L10 RNA Therapeutics Flashcards
Main differences between small molecule drugs and macromolecular drugs
Small molecule drugs have a lower mw, are more stable, are less immunogenic, display linear PK behaviours, are more easily purified, manufacture is readily scaled up, have simpler structures, and are often amenable to oral admin
How many new RNA-based drugs have been approved since 2018?
6
Approved ASOs in the EU
Spinraza, Waylivra
5 siRNA drugs approved
Patisiran, Givosiran, Inclisiran, Lumasiran, Vutrisiran
Challenges for pharmaceutical use of nucleic acids
- Safe & effective delivery to correct target (polyanions, large size, inability to enter cells on their own)
- Delivery systems capable of targeting tissues beyond the liver
- Pre-existing immunity (e.g. anti-PEG Abs, viral vectors)
- Control over dose & duration of therapeutic effect
- Limited admin (low oral bioavailability)
- Shelf-life stability & price
Chemical modifications can protect nucleic acid-based therapies against…
host immunogenic responses to RNA
What are siRNAs?
- short dsRNAs (21-25 nt), 2 nt overhangs at 3’ ends
- generated from long dsRNA fragments or chemically synthesised
siRNAs efficiently load mRNAs onto __ , which leads to mRNA cleavage
RISC complex
Advantage of siRNAs
They have near perfect complementarity with the target sequence - very specific, less immunogenic
The first non-viral siRNA medicine to reach the market
Patisiran (Onpattro)
What is Patisiran?
siRNA-LNP approved for Transthyrin (TTR) amyloidosis
Patisiran is a very effective way to…
reduce serum levels of misfolded amyloid protein
A simple solution to the siRNA delivery problem for hepatocytes
GalNAc-siRNA conjugates
N-acetyl galactosamine (GalNAc) has a high affinity for the __
asialoglycoprotein receptor (ASGPR) expressed on hepatocytes
GalNAc-siRNA conjugates allow for…
s.c. admin & less frequent dosing
What is Givosiran (Givlaari)?
- FDA approved for adults with acute hepatic porphyria
- GalNAc-siRNA conjugate that targets aminolaevulinic acid synthase 1 (ALAS1) mRNA to reduce toxic metabolites
3 common mechanisms utilised by ASOs
- mRNA target degradation via recruitment of RNase H enzyme
- Alternative splicing modification to include or exclude exons
- miRNA inhibition to inhibit miRNA binding to its target mRNA
What is Spinraza (Nusinersen)?
ASO for treatment of spinal muscular atrophy
How does Spinraza treat SMA?
- Targets SMN2 pre-mRNA
- Potent inducer of SMN2 exon 7 inclusion and SMN protein expression
How is Spinraza administered?
Intrathecally
Most developments in ASO therapeutics are directed at __ diseases
neurodegenerative
Advantages of mRNA-based therapy
- Dose control
- No risk of insertional mutagenesis
- Suitable for non-viral delivery by virtue of mRNA cytoplasmic activity
- Enables expression of any therapeutic protein
Structural modifications of mRNA
- 5’ cap modifications
- Coding region (to improve translation)
- PolyA tail (stability)
- 5’ and 3’ UTR (regulates molecule specificity)
- Modified dNTP (modulates innate immunity)
Why is Haemophilia B a good candidate for protein replacement therapy via mRNA treatment?
because it is caused by a single defective protein (Factor IX) normally produced by liver
mRNA can act as an __ to induce IFN responses
adjuvant
What is gene editing?
A type of genetic engineering in which DNA is inserted, deleted, modified or replicated at a specific location in the genome
Gene editing tools can induce __ in the genome that are subsequently repaired
double-stranded breaks
What is non-homologous end joining?
An error prone repair mechanism that results in uncontrolled but predictable indels
What is homology directed repair?
A precise repair mechanism triggered in the presence of homologous donor DNA template that results in insertion of the template into genomic DNA
Biggest breakthrough in gene editing
CRISPR (clustered regularly interspaced short palindromic repeats)
2 nuclease domains of Cas9 that cleave DNA
RuvC- and HNH-like domains
Cas9 nucleases are guided by __ to generate predominantly blunt-end DSBs
single guide RNA (sgRNA)
sgRNA recognises a very specific sequence called __
PAM
What is base editing?
- Mutated Cas9 that creates single-stranded breaks (repaired more easily)
- DNA deaminase allows for point mutations (less dangerous compared to CRISPR)
Prime editing involves Cas9 that also creates a single-stranded break, coupled with __
reverse transcriptase
Ex vivo gene editing being done for __ disease
sickle cell disease
