Gene therapy

GENE THERAPY
INTRODUCTION:-
The new human genome era is creating increasing possibilities for genomics-based
medicine called “personalized medicine.”1 Gene therapy, pharmacogenetics, and
pharmacogenomics are 3 major therapeutic interventions that take into account an individual’s
specifi c .genetic makeup. Genomics involves the study of genes and their surrounding DNA
sequences as well as the structure and function of the human genome.Genes are made up of a
chemical code (DNA) particular to each gene. Human genome discoveries have revealed that
approximately 25,000 genes reside in each individual’s human genome.
The code differs in sequence from gene to gene and directs the composition and production
of proteins that in turn make up living tissues and regulate all of the body’s functions. Genetic
disorders arise when an error in the complex, multistep process of replication and cell division
occurs. The error may be slight—perhaps just one unit of the code is misspelled, repeated, or
deleted—but its corresponding protein will be similarly improperly put together. When the
protein is essential enough, the error may lead to a sequence of events that can cause disability or
even deat Gene therapy interventions are being developed to treat three types of genetically
caused conditions. These are
 Single – gene
 Multi-factorial
 Acquired genetic conditions.
DEFINITION OF GENE THERAPY
Gene therapy involves the correction of defective genes responsible for diseases such as
cancer gene therapy approach seeks to provide therapeutic benefit to a patient by introducing
normal genes into the patient’s cell nuclei to repair, enhance, replace, or compensate for an
altered gene
CLASSIFICATION OF GENETIC DISOREDDER BASED ON GENE INVOLVED

1. Single gene disorders Mendelian genetic disorders and include such conditions as
cystic fibrosis, hemophilia, sickle-cell anemia, and Huntington’s disease
2. Multifactorial genetic disorders:-Conditions caused by a combination of genetic and
environmental influence.
3. Acquired genetic conditions are those that occur as a result of a viral infection such as
hepatitis or acquired immunodeficiency syndrome. In these conditions, the disorder is
caused by the new genetic information the virus carries into the host.
PRINCIPLES OF GENE THERAPY
 Gene therapy approach seeks to provide therapeutic benefit to a patient by introducing
normal genes into the patient’s cell nuclei to repair, enhance, replace, or compensate for
an altered gene.
 Gene therapy strategies under investigation include inserting a new functioning gene into
the cells of a patient to correct a genetic abnormalities or birth defect, thereby providing a
new function for the cell.
 Switching the abnormal gene for a normal gene through a technique called homologous
recombination repairing the abnormal gene to return the gene to its normal function; and
regulating it.
 Gene therapy offers the potential for treating many genetic disorders as well as cancer,
infectious diseases, and autoimmune disorders by genetically modifying cells in the
human body.
 Current gene therapy initiatives are aimed at somatic cells, which are the non
reproductive cells of the body (eg skin, muscle, bone, and liver), somatic gene therapy,
can correct inherited genetic disorders and is limited to only one generation. Gene
therapy aimed at altering sperm and ova (reproductive cells) is called germ-line gene
therapy.
 Enhancement gene therapy and eugenics are two other possible uses of gene therapy:-
 Principle behind enhancement gene therapy is the placement of genes in an embryo or
offspring that would improve a societally desirable trait, such as decreased weight or
increased height.
 Gene therapy used for eugenic purposes involves the introduction of specific genetic
traits into a population to develop “desirable” human attributes such as intelligence
USES OF GENE THERAPY :-
(l) Delivering the gene to the target tissue efficiently
(2) Sustaining long-term gene expression
(3) Ensuring that the gene transfer will not harm the patient in any way
(4) Transferring the corrected gene to non-dividing cells.
GENE TRANSFER

 INVITRO:- It is also called ex vivo. in vitro approach requires that the defective cells or
cells of interest be removed from the patient fi rst. The corrected or marker gene is then
inserted into the cells, and the altered cells are returned to the individual. The cells most
commonly used for this approach include lymphocytes, skin fibroblasts, and tumor and
bone marrow cells. These cells are readily accessible, amenable to manipulation, and able
to survive for long periods of time following reinfusion.
 INVIVO:- In vivo gene transfer involves the direct delivery of therapeutic genes to target
body cells. In vivo approaches to gene therapy have been used in clinical trials for cystic
fibrosis (CF), muscular dystrophy, melanoma, and heart, lung, and metabolic conditions.
With in vivo gene transfer, naked DNA can be delivered without the use of needles, using
a gene gun or jet gun. Both methods use either high pressure helium or liquid to deliver
the DNA to interstitial places.
VECTORS FOR GENE TRANSFER
VIRAL VECTOR:- gene therapy uses viral vectors to deliver the therapeutic gene to the target
tissue. All viruses used have been disabled of any pathogenic effects by removing the genes
required for replication of the virus and replacing them with therapeutic genes and selection
markers. The use of viruses is a potentially powerful technique because many have evolved
specific mechanisms for delivering DNA to cells.
I. RETROVIRAL VECTORS:-A retrovirus is composed of RNA that can insert itself
readily into dividing cells. Retroviruses are considered the most promising gene
transfer vehicle. These RNA viruses are able to carry out effi cient gene transfer into
many types of cells and can integrate into the host cell genes with stability.
Advantage
 The therapeutic gene carried into the cell by the retrovirus will be inherited by all
future generations of the cell and will provide the possibility of long-term gene
expression
Disadvantage:-

 The insertion of the retrovirus will disrupt normal genes essential for proper cell
function, leading to harmful physiological effects that favor cancer development.
II. LENTIVIRAL VECTORS:- Lentiviruses, which belong to the retrovirus family, are
now being used in gene therapy because they can infect both dividing and
nondividing cells and have the ability to provide long-term and stable gene
expression. Human immune deficiency virus (HIV) is the most well-known lentivirus
used for gene transfer in vivo.
Advantage
The use of lentiviruses such as HIV as a vector for gene therapy may not cause problems,
such as cancer, that have arisen with retroviral vectors
III. ADENOVIRAL VECTORS:-Adenoviruses are a family of viruses that cause benign
respiratory tract, intestinal, and eye infections in humans. They also hav the capacity
to infect both dividing and nondividing cells, making them useful for gene therapy.
Advantages
 Adenoviruses are large and can hold large segments of therapeutic DNA.
 They can also be produced in large amounts in culture.
 They have been the vectors of choice for many protocols designed to treat the pulmonary
complications of cystic fibrosis as well as for a variety of clinical protocols to treat
cancer.
 In contrast to retroviruses, which contain RNA, adenoviruses contain DNA and thus do
not integrate into host DNA but instead replicate themselves outside of the nucleus of the
host cell. Because of this limited integration, expression of the therapeutic gene is short-
lived, and regular reapplication of gene therapy using adenovirus vectors is necessary.
 The potential usefulness of adenoviral vectors stems from the fact that they do not require
actively dividing cells to introduce their therapeutic gene.
Disadvantage:-

 Adenoviruses, however, are a common cause of upper respiratory tract infections in
humans. As a result, unfortunately, most of the human population may experience an
active immune response to antibodies from a previous infection, which could reduce the
effectiveness of gene therapy using this vector.
 Another potential concern with using adenoviral vectors is that the integrated gene may
not lead to uniform correction of the gene defect, because it may not remain active in the
host cell. Other viral vectors that may potentially enhance the delivery of therapeutic
genes are therefore being explored.
IV. Newer adenoviral vectors:- The adeno-associated virus (AAV) is one of the newer
viral vectors under investigation; it is a simple, nonpathogenic virus composed of a
single strand of DNA. In order to replicate, AAV needs additional genes. In the past,
a helper virus, usually adenovirus or herpes simplex virus, served this purpose. The
AAV virus can infect a variety of types of cells, and although it appears to integrate
in a nonspecifi c manner, it has been shown to integrate preferentially into
chromosome 19. AAV gene therapy has been investigated for cysticfi brosis and for
factor IX hemophilia.Other viruses that are being considered and developed for use as
vectors for gene therapy are the herpes simplex virus, which infects cells of the
nervous system, and the vaccinia virus. These viral vector systems produce a transient
response, and many people have an immunity to components of the virus from being
infected previously.
CLINICAL PROTOCOLS FOR GENE THERAPY
 Clinical protocol for gene therapy was initiated in 1990.
 Two girls with ADA deficiency, a rare genetic condition that produces severe immune -
defi ciency in children, were injected with white blood cells carrying a therapeutic gene.
The clinical protocol called for inserting the ADA gene into T lymphocytes.
 ADA is an enzyme needed for normal immune system functioning. It prevents the
buildup of deoxy-adenosine, a metabolic product that becomes toxic to immune cells,
especially lymphocytes, when present in high concentrations.
 ADA deficiency accounts for 25% of cases of severe combined immunodeficiency
disease (SCID)
 Treatment for ADA deficiency has also demonstrated the Benefits and risks of gene
therapy. In 2003,
 French researchers reported that two children with SCID who had been treated with
retroviral gene therapy had developed a leukemia-like disorder 30 months after a single
gene therapy treatment. In response to this discovery, the FDA put a hold on clinical
trials that used retroviral vectors to insert the defective gene into hematopoietic cells.
 The American Society of Gene Therapy conducted its own investigation to determine
why this occurred only in patients with SCID, and not in other trials with retroviruses.
Researchers have since discovered that the retrovirus used (Moloney-murine leukemia

virus, or Mo-MuLV) inserted the therapeutic genes next to a gene known to promote
blood cancer.
 Insertional mutagenesis is a recognized complication of retroviral gene transfer attempts
because gene integration occurs randomly. With Mo-MuLV, this appears to occur at the
beginning of the gene, affecting how it works.
GENE THERAPY REGULATION
 Guidelines for clinical gene therapy protocols were established by the National
Institutes of Health (NIH) in the document Points to Consider in the Design and
Submission of Human Somatic Cell Gene Therapy Protocols in 6 parts as given
below:-
 Concern for the clinical benefit of all persons receiving
 gene therapy
 Assurance of informed consent
 Fair selection of persons for gene therapy–research Protocols
 Attention to the need for bio-safety protocols
 Public involvement in genetic research policy
 Attention to long-term consequences of genetic research.
 The Human Gene Therapy Research Subcommittee and the Recombinant DNA
Advisory Committee (RAC) then review the protocol.
 Above mentioned two committees serve in an advisory capacity to the director of
the NIH, who approves all gene transfer and gene-therapy proposals.
 The FDA addresses the scientific methodology and preclinical safety testing and
has created a set of guidelines for the initiation of gene therapy.The FDA’s
guidelines are separate from the Human Genome Research Subcommittee and
RAC guidelines and address the characteristics, production, and certification of
the biological substances being used for gene transfer.
 The “Points to Consider” document is updated regularly and is found as an
appendix of the NIH’s guidelines on recombinant DNA research.
CANCER GENE THERAPY
Introduction to oncogenes :-
Proto-oncogenes are normal cellular genes that are essential for cellular growth and
development. Oncogenes stimulate neoplastic growth and are activated by proto-oncogenes that
encode a growth factor or another protein and disturb normal cell development and regulation.
Antioncogenes are those genes that block the action of growth-inducing proteins. These genes
are also called tumor-suppressor genes to denote their ability to block the action of oncogenes.
When functioning normally, tumor-suppressor genes and proto-oncogenes work together to
enable the body to perform vital functions, such as replacing dead cells and repairing defective
ones.
Definition:- cancer gene therapy is defined as inhibiting oncogene function and restoring
tumor-suppressor function.

Types of gene transfer in clinical cancer gene therapy:-
o Gene marking
o Gene therapy
o Gene marking :- it involves labeling cells for future identifi cation. A gene that has been
genetically marked is introduced into cells, most commonly using a retrovirus as a vector
for the desired gene. Gene marking studies have been used in the treatment of melanoma,
leukemia, neuroblastoma, and stem cell transplantation.
o Gene therapy:- it involve modifi cation of the content or expression of altered genes in
somatic cells by transferring the functional or enhanced genes.
PROTOCOL OF CANCER GENE THERAPY:-
1.Tumor -directed approach:- in this approach therapeutic gene is introduced into tumor cells
to destroy them.
I. “Suicide gene” therapy protocols: suicide gene is that which produces an enzyme
whose activity converts a nontoxic prodrug to its toxic form. The gene transfer is
targeted to tumor cells to make them susceptible to an agent that does not cause harm to
normal cells but kills malignant cells. The suicide gene is toxic to dividing cells only,
thus sparing the normal cells and non dividing tumor cells. A new approach to suicide-
gene therapy uses ultrasoundand nano/microbubbles (NBs) to deliver exogenous
molecules noninvasively into a specifi c target site. Examples :- herpes simplex virus
thymidine kinase gene (HSV-TK) is the one most commonly used. Retroviral vectors
transfer genes to actively dividing cells, making this type of gene therapy well suited for
the treatment of brain tumors.
II. Tumor-suppressor gene therapy: The tumor-suppressor gene, P53, is found in
approximately 50% of all cancers. A normal copy of the gene has been introduced to
restore its function in patients with lung cancer, colorectal cancer, breast cancer, as well
as many others.
III.Antisense oligonucleotides: Genetic therapies for cancer treatment are being developed
that specifi cally target DNA and RNA. The use of specifi c segments of DNA—
antisense oligonucleotides—is one example of this new methodology. Antisense
oligonucleotides are nucleic binding agents. They are short strands of nucleotides that
predictably combine with other nucleotides. This property allows for the design of a
treatment drug that can recognize a unique site on a specifi c gene. Oligonucleotides can
be inserted into cells to interfere with the translation of RNA into an oncogene protein.
When transferred into patients, they prevent the oncogene’s RNA message from being
translated into a functional oncogene protein. This approach is being used in clinical trials
of antiepidermal growth factor receptor in the treatment of head and neck squamous cell
carcinoma.
IV. Oncolytic viral gene therapy: Oncolytic viruses reproduce in tumor cells, causing their
lyses. These viruses can be used to treat cancers with a defective p53 pathway. In normal

cells, p53 is destroyed by adenoviruses, producing a protein called E1B 55K. This protein
binds with p53 and, in synchrony with another viral protein, directs p53 for destruction.
Researchers have been able to modify a strain of adenovirus so that it does not encode
E1B 55K and lose its ability to destroy p53. Normally, p53 activity causes a cascade of
events that lead to the death of the affected cell, and viral replication cannot be
established. In cancer cells that lack p53, however, the normal mechanism that leads to
the death of the infected cells is abolished so that only the modified adenovirus can
replicate and kill tumor cells. Used to treat head and neck cancer and, in some clinical
trials, is being combined with chemotherapeutic agents.
2(A) Active immunotherapy
I. Tumor-infi ltrating lymphocytes: Host immunological responses can be used to alter the
natural course of some cancers, especially malignant melanoma. Tumor-infiltrating
lymphocytes (TILs) that are able to mediate tumor regression in patients with melanoma
have been identified. Administration of an epitope in conjunction with intravenous high-
dose interleukin-2 (IL-2) has been shown to mediate regression in patients with malignant
melanoma.
II. Cytokine genes: Cytokines are molecules that enhance the body’s immune response to
tumor antigens. Cytokine genes are being used to augment the body’s ability to mount an
immune response to tumor cells. Two of the most commonly used cytokine genes are IL-
2 and tumor necrosis factor. These cytokine genes are modified so that they will not
proliferate but can still support expression of the introduced gene products. The modifi ed
cells are then injected into the patient via subcutaneous, intradermal, or intramuscular
routes.
III. DNA vaccines: DNA vaccines that contain tumor associated–antigen genes or cytokine
genes are now being used because the pure DNA encodes only the tumor associated–
antigen gene. DNA vaccines are usually administered intramuscularly, alone or in
combination with cytokine genes.34 Example : Metastatic renal cell carcinoma and
prostate is one cancer that has demonstrated responsiveness to immunotherapeutic
intervention.
2(B) Adoptive immunotherapy:-
Adoptive immunotherapy is a form of gene-transfer therapy in which the patient’s own
lymphocytes (peripheral blood or from TILs) are modifi ed outside of the body with genes
that enhance their antitumor activity. The modifi ed genes are then reinfused back into the
same patient.
I. Chimeric receptors:- it consist of two different molecules brought together to form one
functional molecule—an extracellular antibody molecule linked to intracellular signal
domains of T-cell receptors. In this type of therapy, the patient’s autologous T cells serve
as the vehicle for inserted chimeric antibody molecules. The inserted antibody molecules
have specifi city for a tumor antigen. When transferred into patients, the modified T cells
are redirected to recognize the tumor by virtue of the specific antibody on its surface.
Once the tumor cells are engaged, the T cells are activated via the signaling chain and

mediate antitumor activity. This approach is being used in the treatment of patients with
neuroblastoma, kidney cancer, and metastatic ovarian cancer.34,40
II. Tumor-specifi c T cells: Adoptive immunotherapy using transduction of the IL-2 gene
into antitumor T lymphocytes. Transduction of an IL-2 gene into a TIL has been used
with some success in the treatment of patients with melanoma and renal cell carcinoma.
PHARMACOGENETICS AND PHARMACOGENOMICS: GENE-BASED
TREATMENT OF CANCER
Pharmacogenomics :- comes from the terms pharmacology and genomics and is the intersection
between pharmaceuticals and genomics. Pharmacogenomics leads to the development of drugs
that can be adapted to each person’s specifi c genetic make-up.
Examples :-
 Genetic polymorphism of thiopurine methyltransferase (TPMT). TPMT has been
associated with altered drug metabolism and increased risk for severe toxicity from the
anticancer agent 6-mercaptopurine.
CURRENT CHALANGES FOR GENE THERAPY
 Current viral and nonviral vectors do not yet provide a completely satisfactory means of
propagating the therapeutic genes in proliferating cells. To increase the possibilities of
success, researchers are investigating the introduction of a 47th chromosome (artificial
chromosome) into target cells. This method does not affect the working of the other 46
chromosomes or cause mutations. The artifi cial chromosome can serve as a natural
human vector for therapeutic genes. Nonviral gene therapy methods offer the potential
for therapeutic interventions that may be acceptable to physicians and patients and offer
safety and efficiency similar to those of conventional therapeutic modalities.
FUTURE APPROACHES TO GENE THERAPY:-
A. RNA interference (RNAi):- RNAi is being used to block the expression of a specifi c
gene using this mechanism as a technique for exploring gene function and for treating
disease. Short interfering RNAs (siRNAs) are being used to avoid the problems with long
double stranded RNA molecules that cause the interferon response in some cell types.
There are currently more than 10 clinical trials using this gene therapy technique.
Pharmaceutical companies are also developing RNAi-based treatments for diseases
including
B. hepatitis C, age-related macular degeneration, asthma, and Huntington Disease.
C. Nanotechnology is being applied in cancer diagnostics and treatments. Nanotechnology-
based therapeutics approved for cancer treatment include Doxil, a liposome preparation
of doxorubicin, and Abraxane, which is paclitaxel in nanoparticle formulation. Using
nanoparticles, chemotherapy drugs can now be delivered directly to tumor cells and then
send a signal after the cancer cells are destroyed.
ETHICAL, SOCIAL, AND LEGAL ISSUES IN GENE THERAPY

 Two serious and life-threatening genetic disorders—ADA defi ciency and alpha-
thalassemia—are under consideration for fetal gene therapy. Current issues being debated
include whether it is better to treat a disease for which backup therapies exist, as is the
case for ADA defi ciency, or to go forward with gene treatment for alpha-thalassemia, a
blood disorder that is often fatal to fetuses.
 Genetic testing and gene therapies reveal information about individuals and family
members. This information has the potential to label currently healthy individuals as
being “at risk.” As genetic testing and therapeutics become more common, personal, and
family genetic information may inadvertently become public.
 Cost of genetic therapy.
 Accessibility of genetic services
ROLE AND RESPOSIBITIES OF ONCOLOGY NURSES IN GENE THERAPY AND
ITS CLINICAL IMPLICATION:-
Direct Caregiver
• Provides anticipatory guidance
• Assures informed decision making/consent
• Develops treatment and management plans
• Administers gene therapy
• Observes patients for expected and unexpected side effectsof treatment, including
psychosocial and emotional response
• Participates in developing long-term follow-up plans
• Assures coordination and collaboration of care with allhealthcare providers involved in
patient/family care before,during, and after gene therapy
Educator
• Serves as an information source to patients, families, and the public
• Provides relevant, accurate, and understandable information to patients, in both written
and verbal forms
• Assures that all patient/family questions are answered
Advocate
• Assures privacy and confi dentiality of genetic information
• Protects against discrimination
• Advocates for fair and equitable use of gene therapies for all populations
• Promotes public understanding of somatic gene therapy
General Services Provider
• Gathers relevant family history information
• Identifi es individuals and families in need of further genetic education and counseling
• Assesses psychosocial, ethnoculture, and educational background
• Provides psychosocial support in follow-up to genetic counseling
Research Investigator
• Participates in or conducts clinical-research trials in gene therapy
• Serves as a preceptor to other nurses

• Develops research protocols that will address patient/ family response and adaptation to
genetic information, including gene therapy
Bibliography
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