[Cancer Biology & Therapy 4:5, 512-517; May 2005]; 2005 Landes Bioscience

Vector-Mediated Cancer Gene Therapy

Focused Review

An Overview

Correspondence to: Prem Seth; Laboratory of Gene Therapy; ENH Research

Institute and Department of Medicine; 2650 Ridge Ave.; Room B 643; Evanston
Hospital; Northwestern University; Evanston, Illinios 60201 USA; Tel.:
847.570.2317; Fax: 847.733.5256; Email: pseth@northwestern.edu
Received 03/16/05; Accepted 04/05/05

Moloney murine leukemia virus

adeno-associated virus
herpes simplex virus
human immunodeficiency virus
inverted terminal repeat
vascular endothelial growth factor
interleukin
multi drug resistant-1
plaque forming units
small interfering RNA

OT
D

In principle, gene therapy involves the transfer of foreign genetic material into a patient
in an effort to treat a particular disease. In the past ten years significant progress has been
made in the development of cancer gene therapy as a potential new approach for treating
cancer. Our understanding of the molecular mechanisms underlying the tumor development has played a significant role in developing the various strategies for cancer gene
therapy. Another key factor has been the enormous knowledge of the various viral and
non-viral vectors used as the DNA-delivery vehicles. This chapter provides an overview of
the various strategies of cancer gene therapy and the commonly used viral and non-viral
vectors. Some of the critical issues in cancer gene therapy, and important future directions
are also described. For detailed preclinical and clinical evaluation of vector-based cancer
gene therapy approaches readers are referred to several excellent papers and reviews
published recently.1-6

SC

BIO

ACKNOWLEDGEMENTS

20

05

LA

ND

ES

This work was supported by the Department of

Defense Breast Cancer Research Program grant
#DAMD17-03-1-0703, ENH Auxiliary Breast
Cancer Research Program, and ENH Research
Career Development Award to P.S.
The author is thankful to Dr. Janardan
Khandekar for his enthusiastic support of this
research.

512

INTRODUCTION

IEN

MoMuLV
AAV
HSV
HIV
ITR
VEGF
IL
MDR-1
PFU
siRNA

ON

ABBREVIATIONS

.D

cancer gene therapy, tumor suppressors, antioncogenes, suicide genes, anti-angiogenesis, metastasis, immunomodulation, adenovirus, retrovirus,
lenti-virus, targetable vectors

CE

KEY WORDS

IST

RIB

Previously published online as a Cancer Biology & Therapy E-publication:

http://www.landesbioscience.com/journals/cbt/abstract.php?id=1705

In recent years there has been a dramatic increase in developing gene therapy
approaches for the treatment of cancer. The two events that have permitted the formulation
of concept of cancer gene therapy are the new understanding of the molecular mechanisms underlying oncogenesis, and the development of the DNA-delivery vehicles or
vectors. Many approaches to cancer gene therapy have been proposed, and several
viral and non-viral vectors have been utilized. The purpose of this review article is to
describe the various strategies of cancer gene therapy (transfer of tumor suppressor
genes, suicide genes-enzyme/pro-drug approach, inhibition of dominant oncogenes,
immunomodulation approaches, expression of molecules that affect angiogenesis, tumor
invasion and metastasis, chemosensitization and radiosensitization approaches, and
chemoprotection of stem cells). The chapter also reviews the commonly used vectors (retroviral vectors, adenoviral vectors, adeno-associated viral vectors, pox viruses, herpes simplex
viruses, HIV- vectors, non-viral vectors and targetable vectors) for cancer gene therapy.
Some of the important issues in cancer gene therapy, and the potential future directions
are also being discussed.

Laboratory of Gene Therapy; ENH Research Institute and Department of Medicine;

Evanston Hospital; Northwestern University; Evanston, Illinios USA

ABSTRACT

UT
E

Prem Seth

STRATEGIES FOR CANCER GENE THERAPY

Cancer is a multi-stage process, and involves several molecular alterations such as the
loss of tumor suppressor genes and gain of dominant oncogenes in the malignant cells;
enhanced angiogenesis in the tumor environment; and additional immunological defects
leading to the inability of the immune system to destroy the cancer cells.7,8 Such information has contributed a great deal to devising the various approaches to cancer gene
therapy (Table 1), and some of the approaches that have been explored to date are
described below.
Transfer of tumor suppressor genes. Loss of tumor suppressor function is commonly
associated with many human malignancies.9 Several tumor suppressor genes have been
isolated in recent years. Examples of tumor suppressor genes are - p53, the retinoblastoma
gene pRB and p16INK4A. Although the introduction of tumor suppressor genes in the
tumor cells is intended to be used as gene replacements, the overexpression of these
genes generally induces apoptosis and/or cell cycle arrest in the tumor cells. Moreover,
some tumor suppressor genes such as p53 can also inhibit angiogenesis and tumor invasion, providing additional anti-tumor effects.10,11 Vectors expressing tumor suppressor
genes can be directly injected into the tumor masses; targeted to tumor cells or can be used

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Strategies for Cancer Gene Therapy

ex vivo for killing tumor cells, for example for bone mar- Table 1 Strategies for vector-mediated cancer gene therapy
row purging before autologous bone marrow transplantation.
Toxic transgene products
Suicide genes-enzyme/pro-drug approach. In this
Transfer of tumor suppressor genes
approach, vectors expressing suicide genes whose protein
Suicide genes-Enzyme/pro-drug approach
Expression of antisense, ribozymes or siRNAs for dominant oncogenes
product when simultaneously exposed to small molecular
weight drugs (the enzyme/pro-drug-approach) can be used Immunomodulatory approaches
Expression of cytokines
to kill the cancer cells.12,13 For example, a vector contain Expression of costimulatory molecules
ing bacterial cytosine deaminase in combination with the
Expression of tumor specific antigens
systemic delivery of 5-flouro cytosine can convert the proOther
strategies
drug 5-fluoro-cytosine to the cytotoxic drug, 5-fluoro

Expression of molecules that affect angiogenesis, cell adhesion and metastasis

uracil. Another example is the use of herpes simplex thymi Chemosensitization and radiosensitization approaches
dine kinase in combination with ganciclovir which pro Chemoprotection of stem cells
duces phosphorylated ganciclovir as the toxic species. The
Expression of cell surface receptors/ligands to target cancer cells
bystander effects of the toxic product whereby adjacent
Tumor-specific promoter driven transgene expression
cells are killed by the activated drug further enhance this
Replicating (oncolytic) vectors
approach of cell killing. These vectors can be injected
directly into the tumor mass; used ex vivo to kill tumor
cells, or delivered systemically and targeted to the tumor cells.
tumor invasion and metastasis.16,17 Molecules such as vascular
Inhibition of dominant oncogenes. It is well known that devel- endothelial growth factor (VEGF) are known to enhance the angioopment of many cancers is associated with the expression of domi- genesis, and can be targeted by various means such siRNAs,
nant oncogenes. Examples of oncogenes are ras, HER-2/neu and ribozymes or antisense.16 Many candidate genes involved in invasion
MYC genes.14 Vectors expressing genes that will inactivate the and metastasis are either deficient (e.g., maspin, TIMP-1) or are
dominant oncogenes can be used to inhibit tumor progression. For overexpresed (e.g., metalloproteinase enzymes) in tumor cells.18
example, vectors expressing anti-sense, ribozymes or small interfering Therefore, vectors capable of expressing the TIMP-1 or antagonists
RNAs (siRNAs) directed against ras and HER-2/neu oncogenes have of metalloproteinases can be potentially used to block the invasion
been extensively studied. This approach can be also targeted against and metastasis. This can be conducted by injecting the vectors directly
the tumors in vivo or for killing cells ex vivo.
into the tumors or targeting to vascular endothelial cells lining the
Immunomodulation approaches. The failure of normal immune blood vessels of the tumors.
surveillance mechanisms, leading to an inability to recognize cancer
Chemosensitization and radiosensitization approaches. Gene
cells as foreign cells, is an integral component of the process of therapy approaches can be used to augment the sensitivity of cancer
tumor development. Recent research has identified numerous mech- cells to chemotherapy and radiotherapy.19,20 For example, the delivery
anisms that operate to allow cancer cells to evade host immunity. of wild type p53 has been demonstrated to have a chemosensitizing
These include lower expression of MHC class 1 and class II proteins, effect.21 In another approach, delivery of the liver cytochrome P450
decreased growth and differentiation of effector immune cells and gene to cancer cells has been shown to lead substantial chemosensidefects in expression of costimulatory molecules.15 Thus, utilizing tization to the oxazaphosphorines like cyclophosphamide and ifosvectors expressing genes to activate the host immune system or to famide that require activation by the cytochrome enzymes. Similarly,
attempt to bypass some of these defects by introducing genes that adenoviral E1a gene has been shown to down regulate expression of
alter the local immune microenvironment, is an attractive anti-can- HER-2/neu and lead to increased sensitivity to paclitaxel.22 Clearly
cer strategy.15 The types of genes can be interleukin (IL)-2, IL-4, this is a research area that needs to be further explored.
IL-12, tumor necrosis factor, interferon-; granulocyte-macrophage
Chemoprotection of stem cells. Vector-mediated gene transfer of
colony stimulating factor (GM-CSF); genes which have immunos- drug resistant genes which can be transduced into hematopoietic
timulatory activity (e.g., allogenic major histocompatability complex stem cells to increase their resistance to cytotoxic drugs.23 The most
antigens), or T-cell costimulatory molecules such as B7.1 and B7.2. widely studied gene for this purpose is the multi drug resistant-1
Alternatively, this can be accomplished by using cell-based cancer gene (MDR-1). Bone marrow cells expressing MDR-1 should be
vaccines, in which case the individual can be vaccinated with more resistant to conventional chemotherapy and hence allow
autologous tumor cells expressing vector-mediated cytokines; patients to receive higher doses of chemotherapeutic agents while
immunostimulatory or costimulatory molecules. In another maintaining sensitivity of the tumor cells to the same drugs.24 This
approach, vector- mediated gene transfer to T-lymphocytes or approach therefore has potential to be used in conjunction with high
dendritic cells that will enhance their antitumor effector activity can dose chemotherapy for the treatment of cancers.
be effective. For example, tumor-infiltrating lymphocytes (TILs) can
be transduced with vectors expressing genes encoding cytokines,
VECTORS FOR CANCER GENE THERAPY
such as TNF- to induce anti-tumor activity. Similarly, vectors can
There are essentially two major classes of vectors: viral based and
be used to express variable regions of tumor- specific monoclonal
antibodies to recognize and kill the tumor cells. If successful, non-viral based. Because viruses have evolved natural mechanisms to
immunomodulation approaches have the potential to develop a deliver their genomes into cells, they are excellent vectors to deliver
foreign DNA. Non-viral vectors can also enhance delivery of the
therapy that is truly systemic in scope.
Expression of molecules that affect angiogenesis, tumor inva- nucleic acids to the cells. Following is the brief description of the
sion and metastasis. In recent years, remarkable progress has been commonly used vector for gene transfer. While the choice of vector
made in understanding the molecular mechanism of angiogenesis, will depend upon the experimental or clinical setting; other general
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Table 2

Important considerations for selecting the vector for cancer gene therapy

What are the target cells? These could be tumor cells or host cells such as T-cell, or bone marrow cells. One needs to know the precise developmental
nature of the target cells, i. e. whether these cells are of epithelial origin, hematopoietic stem cells, neuronal cells, skin cells etc.
Are the target cells undergoing active mitotic division or are they quiescent? It might be beneficial to alter the mitotic state of the cells before/during
gene transfer.
How many target cells need to be targeted for successful gene therapy? In some instances, targeting a few cells might be enough, while, in other
instances transfer to a larger populations even 100% may be essential.
The vector should be able to target the tissue/tumor of interest. Should be able to modify vector tropism and tissue/tumor specificity if needed. While
most large vectors are designed to be administered locally, there should be potential to deliver them systemically.
Is short-term expression of the heterologous protein enough or is long-term production of the protein essential for an efficacious result?
What factors might interfere with therapy or produce harmful effects during the treatment? Are there preexisting antibodies to the vector which could
influence the outcome of gene therapy? In the event of any harmful effects, one should be able to immediately stop the vector treatment and the subsequent expression of the foreign gene.
Since gene therapy to only somatic cells is currently being considered, the vector should not reach the gonadal tissues, and if it does, should not be
able to integrate into reproductive cells.

consideration listed in Table 2 could play a role in selecting the optimal vector.
Retroviral vectors. Many of the initial gene therapy studies
utilized retroviruses as the vectors because retroviruses can integrate
into the host genome. The most widely studied vector is the
moloney murine leukemia virus (MoMuLV).25 This retrovirus is
composed of a double stranded RNA genome flanked by 5' and 3'
terminal repeats (LTR). Adjacent to the 5LTR is the region where
the tRNA primer binds and reverse transcription is initiated. Next
to this are splice donor and encapsulation sequences, gag, pol and env
genes which encode for the viral structural proteins ribonucleoprotein
core (gag); protease, reverse transcriptase/RNaseH and integrase
enzymes (pol); and the envelope glycoproteins (env). To generate
recombinant retroviral vectors, the genes gag, pol and env are
replaced by the cDNA of choice (up to 8 kb) in a vector which also
contains the packaging signals. Cell lines which constitutively
express the gag, pol and env genes of MoMuLV provide the necessary
helper function for the propagation of retrovirus vector.26,27
Since retroviruses are capable of integrating into the host
genome, they are suitable vectors when the long-term expression of
the foreign gene is needed.28 The main concerns in using recombinant
retroviruses are accidentally random integration into the host
chromosome resulting in deleterious effects such as the activation of
certain protooncogenes by insertional mutagenesis, or suppression
of other tumor suppressor genes. Another limitation is the requirement
of cell division for the provirus integration. This problem can in
some circumstances be overcome by using lenti viruses (described
below) that are capable of integrating into non-dividing cells.
Adenoviral vectors. Adenoviruses are DNA-containing, nonenveloped viruses. The adenovirus genome consists of a single piece
of linear double stranded DNA, approximately 36 kb long and
flanked by two short-inverted terminal repeats. The genome is
composed of various transcriptional regions which include an early
region (E1 through E4), two delayed early units (IX and 1Va2), a
late region (L1 through L5) and VA regions.29 Adenoviruses enter
cells by receptor-mediated endocytosis.30 Once the virus genome is
released into the nucleus the viral early genes are transcribed, leading
to DNA replication, late transcription, synthesis of viral structural
proteins and virus assembly. Recombinant adenoviruses can be
generated by replacing viral sequences (such as E1, E2, E3 or E4
sequences) in adenovirus DNA by the foreign cDNA.29-32 In one
approach to construct E1 deleted adenovirus, a cDNA of choice is
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cloned into a shuttle vector and cotransfected with the genomic

adenoviral DNA into a packaging cell line, 293, which provides the
E1 proteins in trans. Homologous recombination results in the generation of a recombinant adenovirus devoid of E1 sequences (Fig. 1).
Recombinant adenoviruses can be grown to very high titers (up to
1012 pfu/ml) in the laboratory.
Adenovirus infection produces high-level of gene transfer in the
tumor cells thus making them suitable vectors if high-level gene
expression for the short-term is sufficient for therapy. Because of
their fairly large packaging capacity (up to 35 kb) adenoviruses can
also be used to transfer larger genes. Adenoviruses can infect a variety
of cell types; and produce the heterologous protein of interest in
dividing as well as non-dividing cells.30 The use of adenoviruses as
vectors however, is limited by the fact that they do not integrate into
the host chromosomes. This results in a short-term expression of the
transduced gene. Immunological responses to adenoviruses may also
reduce the duration of expression therapeutic gene expression, and
the immune responses to the adenoviral vectors can potentially cause
harmful effects to the patients.33,34
Adeno-associated viral vectors. Adeno associated viruses (AAV)
are small DNA viruses which belong to the Parvoviridae family.35,36
For productive virus replication, AAV needs a helper virus such as
adenovirus or herpes virus. However, in the absence of a helper virus,
wild type AAV can integrate into the host cell genome, thus making
it an attractive vector for gene therapy. The AAV genome is a linear,
single stranded DNA of 4680 base pairs. Inserted between the two
ITRs are rep, cap and other viral regulatory elements. To generate a
recombinant AAV vectors, in one approach, the cDNA of interest is
inserted between the two AAV ITRs in a plasmid DNA. The second
plasmid is the helper plasmid (AAV/Ad) that supplies the required
AAV coding sequences, cap and rep in trans. The two plasmids are
cotransfected in a permissive cell line (generally 293 cells), and
concomitantly infected with adenovirus. The complementation
between the two vectors allows the production of recombinant AAV.
AAV has been shown to infect a variety of cells including epithelial,
fibroblasts and hematopoietic cells. Like retroviruses, AAVs can integrate into the host chromosome. Thus, it is likely that recombinant
AAVs will find application in gene transfer into hematopoietic cells,
especially stem cells. Contamination with wild type AAV and adenovirus in AAV stocks is sometimes a problem. However, new vector
systems and packaging cell lines have been designed to circumvent
these problems.

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Figure 1. Construction of recombinant adenoviral vectors. cDNA of interest is cloned into a shuttle vector which provides cDNA expression cassette (adenovirus ITR, E1 enhancer, adenovirus encapsidation signal, CMV promoter, and SV40 a polyadenylation signal). Homologous recombination sequences are
also cloned in this vector. Adenovirus genome (e.g., pJM17 shown in the figure) and the shuttle vector containing the cDNA are cotransfected in 293 cells.
Intracellular homologous recombination between the two DNAs results in a E1- recombinant genome; the numbers 0, 20, 100 represent the approximate
map units. This recombinant genome is replication defective. However, in the presence of E1 proteins (provided in trans by 293 cells), the recombinant
genome will replicate and form adenoviral particles.

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Pox viruses. These comprise a large family of complex DNA

viruses that replicate in the cytoplasm. Their genome size varies from
130 to about 300 kb. There are two ITRs at the ends of the large
genome which encodes for at-least 30 proteins. Viruses enter the cell
by fusing with the plasma membrane. After entry into the cytosol,
DNA replicates, virions are assembled and released from the cells.
Foreign DNA can be inserted into the Pox genome, by the process
of homologous recombination or by in vitro ligation.37,38 Advantage
of using Pox virus as a vector include the ease with which recombinant
vectors can be formed and isolated; the large capacity of Pox viruses
for foreign DNA; the relatively high level of transgene expression,
and the wide host range. However, given the vaccination history of
many individual, its application in the clinic is likely to be limited.
Herpes simplex viruses. These viruses belong to the family
Herpesviridae and include Herpes Simplex Virus (HSV) type 1 and 2.
These viral vectors have been primarily developed for targeting the
central nervous system.39 Much of the published work has been
done using HSV-1 based vectors. HSV-1 can either undergo productive infection and express lytic function; or the virus may express
latency genes and stay in the latent state without undergoing viral
replication. HSV-1 is a large, enveloped virus which has a double
stranded DNA of about 150 kb long. Recombinant HSV-1 can be
constructed by replacing the essential early gene such as IE3 gene,
with the cDNA of interest. Instead of the whole virus based vectors,
the use of plasmid based amplicons has also been proposed.40
Features of HSV vectors include their ability to grown to high titers
(1011-1012 pfus/ml); their ability to infect non-dividing cells; and
their ability to package large inserts. Problems associated with the
use of HSV vectors are the difficulty in completely eliminating lytic
viral gene expression; vector induced cytotoxicity, and the transient
nature of gene expression.
HIV-1 vectors. Human immunodeficiency virus-1 (HIV) based
vectors (a sub-class of retroviruses termed lentiviruses) are worthy of
exploring for cancer gene therapy because they can be targeted to
hematopoietic cells, and can deliver foreign genes to dividing as well
non-dividing cells.41 Like MoMuLV, HIV-1 genome also has gag, pol
and env genes and 5'-and 3'-LTRs. However, the genome also contains
six additional genes which are tat, rev, nef, vif, vpr and vpu. The
recombinant HIV based vectors can be generated using similar
strategies as devised for retroviruses.42 The presence of a nuclear targeting signal in the assessory protein Vpr, permits the integration of
the genome into the cell nuclei even in non-dividing cells making
them an attractive vector system. However, the safety of HIV-1
based virus in the clinical setting remains a major concern.
Non-viral vectors. DNA of choice can be also delivered to the
cell nucleus by using non-viral methods. The simplest method to
deliver DNA is by plasmid DNA expression vectors driven by
eukaryotic promoters.43 While this is a simple approach, the efficiency
of gene delivery is poor. To increase the transfection efficiency, many
physicochemical methods have been devised.44 One approach is the
use of liposomes. Since DNA is negatively charged, it can form
complexes with positively charged liposomes. DNA can be also
trapped inside the aqueous interior of the liposomes; in which case
negatively charged pH-sensitive liposomes whose membranes are
destabilized at low pH can be also used.45 DNA entry into cells can
be also accomplished by physical means such as particle bombardment,
commonly referred to as the Gene gun method.46 In this technique
the DNA of choice is coated onto microscopic particles (generally
gold particles) which are accelerated by a motive force to a velocity
sufficient to cause them penetrate cells. It has also been shown that
516

plasmid DNA can be delivered via receptor-mediated endocytosis.

Using this technique, the route of DNA delivery can be controlled
by using specific ligands conjugated to the plasmid DNAs.47 The
key advantage of non-viral methods is that they are safer to administer and do not elicit major immune responses. However, the main
problem is that compared to viral vectors efficiency of gene transfer
is generally low.
Targetable vectors. Because most unmodified viral vectors can
infect normal cells as well the tumor cells, there has been a significant
interest in developing modified viral vectors that will specifically
target the tumor cells. In general, either transduction or transcriptional
targeting can achieve this objective. In transductional targeting the
goal is to enhance the virus infectability of the tumor cells. This can
be accomplished by conjugating the viral capsids directly with the
ligands (e.g., epidermal growth factor, basic fibroblast growth factor,
monoclonal antibodies) or by the genetic manipulations of the viral
genomes.48 Many of the commonly used vectorsadenovirus, AAV,
Herpes and retrovirusescan be modified by these methods.40,49-51
Transcriptional targeting can be achived by using tumor specific
promoters driving the transgene of interest.52 Thus these viruses
though can infect both the tumor and the normal cells; only tumor
cells will express the gene of interest. Several promoters/enhancers
(e.g., Her2/neu promoter, PSA promoter, CEA promoter) have been
successfully used to create recombinant viral vectors including
adenoviruses, retroviruses and AAVs.53 Like viral vectors plasmid
DNAs can be also modified to specifically target the cancer cells, for
example by forcing the DNA to enter the cells by the receptor-mediated endocytosis pathways.54,55

CONCLUSION AND FUTURE DIRECTIONS

Since the original idea of vector-based cancer gene therapy, a great

deal of progress in development of novel strategies and vectors has
been made. However, given that human cancer spreads systemically,
the evolution of gene therapy approaches into curative cancer treatment is likely to be an uphill task. Although the local delivery of the
vectors e.g., for treating residual disease, and other in vitro and ex
vivo purposes should be maximally exploited; the ultimate success of
cancer gene therapy depends upon the induction of the systemic
effects. Perhaps, we need to combine several vector-mediated cancer
gene therapy approaches, for example, the use of an enzyme/
pro-drug system in combination with the components designed to
induce immunomodulation.56 One can also use multiple vectors in
a single regimen. It seems possible that cancer gene therapy in
combination with other conventional cancer treatment approaches
could be also potentially useful.
In general, in vivo transduction efficacy of most vectors is too low
to be effective in cancer gene therapy.57,58 Vectors which induce a
bystander effect or replication-competent vectors (See ref. 59) can in
part overcome this problem, and hence research in this area is likely
to be valuable. Another area where research is much needed is the
development of vectors in which the expression of the transgene is
efficiently controlled. The use of tumor- specific regulatory elements
to drive the transgene expression is worthy of greater research efforts.
The possibility of incorporating regulatory elements that lead to
repression of transgene transcription in specific types of normal cells
should also be considered. There is also a potential in modifying
vectors in a way that they only infect the target cells sparing the nontumor organs such as the liver.
To monitor vector distribution, state of the art imaging technologies need to be developed. Another important development in

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the field is the use of cell-mediated gene therapy. Instead of directly

inoculating the viruses in the body, the cells can be transduced ex
vivo and used as a factory for in vivo gene therapy.
Before specific vectors are taken to the clinical trials, we must
provide evidence that the concept on which they are based is valid
using in vitro, in vivo and ex vivo assays (whichever is applicable).
Better in vivo assays will require the development of more appropriate
animal models. It will be essential for basic scientists to work in close
association with clinical scientists in developing novel vectors and in
designing, executing, and evaluating the results of clinical trials.
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