Lecture 22 Gene Therapy

Question 1

Somatic gene therapy
(Vs germ line gene therapy)
(2 types)

Answer 1

Non-heritable

Ex vivo: remove cells, add therapeutic gene, culture, return.
In vivo: directly introduce wild type gene

Question 2

First gene therapy trial in human
Severe combined immunodeficiency disease (SCID)
Cause
(mechanism)
Disease Phenotype
Protocol
In vivo/ex vivo
(4 corresponding steps)

Answer 2

Lack of T cell, unable to mount normal immune response to infections

Defects in ADA gene

ADA codes for an enzyme.
If it’s missing, the accumulation of metabolite would destroy the T cell.

Ex vivo
Remove ADA deficient lymphocytes from SCID patient,
Culture
Infect cells lymphocytes with viral vector that contains normal ADA gene.
Reinfuse the ADA-gene-corrected lymphocytes back into the SCID patient.

Question 3

Gene therapy challenges
(Four challenges)
Challenge 1
(6 possible approaches and their characteristics)

Challenge 2

(1 solution for sub challenge 1, 1 solutions for sub challenge 2, 2 solutions for the sub challenge 3)

Challenge 3
(2 problems)
Example for p1
Example for p2: OTC/deficiency in ornithine transcarbamylase
Cause
(mechanism)
Disease Phenotype
Protocol
Vector:

Challenge 4
(Risk, 2 problems)
Example for problem 1: Deficiency in IL2 receptor gamma
Cause
(mechanism)
Disease Phenotype
Protocol
Ex vivo/in vivo
Vector:
Result:

Answer 3

1. How to get DNA into the cell?
Viral vector: fast, easy, efficient
Electroporation: harmful
Particle bombardment: harmful
Direct injection: not as efficient
Liposome: not as efficient
CRISPR: off target effect

2. Will the transgene be expressed in the correct cell at the high enough level for an appropriate period of time?
Expressed in the correct cell: ex vivo
Expressed at the high enough level: strong promoter
Expressed for an appropriate period of time: tissue-specific promoter, insulator.

3. Consequences of the immune responses to the vector or transgene product?
Reduced expression
E.g. Adenovirus vector
Lethal
E.g. OTC
Deficiency in ornithine transcarbamylase
OTC breaks down amino acids present in protein
→ Lack of OTC leads to build up of ammonia, damaging brain function.
Treatment: Low-protein diets and ammonia binding drugs.
Gene therapy protocol: Using adenovirus as a vector for the normal OTC gene.
Result: One patient died of a massive immune reaction to the vector virus.

4. Risk of the transgene insertion into a functional gene and what are the consequences?
More common for DNA to insert into actively transcribed regions of the genome

transgene promoter drives expression of a proto-oncogene transgene disrupts a tumour-suppressor gene
E.g. Deficiency in IL2 receptor gamma
IL2 receptor gamma is a cytokine receptor
→ Lack of IL2 receptor gamma leads to the developing lymphocytes unable to respond to cytokine signals, and mature into functional T cells.

Gene therapy protocol: Ex vivo, using CD34 cells as a vector for the normal IL2 receptor gamma.
Result: Three developed T cell lymphoblastic leukemia, the insertion of the transgene into the LMO2 gene,

LMO2 gene codes for a protein essential for the formation of certain transcription factor complexes that boost blood cell proliferation.

LMO previously identified in individual with T cell acute lymphoblastic leukemia
Translocation causes nearby enhancer element to boost the expression of the LMO2 gene.

Question 4

Positive gene therapy trial
Leber congenital amaurosis (LCA)
Causes
(Mechanism)
Phenotypes
(4 phenotypes)
Animal model
Human gene therapy protocol
Ex vivo/in vivo

Answer 4

RPE65 mutation is identified in many cases
RPE65 is an isomerase that, when light is perceived, isomerises molecule essential for vision to occur from trans to cis version.

1. Severe visual impairment in childhood-usually total blindness by 30-40 years of age
2. Abnormal roving eye movements (nystagmus)
3. Abnormal electroretinography (ERG)
4. Poor pupillary light reflexes

Animal model of LCA: Briard dog

in vivo

Question 5

Future of gene therapy
Loss of photoreceptor function
Photoreceptor:
Retinal ganglion cells:

Optogenetics:

Prosthetics:

Answer 5

Loss of photoreceptor function
Photoreceptor: input visual signals
Retinal ganglion cells: output visual signals to the brain

Optogenetics
Gene therapy target retinal ganglion cells introduces components activate their photosensitivity

Prosthetics
Specialized glasses process visual information the way that the photoreceptor would and transmit this information to the retinal ganglion cells.