Gene Therapy pt 2

The overlap between miRNA and siRNAsynthesis

Some shared mechanisms and processes - both involving Dicer and RISK miRNA: encoded in the nucleus Mature miRNA: can have 4 Ago proteins - any of them have microRNA function SiRNA: can only have Ago2 protein - it's the only one that has endonuclease activity in mammals

Telomerase RNA

Template function in telomerase

Messenger RNA (mRNA)

Carries genetic information

Ribosomal RNA (rRNA)

Central to ribosomal function

Transfer RNA (tRNA)

Decodes mRNA in the ribosome

Small nuclear RNAs (snRNAs)

Important in splicing

Viral RNA

Many viruses have RNA genomes

Potential for miRNA-based therapeutics

Modulate levels of miRNAs associated with particular diseases. miRNAs are encoded in genome, so could also use gene therapy/genome modification approaches to increase or decrease expression

Dicer

cleaves double-stranded RNA and pre-microRNA into short single-stranded RNA fragments = siRNA and miRNA

Some other miRNA drugs in phase II eg RG-012 (Regulus):

miR-21 inhibitor to reduce kidney fibrosis development in Alport syndrome.

Many different miRNAs in many different organisms

•2021: miRNAs present in broad range of eukaryotic species: vertebrates, invertebrates, plants •Many show conservation between organisms •2654 miRNAs currently identified in Homo sapiens •Nomenclature miR-1, etc

siRNA and miRNA function: summary

•Both 21-23nt RNAs, processed from larger dsRNA precursors by Dicer •siRNAs derived from external dsRNA (viral infection, dsRNA transfection) •miRNAs encoded in the genome •Both cleave an exactly complementary target RNA •Most animal miRNAs have imperfect complementarity to target sites in 3'UTR of mRNAs, and repress gene expression at the level of translation and/or RNA stability

•Efficiency -siRNAs do not completely knock out a damaging gene (unlike genome modification), but only knock it down -Viruses can evolve to prevent siRNA cleavage (single nucleotide change sufficient) •Combinations of multiple siRNAs targeting same virus can reduce this problem

•Delivery -Some organs are easier to target than others, e.g. liver is easier than brain -For cancer treatment, we want to target the siRNA just to the cancer cells, not to the normal cells surrounding the tumour -Maintenance in cells also difficult

Potential for siRNA-based therapeutics

•Design and deliver siRNAs to target specific mRNAs or specific sites on an mRNA (to reduce production of a protein encoded by that mRNA) •Reduce mRNA levels of an overexpressed gene (e.g. an oncogene) •Specifically reduce mRNA level of a mutant allele •Specifically reduce incorrectly spliced mRNA level •Target viral RNA

How are miRNAs made?

•Encoded in the genome (unlike SiRNAs) •Transcribed as part of longer RNAs (primary miRNAs: pri-miRNAs) •Undergo nuclear processing to a pre-miRNA hairpin by Drosha, and nuclear export by Exportin 5 •In the cytoplasm the dsRNA hairpin is processed by Dicerto a 21-23nt dsRNA duplex •One strand is retained as the mature miRNA

MicroRNAs (miRNAs)

•First example identified in C.elegans (Victor Ambros, 1993) •2001: many miRNAs found in other organisms including human •Endogenous 21-23 nucleotides RNA moleculesthat bind to mRNAs and reduce production of encoded protein

miRNA-targeting drugs

•Hepatitis C virus (HCV) replication requires miR-122 -Liver-specific miRNA. Binds directly to 5'UTR of HCV RNA and positively regulates viral replication

•RNAi machinery conserved across most eukaryotic species

•Higher organisms have more elaborate antiviral mechanisms

•Second siRNA drug = givosiran (Givlaari): Alnylam GalNAc-conjugated siRNA therapy targeting ALAS1 mRNA for acute hepatic porphyria

•NB Inotersen (Tegsedi): Ionis therapy targeting mutant TTR mRNA by gapmer ASO that recruits RNAse H: approved by FDA October 2018

siRNA delivery

•Reminder - mammalian antiviral mechanisms activated by dsRNA longer than 30bp

miRNA inhibitors as drugs

•Single stranded oligonucleotides, complementary to miRNA •Steric block - same principle as ASO regulation of mRNAs

Difficulties with developing siRNAtherapeutics

•Specificity -Off-target binding a problem

Short interfering RNAs(siRNAs)

•dsRNA processed by Dicer -Generates 21-23nt dsRNA duplexes with 2nt 3' overhangs -siRNA

miRNAs and disease

•miRNAs have crucial role in regulation of broad range of cellular processes

All small non-coding RNAs

•piRNAs (piwi-interacting RNA) •lncRNAs (long noncoding RNAs) •eRNAs (enhancer RNAs)

•Overexpress a beneficial miRNA (e.g. tumour suppressor)

-miRNAs are chemically identical to siRNAs, so exactly the sameapproaches and problems as siRNA delivery

•siRNAs are too short to do this, so can be chemically synthesized to target a specific mRNA

-Widely used in research -Therapeutic potential

A microRNA targets ~ 200 mRNAs (siRNA only has one target)

An mRNA can have target sites for several different microRNAs. ~60% of all human mRNAs have microRNA target sites. Difficult to predict targets due to imperfect complementarity. Need to test experimentally

Oligonucleotide delivery (•Encapsulation in lipid nanoparticles)

Applies to siRNA, ASO, miRNA/miRNA inhibitors

Small nucleolar RNAs (snoRNAs)

RNA processing

•Needs to achieve:

-Stability against serum nucleases -Entry into target cells

•Can also encode as short hairpin RNAs (shRNA) in vectors (single-stranded). These are processed by Dicer.

-Allows viral delivery (e.g. AAV) -Potential for targeting specific organs

•Synthetic siRNAs are too short to activate these immune mechanisms

-Can be delivered as naked modified oligonucleotides or in lipid nanoparticles

•They are important in many diseases

-Cancer (Some miRNAs are oncogenes or tumour suppressor genes) -Cardiovascular disease -Diabetes -Neurodegeneration -Viral infection

•Chemical modification

-E.g. Phosphorothioate modification of backbone enhances stability -E.g. Ligand conjugation to enhance uptake into specific cell types -GalNAc (N-acetylgalactosamine). Binds to asialoglycoproteinreceptor on hepatocytes, allowing specific targeting of oligonucleotide drugs to liver

•Same barriers and approaches to delivery as siRNAs

-Encapsulate in lipid nanoparticles -Or conjugate, e.g. to GalNAc -Some organs easier to target

•Patisiran (Onpattro): the first RNAi drug. Alnylam siRNA therapy targeting mutant TTR mRNA approved by FDA August 2018.

-Encapsulated in lipid nanoparticles, requiring lengthy infusionevery 3 weeks -Now developing GalNAc-conjugated siRNAs, allowing efficient delivery to liver and subcutaneous dosing every 3-6 months

•Chemical modification of bases and backbone

-Improves affinity to miRNA -Reduces off-target effects -Improves stability against serum nucleases -E.g. 2'-O-methyl, locked nucleic acid (LNA)

•Most clinical trials target organs:

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

•dsRNA longer than 30 base pairs induces antiviral responses in human cells (not good)

-Long dsRNA delivery not appropriate in medicine

Two different miR-122 inhibitors showed good results in phase II clinical trials

-Miravirsen (Santaris) is a locked nucleic acid (LNA) antagomir. -RG-101 (Regulus) uses cET chemistry for high affinity binding, but also includes GalNAc to direct molecule to hepatocytes -Both discontinued - some side effects and changes to HCV therapeutic landscape

•Hereditary transthyretin amyloidosis (h-ATTR) with polyneuropathy

-Mutant TTR protein is made in the liver from a faulty copy of the gene inherited from a parent (autosomal dominant) -This accumulates in other organs and is eventually fatal.

•This binds to fully complementary RNA sequences and cleaves the RNA

-RNA degraded by cellular machinery following cleavage

•Individual miRNAs show specific expression patterns

-Tissue -Differentiation stage -Important role in normal development

•Inhibit a damaging miRNA (e.g. oncogene)

-chemically modified complementary oligonucleotides('antagomirs')

microRNA activity

1. Complete base pairing to site anywhere in an mRNA -RNA cleavage (similar to siRNAs) followed by RNA degradation Predominant mechanism in plants Very rare in animals 2. Partial base pairing to 3' UTR sites -Translational repression RNA degradation in P bodies Predominant mechanism in animals

How do microRNAs repress protein synthesis?

1. Inhibition of translation initiation 2. removes adenylation - removing the poly A tail, causing the RNA to be less stable de-capped and degraded à causing less protein to be made from the mRNA

How do siRNAs function?

1. siRNAs complementary to target mRNA delivered into cell 2. RISC binds siRNA and selects one strand 3. Selected siRNA strand binds exactly complementary sequence in target mRNA 4. Argonaute 2 (Ago2) endonuclease in RISC cuts target mRNA

RNA interference

1998 Andrew Fire and Craig Mello (Nobel Prize 2006) found that double stranded RNA effectively reduced expression of a gene with matching sequence •Termed RNA interference (RNAi). Mediated by short interfering RNAs (siRNAs) -An antiviral mechanism in lower organisms

small non-coding RNAs: •siRNAs (short interfering RNAs) •miRNAs (microRNAs)

21-23nt RNA molecules Regulate gene expression by binding to complementary targets

•One strand of duplex incorporated into RNAi-induced silencing complex (RISC)

RISC - can silence RNA, cleaving and resulting in degradation

Ribozymes

RNA enzymes