Gene Silencing

Question 1

What is RNA interference? (RNAi) (2)

Answer 1

1. An accurate and potent gene silencing method
2. Silences RNA by breaking it down or stopping translation

Question 2

The key requirement of RNAi is?

Answer 2

dsRNA (double-stranded)

Question 3

Several types of silencing RNAs (RNAis) have been discovered. Name them (4)

Answer 3

1. Small interfering RNAs (siRNAs)
2. MicroRNAs (miRNAs)
3. shRNA (short hairpin)
4. CRISPR

Question 4

The key enzyme in RNAis is?

Answer 4

Dicer

Question 5

How does dicer work?

Answer 5

Has RNAase domains - chops long dsRNA into short RNAs and they will become siRNAs, causing RNAi

Question 6

How does RNAi work? (2)

Answer 6

1. Is a natural cellular process that silences gene expression by promoting the degradation of mRNA
2. It plays an impotant role in gene regulation and innate defense against invading viruses

Question 7

What are the sources of long dsRNA? (3)

Answer 7

1. Hairpin
2. Complementary RNAs
3. RNA dependent RNA polymerases

Question 8

siRNAs have a defined structure. Describe how it is made and what the structure is

Answer 8

Dicer cleaves long dsRNA into siRNA 21-23nt dsRNA
- Symmetric 2nt 3’ overhangs
nt = nucleotide

Question 9

Explain the mechanism of action of siRNA (4)

Answer 9

1. The siRNA interacts with dicer and activates the RNA-induced silencing complex (RISC)
2. The endonuclease argonaute 2 (AGO2) component of the RISC cleaves the passenger strand (sense strand) of the siRNA while the guide strand (antisense strand) remains associated with the RISC.
3. Subsequently, the guide strand guides the active RISC to its target mRNA for cleavage by AGO2.
4. As the guide strand only binds to mRNA that is fully complementary to it, siRNA causes specific gene silencing

Question 10

miRNA is a class of small RNA molecules that __________ regulates gene expression

Answer 10

negatively

Question 11

miRNA gene transcription is carried out by ___ __________ in the nucleus to give _______ _____ with a double-stranded stem-loop structure

Answer 11

RNA polymerase; primary miRNA (pri-miRNA)

Question 12

pri-miRNA is cleaved by a microprocessor complex comprising of ______ to form _________ _____, which is a duplex that contains 70-100 nucleotides with interspersed mismatches and adopts a loop structure

Answer 12

Drosha; precursor miRNA (pre-miRNA)

Question 13

pre-miRNA is subsequently transported by ________ _ from the nucleus to the cytoplasm where what happens? (5)

Answer 13

Exportin 5
1. Where it is further processed by Dicer into a miRNA duplex of 18-25 nucleotides
2. The miRNA duplex then associates with the RISC forming a complex called miRISC
3. The miRNA duplex is unwound, releasing and discarding the passenger strand (sense strand) - unlike in the processing of siRNA, in which the AGO2 of the RISC causes the cleavage of the passenger strand of siRNA
4. The mature single-stranded miRNA guides the miRISC to the target mRNAs.
5. The miRNA binds to the target mRNAs through partial complementary base pairing with the consequence that the
target gene silencing occurs via translational repression, degradation, and/or cleavage.

Question 14

What are the differences between target recognition by siRNA and miRNA? (3)

Answer 14

1. siRNA is usually fully complementary to the coding region of its target mRNA.
2. miRNA is partially complementary to its target mRNA. Complementary binding usually occurs at the seed region (nucleotides (nt) 2–7 of the 5’ end) of miRNA and the 3’ UTR of the target mRNA.
3. Since miRNA-mRNA recognition does not require perfect pairing, one miRNA strand can recognize an array of mRNAs, and hence miRNA has the characteristic of having multiple targets.
   - For example: miRNA-124, which is preferentially expressed in brain tissues, can downregulate 174 annotated genes.

Question 15

What are 2 benefits of RNAi therapeutics?

Answer 15

1. They can overcome the major limitation of traditional small drug molecules, which can only target certain classes of proteins.
2. Even for protein-based drugs including monoclonal antibodies that are highly specific, their targets are mainly limited to cell-surface receptors or circulating proteins.

Question 16

siRNA vs. miRNA:
Prior to Dicer processing - what do these molecules look like/what is their structure prior to Dicer?

Answer 16

siRNA:
- Double-stranded RNA that contains 30 to over 100 nucleotides
miRNA:
- Precursor miRNA (pre-miRNA) that contains 70-100 nucleotides with interspersed mismatches and hairpin structure

Question 17

siRNA vs. miRNA:
What is the structure? (Post Dicer)

Answer 17

siRNA:
- 21-23 nucleotide RNA duplex with 2 nucleotides 3’ overhang
miRNA:
- 19-25 nucleotide RNA duplex with 2 nucleotides 3’ overhang

Question 18

siRNA vs. miRNA:
Complementarity

Answer 18

siRNA:
- Fully complementary to mRNA
miRNA:
- Partially complementary to mRNA, typically targetting the 3’ untranslated region of mRNA

Question 19

siRNA vs. miRNA:
mRNA target

Answer 19

siRNA:
- One
miRNA:
- Multiple (could be over 100 at the same time)

Question 20

siRNA (1) vs. miRNA (3):
Mechanism of gene regulation

Answer 20

siRNA:
- Endonucleolytic cleavage of mRNA
miRNA:
- Translational repression
- Degradation of mRNA
- Endonucleolytic cleavage of mRNA (rare, only when there is a high level of complementarity between miRNA and mRNA)

Question 21

siRNA (1) vs. miRNA (3):
Clinical applications

Answer 21

siRNA:
- Therapeutic agent
miRNA:
- Drug target
- Therapeutic agent
- Diagnostic and biomarker tool.

Question 22

RNAi - stability and delivery. What are the limitations? (4)

Answer 22

1. RNAs are extremely vulnerable to serum nucleases.
2. Although double-stranded RNA is more resistant to nuclease degradation than single-stranded RNA, naked RNAs in their unmodified forms are degraded rapidly following administration. This degradation is done by the abundant nucleases present in the bloodstream, which contributes to a short half-life in vivo.
3. Poor stability is one of the major obstacles toward the successful application of siRNAs and miRNAs as therapeutic agents.
4. Solution - Chemical modifications of RNA

Question 23

Poor delivery is still a major challenge in translating therapeutic siRNAs and miRNAs into the clinic. Why?

Answer 23

Both types of RNA molecules have an intracellular site of action, but their intrinsic properties, including hydrophilic nature, negative charge and high molecular weight (∼14–15 kDa), render them poorly permeable across biological membranes.

Question 24

The primary role of a delivery system is to…?

Answer 24

Facilitate the cellular uptake of siRNAs or miRNAs to their target sites

Question 25

The secondary role of a delivery system is to...?

Answer 25

Protect the nucleic acids from premature nuclease degradation, thereby reducing the need for chemical modifications, which may affect the specificity and functionality of the RNA molecules.

Question 26

Why are viral vectors used as a delivery system?

Answer 26

Lentiviruses, adenoviruses, and adeno-associated viruses (AAVs) are extremely efficient in transferring the RNA-encoding vectors into the nucleus of mammalian cells to ensure high expression of RNA. High transduction efficiency.

Question 27

Viral vectors as a delivery system - how are they designed?

Answer 27

Viruses that are used to carry therapeutic RNA are genetically engineered to remove their virulence, and their tropism can be altered by genetic manipulation of the viral capsid for targeting to specific cell types. In addition, long-term expression can be achieved by using viruses, such as lentiviruses, that can integrate into the host genome

Question 28

What are 3 limitations of viral delivery?

Answer 28

1. Serious safety concerns - high immunogenicity 2. Risk of insertional mutagenesis 3. High production costs have also limited their clinical applications.

Question 29

What are the 2 main categories of non-viral vectors?

Answer 29

1. Polymer-based 2. Lipid-based systems

Question 30

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs: Nature of action

Answer 30

Small molecules: - Activation or inhibition of targets Protein-based drugs: - Activation or inhibition of targets siRNA/mRNA: - Inhibition of targets

Question 31

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs: Site of target proteins

Answer 31

Small molecules: - Extracellular and intracellular Protein-based drugs: - Mainly extracellular siRNA/mRNA: - Virtually any sites

Question 32

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs: Selectivity and potency

Answer 32

Small molecules: - Variable Protein-based drugs: - Highly specific and potent siRNA/miRNA: - Highly specific and potent

Question 33

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs: Lead optimization

Answer 33

Small molecules: - Slow Protein-based drugs: - Slow siRNA/mRNA: - Rapid

Question 34

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs: Manufacture

Answer 34

Small molecules: - Easy Protein-based drugs: - Difficult siRNA/mRNA: - Easy

Question 35

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs: Stability

Answer 35

Small molecules: - Stable Protein-based drugs: - Unstable siRNA/mRNA: - Unstable

Question 36

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs: Delivery

Answer 36

Small molecules: - Easy Protein-based drugs: - Difficult siRNA/mRNA: - Difficult

Question 37

What is CRISPR-Cas9? (2)

Answer 37

A genome editing technique that: 1. Targets a specific section of DNA 2. Makes a precise cut/break at the target site

Question 38

CRISPR-Cas9 can do one of three things:

Answer 38

1. Make gene nonfunctional 2. Replace one version of a gene with another 3. Base editing

Question 39

How does CRISPR-Cas9 work? (5)

Answer 39

1. CRISPR-Cas9 was adapted from a naturally occurring genome editing system in bacteria. 2. The bacteria capture snippets of DNA from invading viruses and use them to create DNA segments known as CRISPR arrays. 3. The CRISPR arrays allow the bacteria to "remember" the viruses (or closely related ones). 4. If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays to target the viruses' DNA. 5. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus

Question 40

What are the features of CRISPR-Cas9? (4)

Answer 40

1. CRISPR/Cas9: Simple to make components: guide RNAs, Cas9, and DNA templates 2. Relatively simple to introduce these into cells and early embryos. 3. Highly specific (off-target events ?) 4. Highly efficient

Question 41

What makes CRISPR-Cas9 versatile? (2)

Answer 41

1. Alterations to DNA: including "indels" and deletions, insertions or substitutions from single base-pairs up to many kilobases 2. Cas9 DNAse activity can be mutated and other proteins linked to it: including transcriptional activators or repressors to manipulate specific gene activity without altering DNA

Question 42

Sickle cell disease is caused by a mutation in the __________-____ gene found on chromosome __

Answer 42

hemoglobin-Beta; 11