Antisense Technology: Theory and Therapy

Question 1

What is antisense technology?

Answer 1

• Synthesize strand of nucleic acid (DNA/RNA) that will bind mRNA produced by gene that causes disease and inactivate it, effectively turing the gene “off”
• Essentially inhibit the protein before it is synthesized

Question 2

What is the advantage of antisense technology over traditional drugs?

Answer 2

• Oligonucleotides are manufactured quickly
• Sensitive, selective, effective, less toxic
• Longer responses (clonal expansion may take longer to produce clinical disease once mRNA is inhibited) therefore lower doses

Question 3

What is the definition of antisense?

Answer 3

• Single stranded oligonucleotide (nucleic acid analog), 15-20 aa in length with sequence complementary to specific target mRNA region called “sense” sequence
• When antisense oligonucleotide and mRNA bind it decreases translation

Question 4

Describe normal transcription/translation

Answer 4

• Antisense DNA transcribed to pre-mRNA
• pre-mRNA –> mRNA
• mRNA –> ribosomes for translation to protein

Question 5

What is one way to prevent transcription in a cell?

Answer 5

• Prevent transcription by DNA targeted agent, such as an antisense agent
• Binds DNA and prevents transcription to pre-mRNA

Question 6

How can you prevent the formation of mature mRNA?

Answer 6

• Block pre-mRNA
• Can use any oligonucleotide-based agent
• Needs to match pre-mRNA sequence

Question 7

How can you prevent translation based on interfering with mRNA?

Answer 7

• Block the formation of proteins by adding antisense agent to mRNA
• Stops formation of ribosomal complex, acts as a steric blocker

Question 8

How can you prevent translation using an enzyme?

Answer 8

• Use RNAse to digest mRNA
• Most important antisense mechanism
• Any oligonucleotide, substrate for nuclease, targeting RNA may destabilize it
• Normal function of RNAses (oligonucleotides) for specific RNase response must be carefully construction

Question 9

What are the two groups of RNAses?

Answer 9

1) RNase H

2) Double-strand RNAses (RNAse III)

Question 10

What is RNase H?

Answer 10

• Ribonuclease that cleaves RNA in a DNA/RNA duplex to produce ssDNA
• RNAse H is a non-specific endonuclease and catalyzes the cleavage of RNA via a hydrolytic mechanism

Question 11

Describe the steps used for RNAse H to break down mRNA

Answer 11

1) Antisense ONT binds target RNA to form heteroduplex substrate
2) RNase H binds via its binding domain at 3’ antisense ONT/ 5’ RNA pole and cleaves target RNA ~7bp from binding site
3) Target mRNA is degraded, whereas antisense ONT stays intact, allowing it to form heteroduplex substrate for induction of RNase H cleavage

Question 12

What are double-stranded RNAses (or RNAse III)?

Answer 12

• Type of ribonuclease that digests dsRNA
• Part of (dsRNS)-Dicer family of RNAse
• Cuts pre-microRNA at a specific site and transforms it into miRNA that is actively involved in regulation of transcription and lifetime of mRNA

Question 13

What are the three new tools to identify new antisense mechanisms, other than RNAse H?

Answer 13

1) ds-RNAses
2) RNAi
3) siRNA

Question 14

What is the cell’s normal mechanism to turn off a gene?

Answer 14

• Cell will synthesize miRNA

- Binds to mRNA and turns off gene

Question 15

What is the siRNA antisense mechanism?

Answer 15

1) ds-ONT enters cytoplasm
2) Helicase separates sense and antisense strands of ONT
3) RISC associates with antisense ONT
4) Antisense ONT binds target mRNA, forming sense-antisense duplex
5) Nuclease component (RNAIII) of RISC degrades target mRNA

Overall, this inhibits target mRNA expression

Question 16

What is the RISC complex?

Answer 16

• RNA interfering silencing complex
• Short dsRNA produced endogenously by DICER or introduced to cell exogenously
• Two strands of dsDNA are separated by an ATP dependent helicase
• One strand of dsDNA remains as RISC, other strand is displaced
• Mature RISC includes single strand of RNA which acts as guide sequence for specific cleavage of targeted mRNA

Question 17

What is RNAi (RNA interference)?

Answer 17

• Conserved biological response to dsRNA
• Natural mechanism for sequence-specific gene silencing
• Many types of small silencing RNAs have been discovered, including siRNAs (small interfering) and miRNAs (micro)
• Process of RNAi can be moderated by either siRNA or miRNA but there are differences between the two

Question 18

What are the differences between siRNA and miRNA?

Answer 18

• miRNA = 19-25nt; endogenous; imperfect match with target; mechanism is through translation repression
• siRNA = 19-21nt; exogenous; exact match to target; mechanism is through mRNA cleavage

Question 19

How do you make dsRNA?

Answer 19

• mRNA is initially single-stranded
• Replicate RNA from mRNA template using RNA replicase
• Two RNA strands are held together by hydrogen bonds
• Now have dsRNA

Question 20

What happens to dsRNA?

Answer 20

• dsRNA is cut by Dicer enzyme
• This leads to formation of either siRNA (21-23nt long) or miRNA
• Cut leads to two overhanging nucleotides at end of strands

Question 21

How does RNAi work?

Answer 21

1) Process dsRNA into 21-23nt fragments using Dicer (produces siRNA)
2) Antisense strand of siRNA guides cleavage - this is guided by base complementarity of the siRNA, RISC targets mRNA for degradation

Question 22

What is the mechanism of miRNA?

Answer 22

1) DNA sequence –> pri-miRNA (ssRNA)
2) Hairpin around miRNA - sequence in pri-miRNA - a signal for ds-ribonuclease (Drosha) to produce pre-miRNA
3) Exportin 5 (carrier protein) leads to nuclear export of pre-miRNA
4) Dicer enzyme cleaves pre-miRNA - releases mature miRNA, which associates with RISC
5) RISC-miRNA complex blocks translation by mRNA by binding to complementary sequence

Question 23

What are some applications for RNAi?

Answer 23

• Clarify gene function in cellular processes
• Find enhancers/suppressors of phenotypes
• Understand biology of different cell lines
• Cell and tissue engineering
• Support miRNA target identification
• Validate drug therapies
• Therapeutics

Question 24

How is antisense gene therapy different from RNA interference?

Answer 24

• Antisense technology destroys target mRNA by recruiting enzyme RNAse H
• RNAi recruits Dicer enzyme
• RNAi molecules are twice as large as antisense ONTs because they are double-stranded, not single-stranded

Question 25

What are three major problems with antisense technology?

Answer 25

1) Stability (the small, single stranded RNA molecules are susceptible to degradation; naked siRNA is degraded by serum endonucleases and efficiently removed by glomerular filtration and therefore has short T1/2)
2) Delivery (problem with cell penetration due to unfavourable physico-chemical properties
3) mRNA may stimulate innate immune responses (longer, dsRNA can rapidly induce interferons, whereas shorter dsRNA do not; immune stimulation by siRNA can be avoided by incorporating 2’-O-methyl uridine/guanosine NTs into one strand of siRNA duplex)

Question 26

What are some challenges in siRNA-based therapy?

Answer 26

1) siRNA design
2) Biological stability, chemistry
3) Cell association and entry
4) Net accumulation
5) Avoid errant compartmentalization
6) Incorporation into RISC
7) Interact with target mRNA
8) Gene silencing

Question 27

What are some siRNA delivery systems?

Answer 27

• Viral vectors

- Non-viral vectors (cationic lipids; cationic cell penetrating peptides; cationic polymers and dendrimers)

Question 28

How are cationic lipids used to deliver siRNA?

Answer 28

• Lipid-based siRNA delivery
• Liposomes = Aqueous core, phospholipid bilayer
• Amphipathic therefore allow incorporation of hydrophilic and hydrophobic drugs
• Can drug load, and actively/passively target cell

Question 29

What are cationic cell penetrating peptides? How are they used to deliver siRNA?

Answer 29

• Conjugated with functional peptides, like cell-penetrating peptide
• Does not require chemical modification of siRNA (preserves activity, decreases purification process)

Question 30

What are cationic polymers and dendrimers?

Answer 30

• Both synthetic and natural polymers
• Positively charged polymers, through electrostatic interactions, form polyplexes with negatively charged RNA phosphates
• Process results in DNA condensation and protects plasmids from nuclease digestion
• Dendrimers = Synthetic macromolecules with highly branched peripheral side chains that can carry many siRNA

Question 31

What are some characteristics of dendrimers?

Answer 31

• Positively charged surface groups - deliver genes, antisense ONTs, siRNA
• Precise core-shell nanostructure enables drug loading by interior encapsulation, surface adsorption, or chemical conjugation
• Biocompatibility of dendrimers is related to structure, size, and surface charge (cytotoxicity and immunogenecity of dendrimers are related to surface charge)

Question 32

What are the two uptake pathways and intracellular trafficking of polymeric delivery systems?

Answer 32

• Endocytosis

- Escape mechanism = proton-sponge hypothesis and flip-flop mechanism

Question 33

Describe the proton-sponge hypothesis

Answer 33

• pH buffering effect
• For cationic polymers, like PEI
• Polyplexes enter cell, trapped by endosome
• Membrane bound ATPase proton pumps actively translocate protons into endosomes. Polymers become protonated, resist acidification of endosomes therefore protons are pumped into endosomes continuously to decrease pH
• Proton pumping action is followed by passive Cl- entry, therefore increasing ionic concentration and water influx
• High osmotic pressure causes swelling and rupture of endosomes, releasing cell contents to cytosol

Question 34

Describe the flip-flop mechanism

Answer 34

• For cationic-lipid based delivery systems
• Lipoplexes (lipids + siRNA) are endocytosed and become entrapped in early endosomes
• Electrostatic interaction between cationic lipoplexes and anionic lipids on cytoplasmic side of endosomal membrane
• Anionic lipids of endosomal membrane laterally diffuse into lipoplexes and form charge-neutralized ion pairs with cationic lipids of lipoplexes
• Nucleic acids are displaced from lipoplexes, allowing nucleic acids entry into cytoplasm

Question 35

What are three FDA approved antisense products? What do they treat?

Answer 35

• RONDEL (cancer)
• MIPOMERSEN/KYNAMRO (homozygous familial hypercholesterolemia)
• FORMIVERSEN/VITRAVINE (CMV rhinitis in AIDS patients)