Study of dendritic cell targeting by engineered lentivectors

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STUDY OF DENDRITIC CELL TARGETING BY ENGINEERED LENTIVECTORS

by

April M Tai

________________________________________________________________________

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 2012

Copyright 2012 April M Tai
ii

ACKNOWLEDGEMENTS


First, I would like to convey my deepest gratitude to my advisor, Dr. Pin Wang,
for providing instruction and guidance to me during every step of this process. I would
not have been able to get this far without his direction. I would also like to thank the
rest of my committee, Dr. Katherine Shing and Dr. Tzung Hsiai, for all the time and
energy they provided to help me.
I would not have been able to complete these projects without the assistance
and knowledge from all of my fellow lab members, particularly Kyeil Joo, Steven
Froelich, Haiguang Yang, and Rigzen Aulakh. They all exhibited overwhelming patience
and kindness towards me.
Lastly, I would like to thank my family: my parents and my brother for their
persistent and unwavering support during this endeavor, P. Tai and M. Hsu for their
comfort and company during the long days and nights, and my husband Jack for doing
everything in his power to make this process as painless as possible—his
encouragement and patience were invaluable.

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

LIST OF TABLES vi

LIST OF FIGURES vii

ABSTRACT xii

CHAPTER 1: INTRODUCTION 1
1.1 Gene Therapy 1
1.2 Gene Delivery Vectors 2
1.2.1 Retroviral Vectors 3
1.2.2 Lentiviral Vectors 6
1.3 Transcriptional Targeting 7
1.4 Transductional Targeting 14
1.4.1 Pseudotyping 14
1.4.2 Bifunctional Adaptors 16
1.4.3 Insertion of targeting molecules 18
1.4.4 Rational point and domain mutagenesis 20
1.5 Two‐molecule method for engineering targeting lentiviral vectors 21
1.6 Overview 22
1.7 Chapter References 24

CHAPTER 2: PRODUCTION OF LENTIVIRAL VECTORS WITH ENHANCED
EFFICIENCY TO TARGET DENDRITIC CELLS BY ATTENUATING MANNOSIDASE
ACTIVITY OF MAMMALIAN CELLS 43
2.1 Introduction 45
2.2 Results and Discussion 49
2.2.1 Transient transfection of 293T cells to produce SVGmu‐
pseudotyped LVs 49
2.2.2 Verification of SVGmu and high‐mannose oligosaccharides on the
vector surface 51
iv

2.2.3 Evaluation of cell‐virus binding to hDCSIGN 53
2.2.4 Transduction of cells with LVs produced with DMJ 57
2.2.5 Transduction of a DC cell line with vectors produced with DMJ 60
2.3 Conclusions 62
2.4 Materials and Methods 63
2.5 Acknowledgements 66
2.6 Chapter References 67

CHAPTER 3: VISUALIZATION OF DC‐SIGN‐TARGETED TRANSDUCTION
BY ENGINEERED LENTIVIRAL VECTORS 71
3.1 Introduction 72
3.2 Results 74
3.2.1 Clathrin‐dependent endocytosis 74
3.2.2 Viral transport is microtubule‐mediated 76
3.2.3 Fusion tracking 78
3.2.4 Tracking with endosomal markers 81
3.2.5 Effect of autophagy on viral infection 86
3.3 Discussion 90
3.4 Materials and Methods 92
3.5 Chapter References 97

CHAPTER 4: TRANSDUCTION OF DENDRITIC CELLS BY A ROSS RIVER
VIRUS‐PSEUDOTYPED LENTIVECTOR 102
4.1 Introduction 103
4.2 Results 104
4.2.1 Design and production of the RRV‐G‐pseudotyped LV 104
4.2.2 Transduction of 3T3 cells expressing cell‐surface DC‐SIGN and L‐SIGN 107
4.2.3 Virus binding to cells is increased in the presence of DC‐SIGN or
L‐SIGN molecules and under conditions of limited carbohydrate
processing 110
4.3 Discussion 112
4.4 Materials and Methods 116
4.5 Chapter References 120

v

COMPREHENSIVE REFERENCES 126

APPENDIX: LENTIVIRAL VECTORS FOR IMMUNE CELLS TARGETING 162

vi

LIST OF TABLES

Table 1.1: Tumour‐specific promoters (Robson and Hirst, 2003) 10

Table 1.2: Tumour environment‐specific promoters
(Robson and Hirst, 2003) 12

Table 1.3: Exogenously controlled inducible promoters 13

vii

LIST OF FIGURES

Figure 1.1: Schematic of retroviral and lentiviral particles
(Schaffer et al., 2008) 4

Figure 1.2: The use of specific tissue/tumour/inducible promoters to
allow targeted transcription in tumour cells (Robson and
Hirst, 2003) 7

Figure 2.1: Chemical structure of DMJ and its inhibition mechanism. (A)
Comparison of the chemical and structural composition of DMJ
to that of mannose. (B) Schematic diagram of the mechanism by
which DMJ inhibits class I α1,2‐mannosidase in the Golgi. α1,2‐
mannosidase I normally trims the α1,2‐linked mannose on
glycoproteins; however, DMJ inhibits α1,2‐mannosidase I activity
and thus restricts the oligosaccharide processing to high‐mannose
forms 48

Figure 2.2: DMJ does not reduce SVGmu production or display on vector‐
producing cells. 293T cells were transiently transfected by the
lentiviral backbone vector encoding the GFP gene (FUGW),
packaging constructs (REV and RRE), and a plasmid encoding either
SVGmu or VSVG. SVGmu‐staining and flow cytometry analysis of the
cells two days post‐transfection revealed that cells cultured with or
without DMJ exhibited similar levels of SVGmu and GFP, indicating
that the presence of DMJ did not restrict either glycoprotein or LV
production. Control cells transfected by VSVG were similarly
unaffected by DMJ and SVGmu‐negative, as expected 50

Figure 2.3: SVGmu is incorporated onto the vector surface for LVs
produced with or without DMJ. Fresh viruses were produced
with the addition of an additional plasmid, GFP‐Vpr to fluorescently
label the vector core. Vectors produced with or without DMJ were
then stained for SVGmu and analyzed by confocal microscopy.
Visualization of the vector particles showed that SVGmu was
efficiently incorporated in both types of vectors. VSVG‐
pseudotyped vectors was included as a control 52
viii

Figure 2.4: LVs produced in DMJ contain more high‐mannose structures.
Vectors were produced with or without DMJ, concentrated, and
digested by EndoH, an enzyme that cleaves high‐mannose
structures. A western blot analysis of the undigested and
digested vectors showed that the vectors produced in DMJ had a
lower molecular weight after EndoH digestion compared to
that of the vectors produced without DMJ. These results infer that
the vector produced with DMJ had more high‐mannose structures
than the vector produced without DMJ 53

Figure 2.5: LVs produced in DMJ bind more readily to human DCSIGN
than vectors produced without DMJ. (A) Vectors produced with
or without DMJ were incubated with fixed 293T.DCSIGN cells, a
cell line that stably displays human DCSIGN. Flow cytometric
analysis of SVGmu‐stained cells revealed that the vectors produced
in DMJ bound to the DCSIGN‐expressing cells over 6 times more
readily than the vectors produced without DMJ. (B) Vectors were
labeled with [35S]‐Trans and incubated with either 293T or
293T.DCSIGN cells. A mannose inhibition assay was also included to
determine the dependency of vector‐receptor binding on the
mannose‐rich structures on the vector. Radioactivity analysis
revealed that the vector produced with DMJ bound much more
readily to the 293T.DCSIGN cells than to the 293T cells, while
the addition of mannose greatly reduced cell‐vector
binding. Vectors produced without DMJ did not exhibit as
much of a difference between binding to the 293T.DCSIGN
cells and the 293T cells, while the presence of mannose reduced
vector levels in both of the cell lines 55

ix

Figure 2.6: LVs produced with DMJ transduced DCSIGN‐expressing cells
much more efficiently than LVs produced without DMJ. (A) LVs
were produced either with or without DMJ and spin‐infected with
293T and 293T.DCSIGN cells. Although FUGW/SVGmu(DMJ–)
preferentially transduced 293T.DCSIGN cells,
FUGW/SVGmu(DMJ+) was over three‐fold more efficient in
transducing 293T.DCSIGN cells while maintaining similar
levels of background transduction to 293T cells. (B) LVs produced
with DMJ yielded higher vector titers for DCSIGN‐expressing cells
compared with LVs produced without DMJ. On 293T.DCSIGN
cells, FUGW/SVGmu(DMJ+) yielded titers over three‐folds higher
than those of FUGW/SVGmu(DMJ–), while titers for 293T cells for
both vectors were similarly low. FUGW/VSVG +/‐ DMJ was included
as a control 58

Figure 2.7: LVs produced with DMJ can transduce a DC cell line more
efficiently than LVs produced without DMJ. A human DC cell line
(MUTZ‐3) was employed to more accurately model human DCs,
as a target for LVs produced with and without DMJ. (A) MUTZ‐3
cells were differentiated for 7 days and stained for DC‐SIGN
expression. (B) The MUTZ‐3 cells were transduced by LVs
produced with or without DMJ. FUGW/SVGmu(DMJ+) transduced
the MUTZ‐3 cells about 25% more efficiently than
FUGW/SVGmu(DMJ–). FUGW/VSVG was included as a
positive control 61

Figure 3.1: Drug treatment to block clathrin‐ and caveolin‐mediated
endocytosis of SVGmu‐ and SFV‐pseudotyped lentiviral vectors, and
dominant‐negative mutant test for dynamin 75

Figure 3.2: Confocal imaging of FUW/SVGmu/GFPVpr viruses (green) with
cells stained for clathrin and caveolin. White arrows indicate the
location of viral particles 76

Figure 3.3: Drug treatments for the effect of actin filaments and
microtubules on viral transduction 77

Figure 3.4: Visualization of virus colocalization with actin
filaments (A) and microtubules (B). The viral particles appear
green through GFPVpr expression 78

x

Figure 3.5: Drug inhibition of low pH‐mediated endosomal fusion 79

Figure 3.6: Representative images of fusion events taken at different
timepoints and quantification of imaging results 80

Figure 3.7: Imaging viral fusion events after NH
4
Cl treatment 81

Figure 3.8: Representative images of cells stained with endosomal
markers at different timepoints and quantification of the
imaging results 83

Figure 3.9: Effect of dominant‐negative mutants on viral transduction
efficiency 85

Figure 3.10: Representative images of virus colocalization with a
lysosome marker 86

Figure 3.11: Effect of autophagy on viral infectivity. Rapamycin enhances
autophage activity while 3‐MA inhibits autophagy 88

Figure 3.12: Imaging and quantification of viruses colocalized with
autophage and lysosome markers in cells treated with rapamycin 89

Figure 4.1: Schematic diagram of the constructs encoding the lentiviral
backbone plasmid FUGW and RRE‐G 105

Figure 4.2: Confocal imaging of viruses stained with glycoprotein
antibodies. Green indicates the GFP+ viruses while red indicates
the stained glycoproteins 106

Figure 4.3: Comparison of viral titers against 293T or 293T.DCSIGN, with
FUGW/RRVG produced with or without DMJ 107

Figure 4.4: Transduction efficiency of RRV‐G‐ and VSV‐G‐pseudotyped
LVs produced with or without DMJ, against the 3T3 cell line
expressing DCSIGN or LSIGN 108

Figure 4.5: Infection of MoDC with RRV‐G‐pseudotyped LV produced with
or without DMJ 110

xi

Figure 4.6: Radiolabeled binding of RRV‐G‐pseudotyped virus compared
to VSV‐G‐pseudotyped LV 111

Figure A.1: Schematic of the retrovirus structure and a lentiviral
vector backbone plasmid, FUW 166

xii

ABSTRACT


Dendritic cell (DC) vaccines have great potential as an emerging form of
immunotherapy, as DCs are potent antigen‐presenting cells, capable of triggering T cell
and B cell responses. Our lab has previously developed an engineered lentiviral vector
(LV) that is pseudotyped with a mutated Sindbis virus glycoprotein (SVGmu), which is
capable of targeting DCs through Dendritic Cell‐specific ICAM3‐grabbing Nonintegrin
(DC‐SIGN), a receptor that is predominantly expressed by DCs.
We hypothesized that SVGmu interacts with DC‐SIGN in a mannose‐dependent
manner, and that increasing the amount of high‐mannose structures on the
glycoprotein surface could result in higher targeting efficiencies of LVs towards DCs. It is
known that 1‐deoxymannojirimycin (DMJ) can inhibit α1,2‐mannosidase I, which is an
enzyme that removes high‐mannose structures during the glycosylation process. Thus,
we investigated the possibility of generating LVs with enhanced capability to modify DCs
by supplying DMJ during vector production. Through western blot analysis and binding
tests, we were able to infer that binding of SVGmu to DC‐SIGN is directly related to
amount of high‐mannose structures on SVGmu. We also found that the titer for the LV
produced with DMJ (FUGW/SVGmu + DMJ) on 293T.DCSIGN, a human cell line
expressing the human DC‐SIGN antibody, was over four times higher than that of vector
xiii

produced without DMJ. In addition, transduction of a human DC cell line, MUTZ‐3,
yielded a higher transduction efficiency for the LV produced with DMJ.
In our next study, we aimed to elucidate the internalization and trafficking
mechanisms of this viral vector through confocal microscopy of GFP‐Vpr‐tagged virus,
drug treatments, and dominant‐negative mutants of GTPases, which are necessary for
endosomal functions. Using these tests, we demonstrated that our engineered lentiviral
vector enters the cell via receptor‐mediated clathrin‐ and dynamin‐dependent
endocytosis, and that microtubule networks were also involved in a productive
infection. Fusion was low‐pH‐dependent and occurred in the early endosomal stage of
transport. Autophagy was also examined for its effect on transduction efficiency. We
observed that enhanced autophage activity reduced viral infectivity, while suppressed
autophagy boosted transduction efficiency. This study gives us insight on the
internalization and trafficking mechanisms used by our engineered vector and gives us
tools to improve the efficiency of this platform.
In our last study, we examined the ability of lentiviruses enveloped with an
alphaviral envelope glycoprotein derived from Ross River virus (RRV) to mediate
transduction of DCs. We found that RRV was only able to specifically mediate
transduction of cells through DC‐SIGN when the viral vectors were produced under
conditions limiting glycosylation to high‐mannose glycans. This suggests that these RRV‐
pseudotyped LVs can be used for DC‐targeting, but would require specific conditions
during vector propagation for effective targeting infections.
1

CHAPTER 1: INTRODUCTION

1.1 Gene Therapy
The delivery of genes of interest to cellular targets has been studied intensively
over the past twenty years in hopes that autoimmune diseases such as Parkinson’s
disease and severe combined immunodeficiency (SCID), as well as cancers, may be
cured. The one of the first gene therapy procedures was performed on a four‐year old
girl in 1990 by Dr. William French Anderson and his colleagues (Anderson et al., 1990).
The patient was born with SCID, which required her to live in relative isolation with
frequent bouts of illnesses and large amounts of antibiotics. For the procedure, the
patient’s white blood cells were removed and grown in the lab. Then the missing genes
were inserted into the cells and the cells were infused back into the patient’s
bloodstream. After this, the patient’s immune system was strengthened by 40% and she
was able to attend school. Although the procedure was not permanent and she had to
have it repeated every few months, she was in good health as of early 2007 and was
able to attend college. There has been some debate whether these results were
obtained solely by the gene therapy procedure versus the other treatments she had
undergone, however, this study was at least able to demonstrate that gene therapy
could be administered to a patient without adverse consequences (Thrasher et al.,
2006).
2

1.2 Gene Delivery Vectors
There are two methods by which these genes may be delivered. First, synthetic
delivery vehicles can be used. These usually consist of either lipids or polymers which
surround the DNA, termed lipoplexes (Dass, 2002) and polyplexes, respectively. There
are three types of lipoplexes: anionic, neutral, and cationic. Anionic and neutral lipids
were initially preferred since they were safer, were more compatible with body fluids,
and had the potential for tissue‐specific gene transfer; however, production of these
lipoplexes was difficult and expression in transduced cells was relatively low (Gardlik et
al., 2005). Cationic liposomes, on the other hand, naturally form complexes with DNA,
which is negatively charged. The positive charge also facilitates the penetration of the
complexes into the negatively charged cellular membrane. Most polyplexes are also
created with cationic polymers. Unlike lipoplexes, though, some polyplexes are unable
to deposit the DNA into the cytoplasm (Forrest and Pack, 2002). Thus, these polyplexes
need to be co‐transfected with endosome‐lytic agents, such as inactivated adenovirus,
to be effective. Although synthetic delivery vehicles have advantages such as safety and
modularity, they are usually too inefficient for clinical applications.
The second type of gene delivery vehicle is the viral vector. These exploit the
natural ability of viruses to efficiently deliver genetic materials to specific cells. With the
tools available to modern biology, therapeutic transgenes can be easily swapped with
the original viral genes, resulting in specialized gene delivery vehicles. Several in vivo
studies, animal disease models, and even clinical trials have been successfully conducted
3

using viral delivery vectors, mostly using adenoviral, adeno‐associated, retroviral, or
lentiviral viral vectors (Edelstein et al., 2007). However, several factors limit the efficacy
of viral vectors for gene delivery. First, systemic barriers, such as pre‐existing immunity,
(Chirmule et al., 1999; Kostense et al., 2004; Muruve, 2004) and cellular barriers, such as
binding to the cell surface, (Howitt et al., 2003; Smith‐Arica et al., 2003) hinder the
efficient delivery of genes into the cells. Also, inefficient production and purification of
the viral vectors (Clemens et al., 1996; Dong et al., 1996), as well as poor transduction
efficiency to the therapeutically relevant cells (Shenk, 2006; Smith‐Arica et al., 2003) are
other barriers that must be overcome for viral gene delivery vectors.

1.2.1 Retroviral Vectors
Retroviruses are enveloped viruses with diploid, single‐stranded, 7‐12 kb positive
sense RNA genomes (Coffin, 1997). This genome contains gag, which encodes the
structural proteins (matrix protein, capsid protein, and nucleocapsid protein), pro and
pol, which encodes enzymatic proteins (protease, reverse transcriptase, and integrase),
and env, which encodes the surface and transmembrane units of the envelope protein
(Figure 1.1)(Schaffer et al., 2008).
4

Figure 1.1 Schematic of the structure of the viral particle and organization of the viral
genome. Representation of key components of retroviral and lentiviral particles along
with the genome organization of each virus (Schaffer et al., 2008).

The genetic material is contained in the nucleocapsid, which is enveloped by a bilipidic
membrane taken from the virus‐producing cell. The glycoproteins are inserted into this
membrane and are responsible for binding the virions to receptors on the cell surface.
The interaction between the envelope glycoprotein and cellular receptors is what
determines viral tropism. Binding leads to conformational changes in the viral envelope
glycoprotein, exposing the hydrophobic fusion peptide. When inserted into cellular
membrane, this peptide will mediate fusion between the viral and cellular membranes.
After fusion occurs, the core nucleoprotein complex is released into the cytoplasm and
5

reverse transcription of the viral genome into DNA occurs. This newly synthesized
double‐stranded DNA is then transported into the nucleus and integrated into to the
host chromosome.
Since retroviruses are able to accommodate extensive changes to their genomes
and integrate efficiently into the genomes of their host cells, they were excellent
candidates for gene transfer vectors. The first retroviral vectors were produced with the
help of replication‐competent or helper viruses. In 1983, retroviral packaging cells were
created that supplied the retroviral proteins but did not produce replication‐competent
viruses. Later, all the viral coding regions were deleted and only the vital viral elements
for high‐efficiency transfer were retained. Since viral entry, reverse transcription, and
genome integration are not dependent on the synthesis of viral proteins, the viral
vectors can contain only the genes of interest.
Retroviral vectors hold many advantages over other gene delivery methods.
First, they can transduce many different types of cells from different species. They can
also integrate their genetic payload into cells with precision and with high expression
levels. Since the virus had been engineered to be replication‐incompetent empty
vectors, there is no danger of the vector spreading to other cells, or of viral proteins
being produced after transduction. Lastly, retroviral vectors have a relatively large
payload capacity and offer low immunogenicity (Coffin, 1997). Drawbacks of retroviral
vectors include the difficulty of efficiently producing vectors, the lack of stability in the
6

envelope proteins, and the risk of insertional mutagenesis due to the semirandom
integration of genes (Hacein‐Bey‐Abina et al., 2003).

1.2.2 Lentiviral Vectors
A subclass of retroviruses that has become studied as another vehicle for gene
delivery is the lentivirus. Lentiviral vectors are derived from the human
immunodeficiency virus (HIV) and are capable of infecting non‐dividing cells through
mitosis‐independent transport of the viral DNA into the nucleus (Lewis et al., 1992;
Weinberg et al., 1991). This feature is particularly useful for gene transfer to non‐
dividing cells, such as hematopoietic stems cells, monocytes, and neurons (Case et al.,
1999; Naldini et al., 1996; Weinberg et al., 1991). Lentiviral vectors also do not tend to
integrate by transcriptional initiation sites, a problem faced by other viral vectors (De
Palma et al., 2005). However, like retroviral vectors, high‐titer production of lentiviral
vectors has been difficult due to the complex nature of the virus. To combat this
problem, vectors have been produced which contain the vesicular stomatitis virus (VSV)
envelope protein with the HIV core proteins. This has resulted in higher‐titer vector
production, as well as higher transduction efficiencies. Development of the lentiviral
vector has been geared towards improving safety by reducing the risk of insertional
mutagenesis. First, the vector was made self‐inactivating (SIN) and the enhancer in the
LTRs has was deleted (Frecha et al., 2008). Second, to prevent read‐through transcripts,
a strong RNA polyadenylation sequence was added to the vector sequence (Zaiss et al.,
7

2002). Lastly, DNA insulators were inserted into the LTR to isolate the internal promoter
from the neighboring genome (Emery et al., 2002).

1.3 Transcriptional Targeting
One way to mediate targeted gene therapy is by using promoters that will only
allow gene expression in target cells. This is demonstrated schematically in Figure 1.2.

Figure 1.2 The use of specific tissue/tumour/inducible promoters to allow targeted
transcription in tumour cells. Tissue‐specific, tumour‐specific, or inducible (eg, by
radiation, drugs, etc) promoters can limit gene expression to target cells which express a
specific transcription factor, or in which the transcription factors are activated
exogenously (Robson and Hirst, 2003).

Since factors specific to different cell types regulate gene expression, transcriptional
targeting can be another useful tool to allow viral vectors to target specific cell types.
8

One way to do this is through tissue‐specific promoters, which are only switched on in
certain tissues. However, this method is has limitations in tumor tissue targeting, since
transgene expression can cause cytotoxic effects in both the normal and tumor cells
derived from the same type of tissue (Robson and Hirst, 2003). Thus, only tissues where
damage to the cells do not put the survival of the host in jeopardy, such as the prostate,
melanocytes, or thyroid, can be targeted therapeutically (Robson and Hirst, 2003).
Other tumors require the direct delivery of the genes to the tumor site or with
retroviruses that are less able to transduce slowly dividing normal tissues (Robson and
Hirst, 2003).
Tissue‐specific promoters have been found and studied for melanocytes,
prostate cells, glial cells, liver cells, and the thyroid. Since cancers cannot be targeted
with tissue‐specific promoters due the effect on normal tissue cells, the telomerase
promoter has been studied as a cancer‐specific promoter. Telomerase is important in
cancer progression because it is a key to cellular immortalization. About 90% of human
cancers have active telomerase activity while the normal tissue counterparts do not
(Broccoli et al., 1995; Kim et al., 1994; Tahara et al., 1999). Tumor‐specific promoters
have also been studied and these include ones that are cancer‐specific and tumor‐type‐
specific. Specific promoters include: carcinoembryonic antigen (CEA) (expressed in
adeno‐carcinomas) (Brand et al., 1998; Cao et al., 1999a; Cao et al., 1999b; Cao et al.,
1998; DiMaio et al., 1994; Humphreys et al., 2001; Koch et al., 2001; Lan et al., 1997;
Nishino et al., 2001; Nyati et al., 2002; Osaki et al., 1994; Richards et al., 1995; Tanaka et
9

al., 2000; Tanaka et al., 1997; Tanaka et al., 1996), α fetoprotein (AFP) (expressed in
hepatocellular carcinomas) (Ido et al., 1995; Ido et al., 2001; Ishikawa et al., 1999;
Ishikawa et al., 2001; Kanai et al., 1997; Kaneko et al., 1995; Maxwell et al., 1996; Su et
al., 1996; Wills et al., 1995), ErbB2 (expressed in a third of breast and pancreatic tumors)
(Harris et al., 1994; Hernandez‐Alcoceba et al., 2001; Maeda et al., 2001; Sikora et al.,
1994; Stackhouse et al., 1999; Takakuwa et al., 1997; Zheng et al., 2000), MUC1/DF3
(overexpressed in breast and cholangiocarcinomas) (Chen et al., 1995; Chen et al., 1996;
Manome et al., 1994; Patterson and Harris, 1999; Stackhouse et al., 1999; Tai et al.,
1999; Zaretsky et al., 1999), Osteocalcin (highly expressed in osteogenic sarcomas, as
well as ovarian, lung, brain, and prostate tumors) (Benjamin et al., 2001; Cheon et al.,
1997; Chung et al., 1999; Ko et al., 1996; Koeneman et al., 2000; Matsubara et al., 2001;
Shirakawa et al., 1998; Yeung et al., 2002), L‐plastin (expressed at high levels in
malignant epithelial cells) (Chung et al., 1999; Peng et al., 2001), and Midkine
(transiently expressed at early stage of embryonal carcinoma cell differentiation , as well
as in many malignant tumors) (Adachi et al., 2002; Adachi et al., 2000; Casado et al.,
2001; Miyauchi et al., 1999; Wesseling et al., 2001). Other tumor‐type‐specific
promoters can target cervical carcinoma cells, lung, breast oropharyngeal, bladder,
endometrial, and colorectal carcinomas (Garver et al., 1994). Cell cycle‐related
promoters can also target cancer cells and tumor endothelial cells. The loss of activity of
the retinoblastoma tumor‐suppressor gene RB allows E2F‐responsive promoters to be
active (Robson and Hirst, 2003). Targeting with these promoters, therefore, has been
10

shown to be able to eradicate established tumors (Parr et al., 1997). Table 1.1 lists some
of the tumor‐specific promoters that have been studied.
Table 1.1 Tumour‐specific promoters (Robson and Hirst, 2003).

11

There are also tumor environment‐specific promoters that result from the
upregulated genes of tumor cells. Since the growth of tumors requires the proliferation
of endothelial cells, promoters of genes that are upregulated in this process, such as the
kinase insert domain receptor (KDR/flk‐1) (Heidenreich et al., 2000; Jaggar et al., 1997;
Modlich et al., 2000; Szary et al., 2001) and E‐selectin (Jaggar et al., 1997; Modlich et al.,
2000; Walton et al., 1998), are attractive for use as transcriptional targets (Robson and
Hirst, 2003). Another type of tumor environment‐specific promoter results from the low
oxygen tension, or hypoxia, present in the disorganized and inadequate tumor
vasculature. Promoters that have been used include that of vascular endothelial growth
factor (VEGF) (Ido et al., 2001; Koshikawa et al., 2000; Shibata et al., 1998; Shibata et al.,
2000; Shibata et al., 2002), erythropoietin (Epo) (Ruan et al., 2001), and
phosphoglycerate kinase‐1 (PGK‐1) (Dachs et al., 1997; Zhang et al., 2002). The targeting
of these cells is especially important because do not respond to other forms of
treatment such as radio and chemotherapy (Robson and Hirst, 2003). Tumors are also
usually deprived of glucose, which causes an upregulation of genes that take part in
glucose metabolism (Robson and Hirst, 2003). Glucose‐responsive promoters
successfully utilized include GRP78 (Little et al., 1994) and hexokinase II (Katabi et al.,
1999). A summary of tumor environment‐specific promoters is shown in Table 1.2.

12

Table 1.2 Tumour environment‐specific promoters (Robson and Hirst, 2003).

Exogenously controlled inducible promoters are another means to control
transcriptional targeting for cancer gene therapy. Radiation‐inducible promoters are
attractive because radiation therapy is widely used for cancer treatment. Some
radiation‐inducible promoters include the EGR1 promoter (Hallahan et al., 1995;
Hallahan et al., 2001; Hanna et al., 1996; Kaminski et al., 2001; Kawashita et al., 1999;
Manome et al., 1998; Marples et al., 2002; Mauceri et al., 1996; Mauceri et al., 1997;
Scott et al., 2000; Seung et al., 1995; Weichselbaum et al., 1994; Weichselbaum et al.,
2001) and the WAF‐1 promoter (Worthington et al., 2000; Worthington et al., 2002).
Since hyperthermia can also improve tumor responses to radiation and
chemotherapeutic treatments (Nielsen et al., 2001), heat‐inducible promoters, or the
heat shock protein (HSP) promoters, are interesting tools as well. HSP70B (Brade et al.,
2000; Braiden et al., 2000; Huang et al., 2000; Lee et al., 2001; Lohr et al., 2000; Lohr et
al., 2001; Madio et al., 1998; Vekris et al., 2000) and Gadd 153 (Ito et al., 2001) are heat‐
13

inducible promoters that have been studied for use with breast, melanoma, and
prostate cancers (Robson and Hirst, 2003). Lastly, strong and can be exogenously
regulated drug‐inducible promoters can be used for transcriptional targeting of cancer
cells (Robson and Hirst, 2003). One example is the tetracycline (tc)‐inducible promoter,
which has been used in breast, melanoma, brain, glioma, and prostate cancers (Baasner
et al., 1996; Chen et al., 1998; Giavazzi et al., 2001; Iida et al., 1996; Maxwell et al.,
1996; Patil et al., 2000; Paulus et al., 1997; Pitzer et al., 1999; Rubinchik et al., 2001; Yao
et al., 1998; Yu et al., 1996). Table 1.3 summarizes the different exogenously‐controlled
inducible promoters that are available for cancer therapy.
Table 1.3 Exogenously controlled inducible promoters (Robson and Hirst, 2003).

The many studies and successes with transcriptional targeting certainly encourage
further research in this area.

14

1.4 Transductional Targeting
1.4.1 Pseudotyping
Another way to target specific cell types is to selectively transduce those cells
with the viral vector. Several different methods have been developed to do this.
Pseudotyping—enveloping the viral vectors with glycoproteins of other viruses to alter
the vector tropism—is one popular and straightforward technique (Cone and Mulligan,
1984; Cronin et al., 2005). The glycoprotein from VSV (VSVG) has been used to
pseudotype retroviral and lentiviral vectors to extend the tropism, as well as enhance
vector stability (Burns et al., 1993; Pan et al., 2002). VSVG seems to attain its broad
tropism by using ubiquitous lipid‐type receptors to transduce cells (Schaffer et al.,
2008). Pseudotyping with other viral glycoproteins confers varying cellular tropisms. For
example, pseudotyping lentiviral vectors based on feline immunodeficiency virus (FIV)
with Ross River virus (RRV) envelope proteins resulted in specific transduction of
astrocytes and oligodendrocytes (Kang et al., 2002), while peudotyping the viruses with
lymphocytic choriomeningitis virus (LCMV) glycoproteins resulted in transduction of
neural stem cells or progenitor cells (Stein et al., 2005). In the same system,
pseudotyping the lentiviral vector with VSVG resulted in the preferential transduction
for mature neurons (Stein et al., 2005). Thus, by only changing the envelope protein
through pseudotyping with different viral glycoproteins, the tropism of the viral vector
can be greatly altered. Pseudotyping vectors with glycoproteins of viruses that have
unique tropisms specific for gene therapy targets enables gene delivery to cell types or
15

tissues that are otherwise difficult to transduce. One such target is polarized airway
epithelial cells from the apical side. Although VSVG‐pseudotyped lentivectors can only
infect the airway epithelial cells through the basolateral side with low efficiency
(Johnson et al., 2000), pseudotyping the vectors with glycoproteins of viruses that
naturally infect respiratory tissues, such as Ebola virus and respiratory syncytial virus
(RSV), can allow the vectors to transduce the cells from the apical side (Kobinger et al.,
2001). In intramuscular injections, lentiviral vectors pseudotyped with VSVG will directly
transduce the muscle cells locally, while vectors pseudotyped with rabies envelope
proteins will be transported to the spinal cord (Mazarakis et al., 2001).
Viral vectors are also able to efficiently incorporate and display proteins that are
overexpressed by the producer cells, which allows for many possible applications.
Studies have shown that the HIV‐1 primary receptors and coreceptors, CD4 and CCR5,
CD4 and CXCR4, or hybrid receptors, incorporated onto lentiviral and retroviral surfaces
allowed the vectors to infect cells with the HIV‐1 envelope protein, as well as cells that
were infected by HIV‐1 (Endres et al., 1997; Somia et al., 2000). Another study showed
that vectors incorporating receptors for Rous sarcoma virus and ecotropic MLV were
able to selectively transduce cells with the corresponding envelope proteins (Balliet and
Bates, 1998). However, vectors pseudotyped in this manner generally have 10‐ to 100‐
fold lower titers than those pseudotyped with the common envelope proteins (Balliet
and Bates, 1998).

16

1.4.2 Bifunctional Adaptors
Another way to transductionally target specific cell types is through using
bifunctional adaptors, which bind to the vectors and have novel functional domains.
Thus, the attachment of these adaptors to the viral surface confers their capabilities to
the vector as well. One way to create these adaptors is by chemically modifying the viral
surface. For example, VSVG‐pseudotyped vectors can be neutralized by their
complements (Croyle et al., 2004); however, by PEGylating the VSVG, VSVG‐
pseudotyped lentivectors are 1000‐fold more protected against serum‐mediated
inactivation (Croyle et al., 2004). Chemical modification of surface glycoproteins can also
allow the vectors to target specific cells types. Galactose‐tagging of ecotropic MLV
vectors allows for specific targeting of asialo‐glycoprotein receptors on a human
hepatoma cell line. These receptors specifically bind to oligosaccharides with terminal
galactose residues (Neda et al., 1991).
Bispecific linker molecules are another kind of adaptor can also be used to target
vectors to specific cell types. These molecules are generally fusion proteins that have a
viral receptor to interact with the envelope protein on the vector particle, and a ligand
that is able to recognize its cognate receptor to target specific cell types (Snitkovsky and
Young, 1998). Researchers had created fusion linkers with either the avian leucosis virus
(ALV) A or B receptor with the EGFP protein. These linkers allowed ALV‐A or ALV‐B
vectors to efficiently transduce cells that expressed EGFR (Snitkovsky and Young, 1998).
Other systems use three linker molecules: two biotinlyated antibodies that are specific
17

for the vector glycoprotein and a surface molecule on the target cells. When these
molecules are connected by streptavidin, the vectors could specifically transduce cells
that expressed major histocompatibility complex class I and II proteins (Roux et al.,
1989).
Linkers can also be directly inserted into envelope proteins, in the receptor
binding domain. Researchers have inserted the ZZ domain into the Sindbis virus E2
glycoprotein to allow targeting antibodies against CD4 or human leukocyte antigen
(HLA) to be incorporated onto the vector surfaces (Morizono et al., 2001). This resulted
in specific transduction of CD4
+
and HLA
+
cells by retroviral vectors (Morizono et al.,
2001). Since the E1 protein is responsible for fusion in the Sindbis envelope protein, and
is independent from the receptor‐binding E2 protein, the insertion of the ZZ domain into
the E2 protein did not hamper the fusogenic activity of the vector. In another study, the
mutation of the non‐specific binding regions of the E2 protein, in conjunction with the
insertion of the ZZ domain, allowed E2 protein‐pseudotyped lentiviral vectors to
specifically transduce metastatic melanoma in the lung in vivo (Morizono et al., 2005).
Some challenges that still remain for the use of bifunctional adaptors in
biological systems include: the dependency of chemical modifications on reaction
conditions and the limitations of controlled site‐specific methods to reduce unwanted
side effects, the dissociation of virus‐adaptor linkages in vivo, the competition between
endogenous antibodies in blood and mAb‐based adaptors, and the difficulty of clinical
approval for studies, which includes the production, purification, and characterization of
18

both the virus and the adaptor (Schaffer et al., 2008). However, despite these
limitations, the studies performed have been shown some promising results and should
be further examined.

1.4.3 Insertion of targeting molecules
Targeting molecules can also be directly incorporated into viral structural
proteins. Short peptides can be inserted into viral envelope proteins without adversely
affecting the protein function. This method has been used to allow the targeted
infection of human melanoma cell lines, and attenuation of the natural tropism of MLV
vectors (Gollan and Green, 2002). However, it has also been shown that insertion of
these peptides could disrupt the trafficking of the envelope proteins in the virus‐
producing cells. Researchers who inserted a peptide into VSVG found that envelope
protein trafficking to the cell surface was blocked at 37°C (Guibinga et al., 2004).
Ligands can also be inserted into envelope proteins with some success (116‐119).
Ligand domains have been incorporated into amphotropic MLV glycoproteins with a
MMP‐cleavable linker, which blocks viral entry. Using these chimeric envelope proteins
to pseudotype retroviral proteins allowed the specific transduction of cells that
expressed MMP, which cleaved off the blocking domain (Peng et al., 1999). However,
the binding of the vector to the target receptors might not be strong enough to cause
the necessary conformational changes in the glycoprotein, which are required for viral
fusion and successful transduction (Lavillette et al., 2001). This reduction in vector
19

efficiency can be amended by incorporating wt envelope proteins into the viral vectors
(Kasahara et al., 1994). HA proteins, mutated to remove binding to sialic acid but
retaining their fusion capabilities, could be added to vectors with MLV envelope
proteins with N‐terminal Flt‐3 ligands to produce vectors capable of efficiently and
specifically transducing cells with Flt‐3 receptors (Lin et al., 2001).
Single‐chain antibodies (scFvs), such as human low‐density lipoprotein receptor
(LDLR)‐specific scFvs, have been inserted into envelope proteins to specifically transduce
cells expressing the cognate receptors, such as LDLR (Somia et al., 1995). Some studies
used ecotropic MLV envelope proteins as a scaffold to insert scFv‐targeting domains to
increase specificity in the transduction of human cells (Marin et al., 1996). These
antibody fragments are usually inserted with a spacer peptide into the N‐terminal
region of envelope proteins. This allows both the scFv domain and the envelope protein
to be in the proper conformations. The advantages of using these scFv fragments to
target specific cell types are their strong binding affinities and ability to have virtually
any specificity; however, they generally large and can therefore disrupt the necessary
conformational changes of the envelope protein required for membrane fusion.
Researchers have circumvented this problem by co‐expressing both engineered and wt
envelope proteins on the viral vectors, but this results in a loss of targeting specificity.
To improve the specificity, additional cell‐specific peptides, such as protease cleavable
peptides, can be added between the scFv and the envelope proteins (Aires da Silva et
al., 2005; Chowdhury et al., 2004; Martin et al., 1999).
20

Difficulties encountered by inserting targeting molecules into viral structural
proteins include the disruption of the multimerization of capsid monomers by peptide
insertion, reduction of the thermostability of the viral vector, and the possibility of
hindered intracellular trafficking of the vector (Guibinga et al., 2004; Shi et al., 2001;
Vellinga et al., 2007; Wickham et al., 1997; Wu et al., 2000). Also, the functionality of
inserted peptides is greatly affected by neighboring amino acids, thus, the insertion of
different peptides in the same location may result in different efficiencies (Shi et al.,
2001; Wickham et al., 1997; Wu et al., 2000). By utilizing high throughput screening or
selecting the correct peptide for the desired function, these techniques can be
incorporated with better efficiency and functionality.

1.4.4 Rational point and domain mutagenesis
There are several reasons why rational point and domain mutations are made on
viral envelope proteins. First, they can improve the properties of the glycoproteins, or
even introduce new functions. By substituting the V3‐loop region of the SIV surface
membrane unit with the corresponding region of T cell tropic HIV‐1, pseudotyped MLV
vectors were able to specifically transduce T cells, despite the presence of anti‐HIV‐1
sera (Steidl et al., 2002). Mutations can also allow the incorporation of envelope
proteins that are difficult to package onto vectors. The truncation of the C‐terminal
region of the HIV‐1 envelope protein resulted in efficient incorporation of the protein by
retroviral vectors (Mammano et al., 1997; Schnierle et al., 1997). Point mutations can
21

also reduce the incidence of insertional mutagenesis by lentiviral vectors and allow
them to become transiently expressing vectors. Mutations made to HIV‐1 integrase
have reduced its ability to integrate. Researchers have mutated RRK to AAH at position
262 (Philippe et al., 2006) and D64V (Yanez‐Munoz et al., 2006) to transiently express
the transgene in dividing cells by nonintegrating HIV vectors with the engineered
integrase (Philippe et al., 2006). However, in non‐dividing cells, longer‐term gene
expression was seen (Philippe et al., 2006; Yanez‐Munoz et al., 2006).

1.5 Two‐molecule method for engineering targeting lentiviral vectors
Our lab has developed a method to pseudotype lentiviral vectors with a 2‐
component targeting system (2CTS)(Yang et al., 2006). By separating the two functions
of fusion and binding, typically undertaken by one molecule, into two distinct molecules,
lentiviral vectors can be engineered to target different cellular receptors without
adversely affecting the fusion ability of the vector. The two different glycoproteins are
expressed on the producer cell surface and incorporated onto the vector surface
through the uptake of the cellular membrane during virus budding. The envelope
glycoprotein of the Sindbis virus, an Alphavirus in the Togaviridae family, has been
mutated to disable binding to heparin sulfate structures but retain the pH‐dependent
fusion function of the protein (Morizono et al., 2005). The Sindbis virus glycoprotein
(SVG) has two transmembrane proteins: E1, which is responsible for cell fusion, and E2,
which is responsible for binding to the cell. Amino acids 61‐64 in the E3 signal sequence
22

were deleted, mutations of 68SLKQ71 to 68AAAA71 and 157KE158 to 157AA158 were
made, and a 10‐residue tag was inserted in the E2 domain between amino acids 71 and
74 to monitor SVG expression (Yang et al., 2006). The incorporation of this SVGmu
fusogen with an antibody to mediate binding lead to targeted transduction of specific
cell types by the lentiviral vector (Yang et al., 2006).
In addition to efficiently mediating fusion of cell‐virus membranes, we have
observed that SVGmu is able to target dendritic cell‐specific intercellular adhesion
molecule‐3‐grabbing non‐integrin (DC‐SIGN), a C‐type lectin expressed by immature
dendritic cells. It has been shown that DC‐SIGN can recognize high‐mannose structures,
including high‐mannose glycoproteins, allowing binding and internalization of viruses
pseudotyped with those proteins (Lozach et al., 2007; McGreal et al., 2005). Since
immature dendritic cells are specialized antigen presenting cells, they are attractive
targets for gene delivery for vaccine purposes.

1.6 Overview
Although we had been able to target human DC‐SIGN with our SVGmu‐
pseudotyped lentivectors, we wanted to further study the mechanisms by which this
interaction is able to mediate specific transduction through DC‐SIGN and to elucidate
possible strategies to enhance both targeting and transduction efficiencies. Thus, in
Chapter 2, we employed 1‐deoxymannojirimycin (DMJ), an inhibitor of ‐mannosidase I,
which removes high‐mannose structures from glycoproteins during glycosylation, to
23

increase the amount of high‐mannose structures on SVGmu. This allowed us to study
the mannose‐dependent interactions between the glycoprotein and the receptor, DC‐
SIGN. In Chapter 3, we utilized confocal microscopy to examine the internalization
mechanisms and intracellular trafficking of SVGmu‐pseudotyped lentivectors, on a DC‐
SIGN‐expressing cell line. Lastly, in Chapter 4, we applied another alphavirus
glycoprotein to pseudotype the lentivector, Ross River Virus (RRV), and examined its
targeting to DC‐SIGN. These studies taken together have helped us better understand
DC‐SIGN targeting by our system, and have aided us in improving our lentiviral vector
transduction efficiency.

24

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43

CHAPTER 2: PRODUCTION OF LENTIVIRAL VECTORS WITH ENHANCED
EFFICIENCY TO TARGET DENDRITIC CELLS BY ATTENUATING MANNOSIDASE
ACTIVITY OF MAMMALIAN CELLS

Portions of this chapter are adapted from:
April Tai, Steven Froelich, Kye‐Il Joo, and Pin Wang, J Biol Eng. 2011. 5:1‐11

Dendritic cells (DCs) are antigen‐presenting immune cells that interact with T
cells and have been widely studied for vaccine applications. To achieve this, DCs can be
manipulated by lentiviral vectors (LVs) to express antigens to stimulate the desired
antigen‐specific T cell response, which gives this approach great potential to fight
diseases such as cancers, HIV, and autoimmune diseases. Previously we showed that LVs
enveloped with an engineered Sindbis virus glycoprotein (SVGmu) could target DCs
through a specific interaction with DC‐SIGN, a surface molecule predominantly
expressed by DCs. We hypothesized that SVGmu interacts with DC‐SIGN in a mannose‐
dependent manner, and that an increase in high‐mannose structures on the
glycoprotein surface could result in higher targeting efficiencies of LVs towards DCs. It is
known that 1‐deoxymannojirimycin (DMJ) can inhibit α1,2‐mannosidase I, which is an
enzyme that removes high‐mannose structures during the glycosylation process. Thus,
44

we investigated the possibility of generating LVs with enhanced capability to modify DCs
by supplying DMJ during vector production.
Through western blot analysis and binding tests, we were able to infer that
binding of SVGmu to DC‐SIGN is directly related to amount of high‐mannose structures
on SVGmu. We also found that the titer for the LV (FUGW/SVGmu) produced with DMJ
against 293T.DCSIGN, a human cell line expressing the human DC‐SIGN antibody, was
over four times higher than that of vector produced without DMJ. In addition,
transduction of a human DC cell line, MUTZ‐3, yielded a higher transduction efficiency
for the LV produced with DMJ.
We conclude that LVs produced under conditions with inhibited mannosidase
activity can effectively modify cells displaying the DC‐specific marker DC‐SIGN. This
study offers evidence to support the utilization of DMJ in producing LVs that are
enhanced carriers for the development of DC‐directed vaccines.


45

2.1 Introduction
Dendritic cells (DCs) are immune cells that are able to present antigens to T cells
in a major histocompatibility complex (MHC)‐restricted manner (Banchereau and
Steinman, 1998; Mellman and Steinman, 2001). These antigens are usually obtained by
phagocytosis of pathogens encountered by the DCs (Mellman and Steinman, 2001). The
naive T cells are activated by the interaction with the antigen‐presenting DCs and are
then able to recognize the corresponding pathogens (Banchereau and Steinman, 1998).
To utilize this mechanism for therapeutic applications such as immunizations and
vaccinations, DCs can be loaded with antigens to stimulate antigen‐specific CD8
+
and
CD4
+
T cell responses (Mellman and Steinman, 2001; Steinman and Banchereau, 2007;
Banchereau and Steinman, 1998; Banchereau et al., 2000). Another method of
modifying DCs to present desired antigens is to genetically alter the cells by using
liposomes or gene‐gun, or by viral transduction with replication‐incompetent viral
vectors (Figdor et al., 2004; Breckpot et al., 2004). The benefits of these strategies are
the increased time of antigen presentation, the ability to present both MHC I and II
epitopes, and the ability to include genes for immomodulatory molecules that may
enhance DC function (Ribas et al., 2007). Currently, adenoviral, gamma‐retrovial, and
lentiviral vectors are studied for the viral vector delivery strategy (Draper and Heeney,
2010; Gardlik et al., 2005; Schaffer et al., 2008; Song et al., 1997). Lentiviral vectors pose
an advantage in their ability to transduce non‐dividing cells, which is beneficial for in
vivo immunization (Esslinger et al., 2003; Palmowski et al., 2004; Pincha et al., 2010; He
46

et al., 2007; Lopes et al., 2008). However, these recombinant viral vectors are known to
have broad specificity and are able to transduce multiple cell types, which can inevitably
result in genetic modification of undesired cells and reduce vaccine efficacy (Follenzi et
al., 2004; Tacken et al., 2007).
A surface molecule present on immature DCs, Dendritic Cell‐specific ICAM3‐
grabbing Nonintegrin (DC‐SIGN), is well‐displayed and a suitable target for DC‐specific
transduction (Tacken et al., 2007; Soilleux et al., 2002). DC‐SIGN is a C‐type (Ca
2+
‐
dependent) lectin that is able to rapidly bind to and endocytose antigenic materials
(Lozach et al., 2004). It is a type II transmembrane protein that is displayed as a
tetramer, and consists of a short, N‐terminal cytoplasmic tail that contains intracellular
sorting motifs, a transmembrane region, an extracellular stalk, and a C‐terminal
carbohydrate‐recognition domain (CRD) (Figdor et al., 2002; Soilleux, 2003; Lozach et al.,
2007). It was reported that the Sindbis virus (SV), a member of the alphavirus genus and
the Togaviridae family, is able to recognize and bind to DCs through DC‐SIGN (Klimstra
et al., 2003). However, the SV glycoprotein (SVG) also has the ability to bind to cell‐
surface heparin sulfate (HS), which is expressed by many cell types, and therefore LVs
pseudotyped with SVG have a broad tropism (Morizono et al., 2001; Cronin et al., 2005).
Further studies showed that the HS binding site of SVG can be mutated (Morizono et al.,
2005) so that the resulting SVGmu glycoprotein can selectively recognize and bind to
DC‐SIGN (Yang et al., 2008). Thus, SVGmu‐pseudotyped LVs can specifically target and
47

recognize DCs, delivering antigens that enable T cell activation for immunization and
vaccine purposes (Yang et al., 2008; Dai et al., 2009; Hu et al., 2010).
The study of DC‐SIGN binding to other proteins has shown that binding occurs in
a carbohydrate‐dependent manner (Lozach et al., 2004; Mitchell et al., 2001); in fact,
Sindbis viruses produced in mosquito cells, which limit glycoprotein processing and
carbohydrate trimming, yielded higher transduction efficiencies for DC‐SIGN‐bearing
cells compared to viruses produced in mammalian cells (Klimstra et al., 2003). The high‐
mannose structures on gp120 have also been studied and have been determined to be
critical for recognition to DC‐SIGN (Geijtenbeek at al., 2000; Feinberg et al., 2001).
Mannosidase is a calcium‐dependent enzyme that removes mannose from N‐linked
glycoproteins in the ER and Golgi (Elbein, 1991). 1‐deoxymannojirimycin (DMJ, Figure
2.1A) is a chemical that can inihibit α‐mannosidase I in the Golgi by binding to the top of
its C‐terminal α‐hairpin, which is located at the bottom of the active site cavity (Elbein,
1991; Elbein et al., 1984; Vallee et al., 2000).
48

Figure 2.1 Chemical structure of DMJ and its inhibition mechanism. (A) Comparison of
the chemical and structural composition of DMJ to that of mannose. (B) Schematic
diagram of the mechanism by which DMJ inhibits class I α1,2‐mannosidase in the Golgi.
α1,2‐mannosidase I normally trims the α1,2‐linked mannose on glycoproteins; however,
DMJ inhibits α1,2‐mannosidase I activity and thus restricts the oligosaccharide
processing to high‐mannose forms.

This effectively halts the processing of the oligosaccharide at Man
9
GlcNAc
2
(Figure 2.1B)
(Balzarini, 2007). It has been reported that DC‐SIGN binds to Man
9
GlcNAc
2
130‐fold
more tightly than it does to mannose (Mitchell et al., 2001). Thus, we hypothesized that
SVGmu also binds to DC‐SIGN through high‐mannose structures, and that the addition
of DMJ inhibits the activities of α1,2‐mannosidase I, which would allow for a greater
amount of high‐mannose structures on the surface of SVGmu‐pseudotyped LVs. In this
study, we test this hypothesis and show that SVGmu‐bearing LVs produced under DMJ
treatment can modify cells expressing the DC‐specific marker DC‐SIGN more efficiently.
49

2.2 Results and Discussion
2.2.1 Transient transfection of 293T cells to produce SVGmu‐pseudotyped LVs
To assess the viability of producing LVs in media containing DMJ, 293T cells were
transiently transfected with a lentiviral backbone plasmid (FUGW) encoding a green
fluorescent protein (GFP) reporter gene driven by the human ubiqutin‐C promoter (Lois
et al., 2002), packaging plasmids, and a plasmid encoding either SVGmu or vesicular
stomatitis virus glycoprotein (VSVG). VSVG has widely been used to pseudotype LVs and
the resulting vectors have a very broad tropism (Cronin et al., 2005). Thus, we included
VSVG in our transfection to produce a control vector that is not DC‐SIGN‐targeting.
Analysis of the transfected cells two days later by flow cytometry showed comparable
results between the samples with and without DMJ added, with slightly higher values
for SVGmu‐staining in the cells transfected with SVGmu and cultured with DMJ (Figure
2.2).
50

Figure 2.2 DMJ does not reduce SVGmu production or display on vector‐producing cells.
293T cells were transiently transfected by the lentiviral backbone vector encoding the
GFP gene (FUGW), packaging constructs (REV and RRE), and a plasmid encoding either
SVGmu or VSVG. SVGmu‐staining and flow cytometry analysis of the cells two days post‐
transfection revealed that cells cultured with or without DMJ exhibited similar levels of
SVGmu and GFP, indicating that the presence of DMJ did not restrict either glycoprotein
or LV production. Control cells transfected by VSVG were similarly unaffected by DMJ
and SVGmu‐negative, as expected.

Cells transfected with SVGmu stained positively for SVGmu and expressed GFP, while
the control cells transfected with VSVG were only GFP‐positive. These results indicate
that the addition of DMJ to the cell culture media does not adversely affect the
transfection of 293T cells.

51

2.2.2 Verification of SVGmu and high‐mannose oligosaccharides on the vector surface
To verify the presence of SVGmu on the vector particle surface produced in
media containing DMJ, we employed a labeling scheme to generate vectors that
encapsulated GFP‐Vpr (GFP fused with the HIV accessory protein Vpr (Joo and Wang,
2008)). A transient co‐transfection protocol similar to what was described above, was
utilized to generate the GFP‐labeled particles, except that the lentiviral backbone
plasmid FUW, which lacks the GFP reporter gene, replaced FUGW, and an additional
plasmid encoding GFP‐Vpr was included in the co‐transfection procedure (Joo and
Wang, 2008). The resulting vectors (FUW‐GFP‐Vpr/SVGmu +/‒ DMJ or FUW‐GFP‐
Vpr/VSVG) were loaded onto cover slips, stained for SVGmu, and analyzed by confocal
imaging. The SVGmu envelope glycoprotein was confirmed to be displayed on the FUW‐
GFPVpr/SVGmu vectors produced both with and without DMJ by the colocalization of
the GFP and SVGmu signals (Figure 2.3).
52

Figure 2.3 SVGmu is incorporated onto the vector surface for LVs produced with or
without DMJ. Fresh viruses were produced with the addition of an additional plasmid,
GFP‐Vpr to fluorescently label the vector core. Vectors produced with or without DMJ
were then stained for SVGmu and analyzed by confocal microscopy. Visualization of the
vector particles showed that SVGmu was efficiently incorporated in both types of
vectors. VSVG‐pseudotyped vectors was included as a control.

As a control, FUW‐GFPVpr/VSVG vector particles were also analyzed. As expected, while
these particles were GFP‐positive, they did not stain for SVGmu.
Next, the SVGmu‐enveloped vector (FUGW/SVGmu) produced either with or
without DMJ was concentrated by ultracentrifugation and then digested by EndoH, an
enzyme that selectively breaks apart high‐mannose structures by cleaving the chitobiose
core from N‐linked glycoproteins (Lozach et al., 2004). A western blot analysis of the
53

EndoH‐treated vector particles confirmed the presence of high‐mannose structures for
the LVs produced with DMJ by yielding a lower molecular weight species (Figure 2.4).

Figure 2.4 LVs produced in DMJ contain more high‐mannose structures. Vectors were
produced with or without DMJ, concentrated, and digested by EndoH, an enzyme that
cleaves high‐mannose structures. A western blot analysis of the undigested and
digested vectors showed that the vectors produced in DMJ had a lower molecular
weight after EndoH digestion compared to that of the vectors produced without DMJ.
These results infer that the vector produced with DMJ had more high‐mannose
structures than the vector produced without DMJ.

In contrast, the vector produced without DMJ did not give as low of a molecular weight
species when digested by EndoH, which is evidence of less high‐mannose structures
present on the vector surface. We believe that this supports our hypothesis that the
untreated vector contains mostly complex sugars on the envelope glycoprotein, along
with some hybrid sugars (both mannose and complex sugars) and/or high‐mannose
sugars.

2.2.3 Evaluation of cell‐virus binding to hDCSIGN
Two experiments were performed to investigate whether SVGmu does in fact
recognize and bind to human DC‐SIGN and whether binding of SVGmu‐bearing LVs
54

(FUGW/SVGmu) to 293T.DCSIGN, a stable 293T cell line expressing human DCSIGN, is
enhanced by the addition of DMJ to vector‐producing cells. In the first experiment,
293T.DCSIGN cells were fixed with 4% paraformaldehyde followed by incubation with
fresh supernatant containing FUGW/SVGmu. The cell‐vector complexes were then
stained for SVGmu and analyzed by flow cytometry. As expected, the vector produced
with DMJ bound more readily to the cells, as shown by the higher percentage of SVGmu
staining (Figure 2.5A).
55

Figure 2.5 LVs produced in DMJ bind more readily to human DCSIGN than vectors
produced without DMJ. (A) Vectors produced with or without DMJ were incubated with
fixed 293T.DCSIGN cells, a cell line that stably displays human DCSIGN. Flow cytometric
analysis of SVGmu‐stained cells revealed that the vectors produced in DMJ bound to the
DCSIGN‐expressing cells over 6 times more readily than the vectors produced without
DMJ. (B) Vectors were labeled with [35S]‐Trans and incubated with either 293T or
293T.DCSIGN cells. A mannose inhibition assay was also included to determine the
dependency of vector‐receptor binding on the mannose‐rich structures on the vector.
Radioactivity analysis revealed that the vector produced with DMJ bound much more
readily to the 293T.DCSIGN cells than to the 293T cells, while the addition of mannose
greatly reduced cell‐vector binding. Vectors produced without DMJ did not exhibit as
much of a difference between binding to the 293T.DCSIGN cells and the 293T cells,
while the presence of mannose reduced vector levels in both of the cell lines.
56

Next, a radioactive‐labeling assay was used to test the cell‐vector binding
responses for LVs produced with and without DMJ, with an additional comparison to
control 293T cells, which lacked DC‐SIGN expression. 293T and 293T.DCSIGN cells were
seeded onto 96‐well plates overnight. The cells were then washed by PBS and incubated
with concentrated,
35
S‐labeled LVs for one hour. A second wash with PBS removed
unbound vectors and the cells were lysed before they were pipetted into scintillation
vials for analysis. A mannose inhibition assay was also employed to test the dependency
of vector binding on the mannose‐rich structures of the envelope glycoprotein.
Mannose was incubated with the cells prior to the addition of LVs to block the binding
sites that are mannose‐linked. A much higher amount of radioactivity was detected in
FUGW/SVGmu(DMJ+) incubated with 293T.DCSIGN as compared to
FUGW/SVGmu(DMJ‒) (Figure 2.5B). In contrast, DMJ did not greatly alter the binding
ability of FUGW/SVGmu to 293T cells; vectors produced with and without DMJ resulted
in a low incidence of binding. Although the addition of mannose lowered the amount of
cell‐vector binding measured with both types of LVs (DMJ+ and DMJ‒) and both types of
cells (293T.DCSIGN and 293T), it had the most significant effect on the binding of
FUGW/SVGmu(DMJ+) to the 293T.DCSIGN cells. This data suggests that the enhanced
ability of the LV to bind to DC‐SIGN we observed was the result of its greater availability
of high‐mannose structures, and that this interaction can be blocked by competitive
inhibition with mannose.

57

2.2.4 Transduction of cells with LVs produced with DMJ
LVs produced with DMJ were used to transduce 293T and 293T.DCSIGN cells to
test the effect of the improved binding on cell transduction. Fresh vector supernatants
were added to cells of each type, followed by spin‐infection. The cells were then
analyzed for GFP expression by flow cytometry after three days of incubation. A p24
ELISA assay was also performed to ensure that the concentrations of the different
viruses were comparable. FUGW/SVGmu(DMJ+) transduced 293T.DCSIGN cells with the
greatest efficiency (25.34%) ‒ more than three‐folds higher than the
FUGW/SVGmu(DMJ‒) vector (7.45%) (Figure 2.6A).
58

Figure 2.6 LVs produced with DMJ transduced DCSIGN‐expressing cells much more
efficiently than LVs produced without DMJ. (A) LVs were produced either with or
without DMJ and spin‐infected with 293T and 293T.DCSIGN cells. Although
FUGW/SVGmu(DMJ–) preferentially transduced 293T.DCSIGN cells,
FUGW/SVGmu(DMJ+) was over three‐fold more efficient in transducing 293T.DCSIGN
cells while maintaining similar levels of background transduction to 293T cells. (B) LVs
produced with DMJ yielded higher vector titers for DCSIGN‐expressing cells compared
with LVs produced without DMJ. On 293T.DCSIGN cells, FUGW/SVGmu(DMJ+) yielded
titers over three‐folds higher than those of FUGW/SVGmu(DMJ–), while titers for 293T
cells for both vectors were similarly low. FUGW/VSVG +/‐ DMJ was included as a control.
59

Both types of LVs had a similar amount of transduction for the control 293T cells. These
results validate our expectation that more highly infective targeting vectors can be
produced with the addition of DMJ in the cell culture media during vector production.
Vector titers were then calculated for FUGW/SVGmu +/‒ DMJ against 293T and
293T.DCSIGN cells. FUGW/VSVG +/‒ DMJ were also included as controls. Triplicate
experiments were performed, where cells were spin‐infected with various fresh vector
supernatants that had been serially diluted. Three days later, the cells were washed and
analyzed for GFP expression by flow cytometry. As expected, FUGW/SVGmu(DMJ+)
yielded a higher transduction titer with 293T.DCSIGN cells, with an average value of 0.26
× 10
6
TU/ml, as compared to FUGW/SVGmu(DMJ‒), which had an average transduction
titer of 0.08 × 10
6
TU/ml (Figure 2.6B). FUGW/SVGmu +/‐ DMJ both transduced 293T
cells similarly with titers of ~0.04 × 10
6
TU/ml. As an additional control, 293T.DCSIGN
cells were also tranduced by FUGW/VSVG +/‐ DMJ and titers of 22.60 × 10
6
TU/ml and
24.60 × 10
6
TU/ml, respectively, were obtained. Thus, our titer measurement confirms
that FUGW/SVGmu(DMJ+) transduced 293T.DCSIGN cells three times more efficiently
than FUGW/SVGmu(DMJ‒) did, and that both FUGW/SVGmu +/‐ DMJ transduced 293T
cells at an equally low rate. The addition of DMJ to the production of the FUGW/VSVG
vector did not significantly alter the vector titer against 293T.DCSIGN.
One group demonstrated that LVs pseudotyped with a similarly modified Sindbis
virus envelope glycoprotein produced without DMJ treatment did not bind to DC‐SIGN
and target DC‐SIGN‐positive cells (Morizono et al., 2010). The mutations used in their
60

envelope protein were identical except for the addition of a ZZ domain versus the HA
tag employed in our system. However, previous studies in our laboratory have shown
that SVGmu binds with DC‐SIGN and targets transduction to DC‐SIGN‐positive cells (Yang
et al., 2008). Also, compared with our data for wild‐type Sindbis envelope‐bearing LVs,
their pseudotyped vectors are less infectious. Perhaps these differences in infectious
vector production are indicative of an abundance of non‐infectious particles that
conceal interactions between the glycoprotein and DC‐SIGN. Other differences, such as
our use of a clonally‐expanded DC‐SIGN‐expressing cell line and other experimental
settings, may further contribute to the reported inability to observe increased
transduction efficiencies for DC‐SIGN bearing cell lines with SVGmu envelope‐bearing
LVs produced without DMJ.

2.2.5 Transduction of a DC cell line with vectors produced with DMJ
To test the transduction efficiency of vectors produced with and without DMJ on
a closer model of dendritic cells, we utilized a DC cell line (MUTZ‐3 cells) that had been
previously shown to closely mirror the behavior of human dendritic cells and are
capable of being differentiated to express human DC‐SIGN (Larsson et al., 2006;
Santegoets et al., 2008). MUTZ‐3 cells were cultured and differentiated for 7 days to
express DC‐SIGN (Figure 2.7A) before they were transduced by concentrated
FUGW/SVGmu vectors produced with or without DMJ.
61

Figure 2.7 LVs produced with DMJ can transduce a DC cell line more efficiently than LVs
produced without DMJ. A human DC cell line (MUTZ‐3) was employed to more
accurately model human DCs, as a target for LVs produced with and without DMJ. (A)
MUTZ‐3 cells were differentiated for 7 days and stained for DC‐SIGN expression. (B) The
MUTZ‐3 cells were transduced by LVs produced with or without DMJ.
FUGW/SVGmu(DMJ+) transduced the MUTZ‐3 cells about 25% more efficiently than
FUGW/SVGmu(DMJ–). FUGW/VSVG was included as a positive control.

Consistent with the results from 293T.DCSIGN transduction, the targeting vector
produced with DMJ transduced the MUTZ‐3 cells more efficiently (87.4%) than the
vector produced without DMJ (62.8%) (Figure 2.7B). Thus, the targeting vector produced
with DMJ, which contained more high‐mannose structures, was able to transduce both
the 293T.DCSIGN cell line and a human DC cell line more efficiently than the vector
produced without DMJ.
62

2.3 Conclusions
DC‐based vaccines have great potential as a new means to fight challenging
diseases, such as cancers, HIV, and autoimmune diseases. Optimization of the efficiency
of the viral vectors used to modify DCs could greatly strengthen this approach and make
it an even more powerful tool for developing novel treatment modalities against various
diseases. Our study aimed to examine the effects of DMJ, which indirectly increases the
amount of high‐mannose structures present on glycoproteins through the inhibition of
class I α1,2‐mannosidase activity in vector‐producing cells, on the efficiency of SVGmu‐
bearing LV transduction of DC‐SIGN‐expressing cells.
We were able to conclude that the FUGW/SVGmu vector made in the presence
of DMJ did in fact have increased high‐mannose structures over those made without
DMJ present. Our results also suggested that binding of SVGmu to DC‐SIGN is directly
related to amount of high‐mannose structures on SVGmu, and that these interactions
can be blocked by competitive inhibition. Furthermore, we found that production of the
targeting vector in DMJ resulted in a three‐fold increase in transduction efficiency in
target cells compared to vectored produced without DMJ. Lastly, the targeting vector
produced in the presence of DMJ was able to transduce MUTZ‐3 cells, a cell line that has
been shown to closely mimic the behavior of human peripheral blood mononuclear cells
(PBMCs) and can be differentiated to cells displaying DC‐SIGN, more efficiently than the
vector produced without DMJ, which strengthens our belief that an increase in high‐
63

mannose structures on the targeting virus surface would result in higher transduction
efficiency of human DCs.
Further investigation into the vector entry mechanism of SVGmu‐pseudotyped
LVs is currently being conducted using confocal microscopy with the aid of drug
treatments. Greater understanding of the pathway of viral uptake and core release into
the cytosol will facilitate the design of more effective and efficient engineered gene
delivery vehicles. Additionally, siRNA can be applied to silence mannosidase in vector‐
producing cells to eliminate the need for DMJ in large‐scale productions of optimized
high‐mannose‐containing engineered LVs.

2.4 Materials and Methods
Production of lentiviral vectors
293T cells were transiently transfected with a standard calcium phosphate
precipitation protocol. 293T cells were seeded onto 6‐cm tissue culture dishes and
transfected with 5 µg of the lentiviral transfer vector plasmid (FUGW or FUW), 2.5 µg of
the envelope plasmid (SVGmu or VSVG), and packaging plasmids (pMDLg/pRRE and
pRSV‐Rev). D10 media (Dulbecco’s modified Eagle’s medium (Mediatech Inc., Manassas,
VA) with 10% fetal bovine serum (Sigma, St. Louis, MO), 2 mM L‐glutamine (Hyclone,
Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin) with and without DMJ was
replaced 4 h later. After 48 h post‐transfection, the vector supernatants were harvested
and filtered through a 0.45‐µm filter (Corning, Acton, MA). To concentrate the vector,
64

the supernatants were ultracentrifugated (Optima L‐80 K preparative ultracentrifuge,
Beckman Coulter, Brea, CA) after filtration at 50,000 × g for 90 min. The viral pellets
were resuspended in 100 µl of cold PBS.

Confocal imaging of lentiviral vectors
LVs were produced with the addition of 2.5 µg of a plasmid encoding GFP‐Vpr.
Vector supernatant was placed on polylysine‐coated coverslips in 6‐well culture dishes
and centrifuged at 3,700 × g at 4°C for 2 h with a Sorvall Legend RT centrifuge (DJB
Labcare, Buckinghamshire, England). The coverslips were then washed twice with cold
PBS and immunostained with anti‐HA‐biotin antibody (Miltenyi Biotec, Bergisch
Gladbach, Germany) and Cy5‐streptavidin (Invitrogen, Carlsbad, CA). The samples were
fluorescently imaged by a Zeiss LSM 510 laser scanning confocal microscope with filter
sets for fluorescein and Cy5 and a plan‐apochromat oil immersion objective (63×/1.4).

Vector digestion by EndoH and western blot
7 µl of concentrated virus was combined with 2 µl of lysis buffer and incubated
at 37°C for 30 min. Next, the glycoprotein was denatured by adding 1 µl of 10×
Glycoprotein Denaturing Buffer to the mixture and heating the reaction to 100°C for 10
min. The protein was then digested by EndoH by adding 2 µl of 10× G5 Reaction Buffer,
5 µl of EndoH (NEB, Ipswich, MA), and 3 µl of H
2
O. This reaction was incubated at 37°C
for 1 h. Protein gels were run and then western blots were performed to transfer the
65

proteins onto membranes. The membranes were then blocked by 5% milk with TBST at
4°C for 1 h. The membranes were washed with TBST and then stained for anti‐HA‐biotin
antibody for 1 h. They were then washed again, stained for streptavidin‐HRP (R&D
Systems, Minneapolis, MN) for 1 hr, and washed. To develop the western blot, TMB
solution was spread onto the surface of the membrane and left for 10 min at room
temperature. The membrane was then imaged.

Radioactive labeling and mannose inhibition of lentiviral vectors
293T cells were transfected by vector plasmids as described earlier, without the
addition of DMJ. 20 h post‐transfection, the media was replaced with methionine‐free
D10 with or without DMJ. After 4 h, 50 µl of [35S]‐Trans (MP Biochemicals, Solon, OH)
was added to 15‐cm tissue culture dishes and incubated at 37°C. 48 h post‐tranfection,
viral supernatants were harvested, filtered, and concentrated. For the mannose
inhibition assay, 10 mM of D‐mannose was added to the 293T.hDCSIGN cells prior to the
cell‐virus binding tests.

Vector transduction of cells
293T or 293T.DCSIGN cells were seeded into a 24‐well culture dish at 0.2 × 10
6

cells per well and spin‐infected with 1 ml of viral supernatant per well at 2,500 rpm and
25°C for 90 min using a Sorvell Legend centrifuge. The cell supernatants were then
replaced with fresh D10 media and incubated at 37°C for 3 days with 5% CO
2
. FACS
66

analysis was used to determine the percentage of GFP+ cells present. To determine
transduction titers, the dilution ranges that showed a linear response were used.

Transduction of a human DC cell line
MUTZ‐3 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen,
Braunschweig, Germany) were cultured in 24‐well tissue culture plates in αMEM media
(BioWhittaker, Walkersville, MD) with 20% FBS (Sigma‐Aldrich, St. Louis, MO) and 40
ng/mL GM‐CSF (PeproTech, Rocky Hill, NJ). To differentiate the cells to express human
DC‐SIGN, MUTZ‐3 cells were cultured in the presence of IL‐4 and GM‐CSF (100 ng/mL of
each; PeproTech) for 7 days. FACS analysis of cells stained with anti‐human DCSIGN‐PE
(BioLegend, San Diego, CA) confirmed the presence of the human DC‐SIGN marker. The
cells (1 × 10
5
) were spin‐infected with concentrated virus and the medium was replaced
with fresh medium containing IL‐4 and GM‐CSF. The cells were analyzed by flow
cytometry for GFP expression 3 days post‐tranduction.

2.5 Acknowledgements
We thank Chi‐Lin Lee for the preparation of figures. This work was supported by
a National Institute of Health grant R01‐AI068978.

67

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CHAPTER 3: VISUALIZATION OF DC‐SIGN‐TARGETED TRANSDUCTION BY
ENGINEERED LENTIVIRAL VECTORS

Dendritic cells (DCs) are potent antigen‐presenting cells and therefore have
enormous potential as vaccine targets. Our lab has previously developed an engineered
lentiviral vector that is pseudotyped with a mutated Sindbis virus glycoprotein (SVGmu),
which is capable of targeting DCs through Dendritic Cell‐specific ICAM3‐grabbing
Nonintegrin (DC‐SIGN), a receptor that is predominantly expressed by DCs. In this study,
we aimed to elucidate the internalization and trafficking mechanisms of this viral vector
through confocal microscopy of GFP‐Vpr‐tagged virus, drug treatments, and dominant‐
negative mutants of GTPases, which are necessary for endosomal functions. We
demonstrated that our engineered lentiviral vector enters the cell via receptor‐
mediated clathrin‐ and dynamin‐dependent endocytosis. Microtubule networks were
also involved in a productive infection. Fusion was low‐pH‐dependent and occurred in
the early endosomal stage of transport. Autophagy was also examined for its effect on
transduction efficiency. We observed that enhanced autophage activity reduced viral
infectivity, while suppressed autophagy boosted transduction efficiency. This study gives
us insight on the internalization and trafficking mechanisms used by our engineered
vector and gives us tools to improve the efficiency of this platform.

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3.1 Introduction
Vaccines are incredibly potent tools in immunology, capable of greatly reducing,
and even eradicating as in the case of smallpox, vaccine‐preventable infections.
Dendritic cells (DCs) are an excellent target for vaccine therapy because they are
considered to be the most potent antigen‐presenting cells of the immune system
(Banchereau and Steinman, 1998; Geijtenbeek et al., 2000a; Geijtenbeek et al., 2000b;
Mellman and Steinman, 2001). Efforts have been made to modify DCs by loading them
with antigens ex vivo, or by genetically altering them using liposomes, gene‐gun, or
transduction by viral vectors (Breckpot et al., 2004; Dullaers and Thielemans, 2006;
Ribas et al., 2002). Genetic alterations are attractive because they allow for longer
periods of antigen presentation, the ability to use both MHC I and II epitopes, and they
can include genes that enhance DC function (Breckpot et al., 2004). Lentiviral vectors
have been an attractive vehicle for gene therapy because they are able to transduce
non‐dividing cells and permanently integrate into the target cell genome (Bukrinsky et
al., 1992; Lewis et al., 1992; Weinberg et al., 1991). These vectors can be efficiently
pseudotyped with other viral glycoproteins to alter their tropism and allow them to
target specific cell types (Cronin et al., 2005; Waehler et al., 2007).
Our lab has developed a method to target lentivectors to specific cell types with
a two‐molecule method: an antibody responsible for binding to the desired cellular
receptor and a pH‐dependent fusion molecule are present as two separate molecules on
the vector surface (Yang et al., 2006). Furthermore, it was found that the glycoprotein of
73

the Sindbis virus, which is an Alphavirus of the Togaviridae family, was a fusion molecule
that could be mutated to eliminate its ability to bind to heparin sulfate structures, while
it retained specific binding to human dendritic cell‐specific intercellular adhesion
molecule‐3‐grabbing non‐integrin (DC‐SIGN), which is a C‐type lectin‐like receptor that is
expressed specifically on DCs (Yang et al., 2008). The study of the pathway of hDC‐SIGN‐
mediated transduction by SVGmu‐pseudotyped lentiviral vectors can allow us to achieve
a greater understanding of the mechanisms involved in this process. Generally,
enveloped viruses utilize receptor‐mediated endocytosis for entry, after which the
viruses travel through endocytic compartments and fuse with the endosomal
membrane, resulting in the release the viral genome into the host cell (Anderson and
Hope, 2005; Gruenberg, 2001; Klasse et al., 1998; Martin and Helenius, 1991). To study
these processes in our engineered lentivectors, we used confocal microscopy to
visualize the fluorescently‐tagged viruses in the stained target cells to determine virus
colocalization with cellular components. Our results suggest that the SVGmu‐
pseudotyped lentivectors enter the hDC‐SIGN‐expressing cell line through clathrin‐
mediated endocytosis, after which they travel intercellularly along microtubule
networks. Fusion of the SVGmu‐pseudotyped lentivectors was pH‐dependent and
occurred for most of the viral particles after 20 min of incubation with the cells. At 20
min, most of the viral particles are localized in early endosomes, which suggests that
fusion is early endosome‐dependent. Autophages were shown to also play a role in viral
infection rates—drug treatments revealed that infection rates decreased as autophagy
74

activity increased. These results give us an idea of what mechanisms are involved in the
internalization and trafficking of the SVGmu‐pseudotyped lentiviral vectors in an hDC‐
SIGN‐expressing cell line, and can help us potentially determine methods to enhance the
transduction efficiency of our DC‐targeting platform.

3.2 Results
3.2.1 Clathrin‐Dependent Endocytosis
First, we sought to determine the mechanism by which the cell endocytosed the
lentiviral vector. Clathrin‐ and caveolin‐mediated pathways of endocytosis are well‐
characterized and were thus examined (Mountain, 2000). A 293T cell line stably
expressing hDC‐SIGN (293T/hDCSIGN) was used to study the trafficking of the targeting
lentivectors. Drug treatments were used to inhibit these two pathways of viral entry:
chlorpromazine, which prevents clathrin polymerization and obstructs internalization
mediated by clathrin‐coated vesicles, was used to block clathrin‐mediated endocytosis
(Rink et al., 2005), while filipin, a drug that depletes cholesterol to inhibit caveolin‐
dependent internalization (Stoorvogel et al., 1991), was used to block caveolin‐
mediated endocytosis. While the chlorpromazine was able to limit transduction of
293T/hDCSIGN cells by SVGmu‐pseudotyped lentiviruses, filipin had no effect on
transduction rates (Figure 3.1).
75


Figure 3.1 Drug treatment to block clathrin‐ and caveolin‐mediated endocytosis of
SVGmu‐ and SFV‐pseudotyped lentiviral vectors, and dominant‐negative mutant test for
dynamin.

Semliki Forest Virus (SFV) is known to enter cells through clathrin‐mediated endocytosis
and was used as a positive control. In addition to the drug treatment tests, a dominant‐
negative (DN) mutant construct was used to disable dynamin function (Joo et al., 2010).
Dynamin is required for the formation of clathrin‐coated vesicles, and is therefore
another indicator of clathrin‐dependency (Van der Bliek et al., 1993). The DN dynamin
construct was used to transfect 293T/hDCSIGN cells 24 h prior to transduction by
FUGW/SVGmu. FACS analysis 3 days post‐transduction was conducted to reveal the
extent to which viral transduction depends on dynamin‐related transport. Compared to
cells transduced by the wild‐type dynamin construct, the cells containing the DN
construct exhibited a reduced level of infection by our targeting viral vector (Figure 3.1).
Thus, the results of the drug treatment and DN mutant tests indicate that our SVGmu‐
pseudotyped lentivector is internalized through clathrin‐mediated endocytosis.
76

To confirm these results by fluorescence imaging, 293T/hDCSIGN cells were
incubated with GFPVpr‐tagged, SVGmu‐pseudotyped lentiviruses, fixed, permeabilized,
and stained with the appropriate clathrin‐ and caveolin‐associated antibodies (Figure
3.2).

Figure 3.2 Confocal imaging of FUW/SVGmu/GFPVpr viruses (green) with cells stained
for clathrin and caveolin. White arrows indicate the location of viral particles.

Consistent with the drug treatment test results, about 77% of the viral particles were
colocalized with clathrin while about 10% were colocalized with caveolin. Thus, we can
confirm that the SVGmu‐pseudotyped lentiviral vector utilizes clathrin‐mediated
endocytosis.

3.2.2 Viral Transport is Microtubule‐Mediated
Next, we aimed to determine whether actin filaments and/or microtubules were
involved with viral transport. Many viruses are known to rely on cellular transport using
77

microtubule or actin networks of the cytoskeleton; for example, Herpes Simplex Virus 1
is known to utilize microtubules, while Epstein‐Barr Virus requires actin for entry into B
cells (Sodeik et al., 1997; Valencia, 2011). 293T/hDCSIGN cells were incubated with the
drugs cyto‐D and nocodazole, used to inhibit actin and microtubules, respectively,
before spin infection with GFP‐expressing, SVGmu‐pseudotyped lentivectors
(FUGW/SVGmu). FACS analysis of the GFP+ cells 3 days post‐transduction revealed that
the actin blocking had no effect on viral infectivity, while microtubule blocking reduced
infection levels to about 40% of the control (Figure 3.3).

Figure 3.3 Drug treatments for the effect of actin filaments and microtubules on viral
transduction.

To visualize viral transport on the cellular cytoskeleton, SVGmu‐pseudotyped
lentiviruses were engineered to express GFPVpr, a protein with green fluorescence
protein (GFP) fused to the N‐terminus of the HIV accessory protein viral protein R, which
was described previously (Joo and Wang, 2008). Hela/hDCSIGN cells, used for the
improved clarity of cytoskeleton structures in Hela cells, were incubated with the
FUW/SVGmu/GFPVpr virus before being stained for actin or microtubules. Visualization
78

of the stained cells revealed that most of the viruses were not on the actin structures,
but were colocalized with the microtubule structures, confirming the results of the drug
treatment tests (Figure 3.4).

Figure 3.4 Visualization of virus colocalization with actin filaments (A) and microtubules
(B). The viral particles appear green through GFPVpr expression.

Taken together, these results suggest that a successful viral infection is dependent on
the microtubule networks in the cell, and not on actin.

3.2.3 Fusion Tracking
Drug treatment was also utilized to confirm the pH‐dependency of viral fusion.
Bafilomycin A1, which specifically inhibits vacuolar proton ATPases (Bowman et al.,
1988), was used to block low‐pH endosomal fusion. Different concentrations of the drug
were used and the results were normalized to the % of GFP
+
cells seen in a control
sample containing no drug (Figure 3.5).
79

Figure 3.5 Drug inhibition of low pH‐mediated endosomal fusion.

Drug treatment of the cells resulted in a marked reduction in transduction efficiency,
which confirmed the low pH‐dependency of the fusion function of SVGmu.
Next, the actual fusion events of the viral vector were examined through
confocal microscopy. The lipophilic dye, 1,1’dioctadecyl‐3,3,3’,3’‐
tetramethylindodicarbocyanine (DiD), can be spontaneously incorporated into viral
membranes to allow viruses to be labeled with both DiD and GFPVpr. At high
concentrations on the viral membrane, DiD can be self‐quenched. When the viral
membrane fuses with the endosomal membrane, however, the dye disperses and
becomes dequenched, resulting in an increase in fluorescence. FUW/SVGmu/GFPVpr
virus was tagged with DiD and incubated with 293T/hDCSIGN cells for various time
periods. Images were then taken and viral fusion events were quantified (Figure 3.6).
80

Figure 3.6 Representative images of fusion events taken at different timepoints and
quantification of imaging results.
The majority (>76%) of the fusion events occurred at 20 min of cell‐virus incubation,
indicating that this was most likely to be the amount of time required between virus
internalization and the release of the viral core from the endosomes. Only 8% and 19%
of the viruses at 10 min and 30 min, respectively, experienced fusion events. The fusion
81

events seen at 30 min could either be some additional fusion events occurring or
perhaps viral cores that had not yet escaped from endosomes.
To reaffirm the low‐pH dependency of viral fusion, NH
4
Cl treatment was used.
The viruses were tagged with DiD and incubated for 20 with the cells. Imaging of the
viruses revealed that only about 26% of the viruses were colocalized with the DiD
dequenching signal, compared to the 76% seen when NH
4
Cl treatment was not used
(Figure 3.7).

Figure 3.7 Imaging viral fusion events after NH
4
Cl treatment.

These results correlate well with the Bafilomycin A1 drug treatment results and confirms
the low‐pH dependency of fusion for our engineered viruses.

3.2.4 Tracking with Endosomal Markers
To associate fusion events with viral presence in early, intermediate, or late
endosomes, we visualized 293T/hDCSIGN cells incubated with FUW/SVGmu/GFPVpr
virus for different time points, which were antibody‐stained for early endosome antigen
82

1 (EEA1) (Lakadamyali et al., 2006; Sieczkarski and Whittaker, 2003; Vonderheit and
Helenius, 2005) and cation‐independent mannose‐6‐phosphate receptor (CI‐MPR)
(Bowman et al., 1988; Lakadamyali et al., 2003; Lakadamyali et al., 2006; Lois et al.,
2002; Marsh et al., 1983; McDonald et al., 2002; Sakai et al., 2006; Sieczkarski and
Whittaker, 2003; Urayama et al., 2004; Vonderheit and Helenius, 2005) for early and
late endosomal markers, respectively. Intermediate endosomes are considered to be
maturing endosomes with both early and late endosomal markers (Rink et al., 2005;
Stoorvogel et al., 1991). Images were taken at 0, 10, 20, and 30 min, correlating with the
timepoints taken for the fusion study, and quantified (Figure 3.8).

83

Figure 3.8 Representative images of cells stained with endosomal markers at different
timepoints and quantification of the imaging results.

84

At 0 min, none of the viral particles were colocalized with the endosomal markers and
most were only bound to the cell surfaces. At 10 min, more viruses had moved into the
endosomes, with 17% of the viruses colocalized with the early endosome marker. At 20
min, also the time when most of the fusion events occurred, over half of the viruses
were colocalized with early endosomes, with about 12% and 6% in intermediate and
late endosomes, respectively. At 30 min, 33% of the viruses are colocalized with both
endosomal markers while 12% and 17% are colocalized with only the early or late
endosomal marker, respectively. Correlation of this data with that of the fusion study
leads us to believe that fusion occurs mostly during the early endosome stage of
endosomal transport for these hDC‐SIGN‐targeting viruses.
For more quantitative data on the dependency of SVGmu‐pseudotyped lentiviral
transduction on late endosome trafficking, DN mutant constructs were used Rab 5 (for
early endosomes), Rab 7 (for late endosomes), and Rab 11 (for recycling endosomes).
Again, 293T/hDCSIGN cells were transfected these constructs 24 h prior to transduction
by FUGW/SVGmu and analyzed by FACS 3 days post‐transduction (Figure 3.9).
85

Figure 3.9 Effect of dominant‐negative mutants on viral transduction efficiency.
From these results, we observed that viral transduction was indeed dependent only on
early endosomes, and not on late and recycling endosomes.
To possibly explain the discrepancy between having a large majority of the
viruses fused by 20 min while quite a few viruses remained in endosomes past 20 min,
we incubated the cells with the virus for 2 h and analyzed the colocalization of the virus
with a lysosome marker (Figure 3.10).
86

Figure 3.10 Representative images of virus colocalization with a lysosome marker.

Viruses that were unable to undergo fusion or endosomal escape would be trafficked to
lysosomes for degradation. About 37% of the GFPVpr+ viral particles were colocalized
with the lysosome markers, which could explain somewhat substantial number of
viruses still colocalized with the endosomal markers at 30 min.

3.2.5 Effect of Autophagy on Viral Infection
Autophagy is the catabolic process by which cellular cytoplasm is enveloped into
a double‐membraned organelle, the autophagosome, and delivered to lysosomes for
degradation (Levine and Klionsky, 2004). This is required for the maintenance of cellular
homeostasis, such as the degradation of protein aggregates from the cytoplasm and the
removal of unwanted or damaged organelles, as well as for the removal of intracellular
pathogens (Levine and Kroemer, 2008).
Several studies have examined the role of autophagy in viral infections, and it is believed
that it has both antiviral (the degradation of viruses) and pro‐viral (aids in the replication
87

or release of viruses from cells) functions (Shoji‐Kawata and Levine, 2009; Kudchodkar
and Levine, 2009). Several studies that have shown links between autophagy and
viruses, such as vesicular stomatitis virus (VSV), coxsackievirus B3 (CVB3), and viral
neurovirulence (Shelly et al., 2009; Wong et al., 2008; Orvedahl and Levine, 2008).
Interestingly, it has been shown the autophagy plays several roles in HIV‐1 infection:
early stages of autophagy enhances HIV yields by promoting productive Gag processing,
later degradative stages of autophagy are prevented through Nef interaction with Beclin
1, and autophagy, and subsequent cell death, is triggered in bystander uninfected CD4 T
cells by HIV‐1‐infected cells (Kyei et al., 2009; Espert et al., 2008; Gougeon and
Piacentini, 2009). On the other hand, there are viruses in which autophagy plays no role,
such as the human rhinovirus (HRV) (Brabec‐Zaruba et al., 2007). Thus, we were
interested in determining the effect of autophagy on infection by our targeting
lentivector.
First, drug treatment was used on 293T/hDCSIGN cells to enhance (rapamycin)
and reduce (3‐methyladenine, 3‐MA) autophage activity before infection by
FUGW/SVGmu (Figure 3.11).
88

Figure 3.11 Effect of autophagy on viral infectivity. Rapamycin enhances autophage
activity while 3‐MA inhibits autophagy.

Inhibition of autophagy by 3‐MA resulted in a clear increase in transduction efficiency
over untreated cells, and the enhancement of autophagy by rapamycin lowered viral
infectivity.
Next, to confirm that autophagy was truly the cause of the reduction in
transduction efficiency, and to check our hypothesis that autophagy resulted in the
transport of more viral vectors to lysosomes, 293T/hDCSIGN cells were treated with
rapamycin, incubated with FUW/SVGmu/GFPVpr virus for the specific amount of time,
and imaged (Figure 3.12).
89

Figure 3.12 Imaging and quantification of viruses colocalized with autophage and
lysosome markers in cells treated with rapamycin.

90

At each of the time points, the addition of rapamycin increased the amount of virus
colocalized with autophage and autophage + lysosome markers. This confirms our belief
that rapamycin treatment increases the activity of autophages, resulting in more viruses
entering the autophagy pathway. The overlap of autophage and lysosome markers can
be interpreted as the end of the autophagy pathway, where the autophagosome fuses
with lysosomes to form autolysosomes, and the contents are degraded (Levine and
Kroemer, 2008; Xie and Klionsky, 2007).

3.3 Discussion
Dendritic cell vaccines have great potential to be potent tools for immunology.
Lentivectors are an attractive vehicle for DC vaccine regimes because they can infect
non‐diving cells and can be efficiently pseudotyped by engineered viral glycoproteins for
specific targeting to cellular markers. Here we have studied a lentivector pseudotyped
with a mutated SVG glycoprotein, which is capable of targeting the DC‐specific marker,
DC‐SIGN. Understanding the infection pathway and mechanisms is useful for optimizing
this platform’s utility as a successful DC vaccine.
We have confirmed that these engineered lentivectors enter cells through
dynamin‐dependent clathrin‐mediated endocytosis, and that they utilize microtubule
networks in the cells. They require low‐pH for endosomal fusion, which occurs during
the early endosome stage of endosomal transport. As we expected, an increase in
autophage activity lowered viral transduction efficienty, and we observed more viruses
91

in autophages after drug treatment with the autophage‐enhancing drug rapamycin.
These viruses are presumably trafficked to lysosomes where they undergo degradation.
Although the fusion function of the engineered glycoprotein should be
unchanged, we were interested in finding out whether the heparin sulfate binding site
mutation affected its endocytic pathway compared to that of wild‐type Sindbis virus.
Alphaviruses, and in particular Sindbis and Semliki Forest virus, have been well‐studied
and characterized (DeTulleo and Kirchhausen, 1998; Spuul et al., 2010; Leung et al.,
2011). The wild‐type Sindbis virus is believed to enter cells through clathrin‐mediated
endocytosis, which is in agreement with our results with the engineered virus. It is
known to undergo low‐pH‐dependent endosomal fusion as well. Thus, it appears that
the glycoprotein mutation has not altered the viral internalization mechanism.
Autophage activity was demonstrated to lower viral transduction efficiency;
however, there is an additional potential concern for this degradation pathway in terms
of vaccine efficiency. It has been shown that autophagosomes may also deliver viral
peptides to MHC class II loading compartments for presentation to CD4 T cells (Shoji‐
Kawata and Levine, 2009; Kudchodkar and Levine, 2009). The development of antivector
immunity to our DC‐directed lentivector‐based vaccine could render this vaccine
modality inefficient. Thus, it is important for both of these reasons to consider the
reduction of autophage activity, in conjunction with this vector‐based platform. Several
cancer studies have utilized chloroquine, an antimalarial drug that works by raising
intralysosomal pH, and thereby halting the last step of the autophagy pathway, to safely
92

reduce autophage activity (Amaravadi et al., 2007; Bellodi et al., 2009; Carew et al.,
2010; Janku et al., 2011). In fact, currently there is a Phase I clinical trial utilizing
chloroquine in the treatment of small cell lung cancer. Incorporation of such a drug in
DC‐targeting lentivector vaccine system may increase the effectiveness and efficiency of
the immunization regime.

3.4 Materials and Methods
Cell lines and antibodies
The 293T/hDCSIGN cell line was generated previously in our lab (Yang et al.,
2008). Hela/hDCSIGN cells were generated in the same manner. Hela/hDCSIGN, 293T,
and 293T/hDCSIGN cells were incubated at 37°C and 5% CO
2
in Dulbecco’s modified
Eagle’s medium (Mediatech Inc., Manassas, VA, USA) with 10% fetal bovine serum
(Sigma, St Louis, MO, USA), 2 mM L‐glutamine (Hyclone, Logan, UT, USA), and 100 U/ml
of penicillin and 100 ug/ml of streptomycin (Gibco‐BRL). Mouse monoclonoal antibody
for EEA1, rabbit polyclonal antibody for CI‐MPR, lysosome‐associated membrane
protein 1 (Lamp‐1), anti‐LC3A/B, and Alexa 647‐conjugated goat anti‐rabbit
immunoglobulin G (IgG) antibody were obtained from Abcam (Cambridge, MA, USA).
Texas red‐labeled goat anti‐mouse IgG was purchased from Molecular Probes (Carlsbad,
CA, USA). Cyto‐D, nocodazole, bafilomycin A1, taxol, chlorpromazine, filipin, rapamycin,
and 3‐MA were obtained from Sigma.

93

Plasmids
GFPVpr was cloned previously in our lab (Joo and Wang, 2008). DsRed‐hDCSIGN
was cloned by PCR‐amplifying hDCSIGN with HindIII and EcoR1 cut sites. The PCR
product was then cloned with these cut sites into the pDsRed‐Monomer‐C1, obtained
from Clontech (Mountain View, CA, USA).

Virus production
GFPVpr‐labeled lentiviruses were made by transiently transfecting 293T cells
with a standard calcium phosphate precipitation method. The cells were transfected at
about 80‐90% confluency in 6 cm culture dishes with 5 µg of the lentiviral backbone
plasmid FUW, with 2.5 µg each of GFPVpr, pSVGmu, and the packaging plasmids
pMDLg/pRRE and pRSV‐Rev. Four hours post‐transfection, the cells were washed with
medium and incubated for 48 h, after which the supernatant was collected and filtered
with a 0.45‐µm pore size filter. The high titer lentiviruses used for confocal imaging were
then concentrated by ultracentrifugation (Optima L‐90 K ultracentrifuge, Beckman
Coulter) for 90 min at 82,700 g and resuspended in 100 µl of Hank’s balanced salt
solution (Hyclone). Concentrated viruses were filtered by a 0.45 µm pore size centrifuge
tube filter (Costar, NY, USA) before experiments were conducted.

94

Viral transduction
293T/hDCSIGN cells (0.2 x 10
6
per well) were plated in a 24‐well culture dish and
spin infected with viral supernatants (2 ml per well) at 2500 rpm and 30°C for 90 min
(Sorval Legend centrifuge). The cells were then washed and cultured for 3 days before
FACS analysis of GFP
+
cells. For drug treatments, cells were incubated with the drugs
(cyto‐D (20 µM), nocodazole (60 µM), bafilomycin A1 (25, 50, and 100 nM),
chlorpromazine (10 nM), filipin (1 nM), 3MA (5 mM), and rapamycin (1 M) for 30 min
at 37°C before spin infection as described earlier. Drug concentration was maintained
during spin infection and incubated with the cells for 60 min at 37°C after infection,
before replacement with fresh D10 medium.

Confocal imaging
A Yokogawa spinning‐disk confocal scanner system (Solamere Technology Group,
Salt Lake City, UT) with a Nikon eclipse Ti‐E microscope equipped with a 60x/1.49 Apo
TIRF oil objective and a Cascade II: 512 EMCCD camera (Photometrics, Tucson, AZ, USA)
was used to aquire the fluorescent images. An AOTF (acousto‐optical tunable filter)
controlled laser‐merge system (Solamere Technology Group Inc.) was used to provide
power for each of the laser lines: 491 nm, 561 nm, and 640 nm solid state lasers (50 mW
for each laser). Hela/hDCSIGN or 293T/hDCSIGN cells were seeded onto glass‐bottom
culture dishes (MatTek Corporation, Ashland, MA, USA) and grown at 37°C overnight.
The cells were then rinsed with PBS and the concentrated viruses were added and
95

incubated with the cells for 30 min at 4°C to synchronize infection. The cells were then
shifted to 37°C for different time periods, washed with PBS to remove unbound and un‐
internalized viruses, and fixed with 4% formaldehyde for 10 min.

Microtubule‐mediated transport
Viruses on microtubule networks were visualized by incubating the cells with
GFPVpr‐labeled viruses for 1 h at 37°C. The cells were then fixed with 4% formaldehyde
and permeabilized with 0.1% Triton X‐100 with 20 µM of taxol. The microtubules were
then stained with anti‐α‐tubulin mAb (Sigma) and Texas red‐conjugated anti‐mouse
secondary antibody.

Actin‐mediated transport
Viruses on actin filaments were visualized by incubating the GFPVpr‐labeled
viruses at 37°C for 1 hr. The cells were then fixed and permeabilized. The actin filaments
were labeled with rhodamine‐conjugated phalloidin (Molecular Probes).

Imaging virus fusion and transport through endosomes
For fusion studies, concentrated viruses were incubated with 100 uM of DiD
(Molecular Probes) for 1 h at room temperature. GFPVpr and DiD were simultaneously
excited with a 488 nm Argon and a 633 nm HeNe laser, respectively. All samples were
scanned under the same magnification, laser intensity, brightness, gain, and pinhole size
96

conditions. For virus tracking with endosomal markers, the cells were incubated with
GFPVpr‐labeled viruses for different time periods at 37°C and fixed. They were then
permeabilized with 0.1% Triton X‐100 and immunostained with EEA1 and CI‐MPR for
early and late endosome markers, respectively. Texas red‐conjugated anti‐mouse IgG
and Alexa 647‐conjugated goat anti‐rabbit IgG antibodies were used as secondary
antibodies. For the visualization of viral particles in lysosomes, permeabilized cells were
immunostained with Lamp‐1 before secondary antibody staining with the Texas red‐
conjugated anti‐mouse IgG. To visualize viral particles in autophages, cells were stained
with the antibody against LC3A/B, followed by secondary staining by anti‐rabbit IgG.

Dominant‐negative mutants for endosomal dependency
293T/hDCSIGN cells were tranfected with either the dominant‐negative mutant
or the wild‐type construct for Rab 5, Rab 7, Rab 11, or Dyn. 24 h post‐transfection, the
cells were seeded at 0.2 × 10
6
cells per well in a 24‐well culture dish and transduced
with 2 ml of viral supernatant. The cells were analyzed for GFP‐expression by FACS 3
days post‐infection.

97

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CHAPTER 4: TRANSDUCTION OF DENDRITIC CELLS BY A ROSS RIVER VIRUS‐
PSEUDOTYPED LENTIVECTOR


Dendritic cells (DCs) are potent antigen‐presenting cells that hold promise as
effective vaccine targets. Lentiviruses (LVs) are excellent candidates as gene delivery
vehicles for dendritic cells due to their ability to be pseudotyped by other viral
glycoproteins, and to transduce non‐dividing cells. Alphaviruses are generally believed
to specifically transduce DCs through DC‐SIGN, a marker found predominantly on DCs.
Here, we examine the ability of lentiviruses enveloped with an alphaviral envelope
glycoprotein derived from Ross River virus (RRV) to mediate transduction of DCs. We
found that RRV was only able to specifically transduce cells through DC‐SIGN when the
viral vectors were produced under conditions limiting glycosylation to high‐mannose
glycans. This suggests that these RRV‐pseudotyped LVs can be used for DC‐targeting,
but may not be the best choice due to the specific conditions required during vector
propagation.
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4.1 Introduction
Gene‐based immunotherapy is a powerful immunological tool that involves the
education of immune cells to achieve a therapeutic benefit, and several approaches
have been successfully documented (Froelich et al., 2010). Dendritic cell (DC)‐targeting,
in particular, has been a focus of this effort due to their role as potent antigen‐
presenting cells of the immune system. Through T and B cell stimulation, DCs are able to
both initiate and maintain immune responses, and genetic modification of DCs has
yielded therapeutic benefits (Ribas et al., 2002).
Viral vectors have been used for genetic modification of immune cells with
varying degrees of success (Dullaers and Thielemans, 2006). Lentiviral vectors (LVs) are
attractive because they can transduce a wide range of cell types, including non‐dividing
cells. In particular, they have been shown to be adept at delivering genes to DCs
(Dullaers and Thielemans, 2006; Lizee et al., 2004; Oki et al., 2001; Schroder et al., 2002;
Sumimoto et al., 2002; Unutmaz et al., 1999). Additionally, gene delivery to DCs by LVs
have resulted in antigen presentation and antigen‐specific responses in vitro (Dyall et
al., 2001; Firat et al., 2002; Gruber et al., 2000; Zarei et al., 2002), after in vivo
transplantation of the DCs (Breckpot et al., 2003; Firat et al., 2002; Metharom et al.,
2001; Zarei et al., 2004), and with direct injection of LV in vivo (Esslinger et al., 2003;
Firat et al., 2002; Palmowski et al., 2004). Another advantage of LVs is their ability to be
easily pseudotyped by other viral glycoproteins, allowing for an altered tropism and
affinity to specific cell types (Cronin et al., 2005). Alphaviruses are in the Togaviridae
104

family and are mosquito‐borne, enveloped, viruses that appear to preferentially
transduce DCs (Gardner et al., 2000; Klimstra et al., 2003; MacDonald and Johnston,
2000). Recent studies have shown that the Sindbis alphavirus glycoprotein has the
capacity to infect DCs through attachment to Dendritic Cell‐specific ICAM3‐grabbing
Nonintegrin (DC‐SIGN), a surface receptor that is predominantly expressed by DCs (Yang
et al., 2008). DC‐SIGN is a tetrameric type II transmembrane protein that has a
carbohydrate recognition domain (CRD) that binds to high‐mannose oligosaccharides,
and a cytoplasmic tail that can activate APCs to result in an immune response to foreign
antigens (Figdor et al., 2002; Lozach et al., 2007; Mitchell et al., 2001; Soilleux, 2003).
Another alphavirus, Ross River Virus (RRV), has been successfully used to
pseudotype lentiviruses (Kahl et al., 2004; Sharkey et al., 2001; Strang et al., 2005). Our
aim is to study whether pseudotyping our LVs with this viral envelope glycoprotein
(RRV‐G) can aid in its transduction of DCs through DC‐SIGN. Evaluation of the binding
and transduction of the RRV‐G‐pseudotyped lentivirus suggests that it only binds to DC‐
SIGN when the virus is produced under high‐mannose restrictive conditions.

4.2 Results
4.2.1 Design and production of the RRV‐G‐pseudotyped LV
We began by standardizing the RRV‐G construct by cloning the expression
cassette into the VSV‐G‐expression vector, which contained the rabbit β‐globin intron
105

and the poly(A) signal sequence (Figure 4.1); the plasmid used for VSV‐G expression has
been used extensively for pseudotyping lentiviruses (Kahl et al., 2004).

Figure 4.1 Schematic diagram of the constructs encoding the lentiviral backbone plasmid
FUGW and RRE‐G.

To produce the LVs, 293T cells were co‐transfected with the lentiviral construct FUGW
(which contains the GFP reporter gene), plasmids that encoded the gag, pol, and rev
genes, and the RRV‐G plasmid (Lois et al., 2002). GFP‐Vpr‐labeled LVs included the
addition of a plasmid that contained GFP fused to the HIV‐1 vpr protein (Joo and Wang,
2008). The LVs were stained for RRV‐G and imaged by confocal microscopy, which
revealed a significant overlap (Mander’s overlap coefficient >0.7) for RRV‐G‐
pseudotyped viruses (Figure 4.2).
106

Figure 4.2 Confocal imaging of viruses stained with glycoprotein antibodies. Green
indicates the GFP+ viruses while red indicates the stained glycoproteins.

VSV‐G‐pseudotyped LVs were included as a negative control. These results indicate that
our RRV‐G construct can effectively pseudotype our LVs.
Next, RRV‐G‐pseudotyped LV were titered on both the parental 293T cells
(negative control) and the 293T.DCSIGN cell line which stably expresses human DC‐SIGN
(Yang et al., 2008). 1‐deoxymannojirimycin (DMJ) is an inhibitor of 1,2‐mannosidase
that arrests glycan processing at Man
8
GlcNAc
2
, and it has been shown to increase the
amount of high‐mannose present on the glycoprotein service when it is added during
107

viral production (Fuhrmann et al., 1984; Tai et al., 2011). Therefore, we included it in
this titer test to assess its affect on the RRV‐G LV (Figure 4.3)

Figure 4.3 Comparison of viral titers against 293T or 293T.DCSIGN, with FUGW/RRVG
produced with or without DMJ.

Thus, we can see that RRV‐G can effectively pseudotype lentivirus to produce infectious
particles, and that these LVs can specifically transduce cells exhibiting DC‐SIGN if they
were produced under high‐mannose conditions (+DMJ).

4.2.2 Transduction of 3T3 cells expressing cell‐surface DC‐SIGN and L‐SIGN
Previous studies have indicated that another type of C‐type lectin, L‐SIGN, is
homologous to DC‐SIGN and also recognizes carbohydrate motifs. To test this receptor
along with DC‐SIGN, the 3T3.DCSIGN and 3T3.LSIGN were transduced with our
FUGW/RRV‐G virus (Figure 4.4).
108

Figure 4.4 Transduction efficiency of RRV‐G‐ and VSV‐G‐pseudotyped LVs produced with
or without DMJ, against the 3T3 cell line expressing DCSIGN or LSIGN.

The parental 3T3 cells and VSV‐G‐pseudotyped LVs were also included as controls. FACS
analysis of the GFP+ cells revealed that neither RRV‐G nor VSV‐G preferentially
transduced the cells containing L‐SIGN or DC‐SIGN (Figure 4.4, DMJ(‐)).
Next, we hypothesized that the ability of DC‐SIGN(R) to promote infection is
achieved through a mechanism utilizing interactions between the carbohydrate
structures on the viral particles and DC‐SIGN(R). To test this, we generated viral particles
containing high‐mannose glycans on their envelope glycoproteins by treating virus‐
producing cells with DMJ. Production of pseudotyped lentiviruses in the presence of
109

DMJ altered their ability to transduce DC‐SIGN(R) expressing cells (Figure 4.4, DMJ (+)),
consistent with previous studies of increased infection of these cells by mannose‐rich,
mosquito‐derived virus (Shabman et al., 2008). When transduced with DMJ‐treated
FUGW/RRVG, the DC‐SIGN/L‐SIGN‐expressing cell lines were preferentially transduced;
the transduction enhancement of DC‐SIGN and L‐SIGN expressing cells was increased to
almost 3‐ and 4‐folds, respectively, compared to that of non‐expressing cells. In
contrast, VSV‐G‐bearing virus did not exhibit any change when produced in DMJ‐
conditioned cells.
We further tested how efficiently the RRV‐G‐pseudotyped lentivirus transduces
primary immune cell targets and the effect of DMJ treatment on transduction efficiency.
Human monocyte derived DCs (MoDCs) were prepared from the PBMC of healthy
human donors and cultured with GM‐CSF and IL‐4 to generate DC‐SIGN
+
DCs (Obermaier
et al., 2003), which were then challenged with the RRV‐G‐pseudotyped lentivirus
produced with and without DMJ treatment. More than 80% of the cultured DCs were
positive for DC‐SIGN expression before infection. Transduction of the MoDCs by
FUGW/RRVG was ~4% GFP+ DCs (Figure 4.5).

110

Figure 4.5 Infection of MoDC with RRV‐G‐pseudotyped LV produced with or without
DMJ.

Similar to DC‐SIGN and L‐SIGN cell lines, transduction by the pseudotyped lentivirus
produced in the presence of DMJ increased its ability to transduce MoDCs (~17%).

4.2.3 Virus binding to cells is increased in the presence of DC‐SIGN or L‐SIGN molecules
and under conditions of limited carbohydrate processing
To determine whether the increase in transduction of pseudotyped lentiviruses
for 3T3 cells expressing DC‐SIGN or L‐SIGN was due to greater cell binding, in vitro
attachment assays were performed with [
35
S]‐methionine‐radiolabeled virus. RRV‐G did
not increase binding of lentiviruses to the DC‐SIGN(R)‐expressing cells unless treated
with DMJ, while VSV‐G‐pseudotyped virus exhibited similar levels of attachment to all
cell types (Figure 4.6).
111

Figure 4.6 Radiolabeled binding of RRV‐G‐pseudotyped virus compared to VSV‐G‐
pseudotyped LV.

This is consistent with the low preferential infectivity observed with FUGW/RRVG on DC‐
SIGN(R)‐expressing cells. As shown in Figure 4.3, the preferential attachment and
transduction of DMJ‐untreated FUGW/RRVG was approximately similar among cells
regardless of DC‐SIGN(R) expression. Our data suggest that mammalian‐produced RRV‐
G‐bearing lentiviruses do not utilize DC‐SIGN or L‐SIGN as attachment receptors and that
there are alternative factors presented on 293T and 3T3 cells capable of facilitating
infection independent of DC‐SIGN(R) expression. However, the preferential transduction
112

of mammalian cell‐produced RRV‐G‐pseudotyped lentiviruses for DC‐SIGN(R) can be
induced by DMJ treatment (Figure 4.3). Therefore, it is likely that a factor required for
efficient infection of DC‐SIGN(R) is high‐mannose carbohydrate

chains on RRV‐G. DMJ
treatment decreases the extent of

processing of glycoprotein carbohydrate
modifications, resulting

in high mannose content and the absence of complex
carbohydrates

containing sialic acid and galactose (Hsieh and Robbins, 1984). These
untrimmed (DMJ treated) high mannose carbohydrates lead to an increase in DC‐
SIGN(R)‐mediated binding and infection for RRV‐G bearing viruses.

4.3 Discussion
We have demonstrated that SFV‐G‐bearing lentiviruses can utilize DC‐SIGN and
L‐SIGN as attachment receptors, resulting in a productive infection in cell lines bearing
these molecules and human DCs. We further found that RRV‐G bearing lentivectors do
not utilize DC‐SIGN(R) as attachment receptors and are less efficient at transducing DCs.
Variations of preferential binding to DC‐SIGN(R) by the RRV‐G glycoprotein has not
previously been reported, indicating that there are unappreciated differences in the
virus‐binding profiles among alphaviral glycoproteins.   Our results suggest that RRV‐G
lentivectors can be used to preferentially transduce antigen‐presenting DCs for gene‐
based immunotherapy after drug treatment during viral production.
113

We found that when FUGW/RRVG was produced in 293T cells, both VSV‐G‐ and
RRV‐G‐pseudotyped viruses exhibited similar binding and transduction of cells
expressing DC‐SIGN, L‐SIGN, or parental 3T3 cells.
RRV‐G‐bearing lentivectors gain the competence to infect DC‐SIGN(R)‐expressing
cells by the incorporation of high‐mannose carbohydrates at N‐linked glycosylation sites
on the envelope glycoproteins. Several reports indicate the presence of high‐mannose‐
content

N‐linked glycans on Sindbis virus, Ebola virus, and West Nile virus (WNV)
enhance the infection of mouse‐derived dendritic cells due to

interactions with the
mannose binding C‐type lectin receptors (Davis et al., 2006, Klimstra et al., 2003; Marzi
et al., 2007). High‐mannose content of viral envelope glycoproteins strongly influences
the efficiency of viral capture by DC‐SIGN and L‐SIGN (Klimstra et al., 2003; Lozach et al.,
2005; Lozach et al., 2003). Similar to findings reported previously for Sindbis virus
(Morizono et al., 2010), when RRV‐G‐bearing lentiviruses were generated in mammalian
cells treated with the mannosidase I inhibitor DMJ, the resulting particles exhibit an
increased capacity to utilize DC‐SIGN and L‐SIGN for infection. The increase in
interaction with these receptors by FUGW/RRVG is presumably mediated by increased
binding by the CRD of the DC‐SIGN(R) to the mannose structures of these glycoproteins.
The RRV‐G has four sites for N‐linked glycosylation (E1‐141, E2‐200, E2‐262, and E3‐14)
(Strauss and Strauss, 1994). The E3 protein is cleaved from the mature RRV particles.
When virus‐producing cells are treated with DMJ, transduction of RRV‐G‐bearing virus
was preferentially increased approximately 3‐ to 4‐fold towards DC‐SIGN‐ and L‐SIGN‐
114

expressing cells as well as MoDCs. The increased infectivity was correlated with an
increase viral binding to the DC‐SIGN(R). The observation that DMJ‐treated but not
untreated virus‐producing cells have the ability to preferentially transduce DC‐SIGN(R)
suggests that modification of the N‐linked glycans on the FUGW/RRVG glycoprotein can
be used to direct the transduction of DCs through DC‐SIGN(R).
We observed that when produced under non‐DMJ‐treated conditions,
FUGW/RRVG and FUGW/VSVG lentivectors exhibited no increase in binding or
transduction with cells expressing DC‐SIGN or L‐SIGN. VSV‐G‐pseudotyped viruses do
not target through DC‐SIGN(R) but rather transduce a wide range of cell types, likely
through a ubiquitous membrane lipid (Schlegel et al., 1983). Similarly, RRV‐G may utilize
similar cellular membrane components to serve as attachment factors to transduce a
wide range of cell types. Although previous studies have found that in mice, RRV infects
macrophages (Way et al., 2002) and RRV‐G pseudotypes of HIV‐1 infected bone
marrow‐derived premature DC (Strang et al., 2005), both of which are C‐type lectin‐
expressing cells (Figdor et al., 2002), FUGW/RRVG produced in human 293T cells
exhibited no preferential transduction towards the human C‐type lectins DC‐SIGN or L‐
SIGN. FUGW/RRVG and FUGW/VSVG lentivectors have a broad tropism and will
transduce multiple cell types. Therefore, they are undesirable for delivering genes in
vivo to APCs. Our results suggest that the transduction by FUGW/RRVG is largely DC‐
SIGN(R)‐independent unless produced under untrimmed (DMJ‐treated) high mannose
conditions. In addition to DC‐SIGN and L‐SIGN, there are other C‐type lectin molecules
115

present on DCs, macrophages, endothelial cells, and other antigen presenting cells, that
might play a role in RRV infection (Figdor et al., 2002). Although we have eliminated DC‐
SIGN and L‐SIGN as major mediators for the infectivity of FUGW/RRVG and
FUGW/VSVG‐bearing viruses, it remains possible these glycoproteins may utilize other
ubiquitous attachment factors to infect the same cell types.
In this study we assessed the relationship of DC‐SIGN and L‐SIGN interactions
with the RRV glycoprotein to mediate the transduction of DCs. DCs are potent antigen‐
presenting cells and play a major role in the activation of both memory and naïve T cells.
Genetically modified DCs have been used to elicit antigen‐specific, major
histocompatibility complex‐restricted cytotoxic T lymphocyte (CTL) responses (Song et
al., 1997; Yang et al., 1008). The development of DC differentiation protocols for PBMC
has facilitated the study of DC biology and the subsequent implementation in clinical DC
vaccination studies.
The results described herein have relevance to the design and production of
vectors used for gene delivery to APC. Targeting of lentivectors to C‐type lectin‐
expressing cells such as DCs can be specified by the choice of a suitable envelope
glycoprotein and further enhanced by production under conditions that limit host‐cell
processing of viral carbohydrate modifications to contain mannose structures.
Enhanced delivery of antigen to immature DCs may provide an opportunity for
improvement of gene‐based vaccination approaches. Our results show that RRV‐G only
binds when high mannose N‐glycans are present.
116

4.4 Materials and Methods
Cell lines
The human embryonic kidney cell line 293T was used to derive 293T.DCSIGN as
previously described (53). Mouse fibroblasts NIH 3T3 cells were obtained from the
American Tissue Culture Collection. 3T3/L‐SIGN and 3T3/DC‐SIGN (51) were obtained
from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID.
These cell lines were maintained in D10 medium (Dulbecco modified Eagle medium,
Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (Sigma‐Aldrich, St.
Louis, MO), 2 mM L‐glutamine, and 100 U/mL of penicillin and 100µg/mL of
streptomycin.

Plasmid construction
The glycoprotein expression plasmids were constructed similarly to previously
reported (Kahl et al., 2004). The cDNA of RRV‐G was amplified from pRR64 (Kuhn et al.,
1991), which contains the full‐length cDNA of the RRV

genome (Sharkey et al., 2001).
The amplified fragment contained the E3‐E2‐6K‐E1 coding regions with a Kozak
sequence at the translational start site. These fragments were cloned using the BamH1
restriction endonuclease on the vesicular stomatitis virus glycoprotein (VSV‐G)
expression plasmid containing the rabbit β‐globin intron and poly (A) signal (Cell
Genesys, Foster City, CA). The resulting plasmid was designated pRRV‐G (Fig. 1). The
lentiviral backbone plasmid (FUGW and its derivatives) used in this study are the third
117

generation HIV‐based lentiviral vectors in which most of the U3 region of the 3’ LTR was
deleted, resulting in a self‐inactivating (SIN) 3’‐LTR (Fig. 1) and have been previously
described (Yang et al., 2006). The integrity of the DNA sequences was confirmed by DNA
sequencing.

Production of pseudotyped viral particles
Recombinant lentiviruses were prepared by transient transfection of 293T cells
using a standard calcium phosphate precipitation protocol (Pear et al., 1993). 293T cells
cultured in 6‐cm tissue culture dishes (BD Biosciences, San Jose, CA ) were transfected
with the lentiviral backbone plasmid FUGW (5 µg), along with 2.5 µg of the envelope
plasmid (pRRV‐G or pVSV‐G) and 2.5 µg the packaging plasmids (pMDLg/pRRE and pRSV‐
REV). The viral supernatants were harvested 48 and 72 hrs post‐transfection and filtered
through a 0.45‐µm filter. To prepare concentrated viruses, the viral supernatants were
ultracentrifugated (Optima L‐80K preparative ultracentrifuge, Beckman Coulter) at
50,000×g for 90 min. The pellets were then resuspended in an appropriate volume of
HBSS. Mammalian cell‐derived viral stocks with high‐mannose glycans were generated
by transient transfection of 293T cells, which were subsequently cultured in 1 mM 1‐
deoxymannojirimycin (DMJ, Sigma‐Aldrich). Viral particles were harvested 48 hrs post‐
transfection, and their titers were determined by GFP expression.


118

Confocal imaging of GFP‐vpr labeled virions
GFP‐vpr‐labeled lentiviral particles were produced as described above with an
additional plasmid encoding GFP‐vpr (2.5 μg). Fresh viral supernatant was overlaid on
polylysine‐coated coverslips in a 6‐well culture dish and centrifuged at 3,700 × g at 4°C
for 2 hrs using a RT Legend centrifuge. The coverslips were washed with cold PBS twice
and incubated with diluted rabbit polyclonal anti‐RRV E1 antibody (1:1000; a gift from
Richard Kuhn, Purdue University) for 40 min at 4°C. After three washes with PBS, the
cells were incubated for 40 min at 4°C with 1:500 dilutions of secondary antibodies
consisting of species‐specific Cy5‐conjugated anti‐immunoglobulin G (Santa Cruz
Biotechnology, Santa Cruz, CA). Fluorescent images were acquired by a Zeiss LSM 510
laser scanning confocal microscope equipped with filter sets for fluorescein and Cy5. A
plan‐apochromat oil immersion objective (63×/1.4) was used for imaging.

Virus attachment assays
Production of [
35
S]‐methionine‐labeled viruses were produced by transfection of
293T cells or 293T cells treated with 1 mM DMJ as described above. Cells were
maintained in DMEM complement for 5 hrs. Cells were then depleted of methionine for
an additional 3 hrs by replacement of media with antibiotics and methionine‐free
αMEM. At 8 hrs post‐transfection, [
35
S]‐methionine was added to a final concentration
of 20 µCi/mL and cells were incubated at 37°C for an additional 12 hrs. [
35
S]‐
radiolabelled virus was purified from cell supernatants by using a discontinuous sucrose
119

gradient (20%/60% [wt/wt] in TNE buffer [50 mM Tris‐HCl, 100 mM NaCl, 1 mM EDTA]),
followed by pelleting through 20% sucrose in TNE buffer. Radiolabeled virus particles
were resuspended in PBS. Approximately 10
5
cpm of each radiolabeled virus diluted in
PBS was mixed with 10
6
cells in 1.5 mL microcentrifuge tubes with a total volume of 50
µL, and this mixture was incubated at 4°C for 1 hr with gentle agitation. Cells were
washed three times with PBS then resuspended in PBS and
35
S radioactivity of the
resuspended cells was quantified with a liquid scintillation counter.

Determination of p24 and infectious titers
To determine infectious titer, 2 × 10
4
293T or 293T.DCSIGN cells were
transduced in triplicate with 100 µl of serially diluted viral supernatants for 1.5 hrs by
spin‐inoculation at 2,500 rpm and 25°C using a RT Legend centrifuge. Following the spin‐
infection, the supernatants were replaced with fresh culture medium and incubated for
an additional 48 hrs at 37°C with 5% CO
2
. The GFP expression was evaluated by flow
cytometry analysis.

Lentivirus‐mediated transduction of cell lines in vitro
Target cells (293T.DCSIGN, 293T, 3T3‐LSIGN, 3T3‐DCSIGN, or 3T3 cells; 0.2 × 10
5

per well) were seeded in 96‐well culture dishs and spin‐infected with viral supernatants
(150 µL per well of p24‐normalized virus) at 2,500 rpm and 25°C for 90 min using a RT
Legend centrifuge. Subsequently, the supernatants were replaced with fresh culture
120

medium and incubated for 48 hrs at 37°C with 5% CO
2
. The GFP expression was
evaluated by flow cytometry.

Transduction of human PBMC‐derived DCs
Peripheral blood mononuclear cells (PBMC) from healthy human donors were
purchased from AllCells (Emeryville, CA). PBMC were allowed to adhere for 2 hrs on
non‐tissue culture‐treated 24 well plates and washed extensively. Adherent monocytes
were plated at a density of 1 million/well and grown in RPMI medium plus 10% FBS,
antibiotics, interleukin‐4 (500 U/mL), and GM‐CSF (1,000 U/mL) as described previously
(Obermaier et al., 2003). After two days of culture, DCs were identified by examining the
surface markers (CD11C
+
, DC‐SIGN
+
) using flow cytometry analysis. Every 3 days new
cytokines were added. Monocyte‐derived DCs (MoDC) at day 2 cells were exposed to
virus at the required MOI based on 293T cells. For comparison between DMJ treated
versus untreated virus cells were spin‐infected at 2,500 rpm and 25°C using a RT Legend
centrifuge. After the spin‐infection, media was changed and cells were cultured. The
cells were analyzed for GFP expression 5 days post‐transduction.

121

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APPENDIX: LENTIVIRAL VECTORS FOR IMMUNE CELLS TARGETING

Portions of this chapter were adapted from:
Steven Froelich, April Tai, and Pin Wang, Immunopharm Immunot. 2010. 32:208‐218

Lentiviral vectors are efficient gene delivery vehicles suitable for delivering long‐
term transgene expression in various cell types. Engineering lentiviral vectors to have
the capacity to transduce specific cell types is of great interest to advance the
translation of lentiviral vectors towards the clinic. Here we provide an overview of
innovative approaches to target lentiviral vectors to cells of the immune system. In this
overview we distinguish between two types of lentiviral vector targeting strategies: 1)
targeting of the vectors to specific cells by lentiviral vector surface modifications, and 2)
targeting at the level of transgene transcription by insertion of tissue‐specific promoters
to drive transgene expression. It is clear that each strategy is of enormous value but
ultimately combining these approaches may help reduce the effects of off‐target
expression and improve the efficiency and saftey of lentiviral vectors for gene therapy.

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Introduction
The delivery of genes of interest to cellular targets has been studied intensively
over the past twenty years in hopes that autoimmune diseases such as Parkinson’s
disease and severe combined immunodeficiency (SCID), as well as cancers, may be
cured. It has been over a decade since the first gene therapy procedure was performed
on a patient (Anderson et al., 1990). The patient was born with SCID, which required
her to live in relative isolation with frequent bouts of illnesses and routine
administration of antibiotics. For the procedure, the patient’s white blood cells were
removed and cultured in the lab. Then, the missing genes were inserted into the cells
and the cells were infused back into the patient’s bloodstream. After this, the patient’s
immune system was strengthened by 40%, although the procedure was not permanent
and so the corrected white blood cells had to be repeatedly infused every few months.
There has been some debate whether these results were obtained solely due to the
gene therapy procedure versus the other treatments which were administered;
however, this study was at least able to demonstrate that gene therapy could be
administered to a patient without adverse consequences (Thrasher et al., 2006).

Gene Delivery Vectors
There are two types of vehicles by which genes may be delivered into cells. First,
synthetic delivery vehicles can be used. These usually consist of either lipids or
polymers which surround the DNA, termed lipoplexes (Dass, 2002) and polyplexes,
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respectively. There are three types of lipoplexes: anionic, neutral, and cationic. Anionic
and neutral lipids were initially preferred since they were safer, more compatible with
body fluids, and had the potential for tissue‐specific gene transfer; however, production
of these lipoplexes was difficult and expression in transduced cells was relatively low
(Gardlik et al., 2005). Cationic liposomes, on the other hand, naturally form complexes
with DNA, which is negatively charged. The positive charge also facilitates the
penetration of the complexes into the negatively charged cellular membrane. Most
polyplexes are created with cationic polymers. Unlike lipoplexes, though, some
polyplexes are unable to deposit the DNA into the cytoplasm (Forrest and Pack, 2002).
Thus, these polyplexes need to be co‐transfected with endosome‐lytic agents, such as
inactivated adenovirus, to be effective. Synthetic, non‐viral vectors have both
advantages and disadvantages. They are capable of being produced on a large scale and
have low host immunogenicity; however, although recent advances in vector technology
have yielded higher transfection efficiencies, they remain too inefficient for most clinical
applications.
The second type of gene delivery vehicle is the viral vector. These exploit the
natural ability of viruses to efficiently deliver genetic materials to cells. With the tools
available to modern biology, therapeutic transgenes can be easily swapped with the
original viral genes, resulting in specialized gene delivery vehicles. Several in vivo
studies, animal disease models, and even clinical trials have been successfully conducted
using viral delivery vectors, mostly using adenoviral, adeno‐associated, retroviral, or
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lentiviral viral vectors (Edelstein et al., 2007). However, several factors limit the efficacy
of viral vectors for gene delivery. First, systemic barriers, such as pre‐existing immunity,
(Chirmule et al., 1999; Kostense et al., 2004; Muruve, 2004) and cellular barriers, such as
binding to the cell surface, (Howitt et al., 2003; Smith‐Arica et al., 2003) hinder the
efficient delivery of genes into the cells. Also, inefficient production and purification of
the viral vectors (Clemens et al., 1996; Dong et al., 1996), as well as poor transduction
efficiency to the therapeutically relevant cells (Shenk, 2006; Smith‐Arica et al., 2003) are
other barriers that must be overcome for viral gene delivery vectors.

Retroviral Vectors
Retroviruses are enveloped viruses with diploid, single‐stranded, 7‐12 kb positive
sense RNA genomes (Coffin, 1997). This genome contains gag, which encodes the
structural proteins (matrix protein, capsid protein, and nucleocapsid protein), pro and
pol, which encodes enzymatic proteins (protease, reverse transcriptase, and integrase),
and env, which encodes the surface and transmembrane units of the envelope protein
(Figure 1) (Schaffer et al., 2008).
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Figure A.1. Schematic of the retrovirus structure and a lentiviral vector backbone
plasmid, FUW.

The genetic material is contained in the nucleocapsid, which is enveloped by a bi‐lipidic
membrane taken from the virus‐producing cell. The glycoproteins are inserted into this
membrane and are responsible for binding the virions to receptors on the cell surface.
The interaction between the envelope glycoprotein and cellular receptors is what
determines viral tropism. Binding leads to conformational changes in the viral envelope
glycoprotein, exposing the hydrophobic fusion peptide. When inserted into cellular
membrane, this peptide will mediate fusion between the viral and cellular membranes.
After virus‐cell membrane fusion occurs, the core nucleoprotein complex is released
into the cytoplasm and reverse transcription of the viral genome into DNA occurs. This
newly synthesized double‐stranded DNA is then transported into the nucleus and
integrated into to the host chromosome.
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Since retroviruses are able to accommodate extensive changes to their genomes
and integrate efficiently into the genomes of their host cells, they are excellent
candidates for gene transfer vectors. The first retroviral vectors were produced with the
help of replication‐competent or helper viruses. In 1983, retroviral packaging cells were
created that supplied the retroviral proteins but did not produce replication‐competent
viruses. Later, all the viral coding regions were deleted and only the vital viral elements
for high‐efficiency transfer were retained. Since viral entry, reverse transcription, and
genome integration are not dependent on the synthesis of viral proteins, the viral
vectors can contain only the genes of interest.
Retroviral vectors hold many advantages over other gene delivery methods. First, they
can transduce many different types of cells from different species. They can also
integrate their genetic payload into cells with precision and produce high levels of
transgene expression. Since the virus had been engineered to be replication‐
incompetent, there is no danger of the vector spreading to other cells, or of viral
proteins being produced after transduction. Lastly, retroviral vectors have a relatively
large payload capacity and offer low immunogenicity (Coffin, 1997).   Drawbacks of
retroviral vectors include the inability to transduce non‐dividing cells, the lack of
stability in the envelope proteins, and the risk of insertional mutagenesis due to the
semirandom integration of genes (Hacein‐Bey‐Abina et al., 2003).

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Lentiviral Vectors
A subclass of retroviruses that has emerged as another vehicle for gene delivery
is the lentivirus. Lentiviral vectors, such as those derived from the human
immunodeficiency virus (HIV) are capable of infecting nondividing cells through mitosis‐
independent transport of the viral DNA into the nucleus (Lewis et al., 1992; Weinberg et
al., 1991). This feature is particularly useful for gene transfer to non‐dividing cells, such
as antigen presenting cells (APCs), monocytes, and neurons (Case et al., 1999; Naldini et
al., 1996; Weinberg et al., 1991). Lentiviral vectors also do not tend to integrate by
transcriptional initiation sites, a problem faced by other viral vectors (De Palma et al.,
2005). However, like retroviral vectors, high‐titer production of lentiviral vectors has
been difficult due to the complex nature of the virus. To combat this problem, vectors
have been produced which contain the vesicular stomatitis virus (VSV) envelope protein
with the HIV core proteins. This has resulted in higher‐titer vector production, as well as
higher transduction efficiencies. Development of the lentiviral vector has been geared
towards improving safety by reducing the risk of insertional mutagenesis. First, the
vector was made self‐inactivating (SIN) and the enhancer in the LTRs has was deleted
(Frecha et al., 2008). Second, to prevent read‐through transcripts, a strong RNA
polyadenylation sequence was added to the vector sequence (Zaiss et al., 2002). Lastly,
DNA insulators were inserted into the LTR to isolate the internal promoter from the
neighboring genome (Emery et al., 2002).

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General Transductional Targeting Strategy: Delivery of Genes to Specific Cells
One strategy for altering the cellular tropism of lentiviruses is through the
construction of phenotypically mixed particles, or pseudotypes, in which heterologous
glycoproteins are incorporated into the viron as it buds out from the producing cell
(Cronin et al., 2005). Pseudotyping lentiviral vectors consists of engineering vector
particles to incorporate envelope glycoproteins (GPs) derived from other enveloped
viruses. Pseudotyped particles adopt the tropism of the virus from which the GP was
derived (Cronin et al., 2005). Perhaps the most prominent glycoprotein used to
pseudotype lentivectors is the vesicular stomatitis virus G (VSV‐G) protein. VSV‐G‐
pseudotyped lentivectors appear to use ubiquitous lipid‐type receptors, such as
phosphatidylserine, resulting in a broad cellular tropism (Carneiro et al., 2006; Coil and
Miller, 2004; Coil and Miller, 2005). This broad tropism, along with good vector stability
(Watson et al., 2002), are reasons why VSV‐G is the most widely used GP for
pseudotyping lentiviral vectors. However, VSV‐G‐pseudotyped vector particles also
have significant shortcomings. VSV‐G expression is toxic to cells if expressed
constitutively and thus complicates the development of stable packaging cell lines (Ory
et al., 1996). Furthermore, VSV‐G‐pseudotyped particles are inactivated by human
serum complements, requiring PEGylation for in vivo applications (DePolo et al., 2000).
Vectors based on HIV and other lentiviruses have also been pseudotyped with various
envelope proteins to expand the host range to a variety of cell types. In addition,
pseudotyping with alternative viral glycoproteins can be used to resolve other
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limitations such as neutralization by host immune responses, inefficiencies in production
and purification, poor specificities, and poor transduction of therapeutically relevant
cells (Schaffer et al., 2008). Thus, pseudotyping techniques to generate viral vectors
with novel and improved gene delivery properties offer a potential system to address
these gene delivery shortfalls.
Entry of pseudotyped viruses is limited to cells and tissues that express the
appropriate cellular receptor. The natural budding mechanism of the lentivirus and the
plasticity of the envelope membrane to be altered allow pseudotyping with surface
glycoproteins from a variety of different enveloped viruses. Previous virus envelopes
used to pseudotype lentivectors have been review elsewhere (Cronin et al., 2005), but
among others include: lyssavirus (Rabies virus), arenavirus (lymphocytic
choriomeningitis virus (LCMV)), alphavirus (Sindbis virus), influenza virus (HA),
coronavirus (SARS‐CoV), Flavivirus (HCV), Filovirus (Ebola), Gammaretrovirus (RD117),
Bacculovirus (GP64), and Measles virus. These pseudotyped vectors vary widely in their
cellular tropism, titer, efficiency of packaging, stability, immune response, and
inactivation by complement. All characteristics should be carefully considered when
choosing a suitable glycoprotein tailored to best fit the experiment. For example, the
superiority of Gibbon Ape Leukemia virus (GALV) and the cat endogenous retroviral
glycoprotein (RD114) for transduction of progenitor and differentiated hematopoietic
cells was established by screening a large library of pseudotyped vectors (Hanawa et al.,
2002; Kelly et al., 2000; Porter et al., 1996; Sandrin et al., 2002). HIV‐1 vectors
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pseudotyped with RD114 and amphotropic murine leukemia virus (MLV) glycoproteins
were more efficient than VSV‐G pseudotypes at transducing human cord blood CD34+
cells and progenitors (Hanawa et al., 2002). When lentiviruses are utilized in the CNS,
additional glycoprotein characteristics such as retrograde transport must be considered.
While envelope proteins from VSV and Rabies come from the same viral family and
exhibit similar tropism, they have very different retrograde transport activities when
injected into the striatum of the mouse brain (Jakobsson and Lundberg, 2006). Whereas
VSV‐G transduces cells locally, equine infectious anemia virus (EIAV) pseudotyped with
rabies envelope proteins undergo retrograde transport to the thalamus upon striatal
injection (Wong et al., 2004). Ultimately, the aim of incorporating alternative envelope
glycoproteins is to produce a therapeutic, safe, and efficient lentiviral vector for clinical
applications.
Alphaviruses exhibit a wide cellular tropism that includes important gene
therapy targets such as antigen‐presenting cells, neurons, and muscle cells. The cellular
receptors for the various alphavirus glycoproteins have not yet been identified;
however, several receptors or receptor‐coreceptor combinations may be involved in
virus entry. This property allows the tropism of HIV‐1‐based lentiviral vectors to be
altered (Kahl et al., 2005). Recently, wild‐type mosquito‐produced Sindbis alphavirus
(Klimstra et al., 2003) was shown to use C‐type lectins as attachment receptors leading
to productive transduction of dendritic cells. Additionally, several reports of efficient
pseudotyping of lentiviral vectors with Ross River virus (RRV), Semliki Forest virus (SFV),
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and Sindbis virus (SIN) glycoproteins have been reported (Kahl et al., 2004; Kang et al.,
2002; Morizono et al., 2001). Pseudotyping is an alternative straightforward method to
utilize the mechanism by which alphaviral glycoproteins can mediate transduction by C‐
type lectins. As an important example, to narrow the tropism of lentiviral vectors and
enhance vector stability, Sindbis virus glycoproteins have been mutated to reduce
binding to heparan sulfate and enhance dendritic cell tropism (Gardner et al., 2000;
Yang et al., 2008b). High affinity interactions of viral glycoproteins with these C‐type
lectins might represent a strategy by which dendritic cells can be targeted by viruses.
Enhanced delivery of antigen to immature dendritic cells may provide an opportunity for
improvement of vaccines, particularly for gene‐based vaccination approaches.
Development of methods capable of engineering lentiviral vectors to be cell
type‐specific receptors could substantially change the current practice of gene therapy
and greatly expand the scope of gene therapy for disease treatment (Lavillette et al.,
2001; Sandrin et al., 2003; Waehler et al., 2007). Cell‐specific transduction can address
most of the side effects of off targeting gene transfer by the precise introduction of the
therapeutic nucleic acid into expected cells (Waehler et al., 2007). A common strategy
is to genetically modify envelope glycoproteins to incorporate targeting ligands into
lentiviral vectors. It was found that several glycoproteins have structures that are able
to tolerate the insertion of binding motifs such as peptide ligands (Gollan and Green,
2002; Guibinga et al., 2004; Han et al., 1995; Valsesia‐Wittmann et al., 1994), single
chain antibodies (Benedict et al., 1999; Jiang et al., 1998; Somia et al., 1995), growth
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factors (Chadwick et al., 1999; Martin et al., 2002; Maurice et al., 2002; Nguyen et al.,
1998), etc. These engineered glycoproteins can retarget vectors to cells expressing their
corresponding target moieties. Another popular approach is to introduce a “molecular
bridge” to direct vectors to specific cells (Waehler et al., 2007). The molecular bridge has
dual specificities: one end can recognize viral glycoproteins and the other end can bind
to the molecular determinant on the target cell. Such a molecule can direct the
attachment of viral vectors to target cells for transduction. To date, ligand‐receptor,
avidin‐biotin, and chemical conjugations have been exploited for the creation of such
molecular bridges to retarget envelope vectors (Boerger et al., 1999; Roux et al., 1989;
Snitkovsky and Young, 1998). Recently, monoclonal antibodies have been introduced as
a new kind of molecular bridge to allow vectors to preferentially transduce cells
expressing cognate surface antigens both in vitro (Morizono et al., 2001) and in vivo
(Morizono et al., 2005). In such studies, the E2 protein of the Sindbis virus glycoprotein
was modified to contain the Fc‐binding domain of protein A. Thus, one end of the
monoclonal antibody could bind to viral vectors and the antigen recognition end could
direct vectors to antigen‐expressing cells. Proteins overexpressed by producer cells can
also be incorporated onto the vector surface. Lentiviral vectors have been produced
that incorporated CD4 and CCR5 or CD4 and CXCR4 to target the HIV primary receptors
and co‐receptors of HIV‐1‐infected cells (Endres et al., 1997; Somia et al., 2000).
Functions of binding and fusion of some natural viruses such as paramyxovirus
are attributed to two proteins: an attachment protein and a fusion protein (Lamb,
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1993). Thus, mimicking such viruses to separate the binding and fusion functions as two
distinct envelope molecules on the surface of enveloped vectors represents another
attractive strategy for targeting. Lin et al. incorporated a binding‐defective but fusion‐
competent hemagglutinin (HA) protein as a fusion protein and a chimeric glycoprotein
engineered to contain specificity for the Flt‐3 receptor as a binding protein, into
gammaretroviral vectors (Lin et al., 2001). It was shown that such two proteins could
complement each other to mediate preferential modification of cells expressing Flt‐3 in
vitro (Lin et al., 2001). We have demonstrated successful targeting lentiviral vectors by
co‐display of membrane‐bound antibody as the binding protein and fusogenic molecule
derived from Sindbis virus glycoprotein as the fusion protein (Yang et al., 2006).
Efficient and specific transduction was accomplished by a two‐stage process:
endocytosis induced by the antibody‐antigen interaction and fusion triggered by the
acidic pH within the endosomal compartment.

General Translational Targeting Strategy: Transgene Expression in Specific Cells
Another strategy for targeting the genetic manipulation of specific cell types is
through the construction of tissue‐specific expression vectors, in which a tissue‐specific
promoter confers restricted transgene expression in only the target cells. When the
transgene is delivered to the affected cell it would encounter the appropriate
transcriptional machinery and theoretically will not be eliminated by degradation or by
an immune response. Tissue‐specific promoters have been widely used to restrict
175

transgene expression using both viral vectors and non‐viral vectors. Most of them aim
at the production of tissue‐specific expression after transduction and differentiation of
hematopoietic stem cells (HSCs).
One focus of gene therapy relies on transduction of HSCs that are self‐renewing
and have the potential to differentiate into all blood cells, which makes them the main
target for the genetic correction of hematopoietic diseases. Recently, transplantation of
genetically modified HSCs has been explored for the treatment of inherited blood
disorders such as SCID resulting from the lack of the common γ chain receptor (X‐linked
SCID) (Cavazzana‐Calvo et al., 2000), adenosine deaminase‐deficient SCID (ADA‐SCID)
(Aiuti et al., 2002), and chronic granulomatous disease (CGD) (Ott et al., 2006). Previous
studies have revealed that MLV‐derived vectors integrate in a nonrandom fashion into
the host genome, favoring transcripitonally active genes, CpG islands, and
transcriptional start sites (Bushman et al., 2005). The occurrence of leukemia‐like
disorders in patients with SCID‐X1 treated by gene therapy has been associated with
insertional activation of protooncogenes (Cavazzana‐Calvo and Fischer, 2007; Hacein‐
Bey‐Abina et al., 2003). The retroviral vectors used in the previous clinical trials
possessed strong enhancer and promoter elements within the integrated viral LTR (Aiuti
et al., 2002; Cavazzana‐Calvo et al., 2000; Gaspar et al., 2004; Ott et al., 2006). The
strong enhancer of the LTR drives expression but is known to be involved in the
overexpression of the LMO2 protooncogene. Improved safety may be achieved by the
third generation self inactivating lentiviral vectors, in which transgene expression is
176

driven by tissue‐specific promoters. Tissue‐specific promoters may prevent oncogenesis
in cells of the relevant lineages by using more tightly regulated protein expression.

Targeting Immune Cells Using Cell‐Specific Promotors
The goal of targeted gene delivery is precise transgene expression. The
technique of driving gene expression using lentiviral vectors with restricted promoters is
amendable to targeting various immune cell types.   Targeted expression in immune
cells must produce an appropriate amount of transcription without inducing an immune
response or gene silencing. Recently, systems have been developed to regulate
transgene expression (Goverdhana et al., 2005). Clearly, transgene expression
restricted to only the target cell type, controlled expression of the gene, and a limited
induced immune response are desirable properties of an immune cell‐specific promotor
gene expression system. Tissue‐specific promoters have been used to restrict transgene
expression to specific cells of both non‐immune and immune systems (Frecha et al.,
2008). To target expression to specific cells of the hematopoietic system, most tissue‐
specific expression has focused on the transduction and differentiation of HSCs.
Previous cell‐specific lentiviral promoters have been shown to result in B cell (Laurie et
al., 2007; Lutzko et al., 2003; Moreau et al., 2004; Taher et al., 2008; Werner et al.,
2004), T lymphoid (Dardalhon et al., 2001; Indraccolo et al., 2001; Lois et al., 2002;
Marodon et al., 2003), and general antigen‐presenting cell‐specific expression (Ageichik
et al., 2008; Bonkobara et al., 2001; Cui et al., 2002; Dresch et al., 2008; Kozmik et al.,
177

1997; Lopes et al., 2008; Morita et al., 2001). However, the study of LV integration has
pointed out the preferential insertion of the transgene in transcriptionally‐active sites of
the cell genome (Schroder et al., 2002). Furthermore, additional genetic elements are
desirable to impede the convolution of the genome environment where the transgene
will be inserted.
For B cells, previously used retro/lentiviral promoters have included an
immunoglobulin (Ig) heavy chain enhancer in combination with a phosphoglycerate
kinase or cytomegalovirus (CMV) promoter to increase expression (Lutzko et al., 2003),
and a CD19 gene promoter to drive expression of a marker gene in mice using a
retroviral vector (Werner et al., 2004) and in human B cells using a lentiviral vector
(Moreau et al., 2004). An Ig kappa (Igκ) light chain promoter and enhancer has been
described as a useful B cell‐specific promoter in a lentiviral vector (Laurie et al., 2007).
Given that T cells, whether CD4
+
or CD8
+
, are prominent players in pathologic

conditions such as viral infection, autoimmunity, and cancer,

specific expression of
therapeutic genes in T cells has important implications for gene therapy strategies.
Using a lentiviral vector with transgene expression restricted through the

CD4 gene
promoter and enhancer sequences (Marodon et al., 2003), expression was restricted to
mature

T cells. A lentiviral vector driven by the T lymphocyte‐specific proximal lck was
also able to restrict expression when injected to mouse embryos (Lois et al., 2002). In
contrast to transducing HSCs then differentiating them into T cells, another strategy
involves direct gene transfer to the target cells. To efficiently transduce T cells with HIV‐
178

1‐based lentivectors, the central DNA flap of the wild‐type virus, which acts as a cis‐
determinant of HIV‐1 nuclear import, is important for efficient gene transfer into
prestimulated CD4
+
, as well as CD8
+
human T cells (Dardalhon et al., 2001). Stable high
level expression of the transgene of interest is a crucial parameter for gene therapy. To
enhance the gene expression of lentiviral vectors in primary T cells, one strategy is to
incorporate the CD2 locus control region (LCR) to regulate gene expression in T cells
(Indraccolo et al., 2001). The use of these vector construction techniques for T cell‐
based gene therapy of genetic disorders appears very promising.
The technique of driving gene expression using lentiviral vectors with restricted
promoters is also amendable to targeting various antigen‐presenting cell types. The 3.2
kb dectin‐2 gene promoter fragment has been used to drive gene expression in a
vaccine construct (Lopes et al., 2008). Because of its tissue distribution, the dectin‐2
gene has been considered a potentially promising method to restrict gene expression to
antigen‐presenting cells (Bonkobara et al., 2001). Lopes and colleagues demonstrated
that lentivectors with gene expression driven by the dectin‐2 promoter exhibited
restricted distribution to CD11c
+
DCs after subcutaneous injection (Lopes et al., 2008).
Additionally, dectin‐2 lentivectors encoding the human melanoma antigen NY‐ESO‐1
stimulated significant CD8
+
and CD4
+
T cell responses in HLA‐A2 transgenic mice (Lopes
et al., 2008). The concept of driving gene expressing using a lentiviral

vector with
transcriptional control of a transgene was alternatively implemented with the DC‐
specific

DC‐STAMP promoter to transduce HSCs and obtain

transgene transcription
179

predominantly in DCs and in some monocytes (Dresch et al., 2008). When injected into
the brain of a mouse, a LV containing the HLA‐DRα promoter was able to target a
population of intraparenchymal microglia APCs (Lesniak et al., 2005). Another potential
application of APC‐gene therapy is to prevent an immune response after the infusion of
gene‐modified autologous stem cells for the treatment of primary hematopoietic
diseases, where the transgene had never been present in the patient. Promoter
targeting can circumvent the expression of transgenes by APCs after hepatocyte gene
therapy in a mouse model (Follenzi et al., 2004). The immune response against the
transgene was much lower in the mice injected with a hepto‐specific promoter versus
an ubiquiotous CMV promoter. Adoptive transfer of transgene‐modified APCs or
transgene‐induced adaptive regulatory T cells together with LVs could induce tolerance
to transgene‐expressing cells (Annoni et al., 2007). However, the efficacy of promoter‐
specific targeting seems to depend on its precise pattern or level of expression.
The administration of lentiviral vectors carrying tissue‐specific promoters should
be directed to the affected tissue where it would encounter the appropriate
transcriptional regulatory machinery and not incur restricted expression or be silenced
by the host cell immune system. This strategy has been proven to be a good method to
restrict transgene expression. However, additional convolution in the genome
environment where the transgene will be inserted can also restrict expression. The
knowledge of LV integration tropism has been advanced enormously and revealed the
preferential insertion of the transgene in transcriptionally‐active sites of the cell
180

genome. Thus, one could expect some transgene expression could take place due to the
transcription of upstream genes, even if the vector contains a tissue‐specific promoter.
The use of tissue‐specific promoters is a good approach to restrict transgene
expression. When combined with additional genetic components such as insulators to
shield the promoter from neighboring regulatory elements (Prioleau et al., 1999;
Recillas‐Targa et al., 2004; Zhang et al., 2007), inducible expression (Goverdhana et al.,
2005), and use of non‐integrating LVs (Negri et al., 2007), the safety and controllability
of tissue‐specific transgene expression can be significantly enhanced leading to better
designed clinical vectors.

Targeting Immune Cells Using Modified Envelope Proteins
The need for efficient and safe gene transfer to immune cells has led to a
growing interest in the development of methods for targeting lentivectors to specific
target cells and tissues. Development of a high titer lentivector to receptor‐specific
immune cells is an attractive approach to restrict gene expression and could potentially
ensure therapeutic effects in the desired cells while limiting side effects caused by gene
expression in non‐target cells. Many attempts have been made to develop targetable
transduction methods by using lentiviral vectors (Lavillette et al., 2001; Sandrin et al.,
2003; Waehler et al., 2007).   Promising targeting methodologies have been developed
for these vectors, but despite enticing results, limitations remain.
181

Lentivectors initiate infection through interactions between their envelope
glycoproteins and specific cellular receptors, and this interaction is a critical determinant
of viral tropism. The natural budding mechanism of the lentivectors and the plasticity of
envelope membrane glycoproteins to be altered allow insertion of ligands, peptides,
cytokines, and single‐chain antibodies that can direct the vectors to specific cell types
(Gollan and Green, 2002; Kueng et al., 2007; Morizono et al., 2005; Schaffer et al.,
2008).   One targeting strategy for gene delivery to immune cells attempts to redirect
the tropism of the envelope glycoprotein of MLVs by the addition of ligands, which bind
to specific molecules associated with the cell membrane (Maurice et al., 1999; Nguyen
et al., 1998). However, this approach has often resulted in relatively inefficient infection
because the function of the chimeric envelope protein was compromised to some
extent (Fielding et al., 1998). Likewise, targeting of enveloped lentiviruses using single‐
chain antibodies fused to the MLV envelope protein has resulted in similar limitations
(Ageichik et al., 2008; Benedict et al., 1999; Chowdhury et al., 2004; Gennari et al., 2009;
Karavanas et al., 2002; Marin et al., 1996). Another strategy involves using “bridging
molecules” to target vectors. In order to target ecotropic MLVs by means of MHC class I
and class II antigens, antibodies against ecotropic MLV GP are bridged by streptavidin to
specific cell membrane markers on the other side (Roux et al., 1989). The MLV‐modified
targeting vector’s transduction remains limited by inefficiency due to the diminished
fusion activity of the engineered surface protein, which reduces endosomal delivery of
the viral capsid into cells. Similar methods have been used to alter the natural tropism
182

of a lentivector surface protein to enhance transduction of MHC‐1‐expressing cells but
have been met with similar difficulties (Dreja and Piechaczyk, 2006). Another approach
circumvents this fusion deficiency by utilizing vectors displaying both MLV glycoproteins
fused to activating ligands and VSV‐G to enhance HIV‐1 lentivector transduction of
resting T cell lymphocytes (Maurice et al., 2002; Verhoeyen et al., 2003; Verhoeyen et
al., 2005). However, these approaches have had limited success and future attempts
must resolve fusion activity while preserving the ligand targeting, thereby producing
efficient high titer lentivectors.
Systemic injection of HIV‐vectors pseudotyped with various envelope proteins
results in predominant transduction of the liver and spleen (Pan et al., 2002). HSCs are
often used as targets for therapy because of their self‐renewal and multi‐lineage
differentiation capabilities. Although HSCs only represent a small fraction of cells in the
bone marrow, they can fully reconstitute all blood cell elements including cells integral
to the immune system such as B cells, T cells, and dendritic cells. A system to deliver
genes specifically to HSCs would be a powerful tool for engineering novel therapies for
the hematopoietic system. To circumvent the need for specific targeting, current
strategies depend upon direct injection to a localized site with cell‐specific
promoters/enhancers or ex vivo isolation, purification, and transduction. While natural
viral variants can offer some desirable properties for the transduction of hematopoietic
cells, they possess several limitations such as poor specificities and poor transduction of
therapeutically relevant cells (Cronin et al., 2005). Recently, RRV‐mediated transduction
183

of human CD34
+
cord blood cells and progenitors was very inefficient (Kahl et al., 2004).
Therefore, protein engineering approaches to generate viral vectors with novel and
improved gene delivery properties offer attractive means to address these gene delivery
problems for HSCs. Interestingly, surface proteins overexpressed by producer cells can
be also efficiently incorporated into virion particles during vector production, facilitating
novel targeted gene delivery opportunities (Kueng et al., 2007; Yang et al., 2006).
However, when both ecotropic MLV glycoproteins and the membrane‐bound form of
stem cell factor (SCF) were produced, the virus transduced cells in a inverse targeting
fashion (Chandrashekran et al., 2004). The SCF‐displaying vectors failed to infect c‐kit‐
positive hematopoietic cells, but efficiently infected c‐kit‐negative epithelial carcinoma
cells. This was because the fusion function of MLV is dependent on the binding of the
glycoprotein to the cell. Similarly, another targeting methodology incorporates avidin or
streptavidin onto the viral surface along with the gp64 glycoprotein. These vectors
conjugated to biotinylated ligands or antibodies can be retargeted to enhance
transduction of target cell types (Kaikkonen et al., 2009). One limitation in using this
strategy is that the viral glycoproteins used for fusion retain their binding potential
creating high levels of non‐targeted infection. However, the fusion function remains
intricately linked with the binding of the viral fusogen glycoprotein, causing problems
when the binding function is separated from the fusion molecule.
One approach to limit the background infection caused by viral fusogen binding is to
create a binding‐defective version of the viral fusogen molecule. Cannon and coworkers
184

created a binding‐defective version of Fowl Plague Virus Rostock 34 (HAmu). When
incorporated into a retrovirus displaying a functionally attenuated envelope
glycoprotein targeted to murine Flt‐3, HAmu could enhance viral transduction efficiency
(Lin et al., 2001). HAmu is thought to mediate fusion independent of receptor binding
and, when targeted to the Flt‐3 receptor, could be useful for vectors directed either to
hematopoietic progenitor cells or myeloid leukemias (Lavillette et al., 2001). In another
strategy to target lentivectors to immune cells, Chen and coworkers created lentivectors
pseudotyped with the Sindbis virus glycoprotein containing the IgG binding domain of
protein A (ZZ domain). When combined with a CD4 antibody, the vectors were able to
specifically transduce CD4
+
lymphocyte subpopulations in human primary peripheral
blood mononuclear cells (PBMCs) (Morizono et al., 2001). To target dendritic cells, the
natural affinity of the Sindbis virus, which naturally binds to DC‐SIGN, was utilized by
eliminating the binding to non‐specific cellular heparan sulfate molecules, thus
restricting SVGmu pseudotyped particles to DCs (Yang et al., 2008b). Further mutations
by Chen and coworkers in the Sindbis glycoprotein made a binding‐deficient and fusion
competent molecule (Morizono et al., 2005), which, when combined with a CD34
antibody, produced vectors which specifically transduced CD34
+
cells in nonpurified
human mobilized PBMCs (Liang et al., 2009). We adapted this form of Sindbis
glycoprotein but decoupled the antibody binding domain and instead separated the
binding and fusion functions into two separate molecules that are inserted into the viral
envelope (Yang et al., 2006). With the addition of membrane‐bound anti‐CD20
185

antibody, vector particles conferred their binding specificity to cells expressing the B cell
marker CD20 (Lei et al., 2009; Yang et al., 2008a; Yang et al., 2006). The efficiency was
further enhanced by engineering several mutant forms of the Sindbis fusogen which
exhibited elevated fusion functions in a pH‐dependent manner (Lei et al., 2009). This
system was further expanded to deliver genes to monospecific immunoglobulin‐
expressing B cells (Ziegler et al., 2008), CD3‐positive T‐cells (Yang et al., 2009), and
CD117‐expressing HSCs (Froelich et al., 2009). In another approach to target CD20
human primary B lymphocytes, the measles virus binding (H protein) and fusion (F
protein) functions were divided and the viral glycoprotein was retargeted using a single‐
chain antibody fused to the mutant binding‐deficient H protein (Funke et al., 2008).
Such lentivectors re‐targeted to specific immune cells are an attractive vehicle for
targeted gene delivery and could potentially ensure therapeutic effects in the desired
cells, while limiting side effects caused by gene expression in non‐target cells.

Conclusion
Several promising targeting methodologies have been developed for
lentivectors. Although this review has focused largely on the final step of achieving
targeted gene expression, other aspects are equally important. These hurdles include
high costs of vector production for clinical use, immune system barriers (antibodies,
complement system), and questions of systemic application and dosage (Waehler et al.,
186

2007). However, despite these hurdles, the enticing results and the promise of cures for
previously incurable diseases warrant further studies and clinical consideration.

187

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Abstract (if available)

Abstract Dendritic cell (DC) vaccines have great potential as an emerging form of immunotherapy, as DCs are potent antigen-presenting cells, capable of triggering T cell and B cell responses. Our lab has previously developed an engineered lentiviral vector (LV) that is pseudotyped with a mutated Sindbis virus glycoprotein (SVGmu), which is capable of targeting DCs through Dendritic Cell-specific ICAM3-grabbing Nonintegrin (DC-SIGN), a receptor that is predominantly expressed by DCs. ❧ We hypothesized that SVGmu interacts with DC-SIGN in a mannose-dependent manner, and that increasing the amount of high-mannose structures on the glycoprotein surface could result in higher targeting efficiencies of LVs towards DCs. It is known that 1-deoxymannojirimycin (DMJ) can inhibit α1,2-mannosidase I, which is an enzyme that removes high-mannose structures during the glycosylation process. Thus, we investigated the possibility of generating LVs with enhanced capability to modify DCs by supplying DMJ during vector production. Through western blot analysis and binding tests, we were able to infer that binding of SVGmu to DC-SIGN is directly related to amount of high-mannose structures on SVGmu. We also found that the titer for the LV produced with DMJ (FUGW/SVGmu + DMJ) on 293T.DCSIGN, a human cell line expressing the human DC-SIGN antibody, was over four times higher than that of vector produced without DMJ. In addition, transduction of a human DC cell line, MUTZ-3, yielded a higher transduction efficiency for the LV produced with DMJ. ❧ In our next study, we aimed to elucidate the internalization and trafficking mechanisms of this viral vector through confocal microscopy of GFP-Vpr-tagged virus, drug treatments, and dominant-negative mutants of GTPases, which are necessary for endosomal functions. Using these tests, we demonstrated that our engineered lentiviral vector enters the cell via receptor-mediated clathrin- and dynamin-dependent endocytosis, and that microtubule networks were also involved in a productive infection. Fusion was low-pH-dependent and occurred in the early endosomal stage of transport. Autophagy was also examined for its effect on transduction efficiency. We observed that enhanced autophage activity reduced viral infectivity, while suppressed autophagy boosted transduction efficiency. This study gives us insight on the internalization and trafficking mechanisms used by our engineered vector and gives us tools to improve the efficiency of this platform. ❧ In our last study, we examined the ability of lentiviruses enveloped with an alphaviral envelope glycoprotein derived from Ross River virus (RRV) to mediate transduction of DCs. We found that RRV was only able to specifically mediate transduction of cells through DC-SIGN when the viral vectors were produced under conditions limiting glycosylation to high-mannose glycans. This suggests that these RRV-pseudotyped LVs can be used for DC-targeting, but would require specific conditions during vector propagation for effective targeting infections.

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