Gene therapy treatment – Introduction:
- Gene therapy is used to
treat large number of diseases.
- Research in the field of
human gene therapy is directed towards correcting genetic defects of
somatic cells i.e., cells that do not contribute to the next
- Human germ line therapy
is not tried due to ethical, safety and technical reasons.
- In human somatic cell
gene therapy the fully functional and expressible gene is inserted
into a target cell, by which that a specific genetic disease can be
- The development of a
therapeutic agent for human passes through four levels of study and
Pre clinical trials that include extensive invitro experiments
and research on laboratory animals.
Phase I trials with a small number (6 to 10) of human subjects.
Phase II trials with an increased number of human subjects to
examine the effect of the drug or therapy.
Phase III trial includes a large human sample and fully
comphrensive analysis of the safety and efficacy of the products.
Characteristic diseases caused by Gene
mutations that code these products
Disease & symptoms
of T & B cells
marrow transplant, adenosine deaminases replacement.
hyper cholesterolemia, elevated blood serum cholesterol level,
coronary artery disease
drugs, liver transplant
disease, Accumulation of glucocerebroside in macrophages causing
liver, spleen and bone damage.
treatment, removal of spleen. Antibiotics, repairing bone damage,
bone marrow transplantation, enzyme replacement.
clotting Factor VIII
A, altered plasma protein that causes defective blood clotting,
chronic internal bleeding into joints, excessive bleeding after
of factor VIII by transfusion. Requires continuous treatment. Risk
of viral infections from transfusions.
Excess phenylalanine in the blood stream of newborns causes mental
of dietary phenylalanine
Deficiency of serum protein protease inhibitor, damage to lungs,
cirrhosis of the liver
therapy, lowering environmental risks.
fibrosis transmembrane regulator
fibrosis multi-system disease, pancreatic insufficiency in some
cases, intestinal blockage, blocked airways of the lung.
physical clearing of the lungs, upgrading diet.
urea cycle defects, ammonia accumulation, arginine deficiency.
protein diet, arginine supplemented diet, drugs, liver transplant.
muscular dystrophy (DME). Progressive muscle wasting.
supportive treatments, good nutrition.
cell disease chronic anemia, multi-system disease, damage to
spleen, heart, kidneys, liver and brain.
transfusions, drugs, analgesics, bone marrow transplant.
Human Gene therapy trials
bone marrow cells.
infiltrating lymphocytes, autologous tumor cells.
glioblastoma, renal cell cancer
tumor cells, tumor cells
locus antigen class –I B7 plus β2-microglobulin
colorectal cancer, renal cell cancer
AIDS, ovarian cancer
cells – T cells
fibrosis transmembrane conductance regulator
and airway epithelium
drug resistance -I
CD 34+ cells
colony stimulating factor
1 receptor antagonist
transduced and exogenic cells.
and neck squamous carcinoma
Gene Augmentation Therapy (GAT):
- Gene augmentation
therapy (GAT), where DNA is added to the genome inorder to replace a
missing gene product.
- Gene targeting to
correct mutant alleles.
- Gene inhibition therapy,
using antisense RNA expression or expression of intracellular
antibodies to treat diseases.
- Targeted ablation of
- Somatic cells can only
be used as Therapeutic gene transfer generates transgenic human cell
- In transient gene
therapy oligonucleotides can be used which can distrupt gene
expression at many levels but does not cause permanent genetic
augmentation therapy for recessive diseases:
- Gene marking is the
first human genetic engineering experiment to demonstrate that an
exogenous gene could be safely transferred into a patient and that
this gene could subsequently be detected in cells removed from the
- Tumor infiltrating
lymphocytes (kills tumor cells) were isolated from patients with
advanced cancer. The cells were then genetically marked with a
neomycin resistance gene and injected back into the same patient.
- Gene augmentation
therapies are being undergone for a small number of recessive single
- Cystic fibrosis
(disorder that affects lungs, liver and pancreas). The disease is
caused by loss of cAMP –regulated membrane spanning chloride
channel, which results in an electrolyte imbalance and accumulation
of mucus leading respiratory failure. Cystic fibrosis is a recessive
disorder which can be altered by introducing a functional copy of
augmentation therapy for cancer:
- Different Gene therapy
strategies are under study for cancer treatment.
- Tumor infiltrating
leucocytes were transformed with a gene for TNF in addition to
neomycin resistance gene with the aim of improving efficiency of
these cells to kill tumors by increasing the amount of TNF they
- Transform the tumor
cells themselves, making them more susceptible to immune system
through expression of cytomines or foreign antigen.
- Transform Fibroblasts,
which are easier to grow in culture and co-inject these together
with tumor cells to provoke an immune response against the tumor.
- Oncogenes (cancer genes)
can be targeted using antisense technology either with antisense
transgenes, oligonucleotides or ribozyme.
- Cancers occurring due to
loss of Tumor suppressor genes function can be altered by delivering
a functional copy of the appropriate gene delivered into affected
- Pro-drug activation
therapy involves activation of a particular enzyme specifically in
cancer cells, which converts a non-toxic pro-drug into toxic
product, so killing the cancer cells, which can be achieved by
driving the expression of so called “suicide gene” selectively
in cancer cells.
- Use of Transcriptional
regulatory elements that are active only in cancer cells.
Gene Therapy and Cancer:
- Cancer is caused by the
somatic mutation of cellular genes.
- These genes include
oncogenes, tumor suppressor genes, and DNA repair genes.
- The generation of cancer
is currently thought to be multi-step process of genetic alterations
that vary according to the type and stage of cancer.
- Since cancer is a
genetic disease, gene therapy could be applied in treatment of
- The most advanced
applications of gene transfer technology in medicine are in gene
therapy for cancer.
– Gene therapy:
- Cells of the immune
system have been found to recognize specific antigens of tumors and
mount both humoral and cellular responses.
- During cancer
development, this response is often limited in intensity and
are being developed to reconstitute an effective antitumor immune
in immunology have made Immunomodulation (immunological approaches
to treatment) the most dominant strategy in gene therapy for cancer.
- These approaches can be
categorized in to three types according to target cells, mode of
delivery, and transferred transgenes.
- Target cells include
tumor cells, T cells, host cells, APCs (antigen presenting cells),
and others. Mode of
delivery refers to choice of vector, in vitro, ex vivo,
or in vivo). Transferred
transgenes refer to cytokines, co-stimulatory molecules, and
- The basic concept of
using prodrug-converting enzymes is to limit the action of a known
cytotoxic drug to local tumor areas.
- Targeted prodrug therapy
includes the delivery of a gene that activates a nontoxic prodrug to
a cytotoxic product by using viral vectors.
- This method maximizes
toxicity at site of vector delivery while minimizing toxic effects
on distant cells.
- The cDNA of the enzyme
is delivered in to the tumor by a vector.
- The corresponding
nontoxic prodrug is applied and is taken up by tumor cells.
- Since these cells have
incorporated the cDNA in to their genome, they express the prodrug-converting
- Therefore, when the
cells take up the drug, it is converted in to a cytotoxic drug that
kills the tumor.
Suppressor Genes and anti-oncogenes:
- Oncogenes, or genes that
promote proliferation, become activated while tumor suppressor
genes, or genes that terminate proliferation, become inactivated.
- Approaches in this
method include the inactivation of oncogenes or the re-constitution
of tumor suppressor genes that have been inactivated by gene
deletion or other mechanisms. The
most effective procedure has been to target genes known to be
most targeted tumor suppressor gene has been p53 because it is the
most commonly mutated gene.
Lysis by Recombinant Viruses:
- Cancer therapy by viral
oncolysis is one of the oldest concepts in gene therapy, and it has
been one of the most challenging strategies.
- The basic concept is to
inject a virus in to the tumor, allowing transduction of cells.
- The virus causes lysis (oncolysis)
of the cancer cells after some method of external disruption.
- Since the viruses have
replicated in the cells, forming new viruses, the infection can be
spread to other cancer cells, which will be lysed at a certain time
in the lytic cycle.
- The adenovirus is a
common virus used in these applications.
- Gene Therapy:
- Cancer cells require
nutrients from the blood stream for proliferation.
Also for cancer to spread, the cells of a tumor must cross
the cellular layers of blood vessel walls and get in to the
- This method by which
tumors spread is called metastasis.
- Antiangiogenic Gene
Therapy targets endothelial cells of blood vessels.
Several angiogenesis inhibitors can inhibit proliferation of
intra-tumoral endothelial cells, therefore inhibiting tumor growth.
- This method is targeted
at preventing side effects of chemotherapy.
- It involves making
normal cells resistant to the toxic effects of chemicals used in
- Anti-mitotic drugs are
currently used to treat cancer.
- These drugs disrupt the
formation of the mitotic spindle during mitosis, thereby eventually
killing dividing cells.
- Since cancer cells are
continuously dividing (uncontrollably), these drugs kill cancer.
However, these drugs can also kill normal cells that are dividing
rapidly, such as skin cells, cells of the intestine, and cells
surrounding hair follicles.
therapy and cardiovascular disease:
- Gene therapy to help
increase blood flow to ischemic tissue.
- Ischemia is a condition
in which the flow of blood, and thus oxygen, is restricted to a part
of the body.
- Limb ischemia and
myocardial ischemia refer to lack of blood flow and oxygen to the
limb and heart muscle.
- Some proteins in the
body can help trigger new blood vessel growth and so increase the
oxygen supply to the ischemic tissue.
- Such angiogenic proteins
include the endothelial growth factors, vascular endothelial growth
factor (VEGF) and fibroblast growth factor (FGF) and hepatocyte
growth factor (HGF).
- In gene therapy trials,
different ways to deliver the genes for VEGF-1, VEGF-2 and FGF4 into
the hearts of patients with advanced myocardial ischemia.
- After gene therapy,
there is less severe angina (chest pain) and their hearts worked
- Gene therapy has also
been successful in preventing re-occlusion, or re-blockage, of
coronary artery bypass grafts and in keeping arteries open
after angioplasty surgery.
therapy and Adenosine Deaminase Deficiency:
- Adenosine deaminase
(ADA) deficiency is inherited as an autosomal recessive disorder.
Defects in the ADA gene can leads to absent or diminished ADA enzyme
activity in all tissues of the body.
- The reversing of this
genetic defect by replacing the defective gene may be possible by
means of gene therapy.
- The newly isolated gene
may then be used for treatment by inserting it into the cells whose
function is dependent on that gene.
- The gene transfer method
used must be highly efficient and able to deliver the corrective
gene to the proper cells to restore its normal function.
Gene therapy and Central Nervous system:
- Gene therapy is used for
direct gene transfer to the affected brain region will carry out a
local therapeutic gene product synthesis.
- Recessive metabolic
diseases of the nervous system caused by defects in single gene
potentially could be cured by replacement of the defective gene with
a correct gene.
neurodegenerative conditions might be improved by the production of
therapeutic products from transferred genes.
Factors hindering gene therapy:
Immune response to
the transferred gene stimulates a potential risk to gene therapy.
- Long lasting therapy is
not achieved by gene therapy; the transferred DNA must remain
functional and stable. But due to rapid dividing of cells long term
benefits of gene therapy is short lived.
Viruses used as
vectors for gene transfer may cause toxicity, immune responses, and
inflammatory reactions in the host. Virus may also become pathogenic to
Disorders caused by
defects in multiple genes cannot be treated effectively using gene
Recent developments in gene therapy:
- Genes are transferred
into the brain using liposome coated in a polymer call 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 technique is used to treat
- RNA interference or
gene silencing may be a new way to treat Huntington's, in which short
pieces of double-stranded RNA (short, interfering RNAs or siRNAs) are
used by cells to degrade RNA of a particular sequence
- 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.
- Gene therapy for
treating children with X-SCID (sever combined immunodeficiency) or the
"bubble boy" disease.
- Tiny liposomes 25
nanometers are created to carry therapeutic DNA through pores in the
- Sickle cell is
successfully treated in mice.
of Gene therapy:
study methods to learn how to turn gene on and off.
insert a gene and regulate its expression through combination of one
- Gene therapy for
- Taming of AIDS virus and
using it for gene transfer.
- Using modified HIV for
treatment of AIDS.
are the different types of gene therapy?
Somatic cell gene therapy is conducted on the somatic cells
(a non-reproductive cell). Germ line gene therapy is performed on
reproductive genes (germ line cells) and the goal is to correct the
individual’s, and their offspring’s, genetic defect.
is genetic engineering?
Genetic engineering is making an alteration in genetic material.
It is used for large scale use and changing
the genetic make up of the entire organism.
It refers to transgenic organisms (humans,
gene therapy include genetic enhancement?
Genetic enhancement does not provide therapeutic benefits.
It is used for overall enhancement of the
genetic make up, like changing the physical characteristics.
Pre-implantation genetic diagnosis, to the select the gender of an embryo.
Is there any genetic enhancement currently done?
Human genetic hormone is a genetically engineered hormone given to
children’s who are deficient in their natural growth hormones.
Does gene therapy transfer genetic changes to next generation?
- In somatic cell gene
therapy it does not pass on to next generation, as it is inserted
into somatic cells and genetic changes occurs only in the patient.
- In Germ cell therapy,
the genetic changes will be passed on to the next generation.
was the first disease treated by gene therapy?
deaminase (ADA) deficiency.
are the risks involved in gene therapy?
- During gene transfer by
virus vector, the virus can infect more than one cell type and the
virus may alter more than the intended cell.
- During gene transfer,
the new gene may be inserted in the wrong place and may lead to
cancer or other damage.
- Over expression of the
inserted gene producing too much of the proteins, inflammation or an
- Transmission of the
virus from the patient to other individuals or into the environment.
Gene therapy related links and
Issues in Human Gene Therapy - A Human Genome News
Report: Ethics of Genetics - From Guardian Unlimited.
Issues in Human Gene Therapy - A Human Genome News
Therapy Clinical Trials
of Pittsburgh Molecular Medicine Institute - Contains
information about ongoing and completed clinical trials.
therapy studies in ClinicalTrials.gov - The U.S. National
Institutes of Health resource for public access to information on
clinical research studies.
Therapy Clinical Trials - Access to a worldwide database of
gene therapy clinical trials at this Web.
Society of Gene Therapy
Gene Therapy Society (AGTS)
Society of Gene Therapy (ESGT)
Cancer Gene Therapy
Current Gene Therapy.
Journal of Gene Medicine