final

Question 1

1. Briefly explain CAR-T cell technology and what it is used for.

Answer 1

Chimeric Antigen Receptor T cells utilize a receptor that responds extracellularly to the target antigen while still containing the intracellular signaling mechanism to activate the T cell.
The CAR-T cell once activated releases granulocytes to kill the target cell. Currently CAR-T cells are FDA approved for several types of blood cancers that have not responded well to traditional therapies.

Question 2

2. Describe how the following decrease the toxicities known to be caused by CAR-T cell therapy: small molecule agents

Answer 2

– These can be used to dynamically control the interaction between the receptor and target antigen, either by direct inhibition of the signaling portion, or requiring a small molecule to engage receptor and target, or by administering a small molecule drug to induce apoptosis

Question 3

2. Describe how the following decrease the toxicities known to be caused by CAR-T cell therapy: altering the gene construct of the CAR-T cell

Answer 3

the cell can be designed to express cytokine antagonists or knockout cytokine genes

Question 4

2. Describe how the following decrease the toxicities known to be caused by CAR-T cell therapy: suicide receptors

Answer 4

these can be expressed to interact with NK cells to halt the activity of the CAR-T cell if toxicity is too high

Question 5

3. Explain how the following increase specificity: targeting multiple antigens

Answer 5

• think of this as a door with two locks vs one, only when both locks are engaged is the system secure. Likewise, if the target has two known antigens, it increases the likelihood that the T cell is engaging with the correct target

Question 6

3. Explain how the following increase specificity: targeting multiple antigens: conditional expression systems

Answer 6

• think of this as two doorways in a hall. Only if the first door opens, will the second door appear in order to let you pass. In this case, only when the first receptor is engaged with a target antigen, will the CAR be expressed in order to engage and induce T cell activation

Question 7

3. Explain how the following increase specificity: PD-1 receptor signaling

Answer 7

• remember PD-1 is immunosuppressive, so it blocks the activity of a CAR. Therefore, a T cell can have two receptors- one to activate the T cell, and one to inhibit (using the intracellular machinery of a PD-1 receptor). The second CAR (PD-1 signaling) engages only with an antigen associated with a healthy cell, thus it will inhibit the T cell activity for healthy cells.

Question 8

4. Explain how bi-specific T cell engagers(BiTEs)/common adapter molecules/antibodies can be used to overcome antigen escape/heterogeneity.

Answer 8

Assuming antigen escape/heterogeneity is a problem that inhibits the T cell activity, designing a whole new CAR-T cell requires an expensive, lengthy, and complex process, considering the administration to the patient. By designing a “general” CAR-T cell that can engage with different antigens upon administration of BiTEs/common adapter molecules/antibodies, this allows for better utility of the treatment.

Question 9

5. What is a PD-1 switch receptor and how is it useful against the tumor microenvironment?

Answer 9

The TME is known to produce cytokines that enhance activity of PD-1 by overexpression of these receptors on tumor cells. Therefore, a CAR-T that expresses PD-1 extracellulary will engage with these receptors, but the intracellular machinery is designed to activate the CAR-T instead of inhibit it.

Question 10

6. Why would a tumor chemokine receptor be utilized for a CAR-T cell?

Answer 10

To enhance tumor penetration. A receptor that is sensitive to the chemokine gradient produced by tumors will act as a “homing signal”, bringing the CAR-T cells to infilitrate the tumor.

Question 11

7. What are the main components of the CRISPR system utilized for therapy and research and what are their respective functions?

Answer 11

Cas protein- recognizes the PAM sequence, cleaves the target DNA/RNA
gRNA or sgRNA – pairs to the target sequence, activates the Cas protein

Question 12

8. What types of disease is CRISPR useful for?

Answer 12

Diseases that are caused by genetic mutations, overexpressed/underexpressed proteins

Question 13

9. What are the advantages of CRISPR over ZFN or TALENs?

Answer 13

9. What are the advantages of CRISPR over ZFN or TALENs?
   Easy to target multiple genes
   Easier to design

Question 14

10. How can Cas9n and Cas12a improve specificity and decrease toxicity?

Answer 14

Since Cas9n and Cas12a can create sticky ends around the target gene, it is easier to repair with the correct template without having the random insertions or deletions caused by double strand break and non-homologous end joining

Question 15

11. Briefly explain the utility of dCas.

Answer 15

dCas (dead Cas) eliminates the cleaving mechanism of Cas but maintains specific targeting for genes. Therefore, other proteins that can alter epigenetic markers can be conjugated to dCas to activate/deactivate specific genes without completely cutting or replacing.

Question 16

12. How can editing of a single base be accomplished?

Answer 16

Using Cas9n conjugated with a deaminase- the deaminase can chemically alter a base (A to I or C to U), while the Cas 9 nickase nicks only one strand. The I is read as G, U is read as T, and the nicked strand is repaired to match the edited base.

Question 17

13. Briefly explain prime editing.

Answer 17

Prime editing involves fusing nCas9 to an engineered reverse transcriptase and a prime editing guide RNA (pegRNA). The pegRNA contains two sections: one that guides to the region of interest, and another that contains the desired substitution/s for repair after the single-stranded cut has been generated. After one strand has been altered by the prime editor, the complementary strand can also be corrected - an additional gRNA and nCas9 will create a nick in the strand and it will be repaired using the previously edited strand as a template

Question 18

14. How do PROTACs work and how can they be more useful than a basic inhibitor?

Answer 18

PROTACs are heterobifunctional molecules that target an E3 ligase and a target protein. The E3 ligase is responsible for the ubiquitination of the target protein, which acts as a molecular tag for degradation by the proteasome.

PROTACS act catalytically so have high potency as well as some other benefits such as mitigating mutational response to inhibitors and increasing selectivity.

Question 19

15. What is the idea behind LYTACs and in what ways do they differ from PROTACs?

Answer 19

Instead of marking the POI for degradation by the proteasome, POIs are degraded within the lysosome. LYTACs contain a moiety for targeting lysosome-targeting receptors linked to another molecule that targets the POI. Although they may have a catalytic mechanism of action, it is likely that they will also be degraded in the lysosome.

Question 20

16. What are AbTACs used for and how is it advantageous over LYTACs?

Answer 20

AbTACs are used to target membrane proteins by targeting the POI and a membrane bound ligase. Since they are bispecific antibodies, they are typically straightforward to engineer particularly for a known antigen.

Question 21

17. AUTACs (Autophagy Targeting Chimeras) have a POI-targeting moiety linked to cGMP. What is the purpose of cGMP?

Answer 21

The cGMP acts as a molecular tag to mark for ubiquitination, which allows for degradation through the autophagosomal pathway.

Question 22

18. How do ATTECs (AuTophagosome TEthering Compound) differ from AUTACs?

Answer 22

They contain a molecular functionality that directly targets a receptor of the autophagosome

Question 23

19. What is required for CMA(chaperone mediated autophagy)-targeting protein degradation?

Answer 23

A specific sequence recognized by the chaperone protein HSC70 needs to be present to allow for HSC70 to interact with the POI and initiate its entry into the lysosome.

Question 24

20. What types of disease could protein-targeted degradation “TACs” be useful for?

Answer 24

Any disease that has a protein known to be one of the causes!