This document discusses small interfering RNA (siRNA) and antisense RNA (asRNA). It explains that siRNA is a class of double-stranded RNA molecules around 21 base pairs long that interfere with gene expression by degrading mRNA. The first FDA-approved siRNA drug was Onpattro in 2018. AsRNA is a single-stranded RNA complementary to mRNA that blocks its translation into protein. AsRNAs can regulate gene expression at the transcriptional, post-transcriptional, and epigenetic levels. They represent potential drug targets due to their small expression levels and ability to regulate genes. However, challenges remain regarding the degradation, off-target effects, and intracellular uptake of artificial asRNAs.

3. Sir David Baulcombe
Prof.ThomasTuschl

4. • Small interfering RNA (siRNA), sometimes known as short interfering
RNA or silencing RNA,
• It is a class of double-stranded RNA at first non-coding RNA molecules,
typically 20-24 (normally 21) base pairs in length,
• similar to miRNA, and operating within the RNA interference (RNAi)
pathway.
• It interferes with the expression of specific genes with complementary
nucleotide sequences by degrading mRNA after transcription,
preventing translation.

5. • Double-stranded RNA (dsRNA) with phosphorylated 5' ends
and hydroxylated 3' ends with two overhanging nucleotides.
• siRNAs can also be introduced into cells by transfection.

6. • siRNAs prevent the
production of specific proteins
based on the nucleotide
sequences of their
corresponding mRNA.
• The process is called
RNA interference (RNAi)
or silencing

7. • In 2018 Alnylam Pharmaceuticals became the first company to have a siRNA therapy approved
by the FDA. Onpattro (patisiran) was approved for the treatment of polyneuropathy of hereditary
transthyretin-mediated (hATTR) amyloidosis in adults. hATTR is a rare, progressively debilitating
condition. It affects 50,000 people worldwide. To deliver the drug directly to the liver, siRNA is
encased in a lipid nanoparticle.
• In 2019, FDA approved the second RNAi therapy, Givlaari (givosiran) used to treat acute hepatic porphyria
(AHP).

8. • Also known as antisense transcript.
• It’s a single stranded RNA that is complementary to a protein
coding messenger RNA (mRNA) with which it hybridizes, and
thereby blocks its translation into protein.

9. • Antisense RNA molecule represents a unique type of DNA
transcript that comprises 19–23 nucleotides and is
complementary to mRNA.

10. • In terms of regulatory mechanisms, some author group
asRNAs into RNA-DNA interactions, RNA-RNA interactions
either in nucleus or cytoplasm and RNA-protein interactions
(epigenetic)
• Cis-acting-Cis-acting asRNAs are transcribed from the
opposite strand of the target gene at the target gene locus.
They often show high degree or complete complementarity
with the target gene. If the cis-acting asRNA regulates gene
expression by targeting mRNA, it can only target individual
mRNA.

11. • Trans-acting-Trans-acting asRNAs are transcribed from loci
that are distal from the targeting genes. In contrast to cis-
acting asRNAs, they display low degree of complementarity
with the target gene but can be longer than cis-acting
asRNAs. They can also target multiple loci. Because of these
properties of trans-acting asRNAs, they form less stable
complexes with their targeting transcripts.

12. • The primary function of asRNA is regulating gene expression.
• Epigenetic regulation
• DNA methylation-DNA methylation can result in long term
downregulation of specific genes. Repression of functional
proteins via asRNA induced DNA methylation has been found
in several human disease, e.g. alpha-thalassemia.

13. • Co-transcriptional regulation
• Post-transcriptional regulation

14. • As a regulatory element, asRNAs bear advantages to be
considered as a drug target.
• First of all, asRNAs regulate gene expression at multiple
levels including transcription, post-transcription and epigenetic
modification.
• Secondly the expression level of asRNAs is very small
therefore, only small amount of asRNAs is required to
produce an effect. In terms of drug targets, this represents a
huge advantage because only a low dosage is required for
effectiveness.

15. • First of all, asRNAs and antagoNATs can be easily degraded by
RNase or other degrading enzymes. To prevent degradation of the
therapeutic oligonucleotides, chemical modification is usually
required. Adverse effects including fever, chills or nausea have
been observed after local injection of phosphrothioate modified
oligonucleotides.
• Secondly, off target toxicity also represents a big problem. Despite
the locus-specific nature of the endogenous asRNAs, only 10–50%
synthesized oligonucleotides showed expected targeting effect.
• Lastly, artificial asRNAs have been shown to have limited
intracellular uptake. Although neurons and glia have been shown
to have the ability to freely uptake naked antisense oligonucleotide.