3.) RNA Therapeutics Flashcards Preview

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Flashcards in 3.) RNA Therapeutics Deck (39):
1

Describe the strategies for gene therapy regarding RNA Therapeutics.

- RNA interference: using siRNA (small/shorts/silencing RNA) to knock down expression of specific gene (specific mRNA/viral RNA)
- microRNA therapeutics: using oligonucleotides (synthetic RNA/DNA) to increase or decrease levels of disease-associated miRNAs

2

Describe the different roles of RNA, aside from mRNA.

mRNA:
- Carries genetic information

tRNA (transfer):
- Decodes mRNA in the ribosome

rRNA (ribosomal):
- Central to ribosomal funciton

Viral RNA:
- Many viruses have RNA genomes

snRNA (small nuclear):
- Important in splicing (introns out)

snoRNA (small nucleolar):
- RNA processing

Telomerase RNA:
- Template function in telomerase

3

Which RNAs are involved in regulating gene expression via binding to complementary targets? Size?

- siRNAs (short-interfering)
- miRNAs (micro)
• 21-23 nucleotide RNA molecules

4

What is RNA interference (RNAi), and how did its discovery come about?

(1998) Andrew Fire and Craig Mello:
- Attempt to reduce expression of C. elegans genes by introduction of antisense RNA
- Unsuccessful
- But dsRNA (double-stranded) contaminant effectively reduced expression of a gene with matching sequence
>>> RNAi

5

What are siRNAs? How do they function?

Short interfering RNAs:
- dsRNA processed (cut) by Dicer enzyme
- Generates 21-23nt dsRNA duplexes with 2nt 3' overhangs
- One strand of dsRNA duplex is incorporated into RNAi-induced silencing complex (RISC); one strand is selected by RISC
- Selected siRNA strand binds exactly complementary sequence in target mRNA
- Target mRNA is cleaved/cut by RISC (guided there by siRNA strand)
- Target mRNA is then degraded by cellular machinery following cleavage
>>> mRNA is degraded after transcription (if RISC complementary), preventing translation to protein = downregulation(?) of protein

6

What are the potential therapeutics for siRNA technology? Give examples.

Fully complementary binding allows targeting of just mutant alleles and not wild-type:
- Design and deliver siRNAs to target specific mRNAs
- Reduce mRNA levels [but not 100% knockdown] of an overexpressed gene (e.g. oncogene, C-myc)
- Specifically reduce mRNA level of a mutant allele (e.g. Huntington's - targets specifically mutant, not wild-type allele)
- Specifically reduce incorrectly spliced mRNA level
- Target viral RNA (and degrade via RISC)

7

What is a RISC?

RNA interference (RNAi)-induced silencing complex
- It is precise, efficient, stable and better than antisense technology for gene suppression.
- Important role in defending cells against parasitic nucleotide sequences – viruses and transposons.
- RISC complex binds siRNA to allow complementary binding to target mRNA and subsequent cleavage and degradation of target mRNA

8

What are the consequences of siRNA binding?

- Decreased mRNA (due to degradation via cleavage/RISC)
- Decreased protein production as a result (translation knockdown from mRNA degradation)

9

How is siRNA technology delivered into cells? What advantage do they have?

• Mammalian antiviral mechanisms are activated by dsDNA longer than 30bp
• Synthetic siRNAs are too short (21bp) to activate these mechanisms
• Thus can be delivered as naked modified oligonucleotides or in nanoparticles

10

What are shRNAs?

Short hairpin synthetic RNAs:
• siRNAs/miRNAs can be expressed as shRNA in viral vector
• Then processed by Dicer enzyme which removes the loop/hairpin
• Acts via RNAi pathway (RISC)
• Allows for viral delivery (e.g. AAV/lentiviral) and potential for targeting specific organs

11

What are the issues surrounding developing siRNA therapeutics?

Specificity:
- Off-target binding is still a problem

Efficiency:
- siRNAs do not completely knock down a damaging gene (unlike genome modification e.g. CRISPR/TALENs), only knockdown
- Viruses can evolve to prevent siRNA/RISC cleavage (single nucleotide change is sufficient - selective pressure)

Delivery:
- Some organs are more accessible e.g. liver easier than brain (BBB, particular issue w/naked siRNAs)
- For cancer treatment, want to target siRNA just to cancer cells and not to normal cells surrounding the tumour
- Maintenance in cells difficult: as opposed to in gene modification editing, siRNA is lost through cell division and get degraded

12

How can potential issues with efficiency of siRNA therapeutics be overcome?

- Viruses can evolve to prevent siRNA cleavage
- Delivery of combination of multiple siRNAs targeting same virus can reduce this issue

13

How can issues regarding the maintenance of siRNA therapeutics be overcome?

Via choice of vectors:
- Using lentiviruses - they integrate into sections of transcriptionally active chromatin and are thus passed on to progeny cells (though > insertional mutagenesis; can overcome by using an integrase-deficient lentivirus.)
- In adeno-associated viruses (AAVs) and adenoviruses, the siRNA/genomes remain episomal (avoiding insertional mutagenesis) though siRNA lost through cell division (repeated dose required)

14

What are the requirements of oligonucleotide delivery/design?

Needs to achieve:
- Stability against serum nucleases (resistant to degradation)
- Entry into target cells

15

What chemical modifications do oligonucleotide therapeutics undergo to assist in delivery and their effects?

- Modify nucleotide (GaINAc conjugation enhances hepatocyte uptake)
- Modify backbone (phosphorothioate modification)

16

What are the effects of introducing a phosphorothioate modification of oligonucleotide backbone?

- Substituting an S instead of O (phosphodiester) in backbone protects against degradation = phosphorothioate modification
- This reduces activity of variety of extra and intracellular nucleases

https://www.sigmaaldrich.com/technical-documents/articles/biology/phosphorothioates.html

17

What do most clinical trials with siRNA therapeutics focus on?

Organs:
- Involved in oligonucleotide clearance after systemic administration (liver and kidney)
- Where local delivery is possible (eye)

18

Describe a disease treatable by siRNA therapeutics, the gene targeted and its role in disease.

Hereditary transthyretin amyloidosis (h-ATTR) with polyneuropathy:
- Mutant TTR protein formed in liver from faulty copy of the gene inherited from a parent (autosomal dominant)
- Mutant TTR accumulates in other organs, eventually fatal
- Hereditary ATTR (hATTR) amyloidosis is an inherited, progressive disease caused by a genetic mutation that results in the misfolding of transthyretin (TTR) proteins.
>>> This results in the formation of amyloid fibrils that could deposit in the nerves, heart, and/or gastrointestinal (GI) tract.

(November 2017)
Patisiran (first RNAi drug):
• Alnylam Pharma's siRNA therapy targeting mutant (only, not wild-type) mutant TTR mRNA successful PIII trials
• Encapsulated in lipid nanoparticles

• Inotersen (Ionis Pharma) also siRNA therapy targeting mutatnt TTR mRNA effective in PIII trials, though problematic side effects

19

Why does Patisiran (RNAi drug for h-ATTR) require a lengthy infusion every 3 weeks?

- As siRNA is lost in cell division; does not integrate into host's genome, just acts on mRNA post-transcription (pre-translation) for cell's lifespan
- siRNA degraded

20

What advantages does GalNAc conjugation confer? Give examples.

(triantennary N-acetyl galactosamine) GalNAc-conjugation yields efficient delivery to liver:

- Many target mRNA are expressed primarily in the hepatocytes in the liver.
- Improves potency in hepatocytes
- Due to interaction with asialoglycoprotein receptor, leading to uptake via endocytosis
- Entry difficult otherwise if not utilising membrane transport proteins due to polarity of siRNAs/antisense oligonucleotides
- E.g. used in Patisiran (Alylam) and Inotersem (Ionis); changing from IV infusion every 3 weeks to S/C dosing every 3-6 months

21

What are miRNAs? How were they discovered?

MicroRNAs:
- Small (22 nt), non-coding RNA molecule
- Central to modulating gene expression post-transcription in eukaryotes, important in disease
- Base-pairing with complementary sequences of mRNA

Discovery:
- FIrst identified in C. elegans (Victor Ambros, 1993)
- Lin-4 gene responsible for repressing lin-14 gene (and thus protein); decrease in LIN-14 protein required for larvae development
- Isolating lin-4 gene found that it coded for a single-stranded, 22nt non-coding RNA (a microRNA)
- Lin-4 microRNA bound partially complentarily to lin-14 3' UTR of lin-14 mRNA, repressing lin-14 expression

22

Do miRNAs only appear in nematodes e.g. lin-4?

Many different miRNAs exist in many organisms:
- 2588 mirNAs identified in Homo sapiens (2018)
- Present in broad range of eukaryotic species: vertebrates, invertebrates, plants
- Nomenclature = miR-1, etc.
- Many miRNAs show conservation between organisms e.g. let-7 (repressed lin-41 to promote later developmental transition in C. elegans)

23

How are miRNAs formed?

- Encoded in the genome (made in the cell)
- Transcribed as part of longer RNAs = pri-mRNAs
- pri-mRNAs undergo nuclear processing to (leave) a pre-miRNA hairpin by Drosha (nuclease enzyme)
- Nuclear export of pre-miRNA out by Exportin 5 (Exp 5)
- In the cytoplasm, the dsRNA hairpin loop is removed and processed by Dicer enzyme with the pre-miRNA being trimmed in length to a 21-23nt ds-miRNA duplex
- One strand is retained as mature miRNA, was the other (passenger) strand is discarded

24

What is the overlap and the differences between miRNA and siRNA - synthesis/ • general?

Overlap:
- Dicer enzyme (w/TRBP cofactor) cleaves pre-miRNA and dsRNA respectively in cytoplasm
- Both miRNA and siRNA complex with Ago protein (argonaute) to form miRISC and siRISC
• Both cleave an exactly complementary target

Differences:
- Pre-miRNA derive from dsRNA transcripts with hairpin loops, siRNA from longer regions of (non-hairpin) dsRNA
- miRNA biogenesis starts off within the nucleus (endogenous), siRNA biogenesis takes place in cytoplasm only (exogenous)
• ONLY Ago2 of siRISC complex has endonuclease (thus RNAi gene-silencing) activity, Ago1 can only load miRNA

25

How do microRNAs bind to and modulate mRNA activity?

Complete base pairing to site anywhere in mRNA:
- RNA cleavage (similar to siRNAs), followed by RNA degradation
- Predominantly in plants, v. rare in animals

Partial (imperfect) base pairing to 3' UTR sites (control elements):
- Translation repression
- RNA degradation (affecting stability) in P bodies (processing bodies that degrade mRNA)
- Predominantly in animals

26

How do microRNAs repress protein synthesis?

Translation repression:
- Inhibition of translation initiation
- Thought to interfere with ribosome recruitment

mRNA decay/degradation:
- mRNA deadenylation, followed by mRNA decapping
- Degradation in P bodies

27

Why are siRNAs only formed exogenously/in the cytoplasm?

siRNAs:
- Derived from external dsRNA e.g. viral infection, dsRNA transfection (lower organisms such as C. elegans)
- Hence exogenous; synthesis in cytoplasm only

miRNAs:
- Encoded in the genome
- Hence endogenous; synthesis starts in nucleus, exported by Exportin-5 for Dicer processing later

28

How do siRNAs and miRNAs differ WRT the number of targets they have?

siRNAs:
- One target
- Fully complementary binding as part of RISC to mRNA

miRNAs:
- Many targets e.g. 200 mRNAs
- mRNA can have target sites for several different miRNAs
- 60% of mRNAs have miRNA target sites
- Due to imperfect complementarity (mostly partial binding to 3' UTR)

29

Describe how miRNAs relate to disease.

Crucial role in regulation of range of cellular processes:
• Individual miRNAs show tissue specific expression patterns e.g. tissue, differentiation stage, role in normal development (think C. elegans lin-14 repression)

Important in many diseases:
- Cancer (miRNAs can be oncogenes/tumour suppressors - latter more likely, dysregulation could lead to uncontrolled proliferation)
- CVD
- DM
- Neurodegeneration
- Viral infection

30

Describe how miRNAs can function as oncogenes and tumour suppressor genes respectively.

Oncogenes:
- Targeting anti-proliferative mRNAs

Tumour suppressors:
- Targeting oncogenic mRNAs

31

How are miRNAs implicated in chronic lymphocytic leukaemia (CLL)?

- Loss of heterozygosity at 13q14 associated with 70% of cases
- Loss of coding genes not responsible
- Missing tumour suppressor genes implicated: miR-15a and miR-16-1
- Usually have role in targeting Bcl-2 oncogene (anti-apoptosis); downregulating Bcl-2 in normal cells

32

How has host miRNA been shown to act detrimentally WRT viral RNA invasion?

Hepatitis C virus (HCV):
- RNA virus establishing persistent infection in liver
- miR-122 (liver specific) interacts directly with 5' UTR (rare) of HCV which is a process required for viral replication
- Novel (detrimental) mechanism for miRNA

33

Describe the potential strategies for miRNA-based therapeutics.

- Modulate levels of miRNAs associated with particular diseases
- Inhibit a damaging miRNA (e.g. oncogene) using chemically modified complementary oligonucleotides (antagomirs - anti-sense oligoNT sequestering/binding miRNA to prevent expression/target binding)
- Overexpress a beneficial miRNA (e.g. tumour suppressor)

34

What are the approaches and issues for miRNA delivery (if wanting to express beneficial miRNA)?

miRNAs chemically identical to siRNAs:
(Same approaches/issues; from siRNA cards:)

Specificity:
- Off-target binding is still a problem

Efficiency:
- siRNAs do not completely knock down a damaging gene (unlike genome modification e.g. CRISPR/TALENs), only knockdown
- Viruses can evolve to prevent siRNA/RISC cleavage (single nucleotide change is sufficient - selective pressure)

Delivery:
- Some organs are more accessible e.g. liver easier than brain (BBB, particular issue w/naked siRNAs - liver easy access, rest difficult)
- For cancer treatment, want to target siRNA just to cancer cells and not to normal cells surrounding the tumour
- Maintenance in cells difficult: as opposed to in gene modification editing, siRNA is lost through cell division and get degraded
- Deliver as naked modified oligonucleotides (e.g. LNA modification) or encapsulate in nanoparticles

35

Explain what place miRNA inhibitors have as drugs, and what form they would take?

Single stranded oligonucleotides would be delivered, complementary to miRNA:
- Same principles as antisense regulation of mRNAs (preventing binding by steric hindrance)

36

Why should miRNA inhibitors be chemically modified (not being just the naked oligonucleotide)?

- Improve affinity to miRNA
- Reduce off-target effects
- Improve stability against serum nucleases (protect from nuclease degradation)
- E.g. LNA modification (2'-O-methyl, locked nucleic acid)

37

Name a disease caused by miRNA and describe how it can be treated.

HCV:
- miR-122 inhibitors (2x in trials)
- Host miR-122 normally required for viral replication, binds to 5'UTR of viral RNA
• Miravisen (Santaris) is a locked nucleic acid (LNA) antagomir
• RG-101 (Regulus) uses cET chemistry for high affinity binding, but also GalNAc conjugation to direct molecule to hepatocytes [jaundice reported in trials though]

38

What are the limitations of miRNA-targeting drugs in the view of Pharma which look promising otherwise?

E.g. HCV treatment:
- miRNA inhibitors (miR-122) not favoured due to new direct acting antiviral drugs
- New drugs interact w/viral proteins directly
- Lost incentive

39

What technological approaches do introducing siRNAs, overexpressing and inhibiting miRNAs share?

- All involve delivery of oligonucleotides (synthetic DNA/RNA)
- Sequence optimisation necessary, off-target effects potential issue