Exam 1: Graduate Students_Viruses and Stuff

Question 1

3 different delivery systems

Answer 1

1) viral vectors
2) non-viral vectors
3) Physical delivery

Question 2

Viral vector examples

Answer 2

retroviral
adenoviral
AAV
herpes simplex-1

Question 3

Non-viral vectors

Answer 3

liposomes
polymersomes
cell-pentrating peptides

Question 4

Physical delivery

Answer 4

electroporation
microinjecting
hydrodynamic delivery

Question 5

What makes a good vector?

Answer 5

1) easy manipulation
2) high payload capacity
3) minimal genetic invasiveness
4) selectivity for cell target
5) absence of immunogenicity
6) stability over time

Question 6

What are the limits of viral vectors?

Answer 6

immunogenicity
limited DNA packaging capacity

broad tropism- can manipulate with coding of viral protein
potential insertional

mutagenesis - incorrect location

Question 7

Gene Therapy

Answer 7

Delivery of therapeutic genes to cell nucleus.

Functional gene
Inhibitory gene
Toxic Gene
Immunogenic gene
Supplementary gene

Question 8

Gene therapy applications

Answer 8

spinal muscular atrophy
Alzheimer’s
epilepsy

Question 9

Challenges of Gene therapy

Answer 9

Only applicable to disease that involve a single gene

Requires delivery of genes to specific cells - vectors, choose the best one and modify

May cause interference with other genes - possible that by adding gene and upregulating one thing regulates others as well

Difficulty of controlling expression of gene - get it in, but how do you get it expressed

Question 10

RNAi

Answer 10

small specially designed RNA fragments

decreases translation of gene of interest

block production outcome

Question 11

How does RNAi work?

Answer 11

• Dicer cleaves dsRNA
  
  • Cleaved dsRNA is bound by Argonaute
  • Guide strand selected
  • RISC is assembled
  • RISC binds target mRNA
  • Target mRNA is cleaved & degraded, or translation is blocked
  • Once you get target mRNA, perfect alignment and target will lead to degradation, but also have slit bind to other locations, which effects rate of translation

Question 12

RNAi applications

Answer 12

Huntington’s AAV
ALS: AAV

TTR amyloidosis

Decrease mRNA translation, stop progression

Question 13

RNAi Challenges

Answer 13

Requires viral or non-viral vectors for delivery to the CNS

Potential for nonspecific degradation of nontarget mRNAs

Saturation of endogenous silencing pathways - can’t be used for silencing, could upregulate other gene regulation within the cell

Question 14

CRISPR

Answer 14

Discovered CRISPR-Cas systems in prokaryotes and vary greatly between species

Serves as adaptive immune response

Cas proteins cleave foreign DNA and integrate into CRISPR array

Crispr RNA (crRNA) serves as guide for identifying future infections

Question 15

CRISPR: revolution in genetic engineering advantages

Answer 15

More robust editing efficiency

Extremely flexible
Easy to use

Only requires design of short guide RNA (sgRNA)

Wide variety of Cas proteins allows for high level of specificity -vary by species

Ability to modify multiple genes as once

Many applications beyond gene editing

Question 16

Cas9 protein

Answer 16

Pam sequence, looks for short guide arm which matches DNA and cleaves.

You can modify Cas9 to be deactivated so it doesn’t cut DNA, effect other activities on the DNA. Add proteins to increase or decrease transcription of the gene. Add methyl groups onto DNA or acetyl groups onto DNA. Increase or decrease gene expression.

Single polymorphisms: change 1 letter which is revolutionary. Do something similar to RNAi

Question 17

CRISPR in neuroscience

Answer 17

• Gene editing in ALS- create survival and muscular control

Epigenome editing in parkinson’s = add factor that can cause methylation. Virus and most factors: does it have the same effect? Methylation =decrease expression of mRNA. Leads to decrease mitochondrial superoxide production.

Gene regulation in Charcot-Marie-Tooth disease type 1A- increase methylation

Question 18

CRISPR challenges

Answer 18

• Target specificity - DNA target selection an sgRNA design: want to only bind to gene of interest
• Off-target cutting
• Selection of Cas
Delivery to cells

Question 19

Controversy: CRISPR Babies

Answer 19

Goal of genetic enhancement rather than therapy

Germline vs. Somatic (body)Cells. Germline cells will be passed on.

Ineffective
○ Lulu: deletion in only 1 copy of CCR5
○ Nana- potential genetic mosaicism

Uncertain effects of mutated protein