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#FME - Gene Therapy, RNA Therapeutics and Stem Cells > 3.) RNA Therapeutics > Flashcards

3.) RNA Therapeutics Flashcards

1
Q

Describe the strategies for gene therapy regarding RNA Therapeutics.

A
  • 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
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2
Q

Describe the different roles of RNA, aside from mRNA.

A

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

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3
Q

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

A
  • siRNAs (short-interfering)
  • miRNAs (micro)
    • 21-23 nucleotide RNA molecules
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4
Q

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

A

(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

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5
Q

What are siRNAs? How do they function?

A

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

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6
Q

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

A

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)
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7
Q

What is a RISC?

A

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
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8
Q

What are the consequences of siRNA binding?

A
  • Decreased mRNA (due to degradation via cleavage/RISC)

- Decreased protein production as a result (translation knockdown from mRNA degradation)

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9
Q

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

A
  • 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
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10
Q

What are shRNAs?

A

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

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11
Q

What are the issues surrounding developing siRNA therapeutics?

A

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
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12
Q

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

A
  • Viruses can evolve to prevent siRNA cleavage

- Delivery of combination of multiple siRNAs targeting same virus can reduce this issue

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13
Q

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

A

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)
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14
Q

What are the requirements of oligonucleotide delivery/design?

A

Needs to achieve:

  • Stability against serum nucleases (resistant to degradation)
  • Entry into target cells
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15
Q

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

A
  • Modify nucleotide (GaINAc conjugation enhances hepatocyte uptake)
  • Modify backbone (phosphorothioate modification)
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16
Q

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

A
  • 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
Q

What do most clinical trials with siRNA therapeutics focus on?

A

Organs:

  • Involved in oligonucleotide clearance after systemic administration (liver and kidney)
  • Where local delivery is possible (eye)
18
Q

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

A

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
Q

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

A
  • 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
Q

What advantages does GalNAc conjugation confer? Give examples.

A

(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
Q

What are miRNAs? How were they discovered?

A

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
Q

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

A

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
Q

How are miRNAs formed?

A
  • 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
Q

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

A

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
Q

How do microRNAs bind to and modulate mRNA activity?

A

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
Q

How do microRNAs repress protein synthesis?

A

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
Q

Why are siRNAs only formed exogenously/in the cytoplasm?

A

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
Q

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

A

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
Q

Describe how miRNAs relate to disease.

A

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
Q

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

A

Oncogenes:
- Targeting anti-proliferative mRNAs

Tumour suppressors:
- Targeting oncogenic mRNAs

31
Q

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

A
  • 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
Q

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

A

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
Q

Describe the potential strategies for miRNA-based therapeutics.

A
  • 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
Q

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

A

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
Q

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

A

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

36
Q

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

A
  • 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
Q

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

A

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
Q

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

A

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
Q

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

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

#FME - Gene Therapy, RNA Therapeutics and Stem Cells (4 decks)

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