Gene Therapy

Question 1

What are the two ways to correct genetic defects?

Answer 1

1. Homologous recombination (best theoretical approach, but low efficacy)
2. Gene addition (add exogenous gene to genome; best practical approach, but used with virus vectors and concerns about insertion site)

Question 2

Name the different types of vectors used in gene transfer and their associated properties.

Answer 2

1. Naked DNA (not effective)
2. DNA in lipid complexes (not effective)
3. Adenoviruses
   
   1. DNA vector
   2. Can cause dangerous immune response (Jesse Gelsinger)
   3. Remains in cytoplasm
4. Adeno-associated virus (AAV)
   
   1. DNA vector
   2. Milder immune response
   3. Remains in cytoplasm
5. Lentivirus
   
   1. RNA vector (reverse transcription)
   2. DNA randomly inserted via integrase
   3. Can integrate into non-dividing cells
6. Oncoretrovirus
   
   1. RNA vector (reverse transcription)
   2. DNA randomly inserted; may lead to oncogene
   3. Can integrate into non-dividing cells

Question 3

What are the applications of gene therapy?

Answer 3

• To correct a single gene defect
  
  • ADA, X-SCID, sickle cell disease, thalassemia
• To add function to a cell
  
  • Growth factors to increase tissue repair (e.g., VEGF in cardiovascular disease)
  • Increase immune responses in cancer (e.g., via growth factors, specific antigens)

Question 4

Explain how gene therapy can be used to treat hemoglobin diseases.

Answer 4

• Beta thalassemia
  
  • Point mutation in/near beta globin gene leads to decreased production, severe anemia
  • Autotransplantation: take cells from bone marrow in patient, insert normal beta globin gene, return corrected cells by vein

Question 5

What immune deficiencies have been treated with gene therapy in children?

Answer 5

• X-linked Severe Combined ImmunoDeficiency
  
  • 9/10 patients cured, but 3 developed leukemia 2 years later
  • Insertion of viral vector near oncogene (unregulated receptor gene used viral promoter/enhancer)
• Adrenoleukodystrophy (ALD)
  
  • 3/3 patients cured, 0 evidence of insertional mutagenesis or clonality

Question 6

What are the implications of inserting a vector into the HMGA2 gene?

Answer 6

This is a proliferative gene normally expressed in transcription. Insertions are associated with tumorigenesis.

Question 7

What is gene transfer RPE65 therapy?

Answer 7

• Utilized in the treatment of Leber Congenital Amaurosis, an early onset photoreceptor degeneration.
• Caused by mutations in 10 genes, but RPE65 is current focus in gene therapy.
• Produces protein for vitamin A metabolism necessary for retinal pigmentary epithelium (detection of light).
• This is an adeno-associated virus gene therapy.

Question 8

What is the future of human globin gene therapy?

Answer 8

• Use differentiated cells (skin cells) from patient to generate embryonic stem cells.
• Grow these cells in large amounts and gene correct by homologous recombination.
• Differentiate corrected cells to hematopoietic stem cells and reinsert.
• This avoids the danger of insertional mutagenesis!