Gene Therapy - Viruses And Human Disease

as well as chimeras that contained only prM and E from JE.

GENE THERAPY

Chimeras containing C, prM, and E from JE were not viable,

whereas chimeras containing only prM and E from JE were

A number of genetic diseases result from the failure to pro-

viable and grew well in culture (Fig. 11.9).

duce a specific protein, usually due to a single defective gene.

The viable chimeras were first tested in mice. The

One of the more exciting possible uses for virus vectors is for

chimera containing the Nakayama strain proteins caused

the expression of a missing protein as a cure for the genetic

lethal encephalitis in mice, as does the YF 17D virus (even

defect associated with its absence. Some of these "monogenic

though it is safe for use in humans). However, the chi-

diseases" that might be curable through the use of gene therapy

mera containing prM and E from the attenuated JE strain

are listed in Table 11.1. For successful treatment, expression

was fully attenuated in mice and did not cause illness. The

of the missing protein must be long-term and preferably

fully attenuated chimera was chosen for testing in mon-

lifelong, the levels of protein produced must be sufficient to

keys, and was found to be safe and to protect monkeys

alleviate the symptoms of the disease, the protein must be

against challenge with JE virus.

expressed in or translocated to those cells that require the nor-

Clinical trials of this candidate vaccine have taken place in

mal protein for function, and infection with the virus vector

humans. The vaccine appears to be safe and more effective than

must be free of disease symptoms. Because of the requirement

the JE vaccines now in use. Furthermore, this approach is appli-

for long-term expression, viruses whose DNA integrates into

cable to other flaviviruses, such as the dengue viruses, for which

the host chromosome, such as the simple retroviruses as well

no licensed vaccines exist, or West Nile virus, which spread

as the lentiviruses, and adeno-associated viruses, offer the most

recently to the Americas where it caused a number of fatal cases

promising system for many diseases. To date, several hundred

of human encephalitis. Recombinant YF 17D expressing the

patients have been treated with vectors based on Moloney

prM and E proteins of all four serotypes of dengue viruses and

murine leukemia virus in clinical trials. Clinical trials have also

recombinant viruses expressing prM and E of West Nile virus

been conducted that use adenovirus. adeno-associated virus,

are also in clinical trials with encouraging results.

poxvirus, and herpesvirus vectors (Fig. 11.10).

Yet another possible approach to developing new gen-

Clinical trials in humans, which require extensive prior

erations of vaccines using the power of biotechnology is to

testing in animals, are divided into three phases. Phase I

attenuate a virus by making changes in the laboratory that

involves relatively few, usually healthy individuals. The

are expected to cripple the virus. Such an approach can be

objective of Phase I trials is to test the safety of a vaccine or

used with virtually any virus. A candidate vaccine strain of

treatment as well as the dosage that is tolerated, and the indi-

dengue virus has been constructed by making deletions in

viduals are closely monitored during the trial. Phase II trials

the 3′ nontranslated region of the genome that attenuate the

involve more individuals and test the efficacy of the treat-

virus, and such viruses are being tested in early trials.

ment, and patients are again closely monitored. If a treatment

TABLE 11.1

Genetic Defects That Are Candidates for Gene Therapy

Disease

Defect

Incidence

Target cells

Severe combined

Adenosine deaminase (ADA)

Rare

Bone-marrow cells or T lymphocytes

immunodeficiency (SCID)

in 25% of SCID patients

Factor VII deficiency

1:10,000 males

Liver, muscle, fibroblasts or bone

Hemophilia

Factor IX deficiency

1:30,000 males

marrow cells

Familial hypercholesterolemia

Deficiency of low-density

1:1 million

Liver

lipoprotein (LDL) receptor

Cystic fibrosis

Faulty transport of salt in lung epithelium

1:3000 Caucasians

Airways in the lungs

(Structural) defects in the α or

Hemoglobinopathies

1:600 in certain

Bone marrow cells that are precursors

β globin gene

thalassemias

ethnic groups

to red blood cells

Gaucher's disease

Defect in the enzyme glucocerebrosidase

1:450 in Ashkenazi Jews

Bone marrow cells, macrophages

α1 antitrypsin deficiency

Lack of α1 antitrypsin

1:3500

Lung or liver cells

inherited emphysema

Duchenne muscular dystrophy

Lack of dystrophin

1:3500 males

Muscle cells

Xeroderma pigmentosa

Impaired DNA repair, leading to

Rare

Fibroblasts

severe sensitivity to sunlight

350

300

250

200

150

100

Adenoviral

Poxviral

AAV

Herpesviral

Plasmid or

Retroviral,

Others

naked

including

DNA

lentiviral

Mode of Gene Delivery

FIGURE 11.10 Vectors used in gene therapy trials as of January 2007. The "others" category includes flavivirus (5),

measles virus (3), Newcastle disease virus (1), poliovirus (1), Semliki Forest virus (1), Sendai virus (1) and SV40 (1).

Data are from Gene Therapy Clinical Trials Worldwide Web site of the Journal of Gene Medicine at: http://www.wiley.

co.uk/genetherapy/clinical/.

passes both of these tests, Phase III trials can begin in which

antigens. Often only the gene of interest and the viral tran-

thousands of individuals are treated to test the efficacy of

scriptional regulatory elements are left, and to prepare the

treatment. Virtually all of the clinical trials in gene therapy

vectors for use in trials, all other functions must be supplied

conducted to date are Phase I or Phase II; fewer than 2% of

by a helper virus or a packaging cell line. Another advantage

trials have progressed to Phase III (Fig. 11.11), and no gene

of such stripped down vectors is the fact that it is improbable

therapy treatments have been licensed to date.

that the vector can recombine with wild-type viruses, either

In addition to the possible treatment of genetic defects,

exogenous or endogenous, to cause disease.

virus vectors may also be useful for the treatment of a number

A partial listing of clinical trials that attempt to treat sev-

of acquired diseases. These include cancer, HIV infection,

eral different genetic defects by using virus vectors to deliver

Parkinson's disease, injuries to the spinal cord, and vascular

specific genes is given in Table 11.5. There have been few

diseases such as restenosis and arteriosclerosis. A partial listing

successes to date and the table is more of a compendium of

of acquired diseases that have been suggested as candidates for

the variety of genes and diseases, as well as the variety of

gene therapy is given in Table 11.2, and the number of trials for

delivery schemes, that are being examined. Also included in

a number of different conditions is shown in Fig. 11.12. Despite

the table is an impending trial for the vaccination of humans

the large efforts to use gene therapy in clinical settings, the

against HPIV-3 using a bovine virus.

progress has been disappointingly slow, and many of the trials

have been aborted due to unforeseen adverse consequences.

Retrovirus Vectors to Genetically Mark Cells

Nevertheless, as infectious clones of viruses continue to

be developed, a large body of research is being devoted to

Retroviruses have been used in a number of clinical

construction of vectors, especially now to second and third

trials to genetically tag cells. Although this use does not fall

generation vectors, as the problems associated with the initial

within the narrow definition of gene therapy, it does provide

systems are becoming clear. A comparison of various virus

background experience in the use of retrovirus vectors in

systems that are being considered for gene therapy is shown

humans. One such use has been in bone marrow transplanta-

in Tables 11.3 and 11.4. Naked DNA has also been used in

tion for leukemia. Severe forms of leukemia can sometimes

a recent trial for coronary artery disease, and the proper-

be treated by ablation of the hematopoietic system with

ties of this system are included in Table 11.4. Most of the

chemotherapy and/or X-rays in order to kill all tumor cells,

modern vectors have had more and more of the dispensable

followed by reconstitution of the system by transplantation

viral genes deleted. Deletion of these genes reduces patho-

of bone marrow from a compatible donor. Although often

genicity, and prevents the production of immunogenic viral

successful, the leukemia sometimes recurs and it is desirable

(775)

(255)

(190)

(27)

(13)

I/II

II/III

III

Clinical phase of trial

FIGURE 11.11 Percent of gene therapy clinical trials that are in each phase as of January 2007. The numbers above

the bars are the actual number of trials. Data from Gene Therapy Clinical Trials Worldwide from the Web site of the

Journal of Gene Medicine at: http://www.wiley.co.uk/genetherapy/clinical/.

to know whether it recurs because of incomplete destruc-

Adenosine is toxic at high concentrations, producing a variety

tion of the patient's leukemic cells or whether the donor cells

of symptoms. The most serious symptom results from the

are the source of the leukemia. Experiments in which the

extreme sensitivity of T cells to elevated adenosine concentra-

donor cells have been tagged using retroviruses that express

tions. Loss of T cells results in SCID, severe combined immu-

a marker gene have been used to answer this question, which

nodeficiency. Both CTL responses (which are T-cell based) and

is important for the design of transplantation protocols.

humoral responses (which require T-helper cells) are impaired.

People with SCID syndrome are unable to mount an immuno-

logic response to infectious agents, and SCID is invariably

Gene Therapy for ADA Deficiency

fatal early in life unless treated in some way. ADA deficiency

Patients who lack the enzyme adenosine deaminase (ADA)

accounts for about 25% of SCID syndromes in humans.

will die early in life unless treated. Lack of ADA results in

SCID can be treated by bone marrow transplantation if

the failure to clear adenosine from the body and, consequently,

a suitable donor can be found. In the case of SCID due to

the accumulation of adenosine in cells throughout the body.

ADA deficiency, weekly or twice weekly injections of ADA

TABLE 11.2

Some Acquired Diseases That Are Candidates for Gene Therapy

Disease

Defect

Incidence

Target cells

Cancer

Many causes, including genetic

1 million/year in

Variety of cancer cell types, in liver,

and environmental

United States

brain, pancreas, breast, kidney

Neurological diseases

Parkinson's, Alzheimer's

1 million Parkinson's and

Neurons, glial cells, Schwann cells

spinal-cord injury

4 million Alzheimer's

patients in the United States

Cardiovascular

Restenosis, arteriosclerosis

13 million in United States

Arteries, vascular endothelial walls

Infectious diseases

AIDS, hepatitis B

Increasing numbers

T cells, liver, macrophages

Rheumatoid arthritis

Autoimmune inflammation of joints

Increasing numbers with

Intra-auricular delivery and expression

of IL-1 and TNF-α inhibitors

aging population

(842)

(113)

(106)

(81)

(50)

(47)

(21)

Healthy

Infectious

Vascular

Others

Gene

Monogenic

Cancer

volunteers

diseases

marking

diseases

Conditions currently addressed by gene therapy clinical trials

FIGURE 11.12

Percent of gene therapy trials which are directed toward various conditions as of January 2007. The

numbers above the bars are the actual number of trials. Data from Gene Therapy Clinical Trials Worldwide from the Web

site of the Journal of Gene Medicine at: http://www.wiley.co.uk/genetherapy/clinical/.

TABLE 11.3 Comparison of Properties of Various Vector Systems Based on RNA Viruses

and Retroviruses

Negavirala

Features

Simple retroviral

Lentiviral

Alphaviral

Coronaviral

~4 kbb

Maximum insert size

77.5 kb

5 kb

2.7 kb

>108

>109

>108

>109 for VSV, PIVs

Concentration in viral

particles/ml

101000 fold lower

Route of gene delivery

Ex vivo

Ex/in vivo

In vivo

Integration

Yes

Duration of expression in vivo

Shorter than theorized

Long

Short

Variable

Not known

Stability

Good

Not tested

Good

Dependent on

Good

background

Ease of preparation scale up

Pilot scale up to

Not known

2050 liters

Preexisting host immunity

Unlikely

Unlikely, except in

Unlikely

AIDS patients

Safety problems

Insertional mutagenesis?

Insertional

Few

Recombination

mutagenesis?

with wild strains

Other advantages

Replicate in

Recombination virtually

nondividing

unknown; naturally

cells

atttenuated viruses exist

Includes consideration of VSV based and PIV based vectors.

Longer inserts are tolerated but vectors are too attenuated in vivo to be useful.

Source: Verma and Somia (1997), Jolly (1994), and Bukreyev et al. (2006).

TABLE 11.4

Comparison of Properties of Various Vector Systems Based on DNA Viruses

Vectors based on DNA viruses

Naked/lipid

Features

Adenoviral

AAV

Herpesviral

Vaccinia

Baculovirus

DNA

Maximum insert size

7.5 kb

<4 kb

~30 kb

2575 kb

>38 kb

Unlimited size

>1010

>1012

>1010

107109

>108

No limitation

Concentration in viral

particles/ml

Route of gene delivery

Ex/in vivo

Ex vivo

Ex/in vivo

Integration

Yes/no

Very poor

Duration of expression

Short

Long

Short/long in CNS?

Short

in vivo

Stability

Good

Unknown

Good

Very good

Ease of preparation

Easy to scale up

Difficult to purify,

Not yet tried

Vaccine

Easy to

Easy to scale up

scale up

difficult to

production

scale up

facilities exist

Immunologic

Extensive

Not known

Extensive

Not known

None

problems

Preexisting host

Yes

Diminishing as

Unlikely

immunity

unvaccinated

population grows

Safety problems

Inflammatory

Neurovirulence?

Disseminated vaccinia

None?

response,

Insertional

in immuno-

toxicity

mutagenesis

compromised hosts

Source: Verma and Weitzman (2005), Jolly (1994), Boulaiz et al. (2005), Hu (2006).

mixed with polyethylene glycol (PEG) have been used to

treatment was available were treated with improved retro-

successfully treat about 60 patients in whom bone marrow

viral therapy. Both patients developed functional immune

transplantation cannot be used because of the lack of com-

systems and no adverse events have been reported. The

patible donors. Of these, about 10 patients have also been

improved results appear to arise from improved protocols

treated with retroviral vectors that express ADA. In these

as well as to selection in the patient for lymphoid progenitor

experiments, T cells were taken from the patient (or in the

cells that expressed adequate amounts of ADA.

case of three newborns, umbilical cord cells were used),

infected ex vivo with the retrovirus vector using a number

Treatment for SCID Caused by IL2RG Deficiency

of different cell culture and infection protocols, and the cells

reinfused into the patient. Many of the patients continue to

SCID disease can also be caused by a failure to produce

produce ADA from the vector several years after treatment.

the receptor for the cytokine interleukin-2 (Chapter 10).

However, all of the patients continue to receive ADAPEG

Thirteen SCID patients with this deficiency were treated in

injections, which is known to be an effective treatment.

two different clinical trials with retroviruses that expressed

Although some patients who have received retroviral ther-

the defective gene (Table 11.5). The results illustrate the

apy have been partially weaned from the supplementary

highs and the lows of gene therapy trials. All 13 patients

ADAPEG, it appears that some of these, and perhaps all,

developed functional immune systems, and the trials at first

do not produce enough ADA to be cured. Thus, although

appeared to be a complete success. However, three of the

no cures were effected in these early trials, the results were

patients later developed T-cell leukemia and one has died

encouraging and suggested that future protocols might be

of the leukemia. The leukemia was at first suspected to arise

more successful. Two areas of retroviral therapy that needed

from insertional mutagenesis, a chronic worry with vectors

improvement were to increase the efficiency with which

that insert into the host DNA, and many gene therapy trials

stem cells are infected, and the need to prevent the retroviral

that used retroviral vectors were suspended. Recent studies

promoter from being downregulated.

indicate that the disease is not due to insertional mutagen-

A more recent trial involving two SCID-ADA patients in

esis, however, but rather due to the oncogenic potential of

Italy was more successful (Table 11.5). Two infants for whom

the IL2RG gene itself, as studies have shown that overex-

no compatible donor existed and for whom no PEG-ADA

pression of this gene in mice results in leukemia.

TABLE 11.5 Recent Human Gene Therapy Trials

Therapeutic

Total

Vector/

Disease

gene

patients

promoter

Method of delivery

Outcome

Monogenic diseases

OTC deficiency

OTC cDNA

E1E4 deleted

In vivo injection to

No clinical benefit; 1 death

adenovirus/CMV

hepatic artery

Factor IX deficiency

Modified Factor

rAAV2/ CMV

In vivo intramuscular

No sustained clinical benefit

(hemophilia B)

IX gene

rAAV2/APOE-

In vivo injection to

Transient Factor IX expression, no

SERPINA1

hepatic artery

sustained benefit

SCID-X1

IL2RG cDNA

Retrovirus/MLV-

Ex vivo transduction of

9 patients developed functional

CD34+ cells

(French trial)

LTR

immune system; 3 developed

T-cell leukemia, 1 death

SCID-X1

IL2RG cDNA

GALV-pseudotyped

Ex vivo transduction of

4 patients developed functional

CD34+ cells

(British trial)

retrovirus/MFG-LTR

immune system; no adverse

results reported in first 2 years

SCID-ADA

ADA cDNA

Retrovirus/MLV-

Ex vivo transduction of

2 patients developed functional

CD34+ cells

(Italian trial)

LTR

immune system; no adverse

results reported in first 4 years

CGD

GP91PHOX

Retrovirus/SFFV-LTR

Ex vivo transduction of

2 patients developed functional

CD34+ cells

neutrophils and clonal myelo-

proliferation (cancer)

Duchenne muscular

Dystrophin

Plasmid/CMV

Intramuscular injection

Low dystrophin expression in 6 of

dystrophy

9, no adverse effects (Phase I)

Microdystrophin

AAV/CMV

Intramuscular biceps injection

Phase I/II ongoing

2OMeAOs

Oligonucleotide

Injection into extensor

Successful non-human primate

digitorum brevis

study; human Phase I/II not yet

opened for patient recruitment

Vaccine for infectious disease

HPIV-3 vaccine

F and HN surface

BPIV-3

Infection

Successful non-human primate

glycoproteins

study; human Phase I trial not

yet opened for patient recruitment

APOE-SERPINA1, α1-antitrypsin promoter linked to APOE enhancer; BPIV-3, bovine parainfluenzavirus 3; CGD, chronic granulomatous disease;

CMV, cytomegalovirus promoter; GALV, gibbon ape leukemia virus; HPIV, human parainfluenzavirus; LTR, long terminal repeat; MFG, derivative

of MLV; MLV, Moloney murine leukemia virus; OTC, ornithine transcarbamylase; rAAV, recombinant adeno-associated virus serotype 2; SCID-

ADA, severe combined immunodeficiency secondary to adenosine deaminase deficiency; SCID-IX, severe combined immunodeficiency secondary to

mutations in the IL2RG gene; SFFV, Friend mink cell spleen focus-forming virus.

Source: Data for this figure came from Porteus et al. (2006), Foster et al. (2006), Bukreyev et al. (2006).

Cystic Fibrosis

mucus that is a characteristic of cystic fibrosis. In addition,

the expression needs to continue for the life of the patient,

Cystic fibrosis results from loss of the cystic fibrosis

which means either a very stable (integrated?) gene being

transmembrane conductance regulator (CFTR), which regu-

expressed, or a system of repeated administration of vector

lates epithelial transport of ions and water. Although lack of

that can be tolerated without immunologic consequences.

this protein results in damage to the epithelium in many parts

Lentiviruses have been proposed as an attractive vector, but

of the body, the most serious manifestation is lung disease

there have been concerns about probable pathogenesis due

accompanied by chronic bacterial infection of the airways.

to the lentivirus itself. However, for this disease, the most

Clinical trials using adenoviral vectors, which infect respi-

promising mode of gene delivery so far developed has been

ratory epithelium, to express CFTR in the lungs have been

DNA compacted into nanoparticles with polycations, in par-

conducted. The first such studies were encouraging, but a

ticular PEG-polylysine, nicknamed "polyplexes." A clini-

more recent trial that was carefully controlled found no relief

cal trial in humans used CFTR polyplexes, and the Phase I

of symptoms. Inflammation produced by the high doses of

trial showed no adverse effects. For this disease a nonviral

adenovirus used in trials is also a problem. It is difficult to

approach may well be the best solution.

get efficient delivery to the lung, especially through the thick

Duchenne Muscular Dystrophy

a frameshift mutation in one of the exons, such that no dys-

trophin is produced. Since severely truncated dystrophin (like

Duchenne muscular dystrophy (DMD) is a severe mus-

micro-dystrophin) can be functional (Fig. 11.13), therapy to

cle wasting disorder due to the lack of functional dystrophin

get rid of the exon causing the problem is being developed.

protein. It occurs in 1/3500 male births. There are a number

Modified antisense oligonucleotides (AOs) can be used to

of difficulties in attempting to cure DMD with gene therapy,

alter the splicing pattern of the gene such that the exon in

including the size of the protein, which is encoded in a cDNA

which the frameshift occurs is skipped, thereby restoring

of 11kb, and the need to deliver the vector to a large propor-

the reading frame. Following success of an AO with a

tion of the body mass, that is to all of the striated muscles and

2′O-methyl-phosphorothiolate backbone (2OMe AOs) to

cardiac muscle. Several approaches have been tried, and the

restore function in the mdx mouse, two clinical Phase I trials

first human clinical Phase I trial has just been completed (see

have been initiated, one in the Netherlands using 2OMe AOs

Table 11.5). In this study, a plasmid containing the entire gene

and one in Great Britain using morpholino AOs. Since both

under the control of a CMV promoter was injected intramus-

are targeting the exclusion of exon 51, it will be possible to

cularly. Although only low levels of dystrophin expression

directly compare the two chemically modified AOs.

were observed in 6 out of 9 patients, there was no evidence

for adverse reactions and the trial was considered a success.

A second approach has been to attempt to introduce the

Rheumatoid Arthritis

gene in an AAV vector, especially in one of the many human

isolates to which most of the population show no preexisting

Rheumatoid arthritis is a chronic, progressive inflamma-

immunity, or into a nonhuman AAV. However, here the size

tory disease of the joints. An estimated 5 million people in

of the gene is a problem, but it has been shown that the

the United States suffer from it. There is no cure. Drug thera-

dystrophin protein contains a large number of repeated ele-

pies are used that ameliorate the symptoms, but most of these

ments (Fig. 11.13) and that "mini-dystrophin" and "micro-

drugs have side effects and cannot be taken indefinitely. If

dystrophin" are functionally active in the mouse model for the

the disease progresses far enough, joint replacement may

disease, the mdx mouse. Low-pressure intravenous injection of

be required. The disease is associated with the release of

AAV6 expressing micro-dystrophin into a mouse could trans-

inflammatory cytokines in the affected joints. Clinical trials

fect 90% of muscle, but high titers of the AAV6 vector were

have started that use retroviruses to deliver the gene for an

required. A Phase I/II clinical trial has been initiated for deliv-

anti-arthritic cytokine gene to the joints. The gene encodes

ery of micro-dystrophin in AAV under the control of a CMV

the interleukin (IL)-1 receptor antagonist, which inhibits the

biological actions of both IL-1α and IL-1β. It is hoped that

promoter into human patients, but there are no results as yet.

A third approach attacks the specific nature of the genetic

such treatment might damp out the disease or at least keep it

defect. It has been shown that 75% of DMD is caused by

from progressing.

cDNA 11 kb

H1 1 2 3 H2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 H3 20 21 22 23 24 H4

cDNA 6.3 kb

H1 1 2 3 19 H3 20 21 22 23 24 H4

Cysteine-rich domains

Carboxy terminal domains

cDNA 3.8 kb

1 2 19 H320 21 22 23 24 H4

Actin binding domains

Hinge regions

Spectrin repeats

FIGURE 11.13 Forms of the dystrophin protein. (A) Full-length dystrophin containing all functional domains.

(B) Mini-dystrophin from a Duchenne muscular dystrophy patient who was only mildly impaired. (C) Micro-dystrophin

engineered for delivery in AAV vectors. Adapted from Figure 1 in Foster et al. (2006).

A Gene Therapy Failure

I/II, but more than 1000 patients were participating in the

trials listed in the table. As shown in Fig. 11.12, the devel-

Patients who have deficiencies in enzymes that participate

opment of gene therapy approaches for the control of can-

in the urea cycle have increased concentrations of ammonia

cers continues to attract much effort, such that the majority

in the blood. High concentrations of ammonia result in vari-

of clinical trails to date have been directed against cancers.

ous symptoms, which can include behavioral disturbances

Although progress has been painfully slow and disappoint-

or coma. Severe deficiencies in these enzymes result in

ing, the prospect remains that effective treatment may yet be

early death, but moderate deficiencies can result in delayed

obtained for at least some cancers using such approaches.

appearance of symptoms and may be partially controlled by

In most of the trials in Table 11.6, viruses are used to

diet. One such enzyme is ornithine transcarbamylase (OTC),

express proteins that control the growth of tumors or that

which is found on the X chromosome. Deficiencies in OTC

are toxic to tumor cells. A number of different cytokines

are therefore more common in males than in females.

are being tried as antitumor agents, such as IFN-γ, IL-2,

Gene therapy trials that use virus vectors recently received

TNF, and GM-CSF. Another approach is to try to repair the

a major setback when a relatively fit 18-year-old male with

defective regulatory gene in the tumor cell, which is often

an inherited deficiency for OTC died 4 days after an ade-

p53. Many other gene products are also being tested. The

novirus vector was injected into his liver. A high dose of

viruses used to express these products include the retrovi-

adenovirus (4 × 1010) that expressed OTC was injected in an

ruses, the adenoviruses, or the poxviruses. More recent tri-

effort to achieve adequate levels of enzyme production. The

als have used additional viruses as well, in particular the

virus unexpectedly spread widely and a systemic inflam-

herpesviruses and the adeno-associated viruses. Herpes

matory response developed, inducing a fever of 40.3°C. He

simplex type 1 would seem to be particularly appropriate

went into a coma, his lungs filled with fluid, and he died of

for control of brain tumors, because the virus is neurotropic

asphyxiation. This unfortunate result makes clear the pos-

but sets up a latent infection in neurons. One idea would

sible drawbacks to experimental treatments and the difficul-

be to engineer herpes to express a protein that is only toxic

ties in designing protocols that allow an adequate margin of

in dividing cells but which would be nontoxic in mature,

safety while trying to achieve a clinically relevant result.

nondividing neurons. The table also lists a number of trials

that use lipofection to introduce the gene of interest into

target cells.

Treatment of Restenosis

Further afield, thought is being given to the possibility of

A gene therapy trial in patients with heart disease gave

using viruses to express proteins that are overexpressed in

very encouraging results. Although this study did not

tumor cells in an attempt to stimulate the immune system to

involve virus vectors, a brief description will be given since

respond by killing tumor cells. This is in essence an attempt

it serves as an incentive for continuation of gene therapy

to vaccinate a person against a tumor. For this approach to

trials. Coronary artery disease is common in older people.

succeed, an antigen overproduced by a tumor cell, such as

Angioplasty or bypass surgery is used to open clogged arter-

a melanoma cell, must be identified, inserted into a suitable

ies, but in many patients the arteries close up again (a process

vector, and the person with the tumor infected with the virus

called restenosis). Thirteen patients with chronic chest pain

vector in an attempt to stimulate the immune system. In prin-

who had failed angioplasty or bypass surgery or both were

ciple, this approach may be feasible, but only time will tell

injected in the heart muscle with DNA encoding vascular

whether it is in fact practical.

endothelial growth factor. This factor promotes the growth

Another approach is to try to direct the virus, more or

of blood vessels, a process called angiogenesis. Two months

less specifically, to infect the tumor cells, so that upon

after treatment, all patients exhibited an improvement in

infection the cells are killed. Cell death might result either

vascularization of damaged areas of the heart, as shown

because the virus itself is cytolytic or because the virus

by imaging and mapping studies. All patients reported a

expresses a protein that renders the cell sensitive to a toxic

decrease in disease symptoms, and all had an improved per-

agent such as BUdR. A number of the trials listed in Table

formance in treadmill tests. Although the number of patients

11.6 use the TK gene for this, since cells that express TK

is small, the uniformly positive results are encouraging.

are sensitive to BUdR. One possible approach is to engineer

the virus so that its surface glycoprotein expresses a

monoclonal antibody directed against an antigen expressed

Viruses as Anticancer Agents

only on the tumor cell, while at the same time causing the

virus to be unable to infect cells that do not express the

A large number of clinical trials have examined the pos-

antigen. Experiments have established the possibility of

sibility of using viruses as anticancer agents. Table 11.6 lists

this approach, at least in principle, with viruses such as

a number of trials that were active in the year 2000, to give

the alphaviruses. Another approach was illustrated by the

a flavor of what is being tried and the number of maligna-

experiments with VSV to design a virus that could infect

cies that are being considered as candidates for treatment

only HIV-infected cells.

using gene therapy approaches. These trials are all Phase I or

TABLE 11.6

Clinical Trials of Gene Transfers for Cancer Therapy in the United States in 2000

Genea

Phaseb

Location

Vector

Number of trials

Number of patients

Brain cancers

IFNγ

Neuroblastoma

Retrovirus

IL-2

Retrovirus

IL-2

Adenovirus

Central nervous system

Adenovirus

Pediatric tumor

Retroviral producing cells

Adult brain tumor

Retroviral producing cells

Ovarian cancer

HSV-TK

Adenovirus

Retroviral producing cells

BRCA-1

Retrovirus

I/II

p53

Adenovirus

Small cell lung cancer

IL-2+NeoR

Lipofection

Anti-sense to k-ras

Retrovirus

p53

Adenovirus

I/II

Prostate cancer

GM-CSF

Retrovirus

I/II

PSA

Poxvirus

HSV-TK

Adenovirus

Breast cancer

BRCA-1

Retrovirus

E1A

Lipofection

MDR-1+NeoR

Retrovirus

CD80

Lipofection

CEA

Poxvirus

CEA

RNA transfer

Melanoma

GM-CSF

Gene gun

GM-CSF

Retrovirus

HLA-B7/β2 m

Lipofection

165

I/II

IL-2+NeoR

Retrovirus

115

IFNγ

Retrovirus

TNF+NeoR

Retrovirus

I/II

MART-1

Adenovirus

MART-1

Poxvirus

gp100

Poxvirus

gp100

Adenovirus

CD80

Lipofection

Miscellaneous carcinomas

p53

Adenovirus

HLA-B7/β2m

Lipofection

IL-2

Lipofection

CEA

Poxvirus

Lymphomas and solid tumors

IL-2

Retrovirus

IL-12+NeoR

Retrovirus

Bladder cancer

p53

Adenovirus

Colo/rectal, renal, and liver cancers

GM-CSF

Retrovirus

HLA-B7/β2m

Lipofection

I/II

Adenovirus

IL-4

Retrovirus

TNF+NeoR

Retrovirus

(Continued)

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