Gene Therapy WRT Viral Vectors

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Gene Therapy - A Brief Review WRT Viral Vectors


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Gene Therapy:

Gene Therapy Department of Pharmacology; GCTS 1 By – Subhajit Hazra (1 st Year, 1 st 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 ::

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 Department of Pharmacology; GCTS 2

Introduction to the Gene Therapy and its Basic Concept:

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 . Department of Pharmacology; GCTS 3

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Department of Pharmacology; GCTS 4 4 The “Central Dogma” 4 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:

“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. Department of Pharmacology; GCTS 5

Features of an “Ideal Carrier Vector”:

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 Department of Pharmacology; GCTS 6

Types of Vectors:

Types of Vectors Vectors are of 2 types – viz : * A Viral Vector * A Non-Viral Vector Department of Pharmacology; GCTS 7

Viral Vectors:

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 Department of Pharmacology; GCTS 8

Basic Principle of Viral Vector Design:

Basic Principle of Viral Vector Design 9 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 Vectors:

RNA Virus Vectors The 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 proteins capsid , 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 mediate virus entry. Department of Pharmacology; GCTS 10

RNA Virus Vectors (Cont.):

RNA Virus Vectors (Cont.) Department of Pharmacology; GCTS 11 Fig 2 - The colored boxes indicate genes elements that are either essential [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 Vectors:

DNA Virus Vectors The 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. Department of Pharmacology; GCTS 12


Adenoviruses Adenoviruses 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. Department of Pharmacology; GCTS 13

Types of Adenoviruses :

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 and late protein synthesis prevented Third Generation - Development of high-capacity , helper-dependent vector Activity regulation Possible Department of Pharmacology; GCTS 14

Adeno-Associated Viruses (AAV):

Adeno -Associated Viruses (AAV) Adeno -associated viruses are nonpathogenic viruses . They contain a single stranded DNA genome of only 4.7 kb . Department of Pharmacology; GCTS 15 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 Vectors:

Other Vectors Vectors derived from Herpes Simplex Viruses (HSVs) are among the most promising because of the size and complexity of its » 150-kb genome. Department of Pharmacology; GCTS 16

Clinical trials:

Clinical trials Worldwide , 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). Department of Pharmacology; GCTS 17


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. Department of Pharmacology; GCTS 18

Selected Bibliography’s:

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 19

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Department of Pharmacology; GCTS 20 Thank You

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