Gene Silencing

Question 1

What is RNAi technology?

Answer 1

RNA interference (RNAi) is an accurate and potent silencing method

RNA is silenced via degradation or stopping translation by specific double strand RNA sequences

Question 2

What are some types of RNAi technologies?

Answer 2

• Small interfering RNAs (siRNAs)
• MicroRNAs (miRNAs)
• shRNA
• CRISPR
• Others

Question 3

What is the key enzyme involved in RNAi technology?

Answer 3

DICER breaks down long sequences of dsRNA into smaller fragments (siRNAs)

Question 4

Where is the dsRNA sourced for RNAi?

Answer 4

Hairpin, complementary RNAs, RNA dependent RNA polymerases

This dsRNA is processed by DICER enzyme into smaller dsRNA fragments known as siRNA (21-23 nucleotides long with 3’ two-nucleotide overhangs)

Question 5

What is the role of siRNA in RNAi?

Answer 5

siRNA interacts with dicer and activates the RISC (RNA-indicing silencing complex)

see slide 10

Question 6

What is the role of RISC in RNAi?

Answer 6

The AGO2 component of RISC cleaves the passenger strand (sense strand) while the guide strand remains associated with the RISC

The guide strand guides the active RISC to its target mRNA for cleavage by AGO2 component of RISC

see slide 10

Question 7

What is miRNA?

Answer 7

miRNA is a class of small RNA molecules that negatively regulate gene expression

Question 8

How can miRNA contribute to gene silencing?

Answer 8

miRNA gene is transcribed to create primary miRNA

Primary miRNA is cleaved by Drosha (protein complex) to form precursor miRNA

Precursor miRNA is further processed by dicer

This sequence of processed pre-miRNA then interacts with RISC and looses passenger strand while guide strand is retained

The RISC-guide strand complex (miRISC) will bind to complementary (usually only partially complementary) mRNA strand and prevent translation of that sequence

Question 9

Do miRISCs have a specific target?

Answer 9

No, unlike siRNA-mRNA recognition, miRNA-mRNA recognition does not require perfect pairing, one miRNA can recognize an array of mRNAs

Question 10

Review slide 19

Question 11

Are free RNA molecules relatively resistant to decompostion?

Answer 11

No, abundant serum nucleases contribute to short half-life in vivo

This instability is resolved through chemical modification of RNA

Question 12

What are the issues for RNA delivery into the cell (site of action)?

Answer 12

Their intrinsic properties, including hydrophillic nature, negative charge, and high molecular weight , render them poorly permeable across biological membranes

Question 13

What is the role of a delivery system in siRNA or miRNA therapeutic agents?

Answer 13

Facilitating the cellular uptake of siRNA or miRNA to their target sites

Protect the nucleic acids from premature nuclease degradation

Question 14

What are some characteristics of viral vectors as RNA delivery systems?

Answer 14

• Lentiviruses, adenoviruses, and adeno-associated viruses can transfer RNA into the nucleus effectively (high transduction efficiency)
• Viral vectors have their virulence removed
• Long-term expression can be achieved by using viruses that integrate into the host genomes (ensure important genes are not unintentionally silenced)

Question 15

What are some limitations associated with viral RNA delivery systems?

Answer 15

• Serious safety concerns (high immunogenicity)
• Risk of insertional mutagenesis
• High production costs
• Non-viral vectors are available (better safety profile and lower cost)

Question 16

What are the main types of non-viral vectors used in RNA delivery systems?

Answer 16

• Polymer-based
• Lipid-based

Question 17

What are the advantages of non-viral RNA delivery systems?

Answer 17

Good safety profile and low production cost

• Easily modified to improve their delivery efficiency to acheive site-specific delivery by incorporating targeting ligands
• Circulation time is enhanced by PEGylation

Question 18

Review slide 26

Question 19

What is CRISPR-Cas 9?

Answer 19

Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9

It is a genome editing technique:
- Targets a specific section of DNA (RNA fragment binds to complementary DNA sequence)
- Makes a precise cut/break at the target site (and does one of the following: makes a gene nonfunctional, replace allele, or base editing)

see slide 31

Question 20

What is the role of CRISPR arrays?

Answer 20

Allow the bacteria to remember the viruses and build a library of RNA sequences to target the viruses DNA

Helps build viral immunity

Question 21

What is the role of Cas9 in normal patient conditions?

Answer 21

The enzyme or similar enzymes cut viral DNA apart, which disables the virus

Question 22

Which mutated gene causes sickle cell disease?

Answer 22

A point mutation on the hemoglobin-beta gene found on chromosome 11

Question 23

What is the impact of the point mutation on the hemoglobin-beta protein structure or function that causes complications associated with sickle cell?

Answer 23

The mutation causes the formation of a protrusion from beta-2 subunit, which binds to a hydrophobic pocket on beta-1

beta-1 and beta-2 subunits polymerize and form long filaments (cause sickling of cells)

see slide 34

Question 24

How is CRISPR-Cas9 therapy used to treat sickle cell disease?

Answer 24

Use CRISPR-Cas9 to eliminate BCL11A genes (represses formation of fetal hemoglobin in adult patients)

Now the patient has fetal hemoglobin in addition to the mutated adult hemoglobin, but they will see improvement in condition

see slide 35 and 36