Gene Therapy - A Novel Approch in Medical Treatment

Gene Therapy

Department of Pharmacology; GCTS 1

By – Subhajit Hazra (1st Year, 1st Semester)

Roll no- 03 Department of Pharmacology

(Masters of Pharmacy) Gupta College of Technological Sciences (GCTS); Asansol

Guided By –

Mr. Avik Das; MS (US) Gupta College of Technological Sciences (GCTS); Asansol

After discussion we would be able to appreciate :

The Basic Concept of Gene Therapy

The Central Dogma

Barrier to Gene Therapy and its resolution

Types of Vectors

Concept of RNA Virus Vectors

Concept of DNA Virus Vectors

Other Vectors

Clinical Trials of Gene Therapy

Conclusion


Introduction to the Gene Therapy and its Basic Concept

Gene therapy Experimental technique

Uses Gene to cure diseases

Thus the basic concept of gene therapy is :

To introduce into target cells a piece of genetic material that will result in either a cure for the

disease or a slowdown in the progression of the disease.


4Department of Pharmacology; GCTS4

The “Central Dogma”

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Central Dogma of Molecular Biology :

Replacing a mutated gene that causes disease with a healthy copy of the gene.

Inactivating, or “knocking out,” a mutated gene that is functioning improperly.

Introducing a new gene into the body to help fight a disease.

“Carrier” – The Primary Stumble

One of the biggest stumbling blocks to successful widespread

application of such genetic treatments is the development of

safe and effective vectors with which to ferry genetic material

into a cell.


Features of an “Ideal Carrier Vector”

The important attributes of an ideal vector are –

Efficient and easy production

Safety aspects

Sustained and regulated transgene expression

Targeting of the viral vectors and Site-specific integration


Types of VectorsVectors are of 2 types – viz : * A Viral Vector * A Non-Viral

Vector


Viral Vectors Viral Vectors : The basic concept of viral vectors is to harness

the innate ability of viruses to deliver genetic material into the infected cell.

Types of Viral Vectors :

RNA Virus Vector

DNA Virus Vectors


Basic Principle of Viral Vector Design

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Fig 1 - (a) Converting a virus into a recombinant vector. (b) The packaging and vector constructs are introduced into the packaging cell by transfection.Both “Packaging Constructs” and “Vector Constructs” express their particular proteins. These expressed proteins are the n encapsulated in into virus particles to generate the recombinant viral vector.

RNA Virus VectorsThe viral RNA contains three essential genes, gag, pol, and env, and

is flanked by long terminal repeats (LTR)

The gag gene encodes for the core proteinscapsid, matrix, and nucleocapsid, which are generated by proteolytic

cleavage of the gag precursor protein.

The pol gene encodes for the viral enzymes protease,reverse transcriptase, and integrase, which are usually derived from

the gag-pol precursor.

The env gene encodes for the envelope glycoproteins, which mediatevirus entry.


RNA Virus Vectors (Cont.)


Fig 2 - The colored boxes indicate genes elements that are eitheressential [and therefore retained in vectors (red) or supplied by packaging constructs or cell lines (green)] or that are nonessential and often deleted

(blue).

DNA Virus VectorsThe currently prominent DNA virus vectors are :

Adeno-associated viruses (AAV) and Adenoviruses.

Adeno-associated viruses (AAVs) contain a single stranded, relatively small (»4.7 kb) genome.

Adenoviruses contain a double-stranded DNA genome .

The basic principals of vector design same as that of RNA virus vectors.


AdenovirusesAdenoviruses are medium-sized DNA

viruses and have been isolated from avian and mammalian species.

The human adenovirus (Ad) family consists

of more than 50 serotypes that can infect and replicate in a wide range of organs.


Types of Adenoviruses Generation of Adenoviruses

Gene/s Targeted Modification Implications

First Generation E1 Viral replication and activation of the

expression of other Ad transcription units

prevented.

Up to 3.2 kb of the E1 region of the viral

vector can be replaced

by trans-genes. Second Generation E2 and/or E4 Viral replication

Prevented (E2). Viral replication as

well as protein synthesis prevented

(E4)

Toxicity of E2 gene expressed protein

prevented. E4 - Viral DNA replication andlate protein synthesis

prevented Third Generation - Development

of high-capacity, helper-dependent

vector

Activity regulation Possible


Adeno-Associated Viruses (AAV)

Adeno-associated viruses are nonpathogenic viruses. They contain a

single stranded DNA genome of only 4.7 kb.


AAV requires co-infection with of helper viruses for efficient, site-specific integration. It binds to human chromosome 19, a highly desirable feature of a gene therapy vector .

Other VectorsVectors derived from Herpes Simplex

Viruses (HSVs) are among the most promising because of the size and

complexity of its »150-kb genome.


Clinical trialsWorldwide, over 400 clinical trials have been conducted,

with enrolment of over 6000 patients. A substantial portion of these clinical trials (over 70%) are cancer related and often carried out on terminal patients.

The most commonly used vectors are retroviral vectors, which were the first viral vectors to be used in a gene

therapy trial.

The targets of that first clinical trial were the T-lymphocytes of two children suffering from severe

combined immunodeficiency (SCID).


Conclusion Geneticists must identify target genes that contribute to specific

diseases or that can influence the disease course.

The task for the virologists is to develop efficient and safe vectors that are able to deliver the genes of interest to the target cells and assure the proper expression of the transferred genetic material.

Clinicians will carry out clinical trials with vectors optimized for the disease and the medical requirements of the patients.


Selected Bibliography’s GENE THERAPY: Promises and

Problems; Alexander Pfeifer and Inder M.Verma.

GENE THERAPY: Twenty-First Century Medicine; Inder M. Verma and Matthew D.Weitzman.


Department of Pharmacology; GCTS

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Gene Therapy - A Novel Approch in Medical Treatment