Lecture 18 - RNA Therapeutics Flashcards
(16 cards)
What are the different types of RNA therapeutics?
A) Antisense RNA (single-stranded RNA)
B) Small interfering RNA (double-stranded RNA)
C) RNA adapter
D) mRNA
What are the key challenges in RNA therapeutics?
- RNA are highly unstable and susceptible to degradation.
- Foreign RNA molecules trigger the activation of the immune response (are immunogenic).
- RNAs are hard to target to specific cell types.
What are some solutions to the challenges faced in RNA therapeutics?
- Adding 5’ cap and Poly-A tail after in-vitro transcription of RNA to enhance stability.
- Extensive purification steps to reduce immunogenicity.
- Chemical modifications of C and U nucleosides (e.g., 1-methylpseudouridine, 5-methylcytidine) to reduce immunogenicity.
- Delivery of RNA using nanoparticles (e.g., lipid nanoparticles).
What experiment led to the 2023 Nobel Prize for Katalin Kariko and Drew Weissman?
They transfected human embryonic kidney cells with various RNAs and measured the expression of TNF-alpha to assess immune response activation.
What controls were used in the experiment for TNF-alpha expression?
- Testing RNA preparation WITH and WITHOUT an RNase enzyme.
- Ensuring that the transfection reagent is not causing the immune response.
What were the observations regarding immune response induction?
The strongest induction of the immune response was observed in in-vitro transcribed RNA, while tRNA did not induce a strong immune response.
What modifications are effective in reducing the immunogenicity of tRNA?
Pseudo-Uridines are the most effective modifications to reduce the immunogenicity of RNAs.
How do lipid nanoparticles function in RNA delivery?
They protect RNAs from degradation, can be delivered intravenously or intramuscularly, and can prevent immune responses to RNA molecules.
How do conventional mRNA vaccines work?
The mRNA molecule is wrapped into a lipid nanoparticle, delivered into the cell, and the cellular machinery produces the protein (antigen) that triggers an immune response.
What are self-amplifying mRNA vaccines?
These vaccines encode not only the protein of interest but also elements of a replicase, allowing for smaller amounts of RNA to be delivered and producing a more persistent immune response.
What is Nusinersen and its role in spinal muscular atrophy?
Nusinersen is an antisense oligonucleotide drug designed to bind to the C to T mutation in the SMN2 gene, preventing the binding of the splicing inhibitor hnRNP, thus producing functional proteins.
What is the mechanism of Patisiran in treating hereditary amyloidosis?
Patisiran is delivered as a lipid nanoparticle and helps eliminate amyloid fibrils by degrading the mutant transthyretin (TTR) protein.
What are circular RNAs (circRNAs) and their advantages?
CircRNAs are formed by back splicing of exon-exon junctions, are endogenously stable, less immunogenic than linear RNAs, and can act as miRNA sponges.
How do circRNA drugs work in neuroblastoma?
CircRNA drugs sponge out the miRNAs activated by MYCN, increasing the expression of tumor suppressor genes to safeguard cells against cancer.
What strategies are used for in-vitro synthesis of circRNAs?
- Flanking the sequence of interest with self-splicing introns.
- In-vitro T4 RNA ligase.
How is the efficacy of circRNA sponges assessed in neuroblastoma?
Delivery using lipid nanoparticles to neuroblastoma cells grown in a 3D bio-printed tumor spheroid to assess cell proliferation and survival.
