up to 17-fold over the original
SB transposase in Hela cells.(Baus et al., 2005) To evaluate the transposition efficacy of
HSB in our target cell types, namely human hematopoietic cells, HSB was compared
with the original SB using a two-plasmid system(Hollis et al., 2006) in K562 human
erythroleukemia cells, Jurkat human T-lymphoid cells, and primary human CD34+
hematopoietic progenitor/stem cells. SB components were delivered in trans into the
target cells by nucleoporation. One plasmid carrying a transposon consisting of the eGFP
reporter gene under transcriptional control of the MNDU3 retroviral LTR promoter and
flanked by the SB IR sequences (pT-MNDU3-eGFP-BGH) was co-delivered with a
transposase-expressing plasmid containing the original SB (CMV-SB), the hyperactive
SB (CMV-HSB), or an inactive mutant SB transposase (CMV-mutSB) (Fig. 2-1).
32

In these studies, 10 μg of the transposon plasmid (pT-MNDU3-eGFP-GBH) and 1
μg of the transposase-expressing plasmid were used. Initial levels of GFP-expressing
cells three days after nucleoporation were consistent for each cell type independently of
which SB plasmid was used (Fig 2-2.). K562 cells showed the highest percentages of
GFP-expressing cells (≥80%) and the Jurkat cells had >50% expressing GFP. The
primary human CD34+ cells had a lower level of 6-12% expressing. In all three cell
types, cells that were electroporated with the mutant inactive SB transposase plasmid
(CMV-mutSB) or with no transposase plasmid did not show any stable eGFP reporter
gene expression, with GFP-expressing cells declining to background levels by 4 weeks
post-electroporation (Fig. 2-2). In contrast, cells receiving the SB or HSB plasmids
persistently expressed GFP out to 4 weeks. This observation indicated that a functional
SB transposase was required to achieve stable transgene persistence.

33

Figure 2-2. Stable long-term HSB16- and SB-mediated gene transfer. Transgene
expression over time in (A) K562, (B) Jurkat, and (C) primary human CD34
+
cells
isolated from cord blood. 10 μg of the transposon plasmid (pT-MND-eGFP-BGH) and 1
μg of either pCMV-SB (filled squares), pCMV-HSB (filled triangles), or pCMV-mutSB
(filled circle) transposase expressing plasmid were co-electroporated into each cell
population. Cells were also electroporated with pT-MND-eGFP-BGH alone (open
squares) or with AMAXA nucleoporation solution only without added DNA (open
diamonds) as controls. Aliquots of cells were analyzed by FACS at each time-point to
determine the percentage of GFP-expressing cells and the percentage of PI (+) cells. Each
experiment was repeated three times (n=3) with 2-3 replicates per experiment. Error bars
represent the standard error of the mean (SEM).
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20 25 30
% eGFP+
Mock
eGFP 10mcg
eGFP/SB 10:1
eGFP/HSB16 10:1
eGFP/mutSB 10:1
Number of day(s)
0
10
20
30
40
50
60
70
80
0 5 10 15 20 25 30
% eGFP+
Mock
eGFP 10mcg
eGFP/SB 10:1
eGFP/HSB16 10:1
eGFP/mutSB 10:1
Number of day(s)
0
2
4
6
8
10
12
14
0 5 10 15 20 25 30
% eGFP+
Number of day(s)
Mock
eGFP 10mcg
eGFP/SB 10:1
eGFP/HSB16 10:1
eGFP/mutSB 10:1
A
B
C
34

The functional assay based on stable eGFP expression showed that HSB was a
more efficient enzyme compared to SB under the same delivery conditions in K562,
Jurkat, and primary human CD34+ cells (Fig. 2-2). K562 cells electroporated with the
HSB-expressing plasmid showed a 2-fold higher level of stable eGFP expression in
comparison to cells electroporated with the original SB at 4 weeks post-electroporation
(64% vs. 37%). Jurkat human T cells that received the HSB plasmid showed a 4.5-fold
increase in stable transgene expression compared to cells receiving the original SB
plasmid (9% vs. 2%). The stable transgene expression detected in primary human
CD34+ cells that were electroporated with HSB plasmid was 6.6-fold higher (4% vs.
<1%) when compared to the CD34+ cells that were electroporated with the original SB
plasmid (Table 2-1). These findings demonstrated that HSB was a superior SB
transposase leading to a higher transposition efficacy than the original SB in these human
hematopoietic cell types.
In addition, the results from the stable transgene expression time-course study
suggested that there might be cell-type-specific differences in SB integration efficiency
as reported by other studies(Izsvak et al., 2000; Berry et al., 2006). K562 cells showed
the highest levels of long-term GFP expression, SB transposition efficiency was
significantly lower in Jurkat human T cells, and lowest in primary human CD34+ cells.
Over a series of experiments, the overall level of stable eGFP expression in K562 cells
using the HSB plasmid was approximately 10 to 16-fold higher than in Jurkat T cells and
primary human CD34+ cells (Table 2-1).

35

Table 2-1. Summary of HSB16 vs. SB-mediated gene transfer in K562, Jurkat,
primary human CD34+.

cultured human CD34+ under directing myeloid differentiation condition. Each
experiment was repeated three or four times (n = 3 or 4) with 2-3 replicates per
experiment. 10 μg of pT-MND-eGFP-BGH-attB plasmid and 1 μg of the transposase
plasmid (either pCMV-SB or pCMV-HSB) were used in each electroporation condition.
LTC-CD34+ referred to long-term Number of fold increase was calculated by dividing
the percent eGFP+ achieved by the HSB-mediated integration over the percent eGFP
achieved by the SB-mediated integration at four weeks post-electroporation. Student t-
test was performed for statistical analyses and the p-values were as indicated. SEM,
standard error of the mean.

Optimization of transposon-mediated gene transfer
Fresh vs. frozen CD34+ cells.
In our previously published studies of gene transfer with SB in human
hematopoietic cells, the SB-mediated gene transfer to primary human CD34+
hematopoietic stem/progenitor cells using electroporation was relatively inefficient,
caused significant cytotoxicity and was not able to achieve any stable transgene
expression in vivo(Hollis et al., 2006). Those studies were done using human umbilical
cord blood (CB) CD34+ cells that were cryopreserved and thawed prior to
electroporation. One of the possible ways to improve stable transgene expression could
36

be by using fresh human CD34+ cells to avoid any possible alterations in cell integrity
that might have occurred during the freeze and thaw process. To investigate the
transposition efficiency of the HSB transposon system using previously frozen human
CD34+, equal number of viable frozen/thawed or fresh primary human CD34+ cells
were co-electroporated with the transposon-containing plasmid (pT-MND-eGFP-BGH)
and the HSB-expressing plasmid. Fresh CD34+ cells showed a 6-fold higher stable
transgene expression than the frozen CD34+ under the same condition (Fig. 2-3A). This
finding suggested that HSB-transposon system delivered by electroporation occurs more
effectively in fresh human CD34+ cells. Therefore, the subsequent experiments using
primary human CD34+ cells in this study were all done with freshly-isolated human
CD34+ cells from CB and were not frozen prior to the experiment.
Nucleoporation program comparison
Stable gene transfer and expression in primary human T cells and hematopoietic
progenitor/stem cells (CD34+) by the SB transposon system have been reported by
utilizing the Amaxa nucleoporator (Amaxa Inc., Walkersville, MD) as the non-viral
deliver system for the SB-mediated gene transfer approach(Hollis et al., 2006; Huang et
al., 2006; von Levetzow et al., 2006; Nakata et al., 2007; Wiehe et al., 2007). The
AMAXA nucleoporator is an electroporation-based technique that augments transfer of
DNA into the nucleus of the target cell. An optimized electrical parameter for each
specific cell type has been pre-programmed into the nucleofection device and cell-
specific solutions are provided by the manufacturer. In an attempt to reduce cell
mortality occurring during electroporation to primary human CD34+ cells, two different
recommended nucleofection programs (U-01 and U-08) were compared. Primary human
37

CD34+ cells were electroporated with 10 μg of pT-MNDU3-eGFP-BGH using either
program U-01 or U-08 of the AMAXA nucleofector and analyzed by flow cytometry for
initial eGFP reporter gene expression and cell viability (by PI staining) three days after
electroporation. Cells electroporated using program U-08 showed a 4-fold higher
transfection efficiency compared to cells treated using program U-01 (10.7% vs. 2.8%)
with only a minimally higher cytotoxicity (32% vs. 28%) (Fig. 2-3B). This suggested
that while program U-01 was a gentler electroporation program, it was also less efficient
in DNA delivery into human CD34+ cells. Subsequent studies (and the ones previously
discussed) were done using the U-08 program.
Optimization of the transposon to transposase plasmid ratio
The quantities of transposon and transposase plasmids have been known to be one
of the major factors that determines the transposition efficiency of the SB transposon
system. An optimal ratio of transposase to transposon is needed to achieve high
transposition levels(Yant et al., 2000; Liu and Visner, 2007). Based on the previously
published quantities of SB plasmids used to achieve stable transgene expression in human
CD34+ cell in vitro (10 μg of the transposon and 10 μg of the transposase SB
plasmid)(Hollis et al., 2006), the amount of pCMV-HSB was varied from 1 to 10 μg with
the amount of transposon plasmid (pT-MND-eGFP-BGH) kept constant at 10 μg.
Populations of human CD34+ cells were electroporated and then analyzed by flow
cytometry to measure eGFP expression after 4 weeks, without any selection. A
transposon to transposase plasmid ratio of 10 μg to 1 μg was determined to be the
optimal ratio for the HSB transposon system to achieve the highest level of stable gene
transfer to the human CD34+ cells (Fig. 2-3C). Lower levels of stable gene transfer were
38

achieved using the SB plasmid at each of the amounts used. The greater activity of HSB
allows the total amount of DNA used in a nucleoporation reaction to be decreased from
20 μg (10 μg transposon and 10 μg SB transposase) to 11 μg (10 μg transposon and 1 μg
HSB transposase).

39

Figure 2-3. Optimization of transposon-mediated gene transfer. (A): 2 x 10
6
viable
frozen/thawed or freshly isolated human CD34+ cells isolated from CB were
electroporated with 10 μg of pT-MNDU3-eGFP-BGH with 1 μg of HSB transposase
expressing plasmid. FACS analysis was performed at four weeks post-electroporation for
stable transgene expression detection. Asterisks indicate significant difference (P < 0.01;
n = 3) between data point marked * and **. (B): Cells were electroporated with 10 μg of
pT-MNDU3-eGFP-BGH with either program U-08 or U-01 of the AMAXA
nucleofector. The percentages of eGFP (+) cells (bars) and the percentages of PI (+) cells
(circles) were determined by FACS analysis at three days post-electroporation for
transgene expression and cell viability, respectively. Asterisks indicate significant
difference (P < 0.05; n = 4) between data point marked * and **. (C): Primary human
CD34+ cells were co-electroporated with 10 μg of pT-MNDU3-eGFP-BGH and
increasing quantities of pCMV-SB (open bar), pCMV-HSB (filled black bar), or pCMV-
mutSB (filled grey bar). FACS analysis was performed at four weeks post-
electroporation for stable transgene expression. n = 2 (D): 10 μg of pT-MNDU3-eGFP-
BGH was co-electroporated with increasing quantities of pCMV-SB (filled squares),
pCMV-HSB (filled triangles), or pCMV-mutSB (filled circles). n = 2. The percentages
of PI (+) cells were determined by FACS analysis at three days post-electroporation. The
error bars represent the standard error of the mean (SEM).
0
1
2
3
4
5
0 1 5 10
% GFP+
Transposase (μ μ μ μg)
SB
HSB
mutSB
0
20
40
60
80
100
No plasmid 0 1 5 10
% PI+
Transposase (μ μ μ μg)
SB
HSB
mutSB
A
B
C
D
40

Interestingly, the levels of stable gene marking in human CD34+ cells were
inversely proportional to the quantity of HSB transposase plasmid used, declining from
3.4% using 1 μg of pCMV-HSB to less than 1% with 10 μg of pCMV-HSB. The
decrease in the level of stable gene expression observed for the largest quantity of
transposase could be either due to toxicity caused by the DNA plasmid(Hollis et al.,
2006), by the activity of the SB transposase per se, or may represent the phenomenon of
“over-production inhibition” where excessive amounts of transposase activity lead to
inhibition of transposition, presumably by increasing the rate of the reverse, excision
reaction. (Yant et al., 2000; Geurts et al., 2003; Hollis et al., 2006; Hackett, 2007)
When cells were mocked nucleoporated without any plasmid added to the
reaction, low levels of PI (+) cells were seen (< 10%) (Fig. 2-3D). Addition of 10 μg of
only the eGFP-expressing transposon plasmid, but no transposase plasmid, led to a large
increase in cytotoxicity, with more than 60% PI (+) cells. Addition of increasing
amounts of any of the transposase-expressing plasmids led to further dose-related
increases in the percentage of PI (+) cells, but these were essentially the same with any of
the HSB, SB and mutSB plasmids, indicating that the presence of functional HSB
transposase did not affect cell viability. Large quantities of plasmid added to the
nucleoporation, in contrast, were responsible for the cytotoxicity, as reported
previously(Hollis et al., 2006). Thus, the ability of the HSB to achieve greater stable
gene transfer with lower amounts of plasmid also moderately reduces acute cytotoxicity.

41

Optimization of Promoter for expression of transposase and transposon reporter

Promoters used in expression plasmids display different expression strengths,
depending on specific cell type.(Fitzsimons et al., 2002; Weber and Cannon, 2007) The
human CMV

early region

enhancer-promoter, for example, expresses strongly in non-
hematopoietic cells(Boshart et al., 1985) but performs poorly in vivo and in
hematopoietic cells.(Scharfmann et al., 1991; Kay et al., 1992; Baskar et al., 1996;
Miyoshi et al., 1999; An et al., 2000) In contrast to the CMV promoter, the human EF1-
α promoter has been identified as a strong promoter in primary human hematopoietic
cells(Ye et al., 1998; Chang et al., 1999) and exhibits high transcription activity in
vivo.(Taboit-Dameron et al., 1999) The MNDU3 LTR promoter, derived from the
MPSV retrovirus, has been shown to express well in murine and human hematopoietic
and lymphoid cells.(Halene et al., 1999; Haas et al., 2003)
To further optimize both the activity of the HSB transposase to mediate stable
gene persistence and the expression level of the reporter eGFP transgene carried by the
transposon in human hematopoietic cells, we evaluated these three different promoters.
Each promoter was cloned into an individual construct to express either the HSB
transposase or the eGFP reporter gene carried by the transposon. The optimization of the
HSB-mediated gene transfer efficacy involved two parts. First, the expression of the
HSB transposase by the different promoters was evaluated, using the pT-MNDU3-eGFP-
BGH-attB reporter transposon plasmid. Second, with the optimized HSB transposase
construct identified, the expression of the eGFP reporter gene contained in the transposon
construct using the different promoters was then examined.

42

HSB transposase expressed by human EF1-α promoter achieved the highest stable
transgene expression

In the comparison of different promoters driving expression of the HSB, K562
cells were much more permissive to HSB-mediated gene integration than Jurkat T cells
and primary human CD34+ cells with any of the promoters used to express HSB (Table
2-2). The long-term stable transgene expressions in K562 cells for all conditions
remained approximately at 80% by four weeks post-electroporation. Both Jurkat and
human CD34+ cells showed lower levels (5 to 14-fold respectively) of HSB-mediated
stable transgene expression than that observed in K562.
The highest level of stable transgene integration were achieved using the EF1-α
promoter to express HSB in all three cell types, compared to either the human CMV or
MNDU3 promoters (Table 2-2). While this effect was not significantly above the already
high level of gene transfer with the CMV-HSB plasmid in K562 cells, there was a 2-fold
increase in the percentages of GFP-expressing Jurkat and primary CD34+ cells when the
EF1-α promoter was used to direct expression of the HSB transposase.

43

Table 2-2. Comparison of the stable transgene expression achieved using HSB
transposon expressed by MNDU3, CMV, or EF1-α promoter in K562, Jurkat, and
primary human CD34+.

Each experiment was repeated 2-3 times (n = 2 or 3) with 1-3 replicates per experiment.
The values for the fold increase were calculated by dividing the percentage of eGFP-
positive cells achieved by pEF1α-HSB over the percentage of eGFP-positive cells
achieved by pCMV-HSB at four weeks post-electroporation. Student t-test was
performed for statistical analyses and p-values were as indicated. SEM, standard error of
the mean.

Optimal eGFP reporter gene expression from the integrated transposon was
influenced by the promoter and the target cell types

Next, we evaluated the MNDU3 and EF1-α promoters for expression of the eGFP
reporter gene from the integrated transposon in K562, Jurkat, and primary human CD34+
cells (Fig 2-4A). The mean fluorescent intensity (MFI) of the eGFP expression by the
MNDU3 promoter was two-fold higher than by the EF1-α promoter in K562 cells (Fig. 2-
4A and 2-4B). In the primary human CD34+ (cultured under conditions directing
myeloid differentiation {LTC-CD34+}), the MFI of eGFP expression was significantly
higher with the MNDU3 promoter than with the EF1-α promoter (P<0.01). In contrast,
the highest MFI of GFP expression in Jurkat human T cells occurred when the EF1-α
44

promoter was used to express the GFP reporter (Fig. 2-4A). Thus, the MND promoter
was more active in myeloid type cells (K562 erythroleukemia cells and cultured CD34+
cells), whereas the EF1-α promoter was more active in the T lymphoid cells.
By incrementally optimizing each element of the SB transposon system we have
been able to significantly increase the efficiency of stable gene delivery to human
hematopoietic cells, including primary human CD34+ hematopoietic progenitor/stem
cells. The level of stable gene expression increased 20-fold by combining the use of the
HSB transposase and the EF1-α promoter to direct HSB expression and the MND
promoter to express the GFP transgene (Fig. 2-5).

45

Figure 2-4. Optimization of promoter transposase and transposon reporter
expression
(A) Representative FACS plots showing stable HSB-mediated eGFP expression after
four weeks in K562, Jurkat, and primary human CD34+ cells (LTC-CD34+) cultured
under conditions directing myeloid differentiation. Average MFI values are indicated on
the upper left corner of each plot. (B) Comparison of the average MFI of the eGFP
expressing cells. LTC-CD34+ is referred to long-term cultured human CD34+ in
myeloid differentiation condition. 10 μg of pT-MND-eGFP-BGH-attB or pT-EF1α-
eGFP-BGH-attB transposon plasmids were co-electroporated with 1 μg of pEF1α-HSB
plasmid in the study. Cells were analyzed at week four post-electroporation for detection
of HSB-mediated stable eGFP reporter gene expression. Each experiment was done in 2-
3 replicates per condition. Error bars represents the SEM. Student t-test was performed
for statistical analyses and p-values were as indicated. ” * ” indicates that p value is less
than 0.01.
46

Figure 2-5. Overall SB-transposon system optimization in primary human CD34+
hematopoietic progenitor/stem cells in vitro. Representative FACS plots elucidating the
progression of optimization of the SB-mediated gene integration system done in this
study. The percentages of eGFP-positive cells at four weeks post-electroporation are
indicated on the upper left corner of each plot. The transposon : transposase plasmid
DNA combinations used for each condition are also denoted on the top of the FACS
plots.

HSB-modified human CD34+ progenitor cells were engrafted and reconstituted in
NSG mice after neonatal transplantation

Primitive human hematopoietic cells can be assayed on the basis of their ability to
repopulate immune-deficient NOD/SCID mice. HSB-mediated gene transfer efficiency
to the human CD34+ hematopoietic cells was therefore evaluated in vivo. Fresh primary
CD34+ hematopoietic progenitor cells were isolated from human cord blood,
electroporated with the optimized HSB plasmids, and transplanted into non-obese
diabetic NOD/SCID/γC(null) (NSG) neonatal mice (Fig. 2-6). To assess primitive
normal and leukemic human stem cells (SCID repopulating cells; SRC), human cell
engraftment (%CD45+) and gene transduction (% eGFP+) were determined in peripheral
blood from NSG mice at 6, 10, and 20 weeks post-transplantation by FACS analyses.

There were total of seven NSG litters (n = 49) received neonatal bone
transplantation with four different treatment groups. Twenty out of those 49 transplanted
neonatal NSG mice did not survive one week post
defect or poor nurturing by their birth parents.
were divided into treatment groups as follow: Group (A) non
(B) mock-transduced (n = 4); Group (C) electroporated with pT
attB transposon plasmid (n = 11); or Group (D) electroporated with pT
BGH-attB transposon plasmid (n = 11). HSB transpoase was also electroporated to the
transplanted human CD34+ cells to facilitate eGFP reporter gene integration in group C
and D.

Figure 2-6. Schematic diagram of the experimental timeline for
SB-mediated gene transfer to human CD34+ cells by NOD/SCID/
transplantation. NSG =
There were total of seven NSG litters (n = 49) received neonatal bone
transplantation with four different treatment groups. Twenty out of those 49 transplanted
neonatal NSG mice did not survive one week post-transplantation due to either birth
poor nurturing by their birth parents. The successfully transpl
were divided into treatment groups as follow: Group (A) non-transduced (n = 3); Group
transduced (n = 4); Group (C) electroporated with pT-MNDU3
attB transposon plasmid (n = 11); or Group (D) electroporated with pT
attB transposon plasmid (n = 11). HSB transpoase was also electroporated to the
transplanted human CD34+ cells to facilitate eGFP reporter gene integration in group C

6. Schematic diagram of the experimental timeline for in
mediated gene transfer to human CD34+ cells by NOD/SCID/
NSG = NOD/SCID/γ γ γ γC
47
There were total of seven NSG litters (n = 49) received neonatal bone marrow
transplantation with four different treatment groups. Twenty out of those 49 transplanted
transplantation due to either birth
The successfully transplanted animals
transduced (n = 3); Group
MNDU3-eGFP-BGH-
attB transposon plasmid (n = 11); or Group (D) electroporated with pT-EF1a-eGFP-
attB transposon plasmid (n = 11). HSB transpoase was also electroporated to the
transplanted human CD34+ cells to facilitate eGFP reporter gene integration in group C

in vivo analysis of
mediated gene transfer to human CD34+ cells by NOD/SCID/γ γ γ γC neonatal
48

Table 2-3 summarized the level of engraftment (% human CD45+) in mouse
peripheral blood and bone marrow at 10 and 20 weeks post-transplantation. Differences
were observed in the engraftment efficiencies of the non-transduced (group A), the mock-
transduced human CD34+ cells (group B), and the human CD34+ electroporated with
either MNDU3 or EF1a expressed eGFP in the presence of HSB (group C and D
respectively). When the cells were electroporated with or without plasmid DNA, the
engraftment efficiency of the transplanted CD34+ was significantly reduced (Fig. 2-7;
p<0.05). At ten weeks post-transplantation, an average 60% initial engraftment level was
observed in animals transplanted with non-transduced human CD34+ cells (group A). In
comparison, mice that received electroporated human CD34+ (group B, C, and D)
showed significantly 5-8 fold lower engraftment levels at a range of 8 – 12%. This
suggested that electroporation DNA delivery method to primary human CD34+ cells
might be detrimental to their engraftment and normal hematopoiesis ability.
Electroporation not only decreased the initial engraftment capacity of the CD34+ cells,
but also reduced to the long-term human hematopoiesis in vivo.
In addition, the level of SCID repopulating cells of the HSB treatment groups
(group C and D) increased to a comparable level (∼ 20%) as detected in the mock-
transduced group (group B) by 5 months post-transplantation despite the lower initial
engraftment levels of the HSB-modified human CD34+ cells (ave. = 3% vs. 7%
respectively). This further suggested that the significant decrease in engraftment level of
the HSB modified CD34+ cells was mostly due to electroporation and not the HSB
transposase or the integration process.

49

Table 2-3. Summary of engraftment level and transgene expression in primary
transplanted NSG mice.

“*” indicate eGFP expression was detected in human repopulating cells of the
transplanted NSG mice by FACS analysis.

%HumanCD45+eGFP+
Treatment Animal no. PBMC BM Spleen BM BM Spleen
week 10 week 20 week 20 week 20 week 20 week 20
1 48.8 4.7 48.5 0.0 0 0
(A) non-transduced; n = 3 2 61.2 59.0 55.1 0.0 0 0
3 63.0 ND ND ND ND ND
1 8.6 1.7 38.4 0.0 0 0
(B) mock-transduced; n = 4 2 1.6 8.4 11.6 0.0 0 0
3 0.0 0.3 12.0 0.0 0 0
4 41.3 48.0 89.0 0.0 0.001 0
1 3.5 58.2 23.0 0.0 0.001 ND
2* 24.9 51.2 92.1 0.6 0.006 0.160
3 0.2 0.0 0.1 0.0 0 0
4 0.0 0.3 3.0 0.0 0.109 0.010
5 0.3 ND ND ND ND ND
(C) pT/MNDU3-eGFP + pEF1- α-HSB 6 30.2 14.2 80.6 0.2 0.011 0.001
n = 11 7 2.9 71.1 79.5 0.0 0 0
8 3.8 55.2 80.7 0.0 0.001 0
9 5.7 8.5 4.1 0.1 0.006 0.012
10 0.5 1.4 3.0 0.2 0.037 0.010
11 34.5 46.1 57.9 0.0 0.004 ND
1 16.3 46.1 48.1 0.0 0.001 0.001
2 5.5 ND ND ND ND ND
3 0.2 1.8 1.2 0.0 0.017 0.025
4 2.3 66.9 63.0 0.0 0.001 0
5* 16.4 48.9 71.4 10.2 0.164 0.102
(D) pT/EF1- α-eGFP + pEF1- α-HSB 6 1.2 42.9 9.6 0.0 0.001 0.032
n = 11 7 21.1 ND ND ND 0.002 0.002
8 4.0 53.6 63.8 0.0 0.001 0
9 0.7 0.5 1.8 0.1 0.065 0.018
10 7.7 21.3 62.5 0.1 0.005 0
11 7.8 19.1 52.3 0.0 0.002 0.001
% Human CD45+ Gene marking (copies/human cell)
50

Figure 2-7. Comparison of engraftment levels of human CD34+ progenitor cells in
NSG mice. At the indicated times after approximately 1 x 10
6
primary human CD34+
cells neonatal transplantation, human CD45+ cells in mouse peripheral blood (PB) were
assayed by FACS analysis. Freshly isolated human CD34+ cells isolated from CB were
electroporated with 10 μg of pT-MND-eGFP-BGH-attB (group C; n = 11) or pT-EF1α-
eGFP-BGH-attB (group D; n = 11) transposon plasmids were co-electroporated with 1 μg
of pEF1α-HSB plasmid. As study controls, “non-transduced” indicates NSG mice that
received human CD34+ only (group A; n = 3), and “mock-transduced” indicates NSG
mice that received human CD34+ that were electroporated without plasmid DNA (group
B; n = 4). Asterisks indicate significant difference (P < 0.01) between data point marked
* and **.

Transgene expression detected in multiple organs of the NSG mice transplanted
with HSB-modified human CD34+ cells
To evaluate the efficiency of the HSB-mediated stable gene transfer to human
CD34+ cells in vivo, eGFP expression in SCID repopulating cells (%CD45+eGFP+) was
determined (Table 2-3). Stable transgene expression up to 2.5% was first observed by
FACS analysis at week 10 post-transplantation in the peripheral blood of the mice that
51

received HSB-modified human CD34+ cells. At 5 months after transplantation, high
eGFP expression level (MFI range from 1000 to 6500; data not show) was detected in
peripheral blood, spleen, bone marrow, and thymus (if present) of two animals, C-2 and
D-5 (Fig. 2-8A). Up to 11.3% human CD45+eGFP+ cells were detected in the bone
marrow of the transplanted NSG mouse 5 months post-transplantation.
eGFP expression detect in SCID repopulating cells of the NSG mice at 5 months
post neonatal bone marrow transplantation
eGFP expression in total SCID repopulating cells was determined in peripheral
blood (PB), bone marrow (BM), thymus, and spleen harvested from the transplanted
NSGs at 5 months post-transplantation. Distinct GFP+ populations were detected by
FACS at 5 months post-transplantation in two NSG mice that were transplanted with
HSB-modified human CD34+ cells. With an average engraftment level of 50% human
CD45+ cells in the BM, stable eGFP expression was detected in T (CD4 and CD8 single
positive), B (CD 19+), NK (CD 56+), and granulocytes (CD 14+) in both C-2 and D-5
animals (Fig. 2-8B). Normal T cell development was observed in the bone marrow of
both eGFP+ animals by 10 weeks post-transplantation. In animal C-2 bone marrow,
eGFP expression was detected in CD8+, CD19+, CD56+, and also CD14+ cells with the
highest %eGFP (13.1%) found in CD56+ NK cell population. Normal thymopoesis was
also observed in C-2 with 0.5% CD8+eGFP+. No eGFP expression detected in the CD
4+ or CD 19+ population of the BM and the thymus of C-2. In comparison to C-2,
animal D-5 showed a high percentage (16.4%) of eGFP+ cells in CD19+ population of
the bone morrow and 3.3% eGFP expression in its CD4+ cells.

52

Figure 2-8. Representative FACS analysis of eGFP expression detected in multi-
organ and multi-lineage differentiated cells from engrafted HSB-modified human
hematopoietic CD34+ cells 5 months post-transplantation. (A): Stable eGFP
expression was detected in peripheral blood, bone marrow, thymus, and spleen from NSG
mice transplanted with modified human CD34+ by HSB-mediated gene transfer. (B):
Stable eGFP expression was detected in CD4+ or CD8+ T cells, CD19+ B cells, CD56+
NK cells, and CD14+ granulocytes of all organs. Data shown here are FACS polts of the
BM and thymus harvested from the C-2 and D-5 NSG mice. The thymus of animal D-5
was not sufficient for immunostaining analysis. (C): Total bone marrow cells harvest
from D-5 NSG mice were successfully engrafted into the secondary adult NSG
recipients. eGFP transgene expression was also detected in NSG human repopulation
cells. Each number represents the percentages of human CD45+ eGFP+ cells in the
specific cell lineage.
A.
B.
C.
53

The eGFP marking (copy/cell) in mouse total bone marrow and spleen at 20
weeks post-transplantation is also summarize in Table 2-3 and Fig. 2-10. DNA from the
total cells harvested from the BM of each transplanted mouse was isolated and detected
for eGFP copy number by quantitative PCR analysis. There were no significant
differences between group C and D when the reporter gene was expressed by either
MNDU3 or EF1a promoter (geometric mean = 6.1 x 10
-4
vs 7.3 x 10
-4
copies per cell
respectively). Animal C-2 with 0.6% of human CD45+eGFP+ cells was detected with
0.003 eGFP copies/cell and animal D-5 with 11.3% of human CD45+eGFP+ cells was
detected with 0.08 eGFP copies/cell in bone marrow. Up to 0.147 copies of eGFP per
cell was detected in spleen of the heterogeneous mouse/SRC cells.
The eGFP expression levels of each cell lineage were also determined in PB, BM,
thymus, and spleen harvested from the transplanted NSGs (Fig. 2-9). The highest eGFP
expression was detected in the NK (21%) and B (25%) cell population in animal C-2
(group C: MNDU3 treatment group). In contrast, the majority of eGFP+ cells (12.5%)
were found in the B cell lineage of animal D-5 (group D: EF1α treatment group).

54

Figure 2-9. Summary of multi-lineage eGFP expression levels determined in
peripheral blood, bone marrow, thymus, and spleen harvested from the NSGs after
5 months neonatal transplantation of HSB-modified human CD34+ cells.

55

Figure 2-10. Summary of eGFP gene marking in human CD45+ cells isolated from
BM and spleen of the NSGs 5 moths post-transplantation. The geometric mean of the
eGFP marking is represented in filled rectangle.

To characterize human hematopoietic stem cells in the SCID repopulating cell
population detect in the grafted NSG mice, secondary transplantation was conducted.
Donor animals were selected based on the engraftment level detected in the peripheral
blood at 10 weeks post-transplantation. Two femur and tibias from each primary mouse
was

injected via retro-orbital venous plexus into individual secondary mice. Total of 8
adult NSG mice were transplanted with bone marrow cells harvested from primary mice
(Table 2-4). At 8 weeks post-transplant, substantial

secondary grafts with eGFP
transgene expression (0.7% human CD45+eGFP+) was detected in an animal that
received bone marrow cells from animal D-5. This indicates that the HSB transposon
system could achieve stable gene transfer to the primitive HSC population

with self-
renewal ability and long-term repopulating capacity (Fig. 2-8C).
56

Table 2-4. Engraftment level and transgene expression in secondary transplanted
NSG mice.

Bone marrow cells were harvested from engrafted NSG mice at 5 months post-neonatal
transplantation as donor cells for the secondary transplantation. Adult NSG mice were
used as recipients for the secondary transplantation. Transplanted number of cells
represents the total cells harvest from the femurs and tibias of the donor mice. Estimated
transplanted human CD45+ cell numbers were calculated based on the % human CD45+
detected in the bone marrow of the donor at 5 months post-transplantation (also see table
3). Engraftment represents the % human CD45+ cells detected in the bone marrow of the
recipient mice at 2 months post-transplantation. ND indicates not done.

57

To characterize human hematopoietic stem cells in the SCID repopulating cell
population detect in the grafted NSG mice, secondary transplantation was conducted.
Donor animals were selected based on the engraftment level detected in the peripheral
blood at 10 weeks post-transplantation. Two femur and tibias from each primary mouse
was

injected via retro-orbital venous plexus into individual secondary mice. Total of 8
adult NSG mice were transplanted with bone marrow cells harvested from primary mice
(Table 2-4). At 8 weeks post-transplant, substantial

secondary grafts with eGFP
transgene expression (0.7% human CD45+eGFP+) was detected in an animal that
received bone marrow cells from animal D-5. This indicates that the HSB transposon
system could achieve stable gene transfer to the primitive HSC population

with self-
renewal ability and long-term repopulating capacity (Fig. 2-8C).

2.4 Discussion
This study reports improved efficacy using the Sleeping Beauty transposon system
for stable gene transduction of human CD34+ hematopoietic stem/progenitor cells
achieved by incrementally optimizing elements of the system. By combining multiple
modifications to the nucleoporation delivery methods we used previously, we improved
gene transduction to CD34+ cells so that we could readily detect expression of the GFP
reporter gene in cells transplanted into immune-deficient mice and beyond into
secondarily transplanted recipients.
We first assessed benefits from using a “hyperactive” version of the SB
transposase (HSB)/ This had been created by a phylogenetic-based comparison approach
to generate a combination of mutations in the N-terminal DNA binding domain, with
58

triple alanine substitutions (K33A, T83A, and L91A),(Geurts et al., 2003; Yant et al.,
2004; Baus et al., 2005) and also in the C-terminal domain with mutations in the
integrase and also the C1 and C2 proteins. We observed that the level of stable gene
transfer to human hematopoietic and lymphoid cells was consistently higher when the
HSB was used, compared to the original SB transposase. The greater activity of the HSB
allowed us to reduce the total amount of plasmid DNA used for nucleoporation, with 1 ug
of HSB plasmid leading to greater gene transfer than 10 ug of the parental SB plasmid.
Using the HSB transposase, we defined an optimal ratio of transposase to transposon
plasmids in the nucleoporation system. Under the conditions we used, the optimal ratio of
transposon plasmid to HSB transposase plasmid was 10 μg to 1 μg. These numbers may
vary for different cell types, different electroporation conditions and with different
plasmids.
With the greater activity of the HSB transposase, we observed the phenomenon of
over-expression inhibition, which has been one of the major limitations of the SB
transposon system for efficient gene delivery. Only a narrow window of transposon to
transposase ratio will allow maximal transposition activity of the SB transposase(Geurts
et al., 2003). SB transposition efficiency depends greatly on the relative availability of
transposase molecules per transposon. Studies demonstrated that a total of four
transposase molecules (two per IR binding site on each end of a transposon) were
required to complete a stable integration of one transposon molecule. The transposase
molecules can bind to each other in a crisscross manner to juxtapose the two ends of the
transposon.(Hartl et al., 1997; Cui et al., 2002; Geurts et al., 2003) Insufficient levels of
transposase will not effectively form a functional synaptic complex for transgene
59

transposition. However, surplus levels of transposase will also inhibit the SB
transposition activity(Lohe et al., 1996; Hartl et al., 1997; Lampe et al., 1998; Geurts et
al., 2003; Wilson et al., 2005) by preventing the juxtaposition of the transposon ends and
inhibiting the transposition process. In this study, an over-expression inhibition effect
was observed when the transposon to transposase ratio increased from 10:1 to 2:1.
As another strategy to increase the efficiency of HSB transposition activity, we
also investigated the effect of different promoters used for the expression of the HSB
transposase and the GFP reporter cassette in the transposon. The promoter from the
ubiquitously-expressed cellular Elongation Factor alpha-1 gene (EF1-α) used to direct
expression of the HSB transposase led to the highest level of stable transgene integration
in each of the cell types examined. The optimal promoter for expression of the transgene
from the integrated transposon varied in the different target cell types studied. We
observed that the MND promoter, from the Myeloproliferative Sarcoma Virus, was more
active for expressing the transgene in human myeloid cells (K562 and primary myeloid
cells derived from transduced CD34+ cells). In contrast, the EF1-α promoter was more
active in the T lymphoid cells. It is important to consider the different expression needs
for the transposase, in the immediate target cells, and the transposon expression cassette,
in the ultimate cell type where transgene expression is intended.
Other important factors that we examined included the quality of the CD34+ cells
used for electroporation, and the specific electroporation parameters encoded by the
Amaxa nucleoporator. Freshly-isolated human CD34+ cells were shown to allow higher
levels of initial and stable transgene expression than cryopreserved cells. There was not
higher immediate cytotoxicity to the frozen cells, so the basis for their poorer
60

transduction is not known. Amaxa nucleoporation program setting U-08 led to a 6-fold
higher transgene expression than did program U-01; there was somewhat more acute
cytotoxicity with the U-08 program than with the more gentle U-01 program, but U-08
nonetheless led to higher DNA delivery efficiency in human CD34+ HSCs.
With the significantly improved gene delivery efficiency achieved in primary
human CD34+ in vitro, these optimized HSB transposon methods were evaluated in a
more stringent model for evaluating transduction of human HSC, by xenografting the
transduced CD34+ cells in the NSG neonatal transplantation model. In our prior study
with the Sleeping Beauty system and human CD34+ cells, we were not able to obtain
engraftment of transduced cells in an immune-deficient mouse model, primarily due to
severe cytotoxicity from the nucleoporation.(Hollis et al., 2006) For the studies reported
here, we made several changes to our approach. First, we used the NSG mouse strain for
these studies, whereas we had used NOD/SCID/β2microglobulin-/- mice in the previous
ones. We have found that the NSG model affords significantly greater human HSC
engraftment (and life expectancy) and supports vigorous T cell production.(Ito et al.,
2002; Ishikawa et al., 2005) Additionally, the NSG mice were transplanted with the
human cells by neonatal transplantation , which also leads to greater engraftment than in
post-natal transplants. The ability of NSG newborn pups to engraft human hematopoietic
cells and support the presence of circulating human cells in the peripheral blood makes
the neonatal transplant model more applicable than the adult transplant model to
investigate the development of rare human cell populations in the transplanted
mice.(Sands et al., 1993; Ishikawa et al., 2005; Park et al., 2008)
61

In addition, the length of culture with cytokines (Stem Cell Factor, flt-3 ligand,
and thombopoietin [SCF/flt-3L/TPO]) for the ex vivo gene modification was also
significantly reduced to less than 12 hours compared to our previous study.(Hollis et al.,
2006) Thes culture conditions have commonly been used for human HSC priming prior
to retroviral vector transduction to facilitate HSC proliferation and survival.(Murphy et
al., 1992; Murray et al., 1999; Ueda et al., 2000; Wu et al., 2001a) In the context of
transfection via electroporation, SCF/flt-3L/TPO pre-stimulation has also been shown to
be necessary and beneficial for transfection efficiency.(Wu et al., 2001a; Nightingale et
al., 2006) Shorter cytokine exposure time may also maintain the stem cell characteristic
of HSCs(Ailles et al., 2002) Lastly, due to the possible electroporation and/or plasmid
DNA related-toxicity involved in our system,(Hollis et al., 2006) the cell dose used for
transplantation was increased to 1 x 10
6
, which was 5–fold higher than the cell dose used
in our previous study.
These modifications of the transplantation protocol augmented the engraftment
level of the HSB-modified human CD34+ in the transplanted NSG mice. Multi-organ
and multi-lineage stable transgene expression was detected in the repopulating human
cells in the NSG mice at 5 months post-transplantation. In addition, persistent transgene
expression was also observed in the secondarily transplanted NSG mice. These results
demonstrated that the optimized HSB transposon protocol delivered by electroporation
could transfer transgenes into primitive human long-term hematopoietic stem cells that
could engraft and sustain expression of the transgene in vivo.

62

During the course of preparation for this manuscript, similar findings have been
published using another version of hyperactive SB transposase mutant (SB100X)
showing similar results to what we report here.(Xue et al., 2009) In their report, the
CMV promoter was used to express the SB transposase and a hybrid CMV enhancer
chicken β-actin promoter (CAGGS) was used to express the transgene expression
encoded in the transposase cassette. Efficient gene transfer and stable transgene
expression was achieved in human CD34+ progenitor cells (HPCs) and also in multiple
cell lineages when differentiated both in vitro and in vivo. However, the “stem-cell
characteristic" of the transgene expressing SRCs was not addressed by serial
transplantation and the SCID repopulating cells (SRCs) detected in their in vivo model
could be more mature, committed hematopoietic progenitor cells.
Furthermore, based on the promoter optimization data showed in our study, the
use of the CMV promoter to express SB100X and CAGGS promoter to express transgene
may be sub-optimal. We found that the EF1-α promoter was more effective to increase
the SB transposition activity when compared to the CMV promoter (Table 2). The
CAGGS promoter has been shown to have lower promoter strength than the EF1-α
promoter in lymphoid cells and lower than the MND promoter in myeloid cells.(Weber
and Cannon, 2007) Therefore, the promoter optimization approaches and findings
describe in our study may further improve the potential stable gene transfer efficiency of
SB100X. Nevertheless, the optimization study reported here together with the work
published by Xue et al.(Xue et al., 2009) further strengthen the possibility of utilizing the
SB transposon system as a non-viral gene transfer system for HSC gene therapy.
63

Despite the significant progress made in these studies, limitations still remain to
be overcome in order for the efficacy of the SB transposon system to be more clinically
relevant for HSC gene therapy. One of the major limitations in the approach is
cytotoxicity to human CD34+ cells from the electroporation per se which is augmented
by the presence of plasmid DNA. In the in vitro studies, toxicity observed during
electroporation of human CD34+ cells was mainly due to the quantity of plasmid DNA in
a concentration-dependent manner, as reported previously.(Wu et al., 2001b; Hollis et al.,
2006) However, electroporation alone seems to be detrimental to the engraftment and
differentiation ability of the human hematopoietic cells in vivo; we observed a significant
decline in the engraftment efficiency of the electroporated cells even in the absence of the
plasmid DNA. Therefore, other methods of DNA delivery with lower toxicity should be
explored. One possible alternative is the use of non-integrating lentiviral (NIL) vectors
as a carrier for SB component delivery. NIL vectors are designed to disable vector
integration but to retain the normal viral entry functions(Nightingale et al., 2006). NIL
vectors have been shown to achieve gene delivery and to mediate transient gene
expression in various cell types, including primary human HPCs and ES cells.(Yanez-
Munoz et al., 2006; Lombardo et al., 2007) In combination with the SB transposon, a
NIL/HSB transposon hybrid gene transfer system could potentially provide not only the
benefit of viral transduction (high-level, minimally cytotoxic transient gene expression)
but also SB-mediated permanent transgene integration. Other delivery methods for SB
plasmids such as hydrodynamic injection, lipofection, and cationic polymer
polythyleneimine (PEI) have also been actively investigated.(Yant et al., 2000; Bell et
al., 2007; Belur et al., 2007; Liu et al., 2009)
64

The efficiency of SB-mediated transposition also depends on the size of the
transposon being delivered. Use of alternative non-viral transposon systems with higher
transgene carrying capacity, such as PiggyBac transposon derived from the cabbage
looper moth Trichoplusia ni. or the bacteriophage φC31 integrase may be more effective
to deliver larger DNA fragments. In addition to their larger carrying capacity, the lack of
over-expression inhibition observed in the PiggyBac transposon system and the site-
specific integration capacity of the φC31 integrase may be advantageous.(Wilson et al.,
2007; Keravala and Calos, 2008)
Although we were able to achieve multi-lineage transgene expression in NSG-
repopulating cells, the engraftment of the electroporated HSB-modified human CD34+
cells showed large variability (0.01 to 60% in BM at 5 months), with only about 10% of
the transplanted mice achieving high level engraftment of human cells with persistent
transgene expression. Because of the low numbers of well-engrafted mice, we were not
able to draw any conclusion on the relative efficacy of the MNDU3 or EF1a promoter on
transgene expression in specific cell lineages in vivo. To improve the inconsistency of
this NSG neonatal transplant model, several modifications could be made to potentially
better evaluate the efficacy of stem cell survival and transduction in vivo. For example,
since cells are relatively fragile immediately after electroporation, the 1-2 hours of
incubation we allowed prior to transplant may not be optimal to allow electroporated
cells to recover fully and may subsequently lead to further cell lost during transplantation.
A longer incubation time after electroporation (i.e. 4 to 5 hours) may be able to reduce
cell death and consequently increase engraftment of stably transduced human CD34+
cells. In addition, the radiation dose given to the animals may be increased to facilitate
65

engraftment ability of the NSG neonates, although neonatal mice may not tolerate much
higher levels of total body irradiation. Alternatively, the electroporated CD34+ cells that
express eGFP could be sorted by flow cytometry and transplanted into the mice to
increase the level of cells expression the transgene in vivo.
In conclusion, the optimized SB transposon system for transduction of primary
human CD34+ hematopoietic progenitors reported here has improved the stable gene
transfer efficiency by at least 20-fold, compared to our prior published data.
Furthermore, this study demonstrated that SB-modified human CD34+ progenitor/stem
cells can be engrafted and differentiated into multi-lineage cell types in vivo, which
shows promise for further advancement of non-viral based HSC gene therapy.

66

Chapter 3 - Hybrid of Sleeping Beauty Transposon System and Non-Integrating
Lentiviral Vectors for Efficient Gene Transfer and Stable Transgene Expression

3.1 Introduction

One of the major limitations in the SB transposon system is the cytotoxicity
caused by both electroporation and plasmid DNA quantity to primary human HSCs. The
quantity of plasmid DNA used during electroporation has been the major cause of the
toxicity observed in vivo.(Wu et al., 2001b; Hollis et al., 2006) In addition,
electroporation alone seems to be detrimental to the engraftment and differentiation
ability of the human hematopoietic cells in vivo based on our findings discussed in
Chapter 2. Significant decline in the engraftment efficiency of the electroporated cells
were observed even in the absence of the plasmid DNA. Furthermore, electroporation
DNA delivery requires a minimum of one million cells per delivery which raises a
constraint when working with usually limited numbers of primary human CD34+ cells.
Therefore, exploring other methods to deliver the SB transposon vector system with
lower toxicity remain necessary.
One possible alternative is using non-integrating lentiviral (NIL) vectors as a
carrier for SB component delivery (Fig. 3-1). Unlike the integration pattern of a lentiviral
vector that integrates preferentially within genes and is strongly biased towards actively
transcribed genes,(Schroder et al., 2002) SB integrates in a fairly random fashion and
may be less likely to cause insertional mutagenesis.(Yant et al., 2005; Berry et al., 2006)
However, SB lacks the ability to achieve efficient cell entry and nuclear translocation.
67

NIL vectors have been shown to achieve gene delivery and mediate transient transduction
in various cell types, including human primary HSCs and ES cells with minimal
toxicity.(Yanez-Munoz et al., 2006; Lombardo et al., 2007) Combining the stable
integration of the SB transposon system (a non-viral vector) with the delivery efficiency
of NIL (a viral vector), termed NILting beauty, could produce a hybrid vector system that
synergizes the advantages of both viral and non-viral vector systems and provide a more
effective and safer approach to genetically modify HSCs.

Figure 3-1. Schematic demonstration of SB delivery using non-integrating lentiviral
(NIL) vectors. Cells are co-transduced with NIL vectors carry the SB transposase and
transposon. Transposase protein is expressed and localized to the nucleus where it binds
to the transposon inverted repeats (IR) and catalyzes excision of the transposon from
episomal lentiviral DNA. The excised transposon is mobile and able to subsequently
integrate into a host chromosome. Figure is taken and modified from Vink et al., 2009.

68

The feasibility and potential of utilizing NILting beauty as an alternative approach
to achieve transgene integration via SB transposition has been evaluated in HeLa
cells.(Vink et al., 2009) Results based on integration analyses indicate that SB
transposition from this hybrid system is achievable and its integration pattern mimics the
characteristic TA dinucleotides integration signature of a SB transposition. The system
allows delivery of a minimal promoter-transgene cassette and avoids genomic integration
of HIV-1 LTRs or other virus-derived sequences that are included for the purpose of titer
enhancement. However, Vink et al. failed to address whether the NIL/SB hybrid system
can achieve stable transgene expression and ultimately if the level of expression can be
physiologically effective to be clinically relevant for therapeutic applications such as
HSB gene therapy.
In this chapter, the NILting beauty hybrid system is evaluated for its capability to
achieve stable gene transfer in K562, Jurkat, and primary human CD34+ cells. Stable
transgene expression up to 6% was achieved at three weeks post-transduction. The
regimen related toxicity of the hybrid system was also determined to be much less than
the electroporation vector delivery. Therefore, while much optimization is still required,
NILting beauty could potentially be a more efficient and less toxic approach for gene
delivery compared to electroporation.

69

3.2 Materials and Methods
Vector construction
The plasmid pNIL-c-MCS was cloned as the backbone for all of the NIL vectors
except pNIL-c-HSB16 used in this study. pNIL-c-MCS was created from digesting
pCCL-c-CMV-Neo-5`(TG)-3`(CA)(Nightingale et al., 2006) digested with EcoRV and
KpnI and a synthetic mutlicloning site was inserted to provide additional unique
restriction sites (5`-ATC CGC GGC TCG AGG TTA ACT CGA GCT AGC TCT AGA
ATT CAT TGG ATC CGC TAG CGG TAC-3` and 5`-CGC TAG CGG ATC CAA TGA
ATT CTA GAG CTA GCT CGA GTT AAC CTC GAG CCG CGG AT-3`) to make
pCCL-c-5`(TG)-3`(CA) backbone. The MCS contains the following restriction sites for
cloning: EcoRV, SacII, XhoI, HpaI, XhoI, NheI, XbaI, EcoRI, BamHI, NheI, KpnI.
The cis NILting beauty vector (Fig. 3-2), pNIL-c-eGFP-muPGK-HSB16,
contained both transposon (eGFP) and transposase (HSB16) in one single vector. It was
derived from VRPGK-eGFP-attB, pCMV-HSB16,(Berry et al., 2006) and pT-MNDU3-
eGFP-BGH-attB(Hollis et al., 2006). The murine PGK promoter was removed from
VRPGK-eGFP-attB with NheI and XhoI and ligated into XbaI site of pNIL-MCS to
create pNIL-c-MCS-muPGK. The HSB16 fragment was isolated from the plasmid
pCMV-HSB16 using BamHI and EcoRI, and was inserted into the BamHI site of pNIL-c-
MCS-muPGK to create pNIL-MCS-muPGK-HSB16.
The plasmid pT-MNDU3-eGFP-BGH containing the transposon cassette IR-
MNDU3-eGFP-BGH-IR was generated by removing the attB site of pT-MNDU3-eGFP-
BGH-attB(Hollis et al., 2006) with SalI and religating to create pT-MNDU3-eGFP-BGH.
Finally, the transposon cassette IR-MNDU3-eGFP-BGH-IR was excised from pT-
70

MNDU3-eGFP-BGH using NdeI and PstI, blunted-ended and ligated into the HpaI site
of pNIL-c-MCS-muPGK-HSB16 to create the cis NILting beauty vector pNIL-c-eGFP-
muPGK-HSB16.
The trans NILting beauty vectors (Fig. 3-2), pNIL-c-MNDU3-eGFP and pNIL-c-
MNDU3-HSB16, were constructed in the following fashion. The plasmid pNIL-c-
MNDU3-eGFP was created from pT-MNDU3-eGFP-BGH and pNIL-MCS. The
transposon cassette IR-MNDU3-eGFP-BGH-IR was removed from pT-MNDU3-eGFP-
BGH using NdeI and PstI, blunted-ended and ligated into the HpaI site of pNIL-MCS to
create pNIL-MNDU3-eGFP. The plasmid pNIL-c-MNDU3-HSB16 used in this study
did not contain a true NIL backbone as the other NILting beauty vector constructed in
that it retained the att sites of the LTR. It was a pCCL-c-MNDU3-HSB16 vector
packaged with the 8.2 int- packaging plasmid. The HSB16 fragment of the plasmid
pCCL-c-MNDU3-HSB16 was excised from pCMV-HSB16(Berry et al., 2006) using
BamHI and EcoRI. To obtain the final product, pNIL-c-MNDU3-HSB16, the HSB16
fragment was ligated into the pCCL-c-MNDU3 fragment removed from pCCL-C-
MNDU3-eGFP (from Aaron Logan) digested with SacI and EcoRI.

71

Supernatant production
Methods for viral supernatant production were based on a three-plasmid transient
transfection technique described previously.(Logan et al., 2004b; Hollis et al., 2006;
Nightingale et al., 2006)
Transduction
Transduction of K562 and Jurkat cells. Cells were cultured in R-10 and expanded to 1
x 10
6
cells/ml in T75 tissue culture flasks. 1 x 10
6
cells were transduced with vectors in a
6-well plate with 8 μg/ml polybrene at 2mL of total volume. Cells were analyzed for
eGFP expression by FACS using CellQuest software on day 2 for initial readout and
weekly thereafter up to 4 weeks. All transductions were performed in duplicate or
triplicate.
Figure 3-2. NILting beauty vectors constructed in this study. The cis
NILting beauty, pNIL-c-eGFP-muPGK-HSB16, contained both transposon and
transposase. The trans NILting beauty, pNIL-c-MNDU3-eGFP and pNIL-c-
MNDU3-HSB16, contained only one of the SB components in each vector. The
NILting beauty vectors were packaged with 8.2 Int- and VSV-G plasmids in
293T cells.
72

Transduction of CD34+ cells. CD34+ cells (1 x 10
5
/well) were pre-stimulated overnight
on retronectin-coated 48-well plates in serum free X-Vivo-15 medium containing 50
ng/ml fit-3 ligand (R&D Systems, Minneapolis, MN, USA), 50 ng/ml stem cell factor
(SCF), and 50 ng/ml thrombopoietin (R&D Systems). After 24-hours of pre-stimulation
period, the cells were treated with 2 x 107 TU/ml of vector in 200 μl in the presence of
8 μg/ml polybrene. At 24-hours post-transduction, the cells were transferred to 24-well
retronectin-coated plates for long-term culture in basal bone marrow medium, BBMM,
(IMDM, 20% FCS, 0.5% BSA) with 5 ng/ml human IL-3, 10 ng/ml IL-6, and 25 ng/ml
SCF (Biosource International). Cells were analyzed for eGFP expression by FACS using
CellQuest software. All transductions were performed in duplicate or triplicate.

3.3 Results
NIL vectors showed less cytotoxicity for SB-transposon system delivery compared to
electroporation

Preliminary study of NILting beauty vector system (cis NIL-eGFP-SB) showed
almost no toxicity in comparson to deliver of SB components by electroporation (Fig. 3-
3A). The transposition efficiency of NILting beauty, however, required further
optimization. The initial eGFP expression of cells transduced with either NIL-eGFP or
NIL-eGFP-SB vectors were 3-fold lower (< 30%) than that observed in K562 cells
electroporation with the SB plasmids. No long-term stable transgene expression was
detected in cells transduced with cis NILting beauty. Level of eGFP expression detected
from the NILting beauty-transduced cells was reduced to zero by day 14 while the
transgene expression persist at 90% in cells that were electroporated with SB plasmids
73

(Fig. 3-3B). Since NIL vectors transduction efficiency is much lower than regular
lentiviral vectors, the NIL viral concentration used (MOI < 1) might be insufficient to
achieve stable transgene integration that lead to lack of long-term transgene expression.
Higher NILting beauty viral concentration per transduction (or MOI) might be required to
achieve higher initial transgene integration and long-term transgene expression.
74

Figure 3-3. Comparison of stable gene transfer efficacy between NILting beauty
viral delivery and electroporation delivery of SB components in K562. (A) transient
eGFP expression and toxicity on day 3 (B) time course of SB-mediated eGFP integration
by NILting beauty viral transduction or electroporation.
A
B
75

The SB transposase and eGFP reporter gene encoded in the cisNILting beauty
vector were expressed and functional
Several reasons could contribute to the lack of long-term transgene expression
observed in Fig. 3-3. First, the design or cloning of cis NIL-eGFP-SB vector construct
might be incorrect. Alternatively, both SB components encoded in one NIL vector might
not express properly and therefore lead to a production of non-functional or insufficient
level of reporter gene and transposase. The cis NILting beauty vector construct was
verified in K562 cells to determine whether SB and eGFP sequences in pNIL-eGFP-SB
were expressed and produced functional enzyme and substrate for SB-mediated
integration (Fig. 3-4). pNIL-eGFP-SB was electroporated into K562 cells in a dose-
escalating manner and assessed for both initial and long-term transgene expression by
FACS analysis. A long-term eGFP expression was expected be observed if both SB
components were expressed and functional.
At 4 weeks post-electroporation, up to 70% of stable eGFP expression was
observed in the cells electroporated with pNIL-eGFP-SB. This indicated that both SB
transposase and eGFP transgene were expressed and functioned properly when delivered
as plasmid DNA by electroporation. There were no evidences of cloning or vector design
errors found in the cis NILting beauty vector. As a result, vector design error could be
excluded from the causes for inadequate stable gene transfer observed in cis NILting
beauty system.

76

SB transposon limitation in cis NILting beauty viral delivery system
The other possible explanations for the lack of transgene integration observed in
cells transduced by NILting beauty viral vector could be due to GFP transposon (as SB
target) or SB transposase (as enzyme) limitation in the “all-in-one” cis vector delivery
system. Either one of the SB components might be limited when delivered by the cis
NILting beauty system. Based on our optimization study described in Chapter 2, the
optimal ratio of SB transposon to transposase ratio (10 ug : 1 ug) indicated that 10-fold
higher level of transposon (target) than SB transposase was required to achieve long-term
transgene expression. In the NIL-eGFP-SB transduced cells, the amount of transposon
might be insufficient for SB transposase to facilitate stable eGFP integration or vice
versa. Thus, the expression levels of the SB transposase and eGFP reporter gene of the
transposon in the cis NIL-eGFP-SB vector required further improvement.
% eG FP + (w eek 4 )
% P I+ (d ay 3)
Mock 1 μ μ μ μg 3 μ μ μ μg 10 μ μ μ μg 20 μ μ μ μg 30 μ μ μ μg
Amount of plasmid DNA (μ μ μ μg)
eGFP
SB
PGK MND
Poly
A
IR
IR
Figure 3-4. Validation of the NILting beauty vector construct. Long term eGFP
expression (week 4) and toxicity (day 3) measured by FACS to validate the cloning
and design of cisNILting beauty vector in K562.
77

Rescue studies were conducted with additional transposon or transposase supplied
to the cells via electroporation to examine whether the additional amount of the SB
components could facilitate stable SB-mediated gene transfer. If one of the SB
components was indeed the limiting factor, an improvement in the stable eGFP
expression should be observed. On a side note, hyperactive SB (HSB16) was also used
instead of the original SB from this point on for the NILting beauty study since HSB has
been determined to be a much more efficient transposase than the original SB (see
Chapter 2; Fig. 2-2). In the NILting beauty rescue study (Fig. 3-5), additional transposon
or HSB transposase was transfected by electroporation in K562 cells 24 hours prior to the
transduction of the cis NILting beauty viral vector. The MOI of each NILting beauty
transduction was increased 10-fold to MOI of 3. The efficacy of stable gene transfer was
determined by weekly FACS analyses for eGFP expression.
Based on the FACS analysis, the addition of an eGFP-containing transposon (pT-
eGFP) prior to NILting beauty transduction increased the long-term stable transgene
expression by approximately 10% at 4 weeks post-transduction (Fig. 3-5). An
approximately 50% of increase in the initial eGFP expression was detected when
additional eGFP encoding transposon was presented at the time of NIL-eGFP-HSB
transduction. In contrast, an additional amount of HSB transposase did not show any
improvement in the SB-mediated gene transfer efficiency. This suggested that the SB
transposon of the NIL-eGFP-HSB16 could be one of the limiting factors in the cis
NILting beauty gene transfer system. However, whether the lack of stable transgene
integration was due to transposon target limitation or poor eGFP transgene expression in
our cis NILting beauty vector still remained to be determined.
78

Day 0
Nucleoporation
--- pGFP
(10 μ μ μ μg)
--- phSB16
(1μ μ μ μg)
pGFP
(10 μ μ μ μg)
pNIL-eGFP-
hSB16 (10 μ μ μ μg)
Day 1
Transduction
--- --- NIL-eGFP-
hSB16
NIL-eGFP-
hSB16
NIL-eGFP-
hSB16
% eGFP+
(MOI of 3)
0
10
20
30
40
50
60
70
80
90
100
% eGFP+ (day 7)
% eGFP+ (week4)

The plasmid of trans NILting beauty vectors, pNIL-eGFP and pNIL-HSB, expressed
functional eGFP and HSB transposase
Since multiple factors (such as transposase to transposon ratio, functionality and
expression level for each component) could be interfered by combining both SB
components in the “all-in-one” cis NILting beauty vector, it might be necessary to
separate SB transposon and SB transposase for further investigations and improvement of
the NILting beauty system. The SB components of the cis NILting beauty system was
cloned into two individual NIL vectors (trans NILting beauty): pNIL-eGFP and pNIL-
HSB16 (Fig. 3-6). To evaluate the transgene expression of each trans construct, K562
cells were transfected with the trans NILting beauty plasmids and followed by weekly
Figure 3-5. NILting beauty rescue study: eGFP expression on day 3 and at week 4
in NIL-eGFP-HSB16 transduced K562 cells supplemented with either transposon
(eGFP) or transposase (HSB16). Additional eGFP prior to NILting beauty
transduction increased the level of stable gene expression. Black font represents the
NILting beauty vector delivered as plasmid DNA and red represents the NILting
beauty delivered as viral vectors.
79

FACS analysis to assess transgene expression. At 5 weeks post-electroporation,
approximately 60% stable eGFP expression was detected when both pNIL-eGFP and
pNIL-HSB16 were present suggesting that trans NILting beauty plasmids express
sufficient and functional SB components resulting a SB-mediated stable integration and
long-term transgene expression (Fig. 3-6).
Transposon
(eGFP, 10μ μ μ μg)
--- pGFP pGFP pGFP pNIL-
eGFP
pNIL
-eGFP
pNIL-
eGFP
Transposase
(hSB16, 1μ μ μ μg)
--- --- phSB16 pNIL-
hSB16
--- phSB16 pNIL-
hSB16
% eGFP+
0
10
20
30
40
50
60
70
80
90

Co-transduction of the trans NILting beauty vectors achieved stable gene expression
in K562 cells
After verifying the integrities of both pNIL-eGFP and pNIL-HSB16 constructs,
both plasmids were packaged as viral vectors to evaluated the gene transfer efficiency of
the transNILting beauty system in K562 cells. K562 cells were co-transduced with NIL-
eGFP and NIL-HSB16 either simultaneously or 24-hour apart. Serial dilutions (5 x 10
5
∼
Figure 3-6. Dissecting the NILting beauty vector system: stable transgene
expression at 5 weeks post-electroporation with trans NILting beauty plasmids in
K562.
80

5 x 10
2
TU/ml) of NIL-HSB16 concentrated viral supernatant were added to the cells
with a constant viral concentration of NIL-eGFP (5 x 10
4
TU/ml). The highest level of
stable transgene expression was 3% GFP+ using 5 x 10
4
TU/ml of NIL-eGFP and 5 x 10
4

TU/ml of NIL-HSB16 viral supernatant at 3 weeks post-transduction (Fig. 3-7). Data
also indicated that no substantial differences (0.5 to 1% variable) in stable transgene
expression between transducing cells with both NIL vectors at the same time or with
delay of the NIL-HSB16 added 24 hours later. Although the stable transgene expression
was much lower than what we observed in an electroporation-based SB delivery, the
observed stable transgene expression was encouraging and suggesting the feasibility to
apply NIL vectors as a carrier for delivering SB-mediated integration system in HSC
gene therapy.

0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
eGFP only
5.00E+05
5.00E+04
5.00E+03
5.00E+02
5.00E+01
5.00E+00
5.00E-01
5.00E-02
%eGFP+
Together
(+)24 hours
Figure 3-7. The trans NILting beauty delivery system in K562: % GFP expression
at week 3 post-transduction. NIL-HSB16 was add1ed either on the same day or 24-
hour post transduction by pNIL-eGFP. The titer for NIL-eGFP was 1 x 10
8
TU/ml
and the titer for NIL-HSB6 was 1 x 10
9
TU/ml. The viral concentration for NIL-
eGFP was kept in constant at 5 x 0
4
TU/ml (MOI 3). This data was prepared by Dr.
Roger Hollis.
NIL-HSB16 (TU/ml)
81

Optimization of trans NILting beauty system in K562 cells
NIL-HSB optimization
Although stable gene expression was observed in the transNILting beauty system,
the level of the stable gene expression was still unsatisfactory. Series of dose escalation
studies were conducted to determine 1) the enzymatic activity of the HSB transposase
and 2) the maximum level of NIL-HSB16 required achieving an optimal stable gene
transfer in target cells. K562 cells were saturated with transposon encoding eGFP
reporter gene by electroporation. Increasing amounts of NIL-HSB16 virus (titer = 1 x
10
9
TU/ml) were then added to the electroporated cells 24 hours later and transgene
expression was determined by weekly FACS analyses.
The transposition activity of HSB16 expressed from NIL-HSB was sufficient for
transgene integration (Fig. 3-8). The stable gene expression showed a dose dependence
on the amount of NIL-HSB viral supernatant added during the transduction. The SB-
mediated eGFP expression plateau after NIL-HSB16 (titer = 1 x 10
9
TU/ml) viral
concentration was reached using higher than 2 x 10
7
TU/ml during transduction. The
highest stable eGFP expression achieved was around 65% at 3 weeks post-transduction.
This suggested that the viral concentration of NIL-HSB16 required to achieve > 50%
stable gene integration and long-term transgene expression might be in the range of 2 x
10
7
to 1 x 10
8
TU/ml.

NIL-eGFP optimization
A similar experiment also was conducted to determine the maximum level of
NIL-eGFP necessary to achieve an optimal initial GFP
SB gene transfer efficiency. 1 x 10
of NIL-eGFP viral supernatant (titer = 6.5 x 10
NIL-eGFP required to achieve highest initi
transduction was 3 x 10
7

fold (30%) by day 10 (Fig. 3
Figure 3-8. NIL-HSB16 supernatant
dose escalation study.
amount of NIL-HSB16 virus were
added to cells saturated with eGFP
targets. The titer for NIL
x 10
9
TU/ml. The optimal NIL
HSB16 viral concentration for highest
stable gene expression is in between 2
x 10
7
and 1 x 10
8
TU/ml. This data
was prepared by Dr. Roger Hollis.
NIL-HSB16 supernatant (ul)

eGFP optimization
A similar experiment also was conducted to determine the maximum level of
eGFP necessary to achieve an optimal initial GFP expression and to facilitate higher
SB gene transfer efficiency. 1 x 10
5
K562 cells were transduced with increasing amount
eGFP viral supernatant (titer = 6.5 x 10
7
TU/mL). Maximal viral concentration of
eGFP required to achieve highest initial eGFP expression (>90%) at day 2 post
TU/ml in K562 cells. The transgene expression level reduced 3
fold (30%) by day 10 (Fig. 3-9).
HSB16 supernatant
dose escalation study. Increasing
HSB16 virus were
added to cells saturated with eGFP
targets. The titer for NIL-HSB6 was 1
TU/ml. The optimal NIL-
HSB16 viral concentration for highest
e expression is in between 2
TU/ml. This data
was prepared by Dr. Roger Hollis.
HSB16 supernatant (ul)
Figure 3-9. Optimization of NIL
eGFP for the trans NILting beauty
system in K562 cells.
amount of NIL-eGFP virus were added
to determine the maximum viral
concentration of NIL-eGFP required for
highest initial eGFP expression. The
titer for NIL-eGFP was 6.5 x 10
TU/ml. The optimal NIL
concentration for highest initial eGFP
expression is around 3 x 10
(MOI 300) in K562.
NIL-eGFP supernatant (MOI)
82

A similar experiment also was conducted to determine the maximum level of
expression and to facilitate higher
K562 cells were transduced with increasing amount
TU/mL). Maximal viral concentration of
al eGFP expression (>90%) at day 2 post-
TU/ml in K562 cells. The transgene expression level reduced 3-
9. Optimization of NIL-
NILting beauty
system in K562 cells. Increasing
eGFP virus were added
the maximum viral
eGFP required for
highest initial eGFP expression. The
eGFP was 6.5 x 10
7

TU/ml. The optimal NIL-eGFP viral
concentration for highest initial eGFP
expression is around 3 x 10
7
TU/ml
eGFP supernatant (MOI)
83

trans NILing beauty transduction with the determined optimal viral concentrations
in K562
Both NIL-HSB and NIL-eGFP were used to transduce cells simultaneously with
the optimal viral concentrations determined in our optimization studies (Fig. 3-8 and 3-9).
Despite finding that the highest stable GFP expression (9.5%) achieved with the trans
NILting beauty system, the average stable transgene expression achieved with the
optimized viral concentrations remained low (range of 1 – 1.5 %; Fig. 3-10). The viral
concentration of 3 x 10
7
TU/ml (MOI = 1200) of NIL-HSB supernatant was used to
transduce with increasing amount of NIL-eGFP in K562 cells. The highest MOI for NIL-
eGFP viral concentration used in this experiment equaled to 2.5 x 10
7
TU/mL per
transduction. The highest initial eGFP expression detected was 16% at 3 days post-
transduction with around 2.5 x 10
7
TU/ml (MOI 1000) and 3 x 10
7
TU/ml (MOI 1200) of
NIL-HSB16 in K562. However, by 4 weeks post-transduction, the detectable eGFP
expression was reduced by 16-fold to around 1%. This poor gene transfer efficiency
observed at the expense of an extremely high viral concentration indicated that other
factors might inhibit the gene transfer efficiency of our NILting beauty system.

84

Defective HIV-1 integrase used during NILting beauty viral vector packaging was
not detrimental to the gene transfer efficiency of trans NILting beauty
The defected integrase and the absence of other viral accessory proteins during
NILting beauty viral vector packaging might also hindered the gene transfer efficiency of
NILting beauty. To evaluate this hypothesis, NILting beauty vectors were packaged with
8.2 (accessory proteins with intact intergrase), 8.2- (accessory proteins with defective
intergrast), and 8.9 (no accessory protein with intact integrase) packaging plasmid and
used to transduce K562 cells for comparison. Figure 3-11 showed that there were only
about 2 – 3 % differences among cells transduced with NILting beauty packaged with
Figure 3-10. Stable eGFP transgene expression achieved by the trans NILting
beauty system in K562 cells. The titer for NIL-eGFP was 2.8 x 10
7
TU/ml and 6.1 x
10
8
TU/mL for NIL-HSB16. The highest viral concentrations used during NILting
beauty transduction were 2.5 x 10
7
TU/ml (MOI 1000) of NIL-eGFP and 3 x 10
7
TU/ml (MOI 1200) of NIL-HSB16 in K562. Two individual experiments were
conducted. The GFP-positive cells were determined by FACS analyses.
85

8.2, 8.2-, and 8.9 packaging plasmid. However, the involvements of integrase or any
other lentiviral accessory proteins during NILting beauty gene transfer still remains
inconclusive. Future investigations would be needed to reach any significant conclusion.

NILting beauty in human CD34+ hematopoietic cells
NILting beauty delivery system was also used in primary CD34+ hematopoietic
cells for SB-mediated gene modification. Preliminary studies of NILting beauty in
human CD34+ cells have been conducted using trans NILting beauty vectors to deliver
SB components. Unfortunately, the result from this initial study was unsatisfactory (Fig.
3-12). The highest initial eGFP expression was around 6% but declined down to < 1%
0
1
2
3
4
5
6
7
8
9
10
MOI 1 MOI 10 MOI 30 MOI 100 MOI 300 MOI 300
no SB
% e G F P +
8.2 int -
8.2 wt
8.9 wt
Figure 3-11. Role of defecting integrase in NIL-HSB16 trans system using 4 x 10
7

TU/ml of NIL-HSB supernatant and increasing MOI of eGFP target packaged with 8.2,
8.2-, 8.9 packaging plasmids. Stable eGFP expression in transduced K562 cells at
week 3 is shown in this figure. This data was prepared by Dr. Roger Hollis.

after 10 days even with the presence of HSB. Since this was a preliminary study, all the
conditions were not optimized at
proper controls (such as cells transduced with NIL vectors containing the regular
orientation of eGFP expression cassette, regular MND
control) are still need to be condu

Figure 3-12. The
transduction in human CD34+ cells.
TU/ml. The titer for NIL
used per transduction. Both viral supernatant
after 10 days even with the presence of HSB. Since this was a preliminary study, all the
conditions were not optimized at the time of the experiment. More experiments with
proper controls (such as cells transduced with NIL vectors containing the regular
orientation of eGFP expression cassette, regular MND-eGFP lentiviral as a transduction
control) are still need to be conducted for any further conclusions.
preliminary study of trans NILting beauty double
uman CD34+ cells. The titer for NIL-eGFP used was 1 x 10
TU/ml. The titer for NIL-HSB16 was 6 x 10
8
TU/ml. 1 x 10
5
CD34+
used per transduction. Both viral supernatants were added at the same time
86
after 10 days even with the presence of HSB. Since this was a preliminary study, all the
the time of the experiment. More experiments with
proper controls (such as cells transduced with NIL vectors containing the regular
eGFP lentiviral as a transduction

NILting beauty double
eGFP used was 1 x 10
8

CD34+ cells were
added at the same time
87

3.4 Discussion
In principle, NILting beauty as the delivery method for SB components not only
could achieve a higher transient SB gene expression but also could potentially be less
toxic to the target cells compared to electroporation. The feasibility and potential
utilization of NIL vectors to deliver SB transposon system (NILting beauty) was
examined in K562 and human CD34+ cells. While some level of long-term transgene
expression was observed by NILting beauty transduced K562 cells, the achieved level
was about 30-fold lower than the long-term stable transgene expression achieved by
delivering SB system via electroporation. In addition, extremely high viral
concentrations were also required in a NILting beauty system to reach minor increase in
transgene expression. Therefore, more modifications and improvements of the NILting
beauty system are much needed.
To troubleshoot the current design of NILting beauty, expression data obtained
from rescue studies indicated that there was a target (transposon carried eGFP expression
cassette) limitation in cells transduced by NILting beauty vectors. Delivering both SB
components using a single NIL vector has been demonstrated to not be ideal. The
requirement for the ratio of transposon and SB transposase to achieve stable gene
integration might not be at a 1:1 ratio. SB components were therefore delivered in trans
by two separate NILting beauty vectors to allow easy adjustment of transposon to
transposase ratio.
Linear SB transposon genome produced by NIL vectors might reduce the gene
transfer efficiency of HSB transposase in our NILting beauty system. As reported in the
previous studies, the frequency of the transposition of a supercoiled, circular donor Tc1
SB transposon was reduced 10 to 20-fold upon linearization (Fig. 3-13A).(Vos et al.,
88

1996; Yant et al., 2002) To test this hypothesis, pNIL-eGFP plasmid was linearized by a
single-cut restriction enzyme and purified before transfection. Both linear and circular
form of transposon were co-delivered with pNIL-HSB by electroporation in K562 cells.
The stable transgene expression was measured by FACS at 4 weeks post-electroporation.
The results suggested that HSB transposase favors circularized transposon as substrates
to achieve stable integration. The HSB transposition efficiency of the linearized
transposon was 25% lower than then the circular transposon (Fig. 3-13B). These data
indicated that further components might be required to facilitate the formation of the
circularized NIL-eGFP viral genome to achieve efficient stable gene transfer in NILting
beauty system.
Based on the data presented in this study, a much higher level of eGFP transposon
needed to be delivered by NILting beauty to achieve similar level of transgene integration
as observed in our two-plasmid electroporation system. Similar to optimization
approaches as used in Chapter 2, different promoters could also be used to increase the
transduction efficiency in the NILting beauty vector system. Promoters such as MNDU3,
CMV, and EFα-1 could be cloned into our NILting beauty vectors for further vector
optimization.

89

Figure 3-13. Linear v.s. circular HSB transposon for HSB-mediated gene
transfer in K562. (A) Transposition efficiency from circular and linear
transposon in HeLa cells. Figure was taken from Yant et al. (2002)
6
. (B) 10 μg
of transposon (encoded eGFP reporter gene) was delivered with 1 μg of HSB16
by electroporation. The eGFP-positive cells were determined by FACS analysis
at 4 weeks post-electroporation. Each experiment was performed twice. Error
bar represents the standard error of the mean.
90

Chapter 4 - Low-Dose Busulfan and Fludarabine Conditioning for Non-
Myeloablative Hematopoietic Stem Cell Transplant in Infant Non-human Primates

4.1 Introduction
Evidences have shown that the host immunologic clearance of cells carrying
foreign or new genes influences the outcome of gene therapy. Specific cytotoxic T
lymphocytes (CTLs) against the fusion gene products of the herpersvirus thymidine
kinase (HSV-TK)(Bordignon et al., 1995a) and the hygromycin phosphotransferase
(Hy)(Riddell et al., 1996) or neo(Heim et al., 2000; Berger et al., 2001) gene led to the
drastic reduction of the autologous transduced T cells in humans. In a more clinically
relevant non-human primate model, induction of CTL and antibody responses against the
eGFP transgene product was observed and determined to be responsible for the
disappearance of eGFP-expressing cells in vivo.(Rosenzweig et al., 2001; Morris et al.,
2004) Therefore, eliminating host immune responses against the transgene is crucial for
transgene marking and expression.
There are several strategies to achieve elimination or reduction of host immune
responses against transgene products and ultimately induce immune tolerance to a
therapeutic transgene. High intensity myeloablation such as high dose TBI (1,000 – 1,300
cGY) and chemotherapy (high dosage of busulfan) is used to “make space” in the bone
marrow for HSC engraftment. Immunosuppressants are often added to prevent
immunologic rejection. However, the adverse effects associated with such intensive
cytoreductive regimens are usually high and the high regimen-related toxicity of HSCT
91

often leads to high risk of morbidity and mortality.(Vassal et al., 1990; Vassal et al.,
1996) Tremendous efforts have been devoted into establishing non-myeloablative
conditioning that could achieve similar levels of clinical effect with relatively moderate
adverse effects.(Rosenzweig et al., 1999; Aiuti et al., 2002; Andersson et al., 2003; Kahl
et al., 2006; Kang et al., 2006)
Non-myeloablative conditioning is defined as using low dose of TBI (200 – 400
cGY) or busulfan to create space in the bone marrow micro-environment and thereby
increasing the efficacy of HSCT. Although non-myeloablative TBI has also
demonstrated to induce immune tolerance and increase gene engraftment in nonhuman
primate models, the level of engraftment was still too low to achieve any clinical benefit
for gene therapy.(Rosenzweig et al., 1999; Kang et al., 2001) More importantly, the
sensitivity to irradiation follows a steep dose-response curve.(Giri et al., 2001) In an
ADA-SCID clinical gene therapy trial, a combination of gene therapy with non-
myeloablative busulfan (4 mg/kg) conditioning has demonstrated an increased level (5%
- 15%) of gene engraftment.(Aiuti et al., 2002) The success of this ADA-SCID gene
therapy trial has demonstrated the feasibility of using a lower dose of busulfan as the
non-myeloablative conditioning for gene therapy.
Busulfan is a bifunctional alkylating agent, which has been used clinically as an
antitumor agent since 1959 and has been a common conditioning treatment for
HSCT.(Grochow, 1993; Bhagwatwar et al., 1996) After systemic absorption of busulfan,
carbonium ions are rapidly formed leading to alkylation of DNA. This results in DNA
breakage as well as cross-linkage of DNA strands interfering with DNA replication and
RNA transcription. The optimal busulfan dosage to achieve stable long-term gene
92

marking and minimal toxicity has recently been established in a busulfan dose escalation
study.(Kahl et al., 2006) The investigators demonstrated that sub-myeloablative
busulfan dosages (40 – 160 mg/m
2
) were well-tolerated and that busulfan dosages and
gene marking were positively correlated in an infant rhesus macaque model.
Since busulfan alone is not immunosuppressive, an addition of an immuno-
suppressive chemo agent, fludarabine, can be used to reduce unwanted lymphocyte
proliferation. Busulfan and fludarabine are often used in combination as part of the non-
myeloablative allogeneic transplant regimens.(Bartelink et al., 2008) Fludarabine is used
in conditioning for immune suppression in non-myeloablative allogeneic transplant
regimens.(Terenzi et al., 1996; Gandhi and Plunkett, 2002) It is a purine analogue that
inhibits the deamination of adenine and thereby inhibits DNA synthesis and prevents
DNA strand elongation. The common range used in clinical non-myeloablative
conditioning regimen is between 90 to 180 mg/m
2
. The optimal dose of fludarabine has
not been determined in infant nonhuman primate models.
The feasibility of combining busulfan with fludarabine as an alternative and
potentially more effective conditioning regimen prior to gene transfer/BMT to induce
immune tolerance to a foreign transgene product (eGFP protein) with acceptable clinical
safety was evaluated in this study. The immunosuppressive chemotherapeutic agent
fludarabine was added to better HSC engraftment and long-term transgene expression by
reducing host immunological responses to the foreign transgene product. The addition of
fludarabine to the non-myeloablative busulfan conditioning may contribute to better HSC
engraftment, an induction of immune tolerance, and ultimately immune reconstitution
when used in conjunction with HSCT.

A clinically relevant infant rhesus macaque bone marrow transplant (BMT) model
was used in this study. Our study was design to address three specific aims: (1) to
evaluate and determine the optimal fludarabine dosage required to achieve stable long
term gene marking and minimal toxicity in HSCT; (2) to determine whether the
optimized non-myeloablative conditioning regimen could eliminate or reduce immune
responses to the eGFP transgene product; (3) to investigate if an immune tolerance and
immune reconstitution can be achieved by HSCT after non
A schematic diagram of research design and timeline was illustrated in Fig. 4

Figure 4-1. Study design and outline to achieve immune reconstitution and
tolerance towards transgene product using busulfan and fludarabine conditioning
for non-myeloablative hematopoietic stem cell transplant in infant nonhuman
primates.

A clinically relevant infant rhesus macaque bone marrow transplant (BMT) model
was used in this study. Our study was design to address three specific aims: (1) to
e and determine the optimal fludarabine dosage required to achieve stable long
term gene marking and minimal toxicity in HSCT; (2) to determine whether the
myeloablative conditioning regimen could eliminate or reduce immune
GFP transgene product; (3) to investigate if an immune tolerance and
immune reconstitution can be achieved by HSCT after non-myeloablative conditioning.
A schematic diagram of research design and timeline was illustrated in Fig. 4

tudy design and outline to achieve immune reconstitution and
tolerance towards transgene product using busulfan and fludarabine conditioning
myeloablative hematopoietic stem cell transplant in infant nonhuman
93
A clinically relevant infant rhesus macaque bone marrow transplant (BMT) model
was used in this study. Our study was design to address three specific aims: (1) to
e and determine the optimal fludarabine dosage required to achieve stable long-
term gene marking and minimal toxicity in HSCT; (2) to determine whether the
myeloablative conditioning regimen could eliminate or reduce immune
GFP transgene product; (3) to investigate if an immune tolerance and
myeloablative conditioning.
A schematic diagram of research design and timeline was illustrated in Fig. 4-1.

tudy design and outline to achieve immune reconstitution and
tolerance towards transgene product using busulfan and fludarabine conditioning
myeloablative hematopoietic stem cell transplant in infant nonhuman
94

Briefly, bone marrow was harvested using established techniques from 3-month
old infant rhesus monkeys, followed by CD34
+
immunoselection. The rhesus CD34
+

cells were transduced with equal amounts of two different SIV-based lentiviral vectors: a
non-expressing neomycin (NoN) vector and an eGFP vector to distinguish between HSC
engraftment and host immune responses that may eliminate transgene carrying progeny
cells.
A final dose of 160 mg/m
2
busulfan infusion was used based on the dose
escalation study reported that a maximal level of cytoablation but minimal regiment-
related toxicity could be achieved by infusing 160 mg/m
2
total busulfan in an infant non-
human primate model.(Kahl et al., 2006) However, the pharmacokinetics of the
fludarabine and the sensitivity to the treatment in infant monkeys when co-treated with
busulfan remains unknown. Thus, a dose-escalation study of fludarabine was conducted
to determine its optimal dosage that could induce significant lymphopenia but still remain
within the clinically tolerated range. To assess sufficient levels of immune suppression
for immune tolerance induction achieved in our monkey subjects, three conditioning
regimens were evaluated to date – controls (N=6) received busulfan at the optimized dose
of 160 mg/m
2
(2 x 80 mg/m
2
)(Kahl et al., 2006) and experimental were administered
busulfan in combination with fludarabine at a final dosage of either 90 (2 x 45) (N=3) or
150 (3 x 50) (N=3) mg/m
2
.
At 48 hours post-conditioning, the autologous transduced CD34+ cells were
transplanted. Busulfan and fludarabine pharmacokinetics, complete blood counts (CBC),
peripheral blood mononuclear cells (PBMC), bone marrow gene marking, and humoral
immune responses against the eGFP transgene were assessed. Once the non-
95

myeloablative conditioning by a combination of busulfan and fludarabine is optimized,
this condition will be used in conjunction with HSCT to induce immune tolerance and
immune reconstitution towards a foreign antigen in an infant nonhuman primate model.
Next, to determine whether the optimized non-myeloablative conditioning
regimen could eliminate or reduce immune responses to the eGFP transgene product, we
hypothesized that the lymphodepletion by fludarabine would allow the engraftment and
expansion of gene modified HSCs that produced functional transgene product. In theory,
animals treated with busulfan and fludarabine, not the animals received busulfan alone,
would become anergy to the eGFP protein. Observations such as persistence of GFP-
expression cells and unresponsiveness towards the eGFP immunogenic antigen would be
the indications of the development of immune anergy in treated animals. Finally, the
animals would be re-challenged with recombinant eGFP protein and eGFP-transduced
autologous cells at 6 and 9 months post HPCT respectively to determine whether an
immune reconstitution was achieved by our conditioning regimen.
In this chapter, we demonstrated that the immune responses against the transgene
played a significant role in the successful outcome of HSC gene therapy and that
fludarabine might be able to modulate these responses. In addition, the clearance rate of
fludarabine in infant rhesus monkeys was much faster than that in human. As a result, it
might require much higher doses of fludarabine to show any effect lymphodepletion in
our infant monkey model. Since significant lymphodepletion was not achieved by the
fludarabine treatment, higher doses of fludarabine may need to be evaluated for an effect
on engraftment and long-term transgene expression.

96

4.2 Materials and Methods
Plasmid construction
Proviral maps for two SIV-based lentiviral vectors used in this study are depicted
in Fig. 4-2. The gene encoding neomycin (neo) was cloned into a third-generation self-
inactivating SIV transfer vector.(Mangeot et al., 2000) The vector has a partially deleted
gag-pol region (gag), and contains the central polypurine tract (cPPT) and the rev
responsive element (RRE). A CMV internal promoter was used in SIV-NoN whereas
MNDU3 was used in SIV-eGFP as the internal promoter. The deletion in the U3 region
of both 5’ and 3’ LTR prevents possible formation of replication competent lentiviral
particles. The translation start codon (ATG) was mutated into (CTG) to abolish neo gene
expression.(Hanazono et al., 1999) Since no transgene products should be present to
provoke host immune responses, the non-expressed neomycin resistance gene SIV (SIV-
NoN) vector was used as the internal control for transduction efficiency. In contrast, the
eGFP SIV (SIV-eGFP) vector contains an intact eGFP reporter gene that should be
expressed. More vector construct information is described in Kahl et al., 2006.(Kahl et
al., 2006)

97

Viral supernatant production

Viral vector supernatants were produced by cotransfecting 293T cells with SIV-
NoN and SIV-eGFP transfer vector, SIV4+ packaging plasmid, and pMD.G plasmid
expressing the VSV-G envelope glycoprotiens. Vector supernatant was tittered in HT29
cells and measured using Taqman real-time q-PCR as previously reported(Miller and
Rosman, 1989; Heid et al., 1996; Price et al., 2002). More detail information can also be
found in ref. 82.
Busulfan and fludarabine infusion
A total of twelve three-month-old rhesus monkeys completed the study and were reported
in this study thus far. They were divided into three treatment groups. Three conditioning
regimens were evaluated: controls (N=6) received busulfan at the optimized dose of 160
mg/m
2
(2 x 80 mg/m
2
)(Kahl et al., 2006) and experimental were administered busulfan in
Figure 4-2. Proviral maps of NoN and eGFP SIV-based lentiviral vectors. The
small rectangular box underneath the Neo and eGFP represents the probes used for
real time q-PCR.
98

combination with fludarabine at a final dosage of either 90 (2 x 45) (N=3) or 150 (3 x 50)
(N=3) mg/m
2
. On day -3 and -1, animals in the busulfan group were intravenously
injected with 80 mg/m
2
of busulfan over a time course of 30 minutes for a total dose of
160 mg/m
2
. In the busulfan plus fludarabine group, fludarabine was given to the subjects
on two or three consecutive days (day -3 to -1) for a total dosage of 90 mg/m
2
(45 mg/m
2

x 2) or 150 mg/m
2
(50 mg/m
2
x 3). Busulfan was given after the fludarabine infusion if
the animals were scheduled to both treatement. Transduced CD34
+
cells were re-infused
on day 0 in all groups (Fig. 4-3).

Figure 4-3. Experimental setup for HPCT in infant rhesus macaques with non-
myeloablative conditioning. Symbol ( ) represents the time point which the initial
CD34
+
cells was isolated from bone marrow. Serum and peripheral blood
mononuclear cells (PBMCs) were obtained for sample analyses. The time point
which the fludarabine was infused into the animals is indicated by symbol ( ). The
time point which the busulfan was infused into the animals is indicated by symbol (
). Symbol ( ) indicates the time point which the transduced CD34
+
cells were
reinfused back into the animals.

99

Transduction and autologous rhesus CD34
+
cell transplantation
Bone marrow isolated rhesus CD34
+
cells were plated at 10
5
cells/cm
2
in non-
tissue culture-treated 25 cm
2
flasks that had been coated with 4 mg/cm
2
of the
recombinant fibronectin. Pre-stimulation was then preformed overnight in X-Vivo 15
serum-free medium supplemented with 2 mM L-glutamine, 100 units/mL penicillin, and
100μg/mL streptomycin, and containing 100 ng/mL each of the recombinant human
cytokines: thrombopoietin (TPO), Flt3-Ligand (Flt3-L), and stem cell factor (SCF). The
next morning, cells were resuspended in fresh cyctokine-containing medium and
transduced with SIV vector supernatant with protamine sulfate. After 2 hours the
medium volume was doubled, and cells were incubated at 37°C with 5% CO
2
until the
next day. Cells were than washed and resuspended in PBS + 1% autologous serum
(collected prior to busulfan infusion). Cell counts and viability were determined using
trypan blue exclusion prior to each transplant. Small aliquots of cells were used for
methylcellulose colony assays and quantitative PCR analysis. Autologous cells were
injected IV into each animal in an approximate 2 mL volume, approximately 48 hours
post-busulfan infusion under ketamine (Fig. 4-4).

100

Blood and bone marrow sample collection
Blood samples were collected prior to busulfan infusion for complete blood
counts (CBCs), chemistry panels, peripheral blood mononuclear cells,
immunophenotyping (CD3, 4, 8, 19, 20, 34; ~ 2 mL), then at 4, 7, 11, 15, 21, and 30 days
posttransplantation for CBCs and chemistry panels (~ 1 mL). Bone marrow was also
collected prior to treatment to obtain CD34
+
for HSCT. Blood and marrow (~3 mL) were
collected at monthly intervals beginning at 1 month post-HSCT.
Sample analysis
For evaluation of gene transfer, peripheral blood and bone marrow samples were
collected monthly as noted above. Mononuclear cells (MNCs) and granulocytes from
peripheral blood were isolated by density gradient centrifugation over Ficoll-Hypaque.
Figure 4-4. Flow diagram of rhesus CD34
+
transduction for each monkey subject in
this study. The CD34
+
cells were split for transduction with two SIV lentiviral vectors,
one carrying a non-express neomycin (NoN) resistance gene (SIV-NoN) and the other
carrying a eGFP gene (SIV-eGFP). The transduced CD34
+
cells were combined and re-
infused via autologous BMT.
101

Hematopoietic colony assay were performed on MNCs. Individual hematopoietic
colonies were collected and then lysed for genomic DNA isolation. To detect proviral
sequences, real-time q-PCR using primers and probe for the neomycin and the eGFP gene
was performed. For each sample, parallel reactions were run in triplicate in separate
tubes for the detection of neomycin and eGFP gene sequences in bone marrow and
peripheral blood fractions.
4.3 Results
Busulfan pharmacokinetics: Area-Under-the Curve (AUC)
The total area under the curve (AUC) level represents the busulfan exposure of
the studied animals. The pharmacokinetics (pK) of busulfan was determined for each
animal. In this study, the busulfan AUC level was used as an indication of the amount of
busulfan that was metabolized in each monkey. The AUC of each animal receiving
intravenous busulfan was calculated from their pharmacokinetic (pK) data and tabulized
in Fig. 4-5. The targeted AUC level for a busulfan dosage of 160 mg/m
2
was expected to
be around 2000 mim*μg/mL based on our previously published data.(Kahl et al., 2006)
Inter-animal variations in busulfan absorption were reflected from the calculated AUC
data. With the same busulfan dose, none of our animals researched our targeted AUC
level. Three out of twelve monkeys achieved an AUC level over 1,500 mim*μg/mL and
two were slightly above 1,200 mim*μg/mL. The AUC levels for rest of the subjects all
were under 1,000 mim*μg/mL. Based on the busulfan AUC and CBC data, a higher
dosage of fludarabine might be required to exert its immunosuppressive ability. There
were no major differences in the absolute lymphocyte count (ALC) in the animals from
all three treatment groups (Fig. 4-5).
102

Strong correlation between the busulfan area-under-the curve (AUC) and the
absolute neutrophil count (ANC) nadir
To determine the overall effect of busulfan for each animal, we calculated the
absolute neutrophil count (ANC) and determined the nadir (the lowest data point) of the
neutrophil count from the CBC data (Fig. 4-5 and 4-6). As reported previously,(Kahl et
al., 2006) the busulfan AUC level positively correlated with the level of neutrophil
reduction in our animal model (Fig. 4-6). In addition, a similar correlation between ALC
and AUC was also observed. A statistical linear regression test was performed to
evaluate if the correlation observed between busulfan AUC and ANC nadir was
Figure 4-5. Busulfan pharmacokinetic data. Comparison of the total area under
the curve (AUC), absolute neutrophil count (ANC), and absolute lymphocyte count
(ALC) of each monkey after busulfan infusion. Group I represents animals received
160 mg/m
2
busulfan treatment. Group II represents animals received 160 mg/m
2

busulfan and 90 mg/m
2
fludarabine. Group III represents animals received 160
mg/m
2
busulfan and 150 mg/m
2
fludarabine.
103

statistically significant in our study (Fig. 4-6). An R
2
value of 0.66 was determined
indicating that there was over 50% of relative predictive power in our rhesus model.
ANC nadirs in the three animals with the highest AUC ( >1,500 mim*μg/mL), are more
than 50% lower than the 5
th
percentile of the normal ANC range (950 cells/μL).

Transient neutropenia was observed but not lymphopenia
The effects of busulfan or combination of busulfan and fludarabine on blood
counts (total white blood cell, neutrophil, and lymphocyte) of the animals with the
highest busulfan AUC from both control and experimental group were shown in Fig. 4-7.
An initial drop in the number of total white blood cell, neutrophil, and lymphocyte was
found. Similar changes in cell numbers over time were also observed in both total white
Figure 4-6. Absolute neutrophil count nadir vs. busulfan AUC by 2 months
post-HSCT. An increased busulfan AUC level led to a reduction in neutrophil count
in animals treated with busulfan. The R
2
value was determined by linear regression
statistical analysis. The pink diamond represents animals received both busulfan and
fludarabine infusion. The blue diamond represents the animals received only
busulfan.
104

blood cell and neutrophil counts. When the busulfan AUC levels of the animals reached
>1,500 mim*μg/mL, a reduction in the ANC to below the 5
th
percentile of the normal
range (950 cells/μL) for a transient period of time (2 to 3 weeks) was observed as
expected (Fig. 4-7).
In the busulfan-only control group, animals showed a major reduction in their
neutrophil count that was sustained for about 3 weeks. During this period, the ANC of
the animals with high busulfan AUC values mostly stayed below the 5
th
percentile of its
normal range. In addition to the reduction in the neutrophil counts, the lymphocyte
counts of some animals in the control group also reduced to below the 5
th
percentile of
the normal range (3,472 cells/μL) for about 3 weeks. This coincided with the fact that
busulfan has generally been used as a non-specific myeloablative agent in clinical
treatments.
The effects of fludarabine followed by busulfan infusion on the counts of total
white blood cell, neutrophil, and lymphocyte of the representative animal were also
shown in Fig. 4-7. Due to the immunosuppressive nature of fludarabine, the animals
were expected to show major reductions in both the neutrophil and lymphocyte cell
counts compared to that achieved in the busulfan control group. However, the CBC data
of the experiment groups was inclusive. Only a few time points had ALC dropped below
the 5
th
percentile normal range. The addition of fludarabine did not cause any significant
or transient ablation of lymphocytes. This again suggested that a higher dosage of
fludarabine might be required to achieve lymphopeina within clinically accepted toxicity
range. The fludarabine dose used in this study still remained suboptimal.

105

Figure 4-7. Effect of busulfan and fludarabine on total white blood cell count (♦),
neutrophil count (■), and lymphocyte count (●). One representative animal with the
highest busulfan AUC from each treatment group was shown in this figure. Open
symbols represent values below the 5
th
percentile of the normal range. The net busulfan
AUC, ANC nadir, and ALC nadir of each representative animal was also tabulated as
shown. The 5
th
percentile of the normal range is 5,600 cells/μL for total white blood
cells, 950 cells/μL for neutrophils, and 3,472 cells/μL for lymphocytes.

The highest gene marking observed declined from 0.3% to 0.05% over 6 months
post-HSCT
Non-expressed neo (NoN) is an inactivated form of the neo gene that cannot be
translated into its protein product. Theoretically, no transgene products from SIV-NoN
transduced cells should provoke host immune responses. The level of NoN gene marking
was expected to remain constant, whereas the level of eGFP marking should decrease
over time in animals of the busulfan control group. A comparison of the gene markings
detected in HSC transduced with SIV-NoN and eGFP vector post-HPCT evaluated
106

whether eGFP protein would be immunogenic. Furthermore, the gene marking data could
also demonstrate whether the detected immunogenicity of eGFP could be overcome and
induced an immune tolerance towards eGFP using non-myeloablative conditioning with
busulfan and fludarabine.
Percent gene marking for both NoN and eGFP were determined by real time q-
PCR. Gene marking in the transduced CD34
+
cells post-HSCT was used to evaluate the
HSC engraftment and long-term transgene expression. In contrast to the eGFP marking,
the average NoN gene marking in PBMCs was relatively higher by one log in both
control and experiment groups throughout the course of study (Fig. 4-8). The initial
eGFP gene marking (up to 0.3%) appeared at 2 months post-HPCT in all three groups.
Figure 4-8C summarized the NoN and eGFP percent gene marking in detected in all
animals. Gene marking for eGFP ranged from 0.001 – 0.05 % in all twelve monkeys at 6
months post-HPCT. The maximum % eGFP gene marking was much higher in the
busulfan control group (0.05%) than that detected in the busulfan plus fludarabine
experiment groups (0.01%).
In contrast to the eGFP marking, the NoN gene marking in PBMCs was relatively
higher ranging from 1.7 to 0.01% (Fig. 4-8C). The initial NoN gene marking was
detected as early as the first month after HPCT in the fludarabine plus busulfan group
whereas the initial NoN gene marking in the busulfan group was detected slightly later (2
months post-HSCT). The NoN marking detected from PBMCs generally increased as the
time progressed. One animal from the busulfan control group (group I) showed 1.65% of
NoN gene marking in their PBMCs by 6 months post-HSCT with an undetectable initial
NoN marking at one month post-transplantation.
107

The CD34
+
percent gene marking obtained from both busulfan and busulfan plus
fludarabine groups was also determined. The CD34
+
gene marking was considerably
lower (about 100-fold lower) than the gene marking detected in PBMCs (Fig. 4-8C). The
highest gene marking in CD34
+
cells was 0.27% detected for NoN at 6 months post
HSCT in the control group. While marking at most points has below the limit of detection
(<0.001%), eGFP marking in CD34
+
cells was detected as early as 1 month post BMT.
The highest eGFP gene marking in CD34
+
cells was 0.065% at 6 months post-HSCT.
Together, marking in all twelve monkeys was not as high as expected. The
changes observed in NoN and eGFP gene marking throughout our 6-month study period
were inconclusive. The internal NoN control of this study did not function as designed.
A steady level of the NoN gene marking was not seen in our study. In addition, gene
markings of the transduced CD34
+
cells were considerably lower that of PBMCs. Further
optimizations, such as the transduction efficiency of our SIV-base lentiviral vectors and
the cell dosing of the transduced CD34
+
cells during HSCT, will be investigated to
improve this low gene marking in our study.

Figure 4-8. Average NoN
cells (PBMCs) at 6 months post
detected in monkeys treated with busulfan.
monkeys treated with busulfan plus fludarabine.
log scale. Error bar represents the standard error of the mean.
AUC and percent gene markings in PBMC and CD34+ cells of the animals in this study.

A.
C.
NoN and eGFP gene marking in peripheral blood mononuclear
cells (PBMCs) at 6 months post-bone marrow transplant. (A) percent gene marking
detected in monkeys treated with busulfan. (B) percent gene marking detected in
monkeys treated with busulfan plus fludarabine. Percent gene marking is presented on a
log scale. Error bar represents the standard error of the mean. (C) table summary of the
AUC and percent gene markings in PBMC and CD34+ cells of the animals in this study.
B.
108

gene marking in peripheral blood mononuclear
percent gene marking
percent gene marking detected in
Percent gene marking is presented on a
table summary of the
AUC and percent gene markings in PBMC and CD34+ cells of the animals in this study.
109

The gene marking detected in the PBMCs might be derived from the gene modified
CD34
+
progenitor cells, not from the self-renewed LT- or ST-HSCs
As mentioned above, the CD34
+
gene marking was considerably lower (about
100-fold lower) than the gene marking detected in PBMCs in all our animals (Fig. 4-8C).
The transgene expression detected in the PBMCs was most likely from the infused SIV-
transduced CD34
+
cells not from the cells that were regenerated and differentiated from
the infused progenitor cells. The unstable expression of gene marking detected in the
transduced CD34
+
cells might indicate that only a limited number of early HSCs was
presented in the transduced population. Most of the transduced CD34
+
cells might
possibly be progenitor cells that have already differentiated or committed to differentiate.
Therefore, the lost of pluripotency in the transduced CD34
+
cell pool could lead to low
CD34
+
gene marking observed in our data. More importantly, if only limited number of
transduced CD34
+
cells were early progenitors, a sustained transgene expression and an
ultimate immune reconstitution could most likely be blunted. The CD34
+
isolation and
transduction protocol might be modified to a shorter transduction period to preserve the
pluripotency of the HSCs.
In addition, the transduced CD34
+
cell dosage might also be too low for
engraftment. Transplanted cell dosage has been shown to be a major factor to influence
engraftment and that sufficient quantities of cells can outweigh the need to create a bone
marrow “niche”.(Rao et al., 1997) Different from other HSCT studies in rhesus
model,(Huhn et al., 1999; Rosenzweig et al., 1999) the infant animals used in this study
were much smaller than the adult animals used in those studies. Limited amount of bone
marrow could be harvest in the infant animals. Due to this constraint of our monkey
110

model, relatively small amount of CD34
+
cells (10-fold lower in compare to the Huhn
and Rosenweig studies(Huhn et al., 1999; Rosenzweig et al., 1999)) from limited volume
of bone marrow from each small animal.
The eGFP transgene product was immunogenic and a humoral immune response
was triggered in an infant rhesus monkey model
Anti-eGFP antibody production was monitored by ELISA as an indication of
humoral immune response in this study. Blood serum was obtained at each sampling
time point to detect the presence of anti-eGFP antibody. We hypothesized that the eGFP
protein could be immunogenic to the animals in the busulfan control group but tolerated
in animals of the experiment group. Animals treated with busulfan were expected to
show some level of anti-eGFP antibody production since busulfan alone is not immune
ablative. In contrast, the detection for anti-eGFP antibody should be negative in animals
of the experiment group due to the immunosuppressive nature of fludarabine.
Serum samples were analyzed for anti-eGFP antibody production. The anti-eGFP
antibody detected by ELISA indicated that eGFP was immunogenic and a humoral
immune response was triggered by introducing eGFP transduced CD34
+
cells via
autologous HSCT in an infant rhesus monkey model (Fig. 4-9). Four out of six animals
in the busulfan control group (Group I) had detectable anti-eGFP antibody in their serum.
Only one animal from the experiment group (conditioned with busulfan plus fludarabine;
Group II and III) was detected positive for anti-eGFP antibody.
There was a delay in the time which the anti-eGFP antibody was initially
detected. Humoral immune response usually appears within 1-2 weeks after exposure of
a foreign antigen. This delay in the anti-eGFP response might be due to a molar effect of
111

eGFP antigen present in the animal. If only a few transduced eGFP cells (based on our
low eGFP gene marking data) existed in a large pool of untransduced cells, the time took
to trigger the host immune response against eGFP could be conceivably extended. A
threshold of eGFP level was most likely required before the activation of an immune
response, which could explain the delay humoral response observed in our animals.
The eGFP gene marking correlated to humoral immune response against transgene
products
The eGFP gene marking correlated to humoral immune response against
transgene products. While monkeys with high levels of eGFP gene marking also showed
detectable levels of anti-eGFP antibodies when no fludarabine was given, they lacked
humoral immune responses to eGFP if they received fludarabine (Fig. 4-9). This data
suggested that the immune responses against the transgene may play a significant role in
the successful outcome of HPC gene therapy and that fludarabine may be able to
modulate these responses.

Figure 4-9. Average % eGFP gene marking in PBMC verses humoral immune
response. The presented eGFP gene marking was an average of GFP markings from
month 4 – 6 of each animal. Busulfan AUC level was also included as shown. Group I
represents animals received 160 mg/m
received 160 mg/m
2
busulfan and 90 mg/m
received 160 mg/m
2
busulfan and 150 mg/m
standard error of the mean.

4.4 Discussion
The main focus of this study was to evaluate a non
increase HSC engraftment after HSCT. Busulfan was used to create “space” in the bone
marrow and consequently stimulate the regeneration of HSCs. By removing existing
HSCs in the bone marrow and transplant the gene
repopulate from the transplanted CD34+ and will also carry the transgene. However, if

9. Average % eGFP gene marking in PBMC verses humoral immune
. The presented eGFP gene marking was an average of GFP markings from
6 of each animal. Busulfan AUC level was also included as shown. Group I
represents animals received 160 mg/m
2
busulfan treatment. Group II represents animals
busulfan and 90 mg/m
2
fludarabine. Group III represents animals
busulfan and 150 mg/m
2
fludarabine. Error bar represents the
standard error of the mean.
The main focus of this study was to evaluate a non-myeloablative conditioning to
increase HSC engraftment after HSCT. Busulfan was used to create “space” in the bone
marrow and consequently stimulate the regeneration of HSCs. By removing existing
HSCs in the bone marrow and transplant the gene-modified CD34
+
cells, new HSCs will
repopulate from the transplanted CD34+ and will also carry the transgene. However, if
112

9. Average % eGFP gene marking in PBMC verses humoral immune
. The presented eGFP gene marking was an average of GFP markings from
6 of each animal. Busulfan AUC level was also included as shown. Group I
busulfan treatment. Group II represents animals
fludarabine. Group III represents animals
fludarabine. Error bar represents the
myeloablative conditioning to
increase HSC engraftment after HSCT. Busulfan was used to create “space” in the bone
marrow and consequently stimulate the regeneration of HSCs. By removing existing
cells, new HSCs will
repopulate from the transplanted CD34+ and will also carry the transgene. However, if
113

the host immune system was not suppressed at this time, these transgene-modified HSCs
would be recognized as foreign and be destroyed. In this study, fludarabine was used as
an immune ablative agent but the dosage did not effectively ablate the immune response
against transgene-modified cells. We were unable to achieve significant
lymphodepletion in the conditioned animals. The engrafted cells were most likely
eliminated by existing host immune response even if the HSCs were engrafted in the
animals.
Since significant lymphodepletion was not achieved by the fludarabine treatment,
higher doses of fludarabine may need to be evaluated for an effect on engraftment and
long-term transgene expression. The dose of fludarabine and busulfan conditioning
should also be optimized and tailored to individual animals. A pharmacokinetic (pK)
study of fludarabine to determine the amount of fludarabine metabolized after the
infusion could facilitate a more accurate dosing for each animal. In addition, there were
noticeably inter-animal variations in the efficacy of CD34
+
transduction, engraftment
efficiency following HSCT, and the responses post-busulfan and fludarabine non-
myeloablative conditioning. The transduction efficiency of the CD34
+
cells also needs to
be improved. Higher number of CD34+ cells and infectivity of the viral supernatant with
shorter transduction period could lead to a potentially higher probability in transferring
gene into early progenitor/stem cells and thereby allow expansion of the gene-modified
cells in the HSCT recipients.
Therefore, modifications have been made for our currently ongoing study
including shorter transduction protocol with a second transduction and fludarabine pK
analysis for each treated animals. A total of six infant rhesus monkeys were enrolled in
114

this study. Experiments to evaluated the immune tolerance and reconstitutions in the
busulfan and fludarabine treated animals are currently ongoing. Due to the nature of the
experiment, results will be reported once the study has been completed at the end of year
2009.

115

Chapter 5 – Concluding Remarks and Future Directions

Over the last decade, hematopoietic stem cell (HSC) gene therapy has been
explored as one of the treatments for inherited monogenetic such as SCID(Aiuti, 2004;
Qasim et al., 2004), Wiskott-Aldrich Syndrome (WAS)(Orange et al., 2004), or acquired
diseases such as HIV infection(Engel et al., 2003). HPSCs are defined by their ability to
self-renew and differentiate into multi-lineages of hematopoietic cells. In principle, these
unique qualities of HSCs can be exploited to obtain long-term expression of the
therapeutic transgene in the mature blood cells. As a result, extended effort has been
used to understand and manipulate these pluripotent hematopoietic stem/progenitor cells
for gene therapy.(Bordignon and Roncarolo, 2002)
The most essential goal for HSC gene therapy research is to increase stable
transgene engraftment and maintain the expression and function of the transgene in the
progeny cells of the gene modified HSCs. This, ultimately, can improve the therapeutic
effect of HSC gene therapy to clinically benefit patients. My projects involved two major
research focuses. Part one of my research focused on the development and optimization
of the non-viral SB transposon gene transfer methods to HSCs both in vitro and in vivo.
Part two of my research focused on a novel non-myeloablative conditioning using low-
dose busulfan and fludarabine as an alternative and potentially more effective
conditioning regimen to achieve long-term stable gene expression in an infant rhesus
HSC transplantation model.
116

In studies described in Chapter 2, using a hyperactive version of SB transposase,
HSB16, combined with the incrementally optimized SB transposon gene transfer system,
we were able to achieve a stable long-term transgene expression up to 27% in long-term
cultured human CD34+ hematopoietic cells in vitro and also detect up to 11% in the
animals that were transplanted with HSB-modified human CD34+ HSCs. Most
importantly, our secondary transplant data suggested that the optimized HSB transposon
system was able to mediate stable transgene integration and achieve long-term transgene
expression in the more primitive HSC populations with self renew and long-term
repopulating capacity. This encouraging data further demonstrates the feasibility of using
SB transposon system as an alternative non-viral delivery system for HSC gene therapy.
In addition, based on our promoter optimization study, promoter strength is
clearly dependent on the target cell type. Transgene expression from the integrated
transposon was strongest under the control of a retroviral MNDU3 promoter in myeloid
cells. In contrast, the transgene expressed the strongest when the EF1a promoter was
used for expression in lymphoid cells. This promoter and cell type dependence has also
been observed by other investigators.(Fitzsimons et al., 2002; Weber and Cannon, 2007)
Unfortunately, due to the small sample size in our in vivo study, we were unable to verify
this observation in vivo to date. It would be interesting to see if the animals that received
MND-eGFP transposon would show higher MFI in their myeloid cell lineages and the
animals that received EF1a-eGFP transposon would show brighter GFP expression in the
lymphoid cell lineages.

117

If this observation could be further verified in vivo, different promoter could be
utilized to expression therapeutic gene carried by a SB transposon plasmid and integrated
into specific cell type by SB transposase in diseases such as ADA-SCID. ADA-SCID is
a disease that lack of functional immune system due to a dysfunctional ADA enzyme or
complete absence of ADA that leads to an accumulation of toxic purine metabolites and
inhibits normal lymphoid development. Instead of a viral vector, the non-viral HSB
transposon system could be used to deliver functional ADA gene expressed by an EF1a
promoter into primary human CD34+ cells isolated from ADA-SCID patient. The SB-
mediated gene transfer system could facilitate ADA gene integration and the EF1a
promoter could then drive a strong expression of ADA when the cells differentiated into
lymphoid lineages.
A more efficient version of SB transposase, SB100X, has been developed during
the course of this thesis preparation.(Mates et al., 2009) It would also be very interesting
to see if using SB100X can achieve even higher stable transgene expression in primitive
human HSCs. In addition, it might be possible to reduce the plasmid related toxicity by
using small quantity of SB component with a more efficient transposase.
Finally, as future directions, the feasibility of using HSB transposon system as a
non-viral based gene transfer system for HSC gene therapy should be evaluated in a large
animal model such as canines or more clinically relevant non-human primates. To
eliminate the pre-existing host immune system in the immune competent animals, the
novel non-myeloablative conditioning described in Chapter 4 could be used to condition
the animals prior to HSCT to achieve greater long-term transgene expression with
relatively minimal adverse effects. Undeniably, there are still many obstacles remain to
118

be uncovered and overcome to apply the non-viral SB transposon gene transfer system
for HSC gene therapy. The studies described in this thesis provided evidences showing
that HSB can achieve long-term transgene expression in LT-HSCs on a multi-organ and
also multi-lineage level in vivo. This finding has shown a huge step forward towards
further advancement of non-viral based HSC gene therapy using SB transposon system.
119

Bibliography

AILLES, L., SCHMIDT, M., SANTONI DE SIO, F.R., GLIMM, H., CAVALIERI, S.,
BRUNO, S., PIACIBELLO, W., VON KALLE, C., and NALDINI, L. (2002).
Molecular evidence of lentiviral vector-mediated gene transfer into human self-
renewing, multi-potent, long-term NOD/SCID repopulating hematopoietic cells.
Mol Ther 6, 615-626.

AIUTI, A. (2004). Gene therapy for adenosine-deaminase-deficient severe combined
immunodeficiency. Best practice & research 17, 505-516.

AIUTI, A., BRIGIDA, I., FERRUA, F., CAPPELLI, B., CHIESA, R., MARKTEL, S.,
and RONCAROLO, M.G. (2009). Hematopoietic stem cell gene therapy for
adenosine deaminase deficient-SCID. Immunologic research.

AIUTI, A., SLAVIN, S., AKER, M., FICARA, F., DEOLA, S., MORTELLARO, A.,
MORECKI, S., ANDOLFI, G., TABUCCHI, A., CARLUCCI, F., MARINELLO,
E., CATTANEO, F., VAI, S., SERVIDA, P., MINIERO, R., RONCAROLO,
M.G., and BORDIGNON, C. (2002). Correction of ADA-SCID by stem cell gene
therapy combined with nonmyeloablative conditioning. Science (New York, N.Y
296, 2410-2413.

AN, D.S., KUNG, S.K., BONIFACINO, A., WERSTO, R.P., METZGER, M.E.,
AGRICOLA, B.A., MAO, S.H., CHEN, I.S., and DONAHUE, R.E. (2001).
Lentivirus vector-mediated hematopoietic stem cell gene transfer of common
gamma-chain cytokine receptor in rhesus macaques. Journal of virology 75, 3547-
3555.

AN, D.S., WERSTO, R.P., AGRICOLA, B.A., METZGER, M.E., LU, S., AMADO,
R.G., CHEN, I.S., and DONAHUE, R.E. (2000). Marking and gene expression by
a lentivirus vector in transplanted human and nonhuman primate CD34(+) cells.
Journal of virology 74, 1286-1295.

ANDERSSON, G., ILLIGENS, B.M., JOHNSON, K.W., CALDERHEAD, D.,
LEGUERN, C., BENICHOU, G., WHITE-SCHARF, M.E., and DOWN, J.D.
(2003). Nonmyeloablative conditioning is sufficient to allow engraftment of
EGFP-expressing bone marrow and subsequent acceptance of EGFP-transgenic
skin grafts in mice. Blood 101, 4305-4312.

ANDREWS, R.G., SINGER, J.W., and BERNSTEIN, I.D. (1986). Monoclonal antibody
12-8 recognizes a 115-kd molecule present on both unipotent and multipotent
hematopoietic colony-forming cells and their precursors. Blood 67, 842-845.

BACHTARZI, H., STEVENSON, M., and FISHER, K. (2008). Cancer gene therapy with
targeted adenoviruses. Expert opinion on drug delivery 5, 1231-1240.
120

BALCIUNAS, D., WANGENSTEEN, K.J., WILBER, A., BELL, J., GEURTS, A.,
SIVASUBBU, S., WANG, X., HACKETT, P.B., LARGAESPADA, D.A.,
MCIVOR, R.S., and EKKER, S.C. (2006). Harnessing a high cargo-capacity
transposon for genetic applications in vertebrates. PLoS genetics 2, e169.

BARKLIS, E., MULLIGAN, R.C., and JAENISCH, R. (1986). Chromosomal position or
virus mutation permits retrovirus expression in embryonal carcinoma cells. Cell
47, 391-399.

BARTELINK, I.H., BREDIUS, R.G., VERVERS, T.T., RAPHAEL, M.F., VAN
KESTEREN, C., BIERINGS, M., RADEMAKER, C.M., DEN HARTIGH, J.,
UITERWAAL, C.S., ZWAVELING, J., and BOELENS, J.J. (2008). Once-daily
intravenous busulfan with therapeutic drug monitoring compared to conventional
oral busulfan improves survival and engraftment in children undergoing
allogeneic stem cell transplantation. Biol Blood Marrow Transplant 14, 88-98.

BASKAR, J.F., SMITH, P.P., NILAVER, G., JUPP, R.A., HOFFMANN, S., PEFFER,
N.J., TENNEY, D.J., COLBERG-POLEY, A.M., GHAZAL, P., and NELSON,
J.A. (1996). The enhancer domain of the human cytomegalovirus major
immediate-early promoter determines cell type-specific expression in transgenic
mice. Journal of virology 70, 3207-3214.

BAUS, J., LIU, L., HEGGESTAD, A.D., SANZ, S., and FLETCHER, B.S. (2005).
Hyperactive transposase mutants of the Sleeping Beauty transposon. Mol Ther 12,
1148-1156.

BELL, J.B., PODETZ-PEDERSEN, K.M., ARONOVICH, E.L., BELUR, L.R.,
MCIVOR, R.S., and HACKETT, P.B. (2007). Preferential delivery of the
Sleeping Beauty transposon system to livers of mice by hydrodynamic injection.
Nature protocols 2, 3153-3165.

BELLANTUONO, I. (2004). Haemopoietic stem cells. The international journal of
biochemistry & cell biology 36, 607-620.

BELUR, L.R., PODETZ-PEDERSEN, K., FRANDSEN, J., and MCIVOR, R.S. (2007).
Lung-directed gene therapy in mice using the nonviral Sleeping Beauty
transposon system. Nature protocols 2, 3146-3152.

BERENSON, R.J., ANDREWS, R.G., BENSINGER, W.I., KALAMASZ, D.,
KNITTER, G., BUCKNER, C.D., and BERNSTEIN, I.D. (1988). Antigen
CD34+ marrow cells engraft lethally irradiated baboons. The Journal of clinical
investigation 81, 951-955.

121

BERGER, C., HUANG, M.L., GOUGH, M., GREENBERG, P.D., RIDDELL, S.R., and
KIEM, H.P. (2001). Nonmyeloablative immunosuppressive regimen prolongs In
vivo persistence of gene-modified autologous T cells in a nonhuman primate
model. Journal of virology 75, 799-808.

BERRY, C., HANNENHALLI, S., LEIPZIG, J., and BUSHMAN, F.D. (2006). Selection
of target sites for mobile DNA integration in the human genome. PLoS
computational biology 2, e157.

BHAGWATWAR, H.P., PHADUNGPOJNA, S., CHOW, D.S., and ANDERSSON, B.S.
(1996). Formulation and stability of busulfan for intravenous administration in
high-dose chemotherapy. Cancer chemotherapy and pharmacology 37, 401-408.

BOECKLE, S., and WAGNER, E. (2006). Optimizing targeted gene delivery: chemical
modification of viral vectors and synthesis of artificial virus vector systems. The
AAPS journal 8, E731-742.

BORDIGNON, C., BONINI, C., VERZELETTI, S., NOBILI, N., MAGGIONI, D.,
TRAVERSARI, C., GIAVAZZI, R., SERVIDA, P., ZAPPONE, E., BENAZZI,
E., and ET AL. (1995a). Transfer of the HSV-tk gene into donor peripheral blood
lymphocytes for in vivo modulation of donor anti-tumor immunity after
allogeneic bone marrow transplantation. Human gene therapy 6, 813-819.

BORDIGNON, C., MAVILIO, F., FERRARI, G., SERVIDA, P., UGAZIO, A.G.,
NOTARANGELO, L.D., GILBOA, E., ROSSINI, S., O'REILLY, R.J., SMITH,
C.A., and ET AL. (1993). Transfer of the ADA gene into bone marrow cells and
peripheral blood lymphocytes for the treatment of patients affected by ADA-
deficient SCID. Human gene therapy 4, 513-520.

BORDIGNON, C., NOTARANGELO, L.D., NOBILI, N., FERRARI, G., CASORATI,
G., PANINA, P., MAZZOLARI, E., MAGGIONI, D., ROSSI, C., SERVIDA, P.,
UGAZIO, A.G., and MAVILIO, F. (1995b). Gene therapy in peripheral blood
lymphocytes and bone marrow for ADA- immunodeficient patients. Science
(New York, N.Y 270, 470-475.

BORDIGNON, C., and RONCAROLO, M.G. (2002). Therapeutic applications for
hematopoietic stem cell gene transfer. Nature immunology 3, 318-321.

BOSHART, M., WEBER, F., JAHN, G., DORSCH-HASLER, K., FLECKENSTEIN, B.,
and SCHAFFNER, W. (1985). A very strong enhancer is located upstream of an
immediate early gene of human cytomegalovirus. Cell 41, 521-530.

122

BOUSSIF, O., LEZOUALC'H, F., ZANTA, M.A., MERGNY, M.D., SCHERMAN, D.,
DEMENEIX, B., and BEHR, J.P. (1995). A versatile vector for gene and
oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.
Proceedings of the National Academy of Sciences of the United States of America
92, 7297-7301.

BRUGGER, W., HENSCHLER, R., HEIMFELD, S., BERENSON, R.J.,
MERTELSMANN, R., and KANZ, L. (1994). Positively selected autologous
blood CD34+ cells and unseparated peripheral blood progenitor cells mediate
identical hematopoietic engraftment after high-dose VP16, ifosfamide,
carboplatin, and epirubicin. Blood 84, 1421-1426.

CAO, W., and ZOU, P. (2004). Liposome-mediated functional expression of multiple
drug resistance gene in human bone marrow CD34+ cells. Journal of Huazhong
University of Science and Technology. Medical sciences = Hua zhong ke ji da
xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao 24, 214-
215, 235.

CAVAZZANA-CALVO, M., HACEIN-BEY, S., DE SAINT BASILE, G., GROSS, F.,
YVON, E., NUSBAUM, P., SELZ, F., HUE, C., CERTAIN, S., CASANOVA,
J.L., BOUSSO, P., DEIST, F.L., and FISCHER, A. (2000). Gene therapy of
human severe combined immunodeficiency (SCID)-X1 disease. Science (New
York, N.Y 288, 669-672.

CHANG, L.J., URLACHER, V., IWAKUMA, T., CUI, Y., and ZUCALI, J. (1999).
Efficacy and safety analyses of a recombinant human immunodeficiency virus
type 1 derived vector system. Gene therapy 6, 715-728.

CHEN, L., and WOO, S.L. (2005). Complete and persistent phenotypic correction of
phenylketonuria in mice by site-specific genome integration of murine
phenylalanine hydroxylase cDNA. Proceedings of the National Academy of
Sciences of the United States of America 102, 15581-15586.

CIVIN, C.I., STRAUSS, L.C., FACKLER, M.J., TRISCHMANN, T.M., WILEY, J.M.,
and LOKEN, M.R. (1990). Positive stem cell selection--basic science. Progress in
clinical and biological research 333, 387-401; discussion 402.

CUI, Z., GEURTS, A.M., LIU, G., KAUFMAN, C.D., and HACKETT, P.B. (2002).
Structure-function analysis of the inverted terminal repeats of the sleeping beauty
transposon. Journal of molecular biology 318, 1221-1235.

DIEBOLD, S.S., LEHRMANN, H., KURSA, M., WAGNER, E., COTTEN, M., and
ZENKE, M. (1999). Efficient gene delivery into human dendritic cells by
adenovirus polyethylenimine and mannose polyethylenimine transfection. Human
gene therapy 10, 775-786.
123

DING, S., WU, X., LI, G., HAN, M., ZHUANG, Y., and XU, T. (2005). Efficient
transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell
122, 473-483.

DORIGO, O., GIL, J.S., GALLAHER, S.D., TAN, B.T., CASTRO, M.G.,
LOWENSTEIN, P.R., CALOS, M.P., and BERK, A.J. (2004). Development of a
novel helper-dependent adenovirus-Epstein-Barr virus hybrid system for the
stable transformation of mammalian cells. Journal of virology 78, 6556-6566.

DUNBAR, C.E., COTTLER-FOX, M., O'SHAUGHNESSY, J.A., DOREN, S.,
CARTER, C., BERENSON, R., BROWN, S., MOEN, R.C., GREENBLATT, J.,
STEWART, F.M., and ET AL. (1995). Retrovirally marked CD34-enriched
peripheral blood and bone marrow cells contribute to long-term engraftment after
autologous transplantation. Blood 85, 3048-3057.

ENGEL, B.C., KOHN, D.B., and PODSAKOFF, G.M. (2003). Update on gene therapy
of inherited immune deficiencies. Current opinion in molecular therapeutics 5,
503-507.

ENGELMAN, A. (1999). In vivo analysis of retroviral integrase structure and function.
Advances in virus research 52, 411-426.

ENGELMAN, A., ENGLUND, G., ORENSTEIN, J.M., MARTIN, M.A., and CRAIGIE,
R. (1995). Multiple effects of mutations in human immunodeficiency virus type 1
integrase on viral replication. Journal of virology 69, 2729-2736.

ESSNER, J.J., MCIVOR, R.S., and HACKETT, P.B. (2005). Awakening gene therapy
with Sleeping Beauty transposons. Current opinion in pharmacology 5, 513-519.

FEUER, G., TAKETO, M., HANECAK, R.C., and FAN, H. (1989). Two blocks in
Moloney murine leukemia virus expression in undifferentiated F9 embryonal
carcinoma cells as determined by transient expression assays. Journal of virology
63, 2317-2324.

FITZSIMONS, H.L., BLAND, R.J., and DURING, M.J. (2002). Promoters and
regulatory elements that improve adeno-associated virus transgene expression in
the brain. Methods (San Diego, Calif 28, 227-236.

FORMAN, S.J. (2005). Should patients with acute lymphoblastic leukemia receive
hematopoietic stem-cell transplant from an unrelated donor? Nature clinical
practice 2, 18-19.

GAMBOTTO, A., DWORACKI, G., CICINNATI, V., KENNISTON, T., STEITZ, J.,
TUTING, T., ROBBINS, P.D., and DELEO, A.B. (2000). Immunogenicity of
enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an
H2-Kd-restricted CTL epitope. Gene therapy 7, 2036-2040.
124

GANDHI, V., and PLUNKETT, W. (2002). Cellular and clinical pharmacology of
fludarabine. Clinical pharmacokinetics 41, 93-103.

GASPAR, H.B., BJORKEGREN, E., PARSLEY, K., GILMOUR, K.C., KING, D.,
SINCLAIR, J., ZHANG, F., GIANNAKOPOULOS, A., ADAMS, S.,
FAIRBANKS, L.D., GASPAR, J., HENDERSON, L., XU-BAYFORD, J.H.,
DAVIES, E.G., VEYS, P.A., KINNON, C., and THRASHER, A.J. (2006).
Successful reconstitution of immunity in ADA-SCID by stem cell gene therapy
following cessation of PEG-ADA and use of mild preconditioning. Mol Ther 14,
505-513.

GEURTS, A.M., YANG, Y., CLARK, K.J., LIU, G., CUI, Z., DUPUY, A.J., BELL, J.B.,
LARGAESPADA, D.A., and HACKETT, P.B. (2003). Gene transfer into
genomes of human cells by the sleeping beauty transposon system. Mol Ther 8,
108-117.

GIRI, N., KAUSHIVA, A., WU, T., SELLERS, S.E., and TISDALE, J.F. (2001). The
effects of SCF/G-CSF prestimulation on radiation sensitivity and engraftment in
nonmyeloablated murine hosts. Experimental hematology 29, 779-785.

GLORIOSO, J.C., MATA, M., and FINK, D.J. (2003). Therapeutic gene transfer to the
nervous system using viral vectors. Journal of neurovirology 9, 165-172.

GREINER, D.L., HESSELTON, R.A., and SHULTZ, L.D. (1998). SCID mouse models
of human stem cell engraftment. Stem cells (Dayton, Ohio) 16, 166-177.

GRESCH, O., ENGEL, F.B., NESIC, D., TRAN, T.T., ENGLAND, H.M., HICKMAN,
E.S., KORNER, I., GAN, L., CHEN, S., CASTRO-OBREGON, S.,
HAMMERMANN, R., WOLF, J., MULLER-HARTMANN, H., NIX, M.,
SIEBENKOTTEN, G., KRAUS, G., and LUN, K. (2004). New non-viral method
for gene transfer into primary cells. Methods (San Diego, Calif 33, 151-163.

GROCHOW, L.B. (1993). Busulfan disposition: the role of therapeutic monitoring in
bone marrow transplantation induction regimens. Seminars in oncology 20, 18-
25; quiz 26.

HAAS, D.L., LUTZKO, C., LOGAN, A.C., CHO, G.J., SKELTON, D., JIN YU, X.,
PEPPER, K.A., and KOHN, D.B. (2003). The Moloney murine leukemia virus
repressor binding site represses expression in murine and human hematopoietic
stem cells. Journal of virology 77, 9439-9450.

125

HACEIN-BEY-ABINA, S., VON KALLE, C., SCHMIDT, M., MCCORMACK, M.P.,
WULFFRAAT, N., LEBOULCH, P., LIM, A., OSBORNE, C.S., PAWLIUK, R.,
MORILLON, E., SORENSEN, R., FORSTER, A., FRASER, P., COHEN, J.I.,
DE SAINT BASILE, G., ALEXANDER, I., WINTERGERST, U., FREBOURG,
T., AURIAS, A., STOPPA-LYONNET, D., ROMANA, S., RADFORD-WEISS,
I., GROSS, F., VALENSI, F., DELABESSE, E., MACINTYRE, E., SIGAUX, F.,
SOULIER, J., LEIVA, L.E., WISSLER, M., PRINZ, C., RABBITTS, T.H., LE
DEIST, F., FISCHER, A., and CAVAZZANA-CALVO, M. (2003). LMO2-
associated clonal T cell proliferation in two patients after gene therapy for SCID-
X1. Science (New York, N.Y 302, 415-419.

HACKETT, P.B. (2007). Integrating DNA vectors for gene therapy. Mol Ther 15, 10-12.

HACKETT, P.B., EKKER, S.C., LARGAESPADA, D.A., and MCIVOR, R.S. (2005).
Sleeping beauty transposon-mediated gene therapy for prolonged expression.
Advances in genetics 54, 189-232.

HALENE, S., WANG, L., COOPER, R.M., BOCKSTOCE, D.C., ROBBINS, P.B., and
KOHN, D.B. (1999). Improved expression in hematopoietic and lymphoid cells in
mice after transplantation of bone marrow transduced with a modified retroviral
vector. Blood 94, 3349-3357.

HANAZONO, Y., BROWN, K.E., HANDA, A., METZGER, M.E., HEIM, D.,
KURTZMAN, G.J., DONAHUE, R.E., and DUNBAR, C.E. (1999). In vivo
marking of rhesus monkey lymphocytes by adeno-associated viral vectors: direct
comparison with retroviral vectors. Blood 94, 2263-2270.

HAO, Q.L., SHAH, A.J., THIEMANN, F.T., SMOGORZEWSKA, E.M., and CROOKS,
G.M. (1995). A functional comparison of CD34 + CD38- cells in cord blood and
bone marrow. Blood 86, 3745-3753.

HARTL, D.L., LOHE, A.R., and LOZOVSKAYA, E.R. (1997). Regulation of the
transposable element mariner. Genetica 100, 177-184.

HE, C.X., SHI, D., WU, W.J., DING, Y.F., FENG, D.M., LU, B., CHEN, H.M., YAO,
J.H., SHEN, Q., LU, D.R., and XUE, J.L. (2004). Insulin expression in livers of
diabetic mice mediated by hydrodynamics-based administration. World J
Gastroenterol 10, 567-572.

HEID, C.A., STEVENS, J., LIVAK, K.J., and WILLIAMS, P.M. (1996). Real time
quantitative PCR. Genome research 6, 986-994.

126

HEIM, D.A., HANAZONO, Y., GIRI, N., WU, T., CHILDS, R., SELLERS, S.E.,
MUUL, L., AGRICOLA, B.A., METZGER, M.E., DONAHUE, R.E., TISDALE,
J.F., and DUNBAR, C.E. (2000). Introduction of a xenogeneic gene via
hematopoietic stem cells leads to specific tolerance in a rhesus monkey model.
Mol Ther 1, 533-544.

HOHENSTEIN, K.A., PYLE, A.D., CHERN, J.Y., LOCK, L.F., and DONOVAN, P.J.
(2008). Nucleofection mediates high-efficiency stable gene knockdown and
transgene expression in human embryonic stem cells. Stem cells (Dayton, Ohio)
26, 1436-1443.

HOLLIS, R.P., NIGHTINGALE, S.J., WANG, X., PEPPER, K.A., YU, X.J., BARSKY,
L., CROOKS, G.M., and KOHN, D.B. (2006). Stable gene transfer to human
CD34(+) hematopoietic cells using the Sleeping Beauty transposon. Experimental
hematology 34, 1333-1343.

HORWITZ, M.E., BARRETT, A.J., BROWN, M.R., CARTER, C.S., CHILDS, R.,
GALLIN, J.I., HOLLAND, S.M., LINTON, G.F., MILLER, J.A., LEITMAN,
S.F., READ, E.J., and MALECH, H.L. (2001). Treatment of chronic
granulomatous disease with nonmyeloablative conditioning and a T-cell-depleted
hematopoietic allograft. The New England journal of medicine 344, 881-888.

HUANG, X., WILBER, A.C., BAO, L., TUONG, D., TOLAR, J., ORCHARD, P.J.,
LEVINE, B.L., JUNE, C.H., MCIVOR, R.S., BLAZAR, B.R., and ZHOU, X.
(2006). Stable gene transfer and expression in human primary T cells by the
Sleeping Beauty transposon system. Blood 107, 483-491.

HUHN, R.D., TISDALE, J.F., AGRICOLA, B., METZGER, M.E., DONAHUE, R.E.,
and DUNBAR, C.E. (1999). Retroviral marking and transplantation of rhesus
hematopoietic cells by nonmyeloablative conditioning. Human gene therapy 10,
1783-1790.

ISHIKAWA, F., YASUKAWA, M., LYONS, B., YOSHIDA, S., MIYAMOTO, T.,
YOSHIMOTO, G., WATANABE, T., AKASHI, K., SHULTZ, L.D., and
HARADA, M. (2005). Development of functional human blood and immune
systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice. Blood 106, 1565-
1573.

ITO, M., HIRAMATSU, H., KOBAYASHI, K., SUZUE, K., KAWAHATA, M., HIOKI,
K., UEYAMA, Y., KOYANAGI, Y., SUGAMURA, K., TSUJI, K., HEIKE, T.,
and NAKAHATA, T. (2002). NOD/SCID/gamma(c)(null) mouse: an excellent
recipient mouse model for engraftment of human cells. Blood 100, 3175-3182.

IVANOVA, N.B., DIMOS, J.T., SCHANIEL, C., HACKNEY, J.A., MOORE, K.A., and
LEMISCHKA, I.R. (2002). A stem cell molecular signature. Science (New York,
N.Y 298, 601-604.
127

IVICS, Z., HACKETT, P.B., PLASTERK, R.H., and IZSVAK, Z. (1997). Molecular
reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its
transposition in human cells. Cell 91, 501-510.

IVICS, Z., KAUFMAN, C.D., ZAYED, H., MISKEY, C., WALISKO, O., and IZSVAK,
Z. (2004). The Sleeping Beauty transposable element: evolution, regulation and
genetic applications. Current issues in molecular biology 6, 43-55.

IZSVAK, Z., and IVICS, Z. (2004). Sleeping beauty transposition: biology and
applications for molecular therapy. Mol Ther 9, 147-156.

IZSVAK, Z., IVICS, Z., and PLASTERK, R.H. (2000). Sleeping Beauty, a wide host-
range transposon vector for genetic transformation in vertebrates. Journal of
molecular biology 302, 93-102.

JAENISCH, R., HARBERS, K., JAHNER, D., STEWART, C., and STUHLMANN, H.
(1982). DNA methylation, retroviruses, and embryogenesis. Journal of cellular
biochemistry 20, 331-336.

JAHNER, D., STUHLMANN, H., STEWART, C.L., HARBERS, K., LOHLER, J.,
SIMON, I., and JAENISCH, R. (1982). De novo methylation and expression of
retroviral genomes during mouse embryogenesis. Nature 298, 623-628.

JOHNSON, L.A., MORGAN, R.A., DUDLEY, M.E., CASSARD, L., YANG, J.C.,
HUGHES, M.S., KAMMULA, U.S., ROYAL, R.E., SHERRY, R.M.,
WUNDERLICH, J.R., LEE, C.C., RESTIFO, N.P., SCHWARZ, S.L., COGDILL,
A.P., BISHOP, R.J., KIM, H., BREWER, C.C., RUDY, S.F., VANWAES, C.,
DAVIS, J.L., MATHUR, A., RIPLEY, R.T., NATHAN, D.A., LAURENCOT,
C.M., and ROSENBERG, S.A. (2009). Gene therapy with human and mouse T
cell receptors mediates cancer regression and targets normal tissues expressing
cognate antigen. Blood.

KAHL, C.A., TARANTAL, A.F., LEE, C.I., JIMENEZ, D.F., CHOI, C., PEPPER, K.,
PETERSEN, D., FLETCHER, M.D., LEAPLEY, A.C., FISHER, J., BURNS,
T.S., ULTSCH, M.N., DOREY, F.J., and KOHN, D.B. (2006). Effects of
busulfan dose escalation on engraftment of infant rhesus monkey hematopoietic
stem cells after gene marking by a lentiviral vector. Experimental hematology 34,
369-381.

KAISER, J. (2003). Gene therapy. Seeking the cause of induced leukemias in X-SCID
trial. Science (New York, N.Y 299, 495.

128

KANG, E., GIRI, N., WU, T., SELLERS, S., KIRBY, M., HANAZONO, Y., TISDALE,
J., and DUNBAR, C.E. (2001). In vivo persistence of retrovirally transduced
murine long-term repopulating cells is not limited by expression of foreign gene
products in the fully or minimally myeloablated setting. Human gene therapy 12,
1663-1672.

KANG, E.M., HSIEH, M.M., METZGER, M., KROUSE, A., DONAHUE, R.E.,
SADELAIN, M., and TISDALE, J.F. (2006). Busulfan pharmacokinetics,
toxicity, and low-dose conditioning for autologous transplantation of genetically
modified hematopoietic stem cells in the rhesus macaque model. Experimental
hematology 34, 132-139.

KAY, M.A., BALEY, P., ROTHENBERG, S., LELAND, F., FLEMING, L., PONDER,
K.P., LIU, T., FINEGOLD, M., DARLINGTON, G., POKORNY, W., and ET
AL. (1992). Expression of human alpha 1-antitrypsin in dogs after autologous
transplantation of retroviral transduced hepatocytes. Proceedings of the National
Academy of Sciences of the United States of America 89, 89-93.

KAY, M.A., GLORIOSO, J.C., and NALDINI, L. (2001). Viral vectors for gene therapy:
the art of turning infectious agents into vehicles of therapeutics. Nature medicine
7, 33-40.

KERAVALA, A., and CALOS, M.P. (2008). Site-specific chromosomal integration
mediated by phiC31 integrase. Methods in molecular biology (Clifton, N.J 435,
165-173.

KIM, Y.K., CHOI, J.Y., JIANG, H.L., AROTE, R., JERE, D., CHO, M.H., JE, Y.H., and
CHO, C.S. (2009). Hybrid of baculovirus and galactosylated PEI for efficient
gene carrier. Virology 387, 89-97.

KOHN, D.B. (2008). Gene therapy for childhood immunological diseases. Bone marrow
transplantation 41, 199-205.

KOHN, D.B., and CANDOTTI, F. (2009). Gene therapy fulfilling its promise. The New
England journal of medicine 360, 518-521.

KOHN, D.B., WEINBERG, K.I., NOLTA, J.A., HEISS, L.N., LENARSKY, C.,
CROOKS, G.M., HANLEY, M.E., ANNETT, G., BROOKS, J.S., EL-
KHOUREIY, A., and ET AL. (1995). Engraftment of gene-modified umbilical
cord blood cells in neonates with adenosine deaminase deficiency. Nature
medicine 1, 1017-1023.

129

KOPORC, Z., BIGENZAHN, S., BLAHA, P., FARIBORZ, E., SELZER, E., SYKES,
M., MUEHLBACHER, F., and WEKERLE, T. (2006). Induction of mixed
chimerism through transplantation of CD45-congenic mobilized peripheral blood
stem cells after nonmyeloablative irradiation. Biol Blood Marrow Transplant 12,
284-292.

KUNG, S.K., AN, D.S., BONIFACINO, A., METZGER, M.E., RINGPIS, G.E., MAO,
S.H., CHEN, I.S., and DONAHUE, R.E. (2003). Induction of transgene-specific
immunological tolerance in myeloablated nonhuman primates using lentivirally
transduced CD34+ progenitor cells. Mol Ther 8, 981-991.

LAMPE, D.J., GRANT, T.E., and ROBERTSON, H.M. (1998). Factors affecting
transposition of the Himar1 mariner transposon in vitro. Genetics 149, 179-187.

LEHRMAN, S. (1999). Virus treatment questioned after gene therapy death. Nature 401,
517-518.

LINNEY, E., DAVIS, B., OVERHAUSER, J., CHAO, E., and FAN, H. (1984). Non-
function of a Moloney murine leukaemia virus regulatory sequence in F9
embryonal carcinoma cells. Nature 308, 470-472.

LIU, H., and VISNER, G.A. (2007). Applications of Sleeping Beauty transposons for
nonviral gene therapy. IUBMB life 59, 374-379.

LIU, L., LIU, H., MAH, C., and FLETCHER, B.S. (2009). Indoleamine 2,3-dioxygenase
attenuates inhibitor development in gene-therapy-treated hemophilia A mice.
Gene therapy.

LIU, L., SANZ, S., HEGGESTAD, A.D., ANTHARAM, V., NOTTERPEK, L., and
FLETCHER, B.S. (2004). Endothelial targeting of the Sleeping Beauty
transposon within lung. Mol Ther 10, 97-105.

LOGAN, A.C., HAAS, D.L., KAFRI, T., and KOHN, D.B. (2004a). Integrated self-
inactivating lentiviral vectors produce full-length genomic transcripts competent
for encapsidation and integration. Journal of virology 78, 8421-8436.

LOGAN, A.C., LUTZKO, C., and KOHN, D.B. (2002). Advances in lentiviral vector
design for gene-modification of hematopoietic stem cells. Current opinion in
biotechnology 13, 429-436.

LOGAN, A.C., NIGHTINGALE, S.J., HAAS, D.L., CHO, G.J., PEPPER, K.A., and
KOHN, D.B. (2004b). Factors influencing the titer and infectivity of lentiviral
vectors. Human gene therapy 15, 976-988.

LOHE, A.R., SULLIVAN, D.T., and HARTL, D.L. (1996). Subunit interactions in the
mariner transposase. Genetics 144, 1087-1095.
130

LOMBARDO, A., GENOVESE, P., BEAUSEJOUR, C.M., COLLEONI, S., LEE, Y.L.,
KIM, K.A., ANDO, D., URNOV, F.D., GALLI, C., GREGORY, P.D., HOLMES,
M.C., and NALDINI, L. (2007). Gene editing in human stem cells using zinc
finger nucleases and integrase-defective lentiviral vector delivery. Nature
biotechnology 25, 1298-1306.

LOWENSTEIN, P.R., SOUTHGATE, T.D., SMITH-ARICA, J.R., SMITH, J., and
CASTRO, M.G. (1998). Gene therapy for inherited neurological disorders:
towards therapeutic intervention in the Lesch-Nyhan syndrome. Progress in brain
research 117, 485-501.

LOZIER, J.N., METZGER, M.E., DONAHUE, R.E., and MORGAN, R.A. (1999).
Adenovirus-mediated expression of human coagulation factor IX in the rhesus
macaque is associated with dose-limiting toxicity. Blood 94, 3968-3975.

LUNDQVIST, A., NOFFZ, G., PAVLENKO, M., SAEBOE-LARSSEN, S., FONG, T.,
MAITLAND, N., and PISA, P. (2002). Nonviral and viral gene transfer into
different subsets of human dendritic cells yield comparable efficiency of
transfection. J Immunother 25, 445-454.

LUTZKO, C., OMORI, F., ABRAMS-OGG, A.C., SHULL, R., LI, L., LAU, K.,
RUEDY, C., NANJI, S., GARTLEY, C., DOBSON, H., FOSTER, R., KRUTH,
S., and DUBE, I.D. (1999). Gene therapy for canine alpha-L-iduronidase
deficiency: in utero adoptive transfer of genetically corrected hematopoietic
progenitors results in engraftment but not amelioration of disease. Human gene
therapy 10, 1521-1532.

MANGEOT, P.E., NEGRE, D., DUBOIS, B., WINTER, A.J., LEISSNER, P.,
MEHTALI, M., KAISERLIAN, D., COSSET, F.L., and DARLIX, J.L. (2000).
Development of minimal lentivirus vectors derived from simian
immunodeficiency virus (SIVmac251) and their use for gene transfer into human
dendritic cells. Journal of virology 74, 8307-8315.

MANTOVANI, J., CHARRIER, S., ECKENBERG, R., SAURIN, W., DANOS, O.,
PEREA, J., and GALY, A. (2009). Diverse genomic integration of a lentiviral
vector developed for the treatment of Wiskott-Aldrich syndrome. The journal of
gene medicine.

MATES, L., CHUAH, M.K., BELAY, E., JERCHOW, B., MANOJ, N., ACOSTA-
SANCHEZ, A., GRZELA, D.P., SCHMITT, A., BECKER, K., MATRAI, J.,
MA, L., SAMARA-KUKO, E., GYSEMANS, C., PRYPUTNIEWICZ, D.,
MISKEY, C., FLETCHER, B., VANDENDRIESSCHE, T., IVICS, Z., and
IZSVAK, Z. (2009). Molecular evolution of a novel hyperactive Sleeping Beauty
transposase enables robust stable gene transfer in vertebrates. Nature genetics 41,
753-761.
131

MILLER, A.D., and ROSMAN, G.J. (1989). Improved retroviral vectors for gene
transfer and expression. BioTechniques 7, 980-982, 984-986, 989-990.

MITSUYASU, R.T., MERIGAN, T.C., CARR, A., ZACK, J.A., WINTERS, M.A.,
WORKMAN, C., BLOCH, M., LALEZARI, J., BECKER, S., THORNTON, L.,
AKIL, B., KHANLOU, H., FINLAYSON, R., MCFARLANE, R., SMITH, D.E.,
GARSIA, R., MA, D., LAW, M., MURRAY, J.M., VON KALLE, C., ELY, J.A.,
PATINO, S.M., KNOP, A.E., WONG, P., TODD, A.V., HAUGHTON, M.,
FUERY, C., MACPHERSON, J.L., SYMONDS, G.P., EVANS, L.A., POND,
S.M., and COOPER, D.A. (2009). Phase 2 gene therapy trial of an anti-HIV
ribozyme in autologous CD34+ cells. Nature medicine 15, 285-292.

MIYOSHI, H., SMITH, K.A., MOSIER, D.E., VERMA, I.M., and TORBETT, B.E.
(1999). Transduction of human CD34+ cells that mediate long-term engraftment
of NOD/SCID mice by HIV vectors. Science (New York, N.Y 283, 682-686.

MIZUSHIMA, S., and NAGATA, S. (1990). pEF-BOS, a powerful mammalian
expression vector. Nucleic acids research 18, 5322.

MONTINI, E., HELD, P.K., NOLL, M., MORCINEK, N., AL-DHALIMY, M.,
FINEGOLD, M., YANT, S.R., KAY, M.A., and GROMPE, M. (2002). In vivo
correction of murine tyrosinemia type I by DNA-mediated transposition. Mol
Ther 6, 759-769.

MORRIS, J.C., CONERLY, M., THOMASSON, B., STOREK, J., RIDDELL, S.R., and
KIEM, H.P. (2004). Induction of cytotoxic T-lymphocyte responses to enhanced
green and yellow fluorescent proteins after myeloablative conditioning. Blood
103, 492-499.

MURPHY, M., REID, K., WILLIAMS, D.E., LYMAN, S.D., and BARTLETT, P.F.
(1992). Steel factor is required for maintenance, but not differentiation, of
melanocyte precursors in the neural crest. Developmental biology 153, 396-401.

MURRAY, L.J., YOUNG, J.C., OSBORNE, L.J., LUENS, K.M., SCOLLAY, R., and
HILL, B.L. (1999). Thrombopoietin, flt3, and kit ligands together suppress
apoptosis of human mobilized CD34+ cells and recruit primitive CD34+ Thy-1+
cells into rapid division. Experimental hematology 27, 1019-1028.

NAKAI, H., FUESS, S., STORM, T.A., MEUSE, L.A., and KAY, M.A. (2003). Free
DNA ends are essential for concatemerization of synthetic double-stranded
adeno-associated virus vector genomes transfected into mouse hepatocytes in
vivo. Mol Ther 7, 112-121.

132

NAKAI, H., HERZOG, R.W., HAGSTROM, J.N., WALTER, J., KUNG, S.H., YANG,
E.Y., TAI, S.J., IWAKI, Y., KURTZMAN, G.J., FISHER, K.J., COLOSI, P.,
COUTO, L.B., and HIGH, K.A. (1998). Adeno-associated viral vector-mediated
gene transfer of human blood coagulation factor IX into mouse liver. Blood 91,
4600-4607.

NAKAJIMA, N., LU, R., and ENGELMAN, A. (2001). Human immunodeficiency virus
type 1 replication in the absence of integrase-mediated dna recombination:
definition of permissive and nonpermissive T-cell lines. Journal of virology 75,
7944-7955.

NAKATA, Y., SHETZLINE, S., SAKASHITA, C., KALOTA, A., RALLAPALLI, R.,
RUDNICK, S.I., ZHANG, Y., EMERSON, S.G., and GEWIRTZ, A.M. (2007). c-
Myb contributes to G2/M cell cycle transition in human hematopoietic cells by
direct regulation of cyclin B1 expression. Molecular and cellular biology 27,
2048-2058.

NALDINI, L., BLOMER, U., GALLAY, P., ORY, D., MULLIGAN, R., GAGE, F.H.,
VERMA, I.M., and TRONO, D. (1996). In vivo gene delivery and stable
transduction of nondividing cells by a lentiviral vector. Science (New York, N.Y
272, 263-267.

NIGHTINGALE, S.J., HOLLIS, R.P., PEPPER, K.A., PETERSEN, D., YU, X.J.,
YANG, C., BAHNER, I., and KOHN, D.B. (2006). Transient gene expression by
nonintegrating lentiviral vectors. Mol Ther 13, 1121-1132.

OHLFEST, J.R., FRANDSEN, J.L., FRITZ, S., LOBITZ, P.D., PERKINSON, S.G.,
CLARK, K.J., NELSESTUEN, G., KEY, N.S., MCIVOR, R.S., HACKETT,
P.B., and LARGAESPADA, D.A. (2005). Phenotypic correction and long-term
expression of factor VIII in hemophilic mice by immunotolerization and nonviral
gene transfer using the Sleeping Beauty transposon system. Blood 105, 2691-
2698.

OLDAK, T., KRUSZEWSKI, M., MACHAJ, E.K., GAJKOWSKA, A., and POJDA, Z.
(2002). Optimisation of transfection conditions of CD34+ hematopoietic cells
derived from human umbilical cord blood. Acta biochimica Polonica 49, 625-632.

OLIVARES, E.C., HOLLIS, R.P., CHALBERG, T.W., MEUSE, L., KAY, M.A., and
CALOS, M.P. (2002). Site-specific genomic integration produces therapeutic
Factor IX levels in mice. Nature biotechnology 20, 1124-1128.

ORANGE, J.S., STONE, K.D., TURVEY, S.E., and KRZEWSKI, K. (2004). The
Wiskott-Aldrich syndrome. Cell Mol Life Sci 61, 2361-2385.

133

OTT, M.G., SCHMIDT, M., SCHWARZWAELDER, K., STEIN, S., SILER, U.,
KOEHL, U., GLIMM, H., KUHLCKE, K., SCHILZ, A., KUNKEL, H.,
NAUNDORF, S., BRINKMANN, A., DEICHMANN, A., FISCHER, M., BALL,
C., PILZ, I., DUNBAR, C., DU, Y., JENKINS, N.A., COPELAND, N.G.,
LUTHI, U., HASSAN, M., THRASHER, A.J., HOELZER, D., VON KALLE, C.,
SEGER, R., and GREZ, M. (2006). Correction of X-linked chronic
granulomatous disease by gene therapy, augmented by insertional activation of
MDS1-EVI1, PRDM16 or SETBP1. Nature medicine 12, 401-409.

PANG, S., KOYANAGI, Y., MILES, S., WILEY, C., VINTERS, H.V., and CHEN, I.S.
(1990). High levels of unintegrated HIV-1 DNA in brain tissue of AIDS dementia
patients. Nature 343, 85-89.

PARK, C.Y., MAJETI, R., and WEISSMAN, I.L. (2008). In vivo evaluation of human
hematopoiesis through xenotransplantation of purified hematopoietic stem cells
from umbilical cord blood. Nature protocols 3, 1932-1940.

PAUZA, C.D., GALINDO, J.E., and RICHMAN, D.D. (1990). Reinfection results in
accumulation of unintegrated viral DNA in cytopathic and persistent human
immunodeficiency virus type 1 infection of CEM cells. The Journal of
experimental medicine 172, 1035-1042.

PETERSEN, R., KEMPLER, G., and BARKLIS, E. (1991). A stem cell-specific silencer
in the primer-binding site of a retrovirus. Molecular and cellular biology 11,
1214-1221.

PFEIFER, A., IKAWA, M., DAYN, Y., and VERMA, I.M. (2002). Transgenesis by
lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and
preimplantation embryos. Proceedings of the National Academy of Sciences of
the United States of America 99, 2140-2145.

PHILPOTT, N.J., and THRASHER, A.J. (2007). Use of nonintegrating lentiviral vectors
for gene therapy. Human gene therapy 18, 483-489.

PORTEUS, M.H., and BALTIMORE, D. (2003). Chimeric nucleases stimulate gene
targeting in human cells. Science (New York, N.Y 300, 763.

PORTEUS, M.H., and CARROLL, D. (2005). Gene targeting using zinc finger
nucleases. Nature biotechnology 23, 967-973.

PRICE, M.A., CASE, S.S., CARBONARO, D.A., YU, X.J., PETERSEN, D., SABO,
K.M., CURRAN, M.A., ENGEL, B.C., MARGARIAN, H., ABKOWITZ, J.L.,
NOLAN, G.P., KOHN, D.B., and CROOKS, G.M. (2002). Expression from
second-generation feline immunodeficiency virus vectors is impaired in human
hematopoietic cells. Mol Ther 6, 645-652.

134

QASIM, W., GASPAR, H.B., and THRASHER, A.J. (2004). Gene therapy for severe
combined immune deficiency. Expert reviews in molecular medicine 6, 1-15.

RAMEZANI, A., HAWLEY, T.S., and HAWLEY, R.G. (2000). Lentiviral Vectors for
Enhanced Gene Expression in Human Hematopoietic Cells. Mol Ther 2, 458-469.

RAO, S.S., PETERS, S.O., CRITTENDEN, R.B., STEWART, F.M., RAMSHAW, H.S.,
and QUESENBERRY, P.J. (1997). Stem cell transplantation in the normal
nonmyeloablated host: relationship between cell dose, schedule, and engraftment.
Experimental hematology 25, 114-121.

RIDDELL, S.R., ELLIOTT, M., LEWINSOHN, D.A., GILBERT, M.J., WILSON, L.,
MANLEY, S.A., LUPTON, S.D., OVERELL, R.W., REYNOLDS, T.C.,
COREY, L., and GREENBERG, P.D. (1996). T-cell mediated rejection of gene-
modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients. Nature
medicine 2, 216-223.

ROBINSON, H.L., and ZINKUS, D.M. (1990). Accumulation of human
immunodeficiency virus type 1 DNA in T cells: results of multiple infection
events. Journal of virology 64, 4836-4841.

ROSENZWEIG, M., CONNOLE, M., GLICKMAN, R., YUE, S.P., NOREN, B.,
DEMARIA, M., and JOHNSON, R.P. (2001). Induction of cytotoxic T
lymphocyte and antibody responses to enhanced green fluorescent protein
following transplantation of transduced CD34(+) hematopoietic cells. Blood 97,
1951-1959.

ROSENZWEIG, M., MACVITTIE, T.J., HARPER, D., HEMPEL, D., GLICKMAN,
R.L., JOHNSON, R.P., FARESE, A.M., WHITING-THEOBALD, N., LINTON,
G.F., YAMASAKI, G., JORDAN, C.T., and MALECH, H.L. (1999). Efficient
and durable gene marking of hematopoietic progenitor cells in nonhuman
primates after nonablative conditioning. Blood 94, 2271-2286.

SANDS, M.S., BARKER, J.E., VOGLER, C., LEVY, B., GWYNN, B., GALVIN, N.,
SLY, W.S., and BIRKENMEIER, E. (1993). Treatment of murine
mucopolysaccharidosis type VII by syngeneic bone marrow transplantation in
neonates. Laboratory investigation; a journal of technical methods and pathology
68, 676-686.

SCHARFMANN, R., AXELROD, J.H., and VERMA, I.M. (1991). Long-term in vivo
expression of retrovirus-mediated gene transfer in mouse fibroblast implants.
Proceedings of the National Academy of Sciences of the United States of America
88, 4626-4630.

135

SCHRODER, A.R., SHINN, P., CHEN, H., BERRY, C., ECKER, J.R., and BUSHMAN,
F. (2002). HIV-1 integration in the human genome favors active genes and local
hotspots. Cell 110, 521-529.

SINN, P.L., SAUTER, S.L., and MCCRAY, P.B., JR. (2005). Gene therapy progress and
prospects: development of improved lentiviral and retroviral vectors--design,
biosafety, and production. Gene therapy 12, 1089-1098.

SPANGRUDE, G.J., HEIMFELD, S., and WEISSMAN, I.L. (1988a). Purification and
characterization of mouse hematopoietic stem cells. Science (New York, N.Y
241, 58-62.

SPANGRUDE, G.J., KLEIN, J., HEIMFELD, S., AIHARA, Y., and WEISSMAN, I.L.
(1989). Two monoclonal antibodies identify thymic-repopulating cells in mouse
bone marrow. J Immunol 142, 425-430.

SPANGRUDE, G.J., MULLER-SIEBURG, C.E., HEIMFELD, S., and WEISSMAN, I.L.
(1988b). Two rare populations of mouse Thy-1lo bone marrow cells repopulate
the thymus. The Journal of experimental medicine 167, 1671-1683.

STEVENSON, M., HAGGERTY, S., LAMONICA, C.A., MEIER, C.M., WELCH, S.K.,
and WASIAK, A.J. (1990). Integration is not necessary for expression of human
immunodeficiency virus type 1 protein products. Journal of virology 64, 2421-
2425.

STEWART, C.L., STUHLMANN, H., JAHNER, D., and JAENISCH, R. (1982). De
novo methylation, expression, and infectivity of retroviral genomes introduced
into embryonal carcinoma cells. Proceedings of the National Academy of
Sciences of the United States of America 79, 4098-4102.

STRIPECKE, R., CARMEN VILLACRES, M., SKELTON, D., SATAKE, N.,
HALENE, S., and KOHN, D. (1999). Immune response to green fluorescent
protein: implications for gene therapy. Gene therapy 6, 1305-1312.

TABOIT-DAMERON, F., MALASSAGNE, B., VIGLIETTA, C., PUISSANT, C.,
LEROUX-COYAU, M., CHEREAU, C., ATTAL, J., WEILL, B., and
HOUDEBINE, L.M. (1999). Association of the 5'HS4 sequence of the chicken
beta-globin locus control region with human EF1 alpha gene promoter induces
ubiquitous and high expression of human CD55 and CD59 cDNAs in transgenic
rabbits. Transgenic research 8, 223-235.

TEICH, N.M., WEISS, R.A., MARTIN, G.R., and LOWY, D.R. (1977). Virus infection
of murine teratocarcinoma stem cell lines. Cell 12, 973-982.

136

TERENZI, A., ARISTEI, C., AVERSA, F., PERRUCCIO, K., CHIONNE, F.,
RAYMONDI, C., LATINI, P., and MARTELLI, M.F. (1996). Efficacy of
fludarabine as an immunosuppressor for bone marrow transplantation
conditioning: preliminary results. Transplantation proceedings 28, 3101.

THOMAS, C.E., EHRHARDT, A., and KAY, M.A. (2003). Progress and problems with
the use of viral vectors for gene therapy. Nature reviews 4, 346-358.

THYAGARAJAN, B., OLIVARES, E.C., HOLLIS, R.P., GINSBURG, D.S., and
CALOS, M.P. (2001). Site-specific genomic integration in mammalian cells
mediated by phage phiC31 integrase. Molecular and cellular biology 21, 3926-
3934.

TSYGANKOV, A.Y. (2009). Current developments in anti-HIV/AIDS gene therapy.
Curr Opin Investig Drugs 10, 137-149.

UEDA, T., TSUJI, K., YOSHINO, H., EBIHARA, Y., YAGASAKI, H., HISAKAWA,
H., MITSUI, T., MANABE, A., TANAKA, R., KOBAYASHI, K., ITO, M.,
YASUKAWA, K., and NAKAHATA, T. (2000). Expansion of human
NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand,
thrombopoietin, IL-6, and soluble IL-6 receptor. The Journal of clinical
investigation 105, 1013-1021.

UETSUKI, T., NAITO, A., NAGATA, S., and KAZIRO, Y. (1989). Isolation and
characterization of the human chromosomal gene for polypeptide chain
elongation factor-1 alpha. The Journal of biological chemistry 264, 5791-5798.

VASSAL, G., DEROUSSENT, A., HARTMANN, O., CHALLINE, D., BENHAMOU,
E., VALTEAU-COUANET, D., BRUGIERES, L., KALIFA, C., GOUYETTE,
A., and LEMERLE, J. (1990). Dose-dependent neurotoxicity of high-dose
busulfan in children: a clinical and pharmacological study. Cancer research 50,
6203-6207.

VASSAL, G., KOSCIELNY, S., CHALLINE, D., VALTEAU-COUANET, D.,
BOLAND, I., DEROUSSENT, A., LEMERLE, J., GOUYETTE, A., and
HARTMANN, O. (1996). Busulfan disposition and hepatic veno-occlusive
disease in children undergoing bone marrow transplantation. Cancer
chemotherapy and pharmacology 37, 247-253.

VIGDAL, T.J., KAUFMAN, C.D., IZSVAK, Z., VOYTAS, D.F., and IVICS, Z. (2002).
Common physical properties of DNA affecting target site selection of sleeping
beauty and other Tc1/mariner transposable elements. Journal of molecular biology
323, 441-452.

137

VINCENTI, F., LARSEN, C., DURRBACH, A., WEKERLE, T., NASHAN, B.,
BLANCHO, G., LANG, P., GRINYO, J., HALLORAN, P.F., SOLEZ, K.,
HAGERTY, D., LEVY, E., ZHOU, W., NATARAJAN, K., and
CHARPENTIER, B. (2005). Costimulation blockade with belatacept in renal
transplantation. The New England journal of medicine 353, 770-781.

VINK, C.A., GASPAR, H.B., GABRIEL, R., SCHMIDT, M., MCIVOR, R.S.,
THRASHER, A.J., and QASIM, W. (2009). Sleeping Beauty Transposition From
Nonintegrating Lentivirus. Mol Ther.

VON LEVETZOW, G., SPANHOLTZ, J., BECKMANN, J., FISCHER, J., KOGLER,
G., WERNET, P., PUNZEL, M., and GIEBEL, B. (2006). Nucleofection, an
efficient nonviral method to transfer genes into human hematopoietic stem and
progenitor cells. Stem cells and development 15, 278-285.

VOS, J.C., DE BAERE, I., and PLASTERK, R.H. (1996). Transposase is the only
nematode protein required for in vitro transposition of Tc1. Genes & development
10, 755-761.

WAGNER, E., ZATLOUKAL, K., COTTEN, M., KIRLAPPOS, H., MECHTLER, K.,
CURIEL, D.T., and BIRNSTIEL, M.L. (1992). Coupling of adenovirus to
transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene
delivery and expression of transfected genes. Proceedings of the National
Academy of Sciences of the United States of America 89, 6099-6103.

WALISKO, O., and IVICS, Z. (2006). Interference with cell cycle progression by
parasitic genetic elements: sleeping beauty joins the club. Cell cycle
(Georgetown, Tex 5, 1275-1280.

WALISKO, O., SCHORN, A., ROLFS, F., DEVARAJ, A., MISKEY, C., IZSVAK, Z.,
and IVICS, Z. (2007). Transcriptional Activities of the Sleeping Beauty
Transposon and Shielding Its Genetic Cargo With Insulators. Mol Ther.

WEBER, E.L., and CANNON, P.M. (2007). Promoter choice for retroviral vectors:
transcriptional strength versus trans-activation potential. Human gene therapy 18,
849-860.

WEISSINGER, F., REIMER, P., WAESSA, T., BUCHHOFER, S., SCHERTLIN, T.,
KUNZMANN, V., and WILHELM, M. (2003). Gene transfer in purified human
hematopoietic peripheral-blood stem cells by means of electroporation without
prestimulation. The Journal of laboratory and clinical medicine 141, 138-149.

WEISSMAN, I.L., HEIMFELD, S., and SPANGRUDE, G. (1989). Haemopoietic stem
cell purification. Immunology today 10, 184-185.

138

WEKERLE, T., NIKOLIC, B., PEARSON, D.A., SWENSON, K.G., and SYKES, M.
(2002). Minimal conditioning required in a murine model of T cell depletion,
thymic irradiation and high-dose bone marrow transplantation for the induction of
mixed chimerism and tolerance. Transpl Int 15, 248-253.

WIEHE, J.M., PONSAERTS, P., ROJEWSKI, M.T., HOMANN, J.M., GREINER, J.,
KRONAWITTER, D., SCHREZENMEIER, H., HOMBACH, V., WIESNETH,
M., ZIMMERMANN, O., and TORZEWSKI, J. (2007). mRNA-mediated gene
delivery into human progenitor cells promotes highly efficient protein expression.
Journal of cellular and molecular medicine 11, 521-530.

WILBER, A., LINEHAN, J.L., TIAN, X., WOLL, P.S., MORRIS, J.K., BELUR, L.R.,
MCIVOR, R.S., and KAUFMAN, D.S. (2007). Efficient and stable transgene
expression in human embryonic stem cells using transposon-mediated gene
transfer. Stem cells (Dayton, Ohio) 25, 2919-2927.

WILLIAMS, D.A. (2008). Sleeping beauty vector system moves toward human trials in
the United States. Mol Ther 16, 1515-1516.

WILSON, M.H., COATES, C.J., and GEORGE, A.L., JR. (2007). PiggyBac Transposon-
mediated Gene Transfer in Human Cells. Mol Ther 15, 139-145.

WILSON, M.H., KAMINSKI, J.M., and GEORGE, A.L., JR. (2005). Functional zinc
finger/sleeping beauty transposase chimeras exhibit attenuated overproduction
inhibition. FEBS letters 579, 6205-6209.

WISKERCHEN, M., and MUESING, M.A. (1995). Human immunodeficiency virus type
1 integrase: effects of mutations on viral ability to integrate, direct viral gene
expression from unintegrated viral DNA templates, and sustain viral propagation
in primary cells. Journal of virology 69, 376-386.

WOLF, D., CAMMAS, F., LOSSON, R., and GOFF, S.P. (2008a). Primer binding site-
dependent restriction of murine leukemia virus requires HP1 binding by TRIM28.
Journal of virology 82, 4675-4679.

WOLF, D., and GOFF, S.P. (2007). TRIM28 mediates primer binding site-targeted
silencing of murine leukemia virus in embryonic cells. Cell 131, 46-57.

WOLF, D., and GOFF, S.P. (2008). Host restriction factors blocking retroviral
replication. Annual review of genetics 42, 143-163.

WOLF, D., and GOFF, S.P. (2009). Embryonic stem cells use ZFP809 to silence
retroviral DNAs. Nature 458, 1201-1204.

139

WOLF, D., HUG, K., and GOFF, S.P. (2008b). TRIM28 mediates primer binding site-
targeted silencing of Lys1,2 tRNA-utilizing retroviruses in embryonic cells.
Proceedings of the National Academy of Sciences of the United States of America
105, 12521-12526.

WU, M.H., SMITH, S.L., DANET, G.H., LIN, A.M., WILLIAMS, S.F., LIEBOWITZ,
D.N., and DOLAN, M.E. (2001a). Optimization of culture conditions to enhance
transfection of human CD34+ cells by electroporation. Bone marrow
transplantation 27, 1201-1209.

WU, M.H., SMITH, S.L., and DOLAN, M.E. (2001b). High efficiency electroporation of
human umbilical cord blood CD34+ hematopoietic precursor cells. Stem cells
(Dayton, Ohio) 19, 492-499.

WU, X., LI, Y., CRISE, B., and BURGESS, S.M. (2003). Transcription start regions in
the human genome are favored targets for MLV integration. Science (New York,
N.Y 300, 1749-1751.

WU, Y. (2004). HIV-1 gene expression: lessons from provirus and non-integrated DNA.
Retrovirology 1, 13.

XUE, X., HUANG, X., NODLAND, S.E., MATES, L., MA, L., IZSVAK, Z., IVICS, Z.,
LEBIEN, T.W., MCIVOR, R.S., WAGNER, J.E., and ZHOU, X. (2009). Stable
gene transfer and expression in cord blood-derived CD34+ hematopoietic stem
and progenitor cells by a hyperactive Sleeping Beauty transposon system. Blood.

YANEZ-MUNOZ, R.J., BALAGGAN, K.S., MACNEIL, A., HOWE, S.J., SCHMIDT,
M., SMITH, A.J., BUCH, P., MACLAREN, R.E., ANDERSON, P.N., BARKER,
S.E., DURAN, Y., BARTHOLOMAE, C., VON KALLE, C., HECKENLIVELY,
J.R., KINNON, C., ALI, R.R., and THRASHER, A.J. (2006). Effective gene
therapy with nonintegrating lentiviral vectors. Nature medicine 12, 348-353.

YANT, S.R., EHRHARDT, A., MIKKELSEN, J.G., MEUSE, L., PHAM, T., and KAY,
M.A. (2002). Transposition from a gutless adeno-transposon vector stabilizes
transgene expression in vivo. Nature biotechnology 20, 999-1005.

YANT, S.R., MEUSE, L., CHIU, W., IVICS, Z., IZSVAK, Z., and KAY, M.A. (2000).
Somatic integration and long-term transgene expression in normal and
haemophilic mice using a DNA transposon system. Nature genetics 25, 35-41.

YANT, S.R., PARK, J., HUANG, Y., MIKKELSEN, J.G., and KAY, M.A. (2004).
Mutational analysis of the N-terminal DNA-binding domain of sleeping beauty
transposase: critical residues for DNA binding and hyperactivity in mammalian
cells. Molecular and cellular biology 24, 9239-9247.

140

YANT, S.R., WU, X., HUANG, Y., GARRISON, B., BURGESS, S.M., and KAY, M.A.
(2005). High-resolution genome-wide mapping of transposon integration in
mammals. Molecular and cellular biology 25, 2085-2094.

YE, Z.Q., QIU, P., BURKHOLDER, J.K., TURNER, J., CULP, J., ROBERTS, T.,
SHAHIDI, N.T., and YANG, N.S. (1998). Cytokine transgene expression and
promoter usage in primary CD34+ cells using particle-mediated gene delivery.
Human gene therapy 9, 2197-2205.

YU, S.F., VON RUDEN, T., KANTOFF, P.W., GARBER, C., SEIBERG, M., RUTHER,
U., ANDERSON, W.F., WAGNER, E.F., and GILBOA, E. (1986). Self-
inactivating retroviral vectors designed for transfer of whole genes into
mammalian cells. Proceedings of the National Academy of Sciences of the United
States of America 83, 3194-3198.

ZEITELHOFER, M., KARRA, D., VESSEY, J.P., JASKIC, E., MACCHI, P.,
THOMAS, S., RIEFLER, J., KIEBLER, M., and DAHM, R. (2009). High-
efficiency transfection of short hairpin RNAs-encoding plasmids into primary
hippocampal neurons. Journal of neuroscience research 87, 289-300.

ZEITELHOFER, M., VESSEY, J.P., XIE, Y., TUBING, F., THOMAS, S., KIEBLER,
M., and DAHM, R. (2007). High-efficiency transfection of mammalian neurons
via nucleofection. Nature protocols 2, 1692-1704.

Abstract (if available)

Abstract The pluripotent characteristic of hematopoietic stem cells (HSCs) makes them a good candidate for gene therapy. The safety drawbacks of the commonly used viral gene transfer system have made the search for alternative gene transfer methods such as non-viral or hybrid gene transfer systems became increasingly appealing in the field. One such system is the Sleeping Beauty (SB) transposon-mediated gene transfer system. Using a non-viral approach to delivery SB plasmids we were able to significantly increase the efficiency of stable gene up to 20-fold higher than previously published data by incrementally optimizing each element of the SB transposon system. In vivo studies demonstrated that SB-modified human CD34+ cells were engrafted in NOD/SCID/yC(null) (NSG) mice and differentiated into multi-lineage cell types with stable transgene expression. Transgene expression remained persistent in the secondary transplanted NSG mice indicating a long-term stable integration achieved by HSB-transposon system. Non-integrating lentiviral (NIL) vectors were also investigated as another method for SB plasmid delivery. Combining the stable integration of the SB transposon system with the delivery efficiency of NIL, termed NILting beauty, could produce a hybrid vector system that synergizes the advantages of both viral and non-viral vector systems and provide a more effective and safer approach to genetically modify HSCs. The feasibility and potential of utilizing NILting beauty to achieve stable transgene integration was evaluated using K562 and human HSCs. Up to 7% stable transgene expression was achieved in K562 cells and around 1% for human CD34+ cells when transduced with NILting beauty vectors. The other approach to increase long-term transgene expression with relatively minimal adverse effects in clinical HSC gene therapy is using non-myeloablative conditioning regimen.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Improving adeno-associated viral vector for hematopoietic stem cells gene therapy

PDF

Effects of a GSK-3 inhibitor on retroviral-mediated gene transfer to human CD34+ hematopoietic progenitor cells

PDF

Stem cell and gene transfer-based approaches to generate insulin-producing cells

PDF

Rational selection of CRISPR/Cas9 guide RNAs for homology directed genome editing and its utility in the development of gene therapies

PDF

Gene therapy for the prevention and treatment of leukemia

PDF

Study of dendritic cell-specific lentivector vaccine system

PDF

Pseudotyped viral vectors: HIV gene therapy applications and basic studies of SARS-COV-2

PDF

Engineering viral vectors for T-cell immunotherapy and HIV-1 vaccine

PDF

Engineering lentiviral vectors for gene delivery and immunization

PDF

Engineering nanoparticles for gene therapy and cancer therapy

Asset Metadata

Creator Sumiyoshi, Teiko (author)

Core Title Non-viral and viral hematopoietic progenitor cell gene therapy

School Keck School of Medicine

Degree Juris Doctor / Doctor of Philosophy

Degree Program Molecular Microbiology

Publication Date 08/07/2009

Defense Date 06/09/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag busulfan,fludarabine,gene therapy,hematopoietic stem cells (HSCs),NOD/SCID/gamma C(null) (NSG) mice,Non-integrating lentiviral (NIL) vectors,non-myeloablative conditioning,non-viral or hybrid gene transfer systems,OAI-PMH Harvest,pluripotent,relevant infant rhesus monkey bone marrow transplant (BMT) model,Sleeping Beauty (SB) transposon,viral gene transfer system

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McMillan, Minnie (committee chair), Cannon, Paula M. (committee member), Kohn, Donald B. (committee member), Ou, Jing-Hsiung James (committee member)

Creator Email sumiyosh@usc.edu,tsumiyos@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2515

Unique identifier UC1315244

Identifier etd-Sumiyoshi-3109 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-182101 (legacy record id),usctheses-m2515 (legacy record id)

Legacy Identifier etd-Sumiyoshi-3109.pdf

Dmrecord 182101

Document Type Dissertation

Rights Sumiyoshi, Teiko

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu