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Engineering lentiviral vectors for gene therapy and DC-vaccine

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Engineering lentiviral vectors for gene therapy and DC-vaccine

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ENGINEERING LENTIVIRAL VECTORS FOR GENE THERAPY AND DC-
VACCINE

by

Chi-Lin Lee

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

May 2012

Copyright 2012 Chi-Lin Lee
ii
1 Dedication
This thesis is dedicated to my parents Chun-Ming Lee and E-Shu Huang, my
brother Chi-Chun Lee, and my girlfriend Sungjin Park. Their support throughout this
process has been invaluable.

iii
2 Acknowledgements
First and foremost I would like to thank my advisor Dr. Pin Wang who has
supported and guided me though this process. He has been always there for me when I
had difficulty in research and help me to reach my full potential. I would also like to
thank my dissertation committee Dr. Katherine Shing and Dr. Don Arnold. It was their
insightful comments and gave me lots of advises that help me to finish this thesis.
I have been fortunate to work with talented people and a great team in RTH-515 at
USC. I will never forget the experience I had here at USC. I would like to show my
thanks to Dr. Haiguang, Dr. Leslie, Dr. Yuning for setting up the labs and guided us,
Dr.Kye-Il for the help in confocal imaging, Dr. April, Dr. Steve, and Dr. Paul for critical
reading of the manuscripts, Dr. Bingbing and Xiaoliang for their help in culturing
mammalian cells and in vivo study. I would also like to show my thanks to the
contributions of students; Jason Dang, Michael Chou, Biliang Hu, Laura Liu, and
Jennifer Zhang for both directly and indirectly help this research.

iv
3 Abstract
An important concept of gene therapy is the delivery of genetic materials to target
cells for therapeutic benefit. One of the most important and efficient methods for gene
delivery is the use of viral vectors as transfer vehicles. Lentiviral vectors (LVs) derived
from human immunodeficiency virus type 1 (HIV-1) are promising vehicles for gene
delivery because they not only efficiently transduce both dividing and non-dividing cells,
but also maintain long-term transgene expression. In order to enhance the gene delivery
ability of the viral vector, we designed a strategy by separating binding and fusion ability
of envelope protein into two distinct proteins. By pseudotyping the viral vectors with
both an antibody and a fusogenic molecule, we can target and transduce specific cell
types. Based on this work, we developed a method to create LVs co-enveloped with the
HIV-1 cellular receptor CD4 and a fusogenic protein derived from the Sindbis virus
glycoprotein and tested its efficiency to selectively deliver genes into cells expressing
HIV-1 envelope proteins. In chapter 2, we demonstrated that this engineered LV can
preferentially deliver transgene to HIV-1 envelope-expressing cells in vitro. We conclude
that this target LVs give a potential alternative treatment for eradicating HIV-1-infected
cells that produce drug-resistant viruses after highly active antiretroviral therapy
(HAART). In order to improve the LV transduction efficiency, we introduced the
mutations in the E1 domain of Sindbis virus glycoprotein at residues 75 and 237
individually or in combination. The mutation at residues 75 from a neutral and non-polar
glycine (Gly, G) to a polar and acidic aspartic acid (Asp, D) can enhance the transduction
efficiency by broadening the range of the pH threshold for fusion. In chapter 3 we
demonstrated our effort in enhancing the targeted transduction by genetically engineering
v
the fusion component displayed on the viral membrane. To further test lentiviral vectors
in preclinical or clinical studies, we constructed a stable producer line for synthesizing
DC-SIGN-targeted LVs by a concatemeric array transfection technique could routinely
produce vector supernatants with titers above 10
7
transduction units per milliliter
(TU/mL) during a continuous 3-month cell passage. Based on our studies, this production
method can generate DC-LVs for preclinical and clinical testing of novel DC-based
immunization.

vi
Table of Contents
Dedication ii
Acknowledgements iii
Abstract iv
List
of
Figures

ix

Chapter1. Introduction
1.1 Introduction of Gene Therapy 1
1.2.1 Purified DNA 2
1.2.2 Cationic lipids and liposomes 3
1.2.3 Cationic polymers 4
1.2.4 Virosome 4
1.3 Viral Vector 5
1.3.1 Retroviral vector 6
1.3.2 Lentiviral Vector 7
1.3.3 Adenoviral vectors 9
1.3.4 Adeno-Associated Virus 10
1.4 Project Overview 11
Chapter2. Engineered lentiviral vectors pseudotyped with a CD4 receptor and a
fusogenic protein can target cells expressing HIV-1 envelope proteins 15
2.1 Introduction 16
2.2 Methods 19
2.2.1 Plasmids 19
2.2.2 Cell lines 21
2.2.3 Cell–cell fusion assay 21
2.2.4 Vector production 22
2.2.5 Vector transduction in vitro 22
2.2.6 Cell–vector binding 23
2.2.7 Soluble CD4 competition and NH
4
Cl neutralizing 23
2.2.8 Targeted delivery of suicide gene therapy in vitro 24
2.3 Results 24
2.3.1 Generation of the HIV-1 envelope protein-expressing cell line 24
2.3.2 Generation of recombinant LVs 27
2.3.3 Targeted transduction of HIV-1 Env-expressing cells 28
2.3.4 Targeting a broader tropism of gp120 moiety 32
2.3.5 Specific binding between vectors and cells for targeted transduction 33
2.3.6 Dependence of targeted transduction on the endosomal pH 35
2.3.7 Transduction inhibition by dominant-negative Rab5 and Rab7 37
2.3.8 Targeted transduction by engineered LVs towards a human T cells 38
vii
2.3.9 Evaluation of the potential of LV-mediated suicide gene therapy in
vitro 41
2.4 Discussion 42
Chapter 3. Mutagenesis of the E1 domain of Sindbis virus glycoprotein alters its
pH-dependent fusion 47
3.1 Introduction 48
3.2 Methods 51
3.2.1 Plasmids Preparation 51
3.2.2 Cell lines 52
3.2.3 Virus production 52
3.2.4 Cell-virus binding 53
3.2.5 Confocal imaging 53
3.2.6 Virus transduction 54
3.2.7 NH
4
Cl neutralizing 54
3.2.8 Virus-liposome fusion 55
3.3 Results 57
3.3.1 Creating mutations in the E1 domain of the fuogen 57
3.3.2 Production of lentiviruses containing mutant FMs 57
3.3.3 Co-expression of fusogen and antibody on lentiviruses 61
3.3.4 Interaction between the binding protein and the target receptor 61
3.3.5 Transduction of lentiviruses bearing various FMs 62
3.3.6 Study of virus entry by neutralizing endosomes 64
3.3.7 Liposomal fusion study of pH and cholesterol dependency of FMs 65
3.3.8 Transduction inhibition by dominant-negative Rab5 and Rab7 67
3.4 Discussion 69
Chapter 4. Construction of Stable Producer Cells to Make High-Titer Lentiviral
Vectors for Dendritic Cell-Based Vaccination 74
4.1 Introduction 75
4.2 Methods 78
4.2.1 Plasmids 78
4.2.2 Cell lines 78
4.2.3 Vector production 78
4.2.4 Vector transduction in vitro 79
4.2.5 Mouse bone marrow-derived dendritic cells culture and transduction 79
4.2.6 Mice and immunization in vivo 79
4.3 Results 80
4.3.1 Generation of a Tet-dependent SVGmu Cell Line 80
4.3.2 Construction of DC-LV Producer Cells by Concatemer Array
Transfection 82
4.3.3 Production of DC-LVs by Producer Cells in Serum-free Medium 84
4.3.4 Targeted Transduction of DC-LVs Produced by LV-MGFP in vitro 85
4.3.5 Production of LVs with Enhanced Efficiency to Target DCs via DMJ 87
4.3.6 Construction of Stable Producer Cells with Ubi Internal Promoter 89
viii
4.3.7 Effect of Replacing MSCV with Ubi Promoter 91
4.4 Discussion 93
References 97

ix
List of Figures
Figure 1.1 Structures of lipids (DOTMA) 3

Figure 1.2 Gene therapy vectors used in clinical trials 5

Figure 1.3 Schematic representation of our engineered lentiviruses enveloped
with a CD20-specific surface antibody and a fusion protein 14
Figure 2.1 Schematic representations of the strategies for targeting LVs to
HIV-1 Env. 18
Figure 2.2 Construction of a cell line stably co-expressing an HIV-1 Env and
LNGFR. 26
Figure 2.3 Co-expression of binding and FM on the surface of vector-
producing cells. 29
Figure 2.4 In vitro targeted transduction of HIV-1 Env expressing cells
by LVs. 31
Figure 2.5 Specific vector titers for various engineered LVs. 32
Figure 2.6 Engineered LVs transducing cells expressing different tropisms of
HIV-1. 34
Figure 2.7 Engineered LVs are capable of binding to HIV-1 Env-expressing
cells. 36
Figure 2.8 Clathrin and dynamin-dependent entry of the engineered LV. 38
Figure 2.9 Targeted transduction to a T cell line and suicide gene delivered
by LVs. 40
Figure 3.1 Schematic of the lentivirus platform and key constructs for the
fusogen study. 56

Figure 3.2 Co-expression of antibody and fusogen molecules on virus producing
cell surfaces. 59

Figure 3.3 Co-display of antibody and fusogen molecule on the surface of
lentiviruses. 60

Figure 3.4 Transduction of FM-bearing lentiviruses in vitro. 63

x
Figure 3.5 Effect of the neutralizing agent on the transduction by viruses
bearing different FMs. 65

Figure 3.6 pH- and cholesterol-dependent study of fusion activity of various FMs. 67

Figure 3.7 Transduction inhibition of viruses bearing various FMs by dominant-
negative Rab5 and Rab7. 68
Figure 4.1 Establishment of producer cell lines with the tet-off-regulated system. 81

Figure 4.2 Construction of stable lines to produce DC-LVs. 83

Figure 4.3 Kinetics of DC-LV production using the LV-MGFP cell line at
two different culture conditions. 84

Figure 4.4 DC-LV production by stable producer cells after prolonged culture. 86

Figure 4.5 DC-LVs produced by the LV-MGFP stable clone can selectively
transduce DCs in vitro. 87

Figure 4.6 DMJ can enhance transduction efficiency in vitro and immune responses
in vivo. 89

Figure 4.7 Construction of producer cells for making DC-LVs bearing Ubi internal
promoter. 91

Figure 4.8 Comparison of titer and immune responses with internal promoters. 93
1
1 Chapter1. Introduction
1.1 Introduction of Gene Therapy
Broadly defined, the concept of gene therapy is introduction of therapeutically
gene material into a cell, tissue, or whole organ to either cure a disease or at least
improving the clinical status of a patient (Somia and Verma 2000; Verma and Weitzman
2005). Main goal to develop this technology is to treatment of both inherited and
acquired disease. The therapeutic strategy may be accomplished by, interfering with gene
function, recovering lost function, or introducing a new function in the target cells. The
idea of using gene therapy method to cure the diseases sounds straightforward,
promising, and allowing for the therapeutic interventions at the molecular level. A major
obstacle for the success of gene therapy is the development of capable gen delivery
systems that can efficient transfer therapeutic gene in a variety of tissues, without causing
any associated pathogenic effects (Al-Hendy and Salama 2009). Two different strategy
are now taken for gene therapy which include an in vivo delivery, transfer the function
gene into the cells within the patients body or an ex vivo delivery, involves the
manipulation of the patient’s cells outside of patients body then replanted back to patients
body. However, Both of these gene delivery strategies have their own disadvantages. For
ex vivo gene delivery method, it spends lots of time and money. For in vivo gene delivery
method involves more risks, as transfer gene into non-specific cells causing toxicity or
pathogenic side effects. Thus, to design a targeting vector is one of the most important
key points to perfect gene delivery.
2
Availability of an appropriate vector for gene transfer is the major challenge for
successful gene therapy. Currently popular methods of gene transfer vehicles include the
use of viral and non-viral vectors. Viral vectors achieve gene transfer directly by viral-
mediated infection. Non-viral gene transfer can be attained through chemical or physical
treatment of target cells.
1.2 Non-Viral Vector
Non-viral vectors are one of the most studied fields in the gene delivery system as
there are several advantages over the viral vectors. Compare with viral vector transfer
system, nonviral vectors can be produced in relatively large amounts and are likely to
present minimal toxic or immunological problems (Ledley 1994). Non-viral vectors are
generally cationic in nature for encapsulation of negatively charged DNA. However, the
disadvantage of the method is low efficiency of delivering the genes of interest into the
cells and the gene expression is only transient. The following section will discuss some
example for non-viral vectors used for gene therapy.
1.2.1 Purified DNA
Purified, histone-free DNA, indicated as naked DNA, injected into tissues or
systemic circulation is the simplest and safest physical/mechanical approach for gene
therapy. The skeletal muscle, liver, thyroid, heart muscle, urological organs, skin, and
tumors are injection sites for naked DNA (Nishikwaw and Huang 2001). However, the
method is restricted by its comparatively low efficiency (Munkongea et al. 2003). To
overcome this problem, approach have been made by using nuclear targeting motifs to
direct the pathway of the DNA plasmid across the nuclear membrane into the nucleus
3
(Mattaj and Englmeier 1998). Using the nuclear targeting signal conjugated DNA can
help to improve gene deliver efficiency. In addition, other efforts have been directed
toward physical manipulation to improve the efficiency of gene deliver that includes
microinjection, particle bombardment, and electroporation.
1.2.2 Cationic lipids and liposomes
Lipid formulations, particularly formulations with cationic lipids, have used as
important molecules for mediating gene transfer. The prototype cationic lipid for gene
delivery is assembled by 1,2-dioleyloxypropyl-3-trimethyl ammonium bromide
(DOTMA) (Felgner and Ringold 1989). Cationic lipids are amphiphilic molecules that
can congregate to form a lipid bilayer structure called liposomes. Once liposomes are
formed, DNA can be encapsulated by liposome known as lipoplex (Rädler et al. 1997).
Cationic lipids have also been shown to be effective agents for gene transfer in vivo
(Canonico et al. 1994), but the stability of lipoplex is quite poor and needs to be applied
right after their formation.
Figure 1.1 Structures of lipids (DOTMA)

4
1.2.3 Cationic polymers
The other material used for non-viral gene delivery is a cationic polymer known
as a dendrimer (Haensler and Jr. 1993). Polymers-DNA complexes are formed by ionic
interactions between the positively charged cationic polmers and negatively charged
DNA. Modifications of complexes can be made to conduct different size, different charge
ratios, and even attaching a ligand to the complex for targeting purpose (Fischer et al.
1999). The most widely used cationic polymers are poly L-lysine (PLL) and poly
ethyleimine (PEI). PLL polymers are linear polypeptides with a biodegradable property
that makes it very useful gene deliver vehicle in vivo (Wu and Wu 1986). Another
popular cationic, PEI is capable to condense DNA and destabilize the lysosomal
membrane to release the genetic material into the target cells (Wightman et al. 2001).
However, the toxicity issue of both polymers still bothers the using of cationic polymers
and more researchs need to be done for the cationic polymers.
1.2.4 Virosome
For non-viral gene therapy, virosome also been demonstrated as a deliver vehicle.
The virosomal carrier systems are specific liposomes that contain viral envelope
glycoprotein, such as the Sendai virus hemagglutinin, anchored in the in the lipid
membrane and lack of the viral genetic material (Kato et al. 1991). The virosome are
designed to enter cells by hemagglutinin-mediated fusion of the liposomal bilayer with
the membrane of the target cell. Virosomes containing positively charged lipids form
stable complexes with DNA and enhance the uptake and expression of the encoded
genes. However, the virosome inherent complexity of liposomal assembles formulations,
and difficulty to produce the virosomal carrier vehicles.
5
1.3 Viral Vector
Currently, viral gene delivery still remains one of the most popular ways for gene
therapy clinical trials, having been used in about two-thirds of the trials performed to date
(Edelstein et al. 2007).

Figure 1.2 Gene therapy vectors used in clinical trials

The basic concept of viral vectors is to control the innate ability of viruses to
deliver genetic material into the infected cell (Verma and Weitzman 2005). The high
gene transfer gene efficiency and high level of transgene expression have made viral
vector the most suitable vehicles for gene delivery tool. In general, the major purpose of
viruses is to replicate and produce copious amounts for progeny. Some of the viruses will
kill the host, but most viral infections lead to deleterious effects on the host, accompanied
by destruction of infected host cells. Damaging effects can be caused by induction of
viral genes products are hazardous to the host cells or by alter host genomic material that
can lead to viral replication. The first step to turn these pathogens into gene deliver
systems is to separate the components needed for replication from those capable of
6
causing disease. Viral necessary packaging component can be supplied in trans, which
means the necessary viral protein are only present during the production of the viral
vectors and none of the those toxicity packaging genes are packaged into the viral vector.
The most of the viral vector design is, therefore, to divide the viral sequences into
required for replication, assembly of viral particles, packaging of the viral genome, and
delivery of the transgene into the target cells. Usually, the viral vectors are separated into
two categories, integrating and non-integrating viral vectors. For integrating viral vectors,
the transgene into the host cells genome to holding a life long gene expression. For non-
integrating viral vectors, them only transiently express the foreigner gene within the
infected cells. Here, I will discuss the most commonly used viral vectors today.
1.3.1 Retroviral vector
Viral vectors based on retroviruses were one of the first viral gene deliver system
to be designed, and they have been show an important technology and concept to
development of viral vectors (Mulligan 1993; Verma 1990). Retroviral vectors have been
derived from different kind of oncoretroviruses, including murine leukemia virus (MLV),
spleen necrosis virus, Rous sarcoma virus, and avian leukosis virus (Verma and
Weitzman 2005). This enveloped virus particle contains two copies of the viral RNA
genome, surrounded by a cone-shaped core. These viral vectors have around 7-7.5 kb
transgene capacity. The viral vecotrs composed of three essential genes, gag encodes the
core proteins capsid, matrix, and nucleocapsid, which are generated by cleavage of the
gag precursor protein, pol that encodes the viral enzymes protease, reverse transcriptase,
and integrase, and env encodes the viral envelope glycoprotein, which mediate virus entry.
All these essential genes can be supplied in trans to reduce the possibility of generating
7
RCV (replication component viruses). People also deleted these vectors LTR U3 resion
to prevent the viral promoter driven transcription in the transduced cells, and transgene
expression is induced by an internal promoter, allowing the use of regulated and tissue
specific promoters (Yu et al. 1986). In order to broaden the viral vectors infection tropism,
the envelopes of retroviruses were replaced with other viral envelope protein (Danos and
Mulligan 1988). The two major disadvantages of retroviral vectors are inability to infect
non-dividing cells, and the viral vectors easily to be silenced by the host cells due to the
site of integration. Despite of its advantages, retroviruses still remained one of the most
promising viral vectors used today due to its simplicity for engineering and its ability to
transduce dividing cells such as tumor cells.
1.3.2 Lentiviral Vector
Currently used lentiviral vectors are derived from human immunodeficiency
viruse type-1. It belongs to the retrovirus family with a some uniques properties to make
it more suitable for clinical studies and come out some excited successful results (Cartier
et al. 2009). Compare with retroviruses, lentiviruses encode three to six more additional
viral proteins, which correlated to virus replication and successful infection (Verma and
Weitzman 2005). All of lentiviruses contain two accessory proteins, tat and rev, induced
transactivation of viral transcription and mediated nuclear export of unspliced viral RNA,
respectively. The exact mechanism is still unclear, however, it is known that the
accessory proteins associates with the lentivruses can help to transport across the nucleus.
Unlike retroviruses, these differences help lentiviruses capable of infecting both dividing
and non-dividing cells (Burkeinsky et al. 1993). The lentiviruses can integrate into the
different sites from retroviruses, make it harder to be silenced by host cells, can have
8
more longer transgene expression. The lentiviral vectors derived from HIV-1 gene is lack
of all viral sequences exclude some essential cis-acting sequences, including the viral
3’LTR, 5’LTR and the packaging signal. The rev responsive element (RRE) is also
encoded in the lentiviral vector RNA. The viral rev protein is supplied in trans to help
export of the full-length viral RNA genomes efficiently from nuclear through binding to
the RRE. The vector RNA expression is driven by the viral promoter LTR and triggered
transactivation by the tat protein (Zufferey et al. 1997). The third generations of the
lentiviral vector changed the viral promoter LTR to CMV/LTR hybrid promoter, which
increases vector production and allows vector production to be lack of tat expression
(Follenzi et al. 2000). Furthermore, the biosafety of vectors also be improved by
development of self-inactivating vector with a mutation in the 3’LTR to prevent the viral
RNA being synthesized after integrating into the target host (Miyoshi et al. 1998b).
Another safety mechanism is to separate the essential proteins used, including rev, gag,
pol and env into different constructs and to provide in trans to eliminate the possibility of
assemble the replication competent viruses.
In addition, some more components are added into the lentiviral vectors to
enhance the transduction and expression of viral vector which includes the central
polypurine tract (cPPT), which allows internal initiation of second strand DNA synthesis
and the transport of the pre-integration complex into the nucleus and the woodchuck
post-transcriptional regulatory element (WRE) which improves the translational
efficiency of lentiviral vectors. Due to these properties, lentiviral vector is among one of
the most promising viral vectors in the field of gene therapy.
9
1.3.3 Adenoviral vectors
The human Adenovirus family contains more than 50 serotypes that can infect
and replicate in a wide range of organs, such as the respiratory tract, the eye, urinary
bladder, gastrointestinal tract, and liver. The viral genome consists of a double-stranded
linear DNA molecule (around 36 kb) with overlapping transcription units on both strands.
The Adenovirus is a nonenveloped virus packaged with icosahedral capsid protein. The
fiber protein projects from the viral surface, and the carboxy-terminal knob domain forms
a high-affinity complex with a host cell receptor, coxsackie-adenovirus receptor (CAR)
(Bergelson et al. 1997). However, unlike lentiviruses, and retroviruses, adenoviruses
don’t integrate into the host cells genome, but remains episommal forms in the cells
nucleus. Today, most of the adenoviruses used in the gene therapy are derived from
replication incompetent viruses keep E1A, E1B, E3 gene, and delete E4 and E2A gene
from the viral genome (Yew and Perricaudet 1997). However, one of major concerns of
adenoviruses is generated severe immunogenicity while tranduce with this virus. Thus,
researchers have developed gutless viral vectors, in which viral genes were removed and
supplied in trans to be used in clinical trial setting (Morral et al. 1999). Nevertheless, the
adenoviruses still remain one of the most useful viral vectors for gene therapy especially
when viral vectors can be prepared in high titer and the transient expression of the
transgene might be useful in certain clinical setting, although the viral vector production
is very laborious and the viral particles can still stimulate a severe immune response
(Kochanek et al. 2001).
10
1.3.4 Adeno-Associated Virus
Adeno-associated virus (AAV) is a small, non- pathogenic, single stranded DNA
virus that has become one of efficient and useful deliver vector in gene therapy (Somia
and Verma 2000). Successful AAV infection and replication requires helpers that can be
provided by co-infection with helper viruses, such as Adenovirus and herpesvirus. AAV
can also replicate in cells be treated with irradiation or genotoxic agents. In the absence
of a supported environment for AAV replication, the virus gene has ability to integrate in
a site-specific location on chromosome 19 with the help of rep proteins, however, the
efficiency is not high and still up for debate (Kotin et al. 1990). AAV has two
fundamental genes, Rep contain the gene for viral replication and integration and cap
contain the gene encodes the viral structural components. The viral genome is flanked by
two inverted terminal repeats (ITRs) that contains the DNA sequences needed to induce
the viral replication and packaging of the viral genome. The viral vector backbone is
constructed by removing the rep and cap genes and input the transgene of interest. The
viral rep and cap are supplied in trans for safety concern. As gene deliver system, there
are two major problems of using AAV are that the rep gene is toxic to the producing cells
which make it harder to construct a stable packaging cell lines. The other problem of the
AAV is that the relatively small packaging capacity (~4.5kb). In order to solve this
limitation, AAV extended the encoding capacity by utilizing the concatomerization
nature of AAV. The transgene can divide into two parts and carried by two individual
Adeno-Associated Viral Vectors (Yan et al. 2000). Transgene is obtained after
recombination between the two viral genomes, but the efficiency is often reduced as
compared to single vector transduction. Nevertheless, AAV has earned more attention by
11
the recent success in clinical trial and become one of the most promising new viral
vectors to be utilized.

Table 1 Commonly used viral vectors for gene therapy (Lu and Madu 2010).
1.4 Project Overview
Until 2007, more than one thousands of gene therapy clinical trials have been
completed, are ongoing or have been approved worldwide (Edelstein et al. 2007). To
achieve the successful therapeutically purpose, the efficient gene deliver system is
important. Over the last 15 years, people have tried to engineer viral vectors to delivery
therapeutically genes to the specific cells. However, engineering viral glycoproteins to
redirect the tropism has proven to be difficult. In our previous study, we separated viral
glycoproteins two important functions, binding and fusion, into two different proteins.
The targeting engineered lentiviral vector using specific antibody interact with cell
surface antigen then followed by an endocytosis of the viruses. Once entering into cells
endosome compartment with the drop in pH, our fusogenic molecule derived from
12
sindbis virus glycoprotein would then change its conformation and fuse with the
endosmal membrane to release the viral capsid (Yang et al. 2006b). Base on this strategy
(Fig 1.3), we construct the targeting viral vector can specific interact with cells of interest.
Further to improve the viral vector transduction efficiency, we engineered the fusogen
molecule to enhance fusion ability and determined what is key factor to change the fusion
proteins property.
Since HIV-1 Infected cells will expressed the viral glycoproteins (gp120-gp41
complexes) on the host cells surface. We used human CD4 T cells receptor as our
targeting receptor, incorporated into our engineered lentiviral vectors, make it can
specific transduce the HIV-1 Infected cells. We demonstrated that lentiviral vectors co-
incorporation of the CD4 and fusogenic proteins can specific deliver suicide gene therapy
to HIV-1 envelope-expressing cells. Based on this method, more than 30% HIV Envelope
positive T cell lines been killed in vitro. Similar result was observed in peripheral blood
mononuclear cell (PBMC), which indicated that the system could be further, extended to
primary cells.
In Chapter 3, we have engineered fusogen molecules to pair with our targeting
antibody for enhance of our viral vectors gene deliver efficiency. We hypothesized that
by engineering in the E1 domain of the Sindbis glycoprotein, we could improve the
transduction efficiency of our viral vector. Mutations in the E1 domain of Sindbis virus
glycoprotein at residues 75 and 237 individually or in combination (G75D, S237A,
G75DS237A) can affect the viral can affect lentiviral vectors transduction efficiency
dramatically. After close investigation, we have demonstrated that the G75D mutation
enhances the transduction efficiency by broadening the range of the pH threshold for
13
fusion. Conversely, the S237A mutation impairs the infectivity and its infectivity can be
partially restored by utilizing G75D as second site revertant.
In chapter 4, we have looked forward to analyzed our lentiviral gene deliver
vectors pseudotyped with the fusion molecular derived from sindbis virus glycoprotein
can specific target the Dendritic Cells (DC) and use as vaccine in vivo. In order to solve
the viral vector production problem by transient transfection, we utilized concatemeric
array-based transfection approach as a possible method of constructing stable producer
lines for making DC-directed LVs (DC- LVs). We show that this method can generate
stable lines that produce DC-LVs with high titers (>10
7
transduction units (TU)/mL)
during a continuous 3-month culture. The lentiviral vector produced by the stable line
system could induce a stronger T cell response in vivo. Thus, our results demonstrate that
this method of constructing stable cell lines is a robust and reproducible means for
routinely making DC-LVs with sufficient scalability for vaccine applications.

14

Figure 1.3 Schematic representation of our engineered lentiviruses enveloped with a
CD20-specific surface antibody (αCD20) and a fusion protein (SVG)

αCD20

CD20

Target Cells

15
Chapter 2. Engineered lentiviral vectors pseudotyped with a CD4 receptor and a
fusogenic protein can target cells expressing HIV-1 envelope proteins
Portions of this chapter are adapted from: Chi-Lin Lee, Jason Dang, Kye-il Joo, and Pin
Wang. Virus Research 160 (2011) p.p340–350
Lentiviral vectors (LVs) derived from human immunodeficiency virus type 1
(HIV-1) are promising vehicles for gene delivery because they not only efficiently
transduce both dividing and non-dividing cells, but also maintain long-term transgene
expression. Development of an LV system capable of transducing cells in a cell type-
specific manner can be beneficial for certain applications that rely on targeted gene
delivery. Previously it was shown that an inverse fusion strategy that incorporated an
HIV-1 receptor (CD4) and its co-receptor (CXCR4 or CCR5) onto vector surfaces could
confer to LVs the ability to selectively deliver genes to HIV-1 envelope expressing cells.
To build upon this work, we aim to improve its relatively low transduction efficiency and
circumvent its inability to target multiple tropisms of HIV-1 by a single vector. We
investigated a method to create LVs co-enveloped with the HIV-1 cellular receptor CD4
and a fusogenic protein derived from the sindbis virus glycoprotein and tested its
efficiency to selectively deliver genes into cells expressing HIV-1 envelope proteins. The
engineered LV system yields a higher level of transduction efficiency and a broader
tropism towards cells displaying the HIV-1 envelope protein (Env) than the previously
developed system. Furthermore, we demonstrated in vitro that this engineered LV can
preferentially deliver suicide gene therapy to HIV-1 envelope-expressing cells. We
conclude that it is potentially feasible to target LVs towards HIV-1-infected cells by
functional co-incorporation of the CD4 and fusogenic proteins, and provide preliminary
16
evidence for further investigation on a potential alternative treatment for eradicating
HIV-1-infected cells that produce drug-resistant viruses after highly active antiretroviral
therapy (HAART).
2.1 Introduction
An important aspect of gene therapy is the delivery of genetic materials to target
cells for therapeutic benefit. One of the most important and efficient methods for gene
delivery is the use of viral vectors as transfer vehicles (Verma and Somia 1997; Verma
and Weitzman 2005). Viral vectors are separated into two major groups—integrating
viral vectors and non-integrating viral vectors. Integrating viral vectors are based on
retroviruses, lentiviruses, and adeno-associated viruses while non-integrating viral
vectors include adenoviruses (Somia and Verma 2000). Of these viruses, HIV-based
lentiviral vectors (LVs) are promising as a gene delivery system for certain applications
because of their ability to induce stable transduction, maintain long-term transgene
expression and transduce nondividing cells (Kohn 2007; Naldini et al. 1996; Verma and
Weitzman 2005). Engineering LVs capable of delivering genes of interest to
predetermined cells can reduce off-target effects and improve the safety profile, which
will further enhance the promise of this vector system for gene therapy applications
(Cronin et al. 2005; Waehler et al. 2007).
The entry of HIV-1 into host cells is mediated by interactions between the viral
envelope glycoprotein (Env) and the receptor complex of target cells, which includes the
CD4 receptor and the co-receptor (CCR5 or CXCR4) of the chemokine receptor family
(Moore 1997). The virus initially gains access to the host cells by interactions between
CD4 and the surface unit (gp120) of Env, which triggers conformational changes in
17
gp120 that allows for further binding with co-receptors. Subsequently the membrane unit
(gp41) of Env evokes fusion to release the viral capsid into the cytoplasm of the host cells
(Dimitrov 1997). Based on this membrane fusion mechanism, the inverse fusion strategy
has been developed to target HIV-1-infected cells (Fig. 2.1A, left panel). This strategy
involves the incorporation of the CD4/co-receptor complex into the viral vector
membrane for its specific entry into HIV-1 Env-expressing cells. Several reports have
described their successes of utilizing this inverse fusion method to generate such
targeting vectors (Bittner et al. 2002; Endres et al. 1997; Mebatsion et al. 1997; Peretti et
al. 2006; Schnell et al. 1997; Somia and Verma 2000; Ye et al. 2005).
Previously we showed that recombinant LVs can be engineered by a two-
molecule method to possess a targeting ability for gene delivery to desired cells in vitro
and in vivo (Yang et al. 2006b). To accomplish this, the vector’s binding and fusion
functions are separated into two proteins. Antibodies or ligands are incorporated onto the
vector surface to mediate binding, while a mutant Sindbis viral glycoprotein is co-
displayed on the vector surface to execute its fusion activity. We further incorporated
several different fusogenic molecules (FMs) that were engineered based on Kielian and
co-workers’ study (Lu et al. 1999) and showed that these FMs could significantly
improve the transduction efficiency of targeting vectors (Yang et al. 2008a). An entry
study revealed that the engineered vector particles can be internalized through clathrin-
dependent endocytosis upon binding to target cells and further transported into the
endosomal compartment, where the FMs on the vector surface sense the low pH and
undergo a conformation change to trigger fusion, releasing the viral core into the cytosol
(Joo and Wang 2008).
18

Figure 2.1 Schematic representations of the strategies for targeting LVs to HIV-1 Env-
expressing cells and key constructs for making the recombinant LVs in this study. (A)
Schematics for the inverse fusion method (left), which utilizes CD4 and a co-receptor
(CCR5 or CXCR4) complex, and our method of displaying CD4 and a fusogenic
molecule (FM) on the vector surface (right), to achieve LV targeting. (B) Key constructs
for making LVs. These constructs include the lentiviral backbone plasmid FUGW,
binding molecule CD4, and fusion protein FM. CMV: enhancer/promoter derived from
human cytomegalovirus; Ubi: the human ubiquitin-C promoter; GFP: green fluorescence
protein; WRE: woodchuck responsive element; E3: the leading peptide of the Sindbis
virus glycoprotein; Several alterations, including the deletion of amino acids 61–64 of
wild-type E3, mutations of amino acids 157 and 158 of wild-type E2, and an HA tag
insertion into E2, were introduced to yield a binding-deficient and fusion-competent FM
AKN. Additional mutations in the fusion loop region of E1 (amino acids 226 and 228)
were conducted to generate two FMs, AGM and SGN.
C.-L. Lee et al. / Virus Research 160 (2011) 340–350 341
Fig. 1. Schematic representations of the strategies for targeting LVs to HIV-1 Env-expressing cells and key constructs for making the recombinant LVs in this study. (A)
Schematicsfortheinversefusionmethod(left),whichutilizesCD4andaco-receptor(CCR5orCXCR4)complex,andourmethodofdisplayingCD4andafusogenicmolecule
(FM)onthevectorsurface(right),toachieveLVtargeting.(B)KeyconstructsformakingLVs.TheseconstructsincludethelentiviralbackboneplasmidFUGW,bindingmolecule
CD4,andfusionproteinFM.CMV:enhancer/promoterderivedfromhumancytomegalovirus;Ubi:thehumanubiquitin-Cpromoter;GFP:greenfluorescenceprotein;WRE:
woodchuckresponsiveelement;E3:theleadingpeptideoftheSindbisvirusglycoprotein;E2:theE2proteinoftheSindbisvirusglycoproteinthatbindstocellularreceptors;
E1: the E1 protein of the Sindbis virus glycoprotein that mediates fusion. Several alterations, including the deletion of amino acids 61–64 of wild-type E3, mutations of
157KE158into157AA158ofwild-typeE2,andanHAtag(MYPYDVPDYA)insertionintoE2betweenaminoacids71and74,wereintroducedtoyieldabinding-deficientand
fusion-competent FM AKN (previously denoted SINmu;Yang et al., 2006). Additional mutations in the fusion loop region of E1 (amino acids 226 and 228) were conducted
togeneratetwoFMs,AGMandSGN.
inverse fusion method to generate such targeting vectors (Bittner
etal.,2002;Endresetal.,1997;Mebatsionetal.,1997;Perettietal.,
2006;Schnelletal.,1997;Somiaetal.,2000;Yeetal.,2005).
PreviouslyweshowedthatrecombinantLVscanbeengineered
by a two-molecule method to possess a targeting ability for gene
delivery to desired cells in vitro and in vivo (Yang et al., 2006). To
accomplishthis,thevector’sbindingandfusionfunctionsaresep-
arated into two proteins. Antibodies or ligands are incorporated
ontothevectorsurfacetomediatebinding,whileamutantSindbis
viralglycoproteinisco-displayedonthevectorsurfacetoexecute
its fusion activity. We further incorporated several different fuso-
genic molecules (FMs) that were engineered based on Kielian and
co-workers’study(Luetal.,1999)andshowedthattheseFMscould
significantly improve the transduction efficiency of targeting vec-
tors(Yangetal.,2008).Anentrystudyrevealedthattheengineered
vector particles can be internalized through clathrin-dependent
endocytosis upon binding to target cells and further transported
into the endosomal compartment, where the FMs on the vector
surface sense the low pH and undergo a conformation change to
trigger fusion, releasing the viral core into the cytosol (Joo and
Wang,2008).
In this study, we investigated the application of this two-
molecule method for generating LVs capable of specifically
transducingHIV-1Env-expressingcells.WedemonstratedthatLVs
displaying the HIV-1 primary receptor CD4 and the FM derived
from the mutant Sindbis virus glycoprotein can achieve selective
gene delivery to cells expressing HIV-1 Env in vitro with remark-
ablespecificityandefficiency.SuchanHIV-1Env-specificLVsystem
was shown to be able to deliver a suicide gene into a human T
celllinethatexpressesHIV-1Envandinducethespecifickillingof
envelope-expressingcellscultivatedwithaprodrug.
2. Materialsandmethods
2.1. Plasmids
The FM molecules derived from the Sindbis virus glycopro-
tein, AKN, AGM and SGN have previously been reported by our
19
In this study, we investigated the application of this two-molecule method for
generating LVs capable of specifically transducing HIV-1 Env-expressing cells. We
demonstrated that LVs displaying the HIV-1 primary receptor CD4 and the FM derived
from the mutant Sindbis virus glycoprotein can achieve selective gene delivery to cells
expressing HIV-1 Env in vitro with remarkable specificity and efficiency. Such an HIV-1
Env-specific LV system was shown to be able to deliver a suicide gene into a human T
cell line that expresses HIV-1 Env and induce the specific killing of envelope-expressing
cells cultivated with a prodrug.
2.2 Methods
2.2.1 Plasmids
The FM molecules derived from the Sindbis virus glycoprotein, AKN, AGM and
SGN have previously been reported by our laboratory (Yang et al. 2008a; Yang et al.
2006b). Human CD4, CCR5, and CXCR4 cDNAs were cloned downstream of the CMV
promoter in the pCDNA3 plasmid (Invitrogen, Carlsbad, CA) to create pCD4, pCCR5,
and pCXCR4. The mouse stem cell virus-based retroviral transfer plasmid MIG (Yang
and Baltimore 2005b) was kindly provided by Dr. David Baltimore’s laboratory. The
cDNA for the surface marker, human low-affinity nerve growth factor receptor
(LNGFR), was extracted from the pMACS-LNGFR-IRES vector (Miltenyi Biotec,
Bergisch Gladbach, Germany) using the NcoI and SalI sites and cloned into the MIG
plasmid in place of the GFP gene. The resulting plasmid was referred to as MINFR. The
cDNA of the CCR5-tropic HIV-1 Subtype C envelope glycoprotein was isolated from the
plasmid pcDNA3-gp160C (Gao et al. 2003) (NIH AIDS Research and Reference Reagent
20
Program, Germantown, MD, USA), and inserted into MINFR at a site upstream of IRES.
The resulting plasmid was designated as MINFR-gp160R5. The cDNA of the CXCR4-
tropic HIV-1 subtype B envelope glycoprotein was derived from pcDNA3-gp160HxBc2,
generously provided by Dr. Pamela Bjorkman’s laboratory at the California Institute of
Technology, and cloned into the MINFR upstream of IRES. This plasmid was referred to
as MINFR-gp160X4. The HIV-1-based lentiviral vector FUGW was reported by Dr.
David Baltimore’s Laboratory (Lois et al. 2002) and used in this study. The suicide gene,
the mutant of Herpes Simplex Virus-1 thymidine kinase SR39Tk, was amplified from a
reported construct (Black et al. 2001) and cloned downstream of the human ubiquitin-C
promoter in the lentiviral vector plasmid FUW (Ziegler et al. 2008). The construct was
referred to as FUWSR39TK.
The wild-type Rab5 and Rab7 cDNAs were PCR-amplified and cloned into the
pDsRed-monomer-C1 (Clontech, Mountain View, CA, USA) to form the DsRed-
Rab5WT and DsRed-Rab7WT constructs. The plasmid encoding the dominant-negative
mutant of DsRed-Rab7DN (Rab7T22N) was created by site-directed mutagenesis using
the forward primer (5′-GTC GGG AAG AAC TCA CTC ATG AAC C-3′) and the
backward primer (5′-GGT TCA TGA GTG AGT TCT TCC CGA C-3′). The construct
for the dominant-negative mutant of DsRed-Rab5DN was obtained from Addgene
(Cambridge, MA, USA).
The cDNAs for wild-type dynamin 2 and the dominant-negative dynamin 2 K44A
mutant were derived from the pEGFP-Dyn2 and pEGFP-Dyn2 K44A vectors,
respectively, which were kindly provided by Dr. Okamoto’s laboratory, using the HindIII
and EcoRI restriction sites and cloned into the pDsRed-monomer-C1 construct
21
(Clontech). The resulting plasmids are designated as DsRed-Dyn2WT and DsRed-
Dyn2K44A.
2.2.2 Cell lines
The 293T cells (human kidney embryonic cells with the Simian Virus 40 large T
antigen) and Jurkat cells were obtained from the American Type Culture Collection.
293T, 293T.EnvR5 and 293T.EnvX4 cells were cultured in Dulbecco’s modified Eagle’s
medium (Mediatech Inc., Manassas, VA, USA) with 10% fetal bovine serum (Sigma–
Aldrich, St. Louis, MO, USA) and 2 mm l-glutamine. The human CD4 and CCR5
expressing cell line, TZM-bl, was provided by the NIH AIDS Research & Reference
Reagent Program. The TZM-b1 cell line was cultured in the same medium described
above with the addition of 50µg/mL gentamycin (Sigma). All of the cells were
maintained in a 5% CO2, 37
◦
C environment.
2.2.3 Cell–cell fusion assay
TZM-b1 (0.2 × 10
6
) or HeLa (0.2 × 10
6
) cells were labeled with 20 µM of
octadecyl rhodamnine B chloride, R18 (Molecular Probes, Carlsbad, CA, USA), in
serum-free medium for 30 min at 37 ◦ C and washed with PBS three times. The cells
were then seeded onto glass-bottom culture dishes (MatTek Corporation, Ashland, MA,
USA) and grown at 37
◦
C overnight. 293T.EnvR5 cells (0.2 × 10
6
) labeled with 10µM of
5,6-carboxy-fluorescein diacetate succinimidyl ester, CFSE (Molecular Probes), were
overlaid on TZM-b1 or HeLa cells for 10 min at 37
◦
C. Fluorescent images were acquired
on a Yokogawa spinning-disk confocal scanner system (Solamere Technology Group,
Salt Lake City, UT) using a Nikon eclipse Ti-E microscope equipped with a 60×/1.49
22
Apo TIRF oil objective and a Cascade II: 512 EMCCD camera (Photometrics, Tucson,
AZ, USA). An AOTF (acousto-optical tunable filter) controlled laser-merge system
(Solamere Technology Group Inc., Salt Lake City, UT, USA) was used to provide
illumination power at each of the following laser lines: 491 nm, 561 nm, and 640 nm
solid-state lasers (50 mW for each laser).
2.2.4 Vector production
The 293T cells were seeded in a 6-cm culture dish. After 18–20 h, the seeded
cells were transfected with DNA plasmids using the standard calcium phosphate
precipitation technique when confluency reached approximately 80%. 5µg of the
lentiviral vector plasmid FUGW, 2.5 µg each of the packaging plasmid pMGL and pRev
(Tiscornia et al. 2006), 2.5 µg of the surface-expressing plasmid pCD4 and
pCCR5/pCXCR4, and 2.5µg of the plasmid pFM encoding AKN, AGM, or SGN, were
mixed together with calcium chloride and added to 2×HBS buffer. The vector supernatant
was harvested 48h after transfection and filtered though a 0.45-µm pore size filter.
2.2.5 Vector transduction in vitro
Target cells (293T, 293T.EnvR5, Jurkat, and Jurkat infected with MINFR-
gp160R5; 0.2 × 10
6
) were plated in 24-well culture dishes with vector supernatant (2 mL
per well) and spin-transduced for 90 min at 2500 rpm and 25 ◦ C. After transduction, the
vector supernatant was replaced with fresh media. The transduced cells were cultured for
another 3–5 days at 37
◦
C and 5% CO
2
. The transduction results were determined by flow
cytometry analysis of GFP-positive cells. Vector titer was determined by counting GFP
23
expression in the vector dilution range in which GFP-positive cells and vector volume
exhibit a linear relationship.
For transduction of drug-treated cells, 293T.EnvR5 cells were preincubated with
drugs (chlorpromazine: 10 µg/mL; genistein: 50 µg/mL; nystatin: 25 µg/mL) for 30 min
at 37
◦
C and then spin-transduced with 2 mL vector supernatant for 90 min at 2500 rpm
and 25
◦
C. The drug-containing media were then replaced with flash D10 media 3 h after
transduction. For vector transduction with dynamin or Rab protein-treated cells,
293T.EnvR5 cells were transfected with DsRed-Dyn2, DsRed-5, or DsRed-7 (either
wild-type or dominant-negative mutant), seeded, and spin-transduced with 2 mL vector
supernatant. GFP-positive cells were analyzed by flow cytometry three days post-
transduction.
2.2.6 Cell–vector binding
293T or 293T.EnvR5 cells (0.2 × 10
6
) were incubated with 1.5 mL of the LV
supernatant at 4
◦
C for 5 min. The cell–vector complexes were washed with cold PBS and
fixed by 4% formaldehyde on ice for 10 min. To probe the cell–vector complexes, an
anti-HA antibody (Miltenyi Biotec) was used to stain the HA-tag expressed on the FM,
followed by Alexa594-conjugated streptavidin (Zymed Laboratories, South San
Francisco, CA, USA), and analyzed by flow cytometry (FACSort, BD Bioscience, San
Jose, CA, USA).
2.2.7 Soluble CD4 competition and NH
4
Cl neutralizing
293T.EnvR4 cells (0.2 × 10
6
) were co-incubated with the vector supernatant and
various quantities of soluble CD4 or ammonium chloride (NH
4
Cl) in a 24-well culture
24
dish at 37
◦
C and 5% CO
2
for 8 h. The media was then replaced with fresh media. After
incubation for an additional 4 days, the GFP+ cells were analyzed by flow cytometry.
2.2.8 Targeted delivery of suicide gene therapy in vitro
Jurkat cells were transduced by a retroviral vector, MINFR- gp160R5,
pseudotyped with VSVG to express HIV-1 Env proteins. Three days later, the transduced
cells were incubated with the concentrated LVs encoding SR39Tk
(FUWSR39tk/CD4+SGN) for transduction by the method previously described.
Afterwards, the cells were cultured in media with or without prodrug ganciclovir (GCV;
Sigma–Aldrich, St. Louis, MO, USA) for an additional seven days. The treated cells were
stained with an Alexa594-conjugated CD271 (LNGFR) antibody (Miltenyi Biotec) and
analyzed by flow cytometry.
2.3 Results
2.3.1 Generation of the HIV-1 envelope protein-expressing cell line
We have previously reported that lentiviral vectors (LVs) pseudotyped with an
antibody and a FM can transduce cells in a cell type-specific manner (Yang et al. 2006b).
The FM was derived from the altered Sindbis virus glycoprotein with mutations in the E2
binding domain. Because this method separates the two key functions of viral
glycoproteins (binding and fusion) into two distinctive proteins, we can theoretically
generate a new targeting vector by simply changing the binding protein on the vector
surface. Therefore, we hypothesized that co-incorporation of a human CD4 protein and
an engineered FM could confer upon an LV with the capability to specifically deliver
genes into cells infected with HIV-1 (Fig. 2.1A, right panel).
25
To test this hypothesis, we created a cell line expressing HIV- 1 Env. A mouse
stem cell virus-based retroviral transfer plasmid was constructed to encode an HIV-1
clade C and CCR5-tropic Env gene (gp160R5, from the 96ZM651 strain) and a surface
marker (LNGFR: truncated low affinity nerve growth factor receptor gene); the construct
is designated as MINFR-gp160R5 (Fig. 2.2A). An internal ribosome entry site (IRES)
was used to link gp160R5 and LNGFR to achieve co-expression that was driven by the
5′LTR promoter (Yang and Baltimore, 2005). We have tested several commercially
available antibodies and could not identify one that could be reliably used for flow
cytometry analysis of Env expression. Thus, this design could allow us to readily
quantify the expression of HIV- 1 Env by surface staining of the marker LNGFR. To
generate the HIV-1 Env-expressing cell line, 293T cells were transduced with the
MINFR-gp160R5 vector pseudotyped with the vesicular stomatitis virus glycoprotein
(VSVG).
After a few weeks of cell passaging, individual single cell clones were selected,
stained with an anti-LNGFR antibody, and analyzed by flow cytometry. An LNGFR-
positive clone (Fig. 2.2B) was subsequently tested by a cell–cell fusion assay. The fusion
events between cells expressing HIV-1 Env and cells expressing CD4 and co-receptor
can be monitored by the redistribution of dye molecules; this assay has been widely used
in the observation of cell and viral membrane fusion (Blumenthal et al., 2002). We
labeled HeLa or TZM-b1 (a HeLa cell line expressing CD4 and CCR5) cells with
fluorescent membrane dye, octadecyl rhodamnine B chloride (R-18), and overlaid them
with the selected clonal cells labeled with an intracellular dye, 5,6-carboxy-fluorescein
diacetate succinimidyl ester (CFSE). Redistribution of the red florescence dye (R-18)
26
only from labeled TZM-b1 cells, but not from HeLa cells, to the target cells confirmed
the functional expression of HIV-1 Env on the selected clonal cells (Fig. 2.2C). This
clonal cell line was designated as 293T.EnvR5 and was used throughout this study.

Figure 2.2 Construction of a cell line stably co-expressing an HIV-1 Env and a surface
marker LNGFR. (A) Schematic representation of a retroviral backbone plasmid, MINFR-
Env, that encodes an HIV-1 Env gene. MINFR-gp160R5 and MINFR-gp160X4 are
designated for this construct encoding a CCR5- or CXCR4-tropic Env gene, respectively.
The biocistronic configuration of this construct with an IRES linker can allow for co-
expression of Env and a surface staining marker LNGFR. MSCV: murine stem cell virus;
LTR: long terminal repeat; Env: HIV-1 envelope protein; IRES: internal ribosome entry
site; LNGFR: human low-affinity nerve growth factor receptor. (B and C) The stable cell
line 293T.EnvR5 was constructed by transducing parent 293T cells with MINFG-
gp160R5 pseudotyped with VSVG and passaging the cells for a few weeks. (B) Flow
cytometry analysis of LNGFR expression in a selected single cell clone. (C) TZM-b1 and
HeLa cells were labeled with R18, and seeded onto glass-bottom culture dishes.
293T.EnvR5 cells were labeled by 5,6-carboxy-fluorescein diacetate succinimidyl ester,
CFSE, and co-cultured with TZM-b1 or HeLa cells. Redistribution of the R18
fluorescence dye was imaged by a laser scanning confocal microscope.

C.-L. Lee et al. / Virus Research 160 (2011) 340–350 343
Fig. 2. Construction of a cell line stably co-expressing an HIV-1 Env protein and a surface marker LNGFR. (A) Schematic representation of a retroviral backbone plasmid,
MINFR-Env,thatencodesanHIV-1Envgene.MINFR-gp160R5andMINFR-gp160X4aredesignatedforthisconstructencodingaCCR5-orCXCR4-tropicEnvgene,respectively.
ThebiocistronicconfigurationofthisconstructwithanIRESlinkercanallowforco-expressionofEnvandasurfacestainingmarkerLNGFR.MSCV:murinestemcellvirus;
LTR:longterminalrepeat;Env:HIV-1envelopeprotein;IRES:internalribosomeentrysite;LNGFR:humanlow-affinitynervegrowthfactorreceptor.(BandC)Thestablecell
line293T.EnvR5wasconstructedbytransducingparent293TcellswithMINFG-gp160R5pseudotypedwithVSVGandpassagingthecellsforafewweeks.(B)Flowcytometry
analysisofLNGFRexpressioninaselectedsinglecellclone.(C)TZM-b1andHeLacellswerelabeledwithoctadecylrhodamnineBchloride,R18,andseededontoglass-bottom
culture dishes. 293T.EnvR5 cells were labeled by 5,6-carboxy-fluorescein diacetate succinimidyl ester, CFSE, and co-cultured with TZM-b1 or HeLa cells. Redistribution of
theR18fluorescencedyewasimagedbyalaserscanningconfocalmicroscope.
chloride(NH
4
Cl)ina24-wellculturedishat37
◦
Cand5%CO
2
for8h.
Themediawasthenreplacedwithfreshmedia.Afterincubationfor
anadditional4days,theGFP-positivecellswereanalyzedbyflow
cytometry(FACSort,BDBioscience).
2.8. Targeted delivery of suicide gene therapy in vitro
Jurkat cells were transduced by a retroviral vector, MINFR-
gp160R5, pseudotyped with VSVG to express HIV-1 Env proteins.
Three days later, the transduced cells were incubated with the
concentrated LVs encoding SR39Tk (FUWSR39tk/CD4+SGN) for
transductionbythemethodpreviouslydescribed.Afterwards,the
cells were cultured in media with or without prodrug ganciclovir
(GCV; Sigma–Aldrich, St. Louis, MO, USA) for an additional seven
days.ThetreatedcellswerestainedwithanAlexa594-conjugated
CD271 (LNGFR) antibody (Miltenyi Biotec) and analyzed by flow
cytometry(FACSort,BDBioscience).
3. Results
3.1. Generation of the HIV-1 envelope protein-expressing cell line
Wehavepreviouslyreportedthatlentiviralvectors(LVs)pseu-
dotyped with an antibody and a FM can transduce cells in a cell
type-specific manner (Yang et al., 2006). The FM was derived
from the altered Sindbis virus glycoprotein with mutations in the
E2 binding domain. Because this method separates the two key
functions of viral glycoproteins (binding and fusion) into two dis-
tinctive proteins, we can theoretically generate a new targeting
vector by simply changing the binding protein on the vector sur-
face.Therefore,wehypothesizedthatco-incorporationofahuman
CD4 protein and an engineered FM could confer upon an LV with
the capability to specifically deliver genes into cells infected with
HIV-1(Fig.1A,rightpanel).
To test this hypothesis, we created a cell line expressing HIV-
1 Env. A mouse stem cell virus-based retroviral transfer plasmid
was constructed to encode an HIV-1 clade C and CCR5-tropic Env
gene (gp160R5, from the 96ZM651 strain) and a surface marker
(LNGFR:truncatedlowaffinitynervegrowthfactorreceptorgene);
the construct is designated as MINFR-gp160R5 (Fig. 2A). An inter-
nalribosomeentrysite(IRES)wasusedtolinkgp160R5andLNGFR
to achieve co-expression that was driven by the 5
"
LTR promoter
(YangandBaltimore,2005).Wehavetestedseveralcommercially
available antibodies and could not identify one that could be reli-
ablyusedforflowcytometryanalysisofEnvexpression.Thus,this
design could allow us to readily quantify the expression of HIV-
1 Env by surface staining of the marker LNGFR. To generate the
HIV-1Env-expressingcellline,293Tcellsweretransducedwiththe
MINFR-gp160R5vectorpseudotypedwiththevesicularstomatitis
virusglycoprotein(VSVG).Afterafewweeksofcellpassaging,indi-
vidualsinglecellcloneswereselected,stainedwithananti-LNGFR
antibody,andanalyzedbyflowcytometry.AnLNGFR-positiveclone
(Fig. 2B) was subsequently tested by a cell–cell fusion assay. The
fusioneventsbetweencellsexpressingHIV-1Envandcellsexpress-
ingCD4andco-receptorcanbemonitoredbytheredistributionof
dye molecules; this assay has been widely used in the observa-
tion of cell and viral membrane fusion (Blumenthal et al., 2002).
We labeled HeLa or TZM-b1 (a HeLa cell line expressing CD4 and
CCR5)cellswithfluorescentmembranedye,octadecylrhodamnine
Bchloride(R-18),andoverlaidthemwiththeselectedclonalcells
labeledwithanintracellulardye,5,6-carboxy-fluoresceindiacetate
succinimidylester(CFSE).Redistributionoftheredflorescencedye
(R-18) only from labeled TZM-b1 cells, but not from HeLa cells, to
thetargetcellsconfirmedthefunctionalexpressionofHIV-1Envon
theselectedclonalcells(Fig.2C).Thisclonalcelllinewasdesignated
as293T.EnvR5andwasusedthroughoutthisstudy.
3.2. Generation of recombinant LVs
To generate HIV-1 Env-specific LVs, 293T cells were co-
transfected using the standard calcium phosphate protocol with
thelentiviraltransferplasmidFUGW,thelentiviralvectorpackag-
ingplasmids(pMGLandpRev)(Tiscorniaetal.,2006),theplasmid
encoding CD4 (pCD4), and the plasmid encoding the FM (pFM)
(Fig. 1B). The resulting vector was designated as FUGW/CD4+FM.
FUGWisaself-inactivatingHIV-1-basedtransfervectorbackbone
27
2.3.2 Generation of recombinant LVs
To generate HIV-1 Env-specific LVs, 293T cells were co- transfected using the
standard calcium phosphate protocol with the lentiviral transfer plasmid FUGW, the
lentiviral vector packaging plasmids (pMGL and pRev) (Tiscornia et al. 2006), the
plasmid encoding CD4 (pCD4), and the plasmid encoding the FM (pFM) (Fig. 2.1B).
The resulting vector was designated as FUGW/CD4+FM. FUGW is a self-inactivating
HIV-1-based transfer vector backbone plasmid with a GFP reporter gene encoded
downstream of the human ubiquitin-C promoter (Lois et al. 2002). Three previously
described FMs (AKN, AGM, and SGN) were used in this study (Yang et al. 2008a).
These FMs were derived from the Sindbis virus glycoprotein with different combinations
of mutations to yield proteins with variations in fusion activity. We wanted to test
engineered LVs incorporating various combinations of FMs with CD4 for their
transduction efficiencies and targeting specificities.
We also wanted to compare our approach with the reported inverse fusion method
(Fig. 2.1A, left panel) (Endres et al. 1997) involving co-incorporation of CD4 and a co-
receptor (CCR5 or CXCR4) to generate targeting LVs (designated FUGW/CD4+CCR5
or FUGW/CD4+CXCR4). As a non-targeting control, we generated VSVG-pseudotyped
LVs (FUGW/VSVG) with a broad tropism capable of transducing many different cell
types (Cronin et al. 2005). For negative controls, we prepared LVs displaying either FM
with a non-relevant protein CD20 (FUGW/CD20+FM) or CD4 alone (FUGW/CD4).
Flow cytometry analysis of vector-producing cells showed that all of the transfected live
cells expressed similar levels of GFP. This indicated that the vector backbone FUGW
was compatible with the expression of these proteins (CD4, CCR5, CXCR4, and FMs)
28
and was present in all of the vector-producing cells (Fig. 2.3A–D, upper panel). Gated on
GFP-positive cells, around 40% of the transfected cells co-expressed CD4 and FM (Fig.
2.3A, lower panel). Approximately 17% of the cells co-expressed the non- relevant
protein CD20 and FM (Fig. 2.3B, lower panel). This apparently low CD20 expression
level can be partially attributed to the insensitivity of the anti-CD20 staining antibody,
which was documented in a previous study (Ziegler et al. 2008). As expected from the
previous inverse fusion study, the producing cells could co-express both CD4 and a co-
receptor (CCR5 or CXCR4) on their surfaces (Fig. 2.3C). From this expression pattern
analysis, we found that individual vector-producing cells could be transfected to co-
express CD4 and FMs, a step that was necessary for them to be incorporated into the
lentiviral surface.
2.3.3 Targeted transduction of HIV-1 Env-expressing cells
To test the ability of engineered LVs to specifically transduce cells expressing
HIV-1 Env, the vector particles were harvested from the supernatant of transfected
vector-producing cells and incubated with 293T.EnvR5; the parental 293T cell line was
used as a negative control. Flow cytometry was used five days post-transduction to
determine the transduction efficiency by calculating the percentage of GFP-positive cells.
The transduction magnitude was obtained by measuring the mean fluorescence intensity
(MFI) of the transduced cells. To compare the difference between the target viral vectors,
we utilized integrated MFI (iMFI), which reflects the total intensity of the GFP signals
from the transduced cells by multiplying the MFI with transduction efficiency to quantify
the targeted transduction ability (Lei et al. 2009) (Fig. 2.4). As expected, both
29
293T.EnvR5 and 293T cells could be transduced by the non-targeting vector
FUGW/VSVG (Fig. 2.4B).

Figure 2.3 Co-expression of binding and FM molecules on the surface of vector-
producing cells.293T cells were transiently transfected with the plasmids FUGW, pCD4,
pCD20, pFM, pCCR5, and pCXCR4, along with other standard packaging plasmids to
produce targeting vectors, FUGW/CD4+FM, or inverse fusion vectors,
FUGW/CD4+CCR5 and FUGW/CD4+CXCR4. 293T cells were transfected to produce
the vector FUGW/CD4 displaying human CD4 and FUGW/VSVG pseudotyped with
VSVG to serve as controls. (A and B) Transfected cells were stained by anti-human CD4
(A) or CD20 (B) and anti-HA tag antibodies to detect the CD4 and FM expression,
respectively (Yang et al., 2006). The upper row shows GFP-expression. The shaded area
represents cells without vector backbone transfection, while the solid lines show cells
transfected with FUGW. The lower panel shows co-expression of the CD4 (A) or CD20
(B) and FM on the cells gated with GFP-positive populations. (C) The upper panel shows
GFP-expression. Gated on GFP-positive cells, the lower panel shows co-expression of
CD4 and CCR5 on the left and CD4 and CXCR4 on the right. (D) The GFP signal of
cells producing FUGW/VSVG (left) and co-expression of CD4 and GFP for cells making
FUGW/CD4 (right).

344 C.-L. Lee et al. / Virus Research 160 (2011) 340–350
Fig. 3. Co-expressionofbindingandFMmoleculesonthesurfaceofvector-producingcells.293TcellsweretransientlytransfectedwiththeplasmidsFUGW,pCD4,pCD20,
pFM, pCCR5, and pCXCR4, along with other standard packaging plasmids to produce targeting vectors, FUGW/CD4+FM, or inverse fusion vectors, FUGW/CD4+CCR5 and
FUGW/CD4+CXCR4.293TcellsweretransfectedtoproducethevectorFUGW/CD4displayinghumanCD4andFUGW/VSVGpseudotypedwithVSVGtoserveascontrols.(A
andB)Transfectedcellswerestainedbyanti-humanCD4(A)orCD20(B)andanti-HAtagantibodiestodetecttheCD4andFMexpression,respectively(Yangetal.,2006).
TheupperrowshowsGFP-expression.Theshadedarearepresentscellswithoutvectorbackbonetransfection,whilethesolidlinesshowcellstransfectedwithFUGW.The
lowerpanelshowsco-expressionoftheCD4(A)orCD20(B)andFMonthecellsgatedwithGFP-positivepopulations.(C)TheupperpanelshowsGFP-expression.Gatedon
GFP-positivecells,thelowerpanelshowsco-expressionofCD4andCCR5ontheleftandCD4andCXCR4ontheright.(D)TheGFPsignalofcellsproducingFUGW/VSVG(left)
andco-expressionofCD4andGFPforcellsmakingFUGW/CD4(right).
plasmid with a GFP reporter gene encoded downstream of the
human ubiquitin-C promoter (Lois et al., 2002). Three previously
describedFMs(AKN,AGM,andSGN)wereusedinthisstudy(Yang
etal.,2008).TheseFMswerederivedfromtheSindbisvirusglyco-
proteinwithdifferentcombinationsofmutationstoyieldproteins
with variations in fusion activity. We wanted to test engineered
LVsincorporatingvariouscombinationsofFMswithCD4fortheir
transductionefficienciesandtargetingspecificities.
We also wanted to compare our approach with the reported
inverse fusion method (Fig. 1A, left panel) (Endres et al., 1997)
involving co-incorporation of CD4 and a co-receptor (CCR5 or
CXCR4) to generate targeting LVs (designated FUGW/CD4+CCR5
or FUGW/CD4+CXCR4). As a non-targeting control, we gener-
ated VSVG-pseudotyped LVs (FUGW/VSVG) with a broad tropism
capable of transducing many different cell types (Cronin et al.,
2005). For negative controls, we prepared LVs displaying either
FM with a non-relevant protein CD20 (FUGW/CD20+FM) or CD4
alone (FUGW/CD4). Flow cytometry analysis of vector-producing
cells showed that all of the transfected live cells expressed sim-
ilar levels of GFP. This indicated that the vector backbone FUGW
wascompatiblewiththeexpressionoftheseproteins(CD4,CCR5,
CXCR4, and FMs) and was present in all of the vector-producing
cells(Fig.3A–D,upperpanel).GatedonGFP-positivecells,around
40% of the transfected cells co-expressed CD4 and FM (Fig. 3A,
lowerpanel).Approximately17%ofthecellsco-expressedthenon-
relevantproteinCD20andFM(Fig.3B,lowerpanel).Thisapparently
lowCD20expressionlevelcanbepartiallyattributedtotheinsen-
sitivityoftheanti-CD20stainingantibody,whichwasdocumented
inapreviousstudy(Ziegleretal.,2008).Asexpectedfromtheprevi-
ousinversefusionstudy,theproducingcellscouldco-expressboth
CD4andaco-receptor(CCR5orCXCR4)ontheirsurfaces(Fig.3C).
From this expression pattern analysis, we found that individual
vector-producingcellscouldbetransfectedtoco-expressCD4and
FMs,astepthatwasnecessaryforthemtobeincorporatedintothe
lentiviralsurface.
3.3. Targeted transduction of HIV-1 Env-expressing cells
To test the ability of engineered LVs to specifically transduce
cells expressing HIV-1 Env, the vector particles were harvested
from the supernatant of transfected vector-producing cells and
incubated with 293T.EnvR5; the parental 293T cell line was
used as a negative control. Flow cytometry was used five days
post-transductiontodeterminethetransductionefficiencybycal-
culating the percentage of GFP-positive cells. The transduction
magnitude was obtained by measuring the mean fluorescence
intensity (MFI) of the transduced cells. To compare the differ-
ence between the target viral vectors, we utilized integrated MFI
(iMFI), which reflects the total intensity of the GFP signals from
the transduced cells by multiplying the MFI with transduction
efficiency to quantify the targeted transduction ability (Lei et al.,
2009)(Fig. 4). As expected, both 293T.EnvR5 and 293T cells could
be transduced by the non-targeting vector FUGW/VSVG (Fig. 4B).
TheHIV-1Env-expressingcells(293T.EnvR5)exhibitedsignificant
iMFI signal when exposed to the targeting vector FUGW/CD4+FM
(Fig.4A,left3columns).Asanegativecontrol,theLVdisplayinga
non-relevantproteinFUGW/CD20+FMshowedonlyabackground
level transduction for 293T.Env (Fig. 4A, right 3 columns). The
Env-negative cells (293T) yielded only background levels of iMFI
signal when transduced with both relevant and non-relevant LVs
(FUGW/CD4+FMandFUGW/CD20+FM).TheLVdisplayingonlyCD4
on the surface (FUGW/CD4) could not transduce either cell line,
confirming the requirement of FM to mediate the fusion of LVs to
delivergenes(Fig.4A).
Asreportedpreviously,wealsoobservedtargetedtransduction
withLVsengineeredbytheinversefusionmethodtocarrybothCD4
30
The HIV-1 Env-expressing cells (293T.EnvR5) exhibited significant iMFI signal
when exposed to the targeting vector FUGW/CD4+FM (Fig. 2.4A, left 3 columns). As a
negative control, the LV displaying a non-relevant protein FUGW/CD20+FM showed
only a background level transduction for 293T.Env (Fig. 2.4A, right 3 columns). The
Env-negative cells (293T) yielded only background levels of iMFI signal when
transduced with both relevant and non-relevant LVs (FUGW/CD4+FM and
FUGW/CD20+FM). The LV displaying only CD4 on the surface (FUGW/CD4) could
not transduce either cell line, confirming the requirement of FM to mediate the fusion of
LVs to deliver genes (Fig. 2.4A).
As reported previously, we also observed targeted transduction with LVs
engineered by the inverse fusion method to carry both CD4 and CCR5 (Fig. 2.4A, left 4th
column). Because the Env expressed on 293T.EnvR5 is CCR5-tropic, only background
transduction was seen when FUGW/CD4+CXCR4 was used. The transduction titers of
various LVs for 293T.Env and 293T are summarized in Fig. 2.5. The titers of targeting
vectors remained lower than that of the non-specific vector FUGW/VSVG. Because
FUGW/CD4+SGN gave us the relatively higher transduction and reasonably low
background (Fig. 2.5A), we decided to focus on this vector for the following
investigations. As compared with the inverse fusion vector FUGW/CD4+CCR5,
FUGW/CD4+SGN yielded a higher vector titer against HIV-1 Env-expressing cells.
31

Figure 2.4 In vitro targeted transduction of HIV-1 Env expressing cells by engineered
LVs. 293T.EnvR5 and 293T cells were either transduced with 2mL of fresh,
unconcentrated LVs (FUGW/CD4+FM, FUGW/CD20+FM, FUGW/CD4, or
FUGW/VSVG) or with no vector as a control. The LVs generated by the inverse fusion
method (FUGW/CD4+CCR5 or FUGW/CD4+CXCR4) were included for comparison.
GFP expression was analyzed using flow cytometry. (A) Integrated mean fluorescence
intensity (iMFI) on 293T.EnvR5 or 293T cells transduced by the indicated LVs. (B) iMFI
on the non-specific FUGW/VSVG vector on 293T and 293T.EnvR5 cells.

C.-L. Lee et al. / Virus Research 160 (2011) 340–350 345
Fig.4. InvitrotargetedtransductionofHIV-1Env-expressingcellsbyengineeredLVs.293T.EnvR5and293Tcellswereeithertransducedwith2mLoffresh,unconcentrated
LVs (FUGW/CD4+FM, FUGW/CD20+FM, FUGW/CD4, or FUGW/VSVG) or with no vector as a control. The LVs generated by the inverse fusion method (FUGW/CD4+CCR5 or
FUGW/CD4+CXCR4)wereincludedforcomparison.GFPexpressionwasanalyzedusingflowcytometry.(A)Integratedmeanfluorescenceintensity(iMFI)on293T.EnvR5or
293TcellstransducedbytheindicatedLVs.(B)iMFIonthenon-specificFUGW/VSVGvectoron293Tand293T.EnvR5cells.
andCCR5(Fig.4A,left4thcolumn).BecausetheEnvexpressedon
293T.EnvR5isCCR5-tropic,onlybackgroundtransductionwasseen
whenFUGW/CD4+CXCR4wasused.Thetransductiontitersofvari-
ousLVsfor293T.Envand293TaresummarizedinFig.5.Thetitersof
targetingvectorsremainedlowerthanthatofthenon-specificvec-
tor FUGW/VSVG. Because FUGW/CD4+SGN gave us the relatively
higher transduction and reasonably low background (Fig. 5A), we
decided to focus on this vector for the following investigations.
As compared with the inverse fusion vector FUGW/CD4+CCR5,
FUGW/CD4+SGN yielded a higher vector titer against HIV-1 Env-
expressingcells.
3.4. Targeting a broader tropism of gp120 moiety
One disadvantage of using the inverse fusion method to target
LVsistheneedtoswitchbetweenthetwoco-receptorsCCR5and
CXCR4onthevectorsurfaceinordertotransducecellsexpressing
Env derived from different HIV-1 tropisms. Based on the inverse
fusionmethod,CD4plusCXCR4pseudotypedvectorsarerequired
totransduceX4-tropicHIV-1infectedcells,whileR5-tropicHIV-1
infected cells require a vector bearing a combination of CD4 and
CCR5 (Peretti et al., 2006). Our experimental data confirmed this
tropic-limited LV system. We found that the FUGW/CD4+CXCR4
vector could not effectively transduce 293T.EnvR5 cells express-
ing an Env derived from a R5-tropic HIV-1 subtype C virus strain
96ZM651(Gaoetal.,2003),butthesamecellscouldbetransduced
by the CCR5-carrying vector (FUGW/CD4+CCR5) (Figs. 4 and 5).
SinceourtargetingLVsystemdoesnotinvolveco-receptors,their
ability to transduce cells should be independent on the tropism
ofHIV-1Env.Toconfirmthishypothesis,weusedthesamestrat-
egytoconstructanX4-tropicEnv-expressingcellline.Thenewcell
linedisplayedanEnvfromanX4-tropicstrainofHIV-1subtypeB
virus (HXBc2 stain) and was designated as 293T.EnvX4 (Fig. 6A).
This cell line was challenged with three different targeting LVs
(FUGW/CD4+SGN, FUGW/CD4+CXCR4, or FUGW/CD4+CCR5). iMFI
signalsfrom293T.EnvX4cellsexposedwithbothFUGW/CD4+SGN
and FUGW/CD4+CXCR4 vectors were detected to be at a similar
level(Fig.6B).AloweriMFIsignalswasfoundinFUGW/CD4+CCR5-
transduced cells, demonstrating that X4-tropic Env-expressing
cellspreferredtransductionbytheCXCR4-bearingLVsratherthan
the vector displaying CCR5. This higher than usual background
transductionofFUGW/CD4+CCR5towards293T.EnvX4cellsispos-
sibly due to non-tropism specific membrane fusion induced by
X4-tropicEnv(Pleskoffetal.,1998).
Fig. 5. Specific vector titers for various engineered LVs. (A) Titers of the unconcentrated vectors on 293T and 293T.EnvR5 cells. (B) Titers of the non-specific FUGW/VSVG
vectoron293Tand293T.EnvR5cells.
32

Figure 2.5 Specific vector titers for various engineered LVs (A) Titers of the
unconcentrated vectors on 293T and 293T.EnvR5 cells. (B) Titers of the non-specific
FUGW/VSVG vector on 293T and 293T.EnvR5 cells.

2.3.4 Targeting a broader tropism of gp120 moiety
One disadvantage of using the inverse fusion method to target LVs is the need to
switch between the two co-receptors CCR5 and CXCR4 on the vector surface in order to
transduce cells expressing Env derived from different HIV-1 tropisms. Based on the
inverse fusion method, CD4 plus CXCR4 pseudotyped vectors are required to transduce
X4-tropic HIV-1 infected cells, while R5-tropic HIV-1 infected cells require a vector
bearing a combination of CD4 and CCR5 (Peretti et al. 2006). Our experimental data
confirmed this tropic-limited LV system. We found that the FUGW/CD4+CXCR4 vector
could not effectively transduce 293T.EnvR5 cells expressing an Env derived from a R5-
tropic HIV-1 subtype C virus strain 96ZM651 (Gao et al. 2003), but the same cells could
be transduced by the CCR5-carrying vector (FUGW/CD4+CCR5) (Figs. 2.4 and 2.5).
Since our LV system does not involve co-receptors, their ability to transduce cells should
C.-L. Lee et al. / Virus Research 160 (2011) 340–350 345
Fig.4. InvitrotargetedtransductionofHIV-1Env-expressingcellsbyengineeredLVs.293T.EnvR5and293Tcellswereeithertransducedwith2mLoffresh,unconcentrated
LVs (FUGW/CD4+FM, FUGW/CD20+FM, FUGW/CD4, or FUGW/VSVG) or with no vector as a control. The LVs generated by the inverse fusion method (FUGW/CD4+CCR5 or
FUGW/CD4+CXCR4)wereincludedforcomparison.GFPexpressionwasanalyzedusingflowcytometry.(A)Integratedmeanfluorescenceintensity(iMFI)on293T.EnvR5or
293TcellstransducedbytheindicatedLVs.(B)iMFIonthenon-specificFUGW/VSVGvectoron293Tand293T.EnvR5cells.
andCCR5(Fig.4A,left4thcolumn).BecausetheEnvexpressedon
293T.EnvR5isCCR5-tropic,onlybackgroundtransductionwasseen
whenFUGW/CD4+CXCR4wasused.Thetransductiontitersofvari-
ousLVsfor293T.Envand293TaresummarizedinFig.5.Thetitersof
targetingvectorsremainedlowerthanthatofthenon-specificvec-
tor FUGW/VSVG. Because FUGW/CD4+SGN gave us the relatively
higher transduction and reasonably low background (Fig. 5A), we
decided to focus on this vector for the following investigations.
As compared with the inverse fusion vector FUGW/CD4+CCR5,
FUGW/CD4+SGN yielded a higher vector titer against HIV-1 Env-
expressingcells.
3.4. Targeting a broader tropism of gp120 moiety
One disadvantage of using the inverse fusion method to target
LVsistheneedtoswitchbetweenthetwoco-receptorsCCR5and
CXCR4onthevectorsurfaceinordertotransducecellsexpressing
Env derived from different HIV-1 tropisms. Based on the inverse
fusionmethod,CD4plusCXCR4pseudotypedvectorsarerequired
totransduceX4-tropicHIV-1infectedcells,whileR5-tropicHIV-1
infected cells require a vector bearing a combination of CD4 and
CCR5 (Peretti et al., 2006). Our experimental data confirmed this
tropic-limited LV system. We found that the FUGW/CD4+CXCR4
vector could not effectively transduce 293T.EnvR5 cells express-
ing an Env derived from a R5-tropic HIV-1 subtype C virus strain
96ZM651(Gaoetal.,2003),butthesamecellscouldbetransduced
by the CCR5-carrying vector (FUGW/CD4+CCR5) (Figs. 4 and 5).
SinceourtargetingLVsystemdoesnotinvolveco-receptors,their
ability to transduce cells should be independent on the tropism
ofHIV-1Env.Toconfirmthishypothesis,weusedthesamestrat-
egytoconstructanX4-tropicEnv-expressingcellline.Thenewcell
linedisplayedanEnvfromanX4-tropicstrainofHIV-1subtypeB
virus (HXBc2 stain) and was designated as 293T.EnvX4 (Fig. 6A).
This cell line was challenged with three different targeting LVs
(FUGW/CD4+SGN, FUGW/CD4+CXCR4, or FUGW/CD4+CCR5). iMFI
signalsfrom293T.EnvX4cellsexposedwithbothFUGW/CD4+SGN
and FUGW/CD4+CXCR4 vectors were detected to be at a similar
level(Fig.6B).AloweriMFIsignalswasfoundinFUGW/CD4+CCR5-
transduced cells, demonstrating that X4-tropic Env-expressing
cellspreferredtransductionbytheCXCR4-bearingLVsratherthan
the vector displaying CCR5. This higher than usual background
transductionofFUGW/CD4+CCR5towards293T.EnvX4cellsispos-
sibly due to non-tropism specific membrane fusion induced by
X4-tropicEnv(Pleskoffetal.,1998).
Fig. 5. Specific vector titers for various engineered LVs. (A) Titers of the unconcentrated vectors on 293T and 293T.EnvR5 cells. (B) Titers of the non-specific FUGW/VSVG
vectoron293Tand293T.EnvR5cells.
33
be independent on the tropism of HIV-1 Env. To confirm this hypothesis, we used the
same strategy to construct an X4-tropic Env-expressing cell line. The new cell line
displayed an Env from an X4-tropic strain of HIV-1 subtype B virus (HXBc2 stain) and
was designated as 293T.EnvX4 (Fig. 2.6A). This cell line was challenged with three
different LVs (FUGW/CD4+SGN, FUGW/CD4+CXCR4, or FUGW/CD4+CCR5). iMFI
signals from 293T.EnvX4 cells exposed with both FUGW/CD4+SGN and
FUGW/CD4+CXCR4 vectors were detected to be at a similar level (Fig. 2.6B). A lower
iMFI signals was found in FUGW/CD4+CCR5- transduced cells, demonstrating that X4-
tropic Env-expressing cells preferred transduction by the CXCR4-bearing LVs rather
than the vector displaying CCR5. This higher than usual background transduction of
FUGW/CD4+CCR5 towards 293T.EnvX4 cells is possibly due to non-tropism specific
membrane fusion induced by X4-tropic Env (Pleskoff et al. 1998).
2.3.5 Specific binding between vectors and cells for targeted transduction
The study of binding between vector particles and target cells was performed to
further investigate whether a specific inter- action between the engineered LV and the
Env-expressing cells was necessary to achieve targeted transduction. The LV particles
(FUGW/CD4+SGN) were co-incubated with either 293T.EnvR5 or 293T at 4
◦
C for 5
min, after which the cell–vector complexes were immediately fixed by 4% formaldehyde
to reduce the binding- induced internalization of the LVs. The cell–vector complexes
were then stained with an antibody against the FM. The FM signals were detected on
293T.EnvR5 surfaces, but not on 293T cell surfaces, indicating that the observed binding
requires the presence of Env proteins (Fig. 2.7A).
34
Next, we determined whether the specific interaction between the CD4 displayed
on the vector surface and the HIV-1 Env on the surface of target cells is critical for
mediating the specific transduction. To do this, 293T.EnvR5 cells were incubated with
the LVs (FUGW/CD4+SGN) in the presence of various concentrations of soluble CD4.
We found that the transduction efficiency decreased as the concentration of the soluble
CD4 increased (Fig. 2.7B), suggesting that the presence of soluble CD4 could compete
with the targeting LVs to bind to 293T.EnvR5 and thus block the vector transduction.
These results confirmed that the binding between the HIV-1 Env on the cell surface and
the CD4 on the vector was necessary for the specific transduction.

Figure 2.6 Engineered LVs transducing cells expressing different tropisms of HIV-1 Env.
We constructed a new cell line expressing an X4-tropic HIV-1 subtype B Env protein and
designated it as 293T.EnvX4. (A) 293T.EnvX4 cells were analyzed by flow cytometry
for the surface marker LNGFR as an indication of HIV-1 Env expression. (B) iMFI on
293T.EnvX4 cells transduced with various unconcentrated LVs (FUGW/CD4+CCR5,
FUGW/CD4+CCR4, and FUGW/CD4+SGN).

346 C.-L.Leeetal./VirusResearch160(2011)340–350
Fig.6. EngineeredLVstransducingcellsexpressingdifferenttropismsofHIV-1Env.
We constructed a new cell line expressing an X4-tropic HIV-1 subtype B Env pro-
tein and designated it as 293T.EnvX4. (A) 293T.EnvX4 cells were analyzed by flow
cytometry for the surface marker LNGFR as an indication of HIV-1 Env expres-
sion. (B) iMFI on 293T.EnvX4 cells transduced with various unconcentrated LVs
(FUGW/CD4+CCR5,FUGW/CD4+CCR4,andFUGW/CD4+SGN).
3.5. Specificbindingbetweenvectorsandcellsfortargeted
transduction
The study of binding between vector particles and target cells
was performed to further investigate whether a specific inter-
action between the engineered LV and the Env-expressing cells
was necessary to achieve targeted transduction. The LV particles
(FUGW/CD4+SGN) were co-incubated with either 293T.EnvR5 or
293Tat4
◦
Cfor5min,afterwhichthecell–vectorcomplexeswere
immediately fixed by 4% formaldehyde to reduce the binding-
inducedinternalizationoftheLVs.Thecell–vectorcomplexeswere
thenstainedwithanantibodyagainsttheFM.TheFMsignalswere
detected on 293T.EnvR5 surfaces, but not on 293T cell surfaces,
indicating that the observed binding requires the presence of Env
proteins(Fig.7A).
Next,wedeterminedwhetherthespecificinteractionbetween
theCD4displayedonthevectorsurfaceandtheHIV-1Envonthe
surface of target cells is critical for mediating the specific trans-
duction.Todothis,293T.EnvR5cellswereincubatedwiththeLVs
(FUGW/CD4+SGN)inthepresenceofvariousconcentrationsofsol-
uble CD4. We found that the transduction efficiency decreased as
theconcentrationofthesolubleCD4increased(Fig.7B),suggesting
thatthepresenceofsolubleCD4couldcompetewiththetargeting
LVstobindto293T.EnvR5andthusblockthevectortransduction.
These results confirmed that the binding between the HIV-1 Env
onthecellsurfaceandtheCD4onthevectorwasnecessaryforthe
specifictransduction.
3.6. DependenceoftargetedtransductionontheendosomalpH
The conformation change of the FM derived from the Sind-
bis virus glycoprotein can be triggered within the lumen of the
endosomes by acidic pH to allow for fusion to occur between the
viral particles and the host cell’s endosomal membrane (Glomb-
Reinmund and Kielian, 1998). This fusion is a critical step for the
viralvectortoreleaseitsgeneticcontentintothecell’scytoplasm.
InordertoverifytheacidicpHrequirementforourtargetingLV,we
incubatedFUGW/CD4+SGNwith293T.EnvR5cellsinthepresence
of graded concentrations of ammonium chloride (NH
4
Cl), which
is known to neutralize the pH in the endosomal compartments
(Mellman et al., 1986). The results showed that the transduction
efficiency dramatically decreased with increasing NH
4
Cl concen-
trations (Fig. 7C), thus confirming the low pH dependency of the
incorporated FM in mediating gene delivery by engineered LV to
targetcells.
Fig.7. EngineeredLVsarecapableofbindingtoHIV-1Env-expressingcellsandarecriticalforspecifictransduction.(A)293T.EnvR5and293Tcellswereincubatedwiththe
FUGW/CD4+SGNvectorandthenfixedbyformaldehydeimmediatelyfollowing.Theresultingvector–cellcomplexeswerestainedwithanantibodyagainsttheFM(Yanget
al.,2006)anddetectedbyflowcytometry.(B)VariousconcentrationsofsolublehumanCD4wereaddedto293T.EnvR5cellsalongwithengineeredLVs.Fourdayslater,we
used flow cytometry to detect GFP expression as an indication of inhibited transduction. (C) Effect of the neutralizing agent (NH4Cl) on the pH-dependent transduction of
targeting LVs. Various concentrations of NH4Cl were added into the vector supernatant and incubated together with 293.EnvR5 cells for 8h. The media was replaced with
freshmediaandcellswereculturedfor4moredays.TheGFPexpressionwasdetectedbyflowcytometry.
35
2.3.6 Dependence of targeted transduction on the endosomal pH
The conformation change of the FM derived from the Sindbis virus glycoprotein
can be triggered within the lumen of the endosomes by acidic pH to allow for fusion to
occur between the viral particles and the host cell’s endosomal membrane (Glomb-
Reinmund and Kielian 1998). This fusion is a critical step for the viral vector to release
its genetic content into the cell’s cytoplasm. In order to verify the acidic pH requirement
for our targeting LV, we incubated FUGW/CD4+SGN with 293T.EnvR5 cells in the
presence of graded concentrations of ammonium chloride (NH
4
Cl), which is known to
neutralize the pH in the endosomal compartments (Mellman et al. 1986). The results
showed that the transduction efficiency dramatically decreased with increasing NH
4
Cl
concentrations (Fig. 2.7C), thus confirming the low pH dependency of the incorporated
FM in mediating gene delivery by engineered LV to target cells. Dependence of clathrin
and dynamin for internalization of engineered LVs
Clathrin and caveolin-mediated endocytosis have been well characterized for the
internalization of many viruses into cells (Blanchard et al. 2006; DeTulleo and
Kirchhausen 1998; Doxsey et al. 1987; Joo et al. 2008; Kirchhausen 2000; Nabi and Le
2003; Nichols and Lippincott-Schwartz 2001; Rust et al. 2004). To determine whether
our engineered LVs entered cells through the clathrin or caveolin-mediated endocytosis
pathway, we investigated the effects of different inhibitory drugs on the transduction
efficiency (Fig. 2.8A). Chlorpromazine is a drug known to inactivate clathrin
polymerization and thereby inhibit internalization mediated by clathrin-coated vesicles
(CCV) (Wang et al. 1993), while genistein, a tyrosine phosphatase inhibitor, and nystatin,
a cholesterol-binding reagent, have been shown to disrupt caveolar-mediated endocytosis
36
(Alkhatib et al. 1996; Rothberg et al. 1992). The transduction results showed that the
entry of FUGW/CD4+SGN was markedly inhibited by the chlorpromazine treatment
(Fig. 2.8A), although no inhibitory effect was clearly seen for the treatments by genistein
or nystatin. This indicates that the targeting LV system utilizes the clathrin-mediated
endocytosis to enter the cells.

Figure 2.7 Engineered LVs are capable of binding to HIV-1 Env-expressing cells (A)
293T.EnvR5 and 293T cells were incubated with the FUGW/CD4+SGN vector and then
fixed by formaldehyde immediately following. The resulting vector–cell complexes were
stained with an antibody against the FM (Yang et al., 2006) and detected by flow
cytometry. (B) Various concentrations of soluble human CD4 were added to 293T.EnvR5
cells along with engineered LVs. Four days later, we used flow cytometry to detect GFP
expression as an indication of inhibited transduction. (C) Effect of the neutralizing agent
(NH4Cl) on the pH-dependent transduction of targeting LVs. Various concentrations of
NH4 Cl were added into the vector supernatant and incubated together with 293.EnvR5
cells for 8 h. The media was replaced with fresh media and cells were cultured for 4 more
days. The GFP expression was detected by flow cytometry.

346 C.-L.Leeetal./VirusResearch160(2011)340–350
Fig.6. EngineeredLVstransducingcellsexpressingdifferenttropismsofHIV-1Env.
We constructed a new cell line expressing an X4-tropic HIV-1 subtype B Env pro-
tein and designated it as 293T.EnvX4. (A) 293T.EnvX4 cells were analyzed by flow
cytometry for the surface marker LNGFR as an indication of HIV-1 Env expres-
sion. (B) iMFI on 293T.EnvX4 cells transduced with various unconcentrated LVs
(FUGW/CD4+CCR5,FUGW/CD4+CCR4,andFUGW/CD4+SGN).
3.5. Specificbindingbetweenvectorsandcellsfortargeted
transduction
The study of binding between vector particles and target cells
was performed to further investigate whether a specific inter-
action between the engineered LV and the Env-expressing cells
was necessary to achieve targeted transduction. The LV particles
(FUGW/CD4+SGN) were co-incubated with either 293T.EnvR5 or
293Tat4
◦
Cfor5min,afterwhichthecell–vectorcomplexeswere
immediately fixed by 4% formaldehyde to reduce the binding-
inducedinternalizationoftheLVs.Thecell–vectorcomplexeswere
thenstainedwithanantibodyagainsttheFM.TheFMsignalswere
detected on 293T.EnvR5 surfaces, but not on 293T cell surfaces,
indicating that the observed binding requires the presence of Env
proteins(Fig.7A).
Next,wedeterminedwhetherthespecificinteractionbetween
theCD4displayedonthevectorsurfaceandtheHIV-1Envonthe
surface of target cells is critical for mediating the specific trans-
duction.Todothis,293T.EnvR5cellswereincubatedwiththeLVs
(FUGW/CD4+SGN)inthepresenceofvariousconcentrationsofsol-
uble CD4. We found that the transduction efficiency decreased as
theconcentrationofthesolubleCD4increased(Fig.7B),suggesting
thatthepresenceofsolubleCD4couldcompetewiththetargeting
LVstobindto293T.EnvR5andthusblockthevectortransduction.
These results confirmed that the binding between the HIV-1 Env
onthecellsurfaceandtheCD4onthevectorwasnecessaryforthe
specifictransduction.
3.6. DependenceoftargetedtransductionontheendosomalpH
The conformation change of the FM derived from the Sind-
bis virus glycoprotein can be triggered within the lumen of the
endosomes by acidic pH to allow for fusion to occur between the
viral particles and the host cell’s endosomal membrane (Glomb-
Reinmund and Kielian, 1998). This fusion is a critical step for the
viralvectortoreleaseitsgeneticcontentintothecell’scytoplasm.
InordertoverifytheacidicpHrequirementforourtargetingLV,we
incubatedFUGW/CD4+SGNwith293T.EnvR5cellsinthepresence
of graded concentrations of ammonium chloride (NH
4
Cl), which
is known to neutralize the pH in the endosomal compartments
(Mellman et al., 1986). The results showed that the transduction
efficiency dramatically decreased with increasing NH
4
Cl concen-
trations (Fig. 7C), thus confirming the low pH dependency of the
incorporated FM in mediating gene delivery by engineered LV to
targetcells.
Fig.7. EngineeredLVsarecapableofbindingtoHIV-1Env-expressingcellsandarecriticalforspecifictransduction.(A)293T.EnvR5and293Tcellswereincubatedwiththe
FUGW/CD4+SGNvectorandthenfixedbyformaldehydeimmediatelyfollowing.Theresultingvector–cellcomplexeswerestainedwithanantibodyagainsttheFM(Yanget
al.,2006)anddetectedbyflowcytometry.(B)VariousconcentrationsofsolublehumanCD4wereaddedto293T.EnvR5cellsalongwithengineeredLVs.Fourdayslater,we
used flow cytometry to detect GFP expression as an indication of inhibited transduction. (C) Effect of the neutralizing agent (NH4
Cl) on the pH-dependent transduction of
targeting LVs. Various concentrations of NH4
Cl were added into the vector supernatant and incubated together with 293.EnvR5 cells for 8h. The media was replaced with
freshmediaandcellswereculturedfor4moredays.TheGFPexpressionwasdetectedbyflowcytometry.
37
To further confirm that the clathrin pathway was involved in LV entry, we used a
dominant-negative mutant of dynamin 2 K44A to block the dynamin-dependent
endocytosis pathway (Vidricaire and Tremblay 2007). 293T.EnvR5 cells were transiently
transfected with the inhibitory plasmid DsRed-Dyn2K44A and then exposed to
FUGW/CD4+SGN. At the same time, the wild type dynamin plasmid, DsRed-Dyn2WT,
was used as a control. The Dyn2K44A-treated cells showed a reduction in transduction
efficiency when compared with Dyn2WT -treated cells (Fig. 2.8B). The data revealed
that the entry of the targeting LVs is mediated through a clathrin and dynamin- dependent
endocytosis pathway.
2.3.7 Transduction inhibition by dominant-negative Rab5 and Rab7
Fusion of many viruses is believed to occur either at the early or late stage of
maturing endosomes. In order to examine which stage of the endosomal compartments
was essential for the successful transduction of the targeting LV, 293T.EnvR5 cells were
transfected with either the wild type (Rab5WT and Rab7WT) or dominant-negative
mutants (Rab5DN and Rab7DN) of Rab5 and Rab7 to disable the early endosome
(Stenmark et al. 1994) or late endosome (Press et al. 1998) functions, respectively. Cells
with the dominant-negative mutant Rab5 showed a significant reduction in transduction
rate, by almost 30%, as compared with the wild type Rab5-expressing cells (Fig. 2.8C),
suggesting that successful transduction is at least in part associated with the early stage of
LV-containing endosomes. However, expression of the dominant-negative Rab7 in
293T.EnvR5 did not markedly alter the transduction by FUGW/CD4+SGN (Fig. 2.8C),
indicating that late endosome trafficking is unlikely to be involved in successful LV-
infection of target cells.
38

Figure 2.8 Clathrin and dynamin-dependent entry of the engineered LV. (A–C) Relative
transduction efficiency measured by flow cytometry. (A) 293T.EnvR5cellswereincubated
with three different drugs chlorpromazine to test for clathrin dependent entry, and
genistein and nystatin to test for caveolin dependent internalization for 30 min at 37
◦
C
(chlorpromazine: 10 µg/mL; genistein: 50 µg/mL; nystatin: 25 µg/mL). The drug-treated
cells were transduced with FUGW/CD4+SGN. (B and C) 293T.EnvR5 cells were
transiently transfected either with wild-type or dominant-negative mutant dynamin2 (B),
or with wild-type or dominant-negative mutants of Rab5 and Rab7 (C), and then
transduced with FUGW/CD4+SGN.

2.3.8 Targeted transduction by engineered LVs towards a human T cells
HIV-1 naturally infects human T cells; therefore we investigated the selective
transduction of a human T cell line expressing R5-tropic HIV-1 Env by the LV
(FUGW/CD4+SGN). We generated Jurkat cells that expressed Env by infecting parent
Jurkat cells, a CD4-positive T cell leukemia cell line, with the VSVG-pseudotyped
retroviral vector MINFR-gp160R5. After four days, these treated Jurkat cells were
analyzed by surface staining of LNGFR as an indication of Env expression. It was found
C.-L.Leeetal./VirusResearch160(2011)340–350 347
3.7. Dependenceofclathrinanddynaminforinternalizationof
engineeredLVs
Clathrin and caveolin-mediated endocytosis have been well-
characterized for the internalization of many viruses into cells
(Blanchard et al., 2006; DeTulleo and Kirchhausen, 1998; Doxsey
etal.,1987;Jooetal.,2008;Kirchhausen,2000;NabiandLe,2003;
NicholsandLippincott-Schwartz,2001;Rustetal.,2004).Todeter-
minewhetherourengineeredLVsenteredcellsthroughtheclathrin
or caveolin-mediated endocytosis pathway, we investigated the
effects of different inhibitory drugs on the transduction efficiency
(Fig. 8A). Chlorpromazine is a drug known to inactivate clathrin
polymerization and thereby inhibit internalization mediated by
clathrin-coatedvesicles(CCV)(Wangetal.,1993),whilegenistein,a
tyrosinephosphataseinhibitor,andnystatin,acholesterol-binding
reagent,havebeenshowntodisruptcaveolar-mediatedendocyto-
sis (Alkhatib et al., 1996; Rothberg et al., 1992). The transduction
results showed that the entry of FUGW/CD4+SGN was markedly
inhibited by the chlorpromazine treatment (Fig. 8A), although no
inhibitory effect was clearly seen for the treatments by genistein
ornystatin.ThisindicatesthatthetargetingLVsystemutilizesthe
clathrin-mediatedendocytosistoenterthecells.
To further confirm that the clathrin pathway was involved in
LVentry,weusedadominant-negativemutantofdynamin2K44A
toblockthedynamin-dependentendocytosispathway(Vidricaire
andTremblay,2007).293T.EnvR5cellsweretransientlytransfected
withtheinhibitoryplasmidDsRed-Dyn2
K44A
andthenexposedto
FUGW/CD4+SGN. At the same time, the wild type dynamin plas-
mid, DsRed-Dyn2
WT
, was used as a control. The Dyn2
K44A
-treated
cellsshowedareductionintransductionefficiencywhencompared
withDyn2
WT
-treatedcells(Fig.8B).Thedatarevealedthattheentry
of the targeting LVs is mediated through a clathrin and dynamin-
dependentendocytosispathway.
3.8. Transductioninhibitionbydominant-negativeRab5and
Rab7
Fusion of many viruses is believed to occur either at the early
or late stage of maturing endosomes. In order to examine which
stage of the endosomal compartments was essential for the suc-
cessful transduction of the targeting LV, 293T.EnvR5 cells were
transfected with either the wild type (Rab5
WT
and Rab7
WT
) or
dominant-negative mutants (Rab5
DN
and Rab7
DN
) of Rab5 and
Rab7 to disable the early endosome (Stenmark et al., 1994) or
late endosome (Press et al., 1998) functions, respectively. Cells
with the dominant-negative mutant Rab5 showed a significant
reduction in transduction rate, by almost 30%, as compared with
the wild type Rab5-expressing cells (Fig. 8C), suggesting that suc-
cessful transduction is at least in part associated with the early
stage of LV-containing endosomes. However, expression of the
dominant-negative Rab7 in 293T.EnvR5 did not markedly alter
thetransductionbyFUGW/CD4+SGN(Fig.8C),indicatingthatlate
endosome trafficking is unlikely to be involved in successful LV-
infectionoftargetcells.
3.9. TargetedtransductionbyengineeredLVstowardsahumanT
cells
HIV-1 naturally infects human T cells; therefore we investi-
gated the selective transduction of a human T cell line expressing
R5-tropic HIV-1 Env by the LV (FUGW/CD4+SGN). We generated
Jurkat cells that expressed Env by infecting parent Jurkat cells, a
CD4-positiveTcellleukemiacellline,withtheVSVG-pseudotyped
retroviral vector MINFR-gp160R5. After four days, these treated
JurkatcellswereanalyzedbysurfacestainingofLNGFRasanindi-
cation of Env expression. It was found that approximately 46%
of the cells expressed the surface marker LNGFR (Fig. 9A). We
Fig.8. Clathrinanddynamin-dependententryoftheengineeredLV.(A–C)Relativetransductionefficiencymeasuredbyflowcytometry.(A)293T.EnvR5cellswereincubated
with three different drugs – chlorpromazine to test for clathrin dependent entry, and genistein and nystatin to test for caveolin dependent internalization – for 30min at
37
◦
C(chlorpromazine:10!g/mL;genistein:50!g/mL;nystatin:25!g/mL).Thedrug-treatedcellsweretransducedwithFUGW/CD4+SGN.(BandC)293T.EnvR5cellswere
transientlytransfectedeitherwithwild-typeordominant-negativemutantdynamin2(B),orwithwild-typeordominant-negativemutantsofRab5andRab7(C),andthen
transducedwithFUGW/CD4+SGN.
39
that approximately 46% of the cells expressed the surface marker LNGFR (Fig. 2.9A).
We intentionally maintained this mixed population of cells for the subsequent experiment
because we were interested in testing the specificity of FUGW/CD4+SGN in targeting
Env-expressing T cells. FUGW/CD4+SGN was then used to transduce this T cell
population and the resulting GFP expression was analyzed after culturing the cells for an
additional four days. There were approximately 51% T cells that were GFP-positive, out
of which ∼95% (47.7% of the total cell population) expressed both GFP and LNGFR
(Fig. 2.9B), indicating a strong correlation between LV transduction and the presence of
Env in the T cells. Only a background level of GFP was seen when the Env-negative
Jurkat cells were exposed to FUGW/CD4+SGN (Fig. 2.9B), confirming the
indispensability of Env expression for efficient targeting. We further assessed the ability
of this engineered LV to target human primary T cells. The in vitro activated human
peripheral blood mononuclear cells (PBMC) were engineered to express R5-tropic HIV-1
Env by MINFR-gp160R5/VSVG-mediated retroviral transduction. These treated PBMC
were then exposed to the engineered FUGW/CD4+SGN vector; transduction to non-
treated PBMC was included as a control. As shown in Fig. 2.9C, treated PBMC
transduced with the targeting vector resulted in about 8% GFP+ cells, whereas only a
background level of the GFP signal was detected in the non-treated cells. This result is in
agreement with the T cell line data and confirms that the engineered LV could selectively
modify Env-expressing cells in a mixed population, omitting the conventional step of
purifying target cells.

40

Figure 2.9 Targeted transduction to a T cell line and suicide gene therapy delivered by
LVs in vitro. A CD4-positive T cell leukemia cell line (Jurkat) was infected with an R5-
tropic envelope-encoding retroviral vector (MINFR-gp160R5) pseudotyped with VSVG
and analyzed by flow cytometry for LNGFR expression. (A) Flow cytometry analysis of
Jurkat cells infected with the MINFR-gp160R5 vector. (B) The pretreated and non-
treated Jurkat cell mixture was challenged with an engineered LV (FUGW/CD4+SGN).
The GFP and surface marker (LNGFR) expressions were analyzed by flow cytometry.
(C) The pretreated and non-treated PBMC were challenged with an engineered LV
(FUGW/CD4+SGN). The GFP expression was analyzed by flow cytometry. (D, left)
Mixed Jurkat cells pre-treated to express HIV-1 Env were challenged with the
concentrated LV (FUWSR39tk/CD4+SGN) vector 3 times and then cultured in media
containing GCV. The treated cells were then analyzed for the expression of the surface
marker LNGFR using flow cytometry. The expression of LNGFR is indicative of Jurkat
cells expressing HIV-1 Env. (D, right) Mixed Jurkat cells pre-treated (MINFR-gp160R5)
to express HIV-1 Env were challenged with the LV (FUWSR39tk/CD4+SGN) vector and
cultured in media containing GCV. Jurkat cells without the vector treatment, or only
pretreated to express HIV-1 Env but no LV treatment were included as controls. The
treated cells were analyzed for apoptosis via 7-AAD staining one week post-GCV
treatment.
348 C.-L. Lee et al. / Virus Research 160 (2011) 340–350
Fig. 9. Targeted transduction to a T cell line and suicide gene therapy delivered by LVs in vitro. A CD4-positive T cell leukemia cell line (Jurkat) was infected with an R5-
tropic envelope-encoding retroviral vector (MINFR-gp160R5) pseudotyped with VSVG and analyzed by flow cytometry for LNGFR expression. (A) Flow cytometry analysis
of Jurkat cells infected with the MINFR-gp160R5 vector. (B) The pretreated and non-treated Jurkat cell mixture was challenged with an engineered LV (FUGW/CD4+SGN).
The GFP and surface marker (LNGFR) expressions were analyzed by flow cytometry. (C) The pretreated and non-treated PBMC were challenged with an engineered LV
(FUGW/CD4+SGN).TheGFPexpressionwasanalyzedbyflowcytometry.(D,left)MixedJurkatcellspre-treatedtoexpressHIV-1Envwerechallengedwiththeconcentrated
LV(FUWSR39tk/CD4+SGN)vector3timesandthenculturedinmediacontainingGCV.ThetreatedcellswerethenanalyzedfortheexpressionofthesurfacemarkerLNGFR
usingflowcytometry.TheexpressionofLNGFRisindicativeofJurkatcellsexpressingHIV-1Env.(D,right)MixedJurkatcellspre-treated(MINFR-gp160R5)toexpressHIV-1
Env were challenged with the LV (FUWSR39tk/CD4+SGN) vector and cultured in media containing GCV. Jurkat cells without the vector treatment, or only pretreated to
expressHIV-1EnvbutnoLVtreatmentwereincludedascontrols.Thetreatedcellswereanalyzedforapoptosisvia7-AADstainingoneweekpost-GCVtreatment.
intentionally maintained this mixed population of cells for the
subsequentexperimentbecausewewereinterestedintestingthe
specificity of FUGW/CD4+SGN in targeting Env-expressing T cells.
FUGW/CD4+SGNwasthenusedtotransducethisTcellpopulation
andtheresultingGFPexpressionwasanalyzedafterculturingthe
cells for an additional four days. There were approximately 51% T
cellsthatwereGFP-positive,outofwhich∼95%(47.7%ofthetotal
cellpopulation)expressedbothGFPandLNGFR(Fig.9B),indicating
a strong correlation between LV transduction and the presence of
Env in the T cells. Only a background level of GFP was seen when
the Env-negative Jurkat cells were exposed to FUGW/CD4+SGN
(Fig.9B),confirmingtheindispensabilityofEnvexpressionforeffi-
cient targeting. We further assessed the ability of this engineered
LV to target human primary T cells. The in vitro activated human
peripheral blood mononuclear cells (PBMC) were engineered to
express R5-tropic HIV-1 Env by MINFR-gp160R5/VSVG-mediated
retroviral transduction. These treated PBMC were then exposed
to the engineered FUGW/CD4+SGN vector; transduction to non-
treatedPBMCwasincludedasacontrol.AsshowninFig.9C,treated
PBMC transduced with the targeting vector resulted in about 8%
GFP
+
cells,whereasonlyabackgroundleveloftheGFPsignalwas
detected in the non-treated cells. This result is in agreement with
theTcelllinedataandconfirmsthattheengineeredLVcouldselec-
tivelymodifyEnv-expressingcellsinamixedpopulation,omitting
theconventionalstepofpurifyingtargetcells.
3.10. Evaluation of the potential of LV-mediated suicide gene
therapy in vitro
To demonstrate the potential utility of the Env-specific LV
described above, we constructed the targeting vector encoding a
thymidine kinase gene derived from Herpes Simplex Virus type 1
(HSV1-TK), a widely used suicide gene, and assessed its efficacy
in eradicating HIV-1 Env-expressing cells. The working mecha-
nismofthissuicidegeneapproachisthattheotherwisenon-toxic
prodrug ganciclovir (GCV), when supplied to the target cells, is
transformed into a toxic metabolite by the HSV1-TK expressed in
the cells to mediate specific killing (Blumenthal et al., 2007). The
HSV1-TK-encoding lentiviral backbone plasmid FUWSR39tk, con-
structed in our laboratory and reported previously (Ziegler et al.,
2008),wasusedinthisexperiment.ThemixedpopulationofEnv-
expressing Jurkat cells described above was challenged with the
FUWSR39tk/CD4+SGNvectorthreetimes,followedbyfurthercul-
tureinmediawithorwithoutthesupplementoftheprodrugGCV
foranadditionalsevendays.Itwasobservedthatmorethan30%of
theEnv-positiveTcellswereeradicated,whilethecontrol groups
(cells treated with GCV but without the vector, or cells treated
with the vector but without GCV) maintained a normal presence
of Env-expressing cells (Fig. 9D, left). The 7-aminoactinomycin D
(7-AAD) staining of various treated Jurkat cell groups confirmed
thattheEnv-expressingcellsexposedtoFUWSR39tk/CD4+SGNand
GCV had the highest ongoing apoptosis (Fig. 9D, right). Our data
demonstrated that it is possible to utilize this targeting LV carry-
ingthesuicidegeneincombinationwiththeprodrugtreatmentto
specificallyeliminateHIV-1-infectedcells.
4. Discussion
WehavedevelopedanovelLVthatdisplaystheHIV-1receptor
(CD4) and the FM molecule. This LV/CD4+FM vector is inefficient
in transducing normal cells, but can enter cells expressing HIV-1
Envproteinsandmediategenedeliverywithreasonableefficiency.
The vector can be produced by transient transfection of 293T
cells with an appropriate combination of plasmids harboring the
41
2.3.9 Evaluation of the potential of LV-mediated suicide gene therapy in vitro
To demonstrate the potential utility of the Env-specific LV described above, we
constructed the targeting vector encoding a thymidine kinase gene derived from Herpes
Simplex Virus type 1 (HSV1-TK), a widely used suicide gene, and assessed its efficacy
in eradicating HIV-1 Env-expressing cells. The working mechanism of this suicide gene
approach is that the otherwise non-toxic prodrug ganciclovir (GCV), when supplied to
the target cells, is transformed into a toxic metabolite by the HSV1-TK expressed in the
cells to mediate specific killing (Blumenthal et al. 2007). The HSV1-TK-encoding
lentiviral backbone plasmid FUWSR39tk, constructed in our laboratory and reported
previously (Ziegler et al. 2008), was used in this experiment. The mixed population of
Env- expressing Jurkat cells described above was challenged with the
FUWSR39tk/CD4+SGN vector three times, followed by further culture in media with or
without the supplement of the prodrug GCV for an additional seven days. It was observed
that more than 30% of the Env-positive T cells were eradicated, while the control groups
(cells treated with GCV but without the vector, or cells treated with the vector but
without GCV) maintained a normal presence of Env-expressing cells (Fig. 2.9D, left).
The 7-aminoactinomycin D (7-AAD) staining of various treated Jurkat cell groups
confirmed that the Env-expressing cells exposed to FUWSR39tk/CD4+SGN and GCV
had the highest ongoing apoptosis (Fig. 2.9D, right). Our data demonstrated that it is
possible to utilize this targeting LV carrying the suicide gene in combination with the
prodrug treatment to specifically eliminate HIV-1-infected cells.

42
2.4 Discussion
We have developed a novel LV that displays the HIV-1 receptor (CD4) and the
FM molecule. This LV/CD4+FM vector is inefficient in transducing normal cells, but can
enter cells expressing HIV-1 Env proteins and mediate gene delivery with reasonable
efficiency. The vector can be produced by transient transfection of 293T cells with an
appropriate combination of plasmids harboring the lentiviral backbone, packaging
proteins, CD4 and FM. The flow cytometry analysis confirmed the amazing ability of the
transfected 293T cells to co-express all of necessary proteins for making this designed
LV. The producer cells then bud vector particles containing CD4 and FM in the vector
membrane and release them into the medium supernatant. An in vitro transduction assay
revealed that the harvested supernatant containing LV/CD4+FM could efficiently
transduce HIV-1 Env-expressing cells and only a background level of transduction was
seen when this same vector was exposed to cells lacking HIV-1 Env. Various control
experiments verified that both CD4 and FM are necessary for the Env-targeted
transduction, and only background transduction was observed when vectors bearing
either CD4 alone or CD4 along with a non-relevant protein were used. This specific
transduction can be inhibited by the soluble CD4 protein or an endosome neutralization
reagent (ammonium chloride) added to the cells. In a mixed population of cells of which
half were Env-positive and half were Env-negative, LV/CD4+FM could selectively
modify Env-expressing cells and no transduction was detected towards cells lacking Env.
Several groups have reported various vector systems targeting Env-expressing cells using
a strategy termed inverse fusion, in which the HIV-1 receptor and co-receptor molecules,
CD4 and CCR5/CXCR4, respectively, are introduced into the surface of individual vector
43
particles, directing them to cells positive for HIV-1 Env (Bittner et al. 2002; Endres et al.
1997; Mebatsion et al. 1997; Peretti et al. 2006; Schnell et al. 1997; Somia and Verma
2000; Ye et al. 2005). Coordinated interactions between incorporated CD4 and
CCR5/CXCR4 on the vector surface with cell surface Env are sufficient to trigger Env-
mediated fusion and achieve targeted gene delivery. This mimics the natural infection of
HIV-1 to tar- get cells, but with a swapped configuration. Success of this inverse fusion
method has been demonstrated in vector systems derived from HIV-1 (Endres et al.
1997), rhabdovirus (Mebatsion et al. 1997), vesicular stomatitis virus (Schnell et al.
1997), and murine leukemia virus (Somia and Verma 2000). The designer vectors based
on this method, however, are limited to target cells infected with one tropism of HIV-1.
Vectors pseudotyped with CD4 and CCR5 can only target cells expressing Env derived
from the macrophage- tropic HIV-1 strain (R5 strain), while vectors enveloped with CD4
and CXCR4 are restricted to target cells expressing the T-cell tropic Env derived from the
X4 strain of HIV-1. As a control group in the current study, we confirmed this tropism-
limited targeting using the inverse fusion method (Figs. 4 and 5). In contrast, our system
does not involve the HIV-1 co-receptor and thus can overcome this tropism restriction.
We showed that the single LV/CD4+FM vector could target both the R5- and X4-tropsim
of HIV-1 Env and mediate gene delivery to cells expressing either type of Env with
similar efficiency.
We hypothesized that the entry of the targeting LV is initiated when the vector
carrying CD4 binds to the HIV-1 Env. Our vector cell binding assay confirmed that
vector-incorporated CD4 remained functional and was able to direct LVs to bind to
293T.EnvR5 cells. When soluble CD4 protein was added to the cells at the initial stage of
44
transduction, a dose-dependent reduction of overall transduction was obtained. These
data suggest that the interaction between vector CD4 and cell surface Env play a pivotal
role in initiating the targeting. We further postulated that the binding should induce
receptor-mediated endocytosis to uptake LVs into endosomal compartments, where the
low pH environment can prompt the conformation change of the vector-carrying FM
molecule and induce fusion between endosome and vector membranes (Joo and Wang
2008). Using an endosomal neutralization assay, we found that the transduction
efficiencies of the targeting vectors decreased with increasing concentrations of NH4Cl.
This trend indicates that the drop in pH is important for vector transduction. When the
three FM molecules (AKN, AGM, and SGN) were employed to test their ability to
mediate targeted transduction by LV, we obtained different transduction efficiencies.
Among them, the SGN vector (FUGW/CD4+SGN) was the most efficient vector. Similar
variations in targeting efficiencies were also observed in our previous studies (Yang et al.
2008a). These FM molecules are based on a binding-deficient and fusion-competent
version of the Sindbis virus glycoprotein with additional mutations in the E1 glycoprotein
domain. Although these additional mutations (AGM and SGN) were originally identified
from Sindbis viruses propagated in cholesterol-depleted cells and viruses carrying these
mutations were believed to be less dependent on cholesterol for fusion, we speculate that
these FM molecules might also have different responsiveness to the endosomal pH, with
SGN being the most responsive and fusion-active. Our reported vector–liposome fusion
assay actually supports this speculation, and SGN was found to be the most pH-sensitive
fusogen (Lei et al. 2010a). Nevertheless, the reduction in transduction efficiency by the
endosomal neutralization assay and the variations in transduction efficiencies among the
45
different FM molecules provide sufficient evidence that endocytosis and endosomal
trafficking must also be involved in the transduction process. The inhibition study by
various endocytosis-inhibitory drugs revealed that the targeting LVs enter HIV-1 Env-
expressing cells through clathrin-mediated endocytosis. Cells transfected to express the
dominant-negative dynamin mutant were more resistant to vector transduction, indicating
that the dynamin-dependent endocytosis pathway plays a crucial role in the
internalization of the engineered vector as well. When the dominant mutants of Rab5 and
Rab7 were introduced into target cells to regulate the maturation of endosomes at early
and late stages, respectively, the transduction efficiency was only affected by the Rab5
mutant, suggesting that the success of vector transduction is largely associated with the
trafficking in the early endosomes.
The therapeutic relevance of our HIV-1 Env-specific LV system was
demonstrated in a mixed population of T cells with some of them infected to express Env
proteins. We showed that Env- positive Jurkat cells could be targeted by LV/CD4+FM to
express the suicide gene HSV1-TK, which caused them to be susceptible to the prodrug
treatment. Our preliminary in vitro study showed that the targeting vector
FUWSR39tk/CD4+FM could significantly reduce the surface marker (LNGFR)-positive
subpopulation of human T cells cultivated in the presence of the prodrug GCV, indicative
of the elimination of HIV-1 Env-expressing cells because of the bio-cistronic nature of
the IRES-linked Env and LNGFR. Experiments are under way to further investigate the
reason as to why the targeting vector, in conjunction with GCV treatment, was unable to
completely eliminate LNGFR/Env-expressing cells under our current experimental
condition. This also raises an interesting question that if this method fails to eradicate
46
infected HIV-1 virus completely, what effects might the imposed selective pressure have
on the remaining virus reservoir? One logical possibility is that the selective pressure
may cause the virus to become a CD4-indpendent strain. Hoffman et al. has actually
identified such a strain capable of directly interact with the chemokine receptor CXCR4
to achieve infection (Hoffman et al. 1999); this CD4-independent strain was further
found to be more sensitive to neutralizing anti- body treatment. It will be interesting to
investigate whether such CD4-independent strains can emerge from our suicide gene
therapy.
In conclusion, we have shown that functional incorporation of CD4 and FM into
the envelope of LVs can be accomplished with resulting vectors having a rather selective
ability to target HIV-1 Env-expressing cells. Compared to the inverse fusion method, the
LVs bearing CD4 and FM exhibited higher transduction efficiencies and were able to
target a broader range of HIV-1 Env proteins irrespective of their CCR5 or CXCR4
tropism. Although our preliminary experiment in vitro showed the feasibility of such a
vector to selectively eliminate Env-expressing T cells through suicide gene therapy,
further investigations will reveal whether this gene delivery vector system has a practical
utility to specifically eradicate HIV-1-infected cells in vivo, thereby reducing the virus
load of HIV-1-infected patients.

47
Chapter 3. Mutagenesis of the E1 domain of Sindbis virus glycoprotein alters its pH-
dependent fusion
Portions of this chapter are adapted from: Chi-Lin Lee, Jason Dang, Kye-il Joo, and Pin
Wang
Mutations in the E1 domain of Sindbis virus glycoprotein at residues 75 and 237
individually or in combination can affect the viral pathogenesis. To understand the fusion
properties driven by these mutations, we constructed three fusogen molecules (FMs)
derived from the Sindbis virus glycoprotein (G75D, S237A and G75DS237A), and
investigated their fusion activity using a designed form of lentiviruses displaying
antibodies to exert binding function and these FMs to mediate fusion.
The G75D mutation enhances the transduction efficiency by broadening the range
of the pH threshold for fusion. Conversely, the S237A mutation impairs the infectivity
and its infectivity can be partially restored by utilizing G75D as second site revertant. We
further demonstrated that S237A requires the functional transport of its bearing viruses to
the late endosomes for virus fusion and infection, whereas G75D can initiate virus fusion
at both the early and the late endosomes.
Taken together, we shed some light on the mechanism for the mutagenesis of FMs
to result in changes of fusion behaviors and show that this lentivirus platform with
binding and fusion provided by two distinct molecules can be a useful tool for elucidating
the mechanisms of viral membrane fusion.

48
3.1 Introduction
The Sindbis virus is a prototype member of the genus alphavirus family, a group
of mosquito-transmitted encephalitis viruses that include such human and veterinary
pathogens as eastern, western, and Venezuelan equine encephalitis viruses (Strauss and
Strauss 1994). The Sindbis virus is one of the structurally well-defined enveloped viruses
commonly used in studies of virus entry, membrane fusion, and virus biosynthesis and
assembly. The virus is comprised of three major proteins, including the capsid protein C
and two envelope glycoproteins, E1 and E2 (Kielian 1995; Strauss and Strauss 1994).
Each viral surface contains 240 copies of E1 and E2 proteins organized in 80 spikes; a
single spike consists of a trimer of E2/E1 heterodimers. Sindbis viruses enter the host
cells by E2/E1 heterodimers through endocytosis. Previous studies have shown that the
E2 domain of the alphavirus virus envelope glycoprotein is primarily involved in the
interaction with target cell receptors (Kielian 2006; Kielian and Rey 2006; Sanchez-San
Martin et al. 2009).
After receptor binding, alphaviruses infect their host cells by membrane fusion
(Dimitrov 2004; Harrison 2008; Jahn et al. 2003; Kielian and Rey 2006). Viral envelope
proteins undergo a conformation change triggered by low pH within the lumen of the
endosomes. Various studies have indicated that the E1 domain contains the virus fusion
peptide, which is responsible for mediating fusion (Kielian and Rey 2006;
Mukhopadhyay et al. 2005). The acidic pH environment causes the E1/E2 dimer to
dissociate and then undergo a conformation change to form a stable, trypsin-resistant
homotrimer of E1 fusion proteins. The E1 trimer associates with the target membrane and
mediates the merging of the viral and target membranes. In vitro liposome fusion studies
49
demonstrate that the alphavirus fusion mechanism requires cholesterol (Kielian and
Helenius 1984; White and Helenius 1980) and sphingolipids (Nieva et al. 1994; Wilschut
et al. 1995) on the target membranes.
Johnston and his coworker have studied the mutagenesis of the E1 domain of the
Sindbis virus glycoprotein and identified two distinct El glycoprotein gene sequences
which affect viral pathogenesis in vivo (Polo and Johnston 1990). In the study, they
created constructs with aspartate substituted for glycine at residue 75, alanine for serine at
residue 237, and a combination of both sites in the E1 glycoprotein. Neonatal mice were
subcutaneously inoculated with the Sindbis viruses harboring these mutations to
determine their virulence. When compared with the wild type viral strain, the mutations
increased the mortality of treated mice from 23% to 98% (Rice et al. 1987). These
mutations in the E1 domain appear to play major roles in altering the viral pathogenesis;
however, the details of their fusion properties have not been characterized yet.
Recently, our laboratory has developed a virus platform to investigate molecular
fusion mediated by a variety of fusogen molecules (FMs) (Joo et al. 2010). This platform
is based on recombinant lentiviruses that utilize separate molecules for cell binding and
virus fusion on the same viral particle. As previously reported, this engineered lentivirus
displaying the CD20-specific surface antibody (αCD20) as the binding molecule and a
binding-deficient fusion-competent glycoprotein as the fusogen can achieve the specific
transduction of CD20-expressing cells in vitro and in vivo (Yang et al. 2006b). Based on
this platform design (Figure 3.1A), this separation of fusion and binding allows us to
compare the fusion properties of various FMs by producing viruses with the same
binding proteins but different FMs. This engineered virus system utilizes the same
50
internalization pathway mediated by the interaction between the binding protein and the
target receptor. Thus, we can readily incorporate different FMs in the system to
investigate their different fusion properties.
In this study, we exploited this αCD20-containing lentivirus to study fusion
properties of its displayed FM with mutations in the E1 domain of the Sindbis virus
glycoprotein. We found that the amino acid substitution in the E1 domain at residue 75
from a neutral and non-polar glycine (Gly, G) to a polar and acidic aspartic acid (Asp, D)
(G75D) can improve transduction efficiency on target cells. However, replacing serine
(Ser, S) with alanine (Ala, A) at residue 237 (S237A) decreases the viral infectivity; this
reduced infectivity can be rescued by introduction of G75D as a second-site revertants.
Cell-virus binding results showed that the binding affinity is similar for all of the viral
particles regardless of FM mutations. The liposome-virus fusion assay demonstrated that
the G75D mutant can induce fusion over a broader pH range. Thus, we have determined
that engineering of the fusion domain of the Sindbis virus glycoprotein can change its
fusion function by altering its pH-dependency. We further revealed that the G75D mutant
does not change its cholesterol dependency for fusion and blockade of either early or late
endosomes does not impair the ability of the G75D mutant to mediate the viral
transduction.

51
3.2 Methods
3.2.1 Plasmids Preparation
The plasmid encoding SINmu, the original binding-deficient fusogen molecule
derived from the Sindbis virus glycoprotein, was previously reported by our laboratory
(Yang et al. 2006b). The plasmids to express the human/mouse chimeric antibody against
the human CD20 antigen and the human antibody accessory proteins Igα and Igβ were
constructed previously (Yang et al. 2006b) using the pBudCD4.1 vector (Invitrogen,
Carlsbad, CA, USA). The HIV-1-based lentiviral vector FUGW was generated by Dr.
David Baltimore’s Laboratory (Lois et al. 2002) and used as the lentiviral backbone
plasmid in this study.
Based on the SINmu construct (pSINmu), we performed 4-primer PCR-
mutagenesis to generate mutants G75D, S237A, and G75DS237A. To generate G75D, a
forward primer (BclIfw, 5’-CAA CTC ACC GGA CTT GAT CAG ACA TGA CG-3’)
and a backward primer (G75Dbw, 5’-CCT TGC AGG TAT AGT CTG CAT GAG CG-
3’) were used to amplify the E1 domain containing the BclI restriction site and the
desired G75D mutation at the E1 residue 75. In parallel, a forward primer (G75Dfw, 5’-
CGC TCA TGC AGA CTA TAC CTG CAA GG-3’) and a backward primer (BsiW1bw,
5’-ACA ATT CGA CGT ACG CCT CAC TCA TCT G-3’) were used to amplify the E1
domain containing the desired G75D mutation at the E1 75 region and BsiW1 site. These
two products were PCR-assembled using BclIfw and BsiW1bw as the primer pair and
cloned into pcDNA3 (Invitrogen). The resulting plasmid was referred as pG75D. The
similar protocol was used to construct the mutant S237A and G75DS237A plasmid by
utilizing the following primer BsiW1fw (5’-CAG ATG AGT GAG GCG TAC GTC
52
GAA TTG T-3’), S237Abw (5’-CAT CTC AAA TCC TGA TGC GGC CTG CGT G-3’),
S237Afw (5’-GTA CAC GAG GCC GCA TCA GGA TTT GAG A-3’) and Mfe1bw (5’-
CCT GAA ACA TAT AAA ATG AAT GCA ATG TT-3’).
The wild-type Rab5 and Rab7 cDNAs were amplified by PCR and cloned into the
pDsRed- monomer-C1 (Clontech, Mountain View, CA, USA) to form the DsRed-
Rab5WT and DsRed- Rab7WT constructs. The plasmid encoding the dominant-negative
mutant of DsRed-Rab7DN (Rab7T22N) was created by site-directed mutagenesis using
the forward primer (5’- GTC GGG AAG AAC TCA CTC ATG AAC C-3’) and the
backward primer (5’- GGT TCA TGA GTG AGT TCT TCC CGA C-3’). The construct
for the dominant-negative mutant of DsRed-Rab5DN was obtained from Addgene
(Cambridge, MA, USA).
3.2.2 Cell lines
The 293T cells (human kidney embryonic cells with the Simian Virus 40 large T
antigen) were obtained from the American Type Culture Collection (Manassas, VA,
USA). The cell line 293T.CD20 was generated by transduction of vesicular stomatitis
virus glycoprotein-pseudotyped lentiviruses encoding the cDNA of the human CD20
protein. All of the cells were cultured in Dulbecco's modified Eagle's medium (Mediatech
Inc., Manassas, VA, USA) with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO,
USA) and 2 mm glutamine and maintained in a 5% CO
2
incubator at 37°C.
3.2.3 Virus production
The 293T cells were seeded in a 6-cm culture dish. After 18-20 hours, in which
the confluency reached approximately 80%, the seeded cells were transfected with DNA
53
plasmids using the standard calcium phosphate precipitation technique. The lentiviral
backbone plasmid FUGW (5 µg), the packaging plasmids pMGL (2.5 µg) and pRev (2.5
µg) (Tiscornia et al. 2006), the plasmids encoding the anti-CD20 antibody (pαCD20, 2.5
µg) and antibody accessory proteins (pIgαβ, 2.5 µg), and the plasmid pFM encoding
SINmu, G75D, S237A, or G75DS237A (2.5 µg), were mixed together for the
transfection. The virus supernatant was harvested 48 hours after transfection and filtered
though a 0.45-µm-pore size filter.
3.2.4 Cell-virus binding
293T.CD20 and 293T cells (0.2×10
6
) were incubated with 2 mL of the virus
supernatant (FUGW/αCD20+FM) at 4°C for 5 minutes. The cell-virus complexes were
washed with cold PBS and probed by anti-HA antibody (Miltenyi Biotec, Auburn, CA) to
stain the HA-tagged FM followed by Alexa594-conjugated streptavidin (Zymed
Laboratories, South San Francisco, CA, USA). The fluorescent staining results were
analyzed by flow cytometry (FACSort, BD Bioscience, San Jose, CA, USA).
3.2.5 Confocal imaging
A Zeiss LSM 510 META laser scanning confocal microscope equipped with
Argon, red HeNe, and green HeNe lasers as well as a Coherent Chameleon Ti-Sapphire
laser for multiphoton imaging was used to obtained fluorescent images. To image
individual viral particles, the fresh viral supernatants were overlaid upon poly-lysine
coated coverslips and centrifuged at 4°C for 2 hours. The coverslips were rinsed with
cold PBS twice, and adhered viruses were stained with Alexa594-conjugated anti-human
IgG (Invitrogen) and anti-HA-biotin (Miltenyi Biotec) followed by the secondary staining
54
using Texas red-streptavidin (Zymed Laboratories). The viruses were also
immunostained with a monoclonal antibody specific to the HIV capsid protein p24 (the
NIH AIDS Research and Reference Reagent Program). The coverslips were then
mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images were analyzed
using the Zeiss LSM 510 software version 3.2 SP2.
3.2.6 Virus transduction
The target cells (293T or 293T.CD20, 0.2 million) were plated in 24-well culture
dishes with virus supernatant (2 mL per well) and spin-transduced for 90 minutes at
2,500 rpm and 25°C. After transduction, the virus supernatant was replaced with fresh
media. The transduced cells were cultured for another 3 to 5 days at 37°C and 5% CO
2
.
The number of GFP
+
cells was determined by flow cytometry. Virus titer was estimated
by counting GFP expression in the virus dilution range in which GFP
+
cells and virus
volume exhibit a linear relationship.
For virus transduction with Rab protein-treated cells, 293T.CD20 cells were
transfected with DsRed-Rab5 or DsRed-Rab7 (either the wild-type or the dominant-
negative mutant), seeded, and spin-transduced with 2 mL virus supernatant. The
transduction results were determined by flow cytometric analysis of GFP
+
cells three
days post-transduction.
3.2.7 NH
4
Cl neutralizing
293T.CD20 cells (0.2 × 10
6
) were co-incubated with virus supernatants (FUGW/
αCD20+FM) and various quantities of ammonium chloride (NH
4
Cl) in a 24-well culture
dish at 37°C and 5% CO
2
for 8 hours, after which the media was replaced with fresh
55
media. After incubation for an additional 4 days, the GFP
+
cells were analyzed by flow
cytometry (FACSort, BD Bioscience).
3.2.8 Virus-liposome fusion
Liposomes were prepared by the extrusion procedure (Smit et al. 1999). 1,2-
dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL,
USA). Cholesterol (Chol) and sphingomyelin (SPM) from egg yolk were obtained from
Sigma (St Louis, MO, USA). The lipid mixtures consisted of PC/PE/SPM/Chol or
PC/PE/SPM were dried from a chloroform solution under a stream of argon gas and
further dried under vacuum for more than 3 hours. The lipid mixtures were hydrated in
HNE buffer (5 mM HEPES, 150 mM NaCl, and 0.1 mM EDTA, pH 7.4) and then
extruded 20 times through 0.2 µm pore size polycarbonate filters (Avanti Polar Lipids).
To monitor virus-liposome fusion, the concentrated viruses were incubated with 70 µM
of octadecyl rhodamine B chloride (R18) (Molecular Probes, Carlsbad, CA, USA) in
serum-free medium for 1 hour at room temperature. The labeled viruses were then mixed
with liposomes. The appropriate amount of acetic acid was added into the mixture
solution to achieve the desired pH for triggering the fusion. The dequenching signal of
R18 fluorescence was measured after acidification by Quanta-Master QM-4SE
spectrofluorometer (Photon Technology International, Lawrenceville, NJ, USA). The
initial fluorescence of virus-liposome mixtures was set at 0% fusion, and the 100% fusion
value was obtained by detergent lysis for each experiment using 0.1% of Triton X-100.

56

Figure 3.1 Schematic of the lentivirus platform and key constructs for the fusogen study.
(A) Schematic of the entry mechanism for lentiviruses displaying a CD20-specific
surface antibody (αCD20) and a fusogen molecule (FM). (B) Key constructs for making
the lentiviruses. The constructs include the fusogenic molecule (FM) derived from
Sindbis virus glycoprotein with indicated mutations at residues 75 and 237 of E1 domain,
the lentiviral backbone FUGW, the membrane-bound human/mouse chimeric antibody
against CD20 (αCD20), and the accessory proteins for surface expression of antibody
(Igαβ). CMV: human cytomegalovirus immediate-early gene promoter; E3: leader
peptide of Sindbis virus glycoprotein; E1: E1 protein of Sindbis virus glycoprotein for
mediating fusion; E2: E2 protein of Sindbis virus glycoprotein for binding to the
attachment receptor; HA tag: 10-amino acid epitope hemagglutinin sequences
(MYPYDVPDYA); Ubi: human ubiquitin-C promoter; GFP: green fluorescent protein;
WRE: woodchuck regulatory element; LTR: long-terminal repeat; ΔU3: U3 region with
deletion to disable the transcriptional activity of integrated viral LTR promoter; EF1α:
human elongation factor 1α promoter; αCD20κ and αCD20λ: light and heavy chains of
human/mouse chimeric antibody against CD20; TM: human antibody transmembrane
domain; Igα and Igβ: human antibody accessory proteins Igα and Igβ.

57
3.3 Results
3.3.1 Creating mutations in the E1 domain of the fuogen
We have previously demonstrated that the cell-specific targeted transduction
could be accomplished by incorporating a FM and an antibody into lentiviruses (Yang et
al. 2006b). The incorporated FM is derived from a binding-deficient Sindbis envelope
glycoprotein. The Sindbis envelope glycoprotein consists of two transmembrane domains
(E1 and E2); E1 is responsible for mediating the fusion and E2 is responsible for
directing the binding of virus to its attachment receptor. Our prior work demonstrated that
it is possible to introduce mutations in the E2 domain to engineer this FM to be more
active in triggering fusion (Lei et al. 2009). Polo and coworkers have previously shown
that the mutations in E1 region (Val72, Asp75, and Ala237) of the Sindbis envelope
glycoprotein could result in more virulent viruses in vivo (Polo and Johnston 1990). Their
studies identified residues Asp75 and Ala237 to be the major El determinants affecting
the pathogenesis. To further study the fusion behavior of these mutants using our unique
lentivirus platform (Joo et al. 2010), we introduced these mutations into our original
fusogen SINmu and designated them G75D, S237A, and G75DS237A, according to the
location and the identity of the amino acid change (Figure 3.1B).
3.3.2 Production of lentiviruses containing mutant FMs
To study these new FMs, we utilized a standard calcium phosphate protocol (Pear
et al. 1993) to generate the recombinant lentiviruses enveloped with a αCD20 and one of
engineered FMs. We co-transfected 293T cells with the backbone plasmid of lentiviruses
FUGW, the packaging plasmids (gag, pol and rev), the plasmid encoding αCD20, the
58
plasmid encoding the antibody accessory protein pIgαβ, and the plasmid encoding the
FM (SINmu, G75D, S237A, or G75DS237A) (Figure 3.1B). FUGW is the self-
inactivating lentivirus backbone derived from HIV-1. It contains an internal human
ubiquitin-C promoter driving the expression of a GFP reporter (Lois et al. 2002). As
established previously (Yang et al. 2006b), antibody accessory proteins (Igα and Igβ) are
necessary and sufficient for functional expression of antibodies on the surface of
producing cells for its subsequent incorporation into the viral particles. The resulting
lentiviruses from these transfected cells were designated as FUGW/αCD20+FM.
The negative controls included viruses bearing a FM and a non-relevant antibody
(B12) that is blind to the CD20 protein and were designated as FUGW/B12+FM.
Expression of GFP, antibody and FM were analyzed by flow cytometry (Figure 3.2). The
results showed that all of virus-producing 293T cells were able to express GFP encoded
by FUGW (Figure 3.2A & 3.2B, upper panel). Gating on GFP
+
cells, similar levels of
various FM expressions (~20% for FUGW/αCD20+FM; ~40% for FUGW/B12+FM)
were observed, suggesting that they were likely incorporated into lentiviruses with
similar efficiencies (Figure 3.2A & 3.2B, lower panel).

59

Figure 3.2 Co-expression of antibody and fusogen molecules on virus producing cell
surfaces. 293T cells were transiently transfected with plasmids FUGW, pαCD20, pIgαβ,
pFM, along with other standard packing plasmids to produce lentiviruses
(FUGW/αCD20+FM). The plasmid pB12 was used in transfection to generate control
viruses (FUGW/B12+FM). (A &B) FACS analysis of cells producing
FUGW/αCD20+FM (A) and FUGW/B12+FM (B). Upper panel: GFP expression (solid
line: transfected cells; shaded area, non-transfected controls); lower panel: expression of
the antibody and fusogen molecule in GFP
+
cells. The expression of antibody and
fusogenic molecule was detected by using anti-human IgG antibody and anti-HA
antibody, respectively.
60

Figure 3.3 Co-display of antibody and fusogen molecule on the surface of lentiviruses.
(A) Acquired confocal images of labeled viral particles incorporating FM and αCD20.
FM was stained by biotin-conjugated anti-HA antibody followed by a secondary staining
with Cy5-conjugated streptavidin (blue), and αCD20 stained by Alexa594-conjugated
anti-human IgG (red). The viral particles were also stained by anti-p24 antibody (green).
(B) FACS analysis of 293T and 293T.CD20 cells incubated with FUGW/αCD20+FM or
FUGW/B12+FM. The binding of virus to 293T.CD20 cells was detected by FACS
staining with the antibody against the FM. Solid line, analysis on cells incubated with
indicated viruses; shaded area (control), analysis on binding between viruses and 293T
cells.

61
3.3.3 Co-expression of fusogen and antibody on lentiviruses
To confirm that these mutant FMs were successfully incorporated onto
lentiviruses, we employed a confocal imaging method to analyze the co-incorporation of
FM and αCD20 (Joo and Wang 2008). The viruses were concentrated and adhered onto
glass slides, followed by sequential staining of the FM using an anti-HA-tag antibody
(blue color) and αCD20 using an anti-human IgG antibody (red color). To probe the viral
core, we further co-stained the p24 capsid protein with an anti-p24 antibody (Green
color). The co-localization of fluorescently stained viruses with signals for αCD20, FM,
and p24, shown by the merged colors, indicated that the mutant FM (G75D) along with
αCD20 can be displayed on the same viral particles (Figure 3.3A, upper panel). As a
negative control, no αCD20 and FM signal was observed from viruses pseudotyped with
the vesicular stomatitis virus glycoprotein (VSVG) (Figure 3.3A, lower panel). Similar
results were also observed for viruses bearing the other FMs (SINmu, S237A, or
G75S237A) (data not shown).
3.3.4 Interaction between the binding protein and the target receptor
To study the functional display of αCD20 when co-incorporated with FMs, we
performed a cell-virus binding assay. Various FM-containing viruses
(FUGW/αCD20+FM) were incubated with the target cells (293T.CD20), or the control
cells (293T), at 4 °C for one hour, after which the HA tag in the FM was stained and
analyzed. The flow cytometry signal could be detected when αCD20 and FM were both
incorporated on the same viral surface. As shown in the upper panel of Figure 3.3B, no
detectable signal was obtained when the 293T cells were incubated with
FUGW/αCD20+FM or FUGW/B12+FM viruses, indicating that the viruses were unable
62
to bind to the cells lacking the interaction between αCD20 and CD20. No signal was
detected either when FUGW/B12+FM were incubated with 293T.CD20 cells (Figure
3.3B, lower panel). Clear signals, however, were seen when 293T.CD20 and
FUGW/αCD20+FM were incubated together; the viruses containing different FMs
exhibited the similar binding intensity (Figure 3.3B, upper panel). This data corroborates
with the imaging study, showing the evidence of the co-incorporation of FMs and αCD20
and that expression of FMs dost not alter the binding capability of αCD20.
3.3.5 Transduction of lentiviruses bearing various FMs
Next, we investigated the ability of FM-bearing viruses to transduce CD20-
expressing cells in vitro. Four days post-transduction, GFP
+
cells were analyzed by flow
cytometry. The virus bearing the original fusogen (FUGW/αCD20+SINmu) was able to
transduce ~20% of 293T.CD20 cells, whereas viruses bearing new mutant FMs,
FUGW/αCD20+G75D, FUGW/αCD20+S237A, and FUGW/αCD20+G75DS237A, were
able to achieve the transduction efficiency of 73.6%, 17.5%, and 44.4%, respectively.
Various low levels of transductions (3~23%) of GFP
+
cells were observed when these
viruses were exposed to 293T cells (Figure 3.4A). To confirm that the incorporated
αCD20 on the virus surface was involved in mediating the selective transduction, we
exposed viruses bearing B12, an antibody blind to CD20, to 293T or 293T.CD20 cells
and found that these viruses only generated low levels (2~14%) of transduction (Figure
3.4A). The quantification of virus titers (Figure 3.4B) corroborated with the
aforementioned measurement of transduction percentages, indicating that G75D is the
most active fusogen to mediate lentiviruses for achieving the highest transduction
efficacy to target cells.
63

Figure 3.4 Transduction of FM-bearing lentiviruses in vitro. (A) 293T.CD20 and 293T
cells were transduced with 2 ml of fresh viruses (FUGW/αCD20+FM or
FUGW/B12+FM). Transduction efficiency was calculated as percentage of GFP
+
cells in
the population. Transduction to 293T cells was included as controls. (B) Titers of the
fresh viruses, in transduction units / mL, are shown for 293T and 293T.CD20 cells.

64
3.3.6 Study of virus entry by neutralizing endosomes
The transduction data indicated that the FM is the key determinant of the virus
infectivity and mutations in the E1 domain of the Sindbis virus glycoprotein can affect
the transduction efficiency. We designed an experiment to further investigate the possible
underlying mechanism responsible for the transduction differences among viruses
bearing various FMs. The lentiviruses were incubated with 293T.CD20 cells in the
absence or the presence of a range of concentrations of ammonium chloride (NH
4
Cl).
GFP
+
positive cells were analyzed by flow cytometry and normalized by transduction
levels without adding the chemical. Ammonium chloride is known to neutralize the
acidic endosomal environment (Mellman et al. 1986). In this study, the four viruses
displaying various FMs behaved differently in response to the concentration gradient of
NH
4
Cl (Figure 3.5). The virus bearing the S237A FM was the most sensitive to the
neutralization treatment and its transduction efficiency dropped the fastest with the
increasing environmental pH. The order of FMs for its resistance to NH
4
Cl treatment,
from low to high, was S237A, G75DS237A, SINmu, and G75D, with the G75D fusogen
showing the strongest resistance to NH
4
Cl treatment, which corresponded to the highest
virus transduction.

65

Figure 3.5 Effect of the neutralizing agent on the transduction by viruses bearing different
FMs.The indicated concentrations of NH
4
Cl were added during transduction for 8 hrs,
after which, the supernatants were replaced with fresh media. GFP-expressing cells were
analyzed by FACS 3 day post-transduction. Relative transduction efficiency was
calculated as percentage of GFP
+
cells in each concentration divided by the percentage of
GFP
+
cells in the absence of NH
4
Cl.

3.3.7 Liposomal fusion study of pH and cholesterol dependency of FMs
As shown in Figure 3.4, lentiviruses bearing different FMs exhibited different
transduction efficiencies. To further characterize the fusion properties of these FMs, we
66
adapted an experimental method to utilize a liposome-virus fusion assay to study the
fusion requirement of acidic pH environment and cholesterol within the endosomal
compartment (Smit et al. 1999). About 35 to 50% of the lentiviruses bearing SINmu,
G75D, or G75DS237A fused with liposomal particles at pH=5.5 (Figure 3.6A), whereas
only 25% fusion occurred for the S237A FM at this same pH. When the same experiment
was conducted at pH=6.2, only 10% of the viruses bearing SINmu fused with liposomes,
whereas much higher fusion (45%) was observed for viruses bearing G75D. The fusion
level for S237A and G75DS237A dropped to 20% at pH=6.2. S237A was the worst
fusogen regardless of the low or high pH conditions. Thus, this assay revealed that G75D
is the fusogen which can maintain the high level of fusion activity in low and high pH
conditions. There exists a correlation between transduction efficacy of viruses and the pH
responsiveness of their bearing FMs to trigger fusion; the fusogen with a broader fusion
window of pH displays higher transduction efficacy for its carrying viruses.
Previous studies have demonstrated the requirement of membrane components
such as cholesterol (Lu et al. 1999) and sphingolipids for the entry of Sindbis viruses into
their target cells. We thus evaluated the cholesterol dependence of our FMs. When these
FMs were mixed with the cholesterol-deficient liposomes, a markedly reduced fusion
(~20%) was observed even at low pH (pH=5.5). It seems that our introduced mutations in
E1 domain do not impact the cholesterol-dependent characteristics of the Sindbis virus
glycoprotein (Figure 3.6B).

67

Figure 3.6 pH- and cholesterol-dependent study of fusion activity of various FMs.
Lentiviruses (FUGW/αCD20+FM) bearing the different FMs were labeled with R-18 and
mixed with liposomes for 1 minute. (A) Virus-liposome fusion was induced by adding a
suitable amount of acetic acid and measured by the dequenching of fluorescent R18 using
a spectrofluorometer. (B) Liposomes were made either with or without cholesterol, and
then mixed with viruses bearing different FMs. Fusion activity was triggered by adjusting
the pH to be 5.5. The results of dequenching of fluorescent R18 were measured by a
spectrofluorometer.

3.3.8 Transduction inhibition by dominant-negative Rab5 and Rab7
Sindbis viruses are internalized into endosomal compartments, where they fuse
with either the early or the late endosomes. In order to determine which stage of the
endosomal compartments was essential for the successful transduction of the lentiviruses
bearing different FMs, the function of either the early or the late endosomes were
disabled in 293T.CD20 cells by transfection with the dominant-negative mutants of Rab5
(Stenmark et al. 1994), or Rab7 proteins (Press et al. 1998), respectively. We exposed
transfected 293T.CD20 cells with lentiviruses for transduction and the GFP expression
was analyzed by flow cytometry. Expression of the Rab5 dominant-negative mutant
68
reduced the transduction efficiency of SINmu- and G75DS237A-bearing viruses around
52% as compared to that of the transduction of wild-type Rab5-expressing cells (Figure
3.7A), suggesting that successful transduction for those viruses is at least partially
associated with the early stage of endosomes.

Figure 3.7 Transduction inhibition of viruses bearing various FMs by dominant-negative
Rab5 and Rab7.293T.CD20 cells transiently transfected with the wild-type (WT) or
dominant-negative mutant (DN) Rab5 (A) or Rab7 (B) protein, were spin-infected with
lentiviruses containing various FMs. Transduction efficiency was calculated as the
percentage of GFP
+
cells as determined by flow cytometry analysis. All transduction
efficiencies were normalized to infection with cells transfected with the wild type Rab5
or Rab7.

However, the transduction by G75D- and S237A-bearing viruses did not result in
significant inhibition on dominant-negative Rab5 expressing cells (Figure 3.7A),
indicating that the early endosomes are unlikely to be involved in infection of target cells
by viruses bearing these two FMs (G75D, S237A).
69
As shown in Figure 3.7B, cells expressing the dominant-negative Rab7 mutant
resulted in significant transduction inhibition of viruses bearing S237A, demonstrating
that the S237A virus requires a functional late endosome for successful infection.
Expression of mutant Rab7 also blocked transduction by SINmu-bearing viruses,
although to a less degree of inhibition than that of S237A viruses, suggesting that
infection by the SINmu virus is also associated with the late endosomal trafficking
(Figure 3.7B). We found that expression of the dominant-negative Rab7 in 293T.CD20
cells did not markedly alter the transduction by viruses containing G75D or G75DS237A
(Figure 3.7B), indicating that these viruses are not dependent on the late endosomes to
complete successful infection.
3.4 Discussion
The purpose of this study is to utilize the engineered lentiviruses as a platform to
compare the properties of various FMs derived from the mutagenesis of the Sindbis virus
glycoprotein. Previously we demonstrated a cell-specific targeting strategy by separating
the binding and fusion functions of the Sindbis virus glycoprotein (Yang et al. 2006b).
The entry mechanism of this engineered virus starts with the binding of αCD20 to CD20
antigen on the cell surface, upon which the virus is then internalized via endocytosis into
the acidic endosomes where the viral surface fusogen triggers membrane fusion (Joo and
Wang 2008). We postulated that this viral system could serve as a useful method to
directly study the fusion processes of various fusogens (Joo et al. 2010).
Previously, Polo and coworkers demonstrated three nucleotide differences by
sequence analysis of two recombinant Sindbis viruses, attenuated strain Toto1101 (Rice
70
et al. 1987) and virulent strain TR2000 (Polo et al. 1988), revealing amino acid
differences at residues 72, 75, and 237 in the glycoprotein E1 domain. They showed that
the substitution of Gly with Asp at residue 75 and Ser with Ala at residue 237 of the E1
domain individually or in combination increased the mouse mortality induced by
Toto1101 infection from 23% to 65%, 61% and 98%, respectively (Polo and Johnston
1990). To further characterize how these mutations altered the virulence, we preformed
site-directed mutagenesis to introduce single amino acid substitutions (G75D, S237A,
and G75DS237A) into the E1 domain of SINmu, a binding-deficient and fusion-
competent FM derived from the Sindbis virus glycoprotein of the Toto1101 strain, and
incorporated these mutant FMs into the CD20-containing lentiviruses. The expression of
the various FMs could be detected successfully and equally on the virus producer cells,
indicating that the mutations do not affect the assembly and transport of FMs to the cell
surface. The producer cells then synthesized viruses carrying αCD20 and FM in their
membranes and release them into the medium supernatant. Confirmed by confocal
imaging, these mutant FMs, along with αCD20, were able to be displayed on the surface
of the recombinant lentiviruses. The cell-virus binding assay confirmed that expression of
FMs on viruses did not impact the ability of αCD20 to mediate the selective binding to
CD20-expressing cells.
Our in vitro transduction assay revealed these novel FMs could alter the ability of
their bearing viruses to infect target cells. The highest transduction efficacy and virus titer
was given by the lentivirus bearing G75D, followed by viruses bearing G75DS237A,
SINmu, and S237A, in descending order. Since all viruses in this study utilized the same
binding protein to mediate endocytosis, the difference in transduction efficiency likely
71
resulted from the endosomal fusion kinetics of the different FMs. The endosomal
neutralization assay showed that the transduction of S237A virus is the most sensitive to
the pH increase in the endosomes. The SINmu- and G75D237A-bearing viruses
responded similarly to the neutralization treatment, whereas G75D virus was the least
responsive to the pH change and exhibited infectivity with the broadest pH window. It
appeared that the fusion responsiveness of FMs to the increasing pH inversely correlated
with the transduction of their bearing lentiviruses. To take this study further, we
employed the dominant-negative Rab5 and Rab7 proteins to examine the stages of
endsomes at which various FMs could initiate virus-endosom fusion. The dominant-
negative mutants of Rab proteins can disable the early (Rab5) and the late (Rab7) stages
of endosome function (Press et al. 1998; Stenmark et al. 1994). It was found that SINmu-
and G75DS237A-bearing viruses could fuse at the early endosomes for infection,
whereas the transduction of the S237A virus was largely associated with its functional
transport to the late endosomes. Both dominant-negative mutants failed to inhibit
infection by the G75D-bearing virus, consistent with the finding from the neutralization
assay that the G75D FM can fuse in a broader range of endosome stages with wider pH
conditions.
Previous reports indicated that certain mutations in the E1 domain of alphavirus
glycoproteins might improve the ability of viruses to fuse in endosomes (Gibbons et al.
2004; Kielian and Rey 2006; Lei et al. 2010b; Liu and Kielian 2009). Studies also found
that mutations in the fusion loop of the Semliki Forest virus envelope glycoprotein could
shift the pH threshold of fusion to a more acidic range (Levy-Mintz and Kielian 1991).
We postulated that those virulence-enhancing mutations for the Sindbis virus
72
glycoprotein might play similar roles in changing the energy barrier for the fusion event
and altering the pH threshold. Although our endosomal neutralization assay supports this
hypothesis, the direct evidence comes from the liposome-virus fusion assay, which
showed that while most of FMs (SINmu, G75DS237A, and S237A) were not very fusion-
active at pH=6.2, fusion of the G75D FM could be efficiently induced in this pH
condition. Comparing the results between the pH of fusion and the transduction
efficiency suggests that the FM that is more active at a higher pH can have better capacity
to mediate viral transduction. S237A has the lowest fusion activity and its bearing virus
infected cells poorly, suggesting that the amino acid at the 237 position of the E1 domain
plays an important role in fusion function and mutation in this position could have
detrimental effects. Interestingly, additional G75D mutation could partially rescue the
fusion function of the FM, resulting in the better transduction of the virus bearing
G75DS237A as compared with that of the virus bearing the original fusogen (SINmu).
The similar second-site revertant effect was also observed for the glycoprotein derived
from Semliki Forest virus (Chanel-Vos and Kielian 2006).
Some studies have demonstrated that the mutation on E1 domain of alphaviruses
could result in less dependence on cholesterol for viral transduction (Liu and Kielian
2009). We therefore conducted experiments to investigate the effects of these mutations
on the dependence of cholesterol for triggering fusion. Most mutations (SINmu, G75D,
or G75DS237A) required cholesterol for successful fusion induction at pH=5.5. No
obvious difference was found for the S237A-bearing virus to fuse with either cholesterol-
containing or cholesterol-null liposomes. This is likely due to its intrinsically low fusion
73
so that the liposome-virus fusion assay is not sensitive enough to detect their possible
differences.
In summary, we exploited the lentivirus platform to investigate the change of
fusion properties for the Sindbis virus glycoprotein with mutations, individually or in
combination, at E1 residues 75 and 237. The mutant fusogens have different pH
requirements for membrane fusion and exhibit different needs for their bearing viruses to
transport to distinct endosomal compartments for successful transduction. The G75D
mutation widens the pH threshold of fusion to a broader pH range and rescues the
impaired mutation at residue 237 (S237A). In addition to shedding some light on the
mechanism for the mutagenesis to result in changes of fusion behaviors, we have
demonstrated that this lentivirus system with binding and fusion functions provided by
two distinct molecules can be a useful technological platform to study the mechanisms of
viral membrane fusion and to design more efficient gene delivery vehicles.

74
Chapter 4. Construction of Stable Producer Cells to Make High-Titer Lentiviral Vectors
for Dendritic Cell-Based Vaccination
Portions of this chapter are adapted from: Chi-Lin Lee, Michael Chou, Bingbing Dai,
Liang Xiao, and Pin Wang. Biotechnology and Bioengineering (2012)
Lentiviral vectors (LVs) enveloped with an engineered Sindbis virus glycoprotein
can specifically bind to dendritic cells (DCs) through the surface receptor DC-SIGN and
induce antigen expression, thus providing an efficient method for delivering DC-directed
vaccines. In this study, we constructed a stable producer line (LV-MGFP) for
synthesizing DC-SIGN-targeted HIV-1-based LVs (DC-LVs) encoding green fluorescent
protein (GFP) by a concatemeric array transfection technique. We demonstrated that the
established stable clones could routinely produce vector supernatants with titers above
10
7
transduction units per milliliter (TU/mL) during a continuous 3-month cell passage.
The producer cells were also capable of generating similar titers of DC-LVs in serum-
free medium. Moreover, the addition of 1-deoxymannojirimycin (DMJ) enabled the
producer cells to manufacture DC-LVs with both improved titers and enhanced potency
to evoke antigen-specific CD8
+
T cell responses in mice. The stable lines could
accommodate the replacement of the internal murine stem cell virus (MSCV) promoter
with the human ubiquitin-c (Ubi) promoter in the lentiviral backbone. The resulting DC-
LVs bearing Ubi exhibited the enhanced potency to elicit vaccine-specific immunity.
Based on accumulated evidence, our studies support the application of this production
method in manufacturing DC-LVs for preclinical and clinical testing of novel DC-based
immunization.
75
4.1 Introduction
Among gene delivery systems, lentiviral vectors (LVs) derived from human
immunodeficiency virus type 1 (HIV-1) have gained considerable status in a variety of
applications by their capacity to achieve stable infection, maintain long-term transgene
expression, and transduce both dividing and nondividing cells (Kohn 2007; Naldini et al.
1996; Verma and Weitzman 2005). Since HIV-1 is the etiologic agent of AIDS, several
modifications have been made to improve the safety of HIV-1-based LVs by minimizing
the use of viral genes, thereby preventing the chance of recombination with a split-
genome design and avoiding the risk of replication with a self-inactivating (SIN)
configuration. SIN-based LVs, when paired with appropriate internal promoters, can
mitigate the risk of provirus mobilization and insertional mutagenesis (Hacein-Bey-Abina
et al. 2008; Howe et al. 2008) through deletion of the viral enhancer and promoter
sequences (Miyoshi et al. 1998a; Zychlinski et al. 2007). Such adjustments make LVs
more suitable for clinical studies.
Generally, HIV-1-based LVs are produced by transient transfection of the
packaging envelope and lentiviral transfer plasmids into mammalian cells, such as 293T.
Because of easy combination of different transfer plasmids with the packaging plasmids,
transient transfection endows enough flexibility of viral production to allow for the
testing of different vectors in a laboratory setting. However, such a production method is
cumbersome, and it is difficult to scale-up for preclinical and clinical applications
requiring large amounts of vectors, particularly those involving LV-based vaccine
delivery (Broussau et al. 2008; Hu et al. 2011). Several early reports have described some
successes in generating stable packaging and producer cell lines for the assembly of LVs
76
(Broussau et al. 2008; Cockrell et al. 2006; Ikeda et al. 2003; Kafri et al. 1999; Strang et
al. 2004; Strang et al. 2005). However, these systems cannot continuously produce high-
titer self-inactivating (SIN) vectors, and they lack an efficient method of integrating a
sufficient quantity of the transfer vector cassette into the packaging cells. To overcome
this hurdle, Gary and his co-workers created a new lentiviral packaging cell line termed
GPR, followed by the development of the concatemeric array-based transfection
approach to generate producer cell lines capable of stably producing high-titer SIN-based
LVs (Throm et al. 2009). The GPR packaging cell line utilizes an inducible tetracycline-
off (tet-off) system to limit the cytotoxic effect associated with the expression of rev
during the non-vector production phase (Blau and Rossi 1999; Lever et al. 2004). This
system was demonstrated to be efficient and robust for generating SIN-based LVs at
clinical scales (Throm et al. 2009).
Accumulating evidence suggests that LVs could be potent vaccine carriers to
induce antigen-specific immunity against infectious diseases and cancer (He et al. 2007;
Hu et al. 2011; Pincha et al. 2010). We have recently developed such a vectored vaccine
system and observed durable and robust immunity against the delivered immunogens
(Yang et al. 2008c). This LV system is unique in its directed delivery of antigens to
dendritic cells (DCs), which are the most powerful antigen-presenting cells (APCs) for
immediate immune responses. The targeting feature is accomplished by pseudotyping
LVs with an engineered Sindbis virus glycoprotein (designated as SVGmu) capable of
specifically binding to the DC-SIGN protein that is predominantly expressed on DC
surfaces (Byrnes and Griffin 1998; Strauss et al. 1994). Although the results from mice
are promising (Dai et al. 2009b; Yang et al. 2008c), we need to carry out vaccine-based
77
investigations in larger animals, such as non-human primates (NHPs) and humans, in
order to judge the full potential of this vector. This requires the development of a scalable
and reliable production method allowing the generation of sufficient vector materials to
conduct these intended studies.
In this report, we tested the concatemeric array-based transfection approach as a
possible method of constructing stable producer lines for making DC-directed LVs (DC-
LVs). We show that this method can generate stable lines that produce DC-LVs with high
titers (> 10
7
transduction units (TU)/mL) during a continuous 3-month culture.
Furthermore, these cell lines have the capacity to produce high-titer DC-LVs in culture
media with or without serum. The production of DC-LVs by inhibiting mannosidase in
producer cells by 1-deoxymannojirimycin (DMJ) resulted in even more potent vectors,
which translated into better antigen-specific T cell responses in vivo, as compared to
vectors produced without DMJ. Finally, we compared the DC-LV-producing stable lines
between those carrying the human ubiquitin-c (Ubi) promoter and those carrying the
murine stem cell virus (MSCV) promoter, and we found that they yielded vectors with
similar titers. Interestingly, however, when compared with the MSCV-driven vector, the
Ubi-driven vector could induce a stronger T cell response in vivo. Thus, our studies
demonstrate that this method of constructing stable cell lines is a robust and reproducible
means for routinely making DC-LVs with sufficient scalability for vaccine applications.

78
4.2 Methods
4.2.1 Plasmids
The cDNA of the DC-specific glycoprotein SVGmu was described previously
(Yang et al. 2006a; Yang et al. 2008b) and cloned downstream of the tTA-advanced
promoter in the retroviral plasmid pRetroX-Tet-Off (Clontech, Mountain View, CA). The
lentiviral plasmid TL20-GFP and bleomycin resistance (ble) cassette PGK-ble were
described previously (Throm et al. 2009) and kindly provided by Dr. John Gray. TL20-
Ubi-GFP was constructed by insertion of the human ubiquitin-C promoter (Lois et al.
2002) into the TL20-GFP to replace the MSCV promoter.
4.2.2 Cell lines
GPR cells were generously provided by Dr. John Gray. GPR and GPRS cells
were cultured in D10 (Dulbecco's modified Eagle's medium [Mediatech Inc., Manassas,
VA] with 10% fetal bovine serum [Sigma-Aldrich, St. Louis, MO], 2 mM L-glutamine,
1ng/mL doxycycline, and 2 µg/mL puromycin). The producer cells were maintained in
D10 with 50 µg/mL Zeocin (Invitrogen, Carlsbad, CA) and 1 ng/mL doxycycline
(Clontech Laboratories, Palo Alto, CA).
4.2.3 Vector production
The producer cells were washed with PBS buffer and seeded in D10 without
doxycycline. The seeded cells were supplemented with fresh medium daily. The vector
supernatant was harvested 72 hours after induction and filtered though a 0.45-µm pore
size filter.

79
4.2.4 Vector transduction in vitro
Target cells (293T.DCSIGN or 293T; 0.2 × 10
5
) were plated in 96-well culture
dishes with vector supernatant (100 µL per well) and spin-transduced for 90 minutes at
2,500 rpm and 25°C. The results were measured by flow cytometry analysis of GFP-
positive cells 4 days post-transduction. Vector titer was counted by GFP expression in the
vector dilution range when GFP-positive cells and vector volume exhibit a linear
relationship.
4.2.5 Mouse bone marrow-derived dendritic cells culture and transduction
The bone marrow cells were harvested from B6 mice to generate mBMDCs as
described before (Yang and Baltimore 2005a). mBMDCs were plated in a 24-well culture
dish and spin-infected with viral supernatant at 2,500 rpm and 25°C for 90 min. The
infected cells were cultured in fresh RPMI medium containing 10% FBS and GM-CSF
(1:20 J558L conditioned medium). The results were analyzed by flow cytometry.
4.2.6 Mice and immunization in vivo
Six- to eight-week-old female BALB/c mice were purchased from Charles River
Laboratories. The mice were injected with DC-LVs via a footpad route with the indicated
dose. Two weeks after immunization, the spleen cells were harvested and analyzed for
the presence of GFP-specific CD8
+
T cells using ICCS as previously described (Dai et al.
2009b).

80
4.3 Results
4.3.1 Generation of a Tet-dependent SVGmu Cell Line
HIV-1-based LVs pseudotyped with the SVGmu derived from the mutant Sindbis
virus glycoprotein targeting DC-SIGN-expressing cells have previously been produced
by the transient transfection method. These DC-LVs can efficiently deliver genetic
materials to DCs in vitro and induce a strong antigen-specific T cell response in vivo (Dai
et al. 2009b; Yang et al. 2008c). However, in order to develop a scalable production
system to meet the need for further vaccine investigations, we sought to construct stable
producer cells for making DC-LVs using the GPR packaging cell line. The GPR line is an
HIV-1-based packaging cell line derived from 293T cells with the necessary viral
components gagpol and rev (Throm et al. 2009).
To adapt the GPR to produce DC-LVs, we introduced the DC-specific envelope
(SVGmu) through γ-retrovirus-based transduction. To minimize the cytotoxicity of the
envelope protein, we constructed the γ-retroviral transfer plasmid Retro-SVGmu
encoding the SVGmu gene to be tightly regulated by the tet-off system (Blau and Rossi
1999) (Fig. 4.1B). The GPR cells were transduced with the Retro-SVGmu vector
pseudotyped with the vesicular stomatitis virus glycoprotein (Retro-SVGmu/VSVG) and
then maintained with doxycycline (Dox)-supplemented medium. The transduced cells
were stained with an anti-Sindbis serum and analyzed by flow cytometry. As shown in
Figure 4.1C, almost 100% of cells expressed SVGmu after the withdrawal of Dox. Still,
approximately 5% of the cells expressed SVGmu under Dox condition, possibly resulting
from leaky expression of the tet-off system (Farson et al. 2001). This cell line was
designated as GPRS and was used throughout this study.
81

Figure 4.1 Establishment of producer cell lines with the tet-off-regulated system. A:
Schematic diagram of the procedure to generate the packaging and producer cells for
making DC-LVs. B: Schematic representation of the retroviral plasmid Retro-SVGmu.
CMV: the cytomegalovirus promoter; P
Tight
: tet-responsive element; ΔU3: 3' moloney
murine leukemia virus LTR with a deletion in U3 region. C: GPRS and 293T cells were
induced by washing with PBS and culturing in medium without Dox. The induced cells
were stained by anti-Sindbis serum to detect the SVGmu expression and measured by
flow cytometry.
82

4.3.2 Construction of DC-LV Producer Cells by Concatemer Array Transfection
To generate DC-LV producer cells, the GPRS packaging cells were transfected
with lentiviral transfer plasmid TL20-GFP (Fig. 4.2A) using the standard calcium
phosphate protocol and concatemer array technique, followed by a drug selection method
(Throm et al. 2009). TL20-GFP is a self-inactivating lentiviral transfer vector plasmid
based on pCL20c-MSCV-GFP with a Dox-regulatable viral RNA genome expression
system, which was created by replacing the cytomegalovirus (CMV) enhancer with 7 tet
operators (Hanawa et al. 2004; Throm et al. 2009). The concatemer array technique was
exploited to optimize the transfected DNA to achieve a high integration number by
ligating the vector plasmid with a zeocin resistance plasmid, PGK-ble, in vitro at a 25:1
molar ratio. After transfection and drug selection, individual single cell clones were
evaluated for their ability to produce DC-LVs. As indicated in Figure 4.2B, significantly
higher titers of cell clones could be obtained from the first group (mean titer = 9.04 × 10
6

TU/mL), where these cell clones achieved 80% confluency in 10-cm culture dishes 7
days after single cell-derived colonies were picked. This was followed by a decrease of
average viral titers in the second (mean titer = 2.03 × 10
6
TU/mL) and third (mean titer =
1.78 × 10
6
TU/mL) groups, cultured for 11 and 15 days, respectively. A flow cytometric
histogram analysis was used to confirm the transduction property of the highest titer
clone (~ 3.3 × 10
7
TU/mL) by incubation of the viral supernatant with 293T.DCSIGN
cells (Fig. 4.2C). This clone was termed LV-MGFP and used for further studies.
83

Figure 4.2 Construction of stable lines to produce DC-LVs. A: Schematic diagram of
HIV-1-based lentiviral transfer plasmid TL20-GFP. GFP: enhanced green fluorescence
protein. TetO: Dox-repressible promoter; MSCV: murine stem cell virus promoter; ΔU3:
self-inactivating LTR. B: Distribution of vector titers of the culture supernatants
harvested from 26 selected producer clones. The amount of DC-LVs in the induced
culture medium was titrated on 293T.DCSIGN cells and analyzed by flow cytometry.
The mean titers for each group of cells with similar growth rate are indicated. C: Flow
cytometric analysis of 293T.DCSIGN incubated with either fresh medium (no vector) or
LV-MGFP/SVGmu produced by the most potent producer cell clone.

84

Figure 4.3 Kinetics of DC-LV production using the LV-MGFP cell line at two different
culture conditions. The producer cells were induced by withdrawal of Dox. The vector
supernatants were harvested every day and titrated against 293T.DCSIGN. A: Cultured in
serum-containing medium. B: Cultured in serum-free medium.

4.3.3 Production of DC-LVs by Producer Cells in Serum-free Medium
For clinical applications, culture medium containing no animal serum has several
marked advantages, including well-defined compositions, reduced contamination, and
lower costs (Broedel and Papciak 2003). In order to examine if our producer cell line LV-
MGFP could be adapted to make DC-LVs (designated LV-MGFP/SVGmu) in serum-free
conditions, LV-MGFP was induced by removing Dox and then tested in either serum-
containing or serum-free medium. The culture supernatant was harvested on a daily basis
and titrated on 293T.DCSIGN to assay the amount of infectious particles. The kinetics of
vector productivity was quite similar between serum-containing (Fig. 4.3A) and serum-
85
free (Fig. 4.3B) conditions. The vector titers were high in both culture media on the
second through fourth days post-induction with average titers above 10
7
TU/mL.
To confirm the long-term stability of vector production of our producer cell line, LV-
MGFP cells were continuously passaged for 3 months in the presence of Dox, during
which they were induced for DC-LV production by removal of Dox at two-week
intervals. The vector titers on day 3 post-induction were determined for each time point
tested. As shown in Figure 4.4, the vector titers did not change significantly, even after
94 days of culture, suggesting that the producer line was relatively stable throughout
prolonged culture. In addition, we implemented a protocol (Ikeda et al. 2003) to
determine the presence of replication-competent lentiviruses (RCLs) by transduction of
the GPRS cells with harvested DC-LVs and detected no RCL signal, confirming the
replication-incompetent nature of vectors produced by the LV-MGFP stable line.
4.3.4 Targeted Transduction of DC-LVs Produced by LV-MGFP in vitro
To evaluate the specificity of the DC-LVs (LV-MGFP/SVGmu) to transduce DC-
SIGN-expressing cells, the vector particles were harvested from producer cells 3 days
post-Dox removal and incubated with 293T.DCSIGN cells; the parental DC-SIGN
‒
293T
cells were used as a negative control. The percentage of GFP-positive cells was analyzed
by flow cytometry 5 days post-transduction to determine the vector titer. As shown in
Figure 4.5A, the titer of fresh, unconcentrated LV-MGFP/SVGmu was around 3 × 10
7

TU/mL on the 293T.DCSIGN cells, approximately 10-fold higher than that of the 293T
cells, indicating that vectors produced by the producer cells retained the capacity to
preferentially transduce DC-SIGN-expressing cells.
86
Next, we examined the ability of LV-MGFP/SVGmu to transduce mouse bone
marrow-derived DCs (mBMDCs). Flow cytometric analysis showed that LV-
MGFP/SVGmu could only transduce CD11c
+
DCs (Fig. 4.5B). To further confirm the
differences in the total functional GFP expression in CD11c
+
and CD11c
‒
cells, we
utilized the metric known as integrated mean fluorescence intensity (iMFI), which is
calculated by multiplying the percentage of GFP
+
cells with the mean fluorescence
intensity (MFI) to quantify the transduction efficiency (Lei et al. 2009). Compared to
CD11c
‒
mBMDCs, LV-MGFP/SVGmu demonstrated a 1000-fold higher iMFI in the
CD11c
+
mBMDCs (Fig. 4.5C), confirming their ability to selectively transduce DCs.
Figure 4.4 DC-LV production by stable producer cells after prolonged culture. LV-
MGFP cells were passaged for 3 months and induced by removal of Dox at 2-week
intervals. The vector supernatants were harvested 3 days after Dox removal and the titer
analyzed.

87

Figure 4.5 DC-LVs produced by the LV-MGFP stable clone can selectively transduce
DCs in vitro. A: 293T.DCSIGN (dark grey bar) or 293T (light grey bar) cells were
transduced with 100 µL of fresh unconcentrated viral vectors. Flow cytometric analysis
was used to analyze and calculate vector titers. B: Murine bone marrow-derived dendritic
cells (mBMDCs) were generated by culturing fresh bone marrow cells in the presence of
cytokines GM-CSF/IL-4 for 6 days. mBMDCs were transduced with the fresh vector
supernatant and then analyzed by flow cytometry for their GFP and CD11c expression.
C: Analysis of integrated mean fluorescence intensity (iMFI) on mBMDCs transduced by
the viral vector (LV-MGFP/SVGmu).

4.3.5 Production of LVs with Enhanced Efficiency to Target DCs via DMJ
As demonstrated from our previous study, addition of 1-deoxymannojirimycin
(DMJ) into cell culture during vector production can yield vectors with improved
capability to transduce DCs (Tai et al. 2011). This was accomplished by increasing the
amount of high-mannose structures present on viral envelope glycoproteins through
DMJ-mediated inhibition of class I α(1,2)-mannosidase activity. We postulated that DC-
LVs produced by the stable line under DMJ treatment could also enhance the capability
88
of vectors to target DC-SIGN-expressing cells. To test our hypothesis, DC-LVs were
produced with or without DMJ and incubated with 293T.DCSIGN. As shown in Figure
4.6A, FACS analysis confirmed that the transduction titer of LV-MGFP/SVGmu with
DMJ treatment was 9-fold higher than that of the vector without DMJ.
To determine whether LV-MGFP/SVGmu generated by producer cells could be
used to deliver immunogens to DCs for stimulating antigen-specific CD8
+
T cell
responses, naive mice were immunized with a single subcutaneous injection of GFP-
encoding vectors produced with or without DMJ. The GFP-specific CD8
+
T cells were
measured by intracellular cytokine staining (ICCS) of IFN-γ upon peptide restimulation.
A significant frequency (~ 3.7%) of IFN-γ-secreting CD8
+
T cells was detected at the
dose of 20 × 10
6
TU 2 weeks post-immunization (Fig. 4.6B). Furthermore, we found that
the production of the vector with DMJ resulted in a 33% increase in immune responses
compared to the vector produced without DMJ (Fig. 4.6B). These results indicate that 1)
LV-MGFP-based production of DC-LVs could be an effective method to elicit cellular
immune responses against the delivered antigen and 2) stronger antigen-specific T-cell
responses could be achieved by the treatment of stable producer cells with DMJ during
vector production.
89
Figure 4.6 DMJ can enhance transduction efficiency in vitro and immune responses in
vivo.A: The vector titers against 293T.DCSIGN for LV-MGFP/SVGmu produced either
with (dark grey bar) or without DMJ (light grey bar) treatment. B: BALB/c mice were
immunized with 20 × 10
6
TU of LV-MGFP/SVGmu produced either with or without
DMJ via the subcutaneous injection route. Two weeks post-immunization, spleen cells
were restimulated in vitro with the GFP-dominant peptide, and the IFN-γ response was
evaluated by ICCS.

4.3.6 Construction of Stable Producer Cells with Ubi Internal Promoter
Previous studies have demonstrated that LV transduction efficiency may vary,
depending on target cell types, viral backbone, and the encoded internal promoter (Dupuy
et al. 2005; Logan et al. 2004). We have shown that DC-LVs carrying a human ubiqutin-c
promoter (Ubi) as the internal promoter can modifiy DCs to efficiently express
transgenes in vitro and in vivo (Dai et al. 2009b; Yang et al. 2008c). Consequently, we
are interested in the possibility of replacing the internal murine stem cell virus (MSCV)
promoter with the Ubi promoter in the lentiviral backbone and investigating whether such
alteration could affect the construction of producer cells and their yield for making C-
LVs. Therefore, we constructed a new lentiviral transfer plasmid by replacing the internal
90
MSCV of TL20-GFP with the Ubi promoter and designated it TL20-Ubi-GFP (Fig.
4.7A). TL20-Ubi-GFP was introduced into the GPRS packaging cells by the same
method previously described and was followed by drug selection. Eleven isolated
colonies were picked, expanded, induced by removal of Dox, and screened by
transduction of 293T.DCSIGN. The vector titer from these 11 stable clones ranged from
5.56 × 10
3
to 1.83 × 10
7
TU/mL, with a mean of 8.51× 10
6
TU/mL (Fig. 4.7B). Most of
the clones yielded higher titers than the titer of Ubi-driven DC-LVs using the
conventional 4-plasmid transfection of 293T cells (1.72 × 10
6
TU/mL, Fig. 4.7B, dashed
line). The clone that made the highest titer vector (LV-UGFP/SVGmu) was designated as
LV-UGFP, and the ability of its produced vector for transducing 293T.DCSIGN was
validated by flow cytometric analysis of GFP expression (Fig. 4.7C).

91

Figure 4.7 Construction of producer cells for making DC-LVs bearing Ubi internal
promoter. A: Schematic diagram of TL20-Ubi-GFP lentiviral transfer plasmid. GFP:
enhanced green fluorescence protein; TetO: Dox repressible promoter; Ubi: human
ubiquitin-C promoter; ΔU3: self-inactivating LTR. B: Distribution of measured vector
titers of supernatants from 11 independent producer clones for making the LV-
UGFP/SVGmu vector. The vectors were titrated on 293T.DCSIGN cells and analyzed by
flow cytometry. The highest titer achieved for Ubi-bearing DC-LVs prepared using 4-
plasmid transient transfection of 293T cells is indicated by dashed line. C: Flow
cytometric analysis of 293T.DCSIGN incubated with either fresh medium (no vector) or
LV-UGFP/SVGmu harvested from the most potent producer clone.

4.3.7 Effect of Replacing MSCV with Ubi Promoter
To further investigate whether the different internal promoters encoded in vector
genome affect productivity of producer cells, the viral titers and immune responses of
DC-LVs generated by the stable lines bearing either internal MSCV or Ubi promoter
were directly compared. Vector production of LV-MGFP and LV-UGFP were induced by
Dox withdrawal. The vector particles were harvested and incubated with 293T.DCSIGN,
92
and the transduction titers were measured (Fig. 4.8A). The titer of infectious particles
produced by LV-MGFP was 70% higher than that produced by LV-UGFP (3.13 × 10
7

TU/mL vs. 1.82 × 10
7
TU/mL).
We next assessed the GFP-specific CD8
+
T cell immune responses evoked by
DC-LVs produced by the new stable lines (LV-MGFP and LV-UGFP). Naive mice were
immunized with a dose of 10 × 10
6
TU of LV-MGFP/SVGmu or LV-UGFP/SVGmu via
a single subcutaneous injection. The splenocytes harvested from vaccinated mice were
restimulated in vitro with the GFP-dominant peptide. The frequency of IFN-γ-producing
CD8
+
T cells was quantified by ICCS 2 weeks post-immunization. As shown in Figure
4.8B, vaccination by LV-UGFP/SVGmu resulted in stronger GFP-specific CD8
+
T cell
responses (~5%) than that by LV-MGFP/SVGmu (~3.3%). Collectively, our data
suggested that replacement of the internal MSCV promoter derived from viruses with the
non-viral Ubi promoter in the vector expression cassettes can slightly alter productivity of
producer lines and that the Ubi-bearing DC-LVs are more potent for eliciting transgene-
specific CD8
+
T cell responses in mice.

93

Figure 4.8 Comparison of transduction titer and immune responses with different internal
promoters. A: Producer cells LV-MGFP and LV-UGFP were induced by removal of Dox,
and supernatants were harvested at 3 days post-induction. The resulting LV-
MGFP/SVGmu and LV-UGFP/SVGmu were titrated on 293T.DCSIGN and analyzed by
flow cytometry. B: BALB/c mice were immunized with either 10 × 10
6
TU of LV-
MGFP/SVGmu or LV-UGFP/SVGmu via subcutaneous injections. Two weeks after
immunization, spleen cells were harvested, stimulated with the GFP-dominant peptide,
and analyzed for the frequency of IFN-γ
+
CD8
+
T cells by ICCS.

4.4 Discussion
In this study, we described a method to generate producer cell lines capable of
producing high-titer replication-deficient DC-LVs that specifically target DC-SIGN-
expressing cells and efficiently induce antigen-specific CD8
+
T cell responses in vivo.
DC-SIGN is one of major receptors predominantly expressed on the surface of DCs for
antigen uptake and viral entry (Zhou et al. 2006). As a vaccine carrier, LVs enveloped
with SVGmu derived from Sindbis virus glycoprotein have been shown to selectively
deliver genetic materials to DCs and potently elicit high levels of immunogen-specific T
cells responses (Dai et al. 2009b; Yang et al. 2008c).
94
Standard calcium phosphate transient transfection is the most widely used
technique for the production of LVs. However, variation in transfection efficiency and
large quantities of transfected plasmid DNA exacerbate the risk of assembling RCLs,
thus creating hurdles for the use of plasmid-based transient transfection methods for
generating clinical grade vectors (Pear et al. 1993). Scaled-up production requires large,
reproducible, and safe stocks of LVs (Cockrell and Kafri 2007). Such requirements could
be achieved by utilization of stable producer cell lines. Several groups have reported
various LV producer cell line systems, but most have proved to be unsuitable for the
production of SIN vectors as a consequence of their dependence on viral transduction to
introduce the vector backbone into packaging cells. However, this situation has been
changed by a recent report describing the new concatemeric array transfection method to
construct stable lines for the production of SIN LVs (Throm et al. 2009).
We therefore adapted this method to construct a packaging cell line for making
DC-LVs by retrovirus-mediated transduction of the parental GPR packaging cell line to
introduce the expression cassette of SVGmu regulated by the tet-off regulatory system
(Blau and Rossi 1999). The parental GPR line was constructed previously by transducing
293T cells with γ-retroviral vectors encoding HIV-1 helper genetic elements (Ikeda et al.
2003; Throm et al. 2009). The resulting GPRS line could efficiently express SVGmu on
cell surfaces upon the removal of Dox. Utilization of the tet-off regulatory system not
only mitigates the potential toxicity of constant expression of SVGmu, but also allows for
collection of DC-LVs under exogenous antibiotics-free condition. These features render
the GPRS line a promising candidate for generating stable producer cells capable of
making high-titer DC-LVs.
95
After concatemer-based transfection, our clonal selection revealed that vector
production by the producer lines decreased with decreasing cell growth. Thus, for this
stable producer system, vector productivity is largely associated with cell growth rate.
Recent studies of retrovirus-based producer lines reached similar conclusions with
respect to cell growth and vector productivity (Coroadinha et al. 2006; Lee et al. 1996).
The best of our selected producer clones, LV-MGFP, could generate vector titers up to 3
× 10
7
TU/mL for at least 3 months. An in vitro transduction assay revealed that LV-
MGFP/SVGmu produced by LV-MGFP could specifically transduce both 293T.DCSIGN
and mBMDC. Verification of T cell responses after a single subcutaneous injection of
LV-MGFP/SVGmu into naive mice demonstrated that the DC-LVs produced by LV-
MGFP could elicit a significant quantity of GFP-specific CD8
+
T cells. Furthermore, our
stable producer lines can be adapted to serum-free culture environments to make high-
titer vectors, which is an important benefit for facilitating large-scale production of DC-
LVs in a clinical setting.
Additional improvements in the production of potent DC-LVs could be achieved
by supplementing DMJ during vector production or replacing the internal promoter used
within the transfer vector cassette to Ubi. Previously, we showed that DMJ could enhance
the efficiency of SVGmu-bearing LVs for transduction of DCs by increasing the amount
of high-mannose structures present on SVGmu through the inhibition of cellular
mannosidase activity (Tai et al. 2011). In this study, we found that the DC-LVs produced
by LV-MGFP with DMJ resulted in a 9-fold increase in transduction of 293T.hDCSIGN
cells and 2-fold enhancement in GFP-specific CD8
+
T cell response compared to vectors
produced without DMJ. We then switched the internal MSCV promoter in the vector
96
backbone to the Ubi promoter and constructed a new producer line, LV-UGFP. The Ubi-
driven vectors produced by LV-UGFP yielded titers over 9-fold higher than those of Ubi-
driven vectors generated by the conventional 4-plasmid transfection of 293T cells and
also a 2-fold increase in GFP-specific CD8
+
T cell response over that induced by MSCV-
driven vectors in mice with a single injection of the same dosage. Nevertheless, the best
clone for producing Ubi-driven vectors could only achieve 58% of the highest titer of the
best clone for producing MSCV-driven vectors. This decrease of DC-LV productivity for
LV-UGFP could be explained by the difference in transcriptional interference, which is
stronger between Ubi and tetO than between MSCV and tetO. Similar positional effects
affecting transcription of adjacent sequences was also observed in a previous report (Osti
et al. 2006).
In conclusion, we demonstrated a method of generating robust producer cells
capable of making SIN-based LVs for such important applications as DC-based
vaccination. This production system will empower us to reliably produce vectors of high
quantity and quality for future testing of this DC-LV as a vaccine carrier in large animal
models, such as non-human primates. By removing the tedious transient transfection step,
this production method can be readily adapted into a GMP condition to manufacture
clinical grade materials for use in humans. Experiments are underway to evaluate the
therapeutic utility of this method against cancer and infectious diseases by constructing
stable cell lines for producing DC-LVs encoding clinically relevant antigens.

97
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Abstract (if available)

Abstract An important concept of gene therapy is the delivery of genetic materials to target cells for therapeutic benefit. One of the most important and efficient methods for gene delivery is the use of viral vectors as transfer vehicles. Lentiviral vectors (LVs) derived from human immunodeficiency virus type 1 (HIV-1) are promising vehicles for gene delivery because they not only efficiently transduce both dividing and non-dividing cells, but also maintain long-term transgene expression. In order to enhance the gene delivery ability of the viral vector, we designed a strategy by separating binding and fusion ability of envelope protein into two distinct proteins. By pseudotyping the viral vectors with both an antibody and a fusogenic molecule, we can target and transduce specific cell types. Based on this work, we developed a method to create LVs co-enveloped with the HIV-1 cellular receptor CD4 and a fusogenic protein derived from the Sindbis virus glycoprotein and tested its efficiency to selectively deliver genes into cells expressing HIV-1 envelope proteins. In chapter 2, we demonstrated that this engineered LV can preferentially deliver transgene to HIV-1 envelope-expressing cells in vitro. We conclude that this target LVs give a potential alternative treatment for eradicating HIV-1-infected cells that produce drug-resistant viruses after highly active antiretroviral therapy (HAART). In order to improve the LV transduction efficiency, we introduced the mutations in the E1 domain of Sindbis virus glycoprotein at residues 75 and 237 individually or in combination. The mutation at residues 75 from a neutral and non-polar glycine (Gly, G) to a polar and acidic aspartic acid (Asp, D) can enhance the transduction efficiency by broadening the range of the pH threshold for fusion. In chapter 3 we demonstrated our effort in enhancing the targeted transduction by genetically engineering the fusion component displayed on the viral membrane. To further test lentiviral vectors in preclinical or clinical studies, we constructed a stable producer line for synthesizing DC-SIGN-targeted LVs by a concatemeric array transfection technique could routinely produce vector supernatants with titers above 107 transduction units per milliliter (TU/mL) during a continuous 3-month cell passage. Based on our studies, this production method can generate DC-LVs for preclinical and clinical testing of novel DC-based immunization.

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Asset Metadata

Creator Lee, Chi-Lin (author)

Core Title Engineering lentiviral vectors for gene therapy and DC-vaccine

Contributor Electronically uploaded by the author (provenance)

School Andrew and Erna Viterbi School of Engineering

Degree Doctor of Philosophy

Degree Program Chemical Engineering

Publication Date 04/11/2012

Defense Date 02/09/2012

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

Tag human immunodeficiency virus,inducible gene expression,OAI-PMH Harvest,producer cell,self-inactivating lentivirus,T cell vaccine,targeted gene delivery,tetracycline

Language English

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

Creator Email chilinle@usc.edu,stevenlee.cl@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-25314

Unique identifier UC11287964

Identifier usctheses-c3-25314 (legacy record id)

Legacy Identifier etd-LeeChiLin-587.pdf

Dmrecord 25314

Document Type Dissertation

Rights Lee, Chi-Lin

Type texts

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA