Gene Therapy - A Basic Overview

1. 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

2. 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

3. 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

4. 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.

5. “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

6. 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

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

8. 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

9. 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.

10. 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

11. 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).

12. 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

13. 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

14. 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

15. 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 .

16. 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

17. 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

18. 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

19. 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

20. Department of Pharmacology; GCTS 20
Thank You