Gene therapy uses vectors like viruses, human artificial chromosomes, and bone marrow cells to deliver therapeutic genes. Viruses commonly used include retroviruses, adenoviruses, and adeno-associated viruses. Non-viral methods include naked DNA, lipoplexes, and DNA-molecular conjugates. Gene therapy approaches for cancer include restoring tumor suppressor genes, inactivating oncogenes, suicide gene therapy, and tagging cancer cells. Limitations of gene therapy include only being able to treat single gene defects currently.

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Course Code: MPL104T
Lecture No: 32
Gene Therapy
Course Leader:
Dr. Mohammad Azamthulla / Dr Anita Murali

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Content
• Vectors
• In vivo gene therapy

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Lecture objectives
• At the end of this lecture, student will be able to:
– Discuss the vectors used in gene therapy
– Explain about invivo gene therapy

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Vectors
The carrier particles or molecules used to deliver genes to
somatic cells are referred to as vectors
Various vectors used in therapy are
1. Viruses
2. Human Artificial Chromosomes
3. Bone marrow cells

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Viruses
• Viruses attack their hosts, introduce their genetic material into
the host cell as part of their replication cycle. The virus is
made harmless by re-engineering
• This virus has two genes- A and B. Gene A encodes a protein
which allows this virus to insert itself into the host's genome
• Gene B causes the disease this virus is associated with. Gene C
is the "normal" or "desirable" gene we want in the place of
gene B
• By re-engineering the virus - gene B is replaced by gene C,
while allowing gene A to properly function, this virus could
introduce your 'good gene'- gene C into the host cell's genome
without causing any disease

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Retrovirus
General Map of a Typical Retrovirus
Gene Map of a modified Retrovirus for use in gene therapy

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• A retroviral vector can carry a therapeutic DNA of maximum
size of 8kb
• One of the problems of gene therapy using retroviruses is
that the integrase enzyme can insert the genetic material of
the virus in any arbitrary position in the genome of the host.
• If genetic material happens to be inserted in the middle of
one of the original genes of the host cell, this gene will be
disrupted (insertional mutagenesis)
Retrovirus

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• If the gene happens to be one regulating cell division,
uncontrolled cell division (i.e., cancer) can occur
Retrovirus

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Therapy for Adenosine Deaminase Deficiency
Gene therapy

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Vectors -Viruses
1. Retroviral Vector System:-
• Comprised of two copies of a positive single-stranded RNA
genome of 7 to 10kb. Their RNA genome is copied into
double-stranded DNA, which integrates into the host cell
chromosome and is stably maintained
• Retroviruses are ~100 nm in diameter and contain a
membrane envelope. The envelope contains a virus-encoded
glycoprotein that specifies the host range or types of cells that
can be infected by binding to a cellular receptor.

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2. Adenoviral Vector System:-
• Adenoviruses (with a DNA genome) can infect most of the
non-dividing human cells
• This vector system based gene therapy requires periodic
administration of recombinant viruses
• The efficiency of gene delivery by it can be enhanced by
developing a virus that can specifically infect target cells
• This is possible by incorporating a DNA encoding a cell
receptor protein
Vectors -Viruses

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3. Adeno-associated Virus System:-
• It is single-stranded, non-pathogenic small DNA virus
(4.7kb)
• Recombinant viruses are created by using two plasmids
and an adenovirus (i.e., helper virus) by a simple
technique
• They are used for the treatment of the hemophilia and
cystic fibrosis
Vectors -Viruses

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4. Herpes Simplex Virus (HSV) Vector System:-
• HSV have a natural tendency to infect a particular
type of cells
• HSV type I infect and persist in non-dividing nerve
cells
• HSV is a human pathogen that causes (though
rarely) cold sores and encephalitis
• HSV is considered as an ideal vector for in vivo
gene therapy of many nervous disorders
Vectors -Viruses

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Non-viral Vector System
• There are certain limitations in using viral vectors in gene
therapy
• The efficiency of gene delivery by non-viral vectors is mainly
due to the following reasons:
1. The efficiency of transfection is very low
2. The expression of the therapeutic gene is for a
very short period, consequently there is no
effective treatment of the disease

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Pure DNA Constructs
• The direct introduction of pure DNA constructs into the target
tissue is quite simple
• However, the efficiency of DNA uptake by the cells and its
expression are rather low
• Consequently, large quantities of DNA have to be injected
periodically
• The therapeutic genes produce the proteins in the target cells
which enter the circulation and often get degraded

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Lipoplexes
• The lipid-DNA complexes are referred to as lipoplexes or more
commonly liposomes
• They are non-toxic and non-immunogenic
• Plasmids (diameter up to approximately 2 μm) are too big to
package in regular liposomes (diameter 0.025-0.1 μm), but
larger particles can be made using positively charged lipids
(lipoplexes), which interact with negative charges on cell
membranes and negatively charged DNA, improving delivery
into the cell nucleus and incorporation into the chromosome

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• Recent studies have shown lipoplexes to be useful in
transfecting respiratory epithelial cells, so they may be used
for treatment of genetic respiratory diseases such as cystic
fibrosis
• The major limitation with the use of lipoplexes is that as the
DNA is taken up by the cells, most of it gets degraded by the
lysosomes
• Thus, the efficiency of gene delivery by lipoplexes is very low
Lipoplexes

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DNA-Molecular Conjugates
• The use of DNA-molecular conjugates avoids the lysosomal
breakdown of DNA
• Another advantage of using conjugates is that large-sized
therapeutic DNAs (> 10kb) can be delivered to the target
tissues
• The most commonly used synthetic conjugate is poly-L-lysine,
bound to a specific target cell receptor
• The therapeutic DNA is then made to combine with the
conjugate to form a complex

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DNA-Molecular Conjugates

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HAC which can carry a large DNA (one or more therapeutic genes
with regulatory elements) is a good and ideal vector
Human Artificial Chromosome (HAC)

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Gene Therapy Strategies for Cancer
• Gene therapy is the latest and a new approach for cancer
treatment
• Chemotherapy for cancer patients often kills healthy cells as
well as cancer cells
• In an ex vivo trial, bone marrow stem cells from women with
ovarian cancer were infected with a virus carrying a gene to
make them more tolerant to chemotherapy

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Approaches
First Approach
• Restoring protective mechanisms such as p53
• This approach is ongoing clinical trials in head and neck cancer
involving injection into the tumor of recombinant adenoviral
vectors containing the human p53 gene
• It is called Gene Replacement Therapy

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Second Approach
• Inactivating oncogene expression (e.g., by a retroviral vector
bearing a construct that produces an antisense transcript RNA
to the k-ras oncogene)
• It is called Antisense Therapy
Approaches

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Third Approach
• Delivering a gene to malignant cells, thus rendering them
sensitive to drugs (e.g., that encoding thymidylate kinase,
which activates ganciclovir)
• This approach is called Suicide Gene Therapy
Approaches

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Fourth Approach
• Delivery of proteins to healthy host cells in order to protect
them (e.g., addition of the multidrug resistance channel to
bone marrow cells ex vivo thereby rendering them resistant to
drugs used in chemotherapy and protecting the patient from
the neutropenia and thrombocytopenia that is otherwise
predictably caused by such treatment)
Approaches

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Fifth Approach
• Tagging cancer cells with genes that when expressed render
malignant cells more visible to the immune system of the host
and trigger a vigorous defensive response (e.g., GCV-HSVTK
suicide gene is clubbed with interleukin-2 gene)
• It is called Two-gene Cancer Therapy
Approaches

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Gene Therapy for AIDS
• The approaches for gene therapy for AIDS are as follows:-
1. rev and env genes
2. Genes of HIV Proteins
3. Gene to inactivate gp120

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rev and env Genes
• A mutant strain of human immunodeficiency virus (HIV),
lacking rev and env genes have been developed
• The regulatory and envelope proteins of HIV are respectively
produced by rev and env genes
• Due to lack of these genes, the virus cannot replicate

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Limitations & Requirements of Gene Therapy
1. Knowledge of exact cause of disease at DNA level
2. Region bearing disorder must be well defined
3. Only single gene defects can be tackled today
4. Normal counterpart of defective gene is required for gene
therapy
5. Homologous recombination will have to occur for
replacement of defective gene by correct one

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6. Addition of functional gene and its random integration with
genome is possible part today
7. Potentially curable diseases are single gene defects with
recessive nature
8. Affected tissue must be accessible for gene therapy
9. Lasting presence of gene manipulated cells after
retransplantation is important. Self renewable cells are
preferred targets so diseases involving such cells could be
cured
Limitations & Requirements of Gene Therapy

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10. Corrected cells should have some selective advantage in-
vivo over affected cells
11. Spread of retrovirus throughout body and unwanted
transmission of virus is the negative aspect possible
12. The induction of oncogenesis as a result of provirus
integration is another risk
Limitations & Requirements of Gene Therapy

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Summary
• Various vectors are used for gene therapy
• Gene therapy also has its limitations
• Only single gene defects can be tackled today