gene therapy - the transplantation of normal genes into somatic cells in place of missing or defective
ones in order go correct genetic disorders Initially -> replacing a defective/missing gene (adding a wile-
type gene)
More recently -> editing/repairing a defective gene
how does DNA get into nucleus - Electroporation, chemical, gene gun, injections, liposomes, naked DNA,
receptor-mediated endocytosis, viruses
o A lot of these methods kill the cell
o Some of these would only work with dividing cells
4 viral vectors - - Herpes Virus
- Lentivirus
- Adenovirus
- Adeno-associated virus (AAV)
pro and cons of herpes as a viral vector - - Dividing and non-dividing cells (+)
- High levels of expression (+)
- Large insert size (50kB) (+)
- Does not insert into host genome (+/-)
- Large viral genome size (+/-)
- Pathogenic/cytotoxic (-)
- High immune responses (-)
pros and cons of retrovirus/lentivirus as a viral vector - - Dividing and non dividing cells (+)
- Prolonged infection and gene expression (+)
- Low immune response (+)
,- Small insert size (<7kb) (-)
- Expression level depends on where it is inserted (-)
- Insertion into host genome (+/-)
*Commonly used
pros and cons of adeno virus as viral vector - - Dividing and non-dividing cells (+)
- Large insert size (36kb) (+)
- Does not insert into genome (+/-)
o Can get lost during division (-)
- High immune responses (-)
pros and cons of AAV virus - - Infects dividing cells and on dividing cells (+)
- Does not insert into the genome (+/-)
o Can get lost during division (-)
- Low immune responses (+)
- Non-pathogenic (+)
- Small insert size (<5kb) (-)
- Sustained expression (-)
- Low expression levels (-)
*Most studies use AAV due to low immune responses and lack of insertion*
4 viral vector variables - - immune response
- trasngene size
- genome insertion (permanent vs transient expression)
- tissue targeting
3 parent therapy - = mitochondrial replacement
- Pro-nucleus from affected mother is transplanted to the egg of a normal donor
,- Fertilized egg has mitochondria from donor but nuclear DNA from the parents
- Can treat ANY mitochondrial gene disease
Trikafta (new CF treatment) - - Combination therapy (3 drugs) specific for the deltaF508 mutation
o 2 drugs help mutated CFTR protein fold correctly in the ER
o 1 drug that helps it stay open at the cell surface (increased CL conductance)
- In North America 90% of CF patients have at least one deltaF508 allele
- Cost is about $300,000/year
o Most provinces now cover cost
o Much cheaper than having children staying in the hospital frequently
Gendicine (Cancer treatment) - - Approved in China to treat some tumors
- Adenovirus vector expresses p53
o Big insert
- Claimed to work in combination with chemo and radiation therapy
- Immune reaction potentially bad
- Not approved elsewhere
- *Promising
ADA treatment - - Bone marrow transplant may be used if a matched donor can be found
- Enzyme replacement therapy
o >$4 million over 10 years
- Retrovirus gene therapy was used in 1990
o First example of successful gene therapy
o Still requires regular medication
- Strimvelis approved in 2016
o Retroviral vector with ex-vivo stem cell therapy
o Costs >$1 million
, o Hematopoietic stem cells are isolated from the patients after GCSF treatment to enrich stem cells,
endogenous bone marrow cells killed, HSCs treated with virus and re-perfused
o Immunosuppressants are needed
X-SCID treatment - - Bone marrow transplant may be use if donor can be found
- In late 1990s 9 of 10 children were cured using retroviral strategy BUT 3 developed leukemia
o Trial and whole field of gene therapy stopped for 5-10 years
Hemophilia treatment - - Administer missing factors
o Only need 5% of normal levels
- AAV in liver has been used to provide factor IX
- Works for B
o Easy to repeat (+)
o Needs only low expression (+)
o Small insert (+)
- Doesn't work for A
o Big insert 180kb (-)
Glybera (LPLD) - - 1 in 1,000,000
- AAV to deliver LPL gene
o Intramuscular injection
- Worked perfectly, no side effects
- No longer available
o Unprofitable
stem cell therapy - - Stem cells are isolated from the patient, treated with lentiviral vector and
reintroduced into the patient
o Endogenous cells are destroyed prior to re-introduction to eliminate immune response
o Uses lentivirus because AVV would get lose and bone marrow procedure is too big of a stress on the
patient to have to do again
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