Lecture 14

Question 1

What are the 5 gene therapy research examples?

Answer 1

1. Whole cell gene therapy
2. Gene therapy for immunocontraception
3. CRISPR and its applications
4. Clinical trials gene therapy for occular disease
5. Molecular therapies for neuromuscular disorders

Question 2

Define Gene Therapy

Answer 2

A collection of techniques for correcting a defective gene problem

Question 3

What is the most common approach to gene therapy?

Answer 3

A normal gene copy is inserted into a non-specific location within the genome to replace a non-functioning gene

Question 4

What are the 4 viral vectors in gene therapy?

Answer 4

1. Adenoviruses
2. Adeno-associated viruses
3. Herpes Simplex Virus
4. Retroviruses

Question 5

Characteristics of Adenoviruses

Answer 5

• Double-stranded DNA
• Cause o Respiratory infections
  o Intestinal infections
  o Eye infections
• Causes the ‘common cold’

Question 6

Characteristics of Adeno-Associated viruses

Answer 6

• Small
• Single-stranded DNA viruses
• Insert their genetic material at a specific point on chromosome 19

Question 7

Characteristics of Herpes Simplex Virus

Answer 7

• double stranded DNA
• Infect neurons
• HSV1 = cold sore

Question 8

Characteristics of Retroviruses

Answer 8

• Create double-stranded DNA copies of their RNA genomes
• Integrate copies into chromosomes of host cells
• e.g. HIV

Question 9

How does gene therapy of non-viral delivery work?

Answer 9

DNA is transferred by physical means

• direct injection
• electroporation
• gene gun

e.g. o Liposomal DNA delivery
o Whole cell transplantation
o (Mammalian expression) plasmids

Question 10

Characteristics of Duchenne Muscular Dystrophy

Answer 10

• X-linked recessive
• Result of a non-functioning dystrophin gene
• Patients wheelchair bound by 12y.o.
• Death (cardiac/resp. failure) in third decade of life

Question 11

Characteristics of the dystrophin gene

Answer 11

2.4 Mb long
79 exons long
Encodes multiple isoforms

Question 12

What is the full length of the dystrophin protein?

Answer 12

427kDa

Question 13

What are the genetics of DMD?

Answer 13

Nonsense or frameshift mutations causing premature termination of translation = truncation

Question 14

What are the 5 genetic therapy approaches for DMD?

Answer 14

• Mini Dystrophin Gene Delivery
• Transcriptional Read Through
• Exon Skipping
• Homologue Induction
• Cell based whole gene replacement

Question 15

What is the Mini dystrophin gene delivery process?

Answer 15

• smaller dystrophin genes based on Becker examples delivered via plasmid or viral vectors

Question 16

What is the transcriptional Read Through process?

Answer 16

Some antibiotics will cause ribosomes to ignore STOP codons

Question 17

What is the Homologue Induction process?

Answer 17

A dystrophin analogue called utrophin has a promoter which may allow selective upregulation

Question 18

What is the Cell based whole gene replacement process?

Answer 18

Donor muscle precursor cells (myoblasts) which have the function dystrophin gene is transplanted by myoblast transfer therapy (MTT)

Question 19

Define Myoblast Transfer Therapy

Answer 19

The direct injection of dystrophin expressing donor myoblasts into dystrophic muscle

–> provides a cell based gene rescue

Question 20

What do the hinge regions do on a dystrophin gene?

Answer 20

Provide flexibility

Question 21

What does the rod domain do on the dystrophin gene?

Answer 21

Provide stretchability

Question 22

The rod and hinge regions on dystrophin gene do what together?

Answer 22

Protect from the force of contraction and relaxation

Question 23

If you get a mutation in the actin binding regions or cysteine rich regions, which dystrophy would you get?

Answer 23

DMD

Question 24

If a mutation is found in the hinge regions or C terminal domain, which dystrophy would you get?

Answer 24

BMD