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Engineering lentiviral vectors for gene delivery

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Engineering lentiviral vectors for gene delivery

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ENGINEERING LENTIVIRAL VECTORS FOR GENE

DELIVERY

by

Steven Michael Froelich

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

May 2011

Copyright 2011 Steven Michael Froelich

ii

Acknowledgements
I would like to thank my advisor Dr. Pin Wang who has supported and guided me
through this process. I would also like to thank the other members of my dissertation
committee Dr. Katherine Shing, Dr. Jesse Yen and Dr. Michael Jakowec. This work was
also supported by invaluable collaborations in immunology by Dr. Lili Yang and the lab
of Dr. David Baltimore as well as collaborators in neuroscience with Dr. Don Arnold, Dr.
Giselle Petzinger, Dr. Michael Jakowec and Dr. Li Zhang and their students.
Finally I would like to thank all the members of the Wang lab. It was wonderful
to be surrounded by such hardworking and talented people. Dr. Kye-il Joo‟s expertise in
confocal imaging was instrumental. Dr. Alex Lei‟s excellence in molecular biology and
Dr. Haiguang Yang‟s expertise in mammalian cell culture were irreplaceable. April Tai‟s
support in designing and conducting experiments was indispensible. I would also like to
acknowledge the contributions of students; Katie Kennedy, Adnan Zubair, Courtney Van
Cott, Meghan Jenks, Xiao Liang, Biliang Hu, Laura Liu, Steven Lee and Bingbing Dai
whose work both directly and indirectly impacted this research. Collaborating with all of
you has enriched my experience.

iii

Table of Contents
Acknowledgements ............................................................................................................. ii

List of Tables ..................................................................................................................... vi

List of Figures ................................................................................................................... vii

Abstract ............................................................................................................................ viii

Chapter 1. Introduction ....................................................................................................... 1
1.1 Gene Delivery Vectors .............................................................................................. 1
1.1.1 Retroviral Vectors ............................................................................................... 3
1.1.2 Lentiviral Vectors ............................................................................................... 5
1.2 General Transduction Strategy .................................................................................. 6
1.3 General Translational Targeting Strategy ............................................................... 11
1.4 Targeting Immune Cells Using Cell Specific Promotors ........................................ 13
1.5 Targeting Immune Cells Using Modified Envelopes Proteins................................ 17
1.6 Conclusion ............................................................................................................... 22
1.7 Acknowlegements ................................................................................................... 23

Chapter 2. Targeted Gene Delivery to CD117-Expressing Cells In Vivo with
Lentiviral Vectors Co-displaying Stem Cell Factor and a Fusogenic Molecule .............. 24
2.1 Introduction ............................................................................................................. 24
2.2 Materials and Methods ............................................................................................ 27
2.2.1 Plasmid Construction ........................................................................................ 27
2.2.2 Generation of Cell Lines ................................................................................... 29
2.2.3 Flow Cytometry Analysis ................................................................................. 29
2.2.4 Effects of Blocking SCF and NH
4
Cl on Viral Transduction ............................ 30
2.2.5 Targeted Transduction in vitro ......................................................................... 30
2.2.6 Virus-cell Binding Assay .................................................................................. 30
2.2.7 Mice .................................................................................................................. 31
2.2.8 Targeting Jurkat.hcKIT in vivo ......................................................................... 31
2.3 Results ..................................................................................................................... 32
2.3.1 Targeting of Human c-KIT-expressing Cell Lines In Vitro ............................. 32
2.3.2 Targeting Subcutaneous Tumor Cells In Vivo .................................................. 39
2.3.3 Intra-Bone Marrow Targeted Delivery ............................................................. 41
2.4 Discussion ............................................................................................................... 43
2.5 Acknowledgements ................................................................................................. 47

Chapter 3. Pseudotyping Lentiviral Vectors with Aura Virus Envelope Glycoproteins
for DC-SIGN-mediated Transduction of Dendritic Cells ................................................. 48
3.1 Introduction ............................................................................................................. 48

iv

3.2 Materials and Methods ............................................................................................ 52
3.2.1 Cell lines ........................................................................................................... 52
3.2.2 Plasmid construction......................................................................................... 52
3.2.3 Production of pseudotyped viral particles ........................................................ 53
3.2.4 Confocal imaging of GFP-vpr labeled virions ................................................. 55
3.2.5 Virus attachment assays .................................................................................... 55
3.2.6 Determination of p24 and infectious titers ....................................................... 56
3.2.7 Vector-mediated transduction of cell lines in vitro .......................................... 57
3.2.8 Assays to inhibit pseudotyped virus-mediated infection .................................. 57
3.2.9 Transduction of human PBMC-derived DCs ................................................... 58
3.3 Results ..................................................................................................................... 58
3.3.1 Generation of AURA-G-pseudotyped lentiviral vectors ................................. 58
3.3.3 Cell-surface expression of DC-SIGN and L-SIGN on 3T3 cells mediates
binding and transduction of pseudotyped lentiviral vectors ...................................... 62
3.3.4 AURA-G-mediated infectivity requires acidification ...................................... 66
3.3.5 Infectivity can be blocked with inhibitors of DC-SIGN................................... 67
3.3.6 AURA-G and SIN-G transduce dendritic cells through DC-SIGN(R) ............ 69
3.4 Discussion ............................................................................................................... 70
3.5 Acknowledgements ................................................................................................. 75

Chapter 4. Virus-Receptor Mediated Transduction of Dendritic Cells by Lentiviruses
Enveloped with Glycoproteins Derived from Semliki Forest Virus ................................. 76
4.1 Introduction ............................................................................................................. 76
4.2 Materials and Methods ............................................................................................ 79
4.2.1 Cell lines ........................................................................................................... 79
4.2.2 Plasmid construction......................................................................................... 79
4.2.3 Production of pseudotyped viral particles ........................................................ 81
4.2.4 Confocal imaging of GFP-vpr labeled virions ................................................. 81
4.2.5 Virus attachment assays .................................................................................... 82
4.2.6 Determination of infectious titers ..................................................................... 82
4.2.7 Lentivirus-mediated transduction of cell lines in vitro ..................................... 83
4.2.8 Assays to inhibit pseudotyped virus-mediated infection .................................. 83
4.2.9 Transduction of human PBMC-derived DCs ................................................... 84
4.3 Results ..................................................................................................................... 84
4.3.1 DC-SIGN correlates with increased infection of lentivirus pseudotyped
with SFV-G................................................................................................................ 84
4.3.2 Preferential transduction of DC-SIGN- or L-SIGN-expressing 3T3 cells ....... 87
4.3.3 SFV-G- and VSV-G-mediated transduction requires acidification .................. 89
4.3.4 SFV-G-mediated infectivity can be blocked with inhibitors of DC-SIGN ...... 91
4.3.5 Effects of viral glycosylation ............................................................................ 93
4.4 Discussion ............................................................................................................... 95
4.5 Acknowledgements ................................................................................................. 99

Chapter 5. Engineering Lentiviral Vectors for use in the Central Nervous System ....... 100

v

5.1 Introduction ........................................................................................................... 100
5.2 Materials and Methods .......................................................................................... 103
5.2.1 Cell Lines ........................................................................................................ 103
5.2.2. Dissociated cultures ....................................................................................... 104
5.2.3 Plasmid Construction ...................................................................................... 104
5.2.4 Lentivector Production ................................................................................... 105
5.2.5 Determination of p24 and infectious titers ..................................................... 105
5.2.6 Vector-mediated transduction of cell lines in vitro ........................................ 106
5.2.7 Vector-mediated transduction of primary cell cultures in vitro ..................... 106
5.2.8 Intracellular staining for flow cytometry ........................................................ 107
5.2.9 Immunocytochemistry on slides ..................................................................... 107
5.3 Results .................................................................................................................. 108
5.3.1 Targeting neuronal cell lines in vitro ............................................................. 108
5.3.2 Transduction of rat cortical neurons in vitro .................................................. 110
5.3.3 Transduction of mixed cultures in vitro ........................................................ 112
5.4 Discussion ............................................................................................................ 114
5.5 Acknowledgements ............................................................................................... 117

References ....................................................................................................................... 118

vi

List of Tables
Table 1.1 Targeting of lentiviral vectors to immune cells ................................................ 17
Table 3.1 Infectious titers of pseudotyped lentivectors .................................................... 60

vii

List of Figures
Figure 1.1 Schematic of the retrovirus structure. ................................................................ 3
Figure 2.1 Virus-producing constructs. ............................................................................ 28
Figure 2.2 Engineered lentiviral vector transduction of targeted cells in vitro. ............... 34
Figure 2.3 Study of the properties of in vitro targeted transduction. ................................ 38
Figure 2.4 In vivo targeted transduction of xenografted cells ........................................... 40
Figure 2.5 In vivo targeted transduction by intra-bone marrow injection. ........................ 42
Figure 3.1 Virus-producing constructs used to make pseudotyped LVs.. ........................ 54
Figure 3.2 Lentiviral transduction of DC-SIGN-expressing 293T cells ........................... 61
Figure 3.3 Effects of DC-SIGN or L-SIGN expression on infectivity ............................. 65
Figure 3.4 Specific inhibitors prevent DC-SIGN-mediated enhancement of infection .... 67
Figure 3.5 LVs bearing AURA-G and SIN-G transduce DCs .......................................... 69
Figure 4.1 Virus-producing constructs used to make pseudotyped lentiviruses. .............. 80
Figure 4.2 Lentiviral transduction of DC-SIGN-expressing 293T cells. .......................... 86
Figure 4.3 Effects of DC-SIGN or L-SIGN expression .................................................... 88
Figure 4.4 Specific inhibitors prevent DC-SIGN-mediated infection .............................. 90
Figure 4.5 Transduction of MoDCs is inhibited with anti-DCSIGN antibody ................. 93
Figure 4.6 Transduction by SFV-G lentiviruses produced in DMJ treated cells .............. 95
Figure 5.1 Virus-producing constructs used to make pseudotyped lentiviruses ............. 109
Figure 5.2 Lentiviral transduction of neuroblastoma cells ............................................. 110
Figure 5.3 Transduced cells in primary cultures of rat cortical neurons. ....................... 112
Figure 5.4 Transduced cells in primary cultures of rat cortical cells. ............................. 114

viii

Abstract
The development of lentiviral vectors to deliver genes to specific cell types are
useful tools because they have the ability to produce stable transduction, maintain long-
term transgene expression, and transduce both dividing and non-dividing cells. Despite
the high transduction efficiency of lentiviral vectors, their tropism frequently does not
match the desired gene delivery application. We report herein, strategies to modify
lentiviral vectors using diverse techniques which allow targeting gene delivery to specific
cell types. To target CD117 expressing cells we engineered a lentivector that incorporates
membrane-bound human stem cell factor (hSCF), and for fusion, a Sindbis virus-derived
fusogenic molecule (FM) onto the lentiviral surface. Lentiviral vectors pseudotyped with
envelope proteins of alphaviruses have recently attracted considerable interest for their
potential utilization for immunotherapy due to their capacity to transduce dendritic cells.
We report lentiviral vectors pseudotyped with envelope glycoproteins derived from the
Aura, Sindbis and Semliki Forest alphaviruses have a natural capacity to transduce
dendritic cells through the DC-SIGN receptor. Finally, in this study, we explore the
ability of pseudotyped lentiviral vectors with envelope glycoproteins derived from a
neuroadapted Sindbis virus envelope glycoprotein to specifically transduce neuronal cell
types. The development of engineered lentiviral vectors to achieve targeted transduction
while avoiding transduction of non-target cells will be important tools for future gene
delivery implementation in a wide range of fields.

1

Chapter 1. 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 severe combined
immunodeficiency (SCID), neurological disorders like Parkinson‟s disease, as well as
cancers, may be cured. It has been over two decades 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 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%. 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).
1.1 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, respectively.
There are three types of lipoplexes: anionic, neutral, and cationic. Anionic and neutral

2

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 has 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
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

3

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.
1.1.1 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). The genetic material is contained in the nucleocapsid, which
Figure 1.1 Schematic of the retrovirus structure and a representative
lentiviral vector backbone plasmid, FUW.

4

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.
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

5

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).
1.1.2 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. 1996a; 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,

6

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).
1.2 General Transduction 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

7

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 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, the 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

8

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 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 to have
limited transduction of human CD34+ cord blood cells and progenitors even at high
multiplicities of infection when pseudotyped with Ross River virus (Kahl et al. 2005).

9

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) 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 to target viruses to dendritic cells. 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

10

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 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, an 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).

11

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 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.
1.3 General Translational Targeting Strategy: Transgene Expression to 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 transgene

12

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 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 driven by tissue-

13

specific promoters. Tissue-specific promoters may prevent oncogenesis in cells of the
relevant lineages by using more tightly regulated protein expression.
1.4 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 a 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. 1997; Lopes et al. 2008; Morita et al. 2001).
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,

14

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)
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-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

15

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 amendable to targeting various antigen-presenting cell types. The 3.2-kbps
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
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,

16

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). Thus, 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. The use of tissue promoters is a good alternative 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 advanced enormously and revealed the preferential insertion of the transgene
in transcriptionally-active sites of the cell 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 alternative 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

17

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.
1.5 Targeting Immune Cells Using Modified Envelopes 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 (Table 1.1).
Target cells Envelope Targeting
References
Transcriptional Targeting
References
Antigen Presenting
Cells
(Gennari et al. 2009); (Yang et al.
2008b)

(Ageichik et al. 2008; Bonkobara et al. 2001;
Cui et al. 2002; Dresch et al. 2008; Kozmik
et al. 1997; Lopes et al. 2008; Morita et al.
2001)
T Cells
(Maurice et al. 1999; Maurice et al.
2002; Morizono et al. 2001; Verhoeyen
et al. 2003; Yang et al. 2009)

(Indraccolo et al. 2001) (Dardalhon et al.
2001; Lois et al. 2002; Marodon et al. 2003)

B Cells
(Funke et al. 2008; Lei et al. 2009; Yang
et al. 2008a; Yang et al. 2006; Ziegler et
al. 2008)

(Laurie et al. 2007; Lutzko et al. 2003;
Moreau et al. 2004; Taher et al. 2008;
Werner et al. 2004)
Hematopoietic
Stem Cells
(Chandrashekran et al. 2004; Fielding
et al. 1998; Froelich et al. 2009) (Liang
et al. 2009; Lin et al. 2001; Verhoeyen et
al. 2005)

(Logan et al. 2002)
Macrophage/Other
Chowdhury et al. 2004; Dreja and
Piechaczyk 2006; Marin et al. 1996;
Nguyen et al. 1998; Roux et al. 1989
(Pawliuk et al. 2001) (Lavenu-Bombled et al.
2007)
Table 1.1Targeting of Lentiviral vectors to immune cells using amendable strategies by
modifying the envelope targeting or by transcriptional targeting.

18

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.
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
(Aires da Silva et al. 2005; 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). Similarly, targeting of enveloped
lentiviruses using single-chain antibodies fused to the MLV envelope protein has also
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

19

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 inefficient transduction remains limiting
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 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 neglects 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

20

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
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 an inverse targeting
fashion (Fielding et al. 1998). 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

21

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 created a binding-defective version of Fowl Plague Virus Rostock 34 (HAmu).
When incorporated into 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

22

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 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.
1.6 Conclusion
Several promising targeting methodologies have been developed for lentivectors
to modify immune cells (see Table for the summary). 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

23

application and dosage (Waehler et al. 2007). However, despite these hurdles, the
enticing results and the promise of cures for previously incurable diseases warrant further
studies and clinical consideration.
1.7 Acknowlegements
April Tai contributed equally to this chapter. Parts of this chapter have been
adapted with permission from Steven Froelich, April Tai and Pin Wang. Lentiviral
vectors for immune cells targeting. Immunopharmacology and Immunotoxicology 2010
32:2, 208-218

24

Chapter 2. Targeted Gene Delivery to CD117-Expressing Cells In
Vivo with Lentiviral Vectors Co-displaying Stem Cell Factor and a
Fusogenic Molecule
The development of a lentiviral system to deliver genes to specific cell types
could improve the safety and the efficacy of gene delivery. Previously, we have
developed an efficient method to target lentivectors to specific cells via an antibody-
antigen interaction in vitro and in vivo. We report herein a targeted lentivector that
harnesses the natural ligand-receptor recognition mechanism for targeted modification of
c-KIT receptor-expressing cells. For targeting, we incorporate membrane-bound human
stem cell factor (hSCF), and for fusion, a Sindbis virus-derived fusogenic molecule (FM)
onto the lentiviral surface. These engineered vectors can recognize cells expressing
surface CD117, resulting in efficient targeted transduction of cells in a SCF-receptor
dependent manner in vitro, and in vivo in xenografted mouse models. This study expands
the ability of targeting lentivectors beyond antibody targets to include cell-specific
surface receptors. Development of a high titer lentivector to receptor-specific 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.
2.1 Introduction
The development of gene delivery has facilitated the utilization of nucleic acids as
practical tools to understand and treat diseases (Verma and Weitzman 2005). One of the

25

obstacles for gene delivery is accomplishing targeted gene expression in a specific subset
of cells, which due to the great diversity of cell types continues to be a key challenge
(Jang et al. 2007; Waehler et al. 2007). Furthermore, gene delivery to a particular type of
cells would limit side effects caused by gene expression in non-targeted cells and ensure
therapeutic effects in only the desired cells (Waehler et al. 2007). Efficient targeted
transduction into specific cell types still represents a major barrier to gene therapy.
Previously, we have reported a method to target lentivectors to specific cells via
antibody-antigen mediated targeting (Yang et al. 2006). It remains unknown whether a
natural ligand-receptor interaction can be similarly utilized to engineer lentivectors for
selective modification of the receptor-expressing cells.
c-KIT is a proto-oncogene encoding CD117, a type III cell surface
transmembrane tyrosine kinase receptor, that naturally binds stem cell factor (SCF)
(Hamel and Westphal 1997). CD117 is expressed in many tissues including mast cells,
gastrointestinal stromal tumors (GISTs), melanocytes in the skin, glial tumors, interstitial
cells of Cajal in the digestive tract and is a precise marker in the bone marrow for
hematopoietic progenitor cells (Edling and Hallberg 2007; Miettinen and Lasota 2005).
Surface CD117 expression can serve as a unique molecular determinant to differentiate
between cell types that can be targeted for gene therapy. Due to the prevalence of c-KIT
receptor in associated malignancies, gene delivery to CD117 specific cells is an
interesting target to demonstrate the potential of engineering targeted lentivectors
utilizing cell surface receptor-ligand interactions.

26

The development of gene delivery vehicles that are targeted to CD117 has been
the goal of many investigators working in the field of gene therapy. Several groups have
targeted c-KIT using plasmid DNA complexes as well as modified adenoviruses (Chapel
et al. 2004; Chapel et al. 1999; Itoh et al. 2003; Schwarzenberger et al. 1996; Zhong et al.
2001). However, these methods do not lead to long-term stable gene expression. Others
have manipulated the gamma-retrovirus – an enveloped double stranded RNA virus that
is capable of stable integration in the host genome. The investigations redirecting
gamma-retroviral vectors to deliver genes to CD117 cells have focused on using
membrane-bound SCF with ecotropic or amphotropic envelope glycoproteins of murine
leukemia virus (Chandrashekran et al. 2004; Fielding et al. 1998). The challenge to this
approach is that the native envelope glycoprotein‟s fusion ability remains intimately
linked with receptor binding. The unknown and delicate coupling mechanisms of
binding and fusion make it extremely difficult to reconstitute fusion function once the
binding determinant of the vector has been altered, which has resulted in inconsistent
targeting and low viral titers (Kasahara et al. 1994; Sandrin et al. 2003; Zhao et al. 1999).
To circumvent the need for specific targeting, current strategies depend upon direct
injection to a localized site with cell specific promoters/enhancers (Logan et al. 2002;
Lutzko et al. 2003; Moreau et al. 2004) or, ex vivo isolation, purification and transduction
(Akporiaye and Hersh 1999; Cavazzana-Calvo et al. 2000).
One limitation to the utility of current viral vectors remains producing a high titer,
long-term expressing, cell specific, gene delivery strategy. In this paper, we engineer
lentivectors capable of specifically transducing receptor-specific cells using lentivectors

27

incorporating a cognate native ligand and fusogenic molecule. Previously, we have
reported a method to target lentivectors to specific cells via antibody-antigen mediated
targeting (Yang et al. 2006). We report herein a novel approach to harness the natural
ligand-receptor mechanism for targeted modification of c-KIT receptor-expressing cells.
For targeting, we incorporate membrane-bound human stem cell factor (hSCF), and for
fusion, a Sindbis virus-derived fusogenic molecule (FM) onto the lentiviral surface.
Targeting lentivectors displaying these two proteins are effective for selective
transduction of a c-KIT receptor model target cell line in vitro and in vivo. Development
of an efficient lentivector for receptor-specific gene transfer could potentially limit the
side effects caused by gene integration in non-target cells and ensure therapeutic effects
in the desired cells.
2.2 Materials and Methods
2.2.1 Plasmid Construction
The fusion molecules have been previously described (Yang et al. 2008a).
Briefly, the original binding-deficient Sindbis virus-derived fusogenic molecule (Yang et
al. 2006), designated FM1, was used as a template for PCR mutagenesis to introduce
mutations at the E1 226 domain to replace amino acids AKN in FM1 with AGM, or
SGM, or SGN to generate new mutant forms of fusogen designated FM2, FM3, and FM4,
respectively (Figure 2.1). To generate phSCF, the genomic DNA was extracted from a
cell line SI/SI4 hSCF220 (ATCC CRL-2453; Manassas, VA) (Toksoz et al. 1992). PCR
was performed to amplify the cDNA of the membrane-bound human SCF (hSCF), which
was then sub-cloned downstream of the CMV promoter in the mammalian expression

28

plasmid pcDNA3 (Invitrogen, Carlsbad, CA) (Figure 2.1). To generate the expression
plasmid for CD117-blind ligand CD20, human CD20 cDNA was sub-cloned downstream
of the CMV promoter in the pcDNA3. The integrity of the DNA sequences was
confirmed by DNA sequencing. The lentiviral backbone plasmid FUGW has been
Figure 2.1 Virus-producing constructs used to make the recombinant targeting lentiviral
vectors. Schematic diagrams of constructs encoding transfer lentiviral vector FUGW,
lentivector expressing luciferase reporter FUGL, membrane-bound human stem cell
factor (hSCF), and fusogenic molecules derived from Sindbis virus glycoprotein. CMV
enhancer: the enhancer element derived from human cytomegalovirus; Ubi: the human
ubiquitin-C promoter; GFP: enhanced green fluorescence protein; WRE: woodchuck
responsive element; ΔU3: deleted U3 region that results in the transcriptional activation
of the integrated viral LTR promoter; Luc: firefly luciferase gene; P2A: self-cleaving
linker; CMV: human cytomegalovirus immediate-early gene promoter; pA:
polyadenylation signal; E1, E2, 6κ, E3: glycoproteins of the Sindbis virus (E1 for fusion,
E2 for receptor binding, 6κ a linker, and E3 is a signal sequence); HA tag: 10-residue
epitope HA tag sequence (MYPYDVPDYA). Fusogen molecules were constructed by
making several alterations (Morizono et al. 2005; Yang et al. 2006), including deletion of
amino acids to disrupt the binding to heparin sulfate glycosaminoglycan, resulting in a
binding deficient but fusion-active fusogenic protein FM1. Mutations were then made in
the fusion loop region to create various engineered fusogens (FM2, FM3, FM4) amino
acid sequences start at 226 of wild-type Sindbis E1.

29

previously described (Figure 2.1) (Yang et al. 2006). The lentiviral backbone plasmid
FUGL was constructed by removing the eGFP gene from FUGW and inserting the PCR
assembly of the cDNA of firefly luciferase linked to self-cleaving P2A (Szymczak and
Vignali 2005) followed by eGFP cDNA (Figure 2.1).
2.2.2 Generation of Cell Lines
To create a stable cell line expressing human c-KIT receptor for the testing of
targeted gene delivery, the cDNA of human c-KIT was sub-cloned downstream of the
ubiquitin-C promoter in the lentiviral backbone plasmid FUW. This construct was
termed FUW-hcKIT. Subsequently, VSVG-pseudotyped FUW-hcKIT was prepared
using calcium-phosphate precipitation and the viral supernatant was used to transduce the
parental Jurkat cell line (ATCC TIB-152) by spin infection. After several passages, the
resulting cells were stained with PE-conjugated anti-human CD117 antibody and
subjected to cell sorting to obtain a uniform population of c-KIT
+
cells designated
Jurkat.hcKIT.
2.2.3 Flow Cytometry Analysis
Antibodies and other staining reagents used for this study include: Biotin-
conjugated anti-HA (Miltenyi Biotech, Bergisch Gladbach, Germany), PECy5-
conjugated streptavidin (BD Biosciences, San Jose, CA), anti-hcKIT (Biolegend, San
Diego, CA), anti-hSCF (R&D Systems, Minneapolis, MN). Flow cytometry was
acquired using a FACScan (Becton Dickinson, San Jose, CA) and analyzed using FlowJo
software (Tree Star, Ashland, OR).

30

2.2.4 Effects of Blocking SCF and NH
4
Cl on Viral Transduction
Jurkat.hcKIT cells (0.1  10
6
) and 500 µL of viral supernatant
(FUGW/hSCF+FM3) were co-incubated in the presence of soluble human SCF (R&D
Systems, Minneapolis, MN), soluble IL-3 as a control (R&D Systems), or NH
4
Cl (Fisher
Scientific, Waltham, MA). The culture media were then replaced 8 hours later with fresh
media and incubated for another 4 days at 37°C, 5% CO
2
. Experiments were performed
in triplicate and eGFP expression was analyzed using flow cytometry.
2.2.5 Targeted Transduction in vitro
In a 24-well flat bottom plate, Jurkat.hcKIT or TF1  cells (0.2  10
6
) were plated
and mixed with 2 mL of fresh viral supernatant and 10 µg/mL polybrene (Chemicon
International, Inc., Temecula, CA). Cells were spin-infected for 90 minutes at 2,500 rpm
at 30°C in Jouan CR 412 centrifuge (Jouan S.A., Cedex, France). Subsequently the
supernatant was removed and replaced with fresh culture medium and incubated for a
further 3 days at 37°C with 5% CO
2
. Experiments were performed in triplicate and one
representative result is shown. Viral titers were performed in duplicate in a 96-well flat
bottom plate, Jurkat.hcKIT or Jurkat cells (0.02  10
6
) were plated spin infected with
100µL dilutions of fresh viral supernatant. The transduction titer was measured in
dilution ranges that exhibited a linear response of eGFP expression with viral serial
dilution concentration.
2.2.6 Virus-cell Binding Assay
Jurkat or Jurkat.hcKIT cells (0.1  10
6
) were incubated with 500 µL of viral
supernatant at 4°C for half an hour and washed with 4 mL of cold PBS. After extensive

31

washing using cold PBS, the cells were mixed with an anti-HA tag antibody (Roche
Applied Science, Mannheim, Germany) to stain the fusogen molecule. After staining,
cells were analyzed by flow cytometry. Experiment was conducted in duplicate and a
representative result shown.
2.2.7 Mice
RAG2
-/-
γ
c
-/-
mice (Jackson Laboratory, Bar Harbor, ME) were maintained in the
university‟s animal facility and cared for in accordance to the NIH guideline and institute
regulations. Treated mice were maintained on mixed antibiotics (sulfamethoxazole and
trimethoprim oral suspension; Hi-Tech Pharmacal, Amityville, NY).
2.2.8 Targeting Jurkat.hcKIT in vivo
Mice (8-12 weeks old) were anesthetized with 2.5% isoflurane and injected either
subcutaneously or intrafemorally with Jurkat.hcKIT cells suspended in PBS. For
intrafemoral or intra-bone marrow (i.b.m.) injection, the right knee was shaved and
sterilized. A 27-gauge needle (BD Bioscience) was inserted into the femur through the
flexed knee to complete injection. Intrafemorally injected mice were given 2 mg/kg
ketoprofen (Fort Dodge, Fort Doge, IA) for pain management. Lentivector was
concentrated using ultracentrifugation (Optima L-80 K preparative ultracentrifuge,
Beckman Coulter) for 90min at 50,000  g. Viral particles were then resuspended in an
appropriate volume of cold PBS. Either 8 hours later or 1 week later mice were injected
with concentrated lentivector expressing firefly luciferase and eGFP through
subcutaneous (s.c.) or i.b.m. administration. To analyze targeting efficiency, mice were
anesthetized with 2.5% isoflurane (Abbott Animal Health, Abbott Park, IL) and 1.5 mg

32

D-Luciferin (Xenogen, Hopkinton, MA) in PBS was injected intraperitoneally. After 5
minutes, the mice were imaged using a Xenogen IVIS 200 (Xenogen). The subcutaneous
experiment was repeated three times and the i.b.m. experiment was repeated twice.
Images were analyzed using Living Image 2.50.1 software and a representative of each
group is presented.
2.3 Results
2.3.1 Targeting of Human c-KIT-expressing Cell Lines In Vitro
The natural budding mechanism of the lentivirus and the plasticity of envelope
membrane to be altered allow pseudotyping and 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; Silva et al. 2005). Here we report
engineering lentivectors capable of specifically transducing receptor-specific cells using a
cognate natural receptor ligand and fusogenic molecule. We report herein a study to
evaluate using this approach to harness the natural ligand-receptor binding mechanism
for targeted modification of c-KIT receptor-expressing cells. By co-transfection of
plasmids, a fusion molecule described previously (termed FM1) (Yang et al. 2006) as
well as membrane-bound ligand (hSCF) may be incorporated into the cell membrane,
which will be transferred to the viral envelope as it buds from the producing cell (Morita
and Sundquist 2004). Besides the FM1 as the fusogen, we developed additional fusion
molecules (FM2-4) (Figure 2.1) by performing mutations in a cholesterol fusion
dependent domain (E1 226) of the Sindbis virus envelope glycoprotein (Lu et al. 1999).
To generate lentivectors enveloped with both targeting ligands and fusogenic molecules,

33

293T cells were transiently transfected by a standard calcium phosphate-mediated
precipitation method with lentiviral vector FUGW, which carries the enhanced green
florescent protein (eGFP) reporter gene under control of the human ubiquitin C promoter,
packaging plasmids (encoding viral gag, pol, and rev genes) and one of the fusion
molecules (FM1-FM4) plus the targeting ligand hSCF or a CD117-blind targeting
molecule CD20 to produce FUGW/hSCF+FM or FUGW/CD20+FM.
A stably expressing CD117 cell line was generated for the study of receptor-
targeted transduction. To assess the expression level of CD117, the relative expression of
the Jurkat cells expressing CD117 (Jurkat.hcKIT) was compared to that of the parental
Jurkat cell line by antibody staining and quantified by flow cytometry (Figure 2.2C). The
Jurkat.hcKIT cell line was found to express human c-KIT receptor stably and uniformly
at significant levels above the parental Jurkat cell line. Vectors incorporating one of the
fusogens and either hSCF or CD20 were spin-infected with Jurkat.hcKIT cells. The
transduction efficiency was measured by the difference in mean fluorescence intensity
(MFI) or eGFP fluorescence activity where non-infected cells were used for setting the
fluorescence gate. FUGW/hSCF+FM vector specifically transduced Jurkat.hcKIT cells
with greater than 80% efficiency and MFI 13 times higher than non-targeted vector
(Figure 2A). In contrast, the FUGW/CD20+FM vector was unable to significantly
transduce Jurkat.hcKIT cells (low MFI), indicating that hSCF confers transduction
efficiency of the vector with Jurkat.hcKIT cells (Figure 2.2A). To extend these
observations, we analyzed the relative ability of the eight vector preparations to transduce

34

Figure 2.2 Engineered lentiviral vector transduction of targeted cells in vitro. (A&B) Fresh
unconcentrated recombinant lentiviral vectors (2 mL) either bearing: hSCF and the
indicated fusogen (FUGW/hSCF+FM) (solid line) or CD20 (control ligand, no specificity
to CD117) and the indicated fusogen (FUGW/CD20+FM) (shaded area), were added to
CD117-expressing Jurkat.hcKIT cells (A) or TF-1α (B). Three days post-transduction the
resulting eGFP expression was taken as an indication of transduction efficiency and mean
fluorescence intensity was analyzed by flow cytometry with cell counts normalized and a
representative result of fluorescence shown with gating of FUGW/hSCF+FM transduced
cells set using non-infected cells. (C) Flow cytometry analysis of the Jurkat.hcKIT cell
line by surface staining using anti-CD117 antibody. Solid line: CD117 expression on
Jurkat.hcKIT cell line; Shaded area: CD117 expression on Jurkat cell line (as a control).
(D) Specific viral titers for various engineered lentiviral vectors. Fresh unconcentrated
lentiviral vectors incorporating hSCF and a fusogen were used to transduce (2  10
4
)
Jurkat.hcKIT or Jurkat cells in a 96 well plate. The viral titer was conducted in duplicate
and measured in dilution ranges that exhibited a linear response of eGFP expression with
viral dilution.

35

TF-1α cells, which have been previously been confirmed by flow cytometry to naturally
express high levels of c-KIT (Chandrashekran et al, 2004). Using SCF or CD20-targeted
vectors and TF-1α cells, the FUGW/hSCF+FM vector gave transductions of up to 70%
and conferred a similar increase in MFI beyond that of non-targeted vector. The CD117-
blind FUGW/CD20+FM vector was unable to significantly transduce these cells (Figure
2.2B). All FUGW/hSCF+FM vectors displayed targeting ability with FM3-bearing
vectors resulting in the highest transduction whereas CD20-bearing vectors resulting in
low background transduction.
The titer of FUGW/hSCF+FM3 (fresh viral supernatant) was estimated on
Jurkat.hcKIT to be ~1 × 10
6
transduction units (TU)/mL (Figure 2.2D); the titer was
determined in the dilution ranges that showed a linear response of eGFP expression with
viral serial dilution. In contrast, the FUGW/hSCF+FM3 resulted in a small background
infection level with a titer of ~7.6 × 10
4
TU/mL on Jurkat cells lacking the expression of
CD117 (Figure 2.2D). Each fusion molecule had a slightly different effect on the ability
of the vector to selectively transduce CD117-expressing Jurkat cells. FUGW/hSCF+FM4
resulted in the highest non-targeted titer ~1 × 10
5
TU/mL in Jurkat cells. The titers were
conducted in duplicate experiments with a deviation of ±0.01 × 10
6
in the results.
Furthermore, when comparing the infectivity of the same viral vector on the targeted
Jurkat.hcKIT cell line, the transduction units of FUGW/hSCF+FM4 only increased 4.6
folds compared to a 14-fold increase with FUGW/hSCF+FM3 (Figure 2.2D). Thus, the
mutations which generate each different fusion molecule were found to effect targeted
viral transduction and FUGW/hSCF+FM3 resulted in the highest infectivity in target

36

Jurkat.hcKIT and the lowest background infectivity in the parental Jurkat cell line.
Because of its improved targeted transduction efficiency, the FUGW/hSCF+FM3 vector
was chosen for the further investigations.
The targeted viral vectors must perform two critical functions for efficient, precise
transduction. First, they must specifically bind with the target cell. To confirm that the
recognition is a consequence of interaction between hSCF and CD117, the binding of the
viral particles to the c-KIT receptor was examined. Jurkat.hcKIT or parental Jurkat cells
were mixed at 4°C to prevent internalization of the viral particles, with lentivector
incorporating one type of FM and hSCF. Cells were then stained for the presence of viral
particles on the cell surface using an anti-FM antibody and quantified by MFI using flow
cytometry (Figure 2.3A). Flow cytometry analysis revealed that lentivectors bearing
hSCF and FM were able to bind to CD117-expressing Jurkat cells (Figure 2.3A). The
control parental Jurkat cells with no CD117 expression displayed no detectable FM,
indicating that the viral particles binding to cells are due to a specific interaction between
the cell surface c-KIT receptor and the viral surface hSCF molecules. In another control,
the vector bearing FM and CD20 antigen exhibited no ability to bind either cell line,
indicating that the FM lacked the capacity for cell binding (data not shown). The mean
fluorescent intensity revealed that the difference in FM does not appear to affect the
binding of the targeted virus to CD117, which indicates that the observed differences in
transduction are not a result of the differences in binding moieties.
Efficient transduction of Jurkat cells was dependent upon the expression of
CD117. To further examine how the specific interaction between targeting ligand on the

37

lentiviral surface and c-KIT receptor on the surface of the target cells mediated the
targeted transduction by FUGW/hSCF+FM3, soluble human SCF cytokine was added at
increasing levels to a virus-cell mixture. As measured by eGFP expression, transduction
of FUGW/hSCF+FM3 was inhibited by >60% during incubation with soluble hSCF but
not the negative control interleukin (IL)-3 (Figure 2.3B). Furthermore, soluble SCF-
mediated inhibition of viral transduction

with Jurkat.hcKIT cells was dose-dependent
(Figure 2.3B). These data confirmed that FUGW/hSCF+FM3 transduction is mediated
by binding of the hSCF ligand to the human c-KIT receptor.
The second critical function targeted viral vectors must perform for efficient,
precise transduction is viral endosomal fusion. Upon binding, the vector should then
induce endocytosis, bringing the lentiviral particle into an endosome where the fusogenic
molecule will respond to the low pH environment and trigger membrane fusion, allowing
the viral core to enter the cytosol. To examine whether c-KIT receptor mediated
endocytosis triggers the pH dependent fusion, we incubated FUGW/hSCF+FM3 with
Jurkat.hcKIT cells in the increased presence of ammonium chloride (NH
4
Cl), which can
raise the pH of the endosomal compartments (Mellman et al. 1986). Addition of NH
4
Cl
to cells directly abolished transduction in a dose dependent manner (Figure 2.3C). These
results are consistent with the observed low pH requirement of the Sindbis derived
glycoprotein to trigger membrane fusion and mediate infection (Lu et al. 1999).

38

Figure 2.3 Study of the properties of in vitro targeted transduction. Fresh unconcentrated
lentiviral vectors bearing hSCF and a fusogen (FUGW/hSCF + FM) were produced. (A)
FACS analysis of the targeted virus cell binding by surface staining using anti-HA tag
antibody against fusogen was used to detect the change in MFI due to viral vectors bound
to the cells. Lentivector (FUGW/hSCF+FM) was mixed at 4°C to prevent internalization
with Jurkat.hcKIT (solid line) or parental Jurkat (shaded area) as a control. (B) Effect of
addition of soluble hSCF on targeted transduction. Either soluble hSCF (black square) or
a control cytokine IL-3 (gray diamond) was added to the wells during transduction of
Jurkat.hcKIT with FUGW/hSCF+SGM. Eight hours later, the media was replaced with
fresh media and the cells placed in the incubator for four days before FACS analysis of
eGFP expression. (C) Various concentrations of NH
4
Cl (dissolved in PBS, pH=7.4) were
added into viral supernatant (FUGW/hSCF+SGM) for 8h, after which the medium was
replaced with fresh medium and cells were cultured for 3 days before FACS analysis of
eGFP-positive cells. The data is presented as the percentage of reduced transduction as
compared to the result of transduction without treatment of either soluble cytokine (B) or
NH
4
Cl (C).

39

2.3.2 Targeting Subcutaneous Tumor Cells In Vivo
A subcutaneous tumor model was developed to determine whether the engineered
vectors could specifically modify CD117-expressing cells in vivo. To assess the
transduction efficiency and specificity, lentiviral vector FUGL, which carries genes for
the firefly luciferase linked to eGFP through a self cleaving 2A peptide (Szymczak and
Vignali 2005) under control of the human ubiquitin C promoter to facilitate co-expression
of both factors (Figure 2.1), was used to transduce cells. Jurkat.hcKIT cells (10 × 10
6
per
mouse) were transferred into irradiated immunodeficient RAG2
-/-
γ
c
-/-
mice on the right
flank subcutaneously (s.c.). Eight hours post-cell injection, 10 × 10
6
TU of engineered
lentivector bearing either membrane-bound hSCF and FM3 or vectors pseudotyped with
VSVG (a viral envelope derived from vesicular stomatitis virus that has a broad tropism)
were injected subcutaneously into both flanks. Figure 2.4A illustrates the experimental
design. Fourteen days post-inoculation, the animals were anesthetized with isoflurane
and the reporter substrate D-luciferin was injected intraperitoneally and luciferase activity
was imaged using a Xenogen IVIS 200 system. The experiment was repeated three times
and one representative result was shown (Figure 2.4B). Mice transduced with
FUGL/hSCF+FM3 show an increased tumor luciferase activity (~12× 10
4
p/s) in the right
flank as compared with the left flank, suggesting that only tumor cells were transduced to
express the reporter gene (Figure 2.4B). In contrast, the non-specific FUGL/VSVG
elicited a much lower luciferase activity in the right flank of approximately 1000 p/s
(Figure 2.4B). Finally, mice were euthanized; the tumors were collected and the cells
were analyzed for surface antigen and eGFP expression. The xenograft tumor cells

40

receiving the targeted vector were 10% positive for eGFP whereas the xenograft tumor
cells receiving VSVG-pseudotyped vector were 2% positive for eGFP (Figure 2.4C).
This verifies that the targeted FUGL/hSCF+FM3 vector is more efficient than the VSVG-
pseudotyped vector in a xenografted subcutaneous Jurkat.hcKIT tumor model in vivo.

Figure 2.4 In vivo targeted transduction of Jurkat.hcKIT cells xenografted on
immunodeficient mice by engineered lentiviral vectors. (A) Schematic diagram of the
procedure used to target cells in vivo. Jurkat.hcKIT cells (10  10
6
) were injected
subcutaneously on the right flank of an immunodeficient RAG2
-/-
γ
c
-/-
mouse. After eight
hours, concentrated lentiviral vectors (10  10
6
TU) expressing both firefly luciferase and
eGFP either targeted (FUGL/hSCF+FM3) or non-targeted (FUGL/VSVG) were injected
subcutaneously on both the right and left flank of the mouse. (B) Two weeks later mice
were injected with the substrate D-luciferin and analyzed using bioluminescence
imaging. The experiment was repeated three times and one representative result is shown
with the indicated luminescence quantification. A mouse receiving no lentivector was
used as a negative control. (C) When tumor cells were palpable the mice were culled and
the tumor cells were collected and cultured. FACS analysis was used to determine CD117
and eGFP expression.

41

2.3.3 Intra-Bone Marrow Targeted Delivery
Having confirmed that Jurkat.hcKIT cells could be infected subcutaneously with
targeting lentivector, a bone marrow model was established to determine whether cells
expressing human c-KIT receptor can be specifically transduced after intra-bone marrow
administration. Three mice were injected with 1 × 10
6
Jurkat.hcKIT cells in the right
femur. Seven days later, 1 × 10
6
TU of either targeted lentivector (FUGL/hSCF+FM3) or
a negative control vector with membrane-bound CD20 (FUGL/CD20+FM3) was injected
into mice with or without Jurkat.hcKIT (Figure 5A). After another seven days, mice
were injected with the reporter substrate D-luciferin and imaged. The experiment was
repeated twice and one representative result was shown (Figure 2.5B). The mice
inoculated with Jurkat.hcKIT and targeted lentivector resulted in the highest
luminescence (24000 p/s), implying transduction of the target CD117 expressing cells in
the bone marrow. The mice that received targeted vector but no Jurkat.hcKIT exhibited
the least luminescence due to the low non-specific transduction of the vector. In contrast,
mice receiving the negative control vector, FUGL/CD20+FM3, displayed similar levels
of luminescence regardless of Jurkat.hcKIT injection (Figure 2.5B). To confirm targeted
transduction, the Jurkat.hcKIT cells were collected from the bone marrow and the cells
were analyzed for CD117 and eGFP expression (Figure 2.5C). The eGFP of mouse
tumor cells expressing CD117 receiving the targeted vector was approximately 3.6 times
higher than the mouse receiving the non-targeted vector.

42

Figure 2.5 In vivo targeted transduction of Jurkat.hcKIT cells xenografted by intra-bone
marrow injection with engineered lentiviral vectors. (A) Schematic diagram of the
procedure used to target cell in the bone marrow. Jurkat.hcKIT cells (1  10
6
) were
injected into the right femur of the mice. One week later lentivector (1  10
6
TU)
expressing both firefly luciferase and eGFP either targeted (FUGL/hSCF+FM3) or
control vector (FUGL/CD20+SGM) was injected into the right femur of the mouse. (B)
One week after the injection of lentivector, mice were injected with the substrate D-
luciferin and analyzed using bioluminescence imaging. The experiment was repeated
twice and one representative result is shown with luminescence quantification. A mouse
receiving Jurkat.hcKIT with no lentivector was used as a negative control. (C) Mice were
culled and the cells from the right femur were collected and cultured. FACS analysis was
used to determine CD117 and eGFP expression.

43

2.4 Discussion
The focus of this study is to test the targeting of lentiviral vectors to specific cell
receptors in vivo mediated by membrane-bound natural receptor ligands. Our results
demonstrate that lentivectors bearing both membrane-bound human stem cell factor and a
Sindbis virus derived fusogen molecule can target cells expressing the c-KIT receptor in
vitro and in vivo. To generate targeting lentivectors, the vector-producing cells were
transfected with plasmids to express both membrane-bound human stem cell factor and
fusogen molecules. The natural budding mechanism of the lentivirus produces particles
with an envelope bearing both a fusion molecule and a targeting hSCF ligand.
The specific binding of the vectors bearing hSCF to the c-KIT receptor on the
target cell surface membrane initiates the mechanism for viral entry. Using flow
cytometry, engineered lentivectors were confirmed to selectively bind to cells expressing
CD117. Furthermore, vectors that bound to CD117 cells were stained with anti-fusogen
antibody indicating that CD117 targeting lentivectors particles have both binding and
fusogen capabilities. Engineered vectors were further confirmed to target cells in a
CD117-dependent mechanism by a SCF inhibition assay. Addition of soluble SCF to the
cells inoculated with targeted vector was found to inhibit transduction in a dose
dependent manner, confirming the binding requirement for the observed targeting.
Because transduction is dependent on binding of the engineered lentivector to the c-KIT
receptor, we expect that the level of c-KIT receptor on the cell surface may affect the
transduction efficiency. The targeted transduction of lentivector requires both specific

44

binding by membrane-bound SCF and that the binding should induce endocytosis of
lentivectors initiating viral entry.
Another key step of entry is fusogen-mediated endosomal fusion releasing the
viral core. The viral fusogen molecules derived from Sindbis virus require a low pH in
the early endosome to induce membrane fusion to deliver the viral payload (Joo and
Wang 2008; Lu et al. 1999). The four Sindbis virus-derived fusogen mutants were
adapted to efficiently envelope lentiviral vectors in a binding-deficient and fusion-
competent manner with mutations in the E1 glycoprotein domain that alter fusion activity
in a cholesterol dependent domain (Lu et al. 1999; Yang et al. 2008a). Binding of the
targeted lentivector was similar regardless of which fusion molecules were co-
incorporated on the lentivector surface. Furthermore, the functional effect of the different
fusion mutants resulted in a varied efficiency of transduction on target as well as on non-
target cells in vitro. In vitro titer data for each targeted vector with the respective fusogen
molecules resulted in the highest transduction in target Jurkat.hcKIT and the lowest
background transduction by FUGW/hSCF+FM3. The mechanism for the enhanced viral
transduction was revealed to be dependent on the endosomal pH. When ammonium
chloride was added to neutralize the acidic endosomal compartments, targeted
transduction was reduced. The sharp decrease in transduction with the addition of NH
4
Cl
is indicative of the pH-dependent nature of the fusogenic molecules incorporated on the
lentivector.
Targeted transduction in vitro shows that the targeting lentivector can
preferentially transduce cells expressing c-KIT receptor. The potential of the targeted

45

lentivector for in vivo targeted gene delivery was first evaluated using a subcutaneous
xenografted Jurkat.hcKIT tumor model. The experiment was repeated only three times
and as such the variances might not be fully captured and therefore shown is only one
representative, which illustrates the observed results but not the variance. The VSVG-
pseudotyped vector was compared to the engineered lentivector FUGL/hSCF+FM3 by
measuring bioluminescence in live animals. VSVG is one of the most common
pseudotypes used in lentivector studies due to its ability to efficiently transduce the
majority of human and mouse cells in a non-specific manner. Although in vitro
experiments revealed that VSVG-pseudotyped vectors can efficiently transduce
Jurkat.hcKIT, in vivo the targeted lentivector is more effective at transducing the target
tumor cells. Perhaps the reason VSVG is very efficient in vitro but not in vivo is due to
non-specific binding of its pseudotyped vectors to multiple cell types which results in less
viral vector reaching the intended cells. The difference in tumor transduction may be
further exacerbated by the difference in growth rate of non-transduced versus transduced
progeny. Overall, the FUGL/hSCF+FM3 vector is able to more efficiently transduce c-
KIT expressing cells than a VSVG-pseeudotyped vector in a subcutaneous xenograft
tumor.
Although direct injection of targeted gene delivery vehicles may be a reasonable
approach for treatment of diseases that have well-defined anatomical barriers, the
treatment of many more evasive diseases requires widespread delivery. Current protocols
for delivery of viral vector-mediated gene delivery involve the localized injection of the
vector to the tissue of interest, resulting only in localized gene expression. Restricted

46

viral trophism would be simplest safe and effective method of achieving cell type specific
distribution. Development of a gene delivery vehicle to receptor-specific cells is an
attractive approach to restrict gene expression and to ensure therapeutic effects in the
desired cells while limiting side effects caused by gene expression in non-target cells.
We report herein a novel approach to harness the natural ligand-receptor mechanism for
targeted modification of specific receptor-expressing cells. This new approach for
targeted gene delivery into c-KIT expressing cells may be a useful tool for investigations
focused on transducing specific cell types within a heterogeneous tissue.
Specific gene delivery to c-KIT-expressing cells in the bone marrow is
particularly interesting because of the important role of this receptor in hematopoiesis. c-
KIT expression is known to be a specific marker in the bone marrow for hematopoietic
progenitor cells. To evaluate the targeting potential of the engineered lentivector in the
bone marrow compartment, a Jurkat.hcKIT tumor was established intra-femorally in a
mouse model. When the FUGL/hSCF+FM3 targeted vector was administrated via intra-
bone marrow injection, it efficiently transduced c-KIT-expressing tumor cells, whereas
when no tumor cells were present, the target vector had a no significant activity. In
contrast, the viral vector carrying CD117-blind CD20 molecules and FM3 resulted in
non-targeted transduction in mice with and without tumor cells. Our data shows that the
targeting vectors can efficiently transduce c-KIT-expressing tumor cells when injected
i.b.m. in a xenograft Jurkat.hcKIT intra-femoral tumor mouse model. c-KIT has been a
popular target for gene therapy due to its association with hematopoietic stem cells
(HSCs). However, clinically gene therapy to HSCs as a personal medicine remains time

47

consuming and expensive. Our results demonstrate the potential for targeted stable gene
transfer into c-KIT receptor expressing cells in the bone marrow. Future studies in other
models are warranted to investigate more real-world physiological situations. Utilizing
this CD117-specific lentiviral gene delivery system in other tissues may further broaden
the prospects of c-KIT receptor targeted gene delivery.
Gene delivery to a particular type of cells would limit side effects caused by gene
expression in non-target cells and ensure therapeutic effects in only the desired cells.
Efficient targeted gene delivery to a specific subset of cells continues to be a key
challenge for gene therapy. Previously, we have reported a method to target lentivectors
to specific cells via antibody-antigen mediated targeting (Yang et al. 2006). Our findings
suggest that another way to target specific cells is to engineer lentivectors bearing
cognate natural ligands and fusogenic molecules. These vectors are able to confer
selective transduction of receptor expressing cell type in vitro and in vivo. We
demonstrate our targeting methodology using c-KIT receptor as a model target. Further
implementation of this natural ligand-receptor targeting strategy may be adapted to other
receptor targets thereby targeting various other cell types.
2.5 Acknowledgements
This work was co-authored with Leslie Ziegler and adapted from: Froelich S,
Ziegler L, Stroup K, Wang P. 2009. Targeted gene delivery to CD117-expressing cells in
vivo with lentiviral vectors co-displaying stem cell factor and a fusogenic molecule.
Biotechnol. Bioeng. 104,206-15.

48

Chapter 3. Pseudotyping Lentiviral Vectors with Aura Virus
Envelope Glycoproteins for DC-SIGN-mediated Transduction of
Dendritic Cells
Lentiviral vectors (LVs) pseudotyped with envelope proteins of alphaviruses have
recently attracted considerable interest for their potential as gene delivery tools. We
report the production of human immunodeficiency virus type 1 (HIV-1)-derived LVs
pseudotyped with envelope glycoproteins derived from the Aura virus (AURA). We
found that the AURA-glycoprotein pseudotyped LVs use C-type lectins (DC-SIGN and
L-SIGN) as attachment factors. These interactions with DC-SIGN are specific as
determined by inhibition assays and appear to facilitate transduction through a pH-
dependent pathway. AURA pseudotyped LVs were used to transduce monocyte-derived
dendritic cells (DCs) and the transduction was shown to be DC-SIGN-mediated, as
illustrated by competitive inhibition with DC-SIGN(R) antibodies and yeast mannan.
Comparisons with LVs enveloped with glycoproteins derived from vesicular stomatitis
virus (VSV) and Sindbis virus (SIN) suggests that AURA-glycoprotein bearing LVs
might be useful to genetically modify DCs for the study of DC biology and DC-based
immunotherapy.
3.1 Introduction
The introduction of a functional gene into specific cell types has emerged as a
useful tool for both performing basic scientific research and developing novel

49

therapeutics. Much effort has been focused on engineering viral vectors as gene transfer
vehicles because of their high efficiency (Kootstra and Verma 2003). Among these
vectors, lentiviral vectors (LVs) derived from human immunodeficiency virus type 1
(HIV-1) are promising because they have the ability to produce stable transduction,
maintain long-term transgene expression, and transduce both dividing and non-dividing
cells (Naldini et al. 1996b). Using such viruses to transduce particular cell types has been
restricted due to the natural tropism of HIV-1 for CD4
+
T cells and macrophages. One
mechanism for altering the tropism of LVs is through pseudotyping, incorporating
envelope glycoproteins from other viruses into the lentiviral surface (Cronin et al. 2005).
Due to its very broad tropism and stability, LVs are commonly pseudotyped with the
vesicular stomatitis virus glycoprotein (VSV-G). However, the VSV-G envelope comes
with a number of limitations including the susceptibility to inactivation in human
complement, toxicity of constitutively expressed glycoprotein in producer cell lines and
broad tropism with unknown entry receptor(s) (Coil and Miller 2004; DePolo et al.
2000).
There is a growing supply of alternative glycoproteins and strategies to engineer
envelope glycoproteins for pseudotyping LVs each with specific advantages and
disadvantages. A number of recent reports have focused on enveloping LVs with
glycoproteins derived from alphaviruses (Cronin et al. 2005). These pseudotypes are
particularly promising because they are able to be concentrated to obtain high-titer viral
preparations, resistant to inactivation by components in human sera, and stable packaging
cell lines have previously been generated that could be scaled up to meet the larger

50

volumes for clinical demand (Strang et al. 2005). Recently, pseudotyping of HIV-1
vectors has been shown with glycoproteins from alphaviruses including, Western and
Venezuelan equine encephalitis viruses, Chikungunya virus, Ross River virus, Semliki
Forest virus and Sindbis virus (SIN) (Akahata et al. 2010; Kahl et al. 2004; Kolokoltsov
et al. 2005; Morizono et al. 2001; Poluri et al. 2008). In addition, SIN-pseudotyped LVs
have been directed to specific cell types by inserting ligands (Aires da Silva et al. 2005)
or attaching ligand recognition domains into the glycoprotein (Morizono et al. 2001) and
by co-displaying membrane bound antibodies (Lei et al. 2009; Yang et al. 2009; Yang et
al. 2006) or ligands (Froelich et al. 2009; Ziegler et al. 2008). In this study, we show for
the first time that the Aura virus glycoprotein (AURA-G) can be incorporated into HIV-
1-derived LVs and form infectious pseudotyped particles.
Although the cell surface receptors used by alphaviruses to infect a broad variety
of species have not yet been determined, it has been suggested that several receptors are
involved in virus entry. Alphaviruses are mosquito-borne, enveloped, positive-sense
RNA viruses which appear to have evolved to exploit cells of the macrophage-dendritic
cell (DC) lineage for early dissemination from the site of inoculation in the skin (Gardner
et al. 2000; Klimstra et al. 2003; MacDonald and Johnston 2000). The alphaviral
glycoprotein complex consists of heterodimers of the E1 and E2 glycoproteins that are
organized into trimers upon exposure to low pH (Strauss and Strauss 1994). The E2
glycoprotein mediates interactions with target cell receptors, whereas E1 is thought to
mediate endosomal fusion with the viral membrane in an acidic environment (Dubuisson
and Rice 1993; Garoff et al. 1980; Kielian 1995). The structural proteins of Aura virus

51

are closely related to those of Sindbis virus with 56% identity in glycoprotein E2 and
61% identity in glycoprotein E1 (Rumenapf et al. 1995). Through adaptive mutation in
tissue culture, laboratory alphaviral strains have been shown to acquire binding affinity
for cell surface heparan sulfate which results in broad cellular tropisms (Bernard et al.
2000; Heil et al. 2001; Klimstra et al. 1998; Zhu et al. 2010). As opposed to heparan
sulfate, the mosquito-produced Sindbis alphavirus (Klimstra et al. 2003) and LVs with
modified Sindbis glycoproteins (Yang et al. 2008b) were shown to use C-type lectins as
attachment receptors leading to productive transduction of dendritic cells (DCs).
Therefore, we reasoned that C-type lectins DC-SIGN and L-SIGN (together known as
DC-SIGN(R)) may be important attachment factors for AURA-G-bearing LVs as well.
In this study, we find that AURA-G-pseudotyped LVs have an enhanced
preferential transduction for DC-SIGN(R)-expressing cells and a low transduction
efficiency towards other cell types, which makes AURA-G pseudotypes very useful for
gene delivery to DCs. We conducted a comparative study of the efficiency of
incorporation of the VSV-, SIN- and AURA-glycoproteins into LVs. We demonstrate the
efficient pseudotyping of LVs with the envelope proteins of Aura virus. When the
conditions were optimized, we achieved titers of ~1×10
5
TU/mL for both AURA and
SIN-pseudotypes on cells expressing DC-SIGN with approximately ten times less
transduction units per mL parental (DC-SIGN
-
) cells whereas, VSV-pseudotypes produce
titers of ~10×10
6
TU/mL on both cell lines. We show that AURA-G pseudotyped
lentivectors have an intrinsic tropism for DC-SIGN(R)-expressing cells, including
monocyte-derived DCs (MoDCs). These interactions with DC-SIGN are specific as

52

determined by inhibition assays and appear to transduce cells through a pH-dependent
pathway similar to that of SIN-G-pseudotyped LVs. Our findings indicate that the
AURA-G might be an attractive envelope to pseudotype LVs for transduction of DCs for
gene-transfer applications.
3.2 Materials and Methods
3.2.1 Cell lines
The human embryonic kidney cell line 293T was used to derive 293T.DCSIGN as
previously described (Yang et al. 2008b). Mouse fibroblasts NIH 3T3 cells were obtained
from the American Tissue Culture Collection. 3T3/MX-L-SIGN and 3T3/MX-DC-SIGN
(Wu et al. 2002) 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 bovine serum (Sigma-Aldrich, St. Louis, MO), 2 mM L-glutamine, and 100 U/mL
of penicillin and 100 µg/mL of streptomycin.
3.2.2 Plasmid construction
The glycoprotein expression plasmids were constructed similarly to a previously
reported procedure (Morizono et al. 2001). The cDNA of SIN-G was amplified from the
AR 339 strain, which contains the full-length cDNA of the SIN

genome (GenBank
sequence J02363). The cDNA of AURA-G was amplified from the full-length cDNA of
the Aura virus genome (GenBank sequence AF126284). The amplified fragments for
both glycoproteins contained the E3-E2-6K-E1 coding regions with a Kozak sequence at

53

the translational start site. These fragments were subcloned using BamH1 restriction
endonuclease into 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 plasmids were designated pSIN-G and pAURA-G (Figure 3.1).
The lentiviral backbone plasmid (FUGW and its derivatives) used in this study are the
third generation HIV-based LVs in which most of the U3 region of the 3‟ LTR was
deleted, resulting in a self-inactivating (SIN) 3‟-LTR (Figure 3.1) and has been
previously described (Yang et al. 2006). The integrity of the DNA sequences was
confirmed by DNA sequencing.
3.2.3 Production of pseudotyped viral particles
Recombinant LVs 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
(pSIN-G, pAURA-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. Their titers were determined by flow cytometry
analysis of GFP expression. To prepare concentrated viruses, the viral supernatants were
concentrated 300 fold by ultracentrifugation at 4°C (Optima L-80K preparative
ultracentrifuge, Beckman Coulter) at 50,000×g for 90 min C with a >80% recovery. The
pellets were then resuspended in an appropriate volume of Hank's Buffered Salt Solution
(HBSS) (Lonza, Walkersville, MD).

54

Figure 3.1 Virus-producing constructs used to make pseudotyped LVs. (A) Schematic
diagrams of constructs encoding lentiviral backbone FUGW and alphaviral envelope
glycoproteins. Ubi: the human ubiquitin-C promoter; GFP: enhanced green fluorescence
protein; WRE: woodchuck responsive element; ΔU3: deleted U3 region that results in the
transcriptional activation of the integrated viral LTR promoter; pA: polyadenylation
signal; E1, E2, 6k, E3: alphavirus glycoproteins (E1 for fusion, E2 for receptor binding,
6k a linker, and E3 is a signal sequence). The VSV-G expressing plasmid contains the
rabbit β-globin intron and poly(A) signal. AURA and SIN glycoprotein expression
plasmids were constructed that have both the rabbit β-globin intron and poly(A) signal
from VSV-G expression plasmid to standardize glycoprotein expression cassettes. (B)
Viral supernatants harvested from virus-producing cells transiently transfected with GFP-
vpr, AURA-G or SIN-G, and other necessary packaging constructs, was coated onto a
poly-lysine containing coverslip by centrifugation. The resulting coverslips were then
rinsed and immunostained with Sindbis virus immune ascitic fluid to label the
glycoproteins (red) and imaged using a laser confocal microscope.

55

3.2.4 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 Sindbis virus immune ascitic fluid (ATCC number VR-1248AF) for
40 min at 4°C. After three washes with PBS, the cells were incubated for 40 min at 4°C
with 1:250 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.
3.2.5 Virus attachment assays
Production of [
35
S]-methionine-labeled viruses were carried out by transfection of
293T as described above. Cells were maintained in DMEM complement for 4 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 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

56

radiolabeled virus, diluted in pH=7.4 Phosphate buffered saline (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. Radioactivity of
35
S for the resuspended cells was
quantitated with a liquid scintillation counter. The experiments were performed in
triplicate and the percentage of bound radiolabeled particles was calculated from a cell
free virus only control.
3.2.6 Determination of p24 and infectious titers
To determine p24 titers, supernatants were diluted 1:10,000 and assayed by an
enzyme-linked immunosorbent assay (ELISA) using the p24 ELISA kit from
ImmunoDiagnostics (Woburn, MA) according to the manufacturer‟s instructions. AURA-
G producing cells made 360 ±38 ng/µL of p24 whereas, SIN-G transfected cells
produced 348 ± 19 ng/µL and VSV-G supernatant yielded 308 ±42 ng/µL of p24. To
determine infectious titer, 2 × 10
4
293T or 293T.DCSIGN cells were transduced in
triplicate with 100 µl of serially diluted viral supernatants with 8 µg/mL of polybrene
(Sigma-Aldrich) 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.

57

3.2.7 Vector-mediated transduction of cell lines in vitro
Target cells (293T.DCSIGN, 293T, 3T3-LSIGN, 3T3-DCSIGN, or 3T3 cells; 2 ×
10
4
per well) were seeded in 96-well culture dishes and spin-infected with viral
supernatants (150 µL per well with 50ng 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 medium and incubated for 48 hrs at 37°C with 5% CO
2
. The GFP
expression was evaluated by flow cytometry.
3.2.8 Assays to inhibit pseudotyped virus-mediated infection
In dose-response experiments, triplicate wells of 293T.DCSIGN were incubated
with 0.2 to 200 µg/mL of yeast mannan (Sigma-Aldrich) at 37°C for 30 min. AURA-G-,
SIN-G- or VSV-G-bearing LVs were incubated for 8 hrs, and then supernatant was
replaced with fresh medium. For bafilomycin and NH
4
Cl inhibitions, pseudotyped viral
particles were spin-inoculated in the presence of Bafilomycin A
1
(Sigma-Aldrich) or
increasing concentrations of NH
4
Cl for 90 min at 25°C. Subsequently, the supernatants
were replaced with medium containing Bafilomycin A
1
or NH
4
Cl and incubated for 2 hrs
before being replaced with fresh medium. 293T.DCSIGN cells were incubated with 5
µg/mL of anti-DC-SIGN antibodies (14E3G7, 19F7, DC-28, and isotype control
antibody, Santa Cruz Biotechnology), 5 mM EDTA at 37°C for 30 min, and then
inoculated with 293T-produced AURA-G-, SIN-G- or VSV-G-bearing LVs at an MOI <1
for 8 hrs. The cells were analyzed for GFP expression 2 days post-transduction.

58

3.2.9 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 indicated multiplicity of infection (MOI) based on 293T
cells. For inhibition of DC-SIGN mediated transduction, DCs were incubated with
20µg/mL of anti-DC-SIGN(R) antibody (14E2G7, Santa Cruz Biotechnology) or 200
µg/mL yeast mannan (Sigma-Aldrich) at 37°C for 30 min, and then inoculated with
293T-produced FUGW/AURAG or FUGW/SING LVs at an MOI=10 based on titer on
293T cells for 8 hrs before the media was changed. The cells were analyzed for GFP
expression 5 days post-transduction.
3.3 Results
3.3.1 Generation of AURA-G-pseudotyped lentiviral vectors
It has not been previously shown that AURA-G can pseudotype HIV-1-derived
lentiviral vectors (LVs). We first investigated the pseudotyping efficiency of HIV-1
vectors with AURA-G glycoproteins by transient transfection. We standardized the

59

glycoprotein expression plasmids used in this study by sub-cloning the AURA-G and
SIN-G expression cassettes into the VSV-G expression vector containing the rabbit β-
globin intron and the poly(A) signal sequence (Figure 3.1A); the plasmid used for VSV-
G expression has been used extensively for pseudotyping LVs (Kahl et al. 2004). The
production of LVs pseudotyped with AURA-, SIN-, or VSV-glycoproteins were
generated by the co-transfection of 293T cells with lentiviral construct FUGW (Lois et al.
2002), plasmids encoding viral gag, pol and rev genes (Klages et al. 2000), and the
respective glycoprotein expression plasmid. FUGW carries the GFP reporter gene under
control of the human ubiquitin-C promoter (Figure 3.1A) (Lois et al. 2002). GFP-vpr-
labeled LVs were produced as described above with an additional plasmid transfected
encoding GFP fused to the HIV-1 vpr protein (Joo and Wang 2008). Antibody staining of
GFP-vpr-labeled virus with an AURA and SIN cross reactive antiserum revealed
significant overlap (Mander‟s overlap coefficient >0.9) for both AURA-G- and SIN-G-
pseudotyped viruses (Figure 3.1B). These results indicate a similar level of incorporation
of the respective glycoproteins onto lentiviral particles. In contrast VSV-G-pseudotyped
viruses exhibited no glycoprotein staining using the antiserum (data not shown).
To compare the infectivity of AURA-G-, SIN-G- and VSV-G-bearing LVs, the
infectious titer was determined on 293T cells and the 293T.DCSIGN cell line which
stably expresses human DC-SIGN (Figure 3.2A). Differences in viral titer may be
attributed to several factors: differences in virus-receptor interactions, the efficiency of
production of functional particles into the supernatant, as well as amount of defective
particles which may serve as interfering particles. As shown in Table I, AURA-G can

60

effectively pseudotype HIV-1 vectors to produce infectious particles; these viruses are
designated FUGW/AURAG. For comparison the infectious titer of SIN-G-pseudotyped
LV (FUGW/SING) and VSV-G-pseudotyped LV (FUGW/VSVG) were produced. The
production of FUGW/VSVG was the most efficient and estimated to be ~10×10
6

transduction units (TU)/mL on both 293T and 293T.DCSIGN cells. SIN-G-pseudotyped
LVs exhibit a lower infectious titer on both cell lines but with an observable increase in
infectivity with cells expressing DC-SIGN. The titer on 293T cells was ~6×10
4
(TU)/mL
versus ~1.7 times higher on 293T cells expressing DC-SIGN (Table I). Similarly, when
AURA-G was used as the envelope glycoprotein, the infectious titer was higher with cells
expressing DC-SIGN. Based on 293T.DCSIGN cells, the FUGW/AURAG titer was
~0.8×10
5
TU/mL versus a 10 fold decrease on 293T cells (Table I). The AURA-G-
bearing LV was much less infectious with 293T cells; the titer was approximately 7.5
times lower than that of SIN-G and 125 times lower than that of VSV-G. Furthermore,
the difference in infectious units between cell types is clear evidence that the transduction
by SIN-G and AURA-G-bearing LVs is enhanced by the presence of DC-SIGN.
Glycoprotein
293T 293T.DCSIGN
Mean infectious titer
(TU/mL) ± SD
Mean infectious titer
(TU/mL) ± SD
AURA-G 0.80±0.05×10
4
0.80±0.01×10
5

SIN-G 6.00±0.50×10
4
1.00±0.09×10
5

VSV-G 10.00±0.1×10
6
9.00±0.10×10
6

Table 3.1 Infectious titers of AURA-G-, SIN-G-, and VSV-G-pseudotyped lentivectors

61

Figure 3.2 Lentiviral transduction of DC-SIGN-expressing 293T cells. (A) The
293T.DCSIGN cell line which stably expresses human DC-SIGN was stained with anti-
DC-SIGN-PE (1:5). Flow cytometry revealed significant DC-SIGN expression on
293T.DCSIGN (no-fill) but not parental 293T (filled). Fresh viral supernatants of
FUGW/AURAG, FUGW/SING and FUGW/VSVG were normalized by p24 (70 ng) and
used to transduce 2×10
4
293T.DC-SIGN (open bars) or parental 293T cells (solid bars)
lacking the expression of DC-SIGN. Three days later, the transduction efficiency was
measured by analyzing GFP expression using flow cytometry. Values are given as the
mean of triplicates ± S.E.

62

3.3.2 Cell-surface expression of DC-SIGN correlates with the infection of lentiviral
vector pseudotyped with AURA-G and SIN-G
To evaluate the effect of DC-SIGN on infectivity per particle, virus preparations
were normalized by p24 measurement and used to infect 293T and 293T.DCSIGN cells.
When the same amount of virus was used, there was little difference between 293T and
293T.DCSIGN in their susceptibility to transduction by VSV-G-pseudotyped viruses
(Figure 3.2B). FUGW/VSVG had similar level of transduction toward both cell lines
(Figure 3.2B), indicating that VSV-G has a high infectivity per particle toward both 293T
and cells expressing DC-SIGN on the cell surface. In contrast, the AURA-G-bearing
virus transduced 293T cells with a very low infectivity but the infectivity was
significantly enhanced in the presence of DC-SIGN (Figure 3.2); FUGW/AURAG could
transduce 293T.DCSIGN cells, with an efficiency of ~36%, but with only ~6%
transduction of 293T cells. FUGW/SING had a higher infectivity per particle than
AURA-G-bearing LVs towards 293T cells (~20%). Similar to FUGW/AURAG, SIN-G-
pseudotyped LV exhibited an increased infectivity towards 293T cells expressing DC-
SIGN (Figure 3.2B). The p24-normalized transduction demonstrates differences in the
infectivity per lentiviral particle and further reveals the natural tropism of SIN-G- and
AURA-G-pseudotyped LVs for DC-SIGN-expressing cells.
3.3.3 Cell-surface expression of DC-SIGN and L-SIGN on 3T3 cells mediates binding
and transduction of pseudotyped lentiviral vectors
Previous studies have indicated that the cell type in which DC-SIGN(R) is
expressed can have a significant impact on the efficiency of these lectins to promote viral

63

infection (Trumpfheller et al. 2003; Wu et al. 2004). To study the function of C-type
lectins as AURA-G attachment factors in other cell types, we transduced 3T3/MX-L-
SIGN and 3T3/MX-DC-SIGN cell lines and the corresponding parental 3T3 cells as
controls. Previously, Wu et al. examined the levels of DC-SIGN/L-SIGN expression on
these cell lines using several cross reactive monoclonal antibodies and revealed similar
levels of expression of both C-type lectins (Wu et al. 2002). These cell lines were
exposed to AURA-, SIN- and VSV-glycoprotein-bearing LVs. After 48 hours, cells were
analyzed by flow cytometry for GFP expression. The levels of transduction were
normalized based on 3T3 transduction and then the magnitude of the increase in
transduction was assessed. The transduction efficiency in 3T3 cells of pseudotyped-
lentiviral vectors were; AURA-G 12.1 ±1.3, SIN-G 10.6 ±0.8, VSV-G 72.2 ±1.0. AURA-
G-pseudotyped virus showed an approximate 4-fold increase in transduction with cells
expressing L-SIGN and DC-SIGN (Figure 3.3A). Similarly, SIN-G-enveloped LVs
exhibited an increased transduction of both L-SIGN and DC-SIGN, whereas VSV-G-
bearing particles did not display a significant difference (Figure 3.3A).
To determine if the increase in transduction of pseudotyped LVs 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. The AURA-G-bearing
particles bound 6- to 5-fold more efficiently to DC/L-SIGN-expressing cells than 3T3
controls. Consistent with the infection data, the increase virus binding to L-SIGN and
DC-SIGN-expressing cells correlates to an increase in transduction (Figure 3.3). Results
of assays with SIN-G-bearing particles showed a direct correlation between an increase in

64

binding and the observed increase in infectivity (Figure 3.3B). In the absence of DC-
SIGN expression, AURA-G and SIN-G pseudotypes (~2224 CPM) bound poorly to 3T3
cells, as compared with VSV-G-radiolabeled particles (~13054 CPM). VSV-G-
pseudotyped virus exhibited similar levels of attachment to all cell types (Figure 3.3B).
This suggests that the improvement in infectivity of the virus is due to binding conferred
by the presence DC-SIGN(R). Together, these results reveal that the mammalian-
produced AURA- and SIN-G-bearing LVs are able to bind DC-SIGN(R) receptors
resulting in enhanced transduction of DC-SIGN-expressing cells.

65

Figure 3.3 Effects of DC-SIGN or L-SIGN expression on the infectivity of pseudotyped
LVs. (A) AURA-G-, SIN-G-, and VSV-G-pseudotyped LVs were produced by transient
transfection of 293T cells. Viral supernatants were normalized by p24 and spin-
inoculated with L-SIGN- or DC-SIGN-expressing 3T3 cells; the parental 3T3 cells were
included as controls. Three days later, the transduction efficiency was measured by
analyzing GFP expression. Fold increase in percentage of GFP-positive cells is shown
based on 3T3 cells where values are given as the mean of triplicates ± S.E. (B)
Specificity of binding to DC-SIGN. [
35
S]-methionine-labeled virus was produced by
transfection of 293T cells. Approximately 10
5
cpm of each radiolabeled virus diluted in
PBS was mixed with 10
6
3T3 or DC-SIGN/L-SIGN-expressing cells. This mixture was
incubated at 4°C for 1 hour with gentle agitation. Cells were washed three times with
PBS and resuspended in PBS and
35
S radioactivity of the resuspended cells was
quantitated with a liquid scintillation counter. Fold increase in percent bound of [
35
S]
cpm is shown based on 3T3 cells where values are given as the mean of triplicates ± S.E.

66

3.3.4 AURA-G-mediated infectivity requires acidification
Transduction by VSV-G as well as some alphaviruses is known to involve the
endocytic pathway, in which upon internalization an acidic environment of endosomal
vesicles is required (Strauss and Strauss 1994; Yamada and Ohnishi 1986). Treatment
with pH-interfering drugs bafilomycin A
1
and ammonium chloride (NH
4
Cl), which can
neutralize acidic endosomal compartments, abolished the infectivity in 293T.DCSIGN
cells for AURA-G, SIN-G and VSV-G pseudotyped LVs (Figure 3.4A and 3.4C). These
results are consistent with the low pH requirement of alphaviral and VSV glycoproteins
to trigger membrane fusion. More direct evidence for pH-dependent fusion was provided
by a dose study of increasing concentrations of ammonium chloride (Figure 3.4C). This
suggests that both DC-SIGN-mediated transduction with FUGW/AURAG and
FUGW/SING have similar inhibition kinetics due to equivalent acidification requirement
for the virus-endosome fusion process. Similarly, VSV-G mediated transduction was
inhibited by increased concentrations of ammonium chloride albeit less than alphaviral
glycoproteins at concentrations up to 50mM.

67

Figure 3.4 Specific inhibitors prevent DC-SIGN-mediated enhancement of infection. In
inhibition experiments, 293T.DCSIGN were incubated with antibodies at a concentration
of 5 µg/ml, or 5 mM EDTA, or Bafilomycin A
1
at 37°C for 30 min, and then inoculated
with LVs bearing VSV-G (no fill), SIN-G (dark fill) or AURA-G (gray fill) at an MOI <1
for 8 hours (A). In dose-dependent inhibition assays 293T.DCSIGN was incubated with
LVs in the presence of increasing concentrations of yeast mannan (B) or NH
4
Cl (C).
Subsequently the supernatants were replaced and incubated with fresh medium for two
days before being analyzed for GFP expression where values are given as the mean of
triplicates ± S.E.
3.3.5 Infectivity can be blocked with inhibitors of DC-SIGN
We observed that the infection efficiency of AURA-G- and SIN-G-bearing LVs
correlates with the expression of DC-SIGN on target cells. To further examine the
specificity of virus interaction with these molecules, we performed infectivity
experiments with an MOI ~0.9 in the presence of increasing concentrations of yeast

68

mannan, EDTA, anti-DC-SIGN monoclonal antibodies, or an isotype-matched control
(Figure 3.4A and 3.4B). These treatments disrupt interactions with DC-SIGN molecules
(Davis et al. 2006; Klimstra et al. 2003; Lozach et al. 2007). Incubation with mannan, a
natural ligand for DC-SIGN, during viral inoculation of FUGW/AURAG and
FUGW/SING with 293T.DCSIGN cells resulted in a dose-dependent reduction in the
amount of GFP-positive cells (Figure 3.4B). However, mannan was not effective at
inhibition of VSV-G-bearing LV, suggesting a different receptor-mediated interaction for
VSV-G transduction of 293T.DCSIGN cells. The principal characteristic of C-type
lectins is that they interact with viral glycoproteins in a calcium-dependent manner via
their C-terminal carbohydrate recognition domain (CRD). EDTA is a calcium chelator
and treatment during transduction with FUGW/AURAG and FUGW/SING resulted in a
>70% reduction in GFP-positive cells (Figure 3.4A). In contrast, FUGW/VSVG virus
was not dependent on calcium for infection as revealed by the EDTA inhibition
experiment. Incubation with anti-DC-SIGN antibodies during infection also resulted in a
reduction in numbers of GFP-positive cells with AURA-G- and SIN-G-mediated
transduction, whereas the isotype control antibody had little inhibitory effect. Again, the
FUGW/VSVG virus did not exhibit significant competition with the antibody treatment
of DC-SIGN-expressing cells (Figure 3.4A). The results of these experiments indicate
that the infectivity of AURA-G- and SIN-G-pseudotyped virus is dependent on DC-SIGN
expression as well as calcium for entry, whereas FUGW/VSVG transduction is not
dependent upon DC-SIGN nor calcium.

69

3.3.6 AURA-G and SIN-G transduce dendritic cells through DC-SIGN(R)
We further evaluated how efficiently AURA-G and SIN-G pseudotyped LVs
could transduce primary immune cell targets. Human monocyte derived DCs (MoDCs)
were prepared from the peripheral blood mononuclear cells (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 MOI=10 of AURA- and SIN-G-pseudotyped
LVs. More than 80% of the cultured DCs were positive for DC-SIGN expression before
infection (data not shown). Transduction of MoDCs by FUGW/AURAG was slightly
more efficient than FUGW/SING, transducing ~31% versus ~25% DCs (Figure 3.5).

Figure 3.5 LVs bearing AURA-G and SIN-G transduce DCs and transduction is mediated
by DC-SIGN(R). Human monocyte-derived DCs (MoDCs) were generated by culturing
respective precursor cells in the presence of GM-CSF and IL-4. The adherent cells (1 ×
10
6
) were cultured for 2 days and then incubated for 1 hour with mannan (200 µg/mL),
anti-DC-SIGN(R) antibody (20 µg/mL) or without any reagents. The cells were then
infected with FUGW/AURAG or FUGW/SING (MOI=10) for 8 hours in the presence of
blocking reagents. GFP expression was assayed by flow cytometry five days post-
transduction where one representative figure is shown with values given as the mean of
duplicates ± S.E.

70

We further investigated the viral envelope-receptor interactions which facilitate
MoDC transduction by AURA-G and SIN-G LVs. Based on our previous results with
DC-SIGN(R) expressing cell lines, we chose to evaluated the ability of inhibitors of DC-
SIGN to block infection of human monocyte derived DCs (MoDCs). MoDCs were
challenged with the same multiplicity of infection (MOI=10) of AURA-G- and SIN-G-
pseudotyped LVs incubated in the presence of mannan or anti-DC-SIGN(R) 14E3G7
antibody. Transduction of MoDCs by FUGW/AURAG decreased to ~6% with anti-DC-
SIGN antibody and decreased to ~1% in the presence of yeast mannan (Figure 3.5).
Similar to FUGW/AURAG, when FUGW/SING was incubated with MoDCs in the
presence of anti-DC-SIGN(R) antibody, the transduction dropped to 4%, and with
mannan transduction dropped to 2% (Figure 3.5). These results suggest that AURA-G
and SIN-G transduction of MoDCs is mediated at least in part by interactions with DC-
SIGN(R) but could also require additional cellular lectins.
3.4 Discussion
In this study, we demonstrate that glycoproteins derived from the Aura virus can
pseudotype HIV-1 derived LVs. Aura virus is a new world alphavirus which shares
stereological cross reactivity with the Sindbis virus lineage (Strauss and Strauss 1994).
Since the AURA-G has a 56% amino acid sequence identity in the E2 binding region
with the SIN-G, we chose to compare these pseudotyped LVs. Staining of SIN- and
AURA-G-pseudotyped LVs using cross reactive Sindbis virus immune ascitic fluid
revealed a similar level of LV particles which incorporated the envelope glycoprotein.

71

This is the first time that the Aura virus glycoprotein (AURA-G) has been shown to be
able to incorporate into HIV-1-derived LVs.
We have demonstrated that the incorporation of AURA glycoproteins into
pseudotyped lentiviral particles can render them capable of infecting human cell types.
Although the titer of vectors pseudotyped with the AURA-G was consistently lower than
that obtained with those pseudotyped with VSV-G, the AURA glycoprotein‟s limited
transduction was sufficient to warrant further studies to elicit specific virus-receptor
interactions which may enhance transduction of specific cells types. One potential class
of receptors which has been shown to act as a principal attachment factor for a broad
range of enveloped viruses is C-type lectins (Lozach et al. 2007). The C-type lectins DC-
SIGN and L-SIGN are important targets because they are promiscuous receptors capable
of capturing viruses on antigen presenting cells (APCs) located throughout the body
(Bashirova et al. 2001; Braet and Wisse 2002). Both lectins are tetrameric type II
transmembrane proteins composed of a carbohydrate recognition domain (CRD), which
binds in a calcium-dependent manner, and a short cytoplasmic tail responsible for
signaling and internalization that can activate APCs leading to an immune response to the
viral vectors or their transgene products (Figdor et al. 2002; Lozach et al. 2007; Mitchell
et al. 2001; Soilleux 2003). The mosquito-produced Sindbis virus (Klimstra et al. 2003)
and LVs with modified Sindbis glycoproteins (Yang et al. 2008b) were shown to use DC-
SIGN(R) as attachment receptors leading to productive transduction of dendritic cells
(DCs). Therefore, we reasoned that C-type lectins DC-SIGN and L-SIGN may be
important attachment factors for SIN- and AURA-G-bearing LVs as well.

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In the light of previous findings, we reasoned that the use of C-type lectins for DC
entry might be a general property of AURA-G and that the virus-receptor interactions
might be achieved with pseudotyped LVs derived from 293T cells. Cells stably
expressing C-type lectins DC/L-SIGN were used to quantify the interactions between the
viral envelope and the receptor. When the conditions were optimized, FUGW/AURAG
achieved titers similar to those of the SIN-G pseudotypes on DC-SIGN-expressing cells
but much lower on parental cells without DC-SIGN expression. Radiolabeled virus cell
binding revealed a significant increase in bound viral particles to cells expressing DC-
SIGN and L-SIGN for AURA-G bearing LV particles. The interactions of AURA-G with
DC-SIGN were determined to mediate transduction as revealed by competitive inhibition
assays and to transduce cells through a pH-dependent pathway similar to that of SIN-G-
pseudotyped LVs. Like other alphaviral pseudotypes, FUGW/AURAG was stable during
ultracentrifugation and when viral supernatant was concentrated 300 fold using
ultracentrifugation a titer of approximately 2.0×10
6
TU/mL was obtained. Concentrated
FUGW/AURAG vectors were used to transduce MoDCs and the transduction was
determined to be DC-SIGN-mediated, as demonstrated by the competitive inhibition
using both DC-SIGN(R) antibodies and the yeast mannan. Our findings indicate that the
AURA glycoprotein might be an attractive alternative to the commonly used VSV-G for
transduction of DCs for certain gene transfer applications.
The VSV-G-pseudotyped LVs exhibited similar binding and transduction of cells
expressing DC-SIGN, L-SIGN or parental 3T3 and 293T cells. Although we have shown
that DC-SIGN and L-SIGN are not major mediators for the infectivity of FUGW/VSVG

73

vectors, it remains possible that these glycoproteins may utilize other ubiquitous
attachment factors to infect the same cell types. The broad tropism of FUGW/VSVG has
proven useful for many investigators developing LVs, however, VSV-G LVs transduce
multiple cell types when administered in vivo and could result in “off-target” gene
transfer. In contrast to the broad high titer of VSV-G-pseudotyped LV, the
FUGW/AURAG vector has a much lower infectivity towards cell types lacking DC-
SIGN(R). The low infectivity observed in 293T and 3T3 cell types clearly reflects a
restricted tropism of the AURA-G-pseudotyped LVs and may be useful to alleviate
certain concerns of the “off-target” effect for gene delivery.
In this study we identified DC-SIGN and L-SIGN interactions with AURA- and
SIN-glycoproteins to mediate the transduction of DCs. 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. 2008b). Transduction of
human PBMC-derived MoDCs by the same MOI of FUGW/AURAG was slightly more
efficient than that by FUGW/SING. The transduction by both AURA-G and SIN-G
pseudotyped LVs was DC-SIGN(R)-specific and could be inhibited by both yeast
mannnan and anti-DC-SIGN(R) antibodies. Although these results suggest that DC-
SIGN(R) mediates transduction it remains possible that additional cellular lectins could
play a role in the transduction of DCs. Further experiments are required to investigate
other possible attachment factors. One group recently observed that LVs pseudotyped
with the same wild-type Sindbis virus envelope proteins and produced by mammalian
cells could not use DC-SIGN as a receptor. The SIN-G strain used in that study was

74

identical to that used in our study, but our study clearly shows that SIN-G-bearing LVs
are able to efficiently bind DC-SIGN and employ it to transduce target cells. One
possibility is that transduction in their experimental setting may not have been optimized
to observe differences in DC-SIGN-mediated transduction. Previous studies by this group
have demonstrated that the wild-type Sindbis virus envelope (5.4 x 10
-4
ng HIVp24/cell)
forms infectious pseudotypes with HIV-1 on 293T cells (Morizono et al. 2001).
However, when attempting to quantify the effects of DC-SIGN-mediated transduction
with wild-type unmodified SIN-G pseudotypes, this group later observed negligible
infection towards both the 293T cell line and DC-SIGN expressing cells (Morizono et al.
2010). One possible reason why they observed no effect of DC-SIGN mediated
transduction is that the amount of virus used (4 x 10
-5
ng HIVp24/cell) was outside the
observable infectious range. In our studies we used a higher amount of SIN-G-
pseudotyped LV (3.5 x 10
-3
ng HIVp24/cell) and observed a significant level of infection
of 293T cells and an increase in transduction with cells expressing DC-SIGN(R). We
found that AURA-G and SIN-G pseudotypes have an innate tropism towards DC-
SIGN(R) expressing cells. The specific infectivity of Aura virons is naturally low
(Rumenapf et al. 1995), but we find that AURA-G-pseudotyped LVs have an innate
ability to transduce DC-SIGN(R) expressing cells which can be utilized for directing the
cellular transduction of LVs towards antigen-presenting cells.
In conclusion, Aura virus glycoproteins can pseudotype HIV-1-derived LVs and
can restrict the tropism towards DC-SIGN-expressing cells. LVs bearing either SIN-G or
AURA-G are able to interact with DC-SIGN(R) to mediate preferential transduction of

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these cell types. Targeting of LVs to C-type lectin-expressing cells such as DCs can be
increased by choice of suitable envelope glycoprotein with a low unspecific infectivity
and preferential transduction towards DC-SIGN(R). Further measures to improve AURA-
G pseudotyping utilizing strategies to mutate the receptor-binding sites and mutations in
the fusion loop region of the glycoprotein to enhance the transduction efficiency are
ongoing. Our findings indicate that AURA-G pseudotyped LVs may serve as promising
tools for selected gene transfer to DCs.
3.5 Acknowledgements
We thank Dr. Kye-Il Joo for assistance with confocal imaging. Portions of this
chapter are adapted from: Steven Froelich, April Tai, Katie Kennedy, Adnan Zubair, and Pin
Wang. (Submitted, 2011).

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Chapter 4. Virus-Receptor Mediated Transduction of Dendritic
Cells by Lentiviruses Enveloped with Glycoproteins Derived from
Semliki Forest Virus
Lentiviruses have recently attracted considerable interest for their potential as a
genetic modification tool for dendritic cells (DCs). In this study, we explore the ability of
lentiviruses enveloped with alphaviral envelope glycoproteins derived from Semliki
Forest virus (SFV) to mediate transduction of DCs. We found that SFV glycoproteins
(SFV-G)-pseudotyped lentiviruses use C-type lectins (DC-SIGN and L-SIGN) as
attachment factors for transduction of DCs. Importantly, SFV-G pseudotypes appear to
have enhanced transduction towards C-type lectin-expressing cells when produced under
conditions limiting glycosylation to simple high-mannose, N-linked glycans. These
results, in addition to the natural DC tropism of SFV-G, offer evidence to support the use
of SFV-G-pseudotyped lentiviruses to genetically modify DCs for the study of DC
biology and DC-based immunotherapy.
4.1 Introduction
The fundamental rationale behind gene-based immunotherapy lies in the ability to
modify immune cells to achieve a therapeutic benefit. We and others have developed
methods to target gene delivery to specific immune cell types (Froelich et al. 2010;
Maurice et al. 1999; Morizono et al. 2001). Recent realization that antigen presenting
cells (APCs) are powerful tools for the manipulation of the immune system, has led to

77

new targets for gene-based immunotherapy. The application of gene delivery for
immunization relies on a new strategy in which dendritic cells (DCs), the most powerful
APCs which can initiate and maintain immune responses by stimulating both T and B
cells, are genetically modified to express antigens or produce immunostimulatory
molecules to create a therapeutic advantage (Ribas et al. 2002).
Efficient gene-based immunization has been achieved using a variety of viral
vectors, each of which have specific advantages and drawbacks (Dullaers and Thielemans
2006). Lentiviruses have emerged as a particularly efficient and promising tool for gene
transfer into a wide range of immune cell types. They have been shown to be very
effective in delivering genes into 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). DCs
that are transduced by antigen-encoding lentiviruses are able to efficiently present the
antigens and stimulate antigen-specific responses either in vitro (Dyall et al. 2001; Firat
et al. 2002; Gruber et al. 2000; Zarei et al. 2002), or after in vivo transplantation
(Breckpot et al. 2003; Firat et al. 2002; Metharom et al. 2001; Zarei et al. 2004), and even
through direct injection of the vector in vivo (Esslinger et al. 2003; Firat et al. 2002; Hu et
al. 2011; Palmowski et al. 2004).
One strategy for directing the cellular transduction is through pseudotyping
lentiviruses with glycoproteins from other enveloped viruses which have a natural
tropism for APCs (Cronin et al. 2005). The mosquito-produced Sindbis alphavirus
(Klimstra et al. 2003) and lentiviruses with engineered Sindbis glycoproteins (Yang et al.
2008b) have been shown to use C-type lectins as attachment receptors leading to

78

productive transduction of DCs in vivo. The C-type lectins DC-SIGN and L-SIGN
(together known as DC-SIGN(R)) are important targets because they are promiscuous
receptors capable of capturing viruses on APCs located throughout the body (Bashirova
et al. 2001; Braet and Wisse 2002). Both lectins are tetrameric type II transmembrane
proteins composed of a carbohydrate recognition domain (CRD), which binds to high-
mannose oligosaccharides in a calcium-dependent manner, and a short cytoplasmic tail
responsible for signaling and internalization that can activate APCs leading to an immune
response to the viral vectors or their transgene products (Figdor et al. 2002; Lozach et al.
2007; Mitchell et al. 2001; Soilleux 2003).
Pseudotyped recombinant lentiviruses using the Semiliki Forest (SFV) envelope
glycoproteins have recently been reported (Kahl et al. 2004; Strang et al. 2005). These
pseudotypes are particularly promising because they are able to be concentrated to obtain
high-titer viral preparations, resistant to inactivation by components in human sera, and
stable packaging cell lines have previously been generated that could be scaled up to
meet the larger volumes for clinical demand (Strang et al. 2005). In this study, we
generated recombinant lentiviral particles with envelope glycoproteins of SFV (SFV-G)
and investigated the ability of SFV-G to facilitate transduction of DCs. In light of
previous findings with other alphaviral envelope glycoproteins (Klimstra et al. 2003;
Morizono et al. 2010; Yang et al. 2008b), we investigated the role of C-type lectins to act
as attachment factors for SFV-G. Our current studies identified effects of DC-SIGN and
L-SIGN on binding and transduction for SFV-G-pseudotyped lentiviruses. Our data
evaluating these pseudotyped viral particles suggest that SFV-G facilitates binding and

79

transduction through C-type lectins which is enhanced when produced under high
mannose N-glycan glycosylation conditions.
4.2 Materials and Methods
4.2.1 Cell lines
293T.DCSIGN were derived as previously described (Yang et al. 2008b) and
stained (anti-DC-SIGN antibody from BD Biosciences) to confirm expression of DC-
SIGN. Mouse fibroblasts NIH 3T3 cells were obtained from the American Tissue Culture
Collection (ATCC, Manassas, VA). 3T3-L-SIGN and 3T3-DC-SIGN were obtained from
the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID.
These cell lines were maintained in DMEM 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 100 µg/mL of streptomycin.
4.2.2 Plasmid construction
The glycoprotein expression plasmids were constructed similarly to previously
reported (Kahl et al. 2004). The cDNA of SFV-G was amplified from the pSFV helper
expression vector (a gift from Dr. Robert Chow, University of Southern California). The
amplified fragments for the glycoproteins were subcloned into the vesicular stomatitis
virus glycoprotein (VSV-G) expression plasmid pVSV-G (Cell Genesys, Foster City,
CA). The resulting plasmid was designated pSFV-G (Figure 4.1). The lentiviral backbone
plasmid (FUGW and its derivatives) used in this study have been previously described
(Lois et al. 2002).

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Figure 4.1 Virus-producing constructs used to make pseudotyped lentiviruses. (A)
Schematic diagrams of constructs encoding the lentiviral backbone FUGW and envelope
glycoproteins. Ubi: the human ubiquitin-C promoter; GFP: enhanced green fluorescence
protein; WRE: woodchuck responsive element; ΔU3: deleted U3 region that results in the
transcriptional activation of the integrated viral LTR promoter; pA: polyadenylation
signal; E1, E2, 6k, E3: SFV-glycoproteins (E1 for fusion, E2 for receptor binding, 6k a
linker, and E3 a signal sequence). The VSV-G expressing plasmid contains the rabbit β-
globin intron and poly(A) signal. (B) Viral supernatants harvested from virus-producing
cells transiently transfected with GFP-vpr, SFV-G, or VSV-G, and other necessary
packaging constructs, were coated to a poly-lysine containing coverslip by centrifugation.
The resulting coverslips were then rinsed and immunostained with an anti-SFV-G
antibody (red) to label the glycoproteins and imaged using a laser confocal microscope.

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4.2.3 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). 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.
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).
4.2.4 Confocal imaging of GFP-vpr labeled virions
GFP-vpr-labeled lentiviral particles were produced as previously described (Joo
and Wang 2008). 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-SFV E1/E2 antibody (1:2000; a gift from Margaret Kielian, Albert
Einstein College of Medicine) for 40 min at 4°C. Coverslips were washed with PBS and
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 with a plan-apochromat oil immersion (63×/1.4) objective.

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4.2.5 Virus attachment assays
Production of [
35
S]-methionine-labeled viruses were produced by transfection of
293T cells as described above. Cells were then depleted of methionine and 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 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 and this mixture was incubated at 4°C for 1 hr
with gentle agitation. Cells were washed and
35
S radioactivity was quantitated with a
liquid scintillation counter.
4.2.6 Determination of infectious titers
To determine infectious titer, 2 × 10
4
293T or 293T.DCSIGN cells were
transduced with 100 µl of serially diluted viral supernatants with 8 µg/mL of polybrene
(Sigma-Aldrich) 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.

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4.2.7 Lentivirus-mediated transduction of cell lines in vitro
The 3T3, 3T3-LSIGN and 3T3-DCSIGN cell lines were stained with cross
reactive anti-DCSIGN(R) antibody 14E3G7. Target cells (3T3-LSIGN, 3T3-DCSIGN, or
3T3 cells; 0.2 × 10
5
per well) were seeded in 96-well culture dishes 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 medium and incubated for 48 hrs at 37°C with 5% CO
2
.
4.2.8 Assays to inhibit pseudotyped virus-mediated infection
In dose-response experiments, 293T.DCSIGN (0.2 × 10
5
per well) were incubated
with 0.2 to 200 µg/mL of yeast mannan (Sigma-Aldrich) at 37°C for 30 min. SFV-G- or
VSV-G- lentiviruses (MOI 0.8) were incubated for 8 hrs, and then supernatant was
replaced with fresh medium. For NH
4
Cl inhibitions, pseudotyped viral particles were
spin-inoculated in the presence of increasing concentrations of NH
4
Cl (Sigma-Aldrich)
for 90 min at 25°C. 293T.DCSIGN cells were incubated with 5 µg/mL of anti-DCSIGN
antibodies (14E3G7, 19F7, DC-28, and isotype control antibody, Santa Cruz
Biotechnology), 5 mM EDTA, 15 nM Bafilomycin A
1
, or 10 µg/ml soluble DC-SIGN at
37°C for 30 min, and then inoculated FUGW/SFVG or FUGW/VSVG at an MOI ~0.8 for
8 hrs. Similarly, virus was incubated with 25 µg/mL concavalin A (Sigma-Aldrich) for 1
hr at 37°C then incubated with 293T.DCSIGN cells for 8 hrs before changing to fresh
D10 medium.

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4.2.9 Transduction of human PBMC-derived DCs
Peripheral blood mononuclear cells (PBMC) from healthy human donors were
purchased from AllCells (Emeryville, CA). PBMC were differentiated into DCs 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 on cells stained with anti-CD11c antibody and anti-DCSIGN antibody (BD
Biosciences). Monocyte-derived DCs (MoDC) were exposed to virus on day 2 at the
required MOI based on 293T cells. For inhibition of DC-SIGN-mediated transduction,
DCs were incubated with 20 µg/mL of anti-DC-SIGN(R) antibody (14E2G7, Santa Cruz
Biotechnology) or 200 µg/mL yeast mannan (Sigma-Aldrich) at 37°C for 30 min and
then inoculated with 293T-produced FUGW/SFVG or FUGW/VSVG lentiviruses at an
MOI 10 for 8 hrs before the media was changed.
4.3 Results
4.3.1 DC-SIGN correlates with increased infection of lentivirus pseudotyped with SFV-G
It has been shown that SFV-G can pseudotype HIV-1-derived lentiviruses (Kahl
et al. 2004; Strang et al. 2005). We standardized the constructs for expressing envelope
proteins by sub-cloning the SFV-G expression cassettes into the VSV-G expression
vector containing the rabbit β-globin intron and the poly(A) signal sequence (Figure
4.1A); the plasmid used for VSV-G expression has been used extensively for
pseudotyping lentiviruses (Kahl et al. 2004). Lentiviruses pseudotyped with alphaviral
glycoproteins were generated by co-transfection of 293T cells with the lentiviral

85

construct FUGW, plasmids encoding viral gag, pol, and rev genes, and the envelope
protein expression plasmid. FUGW carries the GFP reporter gene under the control of the
human ubiquitin-C promoter (Figure 4.1A) (Lois et al. 2002). GFP-vpr-labeled
lentiviruses were produced as described above with an additional plasmid encoding the
GFP protein fused to the HIV-1 vpr protein (Joo and Wang 2008). Antibody staining with
anti-SFV-G of GFP-vpr-labeled virus revealed significant overlap (Mander‟s overlap
coefficient >0.7) for SFV-G- but not VSV-G (Mander‟s overlap coefficient <0.1)
pseudotyped viruses (Figure 4.1B). These results indicate that SFV-G is incorporated
onto lentiviral particles.
To characterize the infectivity of FUGW/SFVG and FUGW/VSVG, the infectious
titer was measured on parental 293T cells and the 293T.DCSIGN cell line which stably
expresses human DC-SIGN (Figure 4.2A). Differences in viral titer may be attributed to
several factors: differences in virus-receptor interactions, the efficiency of production of
functional particles into the supernatant, as well as the amount of defective particles
which may serve as interfering particles. Consistent with previous reports (Kahl et al.
2004; Strang et al. 2005), SFV-G and VSV-G can both pseudotype lentivirus to produce
infectious particles; these viruses are designated FUGW/SFVG and FUGW/VSVG,
respectively. The infectious titer of the VSV-G-pseudotyped lentivirus (FUGW/VSVG)
was approximately 10×10
6
transduction units (TU)/mL on both 293T and 293T.DCSIGN
cells. When SFV-G was used as the envelope glycoprotein, the infectious titer based on
293T.DCSIGN cells was ~5-fold lower than VSV-G (Figure 4.2B). However, the
FUGW/SFVG was much less infectious for 293T cells; the titer was ~7-fold lower than

86

that measured on 293T.DCSIGN cells. The difference in infectious units between cell
types is clear evidence that the transduction of FUGW/SFVG is enhanced by the presence
of DC-SIGN.

Figure 4.2 Lentiviral transduction of DC-SIGN-expressing 293T cells. (A) Expression of
DC-SIGN in 293T (solid fill) and 293T.DCSIGN (open fill) was detected by flow
cytometry. (B) Transduction titer of lentiviruses were quantified by serially diluting fresh
viral supernatants of FUGW/SFVG and FUGW/VSVG and used to transduce 2×104
293T.DC-SIGN (open bars) or parental 293T cells (solid bars). Three days later, the
transduction efficiency was measured by analyzing GFP expression using flow cytometry
where the corresponding viral titer was calculated. Values are given as the mean of
triplicates ± S.E.

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4.3.2 Preferential transduction of DC-SIGN- or L-SIGN-expressing 3T3 cells
Previous studies have indicated that the cell type in which DC-SIGN(R) is
expressed can have a significant impact on the efficiency of these lectins to promote viral
infection (Trumpfheller et al. 2003; Wu et al. 2004). To study in alternative cell types the
function of C-type lectins as attachment factors, we transduced the 3T3-L-SIGN and
3T3-DC-SIGN cell lines and the corresponding parental 3T3 cells. The level of DC-
SIGN or L-SIGN expression on these cell lines was confirmed using a cross reactive DC-
SIGN/L-SIGN monoclonal antibody which revealed similar levels of expression of both
C-type lectins (Figure 4.3A). These cell lines were transduced by FUGW/SFVG and
FUGW/VSVG. After 48 hrs, cells were analyzed by flow cytometry for GFP expression.
The levels of transduction were normalized based on 3T3 transduction, and the
magnitude of the increase in transduction was assessed (Figure 4.3B). SFV-G-
pseudotyped virus showed a preferential increase in transduction with cells expressing L-
SIGN (6 folds) and DC-SIGN (3 folds), whereas FUGW/VSVG did not exhibit a
significant preference (Figure 4.3B).

88

Figure 4.3 Effects of DC-SIGN or L-SIGN expression on (A) Expression of L-SIGN and
DC-SIGN in 3T3 (solid fill), 3T3.DCSIGN (open fill) and 3T3.LSIGN (gray fill),
respectively, was detected using cross reactive DC-SIGN/L-SIGN antibody and
quantified by flow cytometry. (B) Effects of DC-SIGN or L-SIGN expression on the
infectivity of pseudotyped lentiviruses. SFV-G- and VSV-G-pseudotyped lentiviruses
were normalized by p24 and spin-inoculated with LSIGN- or DCSIGN-expressing 3T3
cells; the parental 3T3 cells were included as controls. Three days later, the transduction
efficiency was measured by analyzing GFP expression. Fold increase in percentage of
GFP-positive cells is shown based on 3T3 cells; 6 ±0.7% and 72 ±1.1% for
FUGW/SFVG and FUGW/VSVG respectively, where values are given as the mean of
triplicates ± S.E. (C) Specificity of binding to DC-SIGN. [35S]-methionine-labeled
FUGW/SFVG or FUGW/VSVG were incubated with 3T3 or DC-SIGN/L-SIGN-
expressing cells at 4°C. Cells were washed and 35S radioactivity of the resuspended
cells was quantitated with a liquid scintillation counter. Fold increase in [35S] bound
viral particles is shown based on 3T3 cells where FUGW/SFVG and FUGW/VSVG
bound 25.2 ±1.3% and 3.59 ±0.27% CPM respectively, values are given as the mean of
triplicates ± S.E.

89

To determine if 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. Results of assays with
FUGW/SFVG particles showed a direct correlation between an increase in percentage of
virus bound and the observed increase in infectivity (Figure 4.3C). The FUGW/SFVG
particles bound 2- to 6-fold more efficiently to DC/L-SIGN-expressing cells than to 3T3
cells. Consistent with the infection data, the level of increase of binding to DC-SIGN-
expressing cells was generally lower than that to L-SIGN-expressing cells. In the absence
of DC-SIGN expression, SFV-G pseudotypes bound poorly to 3T3 cells with only 2,289
CPM binding to cells out of the 63,701 CPM that were incubated with the cells. VSV-G-
pseudotyped virus exhibited similar levels of attachment to all cell types (Figure 4.3C)
with approximately 25% of the total CPM of virus bound to all cell types. As shown in
Figure 4.3B and 4.3C, the attachment and transduction of FUGW/VSVG was
approximately similar among cells regardless of DC-SIGN(R) expression. This is
consistent with the lack of increased infectivity observed with FUGW/VSVG on DC-
SIGN expressing cells. Our data suggest that FUGW/SFVG utilizes DC-SIGN and L-
SIGN as attachment receptors to facilitate binding and transduction.
4.3.3 SFV-G- and VSV-G-mediated transduction requires acidification
Internalization of SFV-G and VSV-G lentiviruses is known to involve the
endocytic pathway, in which the acidic environment of endosomal vesicles is required.
Treatment with the pH-interfering drugs ammonium chloride caused a dose-dependent
reduction of infectivity in 293T.DCSIGN cells for both viruses (Figure 4.4A). This

90

suggests that both DC-SIGN-mediated infection of FUGW/SFVG and DC-SIGN-
independent infection by FUGW/VSVG are pH-dependent processes, presumably due to
the acidification requirement of the virus-endosome fusion process (Joo and Wang 2008).
Transduction was inhibited by endosomal neutralization using bafilomycin A
1
(Figure
4.4C), verifying pH-mediated transduction with both FUGW/SFVG and FUGW/VSVG.

Figure 4.4 Specific inhibitors prevent DC-SIGN-mediated infection. In dose-response
experiments, 293T.DCSIGN cells were treated with SFV-G (solid circles) or VSV-G
(open triangles) lentiviruses in the presence of increasing concentrations of mannan (A),
or NH4Cl (B). (C) 293T.DCSIGN cells were incubated with antibodies at a concentration
of 5 µg/ml, 15 nM Bafilomycin A1, 5 mM EDTA, or 10 µg/ml soluble DC-SIGN at 37°C
for 30 min, and then inoculated with SFV-G (filled bars) or VSV-G (open bars)
lentiviruses at an MOI ~0.8 for 8 hrs or lentiviruses incubated with 25 µg/mL ConA (1h
at 37ºC). Subsequently, the supernatants were replaced and incubated with fresh medium
for two days before being analyzed for GFP expression. The relative transduction was
determined based on non-treated controls and values are given as the mean of triplicates
± S.E.

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4.3.4 SFV-G-mediated infectivity can be blocked with inhibitors of DC-SIGN
We observed that the infection efficiency FUGW/SFVG correlates with the
expression of DC-SIGN on target cells. To further examine the specificity of virus
interaction with these molecules, we performed infectivity experiments in the presence of
increasing concentrations of yeast mannan, or ethylenediaminetetraacetic acid (EDTA),
soluble recombinant DC-SIGN, anti-DC-SIGN monoclonal antibodies (mAbs), or an
isotype-matched control (Figure 4.4B and 4.4C). These treatments disrupt interactions
with DC-SIGN molecules (Davis et al. 2006; Klimstra et al. 2003; Lozach et al. 2007).
Incubation with mannan, a natural ligand for DC-SIGN, during viral inoculation of
FUGW/SFVG with 293T.DCSIGN cells resulted in a dose-dependent reduction in the
amount of GFP-positive cells (Figure 4.4B). However, mannan was not as effective at
inhibition of FUGW/VSVG, suggesting a different receptor interaction between VSV-G
and 293T.DCSIGN cells. The principal characteristic of C-type lectins is that they
interact with mannose residues of viral glycoproteins in a calcium-dependent manner via
their C-terminal carbohydrate recognition domain (CRD). EDTA is a calcium chelator
and treatment during infection with FUGW/SFVG resulted in a >70% reduction in GFP-
positive cells (Figure 4.4C). In contrast, FUGW/VSVG virus was not dependent on
calcium for infection as revealed by the EDTA inhibition experiment. Incubation with
anti-DC-SIGN mAbs during infection also resulted in a reduction in the numbers of GFP-
positive cells with FUGW/SFVG infection, whereas the isotype control antibody had
little inhibitory effect (Figure 4.4C). Again, the FUGW/VSVG virus did not exhibit
significant inhibition in the presence of DC-SIGN antibodies (Figure 4.4C). Furthermore,

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pre-incubation of FUGW/SFVG lentivirus with concavalin A (ConA), which binds to N-
linked high-mannose structures reduced infectivity by ~90% (Figure 4.4C). The
inhibition by ConA supports the theory that mannose carbohydrate residues present on
SFV-G participate in the attachment to DC-SIGN. Finally, infection of SFV-G- but not
VSV-G- pseudotyped lentivirus was blocked by pre-incubation of the cells with soluble
DC-SIGN (Figure 4.4C). The results of these experiments indicate that the infectivity of
SFV-G-pseudotyped lentivirus is dependent on DC-SIGN expression for transduction,
whereas FUGW/VSVG transduction is not dependent upon DC-SIGN.
We further evaluated the ability of inhibitors of DC-SIGN to block infection of
human monocyte derived DCs (MoDCs). MoDCs were prepared from the peripheral
blood mononuclear cells (PBMC) of healthy human donors and cultured with GM-CSF
and IL-4 to generate DC-SIGN
+
DCs (Obermaier et al. 2003). More than 80% of the
cultured DCs were positive for DC-SIGN expression before infection (Figure 4.5A).
MoDCs were challenged with the same MOI of SFV-G- and VSV-G-pseudotyped
lentiviruses incubated in the presence of mannan or anti-DC-SIGN(R) antibody.
Transduction of MoDCs by FUGW/SFVG decreased from ~28% to ~8% with anti-DC-
SIGN antibody and to ~4% in the presence of yeast mannan (Figure 4.5B). The
transduction efficiency of FUGW/VSVG was lower than that of FUGW/SFVG (~13%
GFP
+
). In contrast to FUGW/SFVG, there was not a significant decrease when
FUGW/VSVG was incubated with MoDCs in the presence of either mannan or anti-DC-
SIGN(R) antibody (Figure 4.5B). These results suggest that DC-SIGN(R) function as a
SFV-G binding molecule that is required for the productive infection of MoDCs.

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Figure 4.5 Transduction of MoDCs by lentiviruses is inhibited with anti-DCSIGN
antibody and mannan. Human monocyte-derived DCs (MoDCs) were generated by
culturing respective precursor cells in the presence of GM-CSF and IL-4. (A) The
adherent cells (1 × 10
6
) were cultured for 2 days and then DC-SIGN expression was
detected by flow cytometry. (B) Human MoDCs (1 × 10
6
) were incubated for 1 hour
with mannan (200 µg/mL), anti-DC-SIGN(R) antibody (20 µg/mL) or without any
reagents. The cells were then infected with FUGW/SFVG or FUGW/VSVG (MOI=10)
for 8 hours in the presence of blocking reagents. GFP expression was assayed by flow
cytometry five days post-transduction where one representative figure is shown with
values given as the mean of triplicates ± S.E.
4.3.5 Effects of viral glycosylation
Next, we evaluated the ability of mannose carbohydrate structures on the SFV-G
lentiviral particles to promote infection through a mechanism of increased interactions
with DC-SIGN(R). To test this, we generated FUGW/SFVG particles containing only

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high-mannose glycan content on their envelope glycoproteins by treating virus-producing
cells with 1-deoxymannojirimycin (DMJ). DMJ is an inhibitor of Golgi mannosidase I
and can arrest glycan maturation primarily at the Man
8
GlcNAc
2
stage (Fuhrmann et al.
1984). Production of pseudotyped lentiviruses in the presence of DMJ altered their ability
to transduce DC-SIGN-expressing 3T3 cells (Figure 4.6, DMJ (+)). A two fold increase
in transduction efficiency of 3T3-DCSIGN cells was observed for DMJ-treated
FUGW/SFVG, whereas no significant transduction increase in parental 3T3 was seen.
We further tested how efficiently SFV-G pseudotyped lentiviruses transduce primary
immune cell targets, MoDCs, and the effect of DMJ treatment on transduction efficiency.
Transduction of MoDCs by DMJ-treated FUGW/SFVG was approximately twice as
efficient as non-treated FUGW/SFVG, transducing ~20% versus ~10% MoDCs (Figure
4.6). Similar to the 3T3-DCSIGN cell line, transduction by SFV-G pseudotyped
lentiviruses produced in the presence of DMJ increased their ability to transduce MoDCs
(Figure 4.6). Together, these results reveal that the 293T-produced FUGW/SFVG
particles are able to innately bind to DC-SIGN(R)-receptors but DMJ-treatment can
enhance transduction of DC-SIGN-expressing cells.

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Figure 2 Transduction by SFV-G lentiviruses produced in DMJ treated cells. 3T3 (2 ×
104), 3T3-DCSIGN (2 × 104) and MoDCs (1 × 106) were spin-infected with
FUGW/SFVG produced in 293T cells without DMJ(‒) or with DMJ(+) treatment. GFP
expression was assayed by flow cytometry three days post-transduction where values are
given as the mean of triplicates ± S.E.
4.4 Discussion
We have demonstrated that SFV-G- pseudotyped lentiviruses can utilize DC-
SIGN and L-SIGN as attachment receptors, resulting in productive infections of cell lines
bearing these molecules and human MoDCs. Preferential binding to DC-SIGN(R) by
SFV-G has not previously been reported, indicating that there is an unappreciated naive
trophism of SFV-G pseudotyped lentivirus for APCs. Our results suggest that by utilizing
the affinity of SFV-G to DC-SIGN(R), lentiviruses can be engineered to preferentially
transduce antigen-presenting DCs for gene-based immunotherapy.

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We found that FUGW/SFVG but not FUGW/VSVG produced in 293T cells bind
to DC-SIGN(R) receptors. When produced in 293T cells, VSV-G-pseudotyped
lentiviruses exhibited similar binding and transduction of cells expressing DC-SIGN, L-
SIGN or parental cell lines. For FUGW/SFVG, cells expressing DC-SIGN or L-SIGN
were more permissive than non-expressing parental cell lines. The increased transduction
was well-correlated with an increase in binding to the cells as measured by radiolabeled
virus binding assays. Specific interaction between DC-SIGN and SFV-G was
demonstrated by blocking the transduction of the DC-SIGN-expressing cells with
inhibitors such as ConA, EDTA, soluble DC-SIGN protein, yeast mannan, or DC-SIGN-
specific mAbs in both 293T.DCSIGN cells and human MoDCs.
SFV-G-pseudotyped lentiviruses have an enhanced ability to transduce DC-
SIGN-expressing cells when produced under conditions arresting the viral glycan
maturation primarily at the high mannose

stage. Several reports indicate that the presence
of high-mannose-content

N-linked glycans on Sindbis virus, Ebola virus and West Nile
virus enhance the infection of mouse-derived DCs due to

interactions with the mannose
binding C-type lectin receptors (Davis et al. 2006; Klimstra et al. 2003; Marzi et al.
2007). In addition to DC-SIGN and L-SIGN, there are other C-type lectin molecules
present on DCs, macrophages, endothelial cells, and other APCs, that might play a role in
FUGW/SFV-G transduction (Figdor et al. 2002). High mannose content of viral envelope
glycoproteins directly 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.), when SFV-G-pseudotyped

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lentiviruses were generated in mammalian cells treated with the mannosidase I inhibitor
DMJ, the resulting particles exhibit an increased capacity to utilize DC-SIGN for
infection. The increase in interaction with DC-SIGN by FUGW/SFVG is presumably
mediated by increased binding by the CRD to the mannose structures of these
glycoproteins. The SFV-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
SFV particles, but remains associated with the virion of SFV (Mancini et al. 2000). When
virus-producing cells are treated with DMJ, transduction by FUGW/SFVG was increased
towards MoDCs as well as DC-SIGN-expressing 3T3 cells but not the parental 3T3 cell
line. The observation that FUGW/SFVG produced by DMJ-treated cells have an
enhanced ability to preferentially transduce DC-SIGN-expressing cells suggests that
modification of N-linked glycans of SFV-G can be used to enhance the transduction of
DCs.
We observed that FUGW/VSVG lentiviruses 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). Although previous studies
have found that VSV-G pseudotypes of HIV-1 infect bone marrow-derived premature
DCs (Strang et al. 2005), we found that FUGW/VSVG exhibited no preferential
transduction towards cells expressing the human C-type lectins DC-SIGN or L-SIGN.
FUGW/VSVG lentiviruses have a broad tropism and can transduce multiple cell types.
Therefore, they are undesirable for delivering genes in vivo to APCs. Our results suggest

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that the transduction by FUGW/VSVG is largely DC-SIGN(R)-independent and less
efficient than FUGW/SFVG at transducing DCs when normalized by infectious particles.
In this study we assessed the relationship of DC-SIGN and L-SIGN interactions
with SFV glycoproteins to mediate the transduction of DCs. DCs are potent APCs 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. 2008b). The development of DC differentiation protocols for PBMC has facilitated the
study of DC biology and the subsequent implementation of clinical DC-based vaccination
studies. Transduction of human MoDC by FUGW/SFVG was more than twice as
efficient as that by FUGW/VSVG when normalized by MOI. Furthermore, the
transduction by FUGW/SFVG was DC-SIGN(R)-specific and could be inhibited by both
yeast mannan and anti-DC-SIGN antibodies. SFV-G pseudotypes preferentially
transduced the DC-SIGN-positive cells, consistent with the theory that DC-SIGN
mediates transduction in DCs. The preferential transduction of DCs can be further
enhanced by production under untrimmed (DMJ-treated) high mannose conditions.
FUGW/SFVG has previously been shown to have low transduction efficiency for a wide
range of cell types (Strang et al. 2005), but we have found that SFV-G has a natural DC-
SIGN(R) tropism, which can be utilized for directing the cellular transduction of
lentiviruses to APCs.
The results described herein have relevance to the design and production of viral
vectors used for gene delivery to APCs. Targeting of lentiviruses to C-type lectin-

99

expressing cells such as DCs can be increased by the choice of a suitable envelope
glycoproteins 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 SFV-G strongly binds to C-
type lectins. The natural tropism of FUGW/SFVG with DC-SIGN(R) represents a new
strategy to genetically modify DCs.
4.5 Acknowledgements
We thank Dr. Margaret Kielian for providing antibodies against the SFV envelope
glycoprotein, Dr. Robert Chow for the pSFV expression vector, Xiao Liang for providing
soluble DC-SIGN proteins, and Kye-Il Joo for assistance with confocal imaging. .
Portions of this chapter are adapted from: Steven Froelich, April Tai, Katie Kennedy,
Adnan Zubair, and Pin Wang. (Submitted, 2011).

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Chapter 5. Engineering Lentiviral Vectors for use in the Central
Nervous System
In this study, we explore the ability of pseudotyped lentiviral vectors with
envelope glycoproteins derived from a neuroadapted Sindbis virus (SVGnu) to
specifically transduce neuronal cell types. In neuroblastoma cell lines and rat cortical
neuronal cultures the SVGnu pseudotyped lentivector exhibited enhanced transduction
compared with the non-engineered Sindbis virus (SVG) pseudotype. To determine if the
transduction by SVGnu was in fact neuron specific, we transduced primary cortical
cultures containing both glial and neuronal cell types. The SVGnu pseudotyped LV was
the only virus to specifically transduce neurons, whereas both vesicular stomatitis virus
glycoprotein (VSV-G) and SVG transduced similar percentages of the glia population as
the neuronal population. The ability of SVGnu pseudotyped lentivectors to selectively
transduce neuronal cell types and generate efficient gene delivery may be a useful tool for
gene therapy in the central nervous system.
5.1 Introduction
The application of molecular genetics to biology has improved our understanding
and ability to treat a variety of diseases. The development of methods to manipulate gene
expression in living organisms has become an invaluable tool in the study of gene
functions. In neurobiology, the typical approach for genetic manipulation or gene
disruption (knockout) is the generation of transgenic animals which rely on regulatory
elements to control the onset of the genetic manipulation and the cell type that is affected

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(Dittgen et al. 2004; Gossen and Bujard 2002; Valjent et al. 2009). However, transgenic
methodologies are limited by the inability to design specific regulatory elements which
direct genetic manipulations to individual neurons in specific regions of the brain. The
development of methods to manipulate genes in individual neurons is a valuable tool for
the application of gene therapy in the central nervous system.
Gene delivery by viral vectors is one of the most promising methods for the
genetic modification of individual cells or specific tissues in the central nervous system
(CNS). HIV-1 derived lentiviral vectors are particularly well-suited for gene transfer in
the CNS due to their ability to infect both dividing and non-dividing cells, deliver a large
transgene, and stably integrate into the host cell genome providing long-term transgene
expression. Numerous strategies have been developed to introduce genes into cells of the
central nervous system using lentiviral vectors (Davidson and Breakefield 2003;
Lowenstein et al. 2003). However, the development of tailored vectors in a way that
renders them specific for target cell types while retaining high transduction efficiency has
largely been neglected. In most studies, lentiviral vectors are pseudotyped with the
envelope glycoprotein of vesicular stomatitis virus (VSV-G) which facilitates non-
selective cell entry leading to unwanted off-target effects due to ubiquitous transduction
of both glia and neuronal cells (Jakobsson et al. 2003). Applications requiring more cell
type specific gene delivery have so far mainly relied on transcriptional controls (Hioki et
al. 2007; Jakobsson et al. 2003). These methods are hindered by the limited availability
of suitable promoters and unwanted off-target effects due to unspecific transduction. To

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overcome these problems, we propose to engineer the lentiviral envelope glycoproteins to
restrict transduction towards neuronal cell types.
Lentiviral vectors pseudotyped with envelope glycoproteins from various viruses
have been evaluated for transduction in the central nervous system, but none demonstrate
a restricted neuronal specificity (Jakobsson et al. 2006; Watson et al. 2002). In a number
of previous reports, envelope glycoproteins from alphaviruses (Cronin et al. 2005), in
particular Sindbis virus (Morizono et al. 2010; Yang et al. 2008b), have been used to
pseudotype lentiviral vectors and were shown to be a promising alternative to the VSV-
G. In light of previous findings that Sindbis virus efficiently infects neurons
(Ehrengruber et al. 1999; Gwag et al. 1998), we chose to investigate the ability of the
Sindbis virus envelope (SVG) to mediate neuronal infection. The Sindbis virus envelope
glycoprotein is initially synthesized as a polyprotein (E3-E2-6k-E1) which is then
proteolytic processed at signalase cleavage sites into two subunits (E1, E3-E2) and
finally the E3 protein is cleaved by furin protease from the mature SVG particles
(Strauss and Strauss 1994). The mature SVG complex consists of heterodimers of the E1
and E2 glycoproteins that are organized into trimers (Strauss and Strauss 1994). The E2
glycoprotein mediates binding with target cell receptors, whereas E1 is thought to
mediate endosomal fusion with the viral membrane in an acidic environment (Dubuisson
and Rice 1993; Garoff et al. 1980; Kielian 1995). Major determinants of the
neurovirulence of neuroadapted isolates of Sindbis virus have been mapped to specific
amino acid mutations in the E2 region (Lee et al. 2002). The engineering of Sindbis virus

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envelope proteins, by mutating the nonspecific heparan sulfate binding sites and adding
mutations for neurotropism, could in principle improve neuronal targeting specificity.
In this study, we explored the ability of pseudotyped lentiviral vectors with
envelope glycoproteins derived from a neuroadapted Sindbis virus to target the
transduction to neuronal cell types. Their ability to selectively transduce neuronal cell
types and generate efficient transduction was investigated. Ultimately, the development
of a neuroadapted lentiviral vector may be a useful tool for gene delivery in the CNS.
5.2 Materials and Methods
5.2.1 Cell Lines
The human embryonic fibroblast cell line HEK 293T and mouse neuroblastoma
Neuro-2a (N2a) were obtained from ATCC and maintained in D10 medium (Dulbecco
modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine
serum (Sigma Aldrich), 2mM L-glutamine (Gibco-BRL), and 100U of penicillin and
100µg/mL of streptomycin (Gibco-BRL). The human neuroblastoma cell lines CHLA-
20, CHLA-90, and SMS- KCNR were obtained from the Children's Oncology Group Cell
Culture and Xenograft Repository. Cells were grown in medium consisting of Iscove's
modified Dulbecco's medium (IMDM, Bio Whittaker, Walkersville, MD) supplemented
with 3 mM L-glutamine (Gibco-BRL), insulin and transferrin 5 μg/ml each and 5 ng/ml
of selenous acid (ITS Culture Supplement, Collaborative Biomedical Products, Bedford,
MA), and 20% fetal bovine serum (Sigma Aldrich) at 37°C with 5% CO
2
.

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5.2.2. Dissociated cultures
Primary cortical neuron cultures were established from cortices dissected from
E18 fetal Sprague-Dawley rats. The cortices of E18 embryos were dissected in Hanks
Balanced Salt Solution, HBSS (Lonza, Walkersville, MD) supplemented with 1 mM
HEPES (Invitrogen). Cortices were dissociated in HBSS plus HEPES with 0.25% trypsin
for 15 min and then washed three times for 5 min each with HBSS plus HEPES. The
dissociated neurons were then plated on coverslips (Fisher) at a density of 5 x 10
4
cells
per well in neurobasal medium (Invitrogen) supplemented with 10 ml/L Glutamax
(Invitrogen), 1 µg/mL gentamicin (Invitrogen), 20 mL/L NS-21 supplement (Chen et al.
2008) and 50 ml/L fetal bovine serum (Sigma Aldrich). After 4 h, the medium was
replaced with serum-free neurobasal medium.
5.2.3 Plasmid Construction
We obtained the cDNA for wild-type Sindbis virus from J. Strauss's laboratory at
the California Institute of Technology. PCR mutagenesis and assembly were used to
generate the mutant Sindbis virus envelope as described by Chen and colleagues
(Morizono et al. 2001), except a 10-residue tag sequence (MYPYDVPDYA) replaced the
ZZ domain of protein A, which is located between amino acids 71 and 74 of the E2
glycoprotein as previously described (Yang et al. 2006). PCR mutagenesis was then used
to introduce mutations at the E2 domain to replace amino acid glutamine at position 55
with histidine to generate neuroadapted Sindbis virus envelope (SVGnu). The cDNA for
SVGnu was then subcloned into the VSV-G expression plasmid containing the rabbit β-

105

globin intron and its poly (A) signal (Cell Genesys, Foster City, CA). The integrity of the
DNA sequences was confirmed by DNA sequencing.
5.2.4 Lentivector Production
The lentiviral vectors used in this study are the third generation HIV-1 based
lentiviral vectors in which most of the U3 region of the 3‟ LTR was deleted, resulting in a
self-inactivating 3‟-LTR (Lois et al. 2002). Lentivectors 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 (SVG, SVGnu, or VSV-G) and 2.5 µg the packaging
plasmids pMDLg/pRRE and pRSV-REV (Dull et al. 1998). 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 concentrated by
ultracentrifugation at 4°C (Optima L-80K preparative ultracentrifuge, Beckman Coulter)
at 50,000×g for 90 min. The supernatant was discarded and the pellets were then
resuspended in an appropriate volume of HBSS (Lonza, Walkersville, MD).
5.2.5 Determination of p24 and infectious titers
To determine p24 concentrations, supernatants were diluted 1:10,000 and assayed
by an enzyme-linked immunosorbent assay (ELISA) using the p24 ELISA kit from
ImmunoDiagnostics (Woburn, MA) according to the manufacturer‟s instructions. SVG
producing cells made 348 ± 19 ng/µL of p24 whereas, SVGnu transfected cells produced

106

458 ± 38 ng/µL and VSV-G supernatant yielded 308 ±42 ng/µL of p24. To determine
infectious titer, 2 × 10
4
293T cells were transduced in triplicate with 100 µl of serially
diluted viral supernatant or concentrated virus 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.
5.2.6 Vector-mediated transduction of cell lines in vitro
Target cells (N2a, CHLA-20, CHLA-90 and SMS-KCNR; 2 × 10
5
per well) were
seeded in 24-well culture dishes and spin-infected with SVG or SVGnu lentivectors
(500ng 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 medium and
incubated for 48 hrs at 37°C with 5% CO
2
. The experiments were performed in triplicate
and GFP expression was evaluated by flow cytometry.
5.2.7 Vector-mediated transduction of primary cell cultures in vitro
Disassociated rat cortical neuronal cells were cultured on poly-D-lysine-coated
coverslips in 6 well plates for 5 days. On day 5 in vitro (DIV5) an MOI =1 of
concentrated lentivectors in 50µL HBSS was added to the cultures and spin-infected at
2,500 rpm and 25°C for 90 min using a RT Legend centrifuge. Subsequently, the
supernatants were replaced with fresh culture medium and incubated at 37°C with 5%

107

CO
2
. Experiments were performed in triplicate and GFP expression was analyzed on
DIV9 using flow cytometry.
Mixed cultures of glia and neurons were produced by prolonged culture of
disassociated rat cortical cells on poly-D-lysine-coated coverslips in 6 well plates for 21
days. On DIV21, concentrated lentivector (4.5µg of p24) in 50uL HBSS was added to
the cultures and spin-infected at 2,500 rpm and 25°C for 90 min using a RT Legend
centrifuge. Subsequently, the supernatants were replaced with fresh culture medium and
incubated at 37°C with 5% CO
2
. Experiments were performed in duplicate and GFP
expression was analyzed on DIV27 using flow cytometry.
5.2.8 Intracellular staining for flow cytometry
Glia and neurons were detached with 0.5% trypsin, washed and triturated to a
single cell suspension and fixed and permeabilized using the BD Cytofix/Cytoperm™ kit
(BD Biosciences). After permeabilization cells were incubated for 30 min on ice with
primary antibodies diluted 1:250 with biotinylated TUJ-1(R&D Systems) and/or anti-
GFAP (Invitrogen). After a further three washes, the cells were incubated for 30 min at
4°C with a PE-anti-Rabbit IgG diluted 1:500 and Streptavidin-Cy5 diluted 1:500. The
cells were then washed and resuspended in PBS prior to flow cytometry analyses.
5.2.9 Immunocytochemistry on slides
Neurons plated onto poly-D-lysine-coated coverslips were fixed with 4%
paraformaldehyde (PFA) for 30 min at room temperature, washed three times with PBS
and incubated for 30 min at room temperature in PBS-NGS (1 X PBS, 0.1% triton and

108

10% normal goat serum). After three rinses, the cells were incubated overnight at 4 °C
with antibodies raised against TUJ-1 (1/250) diluted in PBS. The cells were then washed
three times and incubated for 2 h at RT with Texas red-conjugated anti-mouse IgG
diluted 1:200 in PBS. After three washes in PBS, the cells were fixed mounted with an
anti-fading medium (Dako). 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.
5.3 Results
5.3.1 Targeting neuronal cell lines in vitro
To facilitate the transduction of neuronal cell types, we constructed a Sindbis
derived envelope glycoprotein that contains mutations that enhance neurotropism and
removes non-specific binding to other cells (Figure 5.1). To remove non-specific binding
we engineered the envelope to be deficient of heparan sulfate binding motifs, which are
expressed by many non-neuronal cell types (Byrnes and Griffin 1998; Strauss et al.
1994). We and others have demonstrated that SVG can efficiently pseudotype
lentiviruses and that alterations to SVG, including deletion of amino acids 61-64 of the
E3 domain, mutations of 157KE158 into 157AA158 of the E2 domain, and an insertion
of 10-amino acid tag sequence (MYPYDVPDYA) between amino acids 71 and 74 of the
E2, can disable its binding to heparan sulfate (Morizono et al. 2001; Yang et al. 2006),
while mutating Q to H at amino acid 55 of E2 can enhance neurotropism (Lee et al. 2002;
Lustig et al. 1988; Tucker et al. 1993); we designated this modified SVG as SVGnu

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(Figure 5.1). Once it is incorporated onto a lentiviral surface, this mutant glycoprotein
should be able to mediate infection of neuronal cells but not other cells.

Figure 5.3 Virus-producing constructs used to make pseudotyped lentiviruses. A
schematic representation of the general strategy to engineer a lentivector system capable
of targeting neuronal cells. SVGnu was constructed by making several alterations in the
Sindbis virus envelope glycoprotein (SVG), including deletion of amino acids to disrupt
the binding to heparin sulfate glycosaminoglycan and the neuroadapted mutation of
histidine (H) at E2 position 55, resulting in a binding deficient but neurotropic envelope
SVGnu. Schematic diagrams of expression constructs encoding the lentiviral backbone
FUGW and envelope glycoproteins. CMV enhancer: the enhancer element derived from
human cytomegalovirus; Ubi: the human ubiquitin-C promoter; GFP: enhanced green
fluorescence protein; WRE: woodchuck responsive element; ΔU3: deleted U3 region that
results in the transcriptional activation of the integrated viral LTR promoter; pA:
polyadenylation signal; E1, E2, 6k, E3: SVG-glycoproteins (E1 for fusion, E2 for
receptor binding, 6k a linker, and E3 a signal sequence). The expression plasmids also
contain the rabbit β-globin intron and poly(A) signal.
Previous studies have indicated that neuroadapted mutations in SVG can have a
significant impact on the efficiency to promote viral infection in neuroblastoma cell lines
(Ryman et al. 2007). To evaluate the effect of neurotropic mutations on infectivity per
particle, SVG and SVGnu viral preparations were normalized by p24 measurement and

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used to infect human and mouse neuroblastoma cells. Transduction was reported as fold
increase in GFP positive cells based on FUGW/SVG which transduced 25.6 ±3.46,
4.4±0.64, 5.67±0.66, and 2.53±0.38 for CHLA-20, CHLA-90, SMS-KCNR and N2a cell
lines, respectively. When the same amount of virus was used, the FUGW/SVGnu
transduced 2 to 4 fold times as many cells for each cell line (Figure 5.2). The p24-
normalized transduction demonstrates SVGnu-pseudotyped LVs have an enhanced
infectivity per lentiviral particle for both human and mouse neuroblastoma cell types.

Figure 5.4 Lentiviral transduction of neuroblastoma cells. Lentiviruses were normalized
by 500ng of p24/well of FUGW/SVGnu (dark fill) and FUGW/SVG(gray fill) and used
to transduce 2 × 10
5
per well of neuroblastoma cells (CHLA-20, CHLA-90, SMS-KCNR
and N2a). Two days later, the transduction efficiency was measured by analyzing GFP
expression using flow cytometry. Fold increase in percentage of GFP-positive cells is
shown based on FUGW/SVG transduction, where values are given as the mean of
triplicates ± S.E.
5.3.2 Transduction of rat cortical neurons in vitro
Since our results indicated that the SVGnu mutations increased the neuroblastoma
infectivity, we sought to determine if this effect was also observed in primary neuronal
cell types. To study the infectivity of SVGnu in primary neuronal cell types, we

111

transduced rat cortical neurons with SVG or SVGnu lentivectors. Viral preparations
were normalized by infectious units on 293T cells (MOI=1) and then used to infect DIV5
rat cortical neuron cultures. Immunostaining followed by flow cytometry and confocal
microscopy was used to distinguish transduced neuronal populations (Figure 5.3). We
used an antibody against anti-neuronal class III beta-tubulin (TUJ-1) as a neuronal
marker. The colocalization of expression of GFP with TUJ-1 was confirmed in DIV9
primary cultures by confocal microscopy (Figure 5.3B). Transduced TUJ-1 expressing
cells were observed for both SVGnu and SVG infections (Figure 5.3B). Flow cytometry
was used to quantify the difference in transduction efficiency between SVGnu and SVG
lentivectors. Flow cytometry data were plotted as cell size (FSC) as a function of
fluorescence intensity of GFP where one representative plot is shown (Figure 5.3A).
FUGW/SVGnu exhibited the highest level of transduction 39.16±7.2% versus
13.73±0.5% for FUGW/SVG (Figure 5.3A). Similar to previous studies (Behbahani et al.
2005), analysis by flow cytometry of DIV9 cultures revealed approximately 90% of cells
were TUJ-1-positive (data not shown). These results further support observations that the
neuroadapted SVGnu is more efficient at transducing neuronal cell types.

112

Figure 5.5 Flow cytometry analyses and confocal microscopy of transduced cells in
primary cultures of rat cortical neurons. (A)Transduction of DIV5 cortical neurons with
an MOI of 1 by FUGW/SVGnu or FUGW/SVG. The results are plotted as GFP
fluorescence as a function of FSC. The flow cytometry data presented is a representative
of three different analyses with mean GFP percentage and variation reported. (B)
Representative analysis of GFP (green) and TUJ-1 (red) in primary cortical neurons
transduced by either FUGW/SVGnu (a-c) or FUGW/SVG (d-f). The data presented are
representative of three different analyses.
5.3.3 Transduction of mixed cultures in vitro
To distinguish the neurotropic features of Sindbis virus pseudotypes, we
performed transduction of mixed cultures of rat cortical neurons and glial cells. Primary
cultures derived from embroynic rat cortices were cultured until significant populations
of glia cells were observed on DIV21. FUGW/SVG, SVGnu or VSV-G preparations were
normalized by p24 and used to transduce these mixed cultures. Immunostaining followed

113

by flow cytometry was used to distinguish neurons from glial cell types. We used an
antibody (TUJ-1) as a neuronal marker, and, for detection of glia, a GFAP antibody was
used. On DIV21, flow cytometry analysis showed approximately 9.34% ± 0.7% GFAP
+

cells. On DIV28 we preformed double staining to identify GFAP (Figure 5.4A) and TUJ-
1 (Figure 5.4B) transduced populations expressing GFP. When p24 was used to add the
same amount of physical particles, VSV-G-pseudotyped viruses exhibited the highest
levels of transduction (Figure 5.4). FUGW/VSVG transduced 80.1 ±3.46% of the total
glia cells (GFAP
+
cells) and 92.1 ±1.27% of the total neuronal cells (TUJ-1
+
cells),
indicating that VSV-G has no preference toward either neuronal or glial cells. Similarly,
FUGW/SVG transduced both glia and neurons but at lower levels with 17.9 ±2.47% of
total glia and 15.5 ±0.77% of the total neurons expressing GFP. In contrast,
FUGW/SVGnu transduced only 7.58 ±2.23% of the glia cells and 48.8 ±3.43% of the
neuronal cells indicating that SVGnu preferentially transduces neuron cell types in a
mixed culture system.

114

Figure 5.6 Flow cytometry analyses of transduced cells in primary cultures of rat cortical
glia and neuronal cells. Transduction of DIV21 mixed culture with FUGW/SVGnu,
FUGW/SVG, or FUGW/VSV-G. The results are plotted as percentage of GFP
+
cells of
either (A)GFAP
+
or (B) TUJ-1
+
cells . The flow cytometry data presented is a
representative of two different analyses.
5.4 Discussion
Optimization of lentivector design for gene delivery into neuronal cell types
remains a challenge. By using a vector coated with a protein selectively targeted to
neuronal cell types, we have achieved high specificity, as measured in vitro. One
measure of the effectiveness of SVGnu is the increased transduction of neuroblastoma
cell lines and rat cortical neurons as compared with the SVG pseudotyped lentivirus.
Furthermore, direct comparison with VSV-G pseudotyped transduction of mixed glial
and neuronal cultures demonstrated effective specificity of SVGnu for neuronal cells.
There is a growing supply of alternative envelopes and strategies to engineer
envelope glycoproteins for targeting LVs in the CNS(Jakobsson and Lundberg 2006).

115

Previous studies describe differences in tropism when using various envelopes
(Jakobsson et al. 2006; Watson et al. 2002), however with the exception of engineered
measles virus envelope (Anliker et al. 2010), the transduction is not restricted to specific
cell types. To direct cell entry to neuronal cells we exploited the neuroadapted Sindbis
virus envelope to confer enhanced neurotropism to lentiviral particles and removed
heparan sulfate binding sites to reduce off-target transduction, resulting in SVGnu.
Lentivectors pseudotyped with SVGnu proved to be specific and effective in modifying
neuronal cell types.
To characterize transduction of SVGnu in vitro we utilized both mouse and
human neuroblastoma cell lines. Our results generally confirm previous studies (Lee et al.
2002; Ryman et al. 2007) with respect to the influence of the H55 mutation to enhance
the cell attachment and transduction of SVG to neuroblastoma cells. We extend these
studies to include the effects of the removal of heparan sulfate binding domains which
reduce non-specific background transduction of the laboratory adapted SVG. In order to
determine if the enhanced infectivity of the SVGnu virus is due to the altered interaction
of these viruses with cell surface structures and not simply a function of the presence of
an increased number of lentiviral vectors, particles were normalized by the p24 capsid
protein ensuring equal numbers of physical lentiviral particles in each sample.
Differences in infectivity between standard Sindbis virus laboratory strains (SVG) and
neuroadapted SVGnu were detected with all neuroblastoma cell types examined. Our
results indicate that the combination of H55 with deletion of heparan sulfate binding

116

domains can confer a high degree of infectivity on the SVG envelope that is evident
during transduction of many neuroblastoma cell lines.
Lentiviruses pseudotyped with SVGnu were uniformly more effective at
transducing rat cortical neuronal cultures in comparison with SVG pseudotypes. To
account for differences in infectivity, viral particles were normalized by infectious units
based on 293T cells. Therefore, the differences in transduction efficiency must be due to
specific interactions with neural-derived cellular factors not present on 293T cells. The
SVGnu may increase the efficiency of transduction in neurons in several ways; possibly
by enhancing or altering interactions with a primary receptor, redirecting the entering
virion to a coreceptor structure or promoting endosomal fusion of internalized particles.
The increased transduction efficiency towards neurons appears to be a property of the
mutations made to SVGnu as evident by comparison with the laboratory adapted SVG.
To determine if the transduction of SVGnu was in fact neuron specific, we
transduced primary cortical cultures containing both glial (GFAP
+
) and neuronal (TUJ-
1
+
) cells. Viruses were normalized by the number of physical particles using p24 capsid
levels. The SVGnu pseudotyped LV was the only virus to specifically transduce neurons,
whereas both VSV-G and SVG transduced similar percentage of the glia as neuronal
cells. Our results and previous reports (Dittgen et al. 2004; Jakobsson et al. 2003;
Zufferey et al. 1997), show that VSV-G pseudotyped lentiviral vectors are able to
ubiquitously transduce both glia and neuronal cells. Even though VSV-G pseudotypes
transduce glia and neuronal cells, it does so with paramount efficiency. Our results and
the work of others (Klimstra et al. 1998; Smit et al. 2002) suggest that heparan sulfate

117

binding of the laboratory adapted SVG can lead to promiscuous attachment resulting in
transduction of both neurons and glia. By removing the promiscuous heparan sulfate
binding and incorporating the neurotropic H55 mutation SVGnu attains an enhanced
transduction efficiency and specificity towards neuronal cells which makes it an
attractive alternative to other pseudotypes.
It is increasingly apparent that subtle differences in the configuration,
carbohydrate structure, or charge of the virion's spike proteins may dramatically alter the
infectivity and tropisms of alphaviral pseudotyped lentivectors. We have recently
demonstrated this phenomenon in the C-type lectin receptor usage of various alphaviral
pseudotypes-derived with different carbohydrate structures (Tai et al. 2011). In the
present study, our results demonstrate that lentiviral tropism can be tailored to target
neuronal cell types by engineering the Sindbis virus envelope glycoproteins to eliminate
promiscuous heparan sulfate binding and incorporate the neurotropic E2 H55 mutation.
Although the titer achieved for SVGnu is lower than what can be obtained with VSV-G,
the neuron-specific transduction is at such a high level that it encourages further
investigations.
5.5 Acknowledgements
We thank Dr. Don Arnold‟s lab for providing the rat cortical neuron cultures and
Dr. Kye-Il Joo for assistance with confocal imaging.

118

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Abstract (if available)

Abstract The development of lentiviral vectors to deliver genes to specific cell types are useful tools because they have the ability to produce stable transduction, maintain long-term transgene expression, and transduce both dividing and non-dividing cells. Despite the high transduction efficiency of lentiviral vectors, their tropism frequently does not match the desired gene delivery application. We report herein, strategies to modify lentiviral vectors using diverse techniques which allow targeting gene delivery to specific cell types. To target CD117 expressing cells we engineered a lentivector that incorporates membrane-bound human stem cell factor (hSCF), and for fusion, a Sindbis virus-derived fusogenic molecule (FM) onto the lentiviral surface. Lentiviral vectors pseudotyped with envelope proteins of alphaviruses have recently attracted considerable interest for their potential utilization for immunotherapy due to their capacity to transduce dendritic cells. We report lentiviral vectors pseudotyped with envelope glycoproteins derived from the Aura, Sindbis and Semliki Forest alphaviruses have a natural capacity to transduce dendritic cells through the DC-SIGN receptor. Finally, in this study, we explore the ability of pseudotyped lentiviral vectors with envelope glycoproteins derived from a neuroadapted Sindbis virus envelope glycoprotein to specifically transduce neuronal cell types. The development of engineered lentiviral vectors to achieve targeted transduction while avoiding transduction of non-target cells will be important tools for future gene delivery implementation in a wide range of fields.

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Asset Metadata

Creator Froelich, Steven Michael (author)

Core Title Engineering lentiviral vectors for gene delivery

School Andrew and Erna Viterbi School of Engineering

Degree Doctor of Philosophy

Degree Program Chemical Engineering

Publication Date 05/03/2011

Defense Date 03/30/2011

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

Tag DC-SIGN,dendritic cell,lentivirus,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Wang, Pin (committee chair), Jakowec, Michael W. (committee member), Shing, Katherine S. (committee member), Yen, Jesse T. (committee member)

Creator Email sfroelic@usc.edu,steve.froelich@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3829

Unique identifier UC1139406

Identifier etd-Froelich-4407 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-459666 (legacy record id),usctheses-m3829 (legacy record id)

Legacy Identifier etd-Froelich-4407.pdf

Dmrecord 459666

Document Type Dissertation

Rights Froelich, Steven Michael

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu