Lecture 24

Question 1

What is the most effective vehicle for gene therapy?

Answer 1

Virus - the virulence factors are knocked out and the virus infects cells, bringing with it the therapeutic gene. Adenoviruses and Retroviruses are vectors of choice.

Question 2

What are the disadvantages to using viruses in gene therapy?

Answer 2

1. There’s a transient effect - the virus can’t replicate, so the therapeutic gene only lasts as long as the virus does.
2. The immune system still recognizes the viral particles as a virus and leads to an immune response/inflammation.

Question 3

What is the therapy of OTCase deficiency (urea cycle disorder, so think about which organ is being targeted with this therapy), and what are its advantages and disadvantages? How have we gotten around the disadvantages?

Answer 3

Gene therapy using adenovirus vector that targets the liver –> same disadvantages as described for viral gene therapy in general. However, adenovirus can infect non-growing OR proliferating cells, and its genome does not integrate into the host’s genome –> low risk of mutagenesis. Using Adeno-Associated Virus elicits much lesser immune and inflammatory response.

Question 4

What are disadvantages of using a retroviral vehicle for gene therapy, and which subclass of retrovirus can be used to avoid one of the disadvantages?

Answer 4

They don’t infect non-growing cells well. Lentivirus can be used, and IS effective at infecting non-proliferating cells.

Also, because it integrates its genome into the host genome, there’s high risk of insertional mutagenesis when using retroviruses.

Question 5

What is the therapy for ADA deficiency (which leads to Severe Combined Immuno Deficiency “SCID”)?

Answer 5

Marrow cells are taken from patient, retrovirus is added exvivo (into the cells outside of the patient), and then the infected cells are reinserted into the patient. There was incidence of insertional mutagenesis that led to lukemia.

Question 6

What is the therapy for dominant disorders (disorders where you want to knock down gene function instead of restore gene function)?

Answer 6

shRNA - Short hairpin RNA that mimics the action of miRNA. The shRNA gene is added to retrovirus and the retrovirus infects the patient –> shRNA genes are transcribed and the shRNA is processed and acts as miRNA to silence the target gene (the aberrant dominant disorder gene.)

Question 7

One of the issues with using viral gene therapy for DMD is the cDNA for the dystrophin gene is too ____. An alternative strategy is to us antisense _______ to force _____ skipping. This converts out of frame deletions to in-frame deletions, and thus a conversion from the more deleterious DMD to the less deleterious _____. Remember that the exons of the DMD gene are not all multiples of 3; it’s the combination of exons in mRNA that form a multiple of three. Think about what this means for skipping exons. There is also the possibility of upregulating ______, a dystrophin homolog not normally expressed in muscle cells.

Answer 7

Large

Oligonucleotide

Exon

BMD

Utrophin

Question 8

CRISPR Cas9 is a ribonucleoprotein complex (Cas9 is the protein enzyme and combines with a ___ RNA that is complimentary to the target gene sequence.) The complex binds just upstream of a ____ motif and induces a _____ stranded cut by Cas9. These cuts can be repaired by either _____ end joining or Homologous-directed repair. The latter is used for gene replacement and restoring function, and it requires a homologous DNA template to be introduced with the CRISPR components. The former is used to generate ___ models (because this process is error prone.)

Answer 8

Guide RNA (gRNA)

PAM

Double stranded

Non-homologous end joining

Knockout (KO) models