University of Southern California Dissertations and Theses

Engineering viral vectors for gene and cell targeting

(USC Thesis Other)

Engineering viral vectors for gene and cell targeting

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content ENGINEERING VIRAL VECTORS FOR GENE AND CELL TARGETING

by

Yuning Lei

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMICAL ENGINEERING)

May 2011

Copyright 2011 Yuning Lei

ii

Dedication
This thesis is dedicated to my parents Hsiao-Hung Lei and Ming-Lien Chia, and
my brother Yu-Wei Lei. Their support throughout this process has been invaluable.

iii

Acknowledgements
I have been fortunate to work with talented people. First and foremost I would
like to thank my advisor Dr. Pin Wang who has supported and guided me though this
process. He has been always there for me when I had difficulity in research and help me
to reach my full potential. I would also like to thank my dissertation committee Dr.
Katherine Shing and Dr. Don Arnold. It was their insightful comments that help me to
finish this thesis.
Is is also my pleasure to work with a great team in RTH-515 at USC. I will never
forget the experience I had here at USC. I would like to show my thanks to Haiguang,
Leslie, Taehoon, Eric for setting up the labs together, Kye-Il for the help in confocal
imaging, April and Steve for critical reading of the manuscripts, Laura and Bingbing for
their help in culturing stem cells and Steven for the illustration.

iv

Table of Contents
Dedication ii
Acknowledgements iii
List of Figures vii
Abstract ix
Chapter 1: Introduction 1
1.1 Introduction to Gene Therapy 1
1.2 Cell types targeting 2
1.2.1 Non-viral gene therapy 2
1.2.2 Viral gene therapy 5
1.3 Gene targeting 12
1.4 Summary and Thesis Work 16
Chapter 2: Engineering fusogenic molecules to achieve targeted transduction
111of enveloped lentiviral vectors 20
2.1 Introduction 21
2.2 Results 23
2.2.1 Generation of pH-dependent FMs 23
2.2.2 Production of recombinant lentiviral vectors 25
2.2.3 Co-incorporation of the αCD20 and the FM on lentiviral vectors 29
2.2.4 Targeted transduction of lentiviral vectors to CD20-positive
11111111111cell line 32
2.2.5 pH dependence of various FMs 33
2.2.6 Antibody directed targeted transduction 34
2.2.7 Targeted transduction of lentiviral vectors to unfractionated
11111111111primary B cells 36
2.3 Discussion 38
2.4 Methods 42
2.4.1 Construct preparation 42
2.4.2 Cell line construction 45
2.4.3 Virus production 45
2.4.4 Fluorescent labeling 46
2.4.5 FACS analysis of cell-virus binding 46
2.4.6 p24 analysis of lentiviral vectors 47
2.4.7 Targeted transduction 47
2.4.8 NH
4
Cl neutralizing assay 47
2.4.9 Antibody competition assay 48
2.4.10 Targeted transduction of unfractioned PBMC 48

v

Chapter 3: Targeting lentiviral vector to specific cell types through
surface displayed single chain antibody and fusogenic molecules 49
3.1 Introduction 50
3.2 Results 52
3.2.1 Construction of SCAb for targeting 52
3.2.2 Production of lentiviral vectors 53
3.2.3 Incorporation of SCAb and FM onto lentiviral vectors 55
3.2.4 Targeted transduction of lentiviral vectors 57
3.2.5 Assays for studying the entry mechanism 59
3.2.6 pH dependency study on the FMs 62
3.2.7 Binding avidity of lentiviral vectors to target cells 63
3.3 Discussion 65
3.4 Material and Methods 67
3.4.1 Construct preparation 67
3.4.2 Viral vector production 69
3.4.3 Virus-cell binding assay 70
3.4.4 Confocal imaging 70
3.4.5 Antibody competition assay 70
3.4.6 Neutralization assay 71
3.4.7 Targeted transduction of 293T/CD20 cells 71
3.4.8 Scatchard analysis 71
3.4.9 Virus-liposome fusion assay 72
Chapter 4: Gene editing of human embryonic stem cells via an engineered baculoviral
111vector carrying zinc finger nucleases 74
4.1 Introduction 75
4.2 Results 77
4.2.1 Construction of site-specific vectors 77
4.2.2 ZFN-mediated target disruption by the baculoviral vector 79
4.2.3 Production of the ZFN-lentiviral vector 82
4.2.4 ZFN-mediated targeted gene addition in cell lines 84
4.2.5 ZFN-mediated targeted gene addition in human embryonic stem
1111111111cell 87
4.3 Discussion 89
4.4 Materials & Methods 92
4.4.1 Maintenance and differentiation of the hES cells 92
4.4.2 Plasmid construction 92
4.4.3 Vector production 94
4.4.4 Surveyor nuclease assay 94
4.4.5 Viral transduction 95
4.4.6 p24 analysis of lentiviral vectors 96
4.4.7 Detection of double-strand breaks in ZFN-treated cells 96
4.4.8 Targeted integration analysis 97
4.4.9 Cell sorting and reverse transcription-PCR analysis 98
4.4.10 Immunohistochemistry and Karyotypic analysis 99

vi

References 100

vii

List of Figures
Figure 1.1 Summary of different viral vectors used in the field of gene therapy 11
Figure 1.2 ZFN homodimer binding to DNA 15
Figure 1.3 Proposed mechanism for targeted transduction by recombinant
lentiviral vectors 18
Figure 2.1 Schematic representation of key constructs in this study 25
Figure 2.2 Co-expression of an antibody and a FM on the surface of transfected
vector producing cells 28
Figure 2.3 Co-expression of αCD20 and a FM on the viral vector surface 31
Figure 2.4 Transduction of engineered lentiviral vectors bearing both an
antibody and FM to cell lines 33
Figure 2.5 Examination of addition of NH4Cl or soluble αCD20 on the targeted
transduction results 35
Figure 2.6 Targeted transduction of CD20-positive human primary B cells 37
Figure 3.1 Schematic representation of key constructs in this study 53
Figure 3.2 Co-transfection of virus-producing cells to generate targeting
lentiviral vectors 55
Figure 3.3 Incorporation of both FM and antibody onto the vector surface 57
Figure 3.4 Targeted transduction of lentiviral vectors to 293T/CD20 cells 59
Figure 3.5 Study of the entry mechanism of engineered lentiviral vector for
transducing target cells 61
Figure 3.6 pH-dependent study of the fusion activity of various FMs 63
Figure 3.7 Scatchard analysis of the lentiviral vector binding to 293T/CD20 cells 64
Figure 4.1 Schematic representation of key constructs in this study 79
Figure 4.2 ZFN-mediated disruption of CCR5 by baculoviral vector 81
Figure 4.3 Analysis of ZFN-associated toxicity for producer cells to make
lentiviral or baculoviral vectors 83
Figure 4.4 Targeted gene editing at the CCR5 gene locus in 293T and U87
cell lines 86

viii

Figure 4.5 Targeted gene editing at the CCR5 gene locus in human embryonic
stem cells 88

ix

Abstract
Gene therapy is the introduction of functional genes into dysfunctional cells to
treat potentially incurable diseases. The technique sounds promising, yet there are many
hurdles must be overcome to make it a practical mean of medicine. Viral vector mediated
gene therapy remains one of the most promising gene therapy techniques as its efficiency
and duration is the highest among the different gene delivery vehicles. To further refine
the viral vector to enhance the gene delivery ability, we designed a strategy by
decoupling binding and fusion ability of envelope protein into two distinct proteins. By
pseudotyping the viral vectors with both an antibody and a fusogenic molecule, we can
target and transduce specific cell types. The underlining mechanism of targeted
transduction is that the viral vector will bind to the desirable cell types via the cognate
antibody antigen interaction and further be endocytosised into the endosome. Inside the
endosome compartment, the low pH environment will trigger the conformation change of
the fusogenic molecule which will fuse both the viral and cellular membrane, resulting in
releasing the viral core. To further enhance the targeted transduction, we focused on
engineering the two parameters on the viral vector. In chapter 2, we engineered the fusion
loop of the fusogenic molecule to enhance the activity of the protein. We demonstrated
that the engineered fusogenic molecules can enhance targeted transduction many folds.
We further looked at the other parameter, the targeting antibody in chapter 3. By
designing a single chain antibody, we were able to constructed our targeting virus a
simplifier viral production protocol and showed that the viral vector still exhibit targeted

x

transduction. These two chapters concluded our effort in enhancing the targeted
transduction by genetically engineering the component displayed on the viral membrane.
The forth chapter in this report combines the technology of the zinc finger
nuclease and the baculoviral vector. Currently, one of the drawback of utilizing
integrating viral vector is the possibility of turning on oncogenes when the viral vectors
integrated to undesirable sites. In this chapter, we utilize zinc finger nuclease to generate
double stranded break and further demonstrate the ability of utilizing this technique to
converting the baculoviral vector into gene targeting vector. We showed that our system
can targeted transduce both immortalized cell lines as well as human embryonic stem
cells. Techniques that we developed here can further enhance the safety of viral gene
therapy while retain the efficiency and duration of the viral vectors.

1

Chapter 1
Introduction
1.1 Introduction to Gene Therapy
The basic concept of gene therapy is the introduction of a functional gene into a
dysfunctional cell to either cure or slow down the progression of a disease (Somia and
Verma, 2000; Verma and Somia., 1997). The disease mentioned here includes both
inherited or acquired diseases which could be the result of a simple addition or deletion of
certain function genes. Once the dysfunctional gene has been identified, researchers could
design a vector to either knock out or silence the dysfunctional gene which will eradicate
the disease. The idea of utilizing gene therapy techniques to cure the diseases sounds
straightforward and promising. However, not many successful news has been published.
One of the major hurdles that researchers face today is to design a suitable gene delivery
vector to introduce the functional genes into the desired cells. Two different pathway are
usually taken for gene therapy which include an in vivo delivery of the function gene into
the cells within the patients body or an ex vivo delivery which involves the manipulation
of the patient’s cells outside of his/her body then replanted back to his/her body.
However, there are pitfalls to both of these delivery pathway as ex vivo gene delivery
pathway is both time consuming and expensive whereas in vivo gene delivery involves
more risks as there are currently no targeting vectors specific enough to delivery the gene
of interest to the target cells. Another major concern associated with gene therapy
involves the integrating of the gene of interest into the actively transcribed regions which

2

results in insertional mutagenesis and oncogene activation. Thus, the development of a
targeting vector that is capable of introducing the functional gene into the desired cell
types at a pre-determined location is one of the most important aspect to advance gene
therapy.
1.2 Cell types targeting
One of the obstacles of gene therapy today is how to properly deliver the desirable
gene into the cell types of interest. Currently, many groups have focused on designing
both non-viral gene delivery and viral gene delivery vectors to fulfill this task by either
chemically or biologically modified the gene delivery vectors with targeting ligand,
peptides or antibody to achieve targeted transduction. However, there have not been
many successful stories around the gene therapy field as the modification usually resulted
in decrease transduction efficiency. In the following section, I will briefly mention the
techniques that are commonly used in the gene therapy field today.
1.2.1 Non-viral gene therapy
Non-viral carriers remain one of the most studied vectors in the gene delivery
field as there are several advantages over the viral vectors. Non-viral vectors are
generally cationic in nature for encapsulation of negatively charged DNA. Compared to
viral vectors, non-viral vectors are usually easy to manufacture and scale-up, the size of
DNA being delivered is larger and do not elicit immune response (Behr, 1994; El-Aneed,
2003; Glover, 2005). However, the major disadvantage is the efficiency of delivering the

3

genes of interest into the cells and the gene expression is only transient. I will discuss a
few examples of the non-viral vectors being researched here.
Naked DNA: As the name suggested, gene delivery with naked DNA is to simply inject
plasmid DNA into the cells with mechanical force (Niidome and Huang, 2002). Without
using any carrier molecule, it is the safest gene delivery system today. The conventional
techniques for delivery includes electroporation in which an electric filed is generated
between the cells for DNA uptake (Drabick, 2001; Somiari, 2000). Gene gun is another
technique that is used by shooting gold particles coated with DNA to directly penetrate
the cell membrane (Davidson et al., 2000; Lin et al., 2000). Ultrasound is also one of the
methods to increase the permeability of cell for DNA update (Endoh, 2002; Price and
Kaul, 2002; Taniyama, 2002).
Cationic polymers: Cationic polymers can form a particulate complex with negatively
charged DNA upon mixing. Since they are synthetic compounds, many modifications can
be introduced to this complex including the size, the charge and even attaching a ligand
to the complex for targeting purpose (Fischer et al., 1999; Kircheis et al., 1999; Kunath et
al., 2003). The most commonly used cationic polymers are poly L-lysine (PLL) and poly
ethyleimine (PEI). PLL polymers are linear polypeptides with a biodegradable nature
which makes it very useful for in vivo experiment (Wu and Wu, 1987). Chloroquine and
fusogenic peptides are commonly added to PLL polymers to enhance the endosomal
escape for releasing (Dash et al., 1999; Pouton et al., 1998). Targeting ligands can also be
linked to the polymer chains of PLL for targeted transduction. One key parameter in
designing PLL polymers is that as size of PLL polymers increases, transfection and DNA

4

condensation efficiency increases, but undesirable toxicity also increases. Another
popular cationic polymers is PEI which is capable of destabilizing lysosomal membrane
to deliver the DNA (Ward et al., 2002). PEI polyplexes can be in linear and branch form.
Current study suggests that linear PEI (22 kDa) is the most efficient in delivering DNA as
compared to branched PEI (25 and 800 kDa) (Wightman et al., 2001). However, toxicity
still comes into play with the use of PEI polymers and more studies are need to be
conducted for the use of PEI.
Cationic peptides: Cationic peptides used in gene delivery are amphiphilic peptides that
undergo conformation change upon exposing to acidic environments for the releasing of
DNA complex (Wyman et al., 1997). They contain positively charged amino acids to
encapsulate negatively charged DNA (Haines, 2001). The exact ratio for DNA
condensation and peptide aggregation for gene delivery is not yet clear (McKenzie et al.,
2000). More studies needs to be conducted to optimize the gene delivery efficiency of
cationic peptides. Similar to other non-viral gene delivery vehicles, targeting ligand can
be linked to cationic peptides tor targeted transduction.
Cationic lipids: Cationic lipids are amphiphilic molecules that can assembled into a lipid
bilayer structure called liposomes (Lasic, 1997). Once liposomes are formed, DNA can
be sandwiched within liposome which is called lipoplex (Radler et al., 1997; Sternberg et
al., 1994), however, the stability of lipoplex is quite poor and needs to be applied right
after their formation. After the lipoplex being endocytosized, neutral lipids will facilitate
conformational change of the lipoplex which allows the release of the DNA into the
cytosol.

5

1.2.2 Viral gene therapy
Currently, viral gene delivery still remains as one of the most promising technique
for gene therapy clinical trials as 70% of the gene therapy trials conducted today involved
the use of viral vectors (Edelstein et al., 2004; Verma, 1990). The high gene transfer
efficiency and high level of transgene expression make viral vector the most suitable
vehicles for gene delivery today. Generally, viral vectors are derived from viruses by
replacing the replication machinery with transgene expression cassette. The essential
packaging component can be supplied in trans, which means the packaging proteins are
only present during the production of the virus and none of the essential packaging genes
are packaged into the viral vector. This method can prevent the generation of replication
competent virus. The viral vectors can be further divided into two categories, integrating
viral vectors and non integrating viral vectors. Integrating viral vectors integrate the
genomic information of themselves (transgene) into the host genome to supply a life long
expression where as non-integrating viral vectors only transiently express the transgene
within the target cells. Here, I will discuss the most commonly used viral vectors today.
Retroviral vector: Retroviral vectors were one of the oldest viral vectors and still one of
the most commonly used viral vector today (Anderson, 1998; Mulligan, 1993; Verma,
1990). It is an integrating virus with a transgene capacity of 7-7.5 kbps and contains two
identical strands of RNA. Retroviruses are composed of three essential genes, gag which
encodes the viral structural proteins (matrix protein, capsid protein, and nucleocapsid
protein), pro and pol which encodes enzymatic proteins (reverse transcriptase, integrase,
and integrase) and env which encodes the viral envelope glycoprotein. The three essential

6

genes can be supplied in trans to eliminate the possibility of generating replication
component retroviruses. Self-inactivating vectors were also designed with a mutation in
the 3’LTR to eliminate the transcription process of the viral genome upon integrating into
the target genome (Yu, 1986). The transduction of retroviral vector begins with the
receptor binding of the viral glycoproteins to the target cell. The interaction then triggers
either an endocytosis event and a conformational change in the endosomal compartment
or a direct fusion event at the cell surface, depending on the envelope glycoprotein.
Following the fusion event, the viral capsid is then unloaded into the cytoplasm and
reverse transcriptased into proviral DNA near nucleus. The newly synthesized DNA is
then transported into the nucleus and integrated into the host genome. In order to
transduce different cell types, the envelope of retroviruses were replaced with envelope
protein from other viruses (Danos and Mulligan, 1988). The disadvantage of retroviruses
lies with its inability to infect non-dividing cells, such as brain, eye, lungs, and neuron
cells. Retroviruses usually have a short term transgene expression despite of its
integrating ability due to the site of integration that tends to be silenced by the host
genome. Despite of its advantages, retroviruses still remained one of the most promising
viral vectors used today due to its simplicity for engineering and its ability to transduce
dividing cells such as tumor cells.
Lentiviral vector: A subclass of retroviruses that has been extensively studied and
engineered is the lentivirus. Most of the lentiviral vectors used today are derived from
human immunodeficiency viruses. It belongs to the retrovirus family with a few unique
properties to make it more suitable for clinical studies. Unlike retroviruses, lentiviruses is
capable of infecting both dividing and non-dividing cells (Bukrinsky, 1993; Lewis et al.,

7

1992). The exact mechanism is still unclear, however, it is known that the accessory
proteins associates with the lentivruses can improve the transport across the nucleus. The
integration site of lentiviruses tend to be within the gene as opposed to transcriptional
initiation sites like retroviral vectors and is less likely to be silenced, hence, longer
transgene expression. Lentiviruses are composed of the usual gag, pol and env proteins,
but differ in terms of accessory proteins, tat, rev, nef, vif, vpu and vpr. The current
generation of lentiviral vectors involved the substitution of env proteins of the HIV’s
gp160 with glycoporteins from other viruses, including VSVG (Naldini, 1996). Self-
inactivating vector was also developed for lentiviral vector with a mutation in the 3’LTR
to prevent the viral RNA being synthesized after integrating into the target host (Miyoshi
et al., 1998). Another safety mechanism employed in the lentiviral vector is the
separating the essential proteins used, including rev, gag, pol and env into different
constructs and supplied in trans to eliminate the possibility of reverting the replication
incompetent viruses back to replication competent viruses. Most of the accessory proteins
of the lentiviral vectors are also removed from production with only 25% of the viral
genome in the packaging construct and 5% of viral genome in the vector construct are
retained (Zufferey et al., 1997). In addition, extra components are added into the lentiviral
vectors to enhance the transduction and expression of viral vector which includes the
central polypurine tract (cPPT), which allows internal initiation of second strand DNA
synthesis and the transport of the pre-integration complex into the nucleus and the
woodchuck post-transcriptional regulatory element (WRE) which improves the
translational efficiency of lentiviral vectors. Due to these properties, lentiviral vector is
among one of the most promising viral vectors in the field of gene therapy.

8

Adeno-associated viral vectors: Adeno-associated virus (AAV) is a small, non-
pathogenic, single stranded DNA virus that has become more popular in the field of gene
therapy (Carter and Samulsky, 2000). As the name suggests, AAV requires the help of
other viruses to replicate, such as adenovirus and herpes simplex virus. AAV itself has
only two essential genes. Rep gene encodes for replication and integration function of the
virus and cap gene encodes for the structural components of the virus. The viral genome
is surrounded by two inverted terminal repeats (ITRs) which contains the DNA sequences
needed to initiate the DNA replication and packaging of the viral genome. The viral
vector is constructed by replacing the rep and cap genes with transgene of interest and rep
and cap are supplied in trans for safety concern. One of the advantage of the AAV is its
ability to integrate in a site-specific location on chromosome 19 with the help of rep
proteins, however, the efficiency is not high and still up for debate (Kotin, 1990).
However, one of the disadvantage of AAV is that the rep gene is toxic to the producing
cells which make it harder to construct a stable packaging cell lines. Another problem of
the AAV is that the encoding capacity is limited to 4.5kb. However, researchers have
extended the encoding capacity of AAV by utilizing the concatomerization nature of
AAV and divided the transgene into two parts supplied by two individual AAV (Yan et
al., 2000). This has dramatically increase the encoding capacity, but the efficiency is high
for only certain transgene and remains unclear the reason of it. Nevertheless, AAV has
gained more and more notice with the recent success in clinical trial and become one of
the most promising new viral vector to be designed.
Adenoviral vectors: Adenoviruses belong to the family of DNA tumor viruses that cause
respiratory tract infections in humans. It is a double stranded linear DNA virus with

9

genomes contains over a dozen genes. Adenoviruses can infect both dividing and non-
dividing cells. However, unlike lentiviruses, and retroviruses, adenoviruses do not
integrate into the host genome, but rather remains episommal in the nucleus. Currently,
most of the adenoviruses used in the gene therapy field are derived from replication
incompetent viruses with E1A, E1B, E3, E4 and E2A being deleted from the viral
genome (Yeh and Perricaudet, 1997). However, one of the major disadvantages of
adenoviruses is the immune response generated from contacting with this virus. Since
most people have contracted adenoviruses early in lives, the pre-existing immune
response generated against the virus is high and also coming from the viral proteins
expressed after infection. Thus, researchers have designed gutless viral vectors, in which
viral genes were replaced with transgenes and supplied in trans to be used in clinical trial
setting (Morral, 1999). Nevertheless, the adenoviruses still remain one of the most useful
viral vector for gene therapy especially when viral vectors can be prepared in high titer
and the transient expression of the transgene might be useful in certain clinical setting.
Baculoviral vectors: Baculovirus is a large enveloped virus with a double stranded,
circular DNA genome. It has a 130-kb double-stranded DNA genome which is capable of
carrying up to 50-kb of transgene (Cheshenko et al., 2001a; Davies, 1994). The
baculovirus viral promoter is only active in insect cells while only marginally functional
in the mammalian cells. One of the best studied virus from this family is the Autographa
californica nuclear polyhedrosis virus (AcMNPV) that has been expensively studied and
tested as a gene delivery vehicle. The insect virus system has been extensively used as a
recombinant proteins expression beginning in the early 1980’s (Pennock et al., 1984;
Smith et al., 1983). Since the initial report, numerous studies have been conducted on

10

baculovirus system to develop it as a protein expression system. The most commonly
used host cell lines to generate baculovirus include the SF9 and SF21 lines that are
derived from Spodoptera Frugiperda pupal ovarian tissue. In the mid 1990s, two groups
reported that recombinant baculoviruses containing a mammalian promoter can be used
to transduce mammalian cells (Boyce and Bucher, 1996; Hofmann et al., 1995). The first
study was conducted on hepatocytes and further expended to a few other cell lines which
include COS-1, T-47D, COS-7, HeLa, porcine kidney cells and 293. However,
transduction to cell lines of hematopoietic origin, such as THP-1, U937, K562, Raw264.7
and P388D1 cells was observed at very low efficiency which prevent the baculoviral
vector for certain therapeutic usage. One of the property of this viral vector system is that
the expression of the transgene is transient, hence can not be utilized as a permanent gene
replacement vector. Currently, there are many groups try to engineer the viral vector to
either convert the viral vector into a integrating vector by utilizing the inverted terminal
repeats (ITR) of adeno-associated virus (AAV) as an integrating machinery (Palombo et
al., 1998; Zeng et al., 2007) or try to extend the expression of the transgene by generating
the transgene cassatte as a large episomal plasmid (Lo et al., 2009).
One of the advantages of baculoviruses is its inherent ability to replicate in
mammalian cells makes them an attractive option for in vivo applications. However,
initial attempts showed no detectable transduction to mace and rats (Sandig et al., 1996).
The failure to transduce mammalian cells in vivo is best explained by the complement
sensitivity generated by the host immune system against the baculovirus. As an
enveloped insect virus, baculovirus carries an envelope protein called GP64, which is
highly immunogenetic and can elicit a strong immune response. To overcome this

11

problem, baculoviruses modified with vesicular stomatitis virus G (VSV-G) protein were
constructed and tested for complement resistance. The transduction efficiency in vivo
showed similar pattern as unmodified baculovirus. However, the virion envelopes
contained both VSV-G and GP64 which might elicit a strong immune response. A recent
report utilizes GP64 null virus to pseudotype baculovirus for transduction in mammalian
cells (Mangor et al., 2001). However, more studies are needed to confirm whether or not
baculoviruses pseudotyped with VSV-G can efficiently transduce cells in vivo.
Baculoviruses enter the cells via a clathrin-mediated endocytosis pathway (Long
et al., 2006). However, the interacting receptor on the cell surface has not been identified.
After the endocytosis event, the envelope protein GP64 will undergo a conformational
change and fuse with the endosomal membrane to release the viral capsid. The viral
capsid is then transported into the nuclease for subsequently viral DNA unloading.

Fig. 1.1 Summary of different viral vectors used in the field of gene therapy. Nature 389: 239-242,
1997

12

1.3 Gene targeting
Gene targeting is the replacement of a dysfunctional endogenous gene with a
functional gene via homologous recombination which represents the most precise and
safe method for gene therapy (Porteus and Carroll, 2005). Gene editing in mammalian
cells have been a long sought tool to provide a better understanding of the complicated
genome and can potentially move forward the technique of current medicine (Porteus,
2005; Porteus and Carroll, 2005). Disease such as sickle cell anemia which is caused by a
single point mutation is a great candidate for the gene targeting technique (Platt, 1994).
Another example to fully utilizing gene targeting technique is to precisely insert
functional genes into the genome of defected cells as opposed to retroviral vectors
integrate at random sites which have caused insertional mutagenesis. Gene correction via
homologous recombination is very unlikely to be silenced overtime as opposed to viral
vector. The expression of the endogenous gene is driven by the internal promoter as
opposed to viral promoter that has a higher chance of being silenced by the host cells.
The idea of gene targeting has been demonstrated in mouse embryonic stem cells to
generate knock out mice to study the function of genes and genetic diseases (Mansour et
al., 1998; Sedivy and Sharp, 1989). It has shown great potential and is recently rewarded
a Nobel Prize for the discovery. However, the limit of gene targeting techniques is the
low efficiency of gene insertion and correction. Although positive selection for the
integrated gene is sufficient to generate corrected cells, the process is both time
consuming and tedious. Thus, designing a more efficient technique to employ the process
of gene targeting is desirable.

13

The limiting step for gene targeting can be overcome by generating a double-
strand break in the site of mutation. Double-strand break is a commonly occurred event in
mammalian cells that is lethal to the cells if not repaired via homologous recombination
pathway (Paques and Haber, 1999; West, 2000). Homologous recombination or non-
homologous end join occur when a DNA double-strand break is formed and several
cellular factors are recruited to the site of lesion for gene correction (Mansour et al.,
1998; Paques and Haber, 1999). Recently, the development of zinc finger nucleases
(ZFNs) has greatly improved gene targeting efficiency (Chandrasegaran and Smith, 1999;
Kim et al., 2004). The C
2
H
2
zinc-finger, one of the most common DNA recognition motif
in eukarya, has been heavily engineered to alter the native recognition sequences to
accommodate different applications (Pavletich and Pabo, 1991). ZFNs is composed of a
zinc finger protein fused to a Fok1 type II restriction endonuclease. Zinc finger proteins is
composed of 30 amino-acids which folds into a ββα configuration and can contacts 3 bps
of DNA (Pavletich and Pabo, 1991). Engineered zinc finger proteins usually contain
either three to four distinct proteins to recognize 9 to 12 distinct DNA nucleotides. Since
Fok1 endonuclease needs to form a dimer to induce a double-stranded break, two ZFNs
are usually generated that bind to either side of the duplex DNA with 18 to 24
recognizing bps (Bibikova, 2001; Smith, 2000). However, even with the advance of
ZFNs, off-targeting that originated from Fok1 endonuclease can still be detected. To
overcome this problem, two groups have developed a obligate endonuclease to minimize
the off-targeting event by engineering the binding domain of the endonuclease (Miller et
al., 2007; Szczepek et al., 2007). High gene disruption efficiency was still observed with
the obligated endonuclease domain while minimum cytotoxicity were observed in the

14

cells that were treated with the ZFNs. Recently, several labs have demonstrated that by
utilizing double strand break via zinc finger nucleases, gene editing can occur with high
frequency and accuracy (Lombardo et al., 2007; Meng et al., 2008a; Moehle et al., 2007;
Santiago et al., 2008) ZFNs have been well tested in other species for gene disruption or
gene addition (Doyon et al., 2008; Meng et al., 2008a; Shukla et al., 2009; Townsend et
al., 2009). The technique has also been extended to human embryonic stem cells and has
shown promising results in gene addition (Hockemeyer et al., 2009; Lombardo et al.,
2007; Zou et al., 2009). Furthermore, by utilizing the gene disruption aspects of the
ZFNs, Holt and co-workers developed a CCR5-negative hematopoietic stem cells that are
capable of preventing HIV-infection (Holt et al., 2010).
One of the limiting steps for gene editing is the efficiency of co-delivering two
ZFNs and a donor DNA into the cells. Different groups have tried to transfect the cell
with the necessary constructs, however, the efficiency of gene correction events remained
low. In a recent report, a group of researchers have designed the ZFNs to demonstrate
the ability to target specific genes with high efficiency in human stem cells. A defective
GFP genes were first introduced into the genome of 293T cells followed by the
introduction of ZFNs. Corrected GFP signals were detected by FACS. IL2Rγ was also
targeted to demonstrate the ability to engineer the ZFNs for different locations (Urnov et
al., 2005). The gene correction efficiency was dramatically improved by this double
stranded mediated gene addition method, but the efficiency was relatively low. A follow
up studied was conducted with non-integrating lentiviral vectors and shown to improve
the efficiency of targeted transduction in vitro (Lombardo et al., 2007). In that report,
Lombardo and co-workers had demonstrated that by delivering the ZFNs and the donor

15

template into the cells via integrase-defective lentiviral vector, the efficiency can be
substantially enhanced. However, the trans-gene capacity of lentiviral vector is restricted
to 10-kb which posed another limitation to this technique. Another problem of utilizing
an integrase-defective lentiviral vector is that the vector itself still retained some residual
integrating ability that might have affected the overall outcome of the delivery system
(Mali et al., 2008; Nightingale et al., 2006).

Fig. 1.2 ZFN homodimer binding to DNA. Shown is a three-finger zinc finger linked to the Fn
domain through a flexible peptide linker. Nature Biotech. 23: 967-973, 2005

16

1.4 Summary and Thesis Work
Over the last 15 years, researchers have tried to engineer viral vectors to delivery
therapeutical genes to cells of interest. However, engineering viral glycoproteins to
redirect the tropism has proven to be difficult. In this report, we designed a HIV-1 based
lentiviral vector bearing both a membrane bound antibody and a fusogenic molecule to
target specific cell types in vitro. Our purposed mechanism for our novel lentiviral vector
includes the binding of our recombinant lentiviral vectors to the desired cells via specific
antibody-antigen interaction followed by an endocytosis of the viruses. Once entering
into endosome with the drop in pH, our fusogenic molecule would then change its
conformation and fuse with the endosmal membrane for capsid release. Since the
fusogenic molecule and antibody play a crucial role in our targeting system, we have thus
focused on engineering these two parameters to improve our targeting system.
In chapter 2, we have engineered fusogenic molecules to pair with our targeting
antibody for targeting transduction. We hypothesized that by engineering in the fusion
loop of the Sindbis glycoprotein, we could improve the transduction efficiency of our
viral vector. We picked CD20-positive cells as our target cells as 90 % of non-Hodgkin’s
lymphomas are CD20-positive and CD20 expression is not observed on precursor B
lymphoid cells or the majority of plasma B cells. These properties make CD20-positive
cells an attractive model to test our system. To validate our hypothesis, we transduced
CD20-positive cells with our lentiviral vector paired with an anti-CD20 antibody as well
as engineered fusogenic molecules. We demonstrated that enhanced transduction to
CD20-positive cells was observed and the enhanced transduction correlated with the

17

improvement of the responsiveness of our fusogenic molecules to pH. Similar effect was
observed in peripheral blood mononuclear cell (PBMC) which indicated that the system
can be further extended to primary cells.
The next parameter that we investigated is the antibody that was used to pair with
the fusogenic molecules. In chapter 3, we constructed single chain antibody to pair with
our existing fusogenic molecules. We hypothesized that by substituting single chain
antibody with the natural form of antibody, we can simplify viral production, thereby
increasing our viral production. Two forms of single chain antibody were generated
which included a HLA-A2 transmembrane or a VSVG transmembrane anchored antibody
was generated. Lentiviral vector pseudotyped with fusogenic molecules and these two
forms of single chain antibody showed targeted transduction to CD20-positive cells,
indicating single chain antibody was successfully generated and incorporated onto the
viral surface. A more comprehensive study on single chain antibody was conducted after
we observed different transduction efficiency when we paired the single chain antibody
to our fusogenic molecules. We then demonstrated that binding affinity of the VSVG
anchored single chain antibody was lowered than the HLA-A2 anchored single chain
antibody which correlated decreased transduction.

18

Fig. 1.3 Proposed mechanism for targeted transduction by recombinant lentiviral
vectors.
The next parameter that can improve for the safety of the viral gene therapy is to
engineer the viral vector to integrate its transgene into a pre-determined site. In chapter 4,
we addressed this issue by generating a baculoviral vector that is capable of performing a
site-specific integrating event with high efficiency. As opposed to non-random
integrating events by other viral vectors, site-specific integrating has the advantages of
both safety and long term gene expression. To test this idea, we constructed baculoviral

19

vector that carried both zinc finger nucleases and a homologous DNA template. The zinc
finger nucleases that are delivered by baculoviral vector can induce double strand break
in the target site and a homologous recombination event with the DNA template will
occur to introduce the transgene into the target site. The target gene of choice is CCR5
gene as the CCR5-null cell types are prevalent and do not pose any safety risk. We first
showed that gene disruption can be achieved by delivering the zinc finger nuclease alone
to trigger the non-homologous end joining. We demonstrated that over 30% of the target
cells showed down expression of CCR5 proteins. We also showed that targeted gene
addition can be observed at up to 10% in immortalized cell lines and 5% in human
embryonic stem cells. The human embryonic stem cells treated with these viral constructs
retained the normal karyotype and pluripotency.

20

Chapter 2
Engineering fusogenic molecules to achieve targeted transduction of
enveloped lentiviral vectors

Portions of this chapter are adapted from: Yuning Lei, Kye-il Joo, and Pin Wang.
Journal of Biological Engineering (2009), 3 (8).

Lentiviral vectors with broad tropism are one of the most promising gene delivery
systems capable of efficiently delivering genes of interest into both dividing and non-
dividing cells while maintaining long-term transgene expression. However, there are
needs for developing lentiviral vectors with the capability to deliver genes to specific cell
types, thus reducing the “off-target” effect of gene therapy. In the present study, we
investigated the possibility of engineering the fusion-active domain of a fusogenic
molecule (FM) with the aim to improve targeted transduction of lentiviral vectors co-
displaying an anti-CD20 antibody (αCD20) and a FM. Specific mutations were
introduced into the fusion domain of a binding-deficient Sindbis virus glycoprotein to
generate several mutant FMs. Lentiviral vectors incorporated with αCD20 and one of the
engineered FMs were successfully produced and demonstrated to be able to preferentially
deliver genes to CD-20-expressing cells. Lentiviral vectors bearing engineered FMs
exhibited 8 to 17-fold enhanced transduction towards target cells as compared to the
parental FM. Different levels of enhancement were observed for the different engineered

21

FMs. A pH-dependent study of vector transduction showed that the broader pH range of
the engineered FM is a possible mechanism for the resulted increase in transduction
efficiency. The fusion domain of Sindbis virus glycoprotein is amenable for engineering
and the engineered proteins provide elevated capacity to mediate lentiviral vectors for
targeted transduction. Our data suggests that application of such an engineering strategy
can optimize the two-molecular targeting method of lentiviral vectors for gene delivery to
predetermined cells.

2. 1 Introduction
Viral gene delivery using retroviral vectors remains one of the most promising
techniques for gene therapy (Verma and Somia, 1997; Verma and Weitzman, 2005b). For
certain situations, one may prefer to deliver genes in a cell-type specific manner,
alleviating the “off-target” effect (Sandrin et al., 2003b; Waehler et al., 2007b). Thus,
many investigations have focused on how to engineer retroviral vectors into targeted gene
delivery vehicles (Sandrin et al., 2003b). Significant works have been reported in which
the viral envelope glycoprotein is engineered to redirect the host tropism by either
inserting a targeting ligand or a single-chain antibody (Ager et al., 1996; Chowdhury et
al., 2004; Han et al., 1995; Kasahara et al., 1994b; Marin et al., 1996; Nilson et al., 1996;
Somia et al., 1995b; Waehler et al., 2007b). Another popular strategy for achieving
targeted transduction is directing the viral vectors to the target cell by an adaptor
molecule (Boerger et al., 1999b; Morizono et al., 2001b; Morizono et al., 2006; Morizono
et al., 2005; Pariente et al., 2008; Pariente et al., 2007; Roux et al., 1989a; Waehler et al.,

22

2007b). Although these approaches can generate vectors that recognize specific cells, the
modification and binding interference introduced to the envelope protein unavoidably
affects the performance of the glycoprotein to mediate transduction (Lavillette et al.,
2001; Waehler et al., 2007b).
Lentiviral vectors, a subfamily of retroviral vectors, have been widely studied for
the purpose of gene delivery because of their ability to transduce both dividing and non-
dividing cells (Kohn, 2007). Like other retroviral vectors, their integration ability has
enabled the vector-transduced cells to maintain a long-term stable expression of
transgenes (Verma and Somia, 1997; Verma and Weitzman, 2005b). Recently, we
developed a novel method to engineer lentiviral vectors that transduce specific cell types
by breaking up the binding and fusion functions of the envelope protein into two distinct
proteins (Yang et al., 2006b). Instead of pseudotyping lentiviral vectors with a modified
viral envelope protein, our lentiviral vectors co-display a targeting antibody and a
fusogenic molecule (FM) on the same viral vector surface. Based on the molecular
recognition, the targeting antibody will direct lentiviral vectors to the specific cell type.
The binding between the antibody and the cognate cellular antigen will induce
endocytosis resulting in the transport of lentiviral vectors into the endosomal
compartment. Once inside the endosome, the FM will undergo a conformation change in
response to the drop in pH, thereby releasing the viral core into the cytosol (Joo and
Wang, 2008).
We previously demonstrated that a binding defective version of the alphavirus
Sindbis glycoprotein was able to envelope lentiviral vectors to mediate fusion of viral

23

membrane and endosomal membrane, a critical step for transduction (Joo and Wang,
2008; Yang et al., 2006b). Kielian and co-workers had studied the cholesterol
dependency of the Sindbis virus and reported several versions of the Sindbis virus
glycoprotein that were less dependent on cholesterol for transduction (Lu et al., 1999b).
We report herein that engineering the fusion domain of the binding defective Sindbis
glycoprotein can enhance fusion function of this protein to pair with an anti-CD20
antibody (αCD20), hence mediating targeted transduction of lentiviral vectors to CD20-
expressing cells. The cellular antigen used in this study is the CD20 protein, whose
expression is B cell specific (Stashenko et al., 1980). It has been shown that 90 % of
non-Hodgkin’s lymphomas are CD20-positive (Anderson et al., 1984; Molina, 2008; Reff
et al., 1994). CD20 is not usually expressed on either precursor B lymphoid cells or the
majority of plasma B cells (Molina, 2008). Thus, this stage-specific expression pattern
makes CD20 an ideal target for therapies against B cell malignancy.

2. 2 Results
2.2.1 Generation of pH-dependent FMs
We previously demonstrated that cell-specific targeted transduction can be
achieved by lentiviral vectors enveloped with αCD20 and a FM (Yang et al., 2006b). The
FM used in that study was a mutant viral glycoprotein derived from the Sindbis virus.
The Sindbis envelope glycoprotein consists of two domains; E1 is responsible for
mediating the fusion between the virus and target cell and E2 is responsible for directing

24

the binding of the virus to the cellular antigen on the target cell surface (Mukhopadhyay
et al., 2005). Chen and co-workers reported a fusion-competent, but binding-deficient
form of the Sindbis envelope glycoprotein which was generated by inserting a ZZ binding
domain into the E2 region of the envelope protein (Morizono et al., 2001b; Morizono et
al., 2005). We further modified this binding-deficient envelope protein by replacing the
ZZ binding domain with a HA tag; the resulting protein was designated SINmu (Yang et
al., 2006b). In addition, Kielian and co-workers have previously shown that a mutation
on E1, at region 266, of the Sindbis virus envelope protein results in viruses that were
less dependent on cholesterol for transduction (Lu et al., 1999b). Their work identified
three distinct mutants with enhanced efficiency to transduce cholesterol-depleted cells
(Lu et al., 1999b). We reasoned that incorporation of these mutations into SINmu might
endow new FMs with enhanced efficiency to induce fusion of antibody-displaying
lentiviral vectors to accomplish targeted transduction. To test this hypothesis, we
generated three FMs designated as SGN, SGM and AGM (Fig. 2.1). These FMs differed
from the parental FM (SINmu) by three amino acids at region 226 of the E1 protein.

25

Figure 2.1 Schematic representation of key constructs in this study encoding the FM derived from
the Sindbis virus glycoprotein, lentiviral transfer vector FUGW, membrane-bound human/mouse
chimeric antibody against CD20 (αCD20), and accessory proteins for surface expression of antibody
(Igαβ). CMV: human cytomegalovirus immediate-early gene promoter; E3: leader peptide of Sindbis
virus glycoprotein; E1: E1 protein of the Sindbis virus glycoprotein for mediating fusion; E2: E2
protein of the Sindbis virus glycoprotein for binding to viral receptor; HA tag: 10-amino acid epitope
hemagglutinin sequences (MYPYDVPDYA); Ubi: human ubiquitin-C promoter; GFP: green
fluorescent protein; WPRE: woodchuck regulatory element; LTR: long-terminal repeat; ΔU3: U3
region with deletion to disable the transcriptional activity of integrated viral LTR promoter; EF1α:
human elongation factor 1α promoter; αCD20κ and αCD20λ: light chain and heavy chain of
human/mouse chimeric antibody against CD20; TM: human antibody transmembrane domain; Igα
and Igβ: human antibody accessory proteins Igα and Igβ. For the FM constructs (SINmu, SGN, SGM
and AGM), the amino acid sequences at E1 226 region are shown. The sequence starts at amino acid
225 and ends with amino acid 234 of the wild-type E1 protein. Specific amino acids involved in
generating new FMs are shown underlined in bold.

2.2.2 Production of recombinant lentiviral vectors
To evaluate the targeting activity of these engineered FMs, we employed a
transient transfection protocol to produce lentiviral vectors enveloped with αCD20 and a

26

specific FM (Pear et al., 1993). We co-transfected 293T cells with FUGW, the self-
inactivating lentiviral transfer construct that is derived from HIV-1, which contains an
internal human ubiquitin-C promoter driving the expression of GFP reporter gene (Fig.
2.1) (Lois et al., 2002a). In addition to FUGW, we supplied the lentiviral packaging
plasmids, the αCD20 construct, the antibody accessory protein construct, and the plasmid
encoding the FM (SINmu, SGN, SGM, or AGM). The αCD20 system has been
previously established in our laboratory and has been shown to display on the surface of
lentiviral vectors for targeted transduction of CD20-expressing cells (Yang et al., 2006b).
Therefore, we used this as a model system for testing FMs. Antibody accessory proteins
(Igα and Igβ) were required for functional expression of the antibody onto the surface of
producing cells for its subsequent incorporation into the viral vector (Fig. 2.1). As a non-
targeting control, a transfection was done to prepare a vector pseudotyped with VSVG
since it has fairly broad specificity and can transduce a variety of cell types (Cronin et al.,
2005b). Negative controls included vectors bearing a FM and an antibody (Ab) that is
blind to CD20 antigen and the vector bearing αCD20 only. Two days after transfection,
expressions of GFP, αCD20 and FM were analyzed by flow cytometry. Virtually all of
the transfected, virus-producing 293T cells expressed GFP which was encoded in FUGW
(Fig. 2.2a). Among these GFP-positive cells, approximately 21-31 % of them expressed
both αCD20 and a FM (FUGW+αCD20+FM, Fig. 2.2b, top left). The viral vectors
produced by these transfections were designated FUGW/αCD20+FM. A similar
percentage of GFP-positive cells were observed to express both a FM and Ab
(FUGW+Ab+FM, Fig. 2.2b, bottom left) and the corresponding viral vectors were
designated FUGW/Ab+FM. As expected, the FM signals were not detected on cells

27

transfected to produce αCD20-bearing, but FM-lacking, FUGW/αCD20 vector
(FUGW+αCD20, Fig. 2.2b, top right), and no signals of the FM and the αCD20 were
seen on cells producing the FUGW/VSVG vector (FUGW+VSVG, Fig. 2.2b, bottom
right). In addition, it appeared from the expression pattern of FM-transfected cells that the
expression level of the four FMs were similar, suggesting that they could be incorporated
into the surface of lentiviral vectors with similar efficiency.

28

Figure 2.2 Co-expression of an antibody and a FM on the surface of transfected vector producing
cells. 293T cells were transiently transfected with plasmids FUGW, pαCD20, pIgαβ, pFM, along with
other standard packing plasmids to produce targeted FUGW/αCD20+FM vectors. The plasmid pAb
was used in transfection to generate control vectors FUGW/Ab+FM. The transfection without FM
plasmid was performed to generate the control vector FUGW/αCD20. The transfection with the
standard envelope plasmid encoding VSVG was conducted to generate the non-targeting control
vector FUGW/VSVG. (a) FACS analysis of GFP expression in vector producing cells. Solid line,
analysis on transfected 293T cells; shaded area, analysis on 293T cells (as control) (b) Gating on
GFP-positive cells, co-expression of an antibody and a FM is shown. Expression of an antibody and a
FM were detected by using anti-human IgG antibody and anti-HA antibody, respectively.

29

2.2.3 Co-incorporation of the αCD20 and the FM on lentiviral vectors
In order for our targeting system to work, lentiviral vectors must be enveloped
with both αCD20 for binding, and a FM for fusion. We employed a confocal imaging
method to analyze the co-incorporation of the αCD20 and the FM. The target cells were
incubated with viral vectors at 4
o
C for 1 hour, followed by sequential staining of the FM
(blue color) and the αCD20 (red color). To label the core of the vectors, we adapted a
previously reported method to synthesize viral vectors encapsulated with a protein (GFP-
Vpr) consisting of GFP fused with viral protein R (Vpr) (McDonald et al., 2002a). It has
been shown that the provision of GFP-Vpr in trans during transfection can allow the
fluorescent protein to be incorporated into the core of HIV-based lentiviral vectors
through the interaction between Vpr and the P6 region of the HIV gag protein (McDonald
et al., 2002b). We harvested GFP-Vpr-tagged lentiviral vectors bearing αCD20 and
SINmu and incubated them with 293T/CD20 cells at 4
o
C for 1 hour. After extensive
washing, the treated cells were subjected to immuno-fluorescence staining and imaging.
GFP-labeled signals were detected on the surface of 293T/CD20 cells and their signals
were co-localized with signals for both αCD20 and SINmu (Fig. 2.3a, top), while no
fluorescence signals were obtained for 293T cells lacking the expression of CD20 (Fig.
2.3a, bottom). The co-localization of GFP, αCD20 and SINmu suggested that the cells
can produce lentiviral vectors displaying both αCD20 and a FM in a single virion. Similar
results were also observed for vectors bearing the other type of FMs (SGN, SGM, or
AGM) (data not shown).

30

We conducted flow cytometry analysis of cells incubated with viral vectors to
further examine their surface properties. Various vectors (FUGW/αCD20+FM) were
allowed to bind to 293T/CD20 target cells at 4
o
C for 1 hour, after which the FM was
stained and analyzed. Only the vectors bearing both αCD20 and a FM could bind to
293T/CD20 cells and be detected by using an antibody against the FM. Therefore, flow
cytometry signals can indicate the presence of both αCD20 and a FM on the same viral
surface. As shown in Fig. 2.3(b), no detectable FM signals were seen when the 293T cells
were incubated with either FUGW/αCD20+FM or FUGW/Ab+FM vectors, indicating
that the lentiviral vectors were unable to bind to 293T cells lacking the expression of
CD20. When FUGW/Ab+FM were incubated with 293T/CD20 cells, no signals were
detected for the FM (Fig. 2.3b). Clear signals were obtained when 293T/CD20 and
FUGW/αCD20+FM were incubated together (Fig. 2.3b). This result confirmed that cell-
virus binding was attributed to the interaction between the αCD20 and CD20, and both a
FM and αCD20 can be incorporated onto the same viral surface.

31

Figure 2.3 Co-expression of αCD20 and a FM on the viral vector surface. (a, left) Schematic
diagrams to illustrate the interaction between αCD20 on the vector surface and CD20 on the cell
surface, and the immuno-fluorescent staining scheme. (a, right) Acquired confocal images of labeled
viral vector binding to cells. (b) FACS analysis of 293T or 293T/CD20 cells incubated with
FUGW/αCD20+FM or FUGW/Ab+FM. The binding of virus to 293T/CD20 cells was detected by
FACS staining with antibody against the FM. Solid line, analysis on cells incubated with indicated
viral vectors; shaded area (control), analysis on cells without incubation with vectors.

32

2.2.4 Targeted transduction of lentiviral vectors to CD20-positive cell line
We then investigated how lentiviral vectors bearing different FMs would
transduce cells by using 293T/CD20 as the target cell line, and the 293T parental cell line
as the negative control. Six days post-transduction, cells were analyzed by flow
cytometry. To quantify the difference of our targeted transduction system, we utilized a
metric that incorporates both the efficiency and magnitude of the GFP signals detected
from the transduced cells (Darrah et al., 2007). The transduction magnitude was obtained
by the mean fluorescence intensity (MFI) of the transduced cells. By multiplying the MFI
by transduction efficiency, we derived a metric termed integrated MFI (iMFI) that
reflects the total intensity of the GFP signals from the virus transduced cells. As indicated
in Fig. 2.4(a), FUGW/αCD20+AGM displayed the highest iMFI in the target cells
(293T/CD20), followed by FUGW/αCD20+SGM, FUGW/αCD20+SGN, and
FUGW/αCD20+SINmu. When the same set of viral vectors were used to transduce the
non-target cells (293T), much lower iMFI signals were detected. The specific
transduction titers of these viral vectors against 293T and 293T/CD20 cells were
measured in Fig. 2.4(b); 8-17-fold increase of preferential transduction of CD20-
expressing cells was achieved, depending on which FM was used (Fig. 2.4b). To confirm
that the specific transduction was mediated by the incorporated antibody on the vector
surface, we made control vectors bearing a FM and Ab. Spin-transduction of these
vectors to 293T/CD20 and 293T cells showed low iMFI signals (Fig. 2.4a). In addition,
to eliminate the possibility that the difference was due to a variance in viral production
from the producing cells, we performed an enzyme linked immunosorbent assay (ELISA)
to detect the p24 levels in the viral supernatants. As indicated in Fig. 2.4(c), the p24

33

levels between the different lentiviral vectors were in the similar range. When comparing
the ability of various FMs to mediate transduction, the FM with higher efficiency for
targeted transduction would always result in higher background transduction.

Figure 2.4 Transduction of engineered lentiviral vectors bearing both an antibody and FM to cell
lines. 293T and 293T/CD20 cells (2 × 10
5
) were transduced with 2 ml of fresh viral vectors
(FUGW/αCD20+FM, or FUGW/Ab+FM). Transduction of 293T cells was included as control. (a)
iMFI on 293T and 293T/CD20 cells transduced by the fresh viral vectors (FUGW/αCD20+FM, or
FUGW/Ab+FM). (b) Titers of fresh viral vectors (FUGW/αCD20+FM) on 293T and 293T/CD20 cells.
(c) p24 amount of fresh viral vectors (FUGW/αCD20+FM, FUGW/Ab+FM, and FUGW/VSVG)

2.2.5 pH Dependence of various FMs
Our transduction experiment clearly showed the important role of FMs in
determining the overall vector infectivity and that the newly engineered FMs exhibited

34

improved ability to induce targeted transduction as compared to original FM (SINmu)
(Fig. 2.4). We designed an experiment to investigate the possible underlying mechanism
responsible for their differences. Lentiviral vectors, FUGW/αCD20+SINmu,
FUGW/αCD20+SGN, FUGW/αCD20+SGM, and FUGW/αCD20+AGM, were incubated
with 293T/CD20 cells in the absence or presence of a graded concentration of ammonium
chloride (NH
4
Cl); NH
4
Cl is known to be able to neutralize the acidic endosomal
environment (Mellman et al., 1986). The changes in transduction were measured by flow
cytometry. As shown in Fig. 2.5(a), the four vectors displaying various FMs behaved
differently in response to different concentration of NH
4
Cl. FUGW/αCD20+SINmu was
the most sensitive to the neutralization treatment and transduction efficiency dropped the
fastest as a result of the changes in pH environment, followed by FUGW/αCD20+SGN,
FUGW/αCD20+SGM, and FUGW/αCD20+AGM. It appeared that the engineered vector
that was more resistant to the NH
4
Cl treatment could transduce target cells with a higher
efficiency.
2.2.6 Antibody directed targeted transduction
To confirm that the targeted transduction was triggered by the specific interaction
between the antibody and the cognate antigen, we performed an antibody competition
assay. Lentiviral vector, FUGW/αCD20+SGN was incubated with 293T/CD20 cells in
the presence of various concentration of either soluble αCD20 antibody or an isotype
control (Fig. 2.5b). We found that the targeted transduction efficiency decreased as the
concentration of soluble αCD20 antibody increased. When the isotype control was used,
no decrease in transduction efficiency was observed. This suggested that the soluble

35

αCD20 competed with the targeted lentiviral vector leading to a decreased uptake of the
vectors.

Figure 2.5 Examination of addition of NH
4
Cl or soluble αCD20 on the targeted transduction results.
(a) NH
4
Cl was added into indicated viral supernatants during transduction for 8 hours, after which,
the supernatants were replaced with fresh media. GFP expression was analyzed 3 days post-
transduction. (b) Various amount of soluble αCD20 or isotype control were added into indicated viral
supernatants during transduction for 8 hours, after which, the supernants were replaced with fresh
media.

36

2.2.7 Targeted transduction of lentiviral vectors to unfractionated primary B cells
One of the advantages of using targeted vectors is their potential ability to
transduce specific cell types in a mixed population without the need to isolate the target
cells. We tested whether our targeted vectors with engineered FMs can specifically
transduce primary B cells in an unfractionated primary cell population. One million of
fresh, unfractionated human peripheral blood mononuclear cells (PBMC) were
transduced twice with concentrated FUGW/αCD20+FM (2.5 × 10
6
TU), or
FUGW/VSVG (25 × 10
6
TU). The cells were analyzed by flow cytometry 2 days post-
transduction. As shown in Fig. 2.6(a), transduction of bulk PBMC populations with
targeted vectors resulted in specific modification of CD20-positive PBMC, whereas no
GFP signals were detected in CD20-negative cells. In the control experiment where
FUGW/VSVG was used for transducing bulk PBMC populations, GFP signals were
detected in both CD20-positive and CD20-negative cells and a higher iMFI signal was
detected in the CD20-negative PBMC as compared to CD20-positive PBMC.
Quantification of total expression intensity indicated that vector FUGW/αCD20+AGM
attributed the highest iMFI in the CD20-positive PBMC, followed by
FUGW/αCD20+SGM, FUGW/αCD20+SIN, and FUGW/αCD20+SGN (Fig. 2.6b). This
was in good agreement with the specific transduction against 293T/CD20 cells (Fig.
2.4b). The stable integration of the GFP gene was confirmed by the genomic PCR
analysis (data not shown).

37

Figure 2.6 Targeted transduction of CD20-positive human primary B cells. (a) Fresh unfractionated
human PBMCs (1 × 10
6
) were spin-transduced twice with indicated vectors (FUGW/αCD20+FM, 2.5
× 10
6
TU, or FUGW/VSVG, 25 × 10
6
TU). 48 hours later, cells were collected and analyzed by FACS
(b) iMFI on fresh unfractionated human PBMCs transduced by the indicated viral vectors
(FUGW/αCD20+FM, and FUGW/VSVG).

38

2.3 Discussion
Lentiviral vectors pseudotyped with envelope glycoprotein from other genus of
viruses, such as VSVG, have shown to be able to genetically modify cells with great
efficiency and broad tropism (Cronin et al., 2005a). However, certain applications may
require using vector systems with cell-type specificity (Sandrin et al., 2003a; Waehler et
al., 2007a). Previous studies have shown that by breaking up binding and fusion functions
into αCD20 and a FM, targeted transduction can be accomplished (Yang et al., 2006a).
To test whether engineered FMs can improve vector targeting efficiency, we
transduced 293T/CD20 with freshly collected viral supernatants containing targeted
lentiviral vectors. Higher iMFI was observed when lentiviral vector,
FUGW/αCD20+AGM, was used, followed by a decrease in iMFI in
FUGW/αCD20+SGM, FUGW/αCD20+SGN, and FUGW/αCD20+SINmu, respectively
(Fig. 2.4a). These results suggested that FUGW/αCD20+AGM was capable of
transducing target cells with higher efficiency and transgene expression. However, some
background levels of transduction on non-target cells were observed. This is likely
resulted from the endocytosis induced by non-specific cellular receptors, although we did
not observe obvious binding between targeting vectors and non-target cells (Fig. 2.3b).
We then set to examine whether such targeted vectors can achieve similar results in
PBMC. As shown in Fig. 2.6(a), a similar trend was observed in that only CD20-
expressing PBMC were shown to be specifically transduced by concentrated virus,
whereas no transduction was observed in the CD20-negative population. In the control
experiment, targeted lentiviral vectors were substituted with a non-targeted vector,

39

FUGW/VSVG, resulting in detection of GFP in all cell types, regardless of CD20-
expression. It is known that FUGW/VSVG preferentially transduce CD20-negative
populations as compared to CD20-positive population (Serafini et al., 2004).
Furthermore, a higher MOI was needed to achieve similar transduction efficiency
highlighting the advantage of targeting. In agreement with previous cell line data,
lentiviral vector, FUGW/αCD20+AGM, was able to attribute the highest iMFI followed
by a decrease in iMFI in FUGW/αCD20+SGM, FUGW/αCD20+SINmu, and
FUGW/αCD20+SGN respectively (Fig. 2.6a).
In order for our targeting system to work, the virus producing cells must co-
express both an antibody and a FM. These vectors can then bind to the target cells based
on the recognition provided by the displayed antibody. When adding a soluble antibody,
transduction was inhibited whereas addition of the isotype control had no effect on
transduction, indicating the binding requirement for targeted transduction (Fig. 2.5b).
After binding, these vectors are endocytosed and transported to the endosomal
compartments. The changing of the pH environment within the endosomes triggers the
FM to alter its conformation, leading to fusion between the endosomal membrane and the
viral membrane (Joo and Wang, 2008). We believe these steps are required for a
successful targeted transduction and employed various methods to investigate this
targeted transduction pathway. We first analyzed our virus producing cells for the display
of the αCD20 and the FM by flow cytometry (Fig. 2.2b), followed by confocal
microscopic imaging to confirm the co-incorporation of both molecules in a single viral
vector (Fig. 2.3a). GFP-Vpr fusion protein was used to label the viral core, while
Alexa594-conjugated and Cy5 antibodies were used to detect the αCD20 and the FMs,

40

respectively. Co-localization of all three colors was observed, indicating that the viral
vector carried both the αCD20 and the FM. Cell-virus binding assay was employed to
evaluate whether the incorporated antibody can provide the function to direct the viral
vectors to the target cells (Fig. 2.3b). As shown by both flow cytometry analysis and
confocal imaging, only the lentiviral vectors bearing the targeting antibody were able to
bind to the target cells.
Since our engineered lentiviral vectors were enveloped with αCD20 and a FM, it
was conceivable that optimization of these two proteins could potentially improve the
efficiency of this targeting system. Kielian and co-workers generated three different
mutants of the Sindbis virus glycoprotein and found that these mutants could increase the
cholesterol independence of the Sindbis virus (Lu et al., 1999a). We adapted these
mutations into the original FM (SINmu) to generate three new FMs (SGN, SGM, or
AGM) (Fig. 2.1). We found that these new FMs exhibited enhanced ability to mediate
lentiviral vectors to transduce the target cells. We further analyzed the responsiveness of
these FMs to acidic neutralization. The neutralization assay with vectors bearing different
FMs revealed that the original FM (SINmu) was the most sensitive to the pH change in
the endosome, as the infectivity of the vector bearing SINmu dropped the fastest with
respect to the ammonium chloride treatment. The new FMs responded slower than that of
SINmu to the treatment, with AGM being the least pH-sensitive FM (Fig. 2.5a). These
results were consistent with the transduction assay, in which the vector bearing AGM had
the highest transduction titer (Fig. 2.4b).

41

The fusion mechanism for the Sindbis glycoprotein involves a drop in pH that
triggers a conformational change in the E2 and E1 subunits that exposes the previously
hidden E1 hydrophobic domain. The E1 domain then interacts with cholesterol in the
target membrane which leads to the fusion of the viral and cellular membranes (Kielian
and Rey, 2005). Similar to the hemagglutinin glycoprotein of the Influenza virus,
mutations throughout the trimer interface alter the pH threshold required for fusion by
either destabilizing the buried position of the fusion peptide or by modifying salt bridges
and hydrogen bonding between trimer subunits (Daniels et al., 1985). We speculate that
these mutations may destabilize the fusion loop of the E1 domain which lowers the
required activation energy needed for fusion. Since the lentiviral vectors are required to
escape from the endosome in order to transduce the host cells, the FM that is active
throughout a wider pH range could endow the corresponding lentiviral vector with a
larger window to escape from the degradation pathway.
In summary, we have demonstrated in this report that targeted transduction could
be accomplished by enveloping lentiviral vectors with αCD20 and a FM. One of the
major advantages of this system is the flexibility as the vectors can be readily
reengineered to target different cell type. As shown in our previously published data, our
system was capable of targeting antigen specific immunoglobulins by simply swapping
the display antibody with an antigen (Ziegler et al., 2008). We are currently expanding
our targeting strategy to a variety of cell types including gp160 expressing cells. We have
also demonstrated that this targeted system is amenable for further optimization to
improve efficiency. Although higher transduction efficiency could be achieved by pairing
the recognition antibody with novel engineered FMs to improve fusion properties, it also

42

led to higher background transduction efficiency. We are currently studying our
engineered FM in the hope that we would gain some insight in the fusion process
allowing us to design a better FM capable of enhanced targeted transduction with
decreased background transduction. When comparing findings from this paper to our
previously published data (Yang et al., 2008), it becomes apparent that a judicious choice
must be made when it comes to the FM, since FMs behave differently under different
settings. In the case of gamma-retrovirus pseudotyped with FM, one FM showed
enhanced transduction whereas in the case of lentiviral vector, multiple FMs showed
enhanced efficiency. It is noteworthy that one advantage of using lentiviral vector as
compared to gamma-retroviral vector lies in the ability of lentiviral vectors to transduce
non-dividing cells.

2.4 Methods
2.4.1 Construct preparation
The original FM, SINmu, was previously constructed in our laboratory (Yang et
al., 2006b). It was generated by replacing amino acids 157KE158 with 157AA158 of the
E2 protein of the Sindbis virus glycoprotein. Additional deletion was performed to
remove amino acids 61-64 in the E3 protein of the Sindbis virus glycoprotein. We also
inserted a hemagglutinin epitope tag sequence (MYPYDVPDYA) between amino acids
71 and 74 of the E2 proteins for detection purpose (Fig. 2.1). Based on SINmu, we
performed 4-primer PCR-mutagenesis to generate mutants SGN, SGM, and AGM. To

43

construct SGN, a forward primer (BsiW1fw, 5’-GCC AGA TGA GTG AGG CGT ACG
TCG AAT TGT CAG C-3’) and a backward primer (SGNbw, 5’-CAT GCA CGT ACC
GGA GGA AGG CTT GAG TAG CCT AAT GTC TGT GCT G-3’) were used to
amplify the E1 domain containing the BsiW1 site and the desired SGN mutations at the
E1 226 region. In parallel, a forward primer (SGNfw, 5’-AGC CTT CCT CCG GTA
ACG TGC ATG TCC CGT ACA CGC AGG CC-3’) and a backward primer (Mfe1bw,
5’-GCT GCA ATA AAC AAG TTA ACA ACA ACA ATT GCA TTC ATT TTA TG-
3’) were used to amplify the E1 domain containing the desired SGN mutations at the E1
226 region and Mfe1 site. The DNA products from these two reactions were PCR-
assembled using BsiW1fw and Mfe1bw as the primer pair and cloned into pcDNA3
(Invitrogen) to yield SGN. The similar PCR protocol was used to generate mutant SGM
and AGM, except that the primers SGNbw and SGNfw were replaced by (SGMbw, 5’-
CAT GCA CCA TAC CGG AGG AAG GCT TGA GTA GCC TAA TGT CTG TGC
TG-3’) and (SGMfw, 5’-AGC CTT CCT CCG GTA TGG TGC ATG TCC CGT ACA
CGC AGG CC-3’) for construction of SGM, and by (AGMbw, 5’-CAT GCA CCA TAC
CGG CGG AAG GCT TGA GTA GCC TAA TGT CTG TGC TG-3’) and (AGMfw, 5’-
AGC CTT CCG CCG GTA TGG TGC ATG TCC CGT ACA CGC AGG CC-3’) for
construction of AGM.
The construct encoding the membrane bound form of the human/mouse chimeric
antibody against human CD20 antigen (pαCD20) and the construct encoding the human
antibody accessory proteins Igα and Igβ (pIgαβ) were constructed previously in our
laboratory (Yang et al., 2006b). Briefly, the cDNAs of the human κ light chain constant
region, and the membrane bound human IgG1 constant chain region were amplified and

44

inserted downstream of the human CMV and EF1α promoters, respectively, in the
pBudCD4.1 vector (Invitrogen). The light chain variable region from the murine αCD20
was amplified from an αCD20 hybridoma cell line (ATCC, Manassas, VA, HB-9303)
with (CD20Lvfw, 5’-CCC AAG CTT ATG GAA ACC CCA GCG CAG CTT C-3’) and
(CD20Lvbw, 5’-CAG CCA CCG TAC GTT TCA GCT CCA GCT TG-3’) and inserted
directly upstream of the light chain constant region via Hind3 and BsiW1 restriction sites.
In parallel, the heavy chain variable region was amplified with (CD20Hvfw, 5’-GGA
CTC GAG ATG GAG TTT GGG CTG AGC TG-3’) and (CD20Hvbw, 5’-GGT GCT
AGC TGA AGA GAC GGT GAC CGT G-3’) and inserted directly upstream of the
heavy chain constant region via Xho1 and Nhe1 restriction sites. The non-relevant
antibody (Ab) used in this study, B12, was kindly provided by the laboratory of Dr.
Dennis Burton at the Scripps Research Institute (Burton et al., 1994). The membrane
bound B12 antibody was constructed similarly to the αCD20, except that (B12Lvfw, 5’-
CCC AAG CTT ACC ATG GGT GTG CCC ATC C-3’) and (B12Lvbw, 5’-CAC CGT
ACG TTT CCT CTC CAG TTT GGT CCC-3’) were used to amplify the light chain
variable region, and (B12Hvfw, 5’-GGG ACC AAA CTC GAG AGG AAA CGT ACG
GTG-3’) and (B12Hvbw, 5’-GGT GCT AGC TGA GCT CAC GAT GAC CGT GG-3’)
were used to amplify the heavy chain variable region. The HIV-1-based lentiviral transfer
plasmid FUGW was constructed by the laboratory of Dr. David Baltimore at the
California Institute of Technology (Lois et al., 2002b).

45

2.4.2 Cell line construction
The 293T cell line was obtained from ATCC. The cell line 293T/CD20 was
generated by stable transduction of vesicular stomatitis virus glycoprotein (VSVG)
enveloped lentiviral vector encoding the cDNA for the human CD20 protein.
2.4.3 Virus production
Recombinant lentiviral vectors were generated via the standard calcium phosphate
precipitation technique (Pear et al., 1993). The vectors producing 293T cells were seeded
in a 6-cm culture dish with DMEM medium supplemented with 10 % Fetal Bovine
Serum (Sigma, St. Louis, MO), L-glutamine (10 mL L
-1
), penicillin (100 units mL
-1
), and
streptomycin (100 units mL
-1
). After 16-18 hours, when the confluency was about 80 %,
the seeded 293T cells were transfected with plasmid DNAs. The packaging plasmids,
pMDLg/pRRE (2.5 µg) and pRSV-Rev (2.5 µg) (Klages et al., 2000a), the plasmids for
surface display of αCD20, pαCD20 (2.5 µg) and pIgαβ (2.5 µg), the plasmid encoding the
FMs, pFM (2.5 µg), and the lentiviral transfer plasmid FUGW (5 µg) were mixed with
calcium chloride and added drop-wise to 2×HBS solution with constant vortexing
(Klages et al., 2000a). The lentiviral vector FUGW/Ab+FM was produced similarly with
the exception that the plasmid encoding pAb was used instead of pαCD20. Co-
transfection of 293T cells with the lentiviral transfer plasmid FUGW (5 µg), the
packaging plasmids pMDLg/pRRE (2.5 µg) and pRSV-Rev (2.5 µg), and the envelope
plasmid pVSVG (2.5 µg) was performed to generate the lentiviral vector FUGW/VSVG.
Transfected cells were replenished with pre-warmed fresh media 4 hours post-

46

transfection. 48 hours later, viral supernatants were harvested, and filtered through a 0.45
µm pore size filter (Nalgene, Rochester, NY).
2.4.4 Fluorescent labeling
GFP-Vpr-labeled lentiviral vectors were produced by co-transfecting 293T cells
with the plasmid encoding GFP-Vpr (Joo and Wang, 2008) in addition to the plasmids
used in generation of corresponding lentiviral vectors. For imaging cell-virus binding, 5 ×
10
5
cells were seeded onto a 35 mm glass-bottom culture dish (MatTek Corporation) and
grown at 37 °C overnight. The seeded cells were rinsed with cold PBS twice and
incubated with concentrated viral vectors for 1 hour at 4 °C to allow for binding. Cells
were washed with cold PBS to remove unbound viral vectors, fixed for 10 minutes on ice
using 4 % formaldehyde, and then immunostained by Alexa594-conjugated anti-human
IgG (Molecular Probes) and biotin-conjugated anti-HA antibody (Miltenyi Biotec Inc.),
followed by a secondary staining with Cy5-conjugated streptavidin (Zymed
Laboratories). The images were acquired with a Zeiss LSM-510 laser scanning confocal
microscope (Carl Zeiss, Thornwood, NY) using a plan-apochromat 63×/1.4 oil immersion
objective. The images were processed using the LSM 510 software version 3.2 SP2.
2.4.5 FACS analysis of cell-virus binding
0.5 million cells (293T or 293T/CD20) were incubated with 2 mL of various
lentiviral vectors at 4
o
C for 1 hour. Cell-virus complexes were then washed with 4 mL of
cold PBS and spun down at 2,000 rpm for 5 minutes. Cell-virus complexes were then
stained with anti-HA antibody to detect the presence of the FM on the lentiviral vectors

47

bound to the target cells. After staining, cells-virus complexes were analyzed by flow
cytometry (BD Bioscience) to measure the binding.
2.4.6 p24 analysis of lentivral vectors
Various lentiviral vectors (10 µL, fresh supernatant) were lysed with 90 µL of 10
% Triton-X 100 in PBS and the p24 levels were measured by a p24 antigen capture
enzyme linked immunosorbent assay (ELISA) kit (ImmunoDiagnostics, Woburn, MA)
2.4.7 Targeted transduction
Various lentiviral vectors (2 mL, fresh supernatant) were added to 0.2 million
cells (293T or 293T/CD20) plated in a 24-well culture dish and spin-transduced for 90
minutes at 2,500 rpm and 25
o
C. The medium was then removed and replenished with 2
mL fresh media. Treated cells were incubated for 6 days at 37
o
C and 5 % CO
2
. The
percentage of GFP expression was determined by flow cytometry analysis. The titer was
calculated based on the GFP expression in the viral dilution range where the percentage
of GFP-positive cells linearly corresponded to the volume of virus.
2.4.8 NH
4
Cl neutralizing assay
293T/CD20 cells (0.2 million) were incubated with lentiviral vectors
(FUGW/αCD20+FM) in the presence of various amounts of NH
4
Cl in a 24-well culture
dish at 37
o
C and 5 % CO
2
for 8 hours. The medium was then removed and replenished
with fresh medium and incubated for an additional 3 days prior to flow cytometry
analysis.

48

2.4.9 Antibody competition assay
293T/CD20 cells (0.2 million) were incubate with lentiviral vectors
(FUGW/αCD20/SGN) in the presence of various amounts of either soluble αCD20 or
isotype control in a 24-well culture dish at 37
o
C and 5 % CO
2
for 8 hours. The medium
was then removed and replenished with fresh medium and incubated for an additional 3
days prior to flow cytometry analysis.
2.4.10 Targeted transduction of unfractioned PBMC
Lentiviral vectors (FUGW/αCD20+FM) were generated and concentrated by
ultracentrifugation (Optima L-90K Ultracentrifuge, Beckman Coulter, Fullerton, CA) at
25,000 rpm for 90 minutes and resuspended in 100 µl cold PBS after overnight
incubation. The concentrated vectors (2.5 × 10
6
transduction units (TU) of the targeted
lentiviral vectors and 25 × 10
6
of the VSVG pseudotyped lentiviral vector) and 1 million
unfractioned PBMC were spin-transduced for 90 minutes at 2,500 rpm and 25
o
C. The
medium was then removed and replenished with 1mL fresh medium. LPS (50 µg ml
-1
)
was then added to support the survival and growth of B cells. Treated cells were collected
48 hours post-transduction and stained with anti-human CD20 antibody. Flow cytometry
was employed to determine the efficiency of targeted transduction.

49

Chapter 3
Targeting Lentiviral Vector to Specific Cell Types Through Surface
Displayed Single Chain Antibody and Fusogenic Molecule

Portions of this Chapter are adapted from: Yuning Lei, Kye-Il Joo, Jonathan Zarzar,
Clement Wong, and Pin Wang. Virology Journal (2010), 7 (35).

Viral delivery remains one of the most commonly used techniques today in the
field of gene therapy. However, one of the remaining hurdles is the off-targeting effect of
viral delivery. To overcome this obstacle, we recently developed a method to incorporate
an antibody and a fusogenic molecule (FM) as two distinct molecules into the lentiviral
surface. In this report, we expand this strategy to utilize a single chain antibody (SCAb)
for targeted transduction.
Two versions of the SCAb were generated to pair with our various engineered
FMs by linking the heavy chain and the light chain variable domains of the anti-CD20
antibody (αCD20) via a GS linker and fusing them to the hinge-CH2-CH3 region of
human IgG. The resulting protein was fused to either a HLA-A2 transmembrane domain
or a VSVG transmembrane domain for anchoring purpose. Lentiviral vectors generated
with either version of the SCAb and a selected FM were then characterized for binding
and fusion activities in CD20-expressing cells.

50

Certain combinations of the SCAb with various FMs could result in an increase in
viral transduction. This two-molecule lentiviral vector system design allows for parallel
optimization of the SCAb and FMs to improve targeted gene delivery.

3.1 Introduction
Gene therapy is the introduction of a functional gene into a dysfunctional cell for
a therapeutic benefit. To date, viral vectors remain the most commonly used gene
delivery vehicles due to their high transduction efficiencies (Verma and Somia., 1997;
Verma and Weitzman, 2005a). In particular, lentiviral vectors represent one of the most
effective gene delivery vehicles as they allow for stable long-term transgene expression
in both dividing and non-dividing cells. In order to expand the targeted specificity of viral
vectors beyond their natural tropism, numerous studies have been focused on
pseudotyping lentiviral vectors with envelope glycoproteins derived from other viruses,
such as the glycoprotein from vesicular stomatitis virus (VSVG) (Burns et al., 1993;
Cronin et al., 2005a). However, since the VSVG is thought to recognize a ubiquitous
membrane phospholipids instead of a unique cellular receptor, pseudotyping generates
vectors with broad specificities (Hall et al., 1998; Mastromarino et al., 1987). To mitigate
this off-target effect, previous attempts have been devoted to engineer the viral
glycoprotein to recognize a specific cellular target by insertion of ligands, peptides, or
antibodies (Fielding, 2000; Gollan and Green, 2002; Jiang, 1999; Jiang et al., 1998;
Kasahara et al., 1994a; Martin, 2002; Maurice, 2002; Nguyen et al., 1998; Somia et al.,
1995a; Valsesia-Wittmann et al., 1994). Another approach involves bridging the viruses
and the targeted cell with ligand proteins or antibodies (Boerger et al., 1999a; Morizono

51

et al., 2001a; Morizono, 2005; Roux et al., 1989b). However, these modifications to the
surface glycoprotein appear to perturb the natural fusion function of the glycoprotein,
resulting in a reduction of transduction efficiency.
Recently, our lab has developed a strategy to target lentiviral vectors to specific
cell types by incorporating a surface antibody specific to CD20 antigen and a fusogenic
molecule (FM) as two distinct molecules (Yang et al., 2006a). Kielian and co-workers
reported several versions of the Sindbis virus glycoprotein that were less dependent on
cholesterol for transduction (Lu et al., 1999a). We applied these mutations (E1 226) to the
binding defective Sindbis glycoprotein and observed that they were able to enhance
transduction efficiency when paired with an anti-CD20 antibody (αCD20) (Lei et al.,
2009). In this study, we report our attempt to utilize a single chain antibody (SCAb) to
pair with a FM for targeting lentiviral vectors. Our SCAb is composed of variable
domains of the heavy and light chains of αCD20, linked by a GS linker and fused to a
hinge-CH2-CH3 region of human IgG. To anchor the SCAb onto the viral surface, we
conjugated the SCAb with either the HLA-A2 transmembrane domain (SC2H7-A2) or
the VSVG transmembrane domain (SC2H7-GS). We demonstrated that the lentiviral
vector enveloped with either of these antibody configurations could achieve targeted
transduction to CD20-expressing cells. We also compared the targeted transduction
efficiency and the binding avidity of both versions of the SCAb and investigate the
molecular roles of the displayed proteins in mediating lentiviral transduction.

52

3.2 Results
3.2.1 Construction of SCAb for targeting
We have previously demonstrated that targeting lentiviral vectors can be
generated by co-transfecting producer cells with a lentiviral vector backbone plasmid,
FUGW, a plasmid encoding an antibody’s heavy and light chains, a plasmid encoding
antibody accessory proteins, and a plasmid encoding a FM, along with lentiviral
packaging plasmids (Yang et al., 2008; Yang et al., 2006a). In this report, we wanted to
expand the targeting strategy by pairing FMs with SCAbs. To generate the SCAb for this
study, we first PCR-amplified the light chain and heavy chain variable regions of the
αCD20 and linked them with a GS linker. To allow for the formation of disulfide-linked
dimmers to stabilize the SCAb, the hinge-CH2-CH3 region of the human IgG was fused
to the heavy chain variable region (Chou et al., 1999; Liao et al., 2003; Liao et al., 2000;
Roffler et al., 2006). To anchor the SCAb, the HLA-A2 transmembrane domain or the
VSVG transmembrane domain was added to the C-terminal and the resulting constructs
were designated as SC2H7-A2 and SC2H7-GS, respectively (Fig. 3.1).

53

Figure 3.1 Schematic representation of key constructs in this study. These constructs include a
lentiviral backbone vector FUGW, fusogenic molecule (FM) derived from Sindbis virus glycoprotein,
membrane-bound single chain antibody against the CD20 antigen with either a HLA-A2
transmembrane domain (SC2H7-A2) or a VSVG transmembrane domain (SC2H7-GS). CMV
enhancer: the enhancer element derived from human cytomegalovirus; GFP: enhanced green
fluorescence protein; Ubi: the human ubiquitin-C promoter; WRE: woodchuck responsive element;
CMV: human cytomegalovirus immediate-early gene promoter; αCD20 Vκ: the variable domain of
the kappa chain of the mouse anti-CD20 antibody; αCD20 Vγ: the variable domain of the gamma
chain of the mouse anti-CD20 antibody; CH2-CH3 region: the CH2-CH3 region of the human IgG1
antibody; HLA-A2 transmembrane domain: the transmembrane domain of the HLA-A2 protein;
VSVG transmembrane domain: the transmembrane domain of the VSVG protein; E3: the leading
peptide of Sindbis virus glycoprotein; E1: the E1 protein of Sindbis virus glycoprotein for mediating
fusion; E2: the E2 protein of Sindbis virus glycoprotein for binding to cellular receptor; HA tag: 10-
amino acid epitope sequence of hemagglutim; pA: polyadenylation signal.

3.2.2 Production of lentiviral vectors
We generated SCAb-bearing lentiviral vectors (FUGW/SC2H7-A2/FM or
FUGW/SC2H7-GS/FM) by co-transfecting 293T cells with the lentiviral backbone
plasmid FUGW, a FM-encoding plasmid (SINmu, SGN, SGM, or AGM), and a plasmid
encoding the described SCAb (pSC2H7-A2, or pSC2H7-GS) along with other necessary
packaging plasmids (Fig. 3.1). Independently, a lentiviral vector bearing an isotype

54

control antibody, pAB and a FM was produced as a non-target control. Furthermore, we
included a VSVG-pseudotyped lentiviral vector, FUGW/VSVG as an additional positive
control since VSVG-carrying viral vectors are known to transduce a variety of different
cell types (Cronin et al., 2005a). As shown in Fig. 3.2A, FACS analysis of transfected,
virus-producing 293T cells showed that virtually all of the cells were able to be
transfected with the viral backbone plasmid FUGW. Among the GFP-positive cells,
roughly 25% to 40% of the producer cells were positive for both the antibody and the FM
(Fig. 3.2B). As expected, transfection with VSVG as the envelope protein showed no
expression of the FM and the SCAb. The similar levels of transfection and expression of
the four FMs suggests that they could be incorporated into the lentiviral surface with
similar efficiency.

55

Figure 3.2 Co-transfection of virus-producing cells to generate targeting lentiviral vectors. 293T cells
were transiently transfected with, FUGW, pSC2H7-A2, pFM and the other standard packaging
plasmids (pMDLg/pRRE and pRSV-Rev) to make FUGW/SC2H7-A2/FM. Antibody construct
pSC2H7-GS was used to generate FUGW/SC2H7-GS/FM. An isotype control antibody construct
pAB was used in the transfection to produce non-targeting lentiviral vector FUGW/AB/FM.
Transfection with plasmids encoding VSVG was used to generate a control vector, FUGW/VSVG.
(A) FACS analysis of GFP expression on transfected cells. Solid line, analysis on transfected
293T/CD20 cells; shaded area, analysis on 293T cells. (B) Analysis of co-expression of the FM and
antibody on gated GFP-positive cells. FM was stained using an anti-HA antibody and antibody was
stained using an anti-human IgG antibody.

3.2.3 Incorporation of SCAb and FM onto lentiviral vectors
A virus-cell binding assay was performed to evaluate SCAb-mediated binding to
CD20-expressing cells. As a target, we used a 293T cell line stably expressing the CD20
antigen (designated as 293T/CD20). The parental cell line 293T served as a negative
control. The lentiviral vector, FUW/SC2H7-A2/SGN or FUW/VSVG, was incubated
with either the target cell line, 293T/CD20, or the control cell line, 293T, for one hour at
4°C, after which, the cell-virus complex was fixed with 4% formaldehyde and stained by
an anti-p24 antibody to detect the viral core and 4’,6-diamidino-2-phenylindole (DAPI)
for nucleus. As shown in Fig. 3.3A, confocal images revealed that the lentiviral vector

56

(FUW/SC2H7-A2/SGN) was able to bind to 293T/CD20 cell line, but not to the control
293T cell line. In contrast, the lentiviral vector FUW/VSVG was able to bind to both
293T and 293T/CD20 cell lines. In addition, a quantitative virus-cell binding assay was
conducted to evaluate SCAb-mediated binding to CD20-expressing cells. Lentiviral
vectors (FUGW/SC2H7-A2/FM, FUGW/SC2H7-GS/FM, and FUGW/AB/FM) were
incubated with either 293T or 293T/CD20 cells for one hour at 4°C to prevent
internalization of the viral particle. Cells were then stained for the presence of viral
particles on the cell surface using an anti-FM antibody and quantified using flow
cytometry. As shown in Fig. 3.3B, flow cytometry analysis showed that the vector of
either FUGW/SC2H7-A2/FM or FUGW/SC2H7-GS/FM was able to bind to 293T/CD20
cells. FACS analysis also showed that the virus bound to the 293T/CD20 cell surface
displayed the FMs (Fig. 3.3B), suggesting that both SCAb and FM were incorporated on
the same virion. Additionally, the control 293T cells showed no detectable FM,
confirming that the observed viral particle binding to the cells is indeed due to the SCAb-
antigen interaction. Similarly, a non-targeting lentiviral vector FUGW/AB/FM was
unable to bind to either 293T or 293T/CD20 cells.

57

Figure 3.3 Incorporation of both FM and antibody onto the vector surface. (A) 293T (top) and
293T/CD20 (bottom) cells were incubated with either FUGW/SC2H7-A2/SGN (left) or FUGW/VSVG
(right) at 4 °C for 1 hour, fixed and immunostained with anti-p24 antibody (green) and DAPI
nuclear staining (blue). Images were acquired with a laser scanning confocal microscope. Scale bar
represents 2 µm. (B) Co-expression of antibody and FM on the same viral surface. 293T (shaded
area) or 293T/CD20 (solid line) cells were incubated with FUGW/SC2H7-A2/FM, FUGW/SC2H7-
GS/FM or FUGW/AB/FM at 4 °C for 1 hour, followed by staining of FM by anti-HA antibody. The
binding of the virus to the cells was detected by FACS analysis.

3.2.4 Targeted transduction of lentiviral vectors
We conducted transduction experiments to evaluate the efficiency of lentiviral
vectors bearing both SCAb and FM to transduce the CD20-expressing cell line. The
lentiviral vector bearing VSVG was used as a positive control, whereas the lentiviral
vector co-displaying AB and FM was included as a negative control. Cell lines were
transduced by indicated lentiviral vectors and analyzed by FACS five days post-

58

transduction. The GFP expression level was detected to quantify the specificity and
efficiency.
Recombinant lentiviral vectors bearing both SCAb and FM were able to
specifically transduce the 293T/CD20 cell line with various efficiencies (15% ~30 %)
varying upon the choice of the FMs (Fig. 3.4A). In contrast, less than 5% of transduction
efficiency was observed for the 293T cell line. In addition, the titer of FUGW/SC2H7-
A2/SGN was estimated to be ~0.15 × 10
6
transduction units (TU)/mL on the 293T/CD20
cells (Fig. 3.4B); the titer was determined in the dilution ranges that showed a linear
response of GFP expression with viral serial dilution. In another control experiment,
when the lentiviral vector bearing an isotype antibody paired with a FM (FUGW/AB/FM)
were used, less than 5% of cells were transduced to express GFP. This finding further
highlighted the significance of antibody-directed transduction. No transduction was
observed with the lentiviral vector containing only SCAb, indicating the necessity of FM
to complete transduction. Thus, lentiviral vectors must display both SCAb and FM for
efficient transduction to target cells.
Among the various lentiviral vectors bearing the same SCAb but different FMs,
different transduction efficiencies were observed. The lentiviral vector displaying
SC2H7-A2 and SINmu exhibited 15% transduction efficiency. However, lentiviral
vectors displaying other FMs (SGN, SGM, and AGM) resulted in specific transductions
of 25% to 30%. A similar trend was observed in another independent study where the
SCAb with VSVG transmembrane domain was used as the targeting antibody (SC2H7-
GS) (Fig. 4A). In this case, the lentiviral vector bearing SINmu and SC2H7-GS was able
to specifically transduce about 14% of the 293T/CD20 cells, whereas the specific

59

transduction efficiency was increased to 25% when other FMs (SGN, SGM and AGM)
were used in combination with the SC2H7-GS.

Figure 3.4 Targeted transduction of lentiviral vectors to 293T/CD20 cells. (A) 293T/CD20 (black bar)
or 293T (grey bar) cells were transduced with 1.5 mL of fresh unconcentrated viral vectors
(FUGW/SC2H7-A2/FM, FUGW/SC2H7-GS/FM, FUGW/AB/FM, FUGW/SC2H7-A2, or
FUGW/SC2H-GS). FACS analysis was conducted to analyze the percentage of GFP-expressing cells
5 days post-transduction. (B) Transduction titers of fresh viral vectors (FUGW/SC2H7-A2/FM,
FUGW/SC2H7-GS/FM, or FUGW/VSVG) on 293T (grey bar) and 293T/CD20 (black bar) cells.

3.2.5 Assays for studying the entry mechanism
We hypothesized that our engineered lentiviral vector entered cells via receptor-
mediated endocytosis followed by the endosomal fusion leading to the release of the
vector core. To validate our hypothesis, we designed two independent experiments to
study these two critical steps. Since the combination of SGN and SC2H7-A2 showed the
greatest targeting efficiency in the transduction experiment, we chose this combination

60

for the study of the entry mechanism. 293T/CD20 cells were exposed to either
FUGW/SC2H7-A2/SGN or FUGW/VSVG in the presence of various amount of either
soluble αCD20 or isotype control antibody (Fig. 3.5B). As expected, the level of
transduction efficiency (FUGW/SC2H7-A2/SGN) dropped as the concentration of the
soluble αCD20 increased, whereas no noticeable reduction in transduction efficiency was
observed when the isotype control was used. In contrast, transduction efficiency of
FUGW/VSVG was not affected by soluble αCD20.
The second critical step of transduction pathway involved the pH-dependent
fusion event leading to the release of the viral core. To verify the pH requirement, we
incubated either FUGW/SC2H7-A2/SGN or FUGW/GP160 with 293T/CD20 or Ghost-
CCR5 cells in the increased presence of bafilomycin, which can raise the pH of the
endosomal compartment. We observed a dramatic decrease in transduction efficiency
(FUGW/SC2H7-A2/SGN) in response to increasing amount of bafilomycin (Fig. 3.5A).
In a control experiment where a pH-independent virus (FUGW/GP160) was used, an
increase in transduction efficiency was observed, which was consistent with previously
published data (Fredericksen et al., 2002; McClure et al., 1988). Thus, the pH in the
endosomal compartment is critical for viral membrane fusion.

61

Figure 3.5 Study of the entry mechanism of engineered lentiviral vector for transducing target cells.
(A) 293T/CD20 or Ghost-CCR5 cells were pre-incubated with various amount of bafilomycin for 30
minutes and spin-transduced with FUGW/SC2H7-A2/SGN or FUGW/GP160. Cells were incubated
for an additional 3 hours before replenishing with fresh media. Transduction efficiency was
measured by FACS analysis of GFP-positive cells 3 days post-transduction. All data was normalized
to transduction without bafilomycin treatment. (B) Effects of supplement of soluble αCD20 on
targeted transduction. 293T/CD20 cells were incubated with FUGW/SC2H7-A2/SGN or
FUGW/VSVG and various amounts of soluble αCD20 or isotype control for 12 hours, after which
the medium was replaced with fresh medium. Transduction efficiency was measured by FACS
analysis of GFP-positive cells 3 days post-transduction. All data was normalized to transduction
without soluble antibody treatment.

62

3.2.6 pH dependency study on the FMs
As shown from the targeted transduction experiment, lentiviral vectors enveloped
with various FMs resulted in different targeting efficiency (Fig. 3.4). We thus designed a
liposome-virus fusion experiment to characterize the fusion property of these FMs. As
shown in Fig. 3.6, roughly 40% to 50% of the lentiviral vector (FUGW/SC2H7-A2/FM)
fused at pH of 5.6. When the same experiment was performed at pH environment of 6.2,
only 12% of the lentiviral vector bearing SINmu fused, whereas a 40% to 50% fusion
activity was obtained for vectors bearing other FMs (SGN, SGM, and AGM). Correlating
the liposome-virus experiment with the targeted transduction experiment (Fig. 3.4), we
observe a clear trend showing that the higher fusion activity of the FMs results in a higher
transduction efficiency.

63

Figure 3.6 pH-dependent study of the fusion activity of various FMs. R18-labeled lentiviral vectors
(FUGW/SC2H7-A2/FM) were mixed with liposomes (200 µM) for 1 minute. Virus-liposome fusion
was triggered by adding the appropriate volume of acetic acid and measured by dequenching of
fluorescent R18 using a spectrofluorometer.

3.2.7 Binding avidity of lentiviral vectors to target cells
In order to understand the different transduction efficiency of lentiviral vectors
bearing these two different versions of SCAb (SC2H7-A2 and SC2H7-GS), we
conducted a binding avidity assay. Increasing amount of the lentiviral vectors
(FUGW/SC2H7-A2/SGN and FUGW/SC2H7-GS/SGN) were incubated with
293T/CD20 cells followed by the surface staining of the FM. The geometry mean
fluorescence (GMF) intensity was measured and scatchard analysis was performed to

64

determine the avidity of the lentiviral vector to bind to 293T/CD20 cells (Fig. 3.7A). In
agreement with our transduction experiment (Fig. 3.4), the SC2H7-A2-enveloped
lentiviral vector showed slightly better binding avidity to the target cells as compared to
that of the SC2H7-GS-enveloped lentiviral vector. We also noted that when SC2H7-A2
was used to envelope the lentiviral vector, the vector production was increased as
compared to that of SC2H7-GS (Fig. 3.7B). These findings explain the result of
transduction experiment where the lentiviral vector pseudotyped with the SC2H7-A2
antibody showed higher transduction efficiency as compared to that of SC2H7-GS-
bearing vector (Fig. 3.4).

Figure 3.7 Scatchard analysis of the lentiviral vector binding to 293T/CD20 cells. (A) 293T/CD20 cells
were incubated with various concentrations of lentiviral vectors (FUGW/SC2H7-A2/SGN or
FUGW/SC2H7-GS/SGN) and stained with anti-HA antibody. The apparent K
d
value (1/slope) was
derived from the scatchard plot of geometric mean fluorescence (GMF)/concentration versus GMF.
(B) p24 concentration of lentiviral vectors (FUGW/SC2H7-A2/SGN and FUGW/SC2H7-GS/SGN).

65

3.3 Discussion
The purpose of this study is to incorporate both membrane-bound SCAb and FM
on the lentiviral surface to achieve targeted transduction to specific cell types. Previously,
we reported a strategy of separating the binding and fusion functions of viral glycoprotein
for cell specific targeting (Yang et al., 2006a). By pairing the αCD20 with a more fusion
active FM, the resulted lentiviral vectors showed enhanced transduction (Lei et al., 2009).
In this study, we extended the targeting strategy to utilize a membrane-bound SCAb with
the engineered FMs. Insertion of SCAb into the viral glycoprotein has shown to be able
to redirect vector particles to specific cellular target (Jiang, 1999; Jiang et al., 1998;
Somia et al., 1995a). However, these modifications usually resulted in reduced
transduction efficiency. Our strategy of separating binding and fusion functions allows us
to engineer a targeting lentiviral vector system by optimizing these two parameters in
parallel without compromising their functions.
The lentiviral vectors bearing both SCAb and FM can specifically transduce
CD20-expressing cells. The specific transduction occurs through a two-step process. First
the virus must recognize and bind to CD20-expressing cells. Using flow cytometry and
confocal microscopy, we verified that the SCAb was able to mediate the binding of the
vector to the CD20 antigen on the cellular surface. Furthermore, the soluble αCD20
inhibition assay revealed that the targeting kinetics of the SCAb vector was inhibited in a
dose-dependent fashion, confirming the binding requirement for the observed targeting.
The second step for targeted transduction is the FM-mediated endosomal fusion to deliver
the viral payload into the cell. A high titer and efficient transduction demonstrated that

66

the FM was functional when combined with SCAb on the viral surface. Thus, the
targeting lentiviral vector succeeds in these two steps to achieve efficient transduction.
As suggested from our previous studies, two different approaches can be applied
to further optimize this two-molecule targeting strategy. By engineering the fusion loop
of the SINmu, transduction can be enhanced (Lei et al., 2009). Lentiviral vectors
incorporating SCAb and SINmu consistently yielded lower transduction efficiency as
compared to viral vectors with other FMs (SGN, SGM, or AGM). The difference in
transduction efficiency may have resulted from the endosomal fusion kinetics of the
different FMs. Recent studies of alphavirus glycoproteins have indicated that mutation in
the E1 fusion domain might favor an increase in endosomal fusion ability (Gibbons et al.,
2004; Kielian and Rey, 2006; Liu and Kielian, 2009). We suspected that our mutation in
the E1 domain might have a similar role in lowering the activation energy for the fusion
event. Our liposome-virus fusion assay revealed that SINmu was not fusion-active at pH
= 6.2, while other FMs were active at this pH. This direct correlation between the pH of
fusion and the transduction efficiency suggests that the FMs that are more active at a
higher pH can have better capacity to mediate lentiviral transduction. Consequently,
targeted transduction may be further improved by constructing a library of FMs and
screening for a FM with higher pH fusion activity.
Another approach to optimize this two-molecule targeting strategy is to engineer
the targeting antibody to be more efficiently incorporated onto the lentiviral vector
surface. Having the targeting molecule more efficiently incorporated onto the vector
surface could enhance the binding of the vector to the cognate receptor on the target cell
surface, thereby increasing transduction efficiency. To enhance the display of SCAb onto

67

the viral surface, we constructed two SCAbs, each fused to a different transmembrane
domain: the HLA-A2 transmembrane domain or the VSVG transmembrane domain. The
targeted transduction efficiency was consistently higher with the SC2H7-A2-bearing
vector. The binding avidity from the scatchard analysis revealed that the FUGW/SC2H7-
A2/SGN vector exhibited a slightly higher binding avidity as compared to
FUGW/SC2H7-GS/SGN. The higher avidity of the SC2H7-A2-bearing vector may be
due to more efficient incorporation of SC2H7-A2 onto the lentiviral vector surface. It has
been proposed that lipid rafts can serve as assembly sites for the pseudotyped lentiviral
vectors (Leung et al., 2008). Recent studies have demonstrated a correlation between
transmembrane domain and raft association with efficient viral incorporation (Jorgenson
et al., 2009). Although these data indicate a role of the transmembrane interaction to
facilitate more efficient incorporation onto the virus, further understanding is needed to
identify the precise mechanism of the transmembrane to facilitate incorporation of both
the SCAb and FMs.

3.4 Material and Methods
3.4.1 Construct preparation
To generate the SCAb against the CD20 antigen, we first PCR-amplified the light
chain variable region from an αCD20 hybridoma cell line (ATCC, Manassas, VA, HB-
9803) with primers CD20Lvfw (5’-CTG ACC CAG ACC TGG GCG CAA ATT GTT
CTC TCC CAG TCT CCA GCA ATC CTG TC-3’) and CD20LvGSbw (5’-CAC CTC
CTG AAC CAC CGC CGC TAC CGC CTC CGC CTT TCA GCT CCA GCT TGG
TCC CAG CAC C-3’). The HLA-A2 leading peptide sequence was then added to the 5’-

68

end of the light chain variable region with primers HLA-A2 (5’-GAA CAA TTT GCG
CCC AGG TCT GGG TCA GGG CCA GAG CCC CCG AGA GTA GCA GGA CGA
GGG TTC-3’) and HLA-A2fw (5’-CTT AAG CTT ATG GCC GTC ATG GCG CCC
CGA ACC CTC GTC CTG CTA CTC TCG GGG G-3’). We also PCR-amplified the
heavy chain variable region with primers CD20hvGSfw (5’-GGT AGC GGC GGT GGT
TCA GGA GGT GGC GGC AGT GGT GGA GGA TCT CAG GCT TAT CTA CAG
CAG TCT GGG GCT GAG CTG -3’) and CD20hvbw (5’-GTT TTG TCA CAA GAT
TTG GGC TCA ACT GAA GAG ACG GTG ACC GTG GTC CCT GTG -3’). The PCR
product was assembled with the light chain variable region using the primers HLA-A2fw
and CD20hvbw. To fuse the hinge-CH2-CH3 domain to the HLA-A2 transmembrane
domain, we PCR-amplified the hinge-CH2-CH3 domain and the HLA-A2
transmembrane domain using the primer pairs (CH2-CH3-Hingefw, 5’-GTC TCT TCA
GTT GAG CCC AAA TCT TGT GAC AAA ACT CAC ACA TGC CCA CCG TGC
CCA GCA CCT GAA CTC CTG GGG GGA CCG TC -3’; CH2-CH3bw, 5’-CTG GGA
AGA CGG GGC CCC CTG TCC GAT CAT GTT CCT G-3’) and (HLA-A2Tfw, 5’-
GAC AGG GGG CCC CGT CTT CCC AGC CCA CCA TCC CC-3’; HLA-A2Tbw, 5’-
CGA GCG GCC GCT CAC ACT TTA CAA GCT GTG AGA GAC ACA TCA GAG
CCC-3’). The resulting two PCR fragments were assembled using primers CH2-CH3-
Hingefw and HLA-A2Tbw. We then assembled the variable fragments with the CH2-
CH3/transmembrane domain using the primers HLA-A2fw and HLA-A2Tbw. The
assembled DNA was finally cloned into pcDNA3 (Invitrogen) via Hind3 and Not1
restriction sites. To construct a single chain antibody with the VSVG transmembrane
domain, a forward primer (SC2H7fw, 5’-CCC CCA TCC CGG GAT GAG CTG ACC-

69

3’) and a backward primer (SC2H7bw, 5’-AGT ATC ACC GGC CCC CTG TCC GAT
CAT GTT CCT GTA GTC-3’) were used to amplify a portion of the CH2-CH3 domain
of SC2H7-A2. In parallel, a forward primer (GSfw, 5’-ATG ATC GGA CAG GGG GCC
GGT GAT ACT GGG CTA TCC AAA AAT CCA ATC GAG CTT-3’) and a backward
primer (GSbw, 5’-GAT CGA GCG GCC GCT TAC TTT CCA AGT CGG TTC ATC
TCT ATG TCT GTA TAA ATC TGT CTT TTC-3’) were used to amplify the
transmembrane domain of VSVG. The DNA products from these two reactions were
PCR-assembled using SC2H7fw and GSbw as the primer pair and the resulting product
was cloned into pSC2H7-A2 to yield SC2H7-GS. The integrity of these constructs was
confirmed by DNA sequencing.
3.4.2 Viral vector production
293T cells were seeded in a 6-cm culture dish in DMEM medium supplemented
with fetal bovine serum (Sigma, St. Louis, MO, 10 %), L-glutamine (10 mL/L),
penicillin, and streptomycin (100 units/mL) the night prior to transfection. 293T cells
were transfected at a confluence of 80~90 % with 5 µg of lentiviral backbone vector
(FUGW), 2.5 µg each of pMDLg/pRRE, pRSV-Rev, pFM, and a plasmid encoding an
antibody (pSC2H7-A2, pSC2H7-GS or pAB) via the standard calcium phosphate
precipitation technique (Klages et al., 2000b). Cells were replenished with pre-warmed
media 4 hours post-transfection. Vectors were harvested two days post-transfection and
filtered through a 0.45-µm pore size filter (Nalgene, Rochester, NY). Lentiviral vectors
were then further concentrated by ultracentrifugation (Optimal L-90K Ultracentrifuge,
Beckman Coulter, Fullerton, CA) at 4 °C, 25,000 rpm for 90 minutes and resuspended in
appropriate volume of cold PBS.

70

3.4.3 Virus-cell binding assay
293T/CD20 or 293T cells were incubated with 2 mL of lentiviral vectors
(FUGW/SC2H7-A2/FM, FUGW/SC2H7-GS/FM or FUGW/AB/FM) at 4 °C for 1 hour.
After extensive washing with cold PBS, cell-virus complexes were stained with anti-HA
tag antibody (Miltenyi Biotec, Inc.) and analyzed by flow cytometry (FACSort, BD
Bioscience).
3.4.4 Confocal imaging
Fluorescent images were acquired on a Zeiss LSM 510 META laser scanning
confocal microscope equipped with Argon, red HeNe, and green HeNe lasers as well as a
Coherent Chameleon Ti-Sapphire laser for multiphoton imaging. Images were acquired
using a Plan-apochromat 63x/1.4 oil immersion objective. To image virus-cell binding,
cells were seeded into a 35-mm glass-bottom culture dish and grown at 37 ºC overnight.
The seeded cells were rinsed with cold PBS and incubated with concentrated viral
particles for 1 hour at 4 ºC to allow for binding. The cells were washed with cold PBS to
remove unbound particles, fixed with 4% formaldehyde on ice for 10 minutes, and then
immunostained with monoclonal antibody specific for HIV capsid protein p24 and 4’,6-
diamidino-2-phenylindole (DAPI) antibody for nuclear staining. Monoclonal antibody
against HIV-1 p24 (AG3.0) was obtained from the NIH AIDS Research and Reference
Reagent Program (Division of AIDS, NIAID, NIH). Images were analyzed using the
Zeiss LSM 510 software version 3.2 SP2.
3.4.5 Antibody Competition Assay
293T/CD20 cells were incubated with the lentiviral vector (FGUW/SC2H7-
A2/SGN or FUGW/VSVG) and various amount of either the soluble αCD20 (BD

71

Bioscience) or the isotype control antibody overnight. Cells were then replenished with
fresh media and incubated for additional 72 hours before flow cytometry analysis.
3.4.6 Neutralization Assay
293T/CD20 or Ghost-CCR5 (NIH AIDS Research and Reference Reagent
Program) cells were pre-incubated with various amount of bafilomycin for 30 minutes,
after which, the lentiviral vector (FUGW/SC2H7-A2/SGN or FUGW/GP160) was added.
The vector and cell mixture was spun at 25 °C, 2,500 rpm for 90 minutes using a RT
legend centrifuge (Sorval). Cells were then incubated at 37 °C and 5% CO
2
and
replenished with fresh media 3 hours later.

Flow cytometry was then used to analyze the
treated cells 3 days post-transduction.
3.4.7 Targeted transduction of 293T/CD20 cells
293T or 293T/CD20 cells were seeded on a 24-well cell culture plate and spin-
transduced with 1.5 mL of indicated lentiviral vectors (FUGW/SC2H7-A2/FM,
FUGW/SC2H7-GS/FM, FUGW/AB/FM, FUGW/SC2H7-A2, or FUGW/SC2H7-GS) at
25 °C, 2,500 rpm for 90 minutes using a RT legend centrifuge. After replacing with fresh
media, the treated cells were cultured for additional 5 days at 37 °C and 5 % CO
2
. Flow
cytometry was then used to analyze transduction efficiency. The titer was determined by
measuring GFP-positive cells in the dilution range that resulted in a linear relationship
between the percentage of GFP-expressing cells and the amount of vectors added.
3.4.8 Scatchard analysis
293T/CD20 cells were incubated with various amount of lentiviral vectors
(FUGW/SC2H7-A2/SGN or FUGW/SC2H7-GS/SGN). Flow cytometry analysis was
carried out to measure the geometric mean fluorescence (GMF) of the bound viruses

72

stained by anti-HA antibody. The concentration of the lentiviral vectors was measured by
a p24 antigen capture enzyme immunosorbent assay (ELISA) kit (ImmunoDiagnostics,
Woburn, MA). Apparent K
d
value was derived from the negative reciprocal of the slope
of the linear fit to scatchard plots, which is the geometric mean
fluorescence/concentration of lentiviral vector (GMF/concentration) against geometric
mean fluorescence (GMF).
3.4.9 Virus-liposome fusion assay
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL,
USA). Cholesterol (Chol) and sphingomyelin (SPM) from egg yolk were obtained from
Sigma (St Louis, MO, USA). Liposomes were prepared by the extrusion procedure (Smit
et al., 1999). Briefly, lipid mixtures (PC/PE/SPM/Chol molar ratio of 1:1:1:2) were dried
from a chloroform solution under a stream of argon gas and further dried under vacuum
for at least 3 hours. The lipid mixtures were hydrated in HNE buffer (5mM HEPES,
150mM NaCl, and 0.1 mM EDTA, pH 7.4). Subsequently, the lipid mixtures were
extruded 20 times through 0.2 µm pore size polycarbonate filters (Avanti polar lipids). To
monitor virus-liposome fusion, the concentrated viruses were incubated with 70 µM of
octadecyl rhodamine B chloride (R18) (Molecular Probes, Carlsbad, CA, USA) in serum-
free medium for 1 hour at room temperature. R18-labeled viruses were then mixed with
liposomes (200 µM phospholipids) in a final volume of 0.4 mL. Fusion was triggered by
adding the appropriate volume of 0.2 M acetic acid, pretitrated to achieve the desired pH.
The dequenching signal of R18 fluorescence was measured 60 seconds after acidification
with QuantaMaster QM-4SE spectrofluorometer (Photon Technology International,

73

Lawrenceville, NJ, USA). The initial fluorescence of virus-liposome mixtures was set at
0 % fusion, and the 100 % fusion value was obtained by detergent lysis for each
experiment using 0.1 % of Triton X-100 (Wunderli-Allenspach and Ott, 1990).

74

Chapter 4
Gene Editing of Human Embryonic Stem Cells via an Engineered
Baculoviral Vector Carrying Zinc Finger Nucleases

Portions of this Chapter are adapted from: Yuning Lei, Chi-Lin Lee, Kye-Il Joo,
Jonathan Zarzar, Yarong Liu, Bingbing Dai, Victoria Fox,

and Pin Wang. Molecular
Therapy. (Accepted, 2011)

Human embryonic stem (hES) cells are a renewable cell source that has potential
applications in regenerative medicine. The development of permanent and site-specific
genome modifications is in demand to achieve future medical implementation of hES
cells. We report herein that a baculoviral vector (BV) system carrying zinc finger
nucleases (ZFNs) can successfully modify the hES cell genome. BV-mediated transient
expression of ZFNs specifically disrupted the CCR5 locus in 30% of transduced cells and
the modified cells exhibited resistance to HIV-1 transduction. To convert the BV to a
gene targeting vector, a DNA donor template and ZFNs were incorporated into the
vector. These hybrid vectors yielded permanent site-specific gene addition in both
immortalized human cell lines (10%) and hES cells (5%). Modified hES cells were both
karyotypically normal and pluripotent. These results suggest that baculoviral delivering
system can be engineered for site-specific genetic manipulation in hES cells.

75

4.1 Introduction
Permanent transgene expression in human embryonic stem (hES) cells could
potentially be beneficial for basic biological studies and regenerative medicine. One
possible application involves the expression of intracellular factors to provide additional
stimulus to control the differentiation of hES cells. Numerous studies on gene transfer to
hES cells have been reported.(Kobayashi et al., 2005; Yates and Daley, 2006) Currently,
the most efficient methodology to genetically engineer hES cells involves utilizing a viral
vector to introduce transgenes into the host genome. However, integrating vectors such as
retroviral vectors pose the risk of insertional mutagenesis and oncogene
activation.(Hacein-Bey-Abina et al., 2003) The development of a targeting vector that is
capable of integrating into a pre-determined genome sites can be a safer and more
desirable approach.
The insect baculovirus Autographa californica multiple nucleopolyhedrovirus
(AcMNPV) has emerged as a promising gene delivery vector in recent years. This DNA
virus is capable of entering mammalian cells and expressing transgenes under the control
of mammalian promoters.(Boyce and Bucher, 1996; Hofmann et al., 1995; Shoji et al.,
1997) Transduction by baculovirus does not cause observable cytotoxicity at high
multiplicity of infection (MOI), nor does it replicate inside mammalian cells, thereby
reducing the safety risk.(Hofmann et al., 1995; Kost and Condreay, 2002; Sandig et al.,
1996; Shoji et al., 1997) One significant advantage of this double-stranded DNA virus as
a vector is the large AcMNPV genome (130 kb), which has been shown to accommodate
transgenes of up to 38 kb.(Cheshenko et al., 2001b) Recently, baculoviral vectors (BVs)

76

have been shown to be able to transduce human mesenchymal stem cells and hES
cells.(Ho et al., 2005; Zeng et al., 2007) These data revealed that BV is a promising and
safe alternative gene therapy vehicle as compared to other pathogenic viral vectors.
Zinc finger nucleases (ZFNs) have been shown to enhance the gene correction
frequency.(Bibikova et al., 2001; Hockemeyer et al., 2009; Lombardo et al., 2007;
Porteus and Carroll, 2005; Porteus and Baltimore, 2003; Urnov et al., 2005; Zou et al.,
2009) ZFNs are engineered DNA-specific zinc finger binding proteins fused to a
nonspecific DNA endonuclease domain (FokI).(Porteus and Carroll, 2005; Porteus and
Baltimore, 2003; Urnov et al., 2005) Each zinc finger domain recognizes 3 base pairs of
DNA and a four finger ZFN can then recognize a 12 base pairs DNA sequence. The
endonuclease domain of FokI must dimerize to cleave the double strand DNA sequence,
thus ZFNs are designed in pairs that bind to the target sequence in the opposite
orientation with the correct spacer. Double strand break mediated by ZFNs is then
repaired by either nonhomologous end joining (NHEJ), an error-prone repairing process
or homologous recombination (HR), an event that copies the homologous sequence from
an adjacent DNA sequence.(O’Driscoll and Jeggo, 2006) ZFN-induced genetic
modification has been applied to a variety of cell lines and several different
species.(Beumera et al., 2008; Carroll, 2008; Doyon et al., 2008; Meng et al., 2008b;
Shukla et al., 2009; Townsend et al., 2009) Most notably, several groups have utilized
ZFNs to enhance gene targeting efficiency in hES cells.(Hockemeyer et al., 2009;
Lombardo et al., 2007; Zou et al., 2009) A recent report utilizing integrase-defective
lentiviral vectors (IDLVs) delivering ZFNs and a DNA donor template showed high gene
targeting event in the hES cells.(Lombardo et al., 2007) Although the use of IDLVs

77

greatly reduced the risk associated with the integrating event, some residual integration
events remained detected.(Mali et al., 2008; Nightingale et al., 2006) A follow up study
addressed this risk by utilizing a virus-free system to perform ZFNs mediated gene
targeting in hES cells.(Zou et al., 2009) However, the gene editing efficiency was 20-fold
lower than that of the IDLV system. To combine the strength of both systems, we
engineered a BV system to co-deliver both ZFNs and the DNA donor template to induce
ZFN-mediated gene targeting. We reported herein that gene editing efficiency was
comparable to the IDLVs system and no random integration event was observed.

4.2 Results
4.2.1 Construction of site-specific vectors
Site-specific integration to the desirable site has been a long-standing goal in the
field of gene therapy. The CCR5 gene was chosen in this study as a site-specific target to
introduce a foreign gene because the homozygous null mutation is prevalent in a small
population of individuals(SAMSON et al., 1996) and disruption of this gene is well
tolerated.(Perez et al., 2008) The C
2
H
2
ZFN protein was generated by fusing the CCR5-
specific zinc finger proteins to engineered obligate heterodimers of the endonuclease
domain of the FokI enzyme, which could minimize the non-specific cleavage.(Miller et
al., 2007; Szczepek et al., 2007) The Bac-ZFN construct consists of both the right and left
ZFNs linked by a F2A sequence driven by the cytomegalovirus (CMV) internal promoter.
ZFNs (ZFN-R: AAA CTG CAA AAG; ZFN-L: GAT GAG GAT GAC) (Figure 4. 2a)

78

can induce a double-strand break at the CCR5 locus, and then by delivering a suitable
DNA donor template, the occurrence of a HR event can introduce the donor sequence
into the CCR5 locus. The DNA donor template used in this study contains a GFP
expression cassette driven by the human elongation factor-1α (EF1α) promoter flanked
by the CCR5 homology arms to initiate HR. The EF1α promoter has been shown to
efficiently drive the expression of the GFP reporter gene in hES cells.(Zeng et al., 2007)
Utilizing the large transgene capacity of BV, we generated a Bac-ZFN-Donor construct
by inserting the ZFN cassette directly into the Bac-Donor construct to facilitate both
double-strand break and transgene integration. We therefore constructed these three
different versions of BVs to deliver either ZFNs (Bac-ZFN), DNA donor template (Bac-
Donor) or ZFNs and DNA donor template together (Bac-ZFN-Donor) (Figure 4.1).
Previously, IDLVs have been shown to successfully achieve gene modification in
hES cells.(Lombardo et al., 2007) The gene editing process was achieved by co-delivery
of both ZFNs and a DNA donor template to the target cells. To co-deliver both ZFNs, we
constructed a lentiviral vector (FUW-ZFN) encoding a F2A-linked two ZFNs driven by
the human ubiquitin-C promoter;(Lois et al., 2002a) the woodchuck hepatitis virus
posttranscriptional regulatory element (WRE) was included downstream of the ZFNs to
increase the level of transcription (Figure 4. 1).

79

Figure 4. 1. Schematic representation of key constructs in this study. These constructs include three
different versions of baculoviral vectors (BVs) carrying either ZFNs, DNA donor template, or ZFNs
and DNA donor template, and a lentiviral vector encoding ZFNs. CMV: human cytomegalovirus
immediate-early gene promoter; ZFN-L: Zinc finger nuclease targeting the left flank of CCR5 gene;
ZFNR: Zinc finger nuclease targeting the right flank of CCR5 gene; F2A: a 2A sequence derived
from the foot and mouth disease virus; GFP: enhanced green fluorescence protein; Homologous
region: homologous DNA sequence to the exon 3 of CCR5 gene; EF1α: human elongation factor-1α
promoter; LTR: Long terminal repeats; Ubi: the human ubiquitin-C promoter; WRE: woodchuck
responsive element; pA: polyadenylation signal.

4.2.2 ZFN-mediated target disruption by the baculoviral vector
To determine whether CCR5 ZFNs could be efficiently delivered by a BV, we
transduced Ghost-CCR5 cell line, a human cell line that highly expresses autologous
CCR5 genes, with the Bac-ZFN vector. The transduced cells were maintained in the
culture for 5 days. Flow cytometry analysis on transduced cells showed that over 30% of
the cells were CCR5-negative, indicating the success of targeted gene disruption (Figure
4. 2b, left). Further analysis of CCR5 of transduced cells revealed a decrease in mean
fluorescence intensity (MFI), confirming the loss of CCR5 expression (Figure 4. 2b,
right). In addition, we transduced both wild-type and ZFN-treated Ghost-CCR5 cells with
a lentiviral vector enveloped with a CCR5-tropsim HIV-1 glycoprotein (FUGW/GP160),

80

or a control vector FUGW/VSVG, to validate the disruption of the CCR5 gene.
Consistent with previous data, a decrease in transduction efficiency was observed on the
ZFN-treated cells exposed to FUGW/GP160 (Figure 4. 2c). In the control experiment
where ZFN-treated cells were transduced by FUGW/VSVG, transduction efficiency was
unaltered, indicating that the decreased transduction was CCR5-dependent. To confirm
and quantify the ZFN-induced cleavage at the target site, we performed a mismatch-
sensitive Surveyor nuclease assay. The DNA analysis of this assay revealed a 32%
targeted gene disruption in the ZFN-treated population, as compared to less than 1% gene
disruption in the untreated Ghost-CCR5 cell line (Figure 4. 2d). These results
demonstrate that ZFNs can be efficiently delivered by BV to induce double-strand break
at the CCR5 target site.

81

Figure 4. 2. ZFN-mediated disruption of CCR5 by baculoviral vector. (a) Schematic representation of
the strategy for targeted gene disruption by Bac-ZFN. The CCR5 locus is shown and exons are
indicated by arrows. The enlarged views depict the binding sites for CCR5 ZFN pairs. DSB: double
strand break; NHEJ: nonhomologous end joining. (b) Ghost-CCR5 cells were transduced by the Bac-
ZFN vector and the decreased level of CCR5 expression was measured by flow cytometry at day 5
post-treatement. The untreated cells were included as a control. Left: histogram plot of CCR5
expression on treated (open line) and untreated (shaded area) cells. Right: mean fluorescence
intensity of Bac-ZFN-treated and untreated cells. (c) Entry efficiency of lentiviral vectors enveloped
with a CCR5-tropsim HIV-1 glycoprotein (FUGW/GP160) towards Bac-ZFN-treated Ghost-CCR5
cells. Untreated cells and lentiviral vectors enveloped with a CCR5-independent vesicular stomatitis
virus glycoprotein (FUGW/VSVG) were included as controls. GFP expression as an indication of
successful entry was measured by flow cytometry and relative transduction efficiency was shown. (d)
The level of targeted gene disruption in Bac-ZFN-treated Ghost-CCR5 cells was assessed by the
Surveyor assay. The untreated control cells are included as a control. The lower migrating bands
indicate the ZFN-mediated gene disruption. The percentage of disruption was quantified by a
phosphorimager.

82

4.2.3 Production of the ZFN-lentiviral vector
Previously, Lombardo et al. reproted the utilization of IDLVs to deliver ZFNs to
disrupt the CCR5 gene.(Lombardo et al., 2007) However, their approach requires the
target cells to be co-transduced by two vectors for a gene disruption. We hypothesized
that incorporating both ZFNs linked by a 2A sequence in a single IDLV vector could
improve the efficiency of the gene targeting. To determine whether FUW-ZFN could
mediate the enhanced gene disruption, we generated a ZFN-containing vector FUW-
ZFN/VSVG and a ZFN-lacking vector FUW/VSVG. However, the lentiviral vector
containing both ZFNs was not efficiently produced. A p24 assay revealed that the
production level of FUW-ZFN/VSVG was less than half of FUW/VSVG (Figure 4. 3a).
Immunostaining to detect the protein 53BP1, which tends to localize to the DNA damage
sites and form foci, revealed that the FUW-ZFN-transfected 293T cells yielded a higher
amount of 53BP1-stained foci than that of the FUW-transfected cells, suggesting that the
decreased production of FUW-ZFN/VSVG was due to the ZFN-mediated DNA cleavage
(Figure 4. 3b). We further performed immunostaining on mammalian 293T and insect
SF9 cells transduced with Bac-ZFN and found that the 53BP1-stained foci was only
observed in 293T cells. We suspect that the toxicity is due to the presence of ZFNs that
were only effectively translated by the mammalian-active CMV promoter in 293T cell
and the presence of the target sequence (CCR5) in the 293T genome (Figure 4. 3c). These
sets of results indicated that the ZFN-mediated toxicity associated with the lentiviral
vector producing cells (293T) reduced the overall virus production, whereas no such
toxicity was detected in the BV producing cells (SF9).

83

Figure 4. 3. Analysis of ZFN-associated toxicity for producer cells to make lentiviral or baculaviral
vectors. (a) Productivity of mammalian 293T producer cells for making ZFN-containing (FUW-ZFN)
or ZFN-negative (FUW) lentiviral particles. p24 is a key component of the lentiviral capsid and its
concentration is a direct indication of the quantity of vector particles. Concentration of p24 was
measured by ELISA. (b) Representative images of transfected 293T producer cells to make lentiviral
vectors (FUW-ZFN or FUW). These cells were intranuclearly stained with antibodies to 53BP1 (red).
Scale bar represents 50 µm. (c) Representative images of vector producer cells (mammalian 293T or
insect SF9) treated with the DNA-damaging agent etoposide (1µm, as a positive control) or
transfected with Bac-ZFN for 5 days before staining with antibodies to 53BP1 (red). Scale bar
represents 50 µm

84

4.2.4 ZFN-mediated targeted gene addition in cell lines
The nature of transient gene expression delivered by conventional BVs has
limited the usage of this vector system in gene therapy. To extend the duration of BV-
mediated gene expression, we constructed three BVs, Bac-ZFN, Bac-Donor and Bac-
Donor-ZFN, and tested their ability to achieve targeted integration to the CCR5 gene
(Figure 4. 4a). We co-transduced both 293T and U87 cell lines with Bac-ZFN and Bac-
Donor and observed that the GFP reporter gene could be stably expressed at one month
post-transduction, whereas no detectable expression was seen when Bac-Donor was used
alone (Figure 4. 4b). We then co-transduced the cell lines with Bac-ZFN and Bac-Donor
at various MOI combinations to seek for an optimal protocol for achieving higher
efficiency of targeted gene integration. The flow cytometry analysis performed at one
month post-transduction revealed that GFP expression culminated at the MOI of 150 for
Bac-ZFN and the MOI of 500 for Bac-Donor, as evidenced by the high percentage (8-
10%) of the GFP expression (Figure 4. 4c). Transduction with Bac-Donor alone resulted
in less than 1% of GFP-positive cells, indicating the necessity for ZFN-mediated double-
strand break for efficient gene insertion. Similarly, when 293T and U87 cell lines were
transduced by Bac-Donor-ZFN, the baculoviral construct that delivered ZFNs and donor
DNA to the cells, up to 6% of targeted gene integration was observed (Figure 4. 4d). In
all cell types tested, targeted integration into the CCR5 gene was confirmed by PCR
analysis on GFP
+
cells sorted by fluorescence-activated cell sorting (FACS) at both
integration junctions (Figure 4. 4e). To further characterize double strand break-mediated
HR, we performed limiting dilution to isolate single clones. We employed a TaqMan

85

quantitative PCR assay to determine the genome copies of the integrated GFP expressing
cassette. The assay revealed that ~11.1% of the GFP positive cells were homozygous for
the alteration (Figure 4. 4f). This series of experiments confirmed that BVs can mediate
specific gene addition to a predetermined target site at high efficiency.

86

Figure 4. 4. Targeted gene editing at the CCR5 gene locus in 293T and U87 cell lines. (a) Schematic
diagram of the strategy to use baculoviral vectors (Bac-ZFN and Bac-Donor) for achieving the
targeted gene addition to the CCR5 gene locus. The gray arrows indicate the exons in the CCR5
locus. Bac-ZFN and Bac-Donor deliver the CCR5 ZFNs and the donor DNA encoding homologous
arms and a GFP expression cassette, respectively. Expression of a pair of CCR5 ZFNs in human cells
induce double strand break (DSB) at the ZFNs binding site. If the homologous recombination (HR)-
mediated repair occurs, the GFP expression cassette will be added into the CCR5 locus through
targeted integration. EF1α: human elongation factor-1α promoter; pA: polyadenylation signal. (b)
293T or U87 cells were transduced with either Bac-Donor alone or Bac-Donor and Bac-ZFN. Phase
contrast and fluorescence images were acquired by an epifluorescence microscopy and representative
images were shown. The scale bar represents 50 µm. (c) 293T or U87 cells were treated with the
indicated MOI (multiplicity of infections) doses of baculoviral vectors (Bac-Donor and Bac-ZFN) and
their GFP expression was analyzed by flow cytometry at 1 month post-transduction. (d) 293T or U87
cells were treated with the Bac-Donor-ZFN vector at the MOI of 150 or 500 and their GFP
expression was analyzed by flow cytometry at 1 month post-transduction. (e) Top: schematic
diagram of targeted integration into the CCR5 locus. The black arrows indicate the primers used in
the analysis. Bottom: the treated cells were sorted on GFP expression and analyzed by PCR for
targeted integration (TI) using two sets of primers specific for the 5’ or 3’ integration junctions. A
pair of primers to amplify a non-targeted locus (NTL) region was included as a control. (f) Copies of
GFP per cellular genome were detected by a quantitative TaqMan PCR protocol. The copy number
was determined by using a primer-probe set that specifically amplifies the GFP reporter gene, which
is normalized to another probe set that allows for quantitation of genomes using the β-actin gene.

87

4.2.5 ZFN-mediated targeted gene addition in human embryonic stem cells
Stable gene expression is an important tool for future implementation of hES cells
for regenerative medicine. To explore the feasibility of utilizing ZFNs for targeted gene
addition, we dissociated hES cells (H9) into single cells and incubated them with BVs
(Bac-ZFN and Bac-Donor) at a MOI of 500 for 2 hours. We found that up to 4.38% of
hES cells were modified at two weeks post-transduction, whereas only background level
GFP was seen in hES cells modified with the Bac-Donor vector alone (Figure 4. 5a). To
confirm the targeted integration event into the desired CCR5 gene, we performed PCR
amplifications at the junctions of the integration sites and found that the GFP reporter
cassette had undergone the predicted HR event at the CCR5 locus (Figure 4. 5b).
Transduction by BVs caused no microscopically observed cytotoxic effects or
morphological changes on hES cells as compared to non-transduced control hES cells
and retained a normal karyotype (Figure 4. 5c). Immunostaining on the transduced cells
was performed to confirm the undifferentiated state of these modified hES cells.
Transduced hES cells stably expressed the GFP reporter gene and remained
undifferentiated as indicated by the expression of the hES markers OCT4, NANOG,
GCTM2 and SSEA4 (Figure 4. 5d). To confirm the pluripotency of the modified hES
cells, transduced hES cells were sorted based on the GFP expression and these sorted hES
cells were employed to form embryoid bodies (EBs). RT-PCR analysis showed that the
EBs were able to form all three germ layers as the expression of germ layer specific
markers were detected in EBs but absent in undifferentiated hES cells, indicating the
pluripotency of the modified hES cells (Figure 4. 5e). Collectively, these data

88

demonstrate that ZFN-based BV approach allows for stable integration of transgene in
hES cells at high efficiency without apparent detriment to the self-renewal and
pluripotent state of the modified cells.

Figure 4. 5. Targeted gene editing at the CCR5 gene locus in human embryonic stem cells. (a) H9
human embryonic stem (hES) cells were dissociated into single cells and treated with either Bac-ZFN
and Bac-Donor or Bac-Donor alone at the MOI of 500. GFP expression was detected by flow
cytometry at week 3 post-transduction. (b) The treated cells were analyzed by PCR to confirm the
targeted integration (TI) of the GFP cassette into the CCR5 gene using primers specific for the 5’ and
3’ integration junction. A pair of primers to amplify a non-targeted locus (NTL) region was included
as a control. (c) Karyotyping analysis on BV-transduced hES cells after 20 passages demonstrates a
normal karyotype (46, XX). (d) Representative confocal microscopy images of H9 one month post-
transduction for GFP (green), the embryonic stem cell markers, OCT4, NANOG, GCTM2, and
SSEA4 (red), and nuclear DNA (DAPI, blue), showing the undifferentiated stem cells. (e) Left: BV-
transduced hES cells were sorted based on GFP expression. Right: expression of selected markers of
embryonic bodies (EBs) (MAP2, NEUROD1, AFP, DCN, HAND1, IGF2) was examined by RT-PCR.

89

4.3 Discussion
Baculovirus has been shown as a promising transient gene delivery vehicle in
many mammalian cell types.(Kost et al., 2005) The high-level transduction, low
cytotoxicity, lack of replication in mammalian cells and large transgene capacity make
baculovirus a useful tool for delivering genes of interest. However, one major limitation
of the broad applications of the BV system is the lack of permanent, site-specific
transgene expression. To address this challenge, others have generated a
baculovirus/adeno-assoicated virus (AAV) hybrid vector system containing a gene
cassette flanked by the inverted terminal repeats of the AAV to prolong the expression of
the transgene.(Palombo et al., 1998; Zeng et al., 2007) The limitation of this strategy is
that the location of the integration site is restricted to the AAV-preferred integration site
and cannot be used to target specific genes. Permanent modification of the genome via
site-specific integration has been a long-lasting goal in the field. It has significant
advantage over other viral integration approaches. Traditionally, the HR pathway has
been exploited to edit a specific locus. However, this approach is both labor intensive and
time consuming as the efficiency of site-specific HR is very low. To overcome the low
HR efficiency, ZFNs have been used along with a donor template to enhance the gene
targeting efficiency. The double strand break induced by ZFNs triggers two downstream
repair mechanisms. One involves the NHEJ of the recessed strands at the double strand
break and results in localized deletion or insertion of nucleotides that leads to gene
disruption. Another repair mechanism yields targeted gene addition via HR of the
damaged DNA with either a sister chromatin or a homologous DNA donor template. By

90

incorporating ZFNs and a DNA donor template, we investigated the utility of BVs for
achieving site-specific integration of a DNA target sequence.
We demonstrated that the permanent transgene expression can be achieved in hES
cells by delivering ZFNs and donor DNA templates with a baculoviral vector system. We
showed that by transducing CCR5-expressing cells with our engineered BV carrying
ZFNs, we could obtain the gene disruption with the efficiency up to 30% (Figure 4. 2).
We further demonstrated that targeted gene integration can be achieved by transducing
cell lines with BV encoding ZFNs and a donor template (Figure 4. 4). To further expand
the baculoviral delivering system to primary cell lines of therapeutic value, we transduced
the hES cells and showed that the modification efficiency is comparable to IDLV-
delivered ZFNs (5%) and approximately 20-fold higher than virus-free system
(0.25%).(Hockemeyer et al., 2009; Lombardo et al., 2007) The BV-transduced hES cells
displayed neither karyotypic abnormalities nor any alteration to their pluripotent property.
This study also highlighted the advantages of using BV as a gene delivery vehicle.
First, the large cloning capacity of the vector allows us to generate a single construct to
deliver both ZFNs and the donor template. The genome modification efficiency is
comparable to that of the two-construct version (~7%) (Figure 4. 4). Second, since
mammalian promoters, such as the CMV promoter in this study has been shown to
ineffectively drive the expression of transgene in the insect cells, the expression of ZFNs
in these cells is highly restrained, resulting in higher viability of the producing
cells.(Cheng et al., 2004) In contrast, the CMV promoter has been shown to highly
transcribe the transgene in vector-producing mammalian cells. The strong expression of

91

both ZFNs in a single producing mammalian cell could lead to cytotoxicity, resulted in
decrease of vector production. Indeed, with a lentiviral vector carrying 2A-linked ZFNs,
we observed a production issue associated with this type of ZFN-mediated cytotoxicity
(Figure 4. 3).
In summary, we developed a novel BV that confers the permanent gene disruption
and genome modification in various mammalian cell lines as well as in hES cells by
delivering ZFNs and a donor template. This system allows for a safe and efficient
genome modification of the target cell. Traditionally, genome modification via
integrating viral vectors such as retrovirus, and lentivirus tends to integrate into coding or
regulatory regions of transcriptionally active genes, which might lead to gene silencing
and insertional mutagenesis.(Bushman et al., 2005; Schröder et al., 2002) Alternatively,
transponsons such as Sleeping Beauty, PiggyBac, or phage integrase such as ϕ31 could
also introduce the permanent genome modification in the target cell.(Ehrhardt et al.,
2007; Ivics et al., 2007; Wilson et al., 2007) However, modification via these approaches
may still impose position effects, which could still lead to insertional mutagenesis. Our
approach of utilizing site-specific HR via ZFN-mediated double strand break allows us to
pinpoint the exact site of integration, which is less likely to cause gene silencing or/and
insertional mutagenesis. In principle, by engineering the ZFN binding domain and
selecting the appropriate donor sequence, we could engineer BV-ZFN to direct the
modification to any desirable genome site. Moreover, by selecting the target site, it will
be possible to predict the level of transgene expression and exploit endogenous promoter
to drive transgene expression, thus preventing gene silencing. Modification via HR can
also be applied to treat diseased cells by replacing the defected genes with a functional

92

gene. The combination of ZFNs and the BV system can enable rapid generation of new
cell lines and study of functions of specific genes, which can advance novel
developments in biotechnology and medicine.

4.4 Materials & Methods
4.4.1 Maintenance and differentiation of the hES cells.
The hES cell line H9(Thomson et al., 1998) was maintained as described
previously.(Reubinoff et al., 2000) Briefly, hES colonies were maintained on mitotically
inactivated mouse embryonic fibroblasts (MEF) in media consisting of DMEM-F12
(Invitrogen) supplemented with 20% Knockout Serum Replacer (Invitrogen) and FGF-2
(4 ng/ml, Peprotech) plus antibiotics and L-glutamine (1mM). Colonies were passaged
weekly using mechanical dissection. To induce differentiation, hES colonies were
detached by using 1 mg/mL collagenase (Invitrogen). The hES cell clumps were
transferred to suspension culture for 5 days to foster the formation of EB’s. The EBs were
then transferred to cell culture dishes coated with matrigel to promote attachment and
outgrowth of differentiated cells. Differentiated cultures were collected for RT-PCR
analysis 5 days following transfer.
4.4.2 Plasmid construction.
The plasmid pFasBac-Dual from Invitrogen was used as a shuttle vector to
generate the recombinant BVs (pFB-ZFN, pFB-donor, and pFB-ZFN-Donor). The
cDNAs of the reported ZFNs targeting the human CCR5(Lombardo et al., 2007; Perez et

93

al., 2008) were synthesized by GeneArt (Regensburg, Germany). The ZFNs were cloned
into the pcDNA3 vector (Invitrogen) via BamHI and EcoRI restriction sites. The resulting
ZFN expression cassette including a CMV internal promoter and a BGH polyA tail was
then cloned into pFastBac-Dual to yield pFB-ZFN. To generate the donor plasmid, the
ZFN-targeted DNA was amplified with a primer pair (CCR5FW: TAC CGA GCT CGG
ATC CTT AGA CCC TCT ATA ACA GTA ACT TCC TTT TAA AAA AGA CCT CTC
CCA C; CCR5BW: TAG ATG CAT GCT CGA CTA GCG TCA ATA AAA ATG TTA
AGA CTG AGT TGC AGC CG) and cloned into a TOPO plasmid (Invitrogen) via XhoI
and BamHI restriction sites. A reporter expression cassette containing an EF1α promoter,
the GFP cDNA, and the polyA tail was then cloned into an EcoRV site that was inserted
between the CCR5-ZFN binding sites. To generate the pFB-Donor plasmid, the donor
vector was PCR amplified with a pair of primers (CEGAFW: ATG GCT CGA GAT CCC
CTT AGA CCC TCT ATA ACA GTA ACT TCC TTT TAA AAA AGA CCT CTC
CCA C; CEGABW: ACT TCT CGA CAA GCT CTA GCG TCA ATA AAA ATG TTA
AGA CTG AGT TGC AGC CG) and cloned into pFastBac-Dual via SmaI and HindIII
restriction sites using the BD Clontech In-Fusion cloning method. Similarly, the pFB-
ZFN-Donor plasmid was generated by amplifying with primers (CCR5ZFNFW: GCA
ATT GTT GTT GTT GTT GAC ATT GAT TAT TGA CTA GTT ATT AAT AGT AAT
CAA TTA CGG GGT CAT TAG; CCR5ZFNBW: TGC AAT AAA CAA GTT CCA
TAG AGC CCA CCG CAT CCC CAG C) and cloned into pFB-Donor via PvuII
restriction site using the BD Clontech In-Fusion cloning method. To generate the
lentiviral plasmid FUW-ZFN, the DNA of F2A-linked ZFNs were cloned into FUW via
EcoR1 and BamH1 restriction sites.

94

4.4.3 Vector production.
Recombinant BVs with the described expression cassettes were produced and
propagated in SF9 cells according to the manual of the Bac-to-Bac Baculovirus
Expression system from Invitrogen. Budded viruses were filtered through a 0.45-µm pore
size filter (Nalgene, Rochester, NY) to remove any cell debris, and concentrated via
centrifugation at 10,000 rpm for 5 hours and 20
o
C. Viral pellets were resuspended in 1mL
of HBSS (Invitrogen). To determine the titers of the recombinant BVs, we adapted a
qPCR assay to first quantify the physical particles of the viral particles.(Hitchman et al.,
2007) BacPAK Baculovirus Rapid Titer Kit (Clontech) was then used to determine the
infectious units. To generate the lentiviral vector FUW-ZFN, 293T cells were seeded in a
6-cm culture dish in DMEM medium supplemented with fetal bovine serum (Sigma, St.
Louis, MO, 10 %), L-glutamine (10 mL/L), penicillin, and streptomycin (100 units/mL)
the night prior to transfection. 293T cells were transfected at a confluence of 80~90 %
with 5 µg of lentiviral backbone vector (FUW-ZFN or FUW), 2.5 µg each of
pMDLg/pRRE, pRSV-Rev, and pVSVG via the standard calcium phosphate precipitation
technique.(Klages et al., 2000b) Cells were replenished with pre-warmed media 4 hours
post-transfection. Vectors were harvested two days post-transfection and filtered through
a 0.45-µm pore size filter (Nalgene, Rochester, NY)
4.4.4 Surveyor nuclease assay.
Genomic DNA was extracted from modified and control cells using the DNeasy
Blood & Tissue Kit (Qiagen). A 292-bp fragment of the CCR5 locus containing the ZFNs
binding site was amplified with the primer sets ZFNFW: AAG ATG GAT TAT CAA

95

GTG TCA AGT CC and ZFNBW: CAA AGT CCC ACT GGG CG using the Taq DNA
polymerase (Fermentas) and supplemented with 5 µCi α-P
32
dATP and 5 µCi α-P
32

dCTP. The PCR product was then purified with a G-50 column (GE Healthcare),
denatured, re-annealed and digested with Surveyor nuclease (Transgenomic). The
resulting products were resolved on a non-denaturing 10% TBE polyacrylamide gel (Bio-
Rad). The gel was dried and exposed and the resulting bands were quantified by
phosphorimager (Bio-Rad).
4.4.5 Viral transduction.
For viral transduction to Ghost-CCR5 cell line, 0.05 million of Ghost-CCR5 cells
were seeded on a 24-well culture plate and spin-transduced with the BVs (Bac-ZFN,
MOI=500) or lentiviral vectors (FUW-ZFN/VSVG or FUW/VSVG) at 25°C, 2,500 rpm
for 90 minutes using a RT Legend Centrifuge. After replacing with fresh media, the
treated cells were cultured for additional 5 days at 37°C and 5% CO
2
. To analyze the
CCR5 disruption efficiency, the treated cells were stained by biotin-conjugated anti-
CCR5 antibody (Invitrogen), followed by a secondary staining with PE-conjugated
streptavidin (BD Pharmingen). Flow cytometry (BD FACSort) was then used to analyze
the disruption efficiency.
For viral transduction to the 293T or U87 cell line, 0.5 million cells were seeded
on a 24-well cell culture plate and spin-transduced with the indicated BVs (Bac-ZFN,
Bac-Donor, or Bac-ZFN-Donor) at 25°C, 2,500 rpm for 90 minutes using a RT Legend
Centrifuge. After replacing with fresh media, the treated cells were cultured for additional

96

30 days at 37°C and 5% CO
2
. Flow cytometry (BD FACSort) was then used to analyze
gene addition efficiency.
For viral transduction to hES cells, single hES cells in suspension were used to
avoid the effect of virus absorption by the MEF’s. At the day of the transduction, single
cell suspensions were generated by treating hES colonies with 0.05% trypsin solution
containing 0.2g/L EDTA.4Na (Invitrogen) for 3 minutes. Dissociated cells were
collected by centrifugation at 1000 rpm for 1 minute. 2.5 × 10
4
hES cells were
resuspended in 100 µL of knockout DMEM containing 10 µM of p160-Rho-associated
coiled-coil kinase (ROCK) inhibitor (Y-27632, Sigma Aldrich) to prevent apoptosis
during culture in suspension and enable the replating of single cells. BVs (Bac-ZFN or
Bac-Donor) were added at the indicated MOI. Transduction was carried out at 37°C for 2
hours, after which the cells were replated onto fresh MEF, in 12 well plates. The treated
cells were cultured for additional 21 days at 37°C and 5% CO
2
. Flow cytometry (BD LSR
II) was used to analyze gene addition efficiency.
4.4.6 p24 analysis of lentiviral vectors.
Various lentiviral vectors (10 µL, fresh supernatant) were lysed with 90 µL of
10% Triton-X 100 in PBS and the p24 levels were measured by a p24 antigen capture
enzyme linked immunosorbent assay (ELISA) kit (ImmunoDiagnostics, Woburn, MA)
4.4.7 Detection of double-strand breaks in ZFN-treated cells.
SF9 or 293T cells were transfected by Cellfectin (Invitrogen) using the protocol
recommended by the manufacturer. Fluorescent images were acquired on a Yokogawa

97

spinning-disk confocal scanner system (Solamere Technology Group, Salt Lake City,
UT) using a Nikon eclipse Ti-E microscope equipped with a 60×/1.49 Apo TIRF oil
objective and a Cascade II: 512 EMCCD camera (Photometrics, Tucson, AZ, USA). An
AOTF (acousto-optical tunable filter) controlled laser-merge system (Solamere
Technology Group Inc.) was used to provide illumination power at each of the following
laser lines: 491 nm, 561 nm, and 640 nm solid state lasers (50 mW for each laser). To
image 53BP1, both treated and non-treated cells were seeded into a 35-mm glass-bottom
culture dish and grown at 27°C or 37°C overnight. The seeded cells were rinsed with cold
PBS and fixed with 4% formaldehyde on ice for 10 minutes, and then immunostained
with anti-53BP1 rabbit polyclonal antibodies (Bethyl Laboratories) followed by
incubation with Texas-Red anti-rabbit IgG (Invitrogen). TO-PRO-3 iodide (Invitrogen)
was also used for nuclear staining.
4.4.8 Targeted integration analysis.
Genomic DNA was isolated with DNeasy Blood & Tissue Kit (Qiagen). 100 ng of
genomic DNA was subjected to PCR using a primer pair of 5’CCR5FW (5’-CTT AGA
ACA GTG ATT GGC ATC CAG TAT GTG CCC TC-3’) and BGHPABW (5’-GCT
GGG GAT GCG GTG GGC TCT ATG G-3’) to confirm 5’ targeted integration, and a
primer pair of EF1αFW (5’-GCC GAC CCC TCC CCC CAA CTT CTC-3’) and
3’CCR5FW (5’-GGC TTA AAA GAT CTA ATC TAC TTT AAA CAG ATG CCA
AAT AAA TGG ATG-3’) ) to confirm 3’ targeted integration. A primer pair of
CCR5NTFW (5’-CTC TCC CTT CAC TCC GAA AGT TCC TTA TGT ATA TTT AAA
AGA AAG C-3’) and CCR5NTBW (5’-CTT GCA GTG AGG CTT CTG TCT TTG

98

CCA GCA ATA G-3’) was used to amplify non-targeted locus DNA. The PCR amplified
products were resolved on 1% agarose gel and visualized by ethidium bromide staining.
4.4.9 Cell sorting and reverse transcription-PCR analysis.
For sorting the GFP
+
cells, single cell suspensions were generated by treating hES
colonies with 0.05% trypsin solution containing 0.2g/L EDTA.4Na for 3 minutes.
Dissociated cells were collected by centrifugation at 1000 rpm for 1 minute and
resuspended in 2 mL of knockout DMEM containing 10 µM of ROCK inhibitor. Cells
were sorted with FACS Aria SORP equipped with 100µm nozzle.
Total RNA was extracted from hES cells or EBs using the RNeasy kit (Qiagen).
First-strand cDNA was synthesized by PowerScript Reverse Transcriptase system (BD
Biosciences). 50 ng of cDNA reaction mix was subjected to PCR amplification with the
following primer pairs and the resulting products were electrophoresed on a 1% agarose
gel. MAP2: 5’-GAA GCA AAG GCA CCT CAC TG-3’ and 5’-TCT GAG GCA GGT
GAT GGG-3’; NEUROD1: 5’-GTC CTT CGA TAG CCA TTC AC-3’ and 5’-CTT TGA
TCC CCT GTT TCT TCC-3’; AFP: 5’-AGA ACC TGT CAC AAG CTG TG-3’ and 5’-
GAC AGC AAG CTG AGG ATG TC-3’; DCN: 5’-CAC AAC ACC AAA AAG GCT
TC-3’ and 5’-TTG CAG TTA GGT TTC CAG TAT C-3’; HAND1: 5’-TGC CTG AGA
AAG AGA ACC AG-3’ and 5’-ATG GCA GGA TGA ACA AAC AC-3’; IGF2: 5’-TCC
TCC CTG GAC AAT CAG AC-3’ and 5’-AGA AGC ACC AGC ATC GAC TT-3’.

99

4.4.10 Immunohistochemistry and Karyotypic Analysis.
For immunostaining hES cell colonies were washed with PBS and fixed with 4%
paraformaldehyde for 10 minutes. Cells were permeabilized with 0.1% Triton X-100 for
5 minutes at room temperature then treated for 1 hour with the following primary
antibodies; OCT4 (C-10, Santa Cruz Biotechnology, 1: 10 dilution), NANOG (H-155,
Santa Cruz Biotechnology, 1:10 dilution), GCTM2 (A kind gift from Dr. Martin Pera),
and SSEA4 (Millipore,1ug/ml). Cells were washed with staining buffer (PBS
supplemented with 2.5% FBS) then incubated for 1 hour with secondary antibodies:
Texas-Red anti-mouse IgG (Invitrogen, T862, 1:400 dilution) or Alexa Fluor 594 anti-
rabbit IgG (Invitrogen, A11012, 1:1000 dilution). All antibodies were diluted in staining
buffer. Nuclear staining was carried out using Hoechst (33258, Sigma-Aldrich). Images
were acquired on an inverted fluorescent microscope (Axioimager, by Carl Zeiss). The
karyotyping of transduced hES cells was conducted by Cell Line Genetics (Wisconsin).

100

References

Ager, S., Nilson, B.H., Morling, F.J., Peng, K.W., Cosset, F.L., and Russell, S.J. (1996).
Retroviral display of antibody fragments; interdomain spacing strongly influences
vector infectivity. Human gene therapy 7, 2157-2164.

Anderson, K.C., Bates, M.P., Slaughenhoupt, B.L., Pinkus, G.S., Schlossman, S.F., and
Nadler, L.M. (1984). Expression of human B cell-associated antigens on
leukemias and lymphomas: a model of human B cell differentiation. Blood 63,
1424-1433.

Anderson, W. (1998). Human gene therapy. Nature 392, 25-30.

Behr, J. (1994). Gene transfer with synthetic cationic amphiphiles: prospects for gene
therapy. Bioconjug Chem 5, 382-389.

Beumera, K.J., Trautmana, J.K., Bozasa, A., Liub, J.-L., Ruttera, J., Gallb, J.G., and
Carrolla, D. (2008). Efficient gene targeting in Drosophila by direct embryo
injection with zinc-finger nucleases. Proc Nat Acad Sci USA 105, 19821-19826.

Bibikova, M. (2001). Stimulation of homologous recombination through targeted
cleavage by chimeric nucleases. Mol Cell Biol 21, 289-297.

Bibikova, M., Carroll, D., Segal, D.J., Trautman, J.K., Smith, J., Kim, Y.-G., and
Chandrasegaran2, S. (2001). Stimulation of Homologous Recombination through
Targeted Cleavage by Chimeric Nucleases. Mol Cell Biol 21, 289-297.

Boerger, A., Snitkovsky, S., and Young, J. (1999a). Retroviral vectors preloaded with a
viral receptor-ligand bridge protein are targeted to specific cell types. Proc Nat
Acad Sci USA 96, 9867-9872.

Boerger, A.L., Snitkovsky, S., and Young, J.A. (1999b). Retroviral vectors preloaded
with a viral receptor-ligand bridge protein are targeted to specific cell types. Proc
Natl Acad Sci USA 96, 9867-9872.

Boyce, M.F., and Bucher, N.L. (1996). Baculovirus-mediated gene transfer into
mammalian cells. Proc Nat Acad Sci USA 93, 2348-2352.

Bukrinsky, M. (1993). A nuclear localization signal within HIV-1 matrix protein that
governs infection of non-dividing cells. Nature 365, 666-669.

101

Burns, J., Friedmann, T., Driever, W., Burrascano, M., and Yee, J. (1993). Vesicular
stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to
very high titer and efficient gene transfer into mammalian and nonmammalian
cells. Proc Nat Acad Sci USA 90, 8033-8037.

Burton, D., Pyati, J., R Koduri, Sharp, S., Thornton, G., Parren, P., Sawyer, L., Hendry,
R., Dunlop, N., and Nara, P. (1994). Efficient neutralization of primary isolates of
HIV-1 by a recombinant human monoclonal antibody. Science 266, 1024-1027.

Bushman, F., Lewinski, M., Ciuffi, A., Barr, S., Leipzig, J., Hannenhalli, S., and
Hoffmann, C. (2005). Genome-wide analysis of retroviral DNA integration. Nat
Rev Microbiol 3, 848-858.

Carroll, D. (2008). Progress and prospects: Zinc-finger nucleases as gene therapy agents.
Gene Ther 15, 1463-1468.

Carter, P., and Samulsky, R. (2000). Adeno-associated viral vectors as gene delivery
vehicles. Int J Mol Med 6, 17-27.

Chandrasegaran, S., and Smith, J. (1999). Chimeric restriction enzymes: what is next?
Biol Chem 380, 841-848.

Cheng, T., Xu, C.-Y., Wang, Y.-B., Chen, M., Wu, T., Zhang, J., and Xia, N.-S. (2004).
A rapid and efficient method to express target genes in mammalian cells by
baculovirus. World J Gastroenterol 10, 1612-1618.

Cheshenko, N., Krougliak, N., Eiensmith, R., and Krougliak, V. (2001a). A novel system
for the production of fuly deleted adenovirus ectors that does not require helper
adenovirus. Gene Ther 8, 846-854.

Cheshenko, N., Krougliak, N., Eisensmith, R.C., and Krougliak, V.A. (2001b). A novel
system for the production of fully deleted adenovirus vectors that does not require
helper adenovirus. Gene Ther 8, 846-854.

Chou, W., Liao, K., Lo, Y., Jiang, S., Yeh, M., and Roffler, S. (1999). Expression of
chimeric monomer and dimer proteins on the plasma membrane of mammalian
cells. Biotechnol Bioeng 65, 160-169.

Chowdhury, S., Chester, K.A., Bridgewater, J., Collins, M.K., and Martin, F. (2004).
Efficient retroviral vector targeting of carcinoembryonic antigen-positive tumors.
Mol Ther 9, 85-92.

Cronin, J., Zhang, X., and Reiser, J. (2005a). Altering the tropism of lentiviral vectors
through pseudotyping. Curr Gene Ther 5, 387-398.

102

Cronin, J., Zhang, X.Y., and Reiser, J. (2005b). Altering the tropism of lentiviral vectors
through pseudotyping. Current gene therapy 5, 387-398.

Daniels, R., Downie, J., Hay, A., Knossow, M., Skehel, J., Wang, M., and Wiley, D.
(1985). Fusion mutants of the influenza virus hemagglutinin glycoprotein. Cell 40,
431-439.
Danos, O., and Mulligan, R. (1988). Safe and efficient generation of recombinant
retroviruses with amphotropic and ecotropic host ranges. Proc Nat Acad Sci USA
85.

Darrah, P., Patel, D., Luca, P.D., Lindsay, R., Davey, D., Flynn, B., Hof, S., Andersen, P.,
Reed, S., Morris, S., et al. (2007). Multifunctional Th1 cells define a correlate of
vaccine mediated protection against Leishmania major. Nat Med 13, 843-850.

Dash, P., Read, M., Barrett, L., Wolfert, M., and Seymour, L. (1999). Factors affecting
blood clearance and in vivo distribution of polyelectrolyte complexes for gene
delivery. Gene Ther 6, 643-650.

Davidson, J., Krieg, T., and Eming, S. (2000). Particle-mediated gene therapy of wounds.
Wound Repair Regen 8, 452-459.

Davies, A. (1994). Current methods for manipulating baculviruses. Biotechnology 12.

Doyon, Y., McCammon, J.M., Miller, J.C., Faraji, F., Ngo, C., Katibah, G.E., Amora, R.,
Hocking, T.D., Zhang, L., Rebar, E.J., et al. (2008). Heritable targeted gene
disruption in zebrafish using designed zinc-finger nucleases. Nature 26, 702-708.

Drabick, J. (2001). Cutaneous transfection and immue responses to intradermal nucleic
acid vaccination are significantly enhanced by in vivo electropermeabilization.
Mol Ther 3, 249-255.

Edelstein, M., Abedi, M., Wixon, J., and Edelstein, R. (2004). Gene therapy clinical trials
worldwide 1989-2004 - an overview. J Gene Med 6, 597-602.

Ehrhardt, A., Yant, S.R., Giering, J.C., Xu, H., Engler, J.A., and Kay, M.A. (2007).
Somatic integration from an adenoviral hybrid vector into a hot spot in mouse
liver results in persistent transgene expression levels in vivo. Mol Ther 15, 146-
156.

El-Aneed, A. (2003). An overview of current delivery systems in cancer gene therapy. J
Controlled Release 94, 1-14.

Endoh, M. (2002). Fetal gene transfer by intrauterine injection with microbubble
enhanced ultrasound. Mol Ther 5, 501-508.

103

Fielding, A.K., S.C. Fernandes, M. P., Chadwick, F. J., Bullough, and Cosset, F. L.
(2000). A Hyperfusogenic Gibbon Ape Leukemia Envelope Glycoprotein:
Targeting of a Cytotoxic Gene by Ligand Display. Hum Gene Ther 11, 817-826.

Fischer, D., Bieber, T., Li, Y., Elsasser, H., and Kissel, T. (1999). A novel non-viral
vector for DNA delivey based on low molecular weight, branched
polyethylenimine: effect of molecular weight on transfection efficiency and
cytotoxicity. Pharm Res 16, 1273-1279.

Fredericksen, B., Wei, B., Yao, J., Luo, T., and Garcia, J. (2002). Inhibition of
endosomal/lysosomal degradation increases the infectivity of human
immunodeficiency virus. J Virol 76, 11440-11446.

Gibbons, D.L., Vaney, M.C., Roussel, A., Vigouroux, A., Reilly, B., Lepault, J., Kielan,
M., and Rey, F.A. (2004). Conformational change and protein-protein interactions
of the fusion protein of Semliki Forest virus. Nature 427, 320-325.

Glover, D.J., H.J. Lipps and Jans, D.A (2005). Nat Rev Genet 6, 299-311.

Gollan, T.J., and Green, M.R. (2002). Selective targeting and inducible destruction of
human cancer cells by retroviruses with envelope proteins bearing short peptide
ligands. J Virol 76, 3564-3569.

Hacein-Bey-Abina, S., Kalle, C.V., Schmidt, M., McCormack, M.P., Wulffraat, N.,
Leboulch, P., Lim, A., Osborne, C.S., Pawliuk, R., Morillon, E., et al. (2003).
LMO2-associated clonal T cell proliferation in two patients after gene therapy for
SCID-X1. Science 302, 415-419.

Haines, A.M., A.S. Irvines, A. Mountain, J. Charlesworth, N.A. Farrow, R.D. Husain, H.
Hyde, H. Ketteringham, R.H. McDermott, A.F. Mulcahy, T.L. Mustoe, S.C. Reid,
M. Rouquette, J.C. Shaw, D.R. Thatcher, J.H. Welsh, D.E. Williams, W. Zauner,
and Phillips, R. O. (2001). CL22-a novel cationic peptide for efficient transfection
of mammalian cells. Gene Ther, 99-110.

Hall, M., Burson, K., and Huestis, W. (1998). Interactions of a vesicular stomatitis virus
G protein fragment with phosphatidylserine: NMR and fluorescence studies.
Biochim Biophys Acta 1415, 101-113.

Han, X., Kasahara, N., and Kan, Y.W. (1995). Ligand-directed retroviral targeting of
human breast cancer cells. Proc Natl Acad Sci USA 92, 9747-9751.

Hitchman, R.B., Siaterli, E.A., Nixon, C.P., and King, L.A. (2007). Quantitative Real-
Time PCR for Rapid and Accurate Titration of Recombinant Baculovirus
Particles. Biotechnol Bioeng 96, 810-814.

104

Ho, Y.C., Chung, Y.C., Hwang, S.M., Wang, K.C., and Hu, Y.C. (2005). Transgene
expression and differentiation of baculovirus-transduced human mesenchymal
stem cells. J Gene Med 7, 860-868.

Hockemeyer, D., Soldner, F., Beard, C., Gao, Q., Mitalipova, M., DeKelver, R.C.,
Katibah, G.E., Amora, R., Boydston, E.A., Zeitler, B., et al. (2009). Efficient
targeting of expressed and silent genes in human ESCs and iPSCs using zinc-
finger nucleases. Nat Biotechnol 27, 851-857.

Hofmann, C., Sandig, V., Jennings, G., Rudolph, M., Schlag, P., and Strauss, M. (1995).
Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc Nat
Acad Sci USA 92, 10099-10103.

Holt, N., Wang, J., Kim, K., Friedman, G., Wang, X., Taupin, V., Crooks, G.M., Kohn,
D.B., Gregory, P.D., Holmes, M.C., et al. (2010). Human hematopoietic
stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control
HIV-1 in vivo. Nat Biotechnol 10, 1038.

Ivics, Z., Katzer, A., Stuwe, E., Fiedler, D., Knespel, S., and Izsvak, Z. (2007). Targeted
sleeping beauty transposition in human cells. Mol Ther 15, 1137-1144.

Jiang, A., and R. Dornburg. (1999). In vivo cell type-specific gene delivery with
retroviral vectors that display single chain antibodies. Gene Ther 6, 1982-1987.

Jiang, A., Chu, T., Nocken, F., Cichutek, K., and Dornburg., R. (1998). Cell type-specific
gene transfer into human cells with retroviral vectors that display single chain
antibodies. J Virol 72, 10148-10156.

Joo, K.I., and Wang, P. (2008). Visualization of targeted transduction by engineered
lentiviral vectors. Gene therapy.

Jorgenson, R.L., Vogt, V.M., and Johnson, M.C. (2009). Foreign glycoproteins can be
actively recruited to virus assembly sites during pseudotyping. J Virol 83, 4060-
4067.

Kasahara, N., Dozy, A., and Kan, Y. (1994a). Tissu-specific targeting of retroviral
vectors through ligand-receptor interactions Science 266, 1373-1376.

Kasahara, N., Dozy, A.M., and Kan, Y.W. (1994b). Tissue-specific targeting of retroviral
vectors through ligand-receptor interactions. Science (New York, NY 266, 1373-
1376.

Kielian, M., and Rey, F. (2005). Virus membrane-fusion proteins: more than one way to
make a hairpin. Nat Rev Microbiol 4, 67-76.

105

Kielian, M., and Rey, F.A. (2006). virus memrane-fusion proteins: more than one way to
make a hairpin. Nat Rev Microbiol 4, 67-76.

Kim, Y., Cha, J., and Chandrasegaran, S. (2004). Hybrid restriction enzymes: zinc finger
fusions to Fok 1 cleavage domain. Proc Nat Acad Sci USA 93, 1156-1160.

Kircheis, R., Schuller, S., Brunner, S., Ogris, M., Heider, K., Zauner, W., and Wagner, E.
(1999). Polycation-based DNA complexes for tumor-targeted gene delivery in
vivo. J Gene Med 1, 111-120.

Klages, N., Zufferey, R., and Trono, D. (2000a). A stable system for the high-titer
production of multiply attenuated lentiviral vectors. Mol Ther 2, 170-176.

Klages, N., Zufferey, R., and Trono, D. (2000b). A stable system for the high-titer
production of multiply attenuated lentiviral vectors. Mol Ther 2, 170-176.

Kobayashi, N., Rivas-Carrillo, J.D., Soto-Gutierrez, A., Fukazawa, T., Chen, Y.,
Navarro-Alvarez, N., and Tanaka, N. (2005). Gene Delivery to Embryonic Stem
Cells. Birth Defects Res C Embryo Today 75, 10-18.

Kohn, D.B. (2007). Lentiviral vectors ready for prime-time. Nat Biotechnol 25, 65-66.

Kost, T.A., and Condreay, J.P. (2002). Recombinant baculoviruses as mammalian cell
gene delivery vectors. Trends Biotechnol 20, 173-180.

Kost, T.A., Condreay, J.P., and Jarvis, D.L. (2005). Baculovirus as versatile vectors for
protein expression in insect and mammalian cells. Nat Biotechnol 23, 567-575.
Kotin, R. (1990). Site-specific integration by adeno-associated virus. Proc Nat
Acad Sci USA 87, 2211-2215.

Kunath, K., Harpe, A., Fischer, D., Petersen, H., Bickel, U., Voigt, K., and Kissel, T.
(2003). Low-molecular-weight polyethylenimine as a non-viral vector for DNA
delivery: comparsion of physicochemical properties, transfection efficiency and in
vivo distribution with high-molecular-weight polyehylenimine. J Controlled
Release 89, 113-125.

Lasic, D. (1997). Liposomes in gene delivery. CRC Press, Boca Raton.

Lavillette, D., Russell, S.J., and Cosset, F.L. (2001). Retargeting gene delivery using
surface-engineered retroviral vector particles. Current opinion in biotechnology
12, 461-466.

Lei, Y., Joo, K.-I., and Wang, P. (2009). Engineering fusogenic molecules to achieve
targeted transduction of enveloped lentiviral vectors. J Biol Eng 3, 8.

106

Leung, K., Kim, J.-O., Ganesh, L., Kabat, J., Schwartz, O., and Nabel, G.J. (2008). HIV-
1 assembly: viral glycoproteins segregate quantally to lipid rafts that associate
individually with virions. Cell Host Microbe 3, 285-292.

Lewis, P., Hensel, M., and Emerman, M. (1992). Human immunodeficiency virus
infection of cells arrested in the cell cycle. EMBO J 11, 3053-3058.
Liao, K., Chen, B., Liu, T., Tzou, S., Lin, Y., Lin, K., Su, C., and Roffler, S. (2003).
Stable expression of chimeric anti-CD3 receptors on mammalian cells for
stimulation of antitumor immunity. Cancer Gene Ther 10.

Liao, K., Chou, W., Lo, Y., and Roffler, S. (2000). Design of transgenes for efficient
expression of active chimeric proteins on mammalian cells. Biotechnol Bioeng 73,
313-323.

Lin, M., Pulkkinen, L., Uitto, J., and Yoon, K. (2000). The gene gun: current application
in cutaneous gene therapy. Int J Dermatol 39, 161-170.

Liu, C.Y., and Kielian, M. (2009). E1 mutants identify a critical region in the trimer
interface of the semliki forest virus fusion protein. J Virol doi:10.1128/JVI.01147-
09.

Lo, W.-H., Hwang, S.-M., Chuang, C.-K., Chen, C.-Y., and Hu, Y.-C. (2009).
Development of a Hybrid Baculoviral Vector for Sustained Transgene Expression.
Mol Ther 17, 658-666.

Lois, C., Hong, E., Pease, S., Brown, E., and Baltimore, D. (2002a). Germline
Transmission and Tissue-Specific Expression of Transgenes Delivered by
Lentiviral Vectors. Science 295, 868-872.

Lois, C., Hong, E.J., Pease, S., Brown, E.J., and Baltimore, D. (2002b). Germline
transmission and tissue-specific expression of transgenes delivered by lentiviral
vectors. Science (New York, NY 295, 868-872.

Lombardo, A., Genovese, P., Beausejour, C., Colleoni, S., Lee, Y., Kim, K., Ando, D.,
Urnove, F., Galli, C., Gregory, P., et al. (2007). Gene editing in human stem cells
using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat
Biotechnol 25, 1298-1306.

Long, G., Pan, X., Kormelink, R., and Vlak, J. (2006). Functional entry of baculovirus
into insect and mammalian cells is dependent on clathrin-mediated endocytosis. J
Virol 80, 8830-8833.

Lu, L., Cassese, T., and Kielian, M. (1999a). The Chloesterol Requirement for Sindbis
Virus Entry and Exit and Characterization of a Spike Protein Region Involved in
Cholesterol Dependence. J Virol 73, 4272-4278.

107

Lu, Y.E., Cassese, T., and Kielian, M. (1999b). The cholesterol requirement for sindbis
virus entry and exit and characterization of a spike protein region involved in
cholesterol dependence. Journal of virology 73, 4272-4278.

Mali, P., Ye, Z., Hommond, H.H., Yu, X., Lin, J., Chen, G., Zou, J., and Cheng, L.
(2008). Improved efficiency and pace of generating induced pluripotent stem cells
from human adult and fetal fibroblasts. Stem Cells 26, 1998-2005.
Mangor, J., Monsma, S., Johnson, M., and Blissard, G. (2001). A GP64-null
baculovirus pseudotyped with vesicular stomatitis virus G protein. J Virol 75,
2544-2556.

Mansour, S., Thomas, K., and Capecchi, M. (1998). Disruption of the proto-oncogene
int-2 in mouse embryo-derived stem cells: a general strategy for targeting
mutations to non-selectable genes. Nature 336, 348-352.

Marin, M., Noel, D., Valsesia-Wittman, S., Brockly, F., Etienne-Julan, M., Russell, S.,
Cosset, F.L., and Piechaczyk, M. (1996). Targeted infection of human cells via
major histocompatibility complex class I molecules by Moloney murine leukemia
virus-derived viruses displaying single-chain antibody fragment-envelope fusion
proteins. Journal of virology 70, 2957-2962.

Martin, F., S. Chowdhury, S. Neil, N. Phillipps, and M. K. Collins. (2002). Envelope-
targeted retrovirus vectors transduce melanoma xenografts but not sleen or liver.
Mol Ther 5, 269-274.

Mastromarino, P., Conti, C., Goldoni, P., Hauttecoeur, B., and Orsi, N. (1987).
Characterization of membrane components of the erythrocyte involved in
vesicular stomatitis virus attachment and fusion at acidic pH. J Gen Virol 68,
2359-2369.

Maurice, M., E. Verhoeyen, P. Salmon, D. Trono, S. J. Russell, and F.L., Cosset (2002).
Efficient gene transfer into human primary blood lymphocytes by surface-
engineered lentiviral vectors that display a T cell-activating polypeptide. Blood 99,
2342-2350.

McClure, M., Marsh, M., and Weiss, R. (1988). Human immunodeficiency virus
infection of CD4-bearing cells occurs by a pH-independent mechanism. EMBO J
7, 513-518.

McDonald, D., Vodicka, M., Lucero, G., Svitkina, T., Borisy, G., Emerman, M., and
Hope, T. (2002a). Visualization of the intracellular behavior of HIV in living cells.
J Cell Biol 159, 441-452.

108

McDonald, D., Vodicka, M.A., Lucero, G., Svitkina, T.M., Borisy, G.G., Emerman, M.,
and Hope, T.J. (2002b). Visualization of the intracellular behavior of HIV in
living cells. The Journal of cell biology 159, 441-452.

McKenzie, D., Smiley, E., Kwok, K., and Rice, K. (2000). Low molecular weight
disulfide cross-linking peptides as nonviral gene delivery carriers. Bioconjug
Chem 11, 901-909.

Mellman, I., Fuchs, R., and Helenius, A. (1986). Acidification of the endocytic and
exocytic pathways. Annual review of biochemistry 55, 663-700.

Meng, X., Noyes, M., Zhu, L., Lawson, N., and Wolfe, S. (2008a). Targeted gene
inactivation in zebrafish using engineered zinc-finger nucleases. Nat Biotechnol
26, 695-701.

Meng, X., Noyes, M.B., Zhu, L.J., Lawson, N.D., and Wolfe, S.A. (2008b). Targeted
gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat
Biotechnol 26, 695-701.

Miller, J.C., Holmes, M.C., Wang, J., Guschin, D.Y., Lee, Y.-L., Rupniewski, I.,
Beausejour, C.M., Waite, A.J., Wang, N.S., Kim, K.A., et al. (2007). An
improved zinc-finger nuclease architecture for highly specific genome editing.
Nat Biotechnol 25, 778-785.

Miyoshi, H., Biomer, U., Takahashi, M., Gage, F., and Verma, I. (1998). Development of
a self-inactivating lentivirus vector. J Virol 72, 8150-8157.

Moehle, E., Rock, J., Lee, Y., Jouvenot, Y., DeDelver, R., Gregory, P., Urnov, F., and
Holmes, M. (2007). Targeted gene addition into a specified location in the human
genome using designed zinc finger nucleases. Proc Nat Acad Sci USA 104, 3055-
3060.

Molina, A. (2008). A decade of rituximab: improving survival outcomes in non-
Hodgkin's lymphoma. Annual review of medicine 59, 237-250.

Morizono, K., Bristol, G., Xie, Y., Kung, S., and Chen, I. (2001a). Antibody-directed
targeting of retroviral vectors via cell surface antigens. J Virol 75, 8016-8020.

Morizono, K., Bristol, G., Xie, Y.M., Kung, S.K., and Chen, I.S. (2001b). Antibody-
directed targeting of retroviral vectors via cell surface antigens. Journal of
virology 75, 8016-8020.

Morizono, K., Ringpis, G.E., Pariente, N., Xie, Y., and Chen, I.S. (2006). Transient low
pH treatment enhances infection of lentiviral vector pseudotypes with a targeting
Sindbis envelope. Virology 355, 71-81.

109

Morizono, K., Xie, Y., Ringpis, G.E., Johnson, M., Nassanian, H., Lee, B., Wu, L., and
Chen, I.S. (2005). Lentiviral vector retargeting to P-glycoprotein on metastatic
melanoma through intravenous injection. Nature medicine 11, 346-352.

Morizono, K., Xie, Y., G. Ringpis, M. Johnson, H. Nassanian, B. Lee, L. Wu, and I. S. Y.
Chen. (2005). Lentiviral vector retargeting to P-glycoprotein on metastatic
melanoma through intravenous injection. Nat Med 11, 346-352.

Morral, N. (1999). Administration of helper-dependent adenoviral vectors and sequential
delivery of different vector serotype for long-term liver-directed gene transfer in
baboons. Proc Nat Acad Sci USA 96, 12816-12821.

Mukhopadhyay, S., Kuhn, R.J., and Rossmann, M.G. (2005). A structural perspective of
the flavivirus life cycle. Nat Rev Microbiol 3, 13-22.

Mulligan, R. (1993). The basic science of gene therapy. Science 260, 926-932.

Naldini, L. (1996). In vivo gene delivery and stable transduction of non-dividing cells by
a lentiviral vector. Science 272, 263-267.

Nguyen, T., Pages, J.C., Farge, D., Briand, P., and Weber, A. (1998). Amphotropic
retroviral vectors displaying hepatocyte growth factor-envelope fusion proteins
improve transduction eficiency of primary hepatocytes. Hum Gene Ther 17, 2469-
2474.

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.

Niidome, T., and Huang, L. (2002). Gene therapy progress and prospects: nonviral
vectors. Gene Ther 9.

Nilson, B.H., Morling, F.J., Cosset, F.L., and Russell, S.J. (1996). Targeting of retroviral
vectors through protease-substrate interactions. Gene therapy 3, 280-286.

O’Driscoll, M., and Jeggo, P.A. (2006). The role of double-strand break repair - insights
from human genetics. Nat Rev Genet 7, 45-54.

Palombo, F., Monciotti, A., Recchia, A., Cortese, R., Ciliberto, G., and Monica, N.L.
(1998). Site-specific integration in mammalian cells mediated by a new hybrid
baculovirus-adeno-associated virus vector. J Virol 72, 5025-5034.

Paques, F., and Haber, J. (1999). Multiple pahtways of recombination induced by double-
strand break in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 63, 349-404.

110

Pariente, N., Mao, S.H., Morizono, K., and Chen, I.S. (2008). Efficient targeted
transduction of primary human endothelial cells with dual-targeted lentiviral
vectors. The journal of gene medicine 10, 242-248.

Pariente, N., Morizono, K., Virk, M.S., Petrigliano, F.A., Reiter, R.E., Lieberman, J.R.,
and Chen, I.S. (2007). A novel dual-targeted lentiviral vector leads to specific
transduction of prostate cancer bone metastases in vivo after systemic
administration. Mol Ther 15, 1973-1981.

Pavletich, N., and Pabo, C. (1991). DNA recognition by Cys2His2 Zinc finger proteins.
Annu Rev Biophys Biomol Struct 29, 183-212.

Pear, W.S., Nolan, G.P., Scott, M.L., and Baltimore, D. (1993). Production of high-titer
helper-free retroviruses by transient transfection. Proc Natl Acad Sci USA 90,
8392-8396.

Pennock, G.D., Shoemaker, C., and Miller, L.K. (1984). Strong and regulated expression
of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector.
Mol Cell Biol 4, 399-406.

Perez, E.E., Wang, J., Miller, J.C., Jouvenot, Y., Kim, K.A., Liu, O., Wang, N., Lee, G.,
Bartsevich, V.V., Lee, Y.-L., et al. (2008). Establishment of HIV-1 resistance in
CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 26,
808-816.

Platt, O. (1994). Mortality in sickle cell disease: life expectancy and risk factors for early
death. N Engl J Med 330, 1639-1644.

Porteus, M. (2005). Mammalian gene targeting with designed zinc finger nucleases. Mol
Ther 13, 438-446.

Porteus, M., and Carroll, D. (2005). Gene targeting using zinc finger nucleases. Nat
Biotechnol 23, 967-973.

Porteus, M.H., and Baltimore, D. (2003). Chimeric nucleases stimulate gene targeting in
human cells. Science 300, 763.

Pouton, C., Lucas, P., Thomas, B., Uduehi, A., Milroy, D., and Moss, S. (1998).
Polycation-DNA complexes for gene delivery: a comparsion of the
biopharmaceutical propterties of cationic polypetides and cationic lipids. J
Controlled Release 53, 289-299.

Price, R., and Kaul, S. (2002). Contrast ultrasound targeted drug and gene delivery and
update on a new therapeutic modality. J Cardiovasc Pharmacol Ther 7, 171-180.

111

Radler, J., Koltover, I., Salditt, T., and Safinya, C. (1997). Structure of DNA-cationic
liposome complexes: DNA intercalation in multilamellar membranes in distinct
interhelical packing regimes. Science 275, 810-814.

Reff, M.E., Carner, K., Chambers, K.S., Chinn, P.C., Leonard, J.E., Raab, R., Newman,
R.A., Hanna, N., and Anderson, D.R. (1994). Depletion of B cells in vivo by a
chimeric mouse human monoclonal antibody to CD20. Blood 83, 435-445.

Reubinoff, B., Pera, M., Fong, C., Trounson, A., and Bongso, A. (2000). Embryonic stem
cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol
18, 399-404.

Roffler, S., Wang, H., Yu, H., Chang, W., Cheng, C., Lu, Y., Chen, B., and Cheng, T.
(2006). A membrane antibody receptor for noninvasive imaging of gene
expression. Gene Ther 13, 412-420.

Roux, P., Jeanteur, P., and Piechaczyk, M. (1989a). A versatile and potentially general
approach to the targeting of specific cell types by retroviruses: application to the
infection of human cells by means of major histocompatibility complex class I
and class II antigens by mouse ecotropic murine leukemia virus-derived viruses.
Proc Natl Acad Sci USA 86, 9079-9083.

Roux, P., Jeanteur, P., and Piechaczyk, M. (1989b). A versatile and potentially general
approach to the targeting of specific cell types by retroviruses: application to the
infection of human cells by means of major histocompatibility complex class I
and class II antigens by mouse esctropic murine leukemia virus-derived viruses.
Proc Nat Acad Sci USA 86, 9079-9083.

SAMSON, M., LIBERT, F., DORANZ, B.J., RUCKER, J., LIESNARD, C., FARBER,
C.-M., SARAGOSTI, S., LAPOUMÉROULIE, C., COGNAUX, J., FORCEILLE,
C., et al. (1996). Resistance to HIV-1 infection in Caucasian individuals bearing
mutant alleles of the CCR-5 chemokine receptor gene. Nature 382, 722-725.

Sandig, V., Hofmann, C., Steinert, S., Jennings, G., Schlag, P., and Strauss, M. (1996).
Gene transfer into hepatocytes and human liver tissue by baculovirus vectors.
Gene Ther 7, 1937-1945.

Sandrin, V., Russell, S., and Cosset, F. (2003a). Targeting retroviral and lentiviral vectors.
Curr Top Microbiol Immunol 281, 137-178.

Sandrin, V., Russell, S.J., and Cosset, F.L. (2003b). Targeting retroviral and lentiviral
vectors. Curr Top Microbio Immunol 281, 137-178.

112

Santiago, Y., Chan, E., Liu, P., Orlando, S., Zhang, L., Urnov, F., Holmes, M., Guschin,
D., Waite, A., Miller, J., et al. (2008). Targeted gene knockout in mammalian
cells by using engineered zinc-finger nucleases. Proc Nat Acad Sci USA 105,
5809-5814.

Schröder, A.R.W., 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.

Sedivy, J., and Sharp, P. (1989). Positive genetic selection for gene disruption in
mammalian cells by homologous recombination. Proc Nat Acad Sci USA 86, 227-
231.

Serafini, M., Naldini, L., and Introna, M. (2004). Molecular evidece of inefficient
transduction of proliferating human B lymphocytes by VSV-pseudotyped HIV-1-
derived lentivectors. Virology 325, 413-424.

Shoji, I., Aizaki, H., Tani, H., Ishii, K., Chiba, T., Saito, I., Miyamura, T., and Matsuura,
Y. (1997). Efficient gene transfer into various mammalian cells, including non-
hepatic cells, by baculovirus vectors. J Gen Virol 78, 2657-2664.

Shukla, V.K., Doyon, Y., Miller, J.C., DeKelver, R.C., Moehle, E.A., Worden, S.E.,
Mitchell, J.C., Arnold, N.L., Gopalan, S., Meng, X., et al. (2009). Precise genome
modification in the crop species Zea mays using zinc-finger nucleases. Nature 459,
437-441.

Smit, J.M., Bittman, R., and Wilschut, J. (1999). Low-pH-dependent fusion of sindvis
virus with receptor-free cholesterol- and sphingolipid-containing liposomes. J
Virol 73, 8476-8484.

Smith, G.E., Summers, M.D., and Fraser, M.J. (1983). Production of human beta
interferon in insect cells infected with a baculovirus expression vector. Mol Cell
Biol 3, 2156-2165.

Smith, J. (2000). Requirements for double-strand cleavage by chimeric restriction
enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res 28,
3361-3369.

Somia, N., and Verma, I. (2000). Gene therapy: trials and tribulations. Nat Rev Genet 1,
91-99.

Somia, N., Zoppe, M., and Verma, I. (1995a). Generation of targeted retroviral vectors by
using single-chain variable fragment: an approach to in vivo gene delivery. Proc
Nat Acad Sci USA 92, 7570-7574.

113

Somia, N.V., Zoppe, M., and Verma, I.M. (1995b). Generation of targeted retroviral
vectors by using single-chain variable fragment: an approach to in vivo gene
delivery. Proc Natl Acad Sci USA 92, 7570-7574.

Somiari, S. (2000). Theory and in vivo application of electroporative gene delivery. Mol
Ther 255, 249-255.

Stashenko, P., Nadler, L.M., Hardy, R., and Schlossman, S.F. (1980). Characterization of
a human B lymphocyte-specific antigen. J Immunol 125, 1678-1685.

Sternberg, B., Sorgi, F., and Huang, L. (1994). New structures in complex formation
between DNA and cationic liposomes visualized by freeze-fracture electron
microscopy. FEBS Lett 356, 361-366.

Szczepek, M., Brondani, V., ̈chel, J.B., Serrano, L., Segal, D.J., and Cathomen, T. (2007).
Structure-based redesign of the dimerization interface reduces the toxicity of zinc-
finger nucleases. Nat Biotechnol 25, 786-793.

Taniyama, Y. (2002). Development of safe and efficient novel nonviral gene transfer
using ultrasound: enhancedment of transfection efficiency of naked plasmid DNA
in skeletal muscle. Gene Ther 9, 372-380.

Thomson, J., Itskovitz-Eldor, J., Shapiro, S., Waknitz, M., Swiergiel, J., Marshall, V., and
Jones, J. (1998). Embryonic stem cell lines derived from human blastocysts.
Science 282, 1145-1147.

Townsend, J.A., Wright, D.A., Winfrey, R.J., Fu, F., Maeder, M.L., Joung, J.K., and
Voytas, D.F. (2009). High-frequency modification of plant genes using
engineered zinc-finger nucleases. Nature 459, 442-444.

Urnov, F., Miller, J., Lee, Y., Beausejour, C., Rock, J., Augustus, S., Jamieson, A.,
Porteus, M., Gregory, P., and Holmes, M. (2005). Highly efficient endogenous
human gene correction using designed zinc-finger nucleases. Nature 435, 646-651.

Valsesia-Wittmann, S., Drynda, A., Deleage, G., Aumailley, M., Heard, J., Danos, O.,
Verdier, G., and Cosset, F. (1994). Modifications in the binding domain of avian
retrovirus envelope protein to redirect the host range of retroviral vectors. J Virol
68, 4609-4619.

Verma, I. (1990). Gene therapy. Sci Am 263, 68-72.

Verma, I., and Somia., N. (1997). Gene therapy - promises, problems and prospects.
Nature 389, 239-242.

114

Verma, I., and Weitzman, M. (2005a). Gene therapy: twenty-first century medicine.
Annu Rev Biochem 74, 711-738.

Verma, I.M., and Somia, N. (1997). Gene therapy -- promises, problems and prospects.
Nature 389, 239-242.

Verma, I.M., and Weitzman, M.D. (2005b). Gene therapy: twenty-first century medicine.
Annual review of biochemistry 74, 711-738.

Waehler, R., Russell, S., and Curiel, D. (2007a). Engineering targeted viral vectors for
gene therapy. Nat Rev Genet 8, 573-587.

Waehler, R., Russell, S.J., and Curiel, D.T. (2007b). Engineering targeted viral vectors
for gene therapy. Nature reviews 8, 573-587.

Ward, C., Pechar, M., Oupicky, D., Ulbrich, K., and Seymour, L. (2002). Modification of
pLL/DNA complexes with a multivalent hydrophilic polymer permits folate-
mediated targeting in vitro and prolonged plasma circulation in vivo. J Gene Med
4, 536-547.

West, S. (2000). Double-strand break repair in human cells Symp Quant Biol 65, 315-321.

Wightman, L., Kircheis, R., Rossler, V., Carotta, S., Ruzicka, R., Kursa, M., and Wanger,
E. (2001). Different behavior of branched and linear polyethylenimine for gene
deliveryin vitro and in vivo. J Gene Med 3.

Wilson, M.H., Coates, C.J., and Jr., A.L.G. (2007). PiggyBac transposon-mediated gene
transfer in human cells. Mol Ther 15, 139-145.

Wu, G., and Wu, C. (1987). Receptor-mediated in vitro gene transformation by a soluble
DNA carrier system. J Biol Chem 262, 4429-4432.

Wunderli-Allenspach, H., and Ott, S. (1990). Kinetics of fusion and lipid transfer
between virus receptor containing liposomes and influenza viruses as measured
with the octadecylrhodamine B chloride assay. Biochemistry 29, 1990-1997.

Wyman, T., Nicol, F., Zelphati, O., Scaria, P., Plank, C., and Jr, F.S. (1997). Design,
synthesis, and characterization of a cationic peptide that binds to nucleic acids and
permeabilizes bilayers. Biochemistry 36, 3008-3017.

Yan, Z., Zhang, Y., Duan, D., and Engelhardt, J. (2000). Trans-splicing vectors expand
the utility of adeno-associated virus for gene therapy. Proc Nat Acad Sci USA 97,
6716-6721.

115

Yang, H., Zeigler, L., Joo, K.-I., Cho, T., Lei, Y., and Wang, P. (2008). Gamma-
retroviral vectors enveloped with an antibody and an engineered fusogenic protein
achieved antigen-specific targeting. Biotechnol Bioeng 101, 357-368.

Yang, L., Bailey, L., Baltimore, D., and Wang, P. (2006a). Targeting lentiviral vectors to
specific cell types in vivo. Proc Nat Acad Sci USA 103, 11479-11484.

Yang, L., Bailey, L., Baltimore, D., and Wang, P. (2006b). Targeting lentiviral vectors to
specific cell types in vivo. Proc Natl Acad Sci USA 103, 11479-11484.

Yates, F., and Daley, G. (2006). Progress and prospects: gene transfer into embryonic
stem cells. Gene Ther 13, 1431-1439.

Yeh, P., and Perricaudet, M. (1997). Advances in adenoviral vectors: from genetic
engineering to their biology. FASEB J 11, 615-623.

Yu, S. (1986). Self-inactivating retroviral vectors designed for transfer of whole genes
into mammalian cells. Proc Nat Acad Sci USA 83, 3194-3198.

Zeng, J., Du, J., Zhao, Y., Palanisamy, N., and Wang, S. (2007). Baculoviral Vector-
Mediated Transient and Stable Transgene Expression in Human Embryonic Stem
Cells. Stem Cells 25, 1055-1061.

Ziegler, L., Yang, L., Joo, K.i., Yang, H., Baltimore, D., and Wang, P. (2008). Targeting
lentiviral vectors to antigen-specific immuoglobulins. Hum Gene Ther 19, 861-
872.

Zou, J., Maeder, M.L., Mali, P., Pruett-Miller, S.M., Thibodeau-Beganny, S., Chou, B.-
K., Chen, G., Ye, Z., Park, I.-H., Daley, G.Q., et al. (2009). Gene Targeting of a
Disease-Related Gene in Human Induced Pluripotent Stem and Embryonic Stem
Cells. Cell Stem Cell 5, 97-110.

Zufferey, R., Nagy, D., Mandel, R., Naldini, L., and Trono, D. (1997). Multiply
attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat
Biotechnol 15, 871-875.

Abstract (if available)

Abstract Gene therapy is the introduction of functional genes into dysfunctional cells to treat potentially incurable diseases. The technique sounds promising, yet there are many hurdles must be overcome to make it a practical mean of medicine. Viral vector mediated gene therapy remains one of the most promising gene therapy techniques as its efficiency and duration is the highest among the different gene delivery vehicles. To further refine the viral vector to enhance the gene delivery ability, we designed a strategy by decoupling binding and fusion ability of envelope protein into two distinct proteins. By pseudotyping the viral vectors with both an antibody and a fusogenic molecule, we can target and transduce specific cell types. The underlining mechanism of targeted transduction is that the viral vector will bind to the desirable cell types via the cognate antibody antigen interaction and further be endocytosised into the endosome. Inside the endosome compartment, the low pH environment will trigger the conformation change of the fusogenic molecule which will fuse both the viral and cellular membrane, resulting in releasing the viral core. To further enhance the targeted transduction, we focused on engineering the two parameters on the viral vector. In chapter 2, we engineered the fusion loop of the fusogenic molecule to enhance the activity of the protein. We demonstrated that the engineered fusogenic molecules can enhance targeted transduction many folds. We further looked at the other parameter, the targeting antibody in chapter 3. By designing a single chain antibody, we were able to constructed our targeting virus a simplifier viral production protocol and showed that the viral vector still exhibit targeted transduction. These two chapters concluded our effort in enhancing the targeted transduction by genetically engineering the component displayed on the viral membrane.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Dissecting the entry mechanism of targeting lentiviral vectors in living cells and developing quantum dot labeling of viruses for single virus tracking

PDF

Engineering lentiviral vectors for gene delivery

PDF

Engineering lentiviral vectors for gene therapy and DC-vaccine

PDF

Study of dendritic cell-specific lentivector vaccine system

PDF

Engineering lentiviral vectors for gene delivery and immunization

PDF

Engineering nanoparticles for gene therapy and cancer therapy

PDF

Study of dendritic cell targeting by engineered lentivectors

PDF

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

PDF

Non-viral and viral hematopoietic progenitor cell gene therapy

PDF

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

PDF

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

PDF

Improving antitumor efficacy of chimeric antigen receptor-engineered immune cell therapy with synthetic biology and combination therapy approaches

PDF

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

PDF

Genomic stability of transcriptionally targeted replication competent retroviral vectors

PDF

Engineering immunotoxin and viral vectors for cancer therapy

PDF

Entry and intracellular trafficking of measles virus glycoprotein pseudotyping lentivector for transduction in target cells

PDF

Novel roles for Maf1 in embryonic stem cell differentiation and adipogenesis

PDF

Dendritic cell-specific vaccine utilizing antibody-mimetic ligand and lentivector system

PDF

Exploring stem cell pluripotency through long range chromosome interactions

PDF

Using engineered exosomes and gene-editing to target latent HIV

Asset Metadata

Creator Lei, Yuning (author)

Core Title Engineering viral vectors for gene and cell targeting

School Andrew and Erna Viterbi School of Engineering

Degree Doctor of Philosophy

Degree Program Chemical Engineering

Publication Date 01/25/2011

Defense Date 11/18/2010

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

Tag baculoviral vector,CD20 targeting,cell targeting,gene targeting,genetic modification,human embryonic stem cells,lentiviral vector,OAI-PMH Harvest,zinc finger nuclease

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Wang, Pin (committee chair), Arnold, Donald B. (committee member), Shing, Katherine S. (committee member)

Creator Email yuning2000@gmail.com,yuningle@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3627

Unique identifier UC1140997

Identifier etd-Lei-4245 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-430755 (legacy record id),usctheses-m3627 (legacy record id)

Legacy Identifier etd-Lei-4245.pdf

Dmrecord 430755

Document Type Dissertation

Rights Lei, Yuning

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