Gene therapy refers to the insertion of genetic material to correct a genetic defect. In gene therapy, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene

1. Advancements of Medical
Biotechnology in Gene Therapy
Presented by
Dr. B. Victor., Ph. D
Email : bonfiliusvictor@gmail.com
Blog: bonvictor.blogspot.com

2. Presentation outline
 Human genetic disorders-types, features.
 Gene therapy- definition, kinds and history.
 Somatic cell and Germ line gene therapy
 Ex vivo and In vivo gene therapy
 Gene delivery vectors: Viral and Non-viral vectors.
 Gene targeting
 Naked DNA gene therapy, post-natal gene therapy
and pre-natal gene therapy
 Risks and recent developments.
 Conclusion

3. Human genetic disorders

4. Kinds of human genetic
disorders

5. DNA / gene mutation

6. Functional or
non-functional DNA
Genes are sequences of bases.
Genes replicate exactly.
Gene is transcribed into mRNA.
mRNA is translated into Protein.
Proteins do metabolic functions.
Mutated genes are non-
functional (build wrong, interact
wrong).
Mutated genes are inherited;
result is genetic disease.

7. Basis of genetic disorder

8. What is a genetic disorder?

9. What is gene therapy?

10. Concepts of gene therapy
techniques
 Replacement of a
abnormal gene with
normal gene.
 Repairing the
abnormal gene.
 Altering how that
gene is controlled.
 Get other cells to
take over function
of abnormal cells.
 Inserting correct
protein and bypass
gene function.

11. Kinds of Gene Therapy
techniques

12. Technique of gene
therapy

13. Gene therapy :mechanism

14. Steps in gene therapy
technology

15. Different genetic diseases need different gene
therapy approaches

16. History of gene therapy :
In the beginning…
◦ In the 1980s, Scientists inserted
human genes into a bacteria cell.
◦ Then the bacteria cell transcribed
and translated the information into a
protein.
◦ Then they introduced the protein
into human cells.

17. The First gene therapy case was
performed on September 14th,
1990.
 Ashanti De Silva was
treated for
SCID(Sever combined
immunodeficiency).
 Doctors removed her
white blood cells,
inserted the missing
gene into the WBC,
and then put them
back into her blood
stream.
 This strengthened her
immune system
 This only worked for a
few months.

18. 9/17/1999
Jesse Gelsinger, 18 years
high school graduate with
OTC deficiency, died
participating in a gene
therapy experiment at the
University of Pennsylvania in
Philadelphia,

19. The first gene therapy cure
 2000 - The first gene
therapy cure was
reported when Alain
Fischer (Paris)
succeeded in totally
correcting children with
SCID-X1, or “bubble
boy” syndrome

20. Flow chart showing
gene therapy types

21. Types of gene therapy;
Germ line gene therapy:
Healthy gene is introduced into reproductive
cells
E.g., eggs, sperms.
Somatic cell gene therapy:
Healthy gene is introduced into adult somatic
cells(body cells).
E.g., bone marrow cells, hepatic cells, central
nervous system cells.
Gene addition therapy :
Functional gene is introduced into the somatic
cell in addition to defective gene endogenous
to the cell.
Gene targeting :
Inactivate a functional defective endogenous
gene.

22. ex vivo(in vitro) and in
vivo somatic gene therapy
Ex vivo gene therapy- refers to the
transfer of genes in cultured cells (outside
the body) (e.g., bone marrow cells) which
are then reintroduced into the patient.
 This technique is used for treating genetic
diseases of blood system.
In vivo gene therapy- the direct delivery
of genes into the cells of a particular tissue.
 This technique is used for treating tissue –
based genetic diseases e.g., Duchenne
muscular dystrophy (DMD).

23. Steps in somatic cell
gene therapy

24. Human ex vivo gene therapy

25. Gene targeting or
targeted gene transfer
It is a form of in-vivo site directed
mutagenesis involving homologous
recombination between a targeting vector
containing one allele and an endogenous
gene represented by a different allele.
Gene targeting can be used either to
inactivate a functional endogenous gene or
to correct a defective gene.
The first case ( in 1985) was used to disrupt
the human b-globin gene in cultured cells.

26. Problems with ex-vivo method
of gene Therapy
Problems Risks
Not enough cells Cells injected may
get desired gene cause an immune
to correct response
problem Random insertion of
Modified cells retrovirus into host
don’t last long; chromosome- may
need repeat likely to interrupt the
treatments coding DNA.

27. Gene delivery
viral Vectors
Adenoviruses
Retroviruses
Adeno-Associated Viruses (AAV)
Herpes simplex viruses

28. Choices of gene delivery Vectors
Viral vectors:
Retrovirus
Adenovirus
Adeno-associated virus
Herpes Simplex Virus
Non-viral vectors:
Liposome
DNA–polymer conjugates
Naked DNA

29. Characteristics of ideal gene
delivery vector system
an adequate carrying capacity.
to be undetectable by the immune system.
to be non-inflammatory.
to be safe to the patients with pre-existing
lung inflammation.
to have an efficiency sufficient to correct
the genetic disease.
to have long duration of expression.

30. Adenovirus(non-specific
insertion)
Adenoviruses have double-
stranded DNA genomes.
 Adenoviruses cause
respiratory, intestinal, and
eye infections in humans.
 The common cold is caused
by an adenovirus.
 Adenovirus genome can
accept large insertions of
human DNA.
 Penetration into the cell is by
endocytosis.
 The viral core migrates to the
nucleus where the DNA enters
through nuclear pores and
becomes incorporated into the
genome.

31. Retrovirus (non-specific
insertion):
 Retroviruses are group
of RNA viruses.
 Retroviruses contain two
copies of the genome in
each viral particle.
 Human
immunodeficiency virus
(HIV) is a retrovirus.
 On infection the ssRNA is
converted into dsDNA
copy by reverse
transcriptase and is
integrated into the host
cell genome by a viral
integrase enzyme.

32. Adeno-Associated Virus
(specific insertion)
 A class of small, single-stranded
DNA viruses that can insert their
genetic material at a specific site
on chromosome 19. Penetration and
Gene Transfer mechanisms are
similar to the Adenovirus.
 Several genetic disorders are related
to genes on chromosome 19 (70
known genetic disorders):
 for example:
 Alzheimer’s disease
 Leukemia
 Muscular Dystrophy
 Congenital Hypothyroidism
 Several Cancers (ovarian,
colorectal, etc.)

33. Herpes simplex viruses
 A class of dsDNA
viruses that infect a
neurons. It has a 150
kbp dsDNA genome. It
consists of over 70
genes.e.g., Cold sores
virus

34. Non-viral DNA carriers:
Cationic liposomes
 Positively charged lipids interact with negatively charged
DNA. (lipid-DNA complex).The liposome carries the
therapeutic DNA through the target cell membrane.
Advantages:
a. Stable complex
b. Can carry large sized DNA
c. Can target to specific cells
d. Does not induce immunological reactions.
Disadvantages:
a. Low transfection efficiency
b. Transient expression
c. Inhibited by serum
d. Some cell toxicity

35. Naked DNA gene therapy
◦ Intramuscular and Intravascular delivery
(liver and muscle).
◦ covalently closed circular form is more
stable that open plasmid
◦ Results in a prolonged low level expression
in vivo
◦ Very cheap
◦ DNA vaccines based on naked DNA are
unaffected by pre-existing immunity e.g.
due to maternal antibodies

36. Postnatal Gene Therapy
 Correction of the deleterious effects of
genetic disease via long term integration of
gene sequences into a patient’s genome.
 This property makes the use of retroviral
vectors particularly attractive when
considering effective gene delivery to correct
inherited monogenetic disorders.

37. Types of Postnatal Gene
Therapy
 Gene replacement: non-functional or
defective gene is replaced by a new,
functional copy of the gene.
 Can be accomplished by homologous
recombination.
 Gene addition: introduction of a gene
that is able to produce a protein not
normally expressed in the cell.
 i.e. Introduction of a so-called “suicide gene”
into cancer cells

38. Prenatal or in utero gene
therapy
Targets genetic diseases which require lifelong
correction
The concept of fetal gene therapy is based on
the following aims:
 avoiding early-onset manifestation of life-
threatening genetic conditions
 achieving permanent correction of such
diseases by stable transduction.
 Avoiding immune reactions against the
therapeutic vector and transgene.

39. Prenatal gene therapy

40. Benefits of prenatal gene
therapy
Provides early phenotypic correction of
genetic disease.
Demonstration of long-term postnatal
therapeutic protein production.
Tolerance to the transgenic protein
can be induced by in utero expression.

41. Risks of Gene Therapy
 New gene might be inserted into wrong
location in the DNA (misfire).
 Other genes may be accidentally
delivered to the cell.
 The deactivated vector virus may be
contagious.
 The viral vectors cause toxicity and
inflammatory responses.
 The vector viruses can infect more than
one type of cell.
 Over-expression of missing protein.
 Immune system complications.

42. Recent Developments
 Genes get into brain using liposomes
coated in polymer call polyethylene
glycol
 potential for treating Parkinson’s disease
 RNA interference or gene silencing to
treat Huntington’s
 siRNAs used to degrade RNA of particular
sequence
 abnormal protein wont be produced
 Create tiny liposomes that can carry
therapeutic DNA through pores of
nuclear membrane
 Sickle cell successfully treated in mice

43.  Dr.B.Victor is a highly experienced professor,
recently retired from the reputed educational
institution- St. Xavier’ s College,
Palayamkottai, India-627001.
 He was the dean of sciences, IQAC
coordinator and assistant controller of
examinations.
 He has more than 32 years of teaching and
research experience
 He has taught a diversity of courses and
guided 12 Ph.D scholars.
 send your comments to :
bonfiliusvictor@gmail.com