Clinical pharmacology II

Question 1

What are the pitfalls of HIV treatment? (3)

Answer 1

1. Lifelong treatment
2. Many side-effects
3. Not curative

Question 2

What are the main characteristics of HIV? (2)

Answer 2

1. Lentivirus - ssRNA
2. Reverse transcriptase

Question 3

Which receptors can HIV use for entry in the cell?

Answer 3

CCR5 or CXCR4

Question 4

Describe the viral life cycle of HIV briefly (5)

Answer 4

1. Entry
2. RNA -> DNA
3. DNA integration in the nucleus
4. Transcription + translation
5. Budding

Question 5

Which cells can be infected by HIV? (3)

Answer 5

1. CD4+ T cells
2. Macrophages
3. DCs

Question 6

HIV: After how many days are many progeny virions produced?

Answer 6

~2 days

Question 7

Antiretroviral therapy does not eliminate the latent reservoir. How many cells remain latently infected?

Answer 7

1 in 100.000 cells

Question 8

What is meant with the effect of the classical/sterilizing HIV cure?

Answer 8

Replication competent HIV provirus is completely eliminated from the blood

Answer 9

1. Individual who received a SCT from a donor who has natural immunity to HIV
2. Individual who has cleared the virus in the absence of ART

Question 10

Which gene therapy strategies can be explored in combating HIV? (2)

Answer 10

1. RNAi
2. CRISPR-Cas

Question 11

To what end is RNAi used to combat HIV?

Answer 11

Combination of production of HIV-resistant anti-HIV immune effector cells

Question 12

To what end is CRISPR-Cas used to combat HIV?

Answer 12

Specific inactivation of integrated HIV DNA from infected cells

Question 13

Current gene therapy approaches for HIV focus on?

Answer 13

Ex vivo transduction of HPSC or T cells followed by rein fusion of the modified cells -> highly inefficient

Question 14

What is the advantage of using RNAi to combat HIV?

Answer 14

You don’t have to introduce an external protein -> we have all the tools in our body

Question 15

What are miRNAs?

Answer 15

Small molecules (20-25nt) which regulate expression of genes

Answer 16

1. siRNA is complementary to target mRNA
2. siRNA is loaded
3. siRNA is cleaved

Question 17

What are examples of exogenous RNAi inducers? (2)

Answer 17

1. Virus
2. Vector

Question 18

Where can you use RNAi in de viral life cycle? (2)

Answer 18

1. Directly after viral RNA enters the cell (not efficient)
2. Translation after exiting the nucleus

Question 19

miRNA biosynthesis (12:30 min)

Question 20

Partial complementarity vs perfect complementarity

Question 21

Describe the basic CRISPR-Cas mechanism (2)

Answer 21

1. Cas (nuclease) binds to short CRISPR RNA
2. crDNA targets complementary viral RNA/DNA sequences

Question 22

What are the three nucleases?

Answer 22

1. Cas9
2. Cas12
3. Cas13d

Question 23

What are the characteristics of Cas9? (3)

Answer 23

1. Targets dsDNA
2. Blunt ends
3. PAM requirement

Question 24

What are the characteristics of Cas12? (3)

Answer 24

1. Targets dsDNA
2. Stagger ends
3. PAM requirement

Question 25

What are the characteristics of Cas13d? (2)

Answer 25

1. Targets ts ssRNA
2. No PAM requirement

Question 26

How can the CRISPR-Cas cut be repaired? (2)

Answer 26

1. NHEJ
2. Homology-directed repair (HDR)

Question 27

Using either RNAi or CRISPR-Cas, you target either…? (2)

Answer 27

1. Sequences essential for HIV-1
2. Conserved HIV-1 sequences

Question 28

True or false: nearly all sgRNAs to different HIV protein exhibit potent suppressive capacity

Answer 28

True