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Current Gene Therapy, 2005, 5, 25-35 25
1566-5232/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.
Retroviral Gene Therapy: Safety Issues and Possible Solutions
Youngsuk Yi
1
, Sung Ho Hahm
2
and Kwan Hee Lee
1,3,
*
1
TissueGene Inc., Gaithersburg, MD 20877;
2
Omega Biosciences LLC, Baltimore, MD 21227;
3
Clinical Research
Center, College of Medicine, Inha University, Inchon, South Korea 400-711
Abstract: The recent incidents of leukemia development in X-SCID patients after a successful treatment of the
disease with retroviral gene therapy raised concerns regarding the safety of the use of retroviral vectors in clinical
gene therapy. In this review, we have tried to re-evaluate the safety issues related to the use of retroviral vectors in
human clinical trials and to suggest possible appropriate solutions to the issues. As revealed by the X-SCID
incident, oncogenesis caused by retroviral insertional activation of host genes is one of the most prominent risks.
An ultimate solution to this problem will be in re-engineering retroviral vectors so that the retroviral insertion
takes place only at the desired specific sites of the host cell chromosome. This is, however, a technically demanding
tasks, and it will take years to develop retroviral vectors with targeted insertion capability. In the mean time, the use
of chromatin insulators can reduce chances for retrovirus-mediated oncogenesis by inhibiting non-specific
activation of nearby cellular proto-oncogenes. Co-transduction of a suicidal gene under the control of an inducible
promoter could also be one of the important safety features, since destruction of transduced cells can be triggered if
abnormal growth is observed. Additionally, conditional expression of the transgene only in appropriate target cells
via the combination of targeted transduction, cell type-specific expression, and targeted local administration will
increase the overall safety of the retroviral systems. Finally, splitting of the viral genome, use of self-inactivating
(SIN) retroviral vectors, or complete removal of the coding sequences for gag, pol, and env genes is desirable to
virtually eliminate the possibility of generation of replication competent retroviruses (RCR).
Keywords: Retroviral, gene, therapy, safety, insertional, targeted, vectors, insulator.
INTRODUCTION
Retroviral vectors have been the most preferred gene
transfer systems in clinical gene therapy due to its well-
understood biology and its high efficiency of transduction
[Somia and Verma, 2000; Thomas et al., 2003]. In spite of
the popularity, the possibility of oncogenic transformation of
host cells as a result of non-specific insertion of retroviral
DNA into the host chromosome has always been a concern.
Although it is generally believed that a single insertional
mutation is not sufficient to develop a malignant phenotype
by itself, a leukemia induction has been reported in an
animal model [Li et al., 2003]. The potential risk of
insertional oncogenesis was realized in a human trial for the
X-linked severe combined immunodeficiency (SCID)
[Hacein-Bey-Abina et al., 2003; Kaiser, 2003; Kohn et al.,
2003]. In the trial, infants with X-SCID who would
otherwise have little chance of survival were actually cured
by retrovirus-mediated ex-vivo gene transfer, and the trial
was credited as the first unequivocal success for gene therapy
[Cavazzana-Calvo et al., 2000]. Unfortunately, however, two
out of nine successfully treated patients later developed
leukemia, and it is generally believed that leukemeogenesis
was triggered by unexpected activation of a cellular proto-
oncogene as a result of retroviral integration. Even though no
major adverse effects have been reported in many other
retroviral gene therapy clinical trials, including the ones
*Address correspondence to this author at the TissueGene, Inc. 209 Perry
Parkway, Suite 13, Gaithersburg, MD 20877, USA; Tel: 301-921-6000;
Fax: 301-921-6011; E-mail: ortholee@tissuegene.com
using more committed hematopoietic cells and mature
lymphocytes [Anderson, 2000], it is now time to re-evaluate
all the safety issues concerning the use of retroviral vectors
and to address them fully.
Stable incorporation of retroviral DNA into the host
genome is in itself advantageous, as long-term expression of
the transgene is possible which is often required for
achieving therapeutic efficacy. However, a non-specific
incorporation of viral DNA throughout the host genome can
either cause a disruption of a host gene at the site of
incorporation or cause an abnormal expression of nearby host
genes driven by the enhancer of the inserted viral DNA. If
the insertional interference occurs against a host gene
involved in a critical cellular function such as cell cycle
progression, it can become a major cause of cell
transformation and oncogenesis, especially in the presence of
additional physiological and/or genetic insults.
In addition to the risk of insertional mutagenesis, another
safety issue with the retroviral vector is the possibility of
generating replication competent retroviruses (RCR).
Although new generations of retroviral vectors have been
designed to reduce the production of RCR, additional efforts
are required to ensure the complete elimination of the
problem.
The recently developed lentiviral gene transfer system
shares many features of the retroviral system. The viral
genome integrates into host chromosomes, and the inserted
gene can be maintained in the cells permanently. In contrast
to conventional retroviral vectors requiring cell division for
26 Current Gene Therapy, 2005, Vol. 5, No. 1 Lee et al.
infection, lentiviral vectors can infect efficiently non-
dividing cells and dividing cells. Therefore, it can be applied
for transgene expression in neuronal cells. Many of the
lentiviral vectors used in gene therapy are based on the
human immunodeficiency virus (HIV). The major limita-
tions of using lentiviral vectors in clinical trial are the safety
concerns related to their HIV origin. Recent trials of
lentiviral vector system will be described in relevant
chapters.
In this review, we will try to discuss about
aforementioned and additional safety issues relating to the
use of retroviral vectors as a tool for therapeutic gene
delivery, and to suggest possible available solutions to these
issues and future directions for an overall increase in the
safety and efficiency of retroviral gene therapy protocols.
Other important parameters in cell-based ex-vivo retroviral
gene therapy protocols are already well covered elsewhere
[Baum et al., 2003], including cell type and numbers,
observation period, proliferation capacity, age of patients,
immunity, and side effects of transgene expression.
TARGETED RETROVIRAL TRANSDUCTION
Current retroviral transfer system lacks specificity for
target cell types. It makes the system unsuitable for
protocols requiring gene
transfer to particular cell types in a
mixed cell population. Therefore, vector targeting
is highly
desirable and will become a prerequisite for certain ex vivo
gene therapy
protocols.
Infection of target cells by retroviruses is initiated by
binding of the viral envelope protein to cell surface receptors
[Hunter and Swanstrom, 1990]. Infusion of viral and cellular
membranes leads to the internalization of the viral core
[White, 1992]. In the past years, various retrovirus receptors,
coreceptors and cofactors have been identified and studied for
their role in viral entry [Overbaugh et al., 2001], and
attempts have been made to engineer viral envelope proteins
and cellular receptors for attaining changes in the viral
tropism [Ting et al., 1998; Chaudry et al., 1999].
In an approach for a targeted transduction, envelope
protein modification by attaching a peptide ligand such as
epidermal growth factor (EGF) receptor binding domain to
the NH
2
-terminus of the envelope glycoprotein (SU) was
attempted [Cosset et al., 1995; Peng et al., 1997; Peng et
al., 1999]. Incorporation of the chimeric envelope protein
into the viral particle allows binding of retrovirus to the
receptor-positive target cells. The subsequent viral entry
steps are blocked, however, because EGF receptors do not
support these processes. A protease cleavable linker was used
in this chimeric protein to join the peptide ligand and SU,
so that an appropriate cellular protease can cleave the attached
ligand. Factor Xa [Nilson et al., 1996; Fielding et al.,
1998], plasmin [Peng et al., 1999], matrix-metalloproteases
(MMPs) [Peng et al., 1997], or intracellular protein conver-
tases [Buchholz et al., 1998] were used for this purpose.
Because overexpression of MMPs is frequently associated
with angiogenesis, inflammation, and cancer invasion,
MMPs are considered to be interesting targets for the
protease-activatable gene delivery systems. Using MMP-
activatable retroviral vectors, selective transduction of MMP-
rich tumor cells was achieved in a heterogeneous cell
population, but with somewhat reduced efficiency of
transduction [Peng et al., 1999; Schneider et al., 2003].
Similarly, targeted infection for the high-molecular-weight
melanoma-associated antigen (HMWMAA) expressing
tumors was achieved by fusing a single chain antibody
recognizing HMWMAA to the amino terminus of the surface
domain of MLV with a matrix metalloprotease-2 (MMP2)
cleavage site linker [Martin et al., 2002].
Matrix targeting is another approach and matrix-targeted
retroviral vectors were found to be more efficient than
unmodified vectors [Gordon et al., 2000; Hall et al., 2000].
A matrix-targeted retroviral vector was constructed by
attaching collagen-binding polypeptide sequence to the
Table 1. Retroviral Vectors for Targeted Infection
Modification Target cell references
EGF-MMP cleavable linker chimeric env Cancer invasion, angiogenesis, inflammation [Peng et al., 1997; Buchholz et al., 1998; Peng et
al., 1999]
IL-2 chimeric env IL-2 R [Maurice et al., 1999]
EGF chimeric env EGFR [Cosset et al., 1995]
SCF-Factor Xa chimeric env Stem cell (Kit) [Fielding et al., 1998]
vWF (collagen binding) chimeric env Cancer (collagen expressing); vascular lesion [Gordon et al., 2000; Hall et al., 2000]
Single-chain variable fragmented antibody
(scFv) for EGFRvIII
Cancer (brain, breast, lung, ovary) [Lorimer and Lavictoire, 2000]
scFv for HMWMAA Cancer [Martin et al., 2002]
scFv from phage display T cell [Engelstadter et al., 2000]
scFv for Carcino embryonic antigen (CEA) Cancer [Khare et al., 2001]
Receptor pseudotyping (CD4 and CXCR4) HIV-1 infected cell [Somia et al., 2000; Bittner et al., 2002]
Retroviral Gene Therapy Current Gene Therapy, 2005, Vol. 5, No. 1 27
amino-terminal region of the amphotropic 4070A envelope
protein. Because tumor development and accompanied
angiogenesis are associated with remodeling of extracellular
matrix components, these vectors accumulate at sites of
tumor development with newly exposed collagens. When the
matrix-targeted retroviral vector expressing dominant mutant
cyclin G1 was administered by portal vein infusion, vector
particles accumulated in the angiogenic tumor vasculature
within 1 hour of infusion. These vectors transduced tumor
cells with high efficiency and reduced the volume of tumor
[Gordon et al., 2000].
Targeting retroviral delivery to quiescent interleukin-2
(IL-2)-dependent cells was also reported [Maurice et al.,
1999]. In this report, chimeric amphotropic MLV envelope
glycoprotein fused with IL-2 was used for a direct binding of
the viral particles to the IL-2 receptors expressed on G0/G1
arrested cells, resulting in a transient stimulation of cell
proliferation. Subsequent viral entry was mediated by
unmodified envelope proteins co-expressed on the same virus
particles. A 34-fold increase in transduction efficiency was
observed with this method. Additionally, targeting efforts
for T cells [Engelstadter et al., 2000], and other cancer cells
[Lorimer and Lavictoire, 2000; Khare et al., 2001] were
reported. For the specific transduction of HIV-envelope
expressing cells, envelope pseudotyping was used to create
hybrid CD4/CXCR4 receptors for MLV retrovirus [Somia et
al., 2000; Bittner et al., 2002] and lentivirus [Somia et al.,
2000; Bittner et al., 2002], In order to improve transduction
efficiency frequently observed to be low for the targeted
retroviral vectors, binding defective but fusion competent
hemagglutinin (HA) protein has also been tried [Lin et al.,
2001].
LOCAL DELIVERY
If local delivery of retroviral vectors is available for an
effective treatment of a disease, it will be generally safer than
systemic delivery in terms of toxicology and long term side
effects. A number of studies have shown the efficacy and
safety of locally delivered retroviral vectors. A retroviral
vector expressing antiproliferative dominant negative mutant
cyclin G1 (dnG1) was successfully used for the prevention of
eximer laser-induced corneal haze [Behrens et al., 2002].
Biodistribution study after the treatment of surgically
induced rabbits with eye drops containing dnG1 retroviral
vectors showed no evidence of vector dissemination in non-
target organs. Localized delivery of lentiviral vectors into the
substantia nigra of adult rats has also been tried [Deglon et
al., 2000]. In a phase I clinical trial for direct intratumoral
injection of interferon-γ retroviral vectors in advanced
melanoma patients, viral injection was well tolerated and no
toxicity was reported [Nemunaitis et al., 1999]. This
suggests that the direct injection approach is feasible for
treating solid tumors with retroviral vectors.
In terms of potential problems associated with
concomitant transduction of surrounding non-target cells, ex
vivo cell-based gene therapy with local delivery can be a
better choice, if extra time and expenses are tolerated. As an
example, an ex vivo cell-mediated gene therapy trial has been
performed successfully for the treatment of artificially
induced hyaline cartilage damage in animals, by injecting
TGF-β1-retrovirus transduced fibroblasts into the knee joints
[Lee et al., 2001].
INTEGRATION OF RETROVIRUS INTO THE HOST
CHROMOSOME
In the ex-vivo gene therapy trials to treat the rare immune
deficiency disorder X-SCID, patients were treated with
autologous hematopoietic stem cells transduced with a
recombinant retrovirus expressing the common gamma chain
(γ
c
) of interleukin receptor [Cavazzana-Calvo et al., 2000].
Although, nine out of eleven treated children showed
dramatic improvements with almost fully restored immune
systems, two of the nine cured patients developed leukemia
three years after the treatment. A copy of the vector DNA
was found in the first intron of the growth-promoting LMO2
gene of the leukemic clones in the first patient, and
approximately 3 kb upstream of the first exon of the same
gene in the second [Hacein-Bey-Abina et al., 2003; Kaiser,
2003; Kohn et al., 2003]. LMO2 is a LIM domain
transcription regulator involved in hematopoiesis [Yamada et
al., 1998] and is reported to be activated in T cell leukemia
by chromosomal translocation [Royer-Pokora et al., 1991].
Deregulation of LMO2 expression can affect hematopoiesis
and lymphocyte development, and consequently can be the
cause of leukemic transformation. However, it is not yet
clear if deregulation of LMO2 alone was solely responsible
for the oncogenic transformation. Other contributing factors
such as immune deficiency shared by the two X-SCID
patients or the expression of the therapeutic transgene
(interleukin receptor γ
c
) itself, by mediating proliferative
cellular signaling events, might also have contributed in the
transformation process.
The U.S. Food and Drug Administration (FDA) put a
hold on U.S. trials using retroviruses to insert genes into
blood stem cells, and the U.S. National Institutes of Health
(NIH) Recombinant DNA Advisory Committee (RAC) urged
about 90 other retroviral trials targeting blood cells to
consider a pause. Subsequently, a public meeting was held
on February 10, 2003, and the NIH RAC made the
following tentative recommendations [Friedmann, 2003a].
1. Pending further data or extenuating circumstances
reviewed on a case-by-case basis, retroviral gene transfer
studies for X-linked SCID should be limited to patients
who have failed identical or haploidentical stem-cell
transplantation or for whom no suitable stem cell donor
can be identified.
2. There are not sufficient data or reports of adverse events
directly attributable to the use of retroviral vectors at
this time to warrant cessation of other retroviral human
gene transfer studies, including studies for non-X-linked
SCID. Such studies may be justified contingent upon
appropriate risk/benefit analysis accompanied by
implementation of appropriate informed consent and
monitoring plans.
The general consensus of the experts of the review boards
in the US and other countries is that the benefits might
outweigh the risks in most SCID-related retroviral gene
therapy trials. FDA has since announced its willingness to
remove the clinical hold on most retroviral gene therapy
28 Current Gene Therapy, 2005, Vol. 5, No. 1 Lee et al.
trials involving hematopoietic stem cells [Friedmann,
2003a]. These trials will likely be allowed to continue with
several necessary precautions as advised by the NIH RAC
meeting held on June 18-19, 2003 [Friedmann, 2003b].
These include a complete integration analysis, modification
of vector design to become self-inactivating (SIN),
conditional expression of the transgenes, incorporation of
suicidal elements or silencers, longer-term studies in large
animals, and close monitoring of clonally expanding cells.
Although FDA has not yet announced specific
recommendations for other non-SCID retroviral gene therapy
trials, Gene Therapy Advisory Committee (GTAC) and
Committee on Safety of Medicines (CSM) Working Party
on Retroviruses in United Kingdom excluded other retroviral
gene therapy trials from the intensified watch list.
TARGETING RETROVIRAL INTEGRATION
After entering the host cell, a single-stranded retroviral
RNA genome is released into the cytoplasm and converted
into a double-stranded DNA by virus-encoded reverse
transcriptase. The viral DNA then forms a large
nucleoprotein structure, termed pre-integration complex,
containing proteins necessary for nuclear localization and
insertion of viral DNA into the host genome. Although the
protein components and the exact mechanism of action of the
complex is still not completely understood, it has been
demonstrated that viral integrase (IN) catalyzes
the key DNA
cutting and joining reactions for inserting viral
DNA into the
host genome [Katz and Skalka, 1994; Wei et al., 1997].
Recent experimental evidence suggests that retroviral
integration is not a completely random process but favors
sites of active gene expression [Schroder et al., 2002; Wu et
al., 2003]. Murine leukemia virus (MLV) can only infect
actively dividing cells in which proto-oncogenes play major
roles in cell-cycle progression or other important cellular
processes. This means that the risk of retrovirus insertion-
mediated abnormal regulation of cellular proto-oncogenes can
be much higher than anticipated. In addition, while MLV
prefers integration near the start of transcriptional units, HIV-
1 integrates anywhere in the transcriptional units [Schroder et
al., 2002; Wu et al., 2003]. The difference of integration
preferences indicates that there are possible fundamental
differences between MLV and HIV for the mechanism of
integration. Elucidating the mechanisms of integration and
establishing the database for preferred integration sites could
permit a better prediction of the integration sites of retroviral
vectors. This may eventually lead to the development of
retroviral vectors capable of integrating to the specific sites
of the host cell chromosome, providing the ultimate solution
to the problems of insertional mutagenesis.
There have been attempts to target retroviral integration
to pre-selected locations of the host genome by fusing viral
integrase with sequence-specific DNA binding domains
obtained from phage lambda repressor, bacterial LexA, or a
zinc finger protein zif268 [Goulaouic and Chow, 1996; Katz
et al., 1996; Bushman and Miller, 1997]. However, these
trials show only a limited success, as the specificity of
integration was only partially altered. In a separate
experiment, Bovine Leukemia Virus (BLV) integrase was
used for site-specific integration of naked DNA to the pre-
integrated integrase recognition sequence of mouse genome
[Tanaka et al., 1998]. Similarly, site-specific integration of
naked DNA into human chromosome 8 has been attempted
with limited success using modified phage φC-31 integrase
[Sclimenti et al., 2001]. In this study, enhanced sequence
specificity and increased integrase efficiency was achieved
through a directed evolution strategy. It is clear that
concentrated efforts are required in defining the precise
mechanism of action of the retroviral pre-integration complex
and in designing modified integrases with sequence-specific
integration capability. The latter may be accomplished either
by rational modification of the protein or by using the
directed evolution approach [Chen, 2001]. One example of
rational modification is fusing integrase with synthetic zinc
finger motifs with defined sequence specificities [Bushman
and Miller, 1997; Kim and Pabo, 1998; Jamieson et al.,
2003]. Directed evolution utilizes error-prone PCR-driven
mutagenesis, recombination, or DNA shuffling, combined
with a high throughput screening for the selection of
modified proteins with significantly improved function. The
newly developed integrases should also maintain the ability
to form a pre-integration complex with a high-level of
infection capability.
INSULATORS TO PREVENT POSITIONAL
EFFECTS AND INSERTIONAL ONCOGENE ACTI-
VATION
Retroviruses are often susceptible to positional effects
and transcriptional silencing depending on the site of
integration in the chromosome [Pannell and Ellis, 2001]. In
order to overcome positional silencing effect, chromatin
insulators have been used in retroviral vectors. Chromatin
insulators are believed to form expression boundaries [Sun
and Elgin, 1999; Burgess-Beusse et al., 2002] and can block
positive and negative positional effects at the site of
integration when they flank a transgene [Kellum and Schedl,
1991; Chung et al., 1993; Chung et al., 1997]. They
prevent interferences between promoters and enhancers of
adjacent genes [Labrador and Corces, 2002]. As an example,
when a 1.2 kb chromatin insulator obtained from the chicken
β-globin locus control region hypersensitive site 4 (cHS4)
was inserted in the retrovirus 3’ LTR, protection of the
positional effects was observed from transduced cultured
cells and from the mice transplanted with transduced marrow
cells [Emery et al., 2000]. Similarly, cHS4 insulator used
with gamma-globin expression cassette increased the
likelihood of stable gamma-globin expression nearly 10-fold,
allowing for the expression at the therapeutic range for
treating sickle cell anemia and beta thalassemia in mouse
bone marrow transplantation models [Emery et al., 2002].
Because sequences in the 3’ LTR of retroviral vectors are
copied to the 5’ LTR during the processing of viral genome
into the provirus, if an insulator is inserted in the 3’ LTR of
the recombinant vector, a barrier of insulators will be formed
surrounding the transgene (Fig. (1A)). In addition, insulators
are inserted in the place of the U3 region of the 3’ LTR,
which is the viral enhancer region. These vectors will
become self-inactivating (SIN) in the proviral form, as there
is no viral enhancer required to produce replication
competent retrovirus. Therefore, insulator containing
retroviral vectors will be less prone to silencing of the
Retroviral Gene Therapy Current Gene Therapy, 2005, Vol. 5, No. 1 29
transgene expression as a result of chromosome positional
effect [Emery et al., 2000], and at the same time, will have
less chance of causing aberrant induction of host genes near
the site of incorporation as it has no viral enhancer.
Boundaries formed by insulators will also prevent the
influence of an internal heterologous enhancer used to drive
transgene expression on the transcription of nearby host
genes, although it has yet to be experimentally proven.
Although retroviral insertion can cause either a disruption
or an abnormal activation of host genes, the latter is a
primary concern in terms of oncogenesis. This is because, in
most cases, retroviral insertion will occur in only one allele
of the host genome leaving the other locus intact, and
insertional disruption of the host gene will become
problematic only in rare cases where haplo-insufficiency is
phenotypically relevant for oncogenic transformation. Thus,
although chromatin insulators cannot prevent host gene
disruption by retroviral insertion, the benefits associated
with the use of insulators preventing unwanted activation of
host genes will be rather significant.
The efficiency of insulator function is, however,
dependent on several factors including topological
constraints, cell types, and the state of cell differentiation
[Rivella et al., 2000; Yannaki et al., 2002]. Also the size
limitations of retroviral vectors should be considered. A 265
bp sea urchin insulator termed sns (silencing nucleoprotein
structure) was found to be effective for insulator function in
human cells, and thus may be useful in retroviral vectors [Di
Simone et al., 2001]. In a recent report, anti-repressor
elements were identified by screening a library of human
genomic DNA fragments between 500 and 2,000 bp, based
on their ability to relieve LexA-dependent transcription
repression [Kwaks et al., 2003]. These elements can confer
high and stable transgene expression in mammalian cells
when they were used to flank the transgene, suggesting that
they play a similar role as insulators. Additional experiments
are required to further improve the function of these and
other insulator elements for more efficient inhibition of
positional repression of the transgene expression and
inhibition of insertional activation of host genes, at the same
time.
Scaffold (or matrix) Attachment Region (SAR) is another
DNA sequence element that is believed to play an important
role in defining boundaries of independent chromatin
domains [Bode et al., 1992; Namciu et al., 1998]. SARs
bind to the nuclear scaffold or nuclear matrix with high
affinity and are proposed to form chromosomal loops [Bode
et al., 2000]. SARs have been used in retroviral vectors with
an enhancement of transgene expression in several different
cell types [Agarwal et al., 1998; Kurre et al., 2003]. A
recent report shows that a high-level transgene expression can
be achieved from a self-inactivating (SIN) lentiviral vector
containing both the human interferon-beta scaffold
attachment region and the chicken beta-globin insulator
[Ramezani and Hawley, 2003]. The proviral form of this
vector does not contain HIV-1 U3 region transcriptional
regulatory elements and is flanked by the enhancer-blocking
beta-globin insulators. These observations indicate that the
usage of SARs in addition to insulators could significantly
improve transgene expression and lower the risk of
uncontrolled activation of cellular proto-oncogenes at or near
the site of incorporation.
Activation of proto-oncogene may also arise due to the
retroviral RNA processing. A strong internal splice acceptor
(SA) is recommended after the splice donor (SD) of retroviral
vector to reduce the positional effects of inserted retroviral
RNA processing on the expression of the transgene as well
as the disrupted host gene. Combination of strong splice
acceptor, deleting cryptic SD in the transgene [Knipper et.
al., 2001], using an improved polyadenylation signal
[Isamail et al., 2001], the removal of LTR promoter, and the
insulator will largely prevent the interactions of the retroviral
splice donor with downstream chromosome sequences.
TRANSCRIPTIONAL TARGETING
Transcriptional targeting using cell type-specific
promoters and enhancers can be applied either alone or in
combination with targeted transduction to minimize the
expression of transduced genes in non-target cells and
thereby reducing potential side effects. (Table 2) summarizes
examples of cell type-specific promoters and enhancers used
for transcriptional targeting in retroviral gene therapy. As an
example, promoters of oncogenes overexpressed in the tumor
cells can be the target for tumor specific promoters (e.g. c-
erbB2 and c-myc). For the treatment of malignant
melanomas, tyrosinase promoter was used for the expression
of HSV-tk or IL-2 [Vile et al., 1994]. Tyrosinase is rate-
limiting enzyme for melanin production, which is highly
expressed in melanomas. In addition to cell type-specific
promoters, inducible or regulatable expression systems can
also be used for increased safety and efficacy. In the case for
mammary tumor and prostate cancer, the growth of tumor is
hormone dependent. Thus, use of combined steroid
hormone-responsive or cell type specific promoters is an
attractive approach for retroviral gene therapy of these cancers
[Sparmann et al., 1994]. Additionally, genes that are
induced by cancer therapy (e.g. by Gamma-irradiation and
chemotherapy) can also be the targets for the regulatory
elements [Walther et al., 1997; Walther et al., 2000].
Although cell type-specific promoters are used
successfully in many different trials, the overall efficiency of
transcription achieved from these promoters is relatively
weak compared with the generally used viral promoters. In a
recent report, hypoxic and cytokine-inducible enhancers, both
of which are active in a tumor environment, are combined
with endothelial cell-specific E-selectin and VEGF receptor 2
promoters [Modlich et al., 2000] to achieve a maximum
possible tumor endothelium-specific transcri-ption. In
another report, human α-fetoprotein (AFP) enhancer was
combined with a house keeping gene phosphoglycerate
kinase-1 (PGK-1) promoter, to augment the activity of the
weak tumor-selective AFP promoter [Cao et al., 2001].
Rat alpha-fetoprotein promoter was used as a cell type-
specific promoter for a lentivirally transduced expression in
human hepatocarcinoma cells [Uch et al., 2003]. Replace-
ment of U3 region of the lentiviral LTR with an upstream
enhancer (HS2) of the erythroid-specific GATA-1 gene and
HIV-1 promoter showed a high level of transgene
expression specifically in mature erythroblasts [Lotti and
Mavilio, 2003].
30 Current Gene Therapy, 2005, Vol. 5, No. 1 Lee et al.
Finally, in many cases, the size constraints of retroviral
vector limit the use of enhancers, which are generally long in
size. Therefore, construction of minimum enhancer/promoter
cassettes with strategic combinations of different sequence
elements will be required to facilitate the efficacy of gene
therapy trials.
CO-EXPRESSION OF A SUICIDAL GENE
Herpes Simplex Virus thymidine kinase (HSV-tk) has
been used for selective destruction of cells in several different
settings. When anti-viral prodrug nucleobase analogue
ganciclovir (GCV) is applied to HSV-tk expressing cells,
GCV is efficiently converted into monophosphate form by
HSV-tk, and then into cytotoxic triphosphate derivatives by
cellular kinases. Actively dividing cells will be killed as
they incorporate the nucleotide derivatives into their genome.
In allogeneic bone marrow transplantation (BMT), donor T
cells are able to mediate anti-leukemic effects but they can
also induce Graft-vs-Host Disease (GvHD), which is often
fatal. In an attempt to reduce GvHD while maintaining anti-
leukemic effect, scientists have retrovirally transduced HSV-
tk to donor T-cells before being used in animal
myeloablative BMT trials [Drobyski et al., 2003]. At first,
the donor T-cells helped to eliminate residual malignant
leukemic cells, but when signs of GvHD development were
noticed, proliferating donor T-cells were rapidly destroyed by
treating the animals with ganciclovir. When this strategy was
used in a human trial, three out of eight patients treated with
donor lymphocytes transduced with HSV-TK gene could be
effectively controlled by ganciclovir-induced elimination of
the transduced cells when they developed GvHD 12 months
after transduction [Bonini et al., 1997].
Similarly, retroviral vectors can be designed to co-express
HSV-tk suicide gene to be used as a safety switch, in
addition to a therapeutic gene. In this case, only if abnormal
growth of transduced cells, such as the leukemic T-cell
clones shown in the X-SCID case, is observed, treating with
ganciclovir can theoretically eliminate all the transduced
cells. However, constitutive expression of HSV-tk can also
induce the death of neighboring uninfected cells (bystander
effects), when ganciclovir is administered. In order to
minimize unwanted side effects due to bystander effects, the
use of cell type-specific or inducible promoter for the
expression of HSV-tk or the use of other pro-apoptotic genes
with a minimum bystander effect may be advantageous. As
an example, lentivirally transduced expression unit
containing the rat alpha-fetoprotein promoter was used to
restrict the HSV-tk induced GCV sensitivity to human
hepatocarcinoma cells [Uch et al., 2003].
On the other hand, retroviral vectors expressing HSV-tk
have been used in antitumor treatment trials [Greco et al.,
2002; Orchard et al., 2002; Barzon et al., 2003]. In this
case, maximum “bystander effect” is required to kill
neighboring uninfected tumor cells as well as infected cells.
The results of tumor treatment with HSV-tk expressing
Table 2. Cell Type-specific Promoters and Enhancers for Transcriptional Targeting in Retroviral Gene Therapy
Promoter Target cell/tissue Transgene References
PEPCK promoter Hepatocyte Neo, bovine growth hormone [Hatzoglou et al., 1990]
hAAT promoter Hepatocyte Alpha I antitrypsin [Hafenrichter et al., 1994]
MMTV-LTR Mammary gland TNF-α [Sparmann et al., 1994]
MCK promoter Muscle β-galactosidase, dystrophin
minigene
[Ferrari et al., 1995]
AFP promoter Cancer: Hepatocellular carcinomas HSV-tk, VZV-tk [Ido et al., 1995]
Tyrosine promoter Cancer: Melanomas HSV-tk, IL-2 [Vile et al., 1994]
Col1a1 promoter Bone β-geo (β-gal, neo fusion) [Stover et al., 2001]
HSP70 promoter Cancer Dominant negative IGF-IR [Romano et al., 2001]
WAP promoter Cancer: Mammary β-galactosidase [Ozturk-Winder et al., 2002]
ppET1 promoter Cancer: Endothelium β-galactosidase [Mavria et al., 2000]
AFP enhancer; PGK promoter Cancer: Hepatocellular carcinomas HSV-tk [Cao et al., 2001]
HRE, PGK-1 enhancer; E-selectin,
KDR promoter
Cancer: Endothelium TNF-α, luciferase [Modlich et al., 2000]
HRE enhancer; AFP promoter Cancer: Hepatocellular carcinomas HSV-tk, luciferase [Ido et al., 2001]
Rat alpha-fetoprotein Human hepatocarcinoma cell HSV-tk, luciferase [Uch et al., 2003]
HS2 of erythroid-specific GATA-1
gene; HIV-1 promoter
Mature erythroblasts GFP [Lotti and Mavilio, 2003]
Retroviral Gene Therapy Current Gene Therapy, 2005, Vol. 5, No. 1 31
retroviral vectors were, however, not fully successful due to
low infection efficiency and low bystander effects.
One potential obstacle for co-expressing HSV-tk suicide
gene as a safety switch in addition to the therapeutic gene is
the limited insert size constraint of the retroviral vector. In
cell-based ex-vivo gene therapy and when the use of clonally-
derived cells is allowed, selecting for single clones
transduced with both the HSV-tk gene and the therapeutic
gene expressed from separate retroviral vectors can be a
solution for the size problem.
AVOIDING REPLICATION COMPETENT RETRO-
VIRUS (RCR)
Generation of RCR remains a potential safety issue in
retroviral gene therapy. Retroviral vectors transfected into the
packaging cell line can produce RCR by recombination
processes between homologous sequences of the retroviral
vector DNA and the gag, pol, and env coding sequences in
the packaging systems. In order to lower the chances for
recombination, both minimizing the homologous sequences
and physically separating genes for gag, pol and env into
two different expression cassettes have become standard
practices. However, residual gag, pol, and env coding
sequences are frequently included in these vectors in an
attempt to increase transduction efficiency and viral titer,
raising a concern for RCR generation. In a recent report, a
complete removal of residual coding sequences for gag, pol,
and env genes was shown without any detrimental effect on
viral transduction efficiency, and thus these vectors further
reduced the chance for RCR generation [Yu et al., 2000].
Same concept for the safer vector by reducing the chance of
recombination was also tried in the lentiviral vector. To
avoid the production of Replication Competent Lentivirus
(RCL), components required for the production of lentivirus
were divided into at least three parts. Vector plasmid
contains gene of interest and the minimal cis-acting element
of HIV. Packaging plasmid has all HIV viral genes except
the env gene. Envelope proteins were provided from a
plasmid containing the envelope gene via co-transfection.
Typically, the Glycoprotein from Vesicular Stomatitis Virus
(VSV-G) is used as an envelope gene [Burns et al., 1993].
Safety concerns specific to HIV virus are recombination of
the vector sequences with the endogenous HIV sequences in
the HIV positive patients. Even though most endogenous
retroviral sequences in the human genome have large
deletions, the possibility of recombination and mobilization
cannot be overlooked. Because the probability of
recombination during the packaging process through
transient transfection is expected to be much higher than that
occurring when stable producer cell lines are used, stable
lentivirus producer cell line has been tried [Farson et al.,
2001; Xu et al., 2001]. Due to the major concern of using
HIV in humans, researchers are developing nonhuman
lentivirus vector systems. Simian Immunodeficiency Virus
(SIV), Feline Immunodeficiency Virus (FIV), and Equine
Immunodeficiency Virus (EIAV) have been used [Poeschla et
al., 1998; Pandya et al., 2001; Bienemann et al., 2003].
However, the safety features of non-primate lentiviruses in
humans have yet to be determined [Connolly, 2002].
Self-inactivating (SIN) retroviral vector was developed by
introducing a deletion in the U3 region of the 3’ LTR which
contains all the enhancer and promoter activities of the viral
vector (Fig. (1B)) [Yu et al., 1986]. Because the 5’LTR of
the provirus carrying the same deletion will be incapable of
inducing transcription for the production of packagable
RNAs, no active viral particle can be made from this vector
even after successful recombination processes to acquire gag,
pol, and env sequences. Additional advantage of the SIN
vector is that because of the lack of promoter activities of the
viral LTRs, the possibility of LTR-mediated activation of
cellular proto-oncogenes near the site of incorporation is also
minimized. SIN vector approach has been tested more
extensively in lentiviruses [Zufferey et al., 1998]. Although
SIN vector was considered as an ideal vector, a very low
level of SIN vector mobilization was detected [Xu et al.,
2001]. Additionally, one of the drawbacks of the SIN
retroviral vectors is the internal promoter’s low
transcriptional activity compared to the viral LTR in a
number of different cell types. Improvements in the design
of the internal promoter/enhancer will be required to
overcome this obstacle.
Split-intron retroviral vector has been shown to enhance
expression of an inserted gene and improve its safety [Ismail
et al., 2000]. A strong synthetic splice donor site and a
splice acceptor site were inserted between U3 and R of the 3’
LTR and downstream of the packaging signal, respectively.
During the reverse transcription process, the strong synthetic
splice donor site introduced in the 3’ LTR is copied into
5’LTR, and theoretically all the transcripts made in the
transduced cells are spliced and the packaging signal is
removed (Fig. (1C)). Therefore, the possibility of producing
RCR will be greatly reduced.
Finally, activation of proto-oncogene may arise due to
aberrant retroviral RNA processing. A strong internal splice
acceptor (SA) is recommended after the splice donor (SD)
site of retroviral vector to prevent a generation of aberrant
readthrough transcripts containing both the transgene and a
portion of the disrupted host gene. Combination of strong
SA, deleting cryptic SD in the transgene [Knipper et al.,
2001], using an improved polyadenylation signal [Ismail et
al., 2001], the removal of LTR promoter, and the presence
of an insulator will largely prevent the interactions of the
retroviral splice donor with downstream chromosome
sequences.
CONCLUDING REMARKS
We have reviewed safety features for the use of retroviral
vector systems in clinical gene therapy. Targeted infection,
local delivery, targeted retroviral insertion, insulators,
transcriptional targeting, co-transduction with a suicidal
gene, and SIN vectors were suggested as possible solutions
for the potential risks of retroviral gene therapy. Some of
these precautions can be applied to gene therapy protocols
using other viral and non-viral vector systems. One major
immediate concern in terms of retroviral gene therapy, as
revealed by the X-SCID case, is insertional oncogenesis.
Several possible approaches for minimizing, if not
completely eliminating, insertional oncogenesis were
considered in depth. In addition to the more widely used
32 Current Gene Therapy, 2005, Vol. 5, No. 1 Lee et al.
retroviral systems that were discussed above, foamy viruses
may be used as a safe and efficient means of targeting non-
dividing cells [Mergia et al., 2001]. Foamy viruses are
known to have a broad host range, without causing any
disease and persist in infected humans [Schweizer et al.,
1997; Heneine et al., 1998; Callahan et al., 1999].
In the case of ex vivo cell-based gene therapy where the
selection of clonally-derived cells is permissible, transduced
cells can be prescreened to select for clones with the insertion
of the transgene only at a desirable site of the chromosome,
minimizing the chances for insertional oncogenesis.
Applications of gene therapy protocols have been
continuously expanding to include a wide variety of acquired
and inherited diseases, such as cancer, SCID, and other life
threatening diseases. Retroviral gene therapy approaches for
the treatment of each of these diseases will have different
treatment requirements and thus will meet variable
challenges for addressing safety issues. Treatment of
orthopedic indications may be one of the most promising
areas of gene therapy research and trials, despite the general
perception that they are the non-lethal, acquired conditions
[Evans et al., 2000; Evans and Scully, 2000; Lee et al.,
2001; Lee et al., 2002; Lieberman et al., 2002; Palmer et
al., 2002; Nussenbaum et al., 2003; Robbins et al., 2003].
Treatment of these conditions will require only a short-term
expression of the transduced genes with more readily
available options for the delivery. Combined with
aforementioned safety precautions such as incorporation of
suicidal genes or chromatin insulators, cell-based ex vivo
retroviral gene therapy has a unique window of opportunity
to bring about a rewarding outcome.
ACKNOWLEDGEMENTS
We thank Lillian Yun, Vivian Yip, and Jonathan Yun for
proofreading this manuscript.
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