TD Lectures Flashcards
(44 cards)
In RNA Therapeutics, what was Breakthrough 1?
Intracellular RNA delivery: LNP (lipid nanoparticle)
‘tiny carrier bubbles that can be guided’
What are the functions of lipid nanoparticles? (LNP)
- protects the therapeutic RNA from degradation during circulation.
- hides the negative charges of RNA and allows RNA to be taken into cells, across membranes.
-biocompatible; made with building blocks (lipids) that are chemically close to normal cell components.
- can be modified to change the tissue and cell destination of the RNA/.
Why use LNPs?
- Naked RNA is intrinsically unstable and can be easily degraded.
- The negative charge and hydrophilicity of RNA prevent its diffusion across the cell membrane.
- RNAases contribute to the degradation of RNA throughout its circulation.
- Biocompatible vector capable of crossing the cell membrane, releasing nucleic acid (mRNA, siRNA, etc.) into the cytoplasm and escaping degradation for efficiency delivery.
What are the different components of a lipid nanoparticle (LNPs)?
1) Cationic/ionizable lipid for nucleic acid encapsulation.
2) Cholesterol for structural support and fusion.
3) Helper lipids (like DSPC) for membrane interaction.
4) PEG-lipids for stability and prolonged circulation.
Large-Scale LNP-RNA Assembly via Microfluidic Formulation (biomedical engineering)
–> Microfluidic Formulation:
Uses precise control of flow rates and ratios in microfluidic channels.
Chaotic mixing in channels induces interactions between components, forming lipid nanoparticles (LNPs).
–> Key Components:
1) Aqueous Phase:
Contains nucleic acids (e.g., RNA).
2) Organic Phase:
Contains lipids (e.g., cationic/ionizable lipids, cholesterol, helper lipids, PEG-lipids).
–> How It Works:
1) Chaotic Mixing:
Rapid and controlled mixing in microfluidic channels facilitates the interaction of nucleic acids and lipids.
Results in the encapsulation of nucleic acids within LNPs.
2) Controlled Parameters:
Flow Rates: Speed of fluids in channels.
Flow Ratios: Proportion of aqueous to organic phase.
Reagent Concentrations: Amount of nucleic acids and lipids.
–> Outcome:
Efficient, large-scale production of LNPs with consistent size, structure, and encapsulation efficiency.
What was the 1st RNA medicine using the therapeutic modality of small interfering RNAs?
Patisiran/Onpattra.
Targeting:
Transthyretin-mediated hereditary amyloidosis (hATTR)
Caused by mutations in the transthyretin (TTR) gene, resulting in abnormal
aggregation and accumulation of the TTR protein.
In RNA Therapeutics, what was Breakthrough 2?
RNA Chemical Biolopgy
Uridine -> N1-methylpseudouridine
A base modification enables mRNA to be used as a therapeutic.
- Exogenous mRNA is intrinsically
immunostimulatory, as it is recognized by a
variety of innate immune receptors on the cell
surface, both endosomal and cytosolic. - Replacing uridines with N1-methylpseudouridine
(or pseudouridine) can reduce immunogenicity,
improve stability and promote mRNA translation translation (Weissman and Karikó)
What were the effects of breakthrough 2 in RNA chemical biology?
- prevents recognition by the innate immune system.
- improves translation and stability of therapeutic mRNAs.
- can be adapted to improve RNA delivery and mode of action.
What are some of the many modalities of RNA-based medicines?
- Harness the immune system through the generation of antigens (mRNA vaccine)
- Replace the product of a diseased gene (mRNA)
- Silence a diseased mRNA
- Alter the active form of an mRNA
- Most importantly, one can (re)direct any of those activities by changing the sequence of the RNA; an incredible diversity of vaccines and therapeutic targets.
Personalized mRNA Therapies
(1) Applications:
- mRNA vaccines: Target neoantigens to
-stimulate T cells against cancer.
- Therapeutic mRNAs: Replace defective mRNAs.
(2) Mode of Action:
- Cellular uptake of mRNAs.
- Production of healthy proteins.
- Functional replacement of diseased mRNAs.
In Vivo Generation of Modified T Cells (CART-T)
(1) Key Components:
mRNA encodes fibroblast activating protein (FAP)-targeting CAR.
Lipid nanoparticles (NPLs) carry mRNA.
(2) Process:
NPLs injected into mice.
Delivered to T cells.
T cells transiently express anti-FAP CAR.
(3) Outcome:
Anti-FAP CAR T cells eliminate activated fibroblasts.
Attenuates cardiac fibrosis in hypertensive cardiac lesions (mouse model).
What are some new mRNA therapeutic modalities (beyond vaccine and CART)?
- circular RNAs (CircRNA)
- self-amplifying RNAs (SA-RNA)
- CRISPR-CAS9
The many ‘branches’ of the RNAi-related pathways in genome regulation.
In vitro-synthesized double-stranded RNA (dsRNA) molecules, such as miRNA precursors, transgene dsRNA, viral dsRNA, transposon dsRNA, and heterochromatic dsRNA, activate Dicer-associated complexes.
These complexes are subsequently processed into smaller RNA fragments, which are then incorporated into Argonaute-associated complexes.
These complexes play key roles in genome regulation by inducing various processes, including:
- mRNA degradation
- Translational inhibition
- mRNA cleavage
- Heterochromatic domain formation
These events are part of the diverse and interconnected RNA interference (RNAi)-related pathways that regulate gene expression and maintain genomic integrity.
Lin-4 and Lin-14 in C. elegans
1) Lin-4: A heterochronic gene in C. elegans encoding small RNAs with antisense complementarity to lin-14.
2) Let-7: Predicted precursors of let-7 microRNA also play a role in developmental timing.
(1) Lin-4 MicroRNA:
Inhibits lin-14 and lin-28, crucial for regulating developmental events.
Lin-14: A target of Lin-4; its 3’ UTR is specifically inhibited by Lin04, affecting developmental timing.
(2) Bantam MicroRNA.
Inhibits Hid (a pro-apoptopic gene), which prevents apoptosis and supports cell survival.
X: a target of Bantam, inhibits cell proliferation.
MicroRNAs like Lin-4 regulate gene expression by binding to target mRNAs, development and cellular processes.
Lin-4’s inhibition of lin-14 and lin-28 reveals how microRNAs control the timing of developmental transitions.
Bantam microRNA shows microRNA regulation of apoptosis and cell proliferation, influencing cell fat decisions.
MicroRNAs play critical roles in developmental timing, apoptosis, and cell proliferation, with the ability to influence complex gene networks in organisms like C. elegans.
The miRNA biogenesis pathway in vertebrate cells
(1)
Transcription:
Pri-miRNAs are transcribed by RNA polymerase II (Pol II), while artificial shRNAs can be transcribed by Pol III or synthesized as duplexes.
(2)Processing by Drosha:
In the nucleus, Drosha processes pri-miRNAs into pre-miRNAs.
(3)
Export: The pre-miRNA is exported from the nucleus to the cytoplasm by Exportin-5 (Exp5).
(4)Processing by Dicer:
In the cytoplasm, Dicer cleaves pre-miRNAs into a double-stranded siRNA duplex.
(5) RISC Formation:
One strand of the duplex is loaded into Ago2 (part of the RNA-induced silencing complex, RISC), which mediates gene silencing by binding complementary mRNA and causing its degradation or translation repression.
–> Key players: Ago2 (involved in gene silencing), Exp5 (exports pre-miRNA), Drosha (processes pri-miRNA), Dicer (cleaves pre-miRNA).
What is the Function of Argonaut?
- Bind mature, single-stranded 21nt siRNAs & miRNAs
- 5’ phosphate bound by Mid domain
- 5’-most nucleotides: Mid and PIWI domains
- 3’ end bound by PAZ domain
- PIWI domain folds as an RNaseH domain, and carries out the ‘slicer’ activity (the axe).
- When extensive base-pairing with target, base-pairing of nucleotides 10, 11 trigger conformational change and subsequent slicing occuring between the 10th and 11th nucleotides of mRNA.
- ‘Scans’ the RNAs in the cell for complementarity.
Argonaute is the central protein component of RNA-silencing mechanisms. It provides the platform for target-mRNA recognition by short regulatory guide RNA strands and the Slicer catalytic activity for mRNA cleavage in RNA interference.
What is this phrase “if it looks like a duck, and it quacks like a duck, it must be a duck” used to describe?
The phrase “if it looks like a duck, and it quacks like a duck, it must be a duck” means that if something behaves in a certain way or exhibits specific characteristics, it is likely that thing, even without formal identification. In the context of Argonaute-PIWI, RNAse HI, and RNAse HIII, this implies that if a protein or enzyme shows the typical behaviors associated with these families, such as RNA silencing or RNA degradation in RNA-DNA hybrids, it can be identified as part of that group. Essentially, appearance and function serve as indicators of identity.
mRNA Targeting by miRNAs in Animals
- In animals, most mRNA targets do not perfectly match with miRNAs; instead, they form incomplete base-pairing with miRNAs. This partial pairing is enough to regulate gene expression.
Example:
Lin-4 binds imperfectly to the lin-14 mRNA 3’ UTR (untranslated region), where it regulates gene expression, leading to reduced translation of lin-14 protein.
What is the Structure of Argonautes?
- N-terminal: Interacts with other proteins or RNA.
- N-linker 1: Connects N-terminal to PAZ domain.
- PAZ domain: Binds the 3’ end of the guide RNA.
- Linker 2: Connects PAZ domain to MID domain.
- MID domain: Binds the 5’ end of the guide RNA.
- PIWI domain: Binds target mRNA and has slicer activity to cut it.
- Seed region: 5’ part of the guide RNA that pairs with target mRNA.
-Guide RNA: Directs Argonaute to its target mRNA.
- 3’ supplementary center: Ensures precise pairing with the target mRNA.
This structure enables RNA silencing by guiding the cleavage of target mRNAs.
Control of Plant Development by MicroRNAs
- Arabidopsis thaliana uses microRNAs (miRNAs) to regulate mRNA cleavage (slicing) between the 10th and 11th nucleotides, which is crucial for controlling plant development.
- miRNAs play a key role in regulating genes involved in plant growth and development.
- In Arabidopsis thaliana, miRNAs help control processes such as meristem function, leaf patterning, and stress responses.
- Cleavage occurs specifically between the 10th and 11th nucleotides of the guide miRNA.
In MicroRNA targeting, is the seed region important?
Seed region: The 5’ region of the miRNA (usually positions 2-7) that interacts with complementary sequences on the target mRNA.
Key role for the seed: The seed region is critical for target recognition. Even slight differences in this region can change which mRNAs are targeted by the miRNA, making it the main determinant for specificity in gene silencing.
What are parameters for microRNA targeting?
- 3’ UTR RNA Structure
- Distance to the poly(A) tail
- RNA-binding proteins sites in the 3’UTR
- Distance to the ORF
- MicroRNA cooperation
What are the differences between the two mechanisms of miRNA-mediated silencing what were published?
(A) Initiation Block:
Mechanism: miRISC (with AGO and GW182) inhibits translation initiation.
How:
GW182 inhibits PABP, preventing mRNA cap recognition and blocking the 60S ribosomal subunit from joining.
Translation initiation is blocked, preventing protein synthesis.
(B) Postinitiation Block:
Mechanism: miRISC disrupts translation after initiation.
How:
miRISC causes ribosome drop-off during elongation.
Translation is stalled, and nascent peptides are not fully synthesized.
–> Key Difference:
Initiation block prevents translation initiation, while post-initiation block interferes with translation elongation and ribosome dissociation.
Assay for Evidence of MicroRNA Effect on mRNA Translation Initiation
(1) Control without miRNA inhibitor:
- miRISC binds to mRNA and targets eIF4F complex (binds to 5’ cap), this blocks the initiation of translation.
- expected reduced 80S ribosome formation because miRISC blocks translation initiation by targeting the eIF4F complex.
(2) With miRNA inhibitor:
- Inhibition of miRISC: adding the miRNA inhibitor should allow normal translation initiation.
- Ribosome assembly will proceed normally, and the 80S ribosome will form and be detected in the sucrose gradient.
