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Flashcards in Final - Gene Therapy Deck (18):

Gene therapy

- an experimental proedure aimed at replacing, mainpulation, or supplementing nonfunctional or dysfunctional genes with health genes


Goals of Gene Therapy

1. To compensate for a mutant gene that causes a loss of function in a specific protein
2. To replace or inactivate a mutant gene
3. To counteract the effects of disease pathogenesis in acquired disorders (e.g. cancer)


Criteria for Effective Gene Therapy

1. must have sufficient knowledge on molecular basis of the disease to be treated
2. the gene to be transferred must be identified and cloned
3. Must have an effective method of introducing the gene so that it is exposed in a large number of target cells
4. Target cells should have a long half-life or good potential to replicate in vivo
5. The therapy must not harm the patient


Recessive genetic disorder gene therapy

- requires goal #1 (To compensate for a mutant gene that causes a loss of function in a specific protein)
- involves the addition of a normal gene into target cells so that the recessive allele is masked


Dominant genetic disorder gene therapy

- requires goal #2 (To replace or inactivate a mutant gene)
- involves replacement of gene which is harder to do.


Basic Principal of Gene Therapy

- a therapeutic gene inserted into a vector that carries the gene to specific target cells
- can be performed in vivo or ex vivo


Viral Vectors

- modified to remove harmful gene and replaced with desired gene
1. Retrovirus
2. Adenovirus
3. Adeno-Associated Virus (AAV)


- advantages
- limitations

- simple RNA virus with only 3 structural genes
1. enter target cell efficiently
2. Viral DNA integrates into host genome and is stable
3. Can carry a relatively large piece of DNA (8 kb)

1. cannot infect nondividing cells (exception = lentiviruses)
2. potential disruption of host genes = side effects


- advantages
- limitations

- cause common cold, natural tropism for respiratory epithelium
1. Can infect dividing and nondividing cells
2. Carry large piece of DNA (30-35 kb)

1. Don't integrate into host genome = only expressed transiently
2. induce a strong immune response = destroys virus


Adeno-Associated virus (AAV)
- advantages
- limitations

- DNA virus that does not cause disease in humans
1. infects dividing and nondividing cells
2. integrates into host genome, producing latent, long-term expression.
3. induces a milder immune response

1. can only accommodate small pieces of DNA (5 kb)
2. Difficult to produce in large quantities


Non-Viral Vectors
- advantages
- limitations
- types

1. No biological risks
2. Do not induce an immune response
3. Can carry larger amounts of DNA

1. Do not integrate into host genome and therefore, have a poor long-term expression
2. May be difficult to target tissue of interest

A. Naked DNA - cDNA with regulatory elements in a plasmid
B. Liposomes- synthetic lipid vesicles which can transport genes
C. Protein-DNA conjugates - allows binding to a cell surface receptor to facilitate entry into cells


Ex vivo gene transfer

- target cells are removed from the patient, genetically reconstituted with the appropriate gene in vitro and then returned to the patient
-Ex: Gene therapy for ADA defieciency


In vivo gene transfer

- genetically modified vector is introduced directly into the body (e.g. inhaled)
- Ex: Gene therapy for Cystic Fibrosis


ADA Deficiency

- the first disease to be treated with gene therapy
- an autosomal recessive disorder that causes severe immunodeficiency
- example of ex vivo gene transfer: Patients T lymphocytes are removed and incubated with MMLV containing the human ADA gene. Genetically modified T cells are injected IV into patient


Severe combined immunodeficiency

- ex vivo gene transfer
- bone marrow stem cells are genetically altered to include a gene encoding part of a receptor necessary for lymphocyte development


Cystic Firosis

- In vivo gene transfer
- uses adenovirus, AAV, or liposomes
- CFTR gene introduced directly in nasal passages or bronchial epithelium (inhale)



- immunologic gene therapy
- introduce genes coding for molcules of immune system to boost immune response to tumor
- ex: Cytokines (interleukin-2), chimeric antigen receptors, genes coding for HLA proteins


Ethical Issues in Gene Therapy

1. Safety
2. Unequal access to high cost therapies
3. Potential misuse of germline gene therapy