GENE THERAPY

Gene Therapy - the use of genetic manipulation

for treatment of disease.
Gene Delivery System
 Why use viral vectors

Virus are obligate intracellular parasites

Very efficient at transferring viral DNA into host
cells
Specific target cells: depending on the viral
attachment proteins (capsid or glycoproteins)
Gene replacement: non-essential genes of virus are
deleted and exogenous genes are inserted
Retroviruses
can create double-stranded DNA copies of their RNA genomes.
Can integrate into genome. HIV, MoMuLV, v-src, Rous sarcoma
virus

Adenoviruses
dsDNA viruses that cause respiratory, intestinal, and eye
infections in humans. Virus for common cold
Adeno-associated Viruses
ssDNA viruses that can insert their genetic material
at a specific site on chromosome 19

Herpes simplex
viruses
dsDNA viruses that infect a neurons. Cold sores virus
 Retrovirus
• Moloney murine leukemia virus (MuLV)
• Generation of replication defective retroviral vector
– Transfer plasmid vector:
• Gene of interest
– Packaging vector
• Cell line stably transfected with plasmid constructs
containing Gag/pol and Env

• Advantages
– Integration: permanent expression
– Pseudotyped virus
• Disadvantages
– Only infecting dividing cells
– Insertional mutagenesis (tumor formation)
• Activate oncogenes
• Inhibit tumor suppressor genes
 Lentiviral vectors
Lentiviruses are retroviruses
that can infect both dividing and nondividing cells

Preintegration complex of lentiviruses can get through

the intact membrane of the nucleus of the target cell.

Able to infect nondividing or terminally differentiated cells

such as neurons, macrophages, hematopoietic stem cells,
retinal photoreceptors, and muscle and liver cells

Example of lentiviruses:
HIV-1 (infects T-helper cells) –
AIDS.

Good feature – no immune response!

 Adenovirus Associated Virus

• Non-pathogenic human parvovirus, non-enveloped ss DNA

virus, 4.6 kilobases
• Dependent on a helper virus ( adenovirus or herpesvirus)
for replication (dependovirus)
• AAV-2 mostly used for vector

• Advantages
– Integration and persistent expression
– No insertional mutagenesis
– Infecting dividing and nondividing cells
– Safe
• Disadvantages
– Size limitation, 4.9 kb
– Low titer of virus, low level of gene expression
Liposomes
 Electroporation
Electroporation is a method that
uses short pulses of voltage to
carry DNA across the cell
membrane
Patterns of Gene Expression
 GENE THERAPY AND
MOLECULAR MEDICINE
 GENE THERAPY AND
MOLECULAR MEDICINE
 GENE THERAPY AND
MOLECULAR MEDICINE
Uses of Gene Therapy
 Gene Therapy
Molecular medicine is the application of molecular biological techniques to the
treatment and diagnosis of disease. It is derived form the successful
development of human organ transplantation, pharmacotherapy, and
elucidation of the human genome.

• Gene therapy is the use of any of a collection of approaches to the treatment

of human disease based on the transfer of DNA-based genetic material to an
individual.

The successful application of gene therapy requires the achievement of

therapeutic levels of protein along with the long-term regulation of gene
expression. Somatic gene line therapy targets non-germline cells and is
consistent with the extension of biomedical science into medical therapy.
 Gene Therapy
For the immediate future, a major challenge is to develop vectors that can yield
stable therapeutic concentrations of gene products in nondividing cells located deep
within the body. A lingering concern is raised by the possibility that cosuppression
occurs in humans and that the same biochemical machinery that carries out gene
silencing may shut off high-level expression of therapeutic genes. If true, gene
therapies face an unanticipated roadblock that may be difficult to circumvent.

• The key gap in the gene therapy field is our lack of knowledge of exactly what sets
the stage for the serious diseases causing morbidity and mortality in the United
States. At the molecular level, it is not clear what processes go awry. Therefore, it is
not clear which gene products have the greatest potential to be curative.

• A group of promising new tools is emerging that will allow patterns of gene
expression to be compared in healthy and diseased tissue. On the one hand, these
gene-profiling techniques will detect gene therapy targets—genes whose products
contribute to disease. On the other hand, they will identify genes whose products
may be useful when delivered as replacement genes.
 Gene Therapy
• Long-term and complex clinical trials will be needed to optimize and deliver new
therapies..

• Gene therapies to prevent aging await a fuller understanding of biological clocks

and the aging process.
Assignments

Gene Therapy and cancer

Gene therapy and DNA vaccines
Gene therapy and Huntington’s disease
Gene therapy and Cystic fibrosis
Gene therapy and Thalassemia