INTRODUCTION

“ IT IS A MEDICAL TECHNOLOGY THAT AIMS TO PRODUCE A THERAPEUTIC EFFECT

THROUGH THE MANIPULATION OF GENE EXPRESSION OR THROUGH ALTERING
THE BIOLOGICAL PROPERTIES OF LIVING CELLS .”

-IT IS A TYPE OF TREATMENT USED TO TREAT GENETIC DISORDERS AND OTHER MEDICAL
CONDITIONS .
- It is also called as ‘BIOLOGICS’ because they come from living sources and not chemicals .

GOAL OF GENE THERAPY -

It is to change how existing genes in our body operate . There are three ways this can occur –

(i)Gene Inhibition : turning off the gene that is responsible for encouraging disease.

(ii) Gene Augmentation : adding a healthy copy of a gene to a cell where a faulty gene exists, so
the healthy copy can override the negative effect caused by the faulty gene

(iii) Killing disease causing cells : giving an unhealthy cell “ instructions “ (a set of new DNA )
which cause the cell to die.

-Currently , gene therapy is an area that exists predominantly in research laboratories and its
application is still experimental .
-Most trials are conducted in the United States, Europe and Australia. The approach is broad ,
with potential treatment of diseases caused by recessive gene disorders ( cystic fibrosis,
haemophilia, muscular dystrophy and sickle cell anaemia ) acquired genetic diseases such as
cancer and certain viral infections such as AIDS .

HISTORY OF GENE THERAPY

 In 1990 ,FDA for the first time approved a gene therapy experiment on ADA-SCID in the
U.S after the treatment of Ashanti Desilva.
 Many diseases such as ADA-SCID, X- linked SCID, Leberis congenital amaurosis ( a retinal
disease) , Parkinson’s disease ,multiple myeloma , chronic and acute lymphocytic
leukemia , adreno-leukodystrophy have reported of successful clinical trails.
 But these are still not approved by FDA , some other diseases on which gene therapy
based research is going on are Haemophilia , Tyrosinemia , Hyperbilirubinemia, Cystic
Fibrosis and many other cancers.
 After 30 years of research and clinical trials, only one product called Glybera got
approved in November 2012 which was made available in marked in the late 2013. It
has the ability to cure Lipoprotein lipase deficiency [LPLD] a very rare disease.
 In 1960, the concepts of gene therapy was introduced .
 In 1972, Firiedman and Roblin authored a paper in science titled “ Gene Therapy for
Human Genetic Disease”.
 In 1984, a retrovirus vector system was designed that could efficiently insert foreign
genes into mammalian chromosomes.
 In 1992, Dr. Claudio Bordignon performed the first procedure of gene therapy using
Hematopoietic stem cells as vectors to deliver genes inted to correct hereditary diseases.
 In 1999, death of Jesse Gelsinger in a gene therapy experiment.
 In 2003, a research team inserted genes into the brain for the first time, they used
liposomes which unlike viral vectors are small enough to cross the blood-brain barrier.
 In 2006, successful use of gene therapy to treat two adult patients for X-linked chronic
granulomatous disease.
 In 2007, first gene therapy trial for inherited retinal disease.
 In 2010, an 18 year old male patient in France with Beta-thalassemia major had been
successfully treated.
 In 2011,a medical community accepted that it can cure HIV as in 2008,Gero Hutter has
cured a man from HIV using gene therapy.
 From 2011-2015, the research is still ongoing and the number of diseases that has been
treated successfully by gene therapy increases.
 Gene therapy offers a promising new approach to treating a wide range of diseases
including various forms of cancer, inherited diseases and certain viral infections.
- There are basically two types of gene therapy –
(i)GERMLINE THERAPY
(ii) SOMATIC GENE THERAPY

1) GERMLINE THERAPY
 Germline therapy involves the modification of the genes ,inside germ or gamete cells ,
which includes sperm or ova.
 Germline therapy would therefore be administrated during reproduction, where the
modified gamete cells fuse to form a zygote.
 Once fused together, the zygote divides and passes on the modified genes to all other
cells of the body during the development of offspring.
 In this way Germline therapy alters the genome of future generations to come.
 Although theoretically, it could counteract hereditary diseases, jurisdictions in various
countries like Switzerland, Australia and Germany prohibit the use of Germline therapy
due to fears on the unknown risk of this therapy and whether it causes any long term
effects in future generations
 It is also extremely expensive which further limits its practical use for examples ( genes
introduced into eggs and sperms )
2) SOMATIC GENE THERAPY
 Somatic gene therapy involves the insertion of therapeutic DNA into body cells rather
than germ cells and gametes.
 This means that any effects of the therapy are confined to the individual being treated
and are not inherited by future offspring.
 The field of somatic gene therapy is surrounded by fewer ethical issues as compared to
Germline therapy.
 While this may be true, this therapeutic approach remains in the early stages of
development.
 The first hurdle in Somatic gene therapy is the successful incorporation of the new gene
in the genome.
 In fact, integrating the modified gene into the wrong part of the DNA could include
rather than prevent disease.
 In addition to requiring the desired gene needs to be expressed, the gene expression of
the new gene needs to be regulated in order to prevent over-expression that could also
trigger disease. For example ( introduction of genes into bone marrow cells, blood cells,
skin cells etc).
 → Somatic gene therapy is further classified into:-

(i) Ex vivo (ii) In vivo

(i) Ex- vivo : it is a procedure that involves removing cells from a patient modifying them in a lab
and then returning them to the patient.

- This process work by genetically modifying a patients stem cells, which then replace target
cells that have a missing or malfunctioning gene.

- Ex vivo therapy methods include ex vivo gene insertion using CAR-T.

PROCEDURE

(i)Isolate cells with genetic defect from a patient.

(ii) Grow the cells in culture.

(iii) Introduce the therapeutic gene to correct gene defect.

(iv) Select the genetically corrected cells and grow.

(v) Transplant the modified cells to the patient.

EXAMPLE OF EX-VIVO GENE THERAPY

-1st gene therapy to correct deficiency of enzyme Adenosine deaminase [ADA]

- Performed on a 4 year old girl Ashanti Desilva, was suffering from SCID ( Severe Combined
Immuno-deficiency ).

- Caused due to defect in gene coding for ADA.

- Deoxy-adenosine accumulate and destroys T-lymphocytes.

- It disrupts immunity, suffer from infectious diseases and die at young age.

(ii) In-vivo : The direct delivery of the therapeutic gene into the target cells of a particular tissue
of a patient constitutes in vivo gene therapy.

-Many tissues are the potential candidates for this approach.

- These include liver, muscle, skin, spleen, lung, brain and blood cells.

- it depends on the efficiency of the uptake of the remedial therapeutic gene by the target cells,
intracellular degradation of the gene and its uptake by nucleus and the expression stability of
the gene.
EXAMPLE OF IN-VIVO THERAPY

-In patients with cystic fibrosis, a protein called CFTR [Cystic Fibrosis Trans-regulator ]is absent
due to a gene defect.

- In the absence of CFTR chloride ions concentrate within the cells and it draws water from
surrounding.

- This leads to the accumulation of sticky mucous in respiratory tract and lungs.

- Treated by in-vivo replacement of defective gene by Adenovirus vector.

KEY ASPECTS :- Therapeutic gene

Vectors

Target cell

Routes of administration

Animal models
VECTORS IN GENE THERAPY : To transfer the desired gene in to a target cell a carrier is required
such vehicle of gene delivery are known as vectors.

Divided basically into:-

Viral vectors

Non-viral vectors

→ Ideal vector :-

-Target the right cells.

-Integrate the gene in the cells.

-Activate the gene.

-Avoid harmful side effects.

-No universal vector exist.

→VIRAL VECTORS:-

-Viruses introduce their genetic material in to the host cell as part of their replication cycle.

-Remove the viral DNA and using the virus as a vehicle to deliver the therapeutic DNA.

-The viruses used are altered to make them safe, although some risk still exist with Gene
therapy.

> TYPES OF VIRAL VECTORS:-

1) Retro Virus:

-Ability to integrate in to the host genome.

-Can carry a DNA of size less than 3.4kb.

-Target cell dividing .

2) ADENO VIRUS:

-Adenoviral DNA does not integrate into the genome and is not replicated during cell division.

-Humans commonly come in contact with Adenovirus, majority of patient have already
developed neutralising anti-bodies which can inactivate the virus.

-Target- Non-dividing, dividing cells.

3) ADENO ASSOCIATED VIRUS:

-It is a human virus.

-It is single stranded.

-AAV enters host cell, becomes double stranded and gets integrated into chromosomes.

-Target- Non-dividing cells, dividing cells.

4) HERPEX SIMPLEX VIRUS:

-Viruses which have natural tendency to infect a particular type of cell.

-It is a human neurotropic virus, it is mostly examined for gene transfer in the nervous system.

>NON VIRAL VECTOR SYSTEM

- It is more safer, of low cost, more reproducible and do not present DNA size limit.

PURE DNA CONSTRUCT:

-Direct introduction of pure DNA construct in to target tissue.

-Efficiency of DNA uptake by cells and expression rather low.

-Consequently large quantities of DNA have to be injected periodically.

DNA MOLECULAR CONJUGATES:

-Commonly used synthetic conjugate is Poly-L-lysine bound to specific target cell receptor.

-Therapeutic DNA is then made to combine with the conjugate to form a complex.

-It avoids lysosomal breakdown of DNA.

LIPOPLEXES:

-Lipid DNA complexes.

-DNA constructs surrounded by artificial lipid layer.

-Most of it gets degraded by lysosomes.

HUMAN ARTIFICIAL CHROMOSOMES:

-Can carry a large DNA that is with one or more therapeutic genes.
METHOD OF GENE DELIVERY:

[PHYSICAL METHODS]

1)GENE GUN:

-It posses a high pressure delivery system to shoot tissue with gold or tungsten particles that are
coated with DNA.

2)MICRO INJECTION:

-Process of using a micro pipette to insert microscopic substances into a single living cell.

-Normally performed under a specialised optical microscope setup called a micro manipulator.

[CHEMICAL METHODS]

1)USING DETERGENT MIXTURES:

-Certain