Final - Gene Therapy

Question 1

Gene therapy

Answer 1

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

Question 2

Goals of Gene Therapy

Answer 2

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)

Question 3

Criteria for Effective Gene Therapy

Answer 3

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

Question 4

Recessive genetic disorder gene therapy

Answer 4

• 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

Question 5

Dominant genetic disorder gene therapy

Answer 5

• requires goal #2 (To replace or inactivate a mutant gene)

- involves replacement of gene which is harder to do.

Question 6

Basic Principal of Gene Therapy

Answer 6

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

Question 7

Viral Vectors

Answer 7

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

Question 8

Retrovirus

• advantages
• limitations

Answer 8

• simple RNA virus with only 3 structural genes
  Advantages:
  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)

Limitations:

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

Question 9

Adenoviruses

• advantages
• limitations

Answer 9

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

Limitations:

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

Question 10

Adeno-Associated virus (AAV)

• advantages
• limitations

Answer 10

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

Limitations:

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

Question 11

Non-Viral Vectors

• advantages
• limitations
• types

Answer 11

Advantages:

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

Limitations:

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

Types:
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

Question 12

Ex vivo gene transfer

Answer 12

• 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

Question 13

In vivo gene transfer

Answer 13

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

Question 14

ADA Deficiency

Answer 14

• 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

Question 15

Severe combined immunodeficiency

Answer 15

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

Question 16

Cystic Firosis

Answer 16

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

Question 17

Cancer

Answer 17

• 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

Question 18

Ethical Issues in Gene Therapy

Answer 18

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