Lecture 23: Gene Therapy for DMD: Viral-mediated strategies Flashcards Preview

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Flashcards in Lecture 23: Gene Therapy for DMD: Viral-mediated strategies Deck (25):

What are the characteristics of an adenovirus?

Medium sized non-enveloped icosohedral virus.

dsDNA genome 35 kb capacity

Generally cause mild disease in humans

Capable of transducing both proliferating and non proliferating cells

Various adenoviral vectors have been generated based on adenovirus to use tools for gene therapy.


How long is the full length dystrophin gene?

14kb meaning it'll fit easily in an adenovirus


What are the advantages of using adenoviruses for gene therapy?

adenoviruses have a large capacity (up to 30 - 35 kb)

They can transduce both proliferating and non-proliferating muscle cells


What are the three types of adenoviral vectors that have been used to treat DMD?

1st generation

2nd generation

3rd generation


What are the characteristics of the 1st generation adenoviruses?

Generated by substitution of E1 and/or E3 regions with expression cassette.

Lack early viral genes resulting in a carrying capacity of 8 kb

Elicit a significant immune response in vivo due to de novo synthesis of viral proteins. This is even worse in people who have previously been infected by adenovruses

They require local injection due to immune response. And injecting it everywhere would result in sepsis

*Successfully delivered a minidystrophin gene to mouse muscle for long term expression in 1993


What was the experiment conducted to test the effectiveness of adenovirus vectors in producing a functional dystrophin protein?

One leg injected with one virus that expressed a protein called beta galactosidase which glows when produced. The other leg with beta galactosidase as well as dystrophin.

If muscles went through cycles of degeneration and regeneration they would express less beta galactosidase. Adenovirus only infects muscle cells not the satellite cells.

After a long time the left muscle with no dystrophin showed a disappearance of beta galactosidase. in dystrophin expressing muscle the beta galactosidase survived.


How were second generation adenoviruses made from 1st generation adenoviruses?

Deletion of additional viral genes to the extent where carrying capacity of approximately 10 kb.

Immune response was reduced

No reported studies demonstrated delivery of dystrophin


What happened with the use of 1st and 2nd generation adenoviruses?

Both 1st and 2nd generation vectors were demonstrated to have leaky expression of the viral genes resulting in animmune response against the vector.


What was the result of using 3rd generation adenoviral vectors?

Lack all viral genes which increases carrying capacity to 30kb which allows less immune response to occur and expression of the full dystrophin gene.


What is a lentivirus?

Enveloped retrovirus

RNA genome with an 8kb capacity

Capable of trasducing both proliferating and non-proliferating cells

Able to integrate into genome of host


What are the characteristics of lentiviral vectors?

Lentiviral vectors infect both proliferating myoblasts and differentiated myotubs in vitro.

Limited in transducing skeletal muscle in vivo.

Murine retroviral vectors were amongst the first vectors tested for gene replacement therapy

Can infect stem cell population as well as mature fibers


What is the problem with lentivirus? How has this problem been addressed?

Lentivirus ability to transduce muscle in vivo is limited.

Retroviral vectors have been generated based on human and feline lentiviruses


What happens to force production in response to lentivirus transduced BMD variants of dystrophin?

improvement in force was seen

general functional improvements were seen


What is the problem with using lentiviruses?

potential oncogenesis when injected systemically


What is an adeno associated virus?

Small ssDNA parvovirus

Requires the presence of a 'helper' virus in order to replicate

Has infected 80% of the population already

Does not cause disease in humans

Limited carrying capacity of approximately 5 kb


How can AAV viruses be used?

1 vector contains gene of interest: i.e dystrophin flanked by ITRs.

2nd vector has genes for replication and for capsid.

Together they function to be expressed in target cells


How did the mice cells receive the injection?

Better transduction occured in young mice compared to older mice after EDL muscle intramuscular injection.


How good was AAV action?

When treated Dko mice saw dystrophin localization to the sarcolemma

improvement in centrally nucleatid fibers

Significant improvement in force production

Less kyphosis and longer lifespan of treated dko mice


What is the effect of AAV action in late stage disease?

No improvement in heart fibrosis

Small improvements in heart function


Summary for AAV:

AAV can efficiently deliver the DMD gene to skeletal and cardiac tissue in young and old mice. However, efficacy of treatment is limited by disease pathology.


What happened when AAV were used in dogs?

In dogs there was an immune response that decreased efficacy


What are the advantage and disadvantages of AAV?

Capable of systemic delivery

Not able to deliver full-length gene

No transduction of muscle stem cells


What is utrophin?

homologue of dystrophin with structural similarities to dystrophin.

Used in mdx mice which diminishes the severity of dystrophin expression


What happened when microutrophin was used instead of microdystrophin for AAV transmission?

Increased lifespan and effectiveness during lifespan


What is a method by which a full dystrophin protein can be transduced?

segment in different viruses

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