NKJ Lecture-10-MBT-101: Genetic Engineering: (Gene Therapy)

Somatic Cell Gene Therapy

Human somatic cell gene therapy is a form of medical treatment that is being developed as a genetic
approach to disease management. Two basic gene therapy strategies have been investigated;

1) In vivo gene therapy requires that the gene transfer vector be delivered in a cell-type selective manner,
either through direct tissue injection, or perhaps someday, by receptor-mediated processes.

2) Ex-vivo gene therapy involves removing tissue from the patient, transfecting (or virally-infecting) the
cells in culture, and then reimplanting the genetically altered cells to the patient.

In vivo gene therapy is done by targeting the gene delivery system to the desired cell type in the patient
using either physical means such as tissue injection (brain tumor) or biolistics (dermal DNA vaccination),
or potentially in the future, using systemic infusion of cell-specific receptor-mediated DNA carriers
(reconstructed liposomes or viruses). Importantly, neither of these gene therapy strategies involve
reproductive germline cells and therefore the genetic alteration will NOT be transmitted to the next
generation. In many countries, human germline gene therapy is considered unethical or even illegal.

Ex-vivo gene therapy is performed by transfecting or infecting patient-derived cells in culture with
vector DNA and then reimplanting the transfected cells into the patient. Two types of ex-vivo gene
therapies under development are those directed at fibroblasts and hematopoietic stem cells.

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 Monogenic loss-of-function diseases have been used as paradigms to develop gene therapy strategies
using animal models. In this context, gene therapy refers to genotypic alterations (chromosomal or
episomal) of somatic cells, resulting in sustained gene expression. This is to be distinguished from
transient DNA transfection approaches, such as repeated inhalation or infusion of functional
nucleic acids, for example, drug therapies utilizing ribozymes or antisense
oligonucleotides.

Disease Target cells Transfected gene(s)

Hemophilia A liver, muscle, bone marrow cells, fibroblasts Factor VIII
Hemophilia B
Factor IX
Familial liver Low-density lipoprotein receptor
hypercholesterolaemia

Severe combined bone marrow cells, T cells Adenosine deaminase (ADA)

immunodeficiency

Haemoglobinopathies red blood precursor cells a-globin, b-globin

Cystic fibrosis lung airway cells Cystic fibrosis gene (CFTR)

Gaucher’s bone marrow cells, macrophages glucocerebrosidase

Cancer tumor cells p53, Rb, interleukins, growth-inhibitory

genes, apoptosis genes
Fate of gene transfer:
Following gene transfer, the inserted genes may integrate into the chromosomes of the
cell, or remain as extrachromosomal genetic elements (episomes).

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Fig-2. Exogenous genes that integrate into chromosomes can be stably transmitted to all
daughter cells, unlike episomal (extrachromosomal) genes. (two possible fates of genes that
have been transferred into nucleated cells. If the cells are actively dividing, any genes, which
integrate stably into chromosomal DNA, can be replicated under the control of the parent
chromosome (during the S phase of the cell cycle). Following each cell division, an integrated
gene will be stably inherited by both daughter cells. As a result, all cells that descend from a
single cell in which stable integration took place, will contain the integrated gene. Gene therapy
involving chromosomal integration of exogenous genes offers the possibility of continued stable
expression of the inserted gene and a permanent cure, but carries certain risks, notably the
possibility that one of the integration events may result in cancer. By contrast, episomal genes
which do not integrate but replicate extrachromosomally (under the control of a vector origin of
replication) may not segregate to all daughter cells during subsequent mitoses. As a result, this
type of approach has been particularly applied in gene therapies where the target tissue consists
of nondividing cells.)

Chromosomal integration has its disadvantages, however, because normally the

insertion occurs almost randomly. Inserted genes may not be expressed, e.g. they may have
integrated into a highly condensed heterochromatic region. In some cases the integration event
can result in death of the host cell (the insertion may occur within a crucially important gene,
thereby inactivating it).
A greater concern is the possibility of cancer: an integration event in one of the many cells that
are targeted could disturb the normal expression patterns of genes that control cell division
or cell proliferation. For example, the integration can cause activation of an oncogene or it
could inactivate a tumor suppressor gene or a gene involved in apoptosis (programmed cell
death).

Requrements for gene therapy:

Vectors in gene therapy

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1. Viral Vectors:
a. Retroviruses
b. Adenoviruses
c. Adeno-associated viruses
d. Envelope protein pseudotyping of viral vectors
e. Lentiviruses

2. Non-viral methods
a. Naked DNA
b. Oligonucleotides
c. Lipoplexes and polyplexes
d. Hybrid methods

The vector chosen for gene transfer depends on:

1) The nature of the target tissue.
2) Process of gene transfer.

a) Retroviral vectors: Retroviruses are RNA viruses which possess a reverse transcriptase
function, enabling them to synthesize a complementary DNA form ( see Fig -3). Retroviruses are
very efficient at transferring DNA into cells, and the integrated DNA can be stably propagated,
offering the possibility of a permanent cure for a disease. Because of these properties, retroviruses
were considered the most promising vehicles for gene delivery and currently about 60% of all
approved clinical protocols utilize retroviral vectors.

The retrovirus vectors that have traditionally been used in gene therapy are derived from
simple retroviruses.
Since all the viral genes are removed from the vector, the viruses cannot replicate by
themselves.
They can accept inserts of up to 8 kb of exogenous DNA and require a variety of packaging
systems to enclose the viral genome within viral particles (simple injection of retroviral vectors is
usually inappropriate for in vivo gene therapy because they can generally be killed by human
complement).

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Fig-3. Retroviral life cycle. (The virus particle (top) contains the RNA genome and viral reverse
transcriptase within an outer lipoprotein envelope and inner protein capsid. A double-stranded
DNA copy of the viral genome integrates into the host DNA. Here it directs synthesis of viral
RNA and proteins, which self-assemble and bud off from the cell membrane. The host cell is not
killed.)

Gene Therapy for severe combined immunodeficiency (SCID): requirements

The gene must be identified and cloned. This has been done for the ADA gene.
The patient's own cells are removed, treating them in tissue culture, and then returning
them to the patient.
It must be inserted in the DNA so that it will be expressed adequately; that is, transcribed
and translated with sufficient efficiency that worthwhile amounts of the enzyme are produced.
All these requirements seem to have been met for SCID therapy using a retrovirus as the
gene vector. Retroviruses have several advantages for introducing genes into human cells.
Their envelope protein enables the virus to infect human cells.
RNA copies of the human ADA gene can be incorporated into the retroviral genome using a
packaging cell.
Packaging cells are treated so they express:
1) an RNA copy of the human ADA gene along with
a packaging signal (P) needed for the assembly of fresh virus particles
inverted repeats ("R") at each end; to aid insertion of the DNA copies
into the DNA of the target cell.

2) an RNA copy of the retroviral gag, pol, and env genes but with no packaging signal (so these
genes cannot be incorporated in fresh viral particles).

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Treated with these two genomes, the
packaging cell produces a crop of
retroviruses with:
the envelope protein needed to infect the
human target cells
an RNA copy of the human ADA gene,
complete with R sequences at each end
reverse transcriptase, needed to make a
DNA copy of the ADA gene that can be
inserted into the DNA of the target cell
none of the genes (gag, pol, env) that would
enable the virus to replicate in its new host.
Once the virus has infected the target cells,
this RNA is reverse transcribed into DNA
and inserted into the chromosomal DNA of
the host.

Fig-4. Gen therapy for SCID using

retroviral vector.

b) Adenovirus vectors
Adenovirus vectors have been the second most popular delivery system in gene therapy
(with extensive applications in gene therapy for cystic fibrosis and certain types of cancer) and
have several advantages as gene delivery vectors. They are human viruses which can be produced
at very high titers in culture, and they are able to infect a large number of different human cell
types including nondividing cells. Entry into cells occurs by receptor-mediated endocytosis
(Figur-5.) and transduction efficiency is very high (often approaching 100% in vitro). They are
large viruses and so have the potential for accepting large inserts.

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 Figure-5. Adenoviruses enter cells by receptor-mediated endocytosis. (Binding of
viral coat protein to a specific receptor on the plasma membrane of cells is followed by
endocytosis, a process in which the plasma membrane invaginates and then pinches off to form
an intracellular vesicle (endosome). Subsequent vesicle disruption by adenovirus proteins allows
virions to escape and migrate towards the nucleus where viral DNA enters through pores in the
nuclear envelope.)
Major disadvantages of adenovirus vector: 1)The inserted DNA does not integrate, and
so expression of inserted genes can be sustained over short periods only. The first generation
recombinant adenoviruses used in cystic fibrosis gene therapy trials showed that transgene
expression declined after about 2 weeks and was negligible after only 4 weeks.
2) Because they can infect virtually all human cells, adenovirus vectors may conceivably
pose a risk in some therapies that are designed to kill cancer cells without causing toxicity to
normal surrounding cells.
3) Most importantly, first generation adenovirus vectors can generate unwanted immune
responses, causing chronic inflammation.

c) Adeno-associated virus (AAV) vectors:

Adeno-associated viruses (AAVs) are a group of small, single-stranded DNA viruses
which cannot usually undergo productive infection without co-infection by a helper virus, such as
an adenovirus or herpes simplex virus.
Many of these difficulties of adenovirus have been addressed in the construction of
second generation adenovirus vectors (AAV). All of the adenovirus genes have been deleted
from adenovirus vectors ('gutless vectors') which then require the assistance of a helper virus.
Such a virus provides certain viral functions in trans (e.g. enzymes involved in viral DNA
replication etc.) which are essential for productive infection (including viral DNA replication,
viral assembly and infection of new cells) by certain natural viruses, such as AAV (adeno
associated virus), or artificially disabled viruses.
This is an important consideration given the need to administer treatment frequently (because of
the inability of adenovirus to integrate into chromosomal DNA).
Advantage of adeno associated virus vector: 1) They can accept much larger inserts (up
to 35 kb).
2) Deletion of the adenoviral E3 region removes the capacity to encode a protein that
protects the virus from immune surveillance mechanisms in the host.
3) In addition, fully disabled adenoviral vectors have much lower transduction
efficiencies.

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 The risk of immune response to these vectors is negligible.

Fig. 6. Gene therapy using an Adenovirus vector. A new gene is inserted into an
adenovirus vector, which is used to introduce the modified DNA into a human cell. If the
treatment is successful, the new gene will make a functional protein.

d) Herpes simplex virus vectors:

HSV vectors are tropic for the central nervous system (CNS) and can establish lifelong
latent infections in neurons. They have a comparatively large insert size capacity (>20 kb) but are
nonintegrating and so long-term expression of transferred genes is not possible. Their major
applications are expected to be in delivering genes into neurons for the treatment of neurological
diseases, such as Parkinson's disease, and for treating CNS tumors.)

e) Envelope protein pseudotyping of viral vectors:

The viral vectors described above have natural host cell populations that they infect most
efficiently. Some cell types are refractory to infection by these viruses as well. Entry into
potential host cells requires a favorable interaction between a protein on the surface of the virus
and a protein on the surface of the cell. For the purposes of gene therapy, many vectors have been
developed in which the endogenous viral envelope proteins have been replaced by either
envelope proteins from other viruses, or by chimeric proteins. Such chimera would consist of
those parts of the viral protein necessary for incorporation into the virion as well as sequences
meant to interact with specific host cell proteins. Viruses in which the envelope proteins have
been replaced as described are referred to as pseudotyped viruses. For example, the most
popular retroviral vector for use in gene therapy trials has been the lentivirus Simian
Immunodeficiency virus (SIV) coated with the envelope proteins, G-protein, from Vesicular
Stomatitus virus (VSV). This vector is referred to as VSV G-pseudotyped lentivirus, and infects
an almost universal set of cells.

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f) Lentiviruses;
The lentivirus family, which includes HIV (human immunodeficiency virus), are complex
retroviruses that infect macrophages and lymphocytes. Unlike oncoretroviruses, lentiviruses are
able to transduce nondividing cells. Because of their ability to infect nondividing cells and to
integrate into host cell chromosomes, considerable efforts are now being devoted to making
lentivirus vectors for gene therapy.

Gene therapy trials to treat severe combined immunodeficiency (SCID) were halted or restricted
in the USA when leukemia was reported in three of eleven patients treated in the French Therapy
X-linked SCID (XSCID) gene therapy trial. Ten XSCID patients treated in England have not
presented leukemia to date and have had similar success in immune reconstitution. Gene therapy
trials to treat SCID due to deficiency of the Adenosine Deaminase (ADA) enzyme continue with
relative success in the USA, Italy and Japan.

Non-viral methods:
Non-viral methods present certain advantages over viral methods;
1) simple large scale production and
2) low host immunogenicity being.

Disadvantage: Low levels of transfection and expression of the gene held in non-viral
methods. However recent advances in vector technology has yielded molecules and
techniques with transfection efficiencies similar to that of viruses.

a) Naked DNA
This is the simplest method of non-viral transfection. Clinical trials carried out of
intramuscular injection of a naked DNA plasmid have occurred with some success, however the
expression has been very low in comparison to other methods of transfection. In addition to trials
with plasmids, there have been trials with naked PCR product, which have had similar or greater
success. This success, however, does not compare to that of the other methods, leading to
research into more efficient methods for delivery of the naked DNA such as electroporation and
the use of a "gene gun", which shoots DNA coated gold particles into the cell using high pressure
gas.

b) Oligonucleotides
The use of synthetic oligonucleotides in gene therapy is to inactivate the genes involved
in the disease process. There are several methods by which this is achieved. One strategy uses
antisense specific to the target gene to disrupt the transcription of the faulty gene. Another uses
small catalytic molecules of RNA called siRNA to cleave specific unique sequences in the mRNA
transcript of the faulty gene, disrupting translation of the faulty mRNA, and therefore expression
of the gene. A further strategy uses double stranded oligodeoxynucleotides as a decoy for the
transcription factors that are required to activate the transcription of the target gene. The
transcription factors bind to the decoys instead of the promoter of the faulty gene which reduces
the transcription of the target gene, lowering expression.

c) Lipoplexes and polyplexes

To improve the delivery of the new DNA into the cell, the DNA must be protected from
damage and its entry into the cell must be facilitated. To this end new molecules, lipoplexes and
polyplexes, have been created that have the ability to protect the DNA from undesirable
degradation during the transfection process.
Plasmid DNA can be covered with lipids in an organized structure like a micelle or a liposome.
When the organized structure is complexed with DNA it is called a lipoplex. There are three types

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of lipids, anionic (negatively charged), neutral, or cationic (positively charged). Initially, anionic
and neutral lipids were used for the construction of lipoplexes for synthetic vectors. However, in
spite of the facts that there is little toxicity associated with them, that they are compatible with
body fluids and that there was a possibility of adapting them to be tissue specific; they are
complicated and time consuming to produce so attention was turned to the cationic versions.
Cationic lipids, due to their positive charge, naturally complex with the negatively charged DNA.
Also as a result of their charge they interact with the cell membrane, endocytosis of the lipoplex
occurs and the DNA is released into the cytoplasm. The cationic lipids also protect against
degradation of the DNA by the cell.
The most common use of lipoplexes has been in gene transfer into cancer cells, where the
supplied genes have activated tumor suppressor control genes in the cell and decrease the activity
of oncogenes. Recent studies have shown lipoplexes to be useful in transfecting respiratory
epithelial cells, so they may be used for treatment of genetic respiratory diseases such as cystic
fibrosis.
Complexes of polymers with DNA are called polyplexes. Most polyplexes consist of cationic
polymers and their production is regulated by ionic interactions. One large difference between the
methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA load
into the cytoplasm, so to this end, co-transfection with endosome-lytic agents (to lyse the
endosome that is made during endocytosis, the process by which the polyplex enters the cell)
such as inactivated adenovirus must occur. However this isn't always the case, polymers such as
polyethylenimine have their own method of endosome disruption.
Hybrid methods
Due to every method of gene transfer having shortcomings, there have been some hybrid
methods developed that combine two or more techniques. Virosomes are one example; they
combine liposomes with an inactivated HIV or influenza virus. This has been shown to have more
efficient gene transfer in respiratory epithelial cells than either viral or liposomal methods alone.
Other methods involve mixing other viral vectors with cationic lipids or hybridising viruses.

Recent developments in gene therapy

Scientists at the National Institutes of Health (Bethesda, Maryland) have successfully
treated metastatic melanoma in two patients using killer T cells genetically retargeted to attack
the cancer cells. This study constitutes the first demonstration that gene therapy can be effective
in treating cancer. The study results have been published in Science (October 2006).
In May 2006 a team of scientists led by Dr. Luigi Naldini and Dr. Brian Brown from the San
Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) in Milan, Italy reported a
breakthrough for gene therapy in which they developed a way to prevent the immune system
from rejecting a newly delivered gene. Similar to organ transplantation, gene therapy has been
plagued by the problem of immune rejection. So far, delivery of the 'normal' gene has been
difficult because the immune system does not recognize the new gene and rejects the cells
carrying it. To overcome this problem, the HSR-TIGET group utilized a newly uncovered
network of genes regulated by molecules known as microRNAs. Dr. Naldini's group reasoned that
they could use this natural function of microRNA to selectively turn off the identity of their
therapeutic gene in cells of the immune system and prevent the gene from being found and
destroyed. The researchers injected mice with the gene containing an immune-cell microRNA
target sequence, and spectacularly, the mice did not reject the gene, as previously occurred when
vectors without the microRNA target sequence were used. This work will have important
implications for the treatment of hemophilia and other genetic diseases by gene therapy.
In March 2006 an international group of scientists announced the successful use of gene therapy
to treat two adult patients for a disease affecting myeloid cells. The study, published in Nature
Medicine, is believed to be the first to show that gene therapy can cure diseases of the myeloid
system.

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 In 2003 a University of California, Los Angeles research team inserted genes into the
brain using liposomes coated in a polymer called polyethylene glycol (PEG). The transfer of
genes into the brain is a significant achievement because viral vectors are too big to get across the
"blood-brain barrier." This method has potential for treating Parkinson's disease. See Undercover
genes slip into the brain at NewScientist.com (March 20, 2003).
RNA interference or gene silencing may be a new way to treat Huntington's. Short pieces
of double-stranded RNA (short, interfering RNAs or siRNAs) are used by cells to degrade RNA
of a particular sequence. If a siRNA is designed to match the RNA copied from a faulty gene,
then the abnormal protein product of that gene will not be produced. See Gene therapy may
switch off Huntington's at NewScientist.com (March 13, 2003).
New gene therapy approach repairs errors in messenger RNA derived from defective
genes. Technique has potential to treat the blood disorder thalassaemia, cystic fibrosis, and some
cancers. See Subtle gene therapy tackles blood disorder at NewScientist.com (October 11,
2002).
Researchers at Case Western Reserve University and Copernicus Therapeutics are able to
create tiny liposomes 25 nanometers across that can carry therapeutic DNA through pores in the
nuclear membrane. See DNA nanoballs boost gene therapy at NewScientist.com (May 12,
2002).
Sickle cell is successfully treated in mice. See Murine Gene Therapy Corrects
Symptoms of Sickle Cell Disease from March 18, 2002, issue of The Scientist.
The success of a multi-center trial for treating children with SCID (severe combined immune
deficiency or "bubble boy" disease) held from 2000 and 2002 was questioned when two of the ten
children treated at the trial's Paris center developed a leukemia-like condition. Clinical trials were
halted temporarily in 2002, but resumed after regulatory review of the protocol in the United
States, the United Kingdom, France, Italy, and Germany. (V. Cavazzana-Calvo, Thrasher and
Mavilio 2004; see also 'Miracle' gene therapy trial halted at NewScientist.com, October 3,
2002).
Problems and ethics
For the safety of gene therapy, the Weismann barrier is fundamental in the current
thinking. Soma-to-germline feedback should therefore be impossible. However, there are
indications that the Weissman barrier can be breached. One way it might possibly be breached is
if the treatment were somehow misapplied and spread to the testes and therefore would infect the
germline against the intentions of the therapy.
Some of the problems of gene therapy include:
Short-lived nature of gene therapy - Before gene therapy can become a permanent cure
for any condition, the therapeutic DNA introduced into target cells must remain functional and
the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating
therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene
therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of
gene therapy.
Immune response - Anytime a foreign object is introduced into human tissues, the
immune system is designed to attack the invader. The risk of stimulating the immune system in a
way that reduces gene therapy effectiveness is always a potential risk. Furthermore, the immune
system's enhanced response to invaders it has seen before makes it difficult for gene therapy to be
repeated in patients.
Problems with viral vectors - Viruses, while the carrier of choice in most gene therapy
studies, present a variety of potential problems to the patient --toxicity, immune and inflammatory
responses, and gene control and targeting issues. In addition, there is always the fear that the viral
vector, once inside the patient, may recover its ability to cause disease.
Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the
best candidates for gene therapy. Unfortunately, some of the most commonly occurring disorders,

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 such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused
by the combined effects of variations in many genes. Multigene or multifactorial disorders such
as these would be especially difficult to treat effectively using gene therapy.
Chance of inducing a tumor - If the DNA is integrated in the wrong place in the genome, for
example in a tumor suppressor gene, it could induce a tumor.
Gene therapy plays a major role in the sci-fi series Stargate Atlantis, as a certain type of
alien technology can only be used if one has a certain gene which is given to the members of the
team through gene therapy.

Each of these gene delivery strategies have their own advantages and disadvantages.

Gene delivery system Advantages Disadvantages

Retrovirus integrates into host cell genome providing stable random integration may cause insertional
gene expression mutations

Adenovirus contains >30 kb of non-viral DNA, infects non- does not provide long term gene expression; no
dividing and dividing cells integration

Adeno-associated contains no viral genes, non-pathogenic, no small capacity for gene sequences, difficult to
virus immunity problems obtain large viral stocks

Liposomes non-pathogenic, no immunity problems, no limit low transfection efficiency, low rate of stable
to size of gene integration

Biolistics same as liposome-mediated transfer, promising limited to dermal tissue, low rate of stable
as a vaccination method integration, difficult to QC

In addition to being safe and cost-effective, the most important properties of an efficacious gene transfer
system will be;

1) target cell selective.

2) transcriptionally competent for the desired length of time.

3) available in a highly concentrated active form.

4) immunologically neutral.

While it is still too early to know how useful somatic cell gene therapy will be as a routine treatment for
human genetic diseases, most biomedical researchers agree that it will represent just one of the many new
molecular genetic tools that 21st century physicians will have at their disposal to better prevent, diagnose
and treat their patients.

An example of ex vivo gene therapyis used by Transkaryotic Therapies, Inc., to treat hemophilia A (lack of
Factor VIII) and was recently reported in a New England Journal of Medicine article:
Some background on using gene therapy to treat hemophilia A as described in a review that accompanied

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the Tkt publication in the same issue of the New England Journal of Medicine.

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The Tkt primary journal article:
New England Journal of Medicine, June 7, 2001 (vol. 344, pages 1735-1742)
"Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A"
Roth et al. (supported by TKT).

This study was conducted on six individuals with severe hemophilia A all of which received the gene
therapy treatment knowingly (open label not double blind). The results showed that a few of the patients
did indeed express higher levels of Factor VIII and did not need as much exogenous treatment. However,
the effect lasted for less than a year and in several patients there was not much benefit. Let's look at the
data:

First off, justification for using this ex vivo gene therapy approach for hemophilia A (factor VIII):

- Factor VIII production is not regulated in response to bleeding

- The broad therapeutic index of factor VIII minimizes risk of overdose

- Delivery of factor VIII into the bloodstream does not require cell-specific expression

- Even low levels of the protein can be beneficial to the patient

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The fibroblasts from the skin-biopsy specimen were transfected with a plasmid containing the
gene encoding human factor VIII from which the B domain had been deleted. Prior basic science studies
had shown the the B domain is glycosylated and is involved in secretion. Recombinant factor VIII lacking
the B domain still functions to stimulate coagulation and has a longer half-life because it is more resistant
to protein degradation. The function of the B domain is not known.

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Conclusions from this small pilot study (albeit high profile and of great benefit to Tkt stock value) is that it
shows the benefit of a non-viral ex vivo approach. The cells come from the patient and the site of
placement is relatively simple (like a navel pierce by Kraiger but worse). The down side of course is that it
didn't work that well and many more studies are needed.

Other human gene therapy companies

Cell Genesys in Foster City, California.

Vical Gene Delivery Systems in San Diego, California.

Introgen Therapeutics in Austin, Texas.

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