RNAi Mechanisms Explained PDF

Initial indications came from plants: Overexpression of chalcone synthase an enzyme required for colour pigment production rather reduced the expression of the colour temporarily. This is said to have been RNAi because over expression of the RNA sequence caused RNA to overlap and make dsRNA which is...

Initial indications came from plants: Overexpression of chalcone synthase an enzyme required for colour pigment production rather reduced the expression of the colour temporarily. This is said to have been RNAi because over expression of the RNA sequence caused RNA to overlap and make dsRNA which is cleaved into siRNA(small interfering RNA) that can, in the RNAi pathway, break down mRNA, leading to a knockdown. Basic Concept: Antisense mRNA can be used to generate mutants Effects: Vector DNA has two promoter regions starting the coding of the RNA but in the opposite direction. Sense RNA promoter region is T3 polymerase and antisense is T7 promoter for T7 polymerase. The sense and antisense strands hybridize (bind together), which prevents the translation of the sense mRNA into protein. This is crucial for gene silencing causing the same effect as a null mutation(gene does not function). Working more into this lead, is was also that RNA demonstrated that the control (sense strand injected only) also caused a loss of function. It was now known that ss-anti-RNA can cause the disfunction but ds-RNA also leads to the same effect as seen in the plant overexpression of chalcone synthase What is the mechanism of dsRNA? Experiments use the classical RNAi pathway. siRNAs (small interfering RNA) or anti-viral mechanism RNA virus or a transposon is in the process of copying itself produces dsRNA Classic RNAi pathway needs: Dicer, Small interfering RNA (siRNA), RNA- Induced Silencing Complex (RISC) Steps: Dicer enzyme, an RNase III endonuclease, recognizes and cleaves the dsRNA into small fragments of around 21-23 nucleotides called small interfering RNAs (siRNAs have 2- nucleotide 3’ overhangs) having the guide strand(antisense) and passenger strand(sense) RISC is composed of multiple proteins, including a Slicer (Argonaute (Ago)), which plays a key role in RNA cleavage using the guide strand from the unwounded siRNA. The guide strand within RISC base-pairs with its complementary target mRNA, based on sequence complementarity. mRNA Degradation: Argonaute, as part of the RISC complex, cleaves the target mRNA, leading to its degradation. Major classes of small RNAs: siRNA: produced by cleavage of long endogenous or exogenous dsRNA or miRNA: noncoding regulatory RNAs that are single stranded that occasionally fold form ds regions(hairpins) by a complex consisting a(Drosha) and (Pasha), processed by Dicer to from dsRNA for incorporation into RISC. miRNA is used for different roles such as brain and heart development and is a regulatory RNA. Uses of dsRNA: interfering agent, Sequence-specific loss of mRNA and protein, Effects can cross cell barriers. Problem: Not all cells take up the dsRNA or siRNA, Problems with abundant proteins or proteins with low turnover(meaning it only downregulates not deltes thus sometimes only a small portion of protein is needed to continue the function even if 90% does not function) Conditional activation: Generate a cell line that express a T7 polymerase (to transcribe the target RNA) and a mechanism to control the polymerase usually a tetracycline operator: Tet on/off switch (transfection) with the induction of tetracycline. A marker to monitor if the gene was taken in by the cell. Introduce a construct where the target sequence GREEN (of gene you wish to knockdown) is inserted between flanking T7 promotors which drives transcription of the both strands(sense, anti-sense) double T7 promoters that flank the target sequence. Another method is to produce stem-loop (hairpin) structures which can be transcribed off a single T7 promotor: transcript fold s back to create stem/loop Analysis of phenotype: comparison of cells grown in presence and absence of inducer +/- tetracycline: Growth curve analysis for potential growth phenotypes 2) Morphological analysis, very often there are clear phenotyes, e.g. motility defects, abnormal shape etc 3) Confirm production of DS RNA/loss of target mRNA Northern blotting or QRT-PCR 4) Loss of protein requires some way to detect gene product: Antibody based Western blots/ELISA or direct assay for activity if known. 5) Controls for comparison extremely important: loading etc. 6) Important to monitor effects over time: phenotypes may be early or late in RNAi experiments e.g. protein stability. Source of siRNA: Vector-based RNAi uses plasmids or viral vectors to express short hairpin RNAs (shRNAs) that are processed into siRNAs within the cell, while synthetic siRNA-based RNAi involves the direct introduction of chemically synthesized double-stranded RNA molecules that are ready for incorporation into the RNA-induced silencing complex (RISC) without the need for transcription within the cell. Problems with siRNA: : lack of tight off control thus production of dsRNA in the absence of the inducer. Solution: check for production of dsRNA, better regulated system, target transcriptionally silent sites. OR affecting other genes and secondary knockdown of related proteins. Solution: Always check for sequence similarity in target region, check related mRNA levels as a control. OR These include stimulation of a subset of genes involved in the IFN response which can leads to effects on other targets. Presence of dsRNA can also lead to activation of TLRs VIVO RNAi: Transgenic RNAi: transgenic mice that express RNAi constructs, typically through the introduction of short hairpin RNAs (shRNAs) or other RNAi elements. Local RNAi: Deliver RNAi constructs specifically to targeted tissues or cells. Systemic RNAi: administration of RNAi agents (like synthetic siRNAs or direct dsRNAs) that circulate throughout the body and can target multiple tissues challenges for RNAi based therapeutics: size and structure prevents them from diffusing readily across membranes. elivery systems face issues with excipient toxicity, which can limit drug doses and cause harmful effects in nanoparticle formulations. break down of siRNA quickly, reducing its effectiveness in silencing target genes, matches between RNAi guide strands and non- targeted mRNAs the only way to ensure safety is via extensive testing ; some off- target RNAi effects may be unavoidable. Chemical modifications : essential for RNAi: attenuating activation of innate sensors that detect Nucleic acids/PAMPs dsRNA to resist degradation AND enhance antisense strand selectivity for RISC and reduce off- target RNAi Delivery systems under investigation: Naked siRNA Small - molecule ligand conjugated to RNAi agent Lipid nanoparticles Known mechanisms: Patisiran consists of the small interfering RNA (siRNA) shown in complex with lipid excipients. The components are assembled under acidic pH into lipid nanoparticles (LNPs) and injected intravenously every 3 weeks at dosages of 0.3 mg per kg. The siRNA targets the 3ʹ untranslated region (UTR) of the TTR gene to silence all possible mRNAs with coding region mutations. RNA interference (RNAi) silencing results in sustained >70% reductions of circulating TTR proteins, effectively stopping deposition of TTR TTR misfolding and aggregation is known to be associated with several amyloid diseases; senile systemic amyloidosis (SSA). Experiment to check if the insert and check the genome Use PCR to amplify the region of interest (ORF/UTR). Insert this amplified region into an RNAi vector and pick clones. Prepare a large amount of the vector (Maxiprep). Linearize the vector (cut it at a specific site like Not-1) for integration. Transfect cells with the DNA using electroporation. After 12-24 hours, apply selection using antibiotics (like Phleomycin or Hygromycin). Expand the surviving clones. Analyze the cells by growing them with or without an inducer (like tetracycline). RNA Interference Overview 1. How was classical RNA interference and the significance of dsRNA discovered? o The initial indications of RNA interference (RNAi) came from plant studies, particularly the overexpression of the chalcone synthase gene, which unexpectedly reduced color pigment expression. This phenomenon suggested that excess RNA caused overlapping sequences, resulting in double-stranded RNA (dsRNA) formation. This dsRNA was then cleaved into small interfering RNAs (siRNAs), which could degrade mRNA and lead to gene knockdown. 2. Who were Fire and Mello? o Andrew Fire and Craig Mello are scientists credited with the discovery of RNA interference. They conducted pioneering experiments demonstrating that introducing dsRNA into nematodes resulted in the specific silencing of genes, significantly contributing to the understanding of RNAi. 3. What is Dicer? o Dicer is an RNase III endonuclease that plays a crucial role in the RNAi pathway. It recognizes and cleaves long dsRNA into smaller fragments of approximately 21-23 nucleotides known as siRNAs. These siRNAs have 2-nucleotide 3’ overhangs. 4. What is the RISC? o The RNA-Induced Silencing Complex (RISC) is a multiprotein complex essential for RNAi. It incorporates one strand of the siRNA (the guide strand) and facilitates the base pairing of this strand with complementary target mRNA, leading to its degradation by Argonaute, a key protein within RISC. 5. What are the key features of a small interfering RNA? o Small interfering RNAs (siRNAs) are typically 21-23 nucleotides long, have 2-nucleotide 3' overhangs, and are derived from the cleavage of long dsRNA. They are critical for the RNAi mechanism, mediating sequence-specific degradation of target mRNA. 6. miRNA vs. siRNA: o miRNA (microRNA): Noncoding regulatory RNAs that are usually single-stranded and can form hairpin structures. They play roles in regulating gene expression and are processed by Dicer. o siRNA (small interfering RNA): Typically derived from long dsRNA, siRNAs are primarily involved in the degradation of complementary mRNA and are essential for the classical RNAi pathway. 7. What are the key features of RNAi? Why is it useful? o RNA interference allows for specific and sequence-dependent gene silencing. Key features include the ability to target and degrade specific mRNA, cross-cell barrier effects, and potential therapeutic applications. It is useful for functional genomics, gene knockdown studies, and developing RNAi- based therapies. 8. How would you perform an RNAi experiment? o To perform an RNAi experiment, design and synthesize specific siRNAs targeting the gene of interest. Introduce these siRNAs into the cell using transfection methods. Assess the knockdown efficiency by measuring levels of mRNA (using qRT-PCR) and protein (using Western blotting or ELISA). 9. What do we mean by vector-based and siRNA-based approaches? o Vector-based RNAi: Utilizes plasmids or viral vectors to express short hairpin RNAs (shRNAs), which are processed into siRNAs within the cell. o siRNA-based approaches: Involve the direct introduction of synthetic siRNAs into the cell, which are ready to incorporate into RISC without the need for transcription. 10. What is the difference between opposing T7 and hairpin-based RNAi? o Opposing T7 RNAi: Involves using two promoter regions (T7) in opposite directions to generate dsRNA from sense and antisense strands. o Hairpin-based RNAi: Involves designing RNA constructs that form a hairpin structure, allowing a single promoter to drive transcription and subsequent processing by Dicer. 11. What is conditional RNAi, and how does Tet on/off work? o Conditional RNAi involves a system where the expression of RNAi constructs can be controlled, such as the Tet on/off system. In this method, a tetracycline operator regulates T7 polymerase activity, allowing for controlled transcription of the target RNA in response to tetracycline presence. 12. Lentivirus and RNAi in mammals: why use a virus? o Lentiviral vectors can efficiently deliver RNAi constructs into mammalian cells, allowing for stable integration and long-term gene silencing. Their ability to infect dividing and non-dividing cells makes them a powerful tool for RNAi-based therapies. 13. Could you describe an RNAi experiment? o In an RNAi experiment, you would design specific siRNAs targeting a gene of interest. Introduce these siRNAs into cultured cells via transfection. Monitor the effects by assessing mRNA and protein levels through qRT-PCR and Western blotting, respectively, comparing treated versus control groups. 14. Knockdown vs. knockout: o Knockdown refers to the reduction of gene expression through RNAi, which is often temporary and does not completely eliminate the gene’s function. o Knockout involves the complete and permanent loss of gene function, typically through genetic engineering techniques. 15. How would you assess whether an RNAi experiment is working? Levels of mRNA, protein, etc.? o Assess RNAi efficacy by measuring target mRNA levels using qRT-PCR and protein levels using Western blotting or ELISA. Additional methods include Northern blotting to confirm dsRNA production. 16. What are the difficulties with RNAi? How can I be certain that I have no off-target effects? o Challenges with RNAi include off-target effects, incomplete knockdown, and the potential for activating immune responses. To minimize off-target effects, perform thorough sequence analysis of target regions and use multiple siRNAs targeting different regions of the same gene for validation. 17. Any idea about RNAi libraries? o RNAi libraries consist of collections of siRNAs targeting a wide range of genes, allowing for high-throughput screening to identify gene functions and potential therapeutic targets. 18. RNAi therapies? o RNAi therapies aim to treat diseases by specifically silencing disease-causing genes. Examples include Patisiran, which uses lipid nanoparticles to deliver siRNAs targeting the TTR gene, reducing circulating TTR protein levels in patients with amyloid diseases. Class 4 Types of Viral vectors: adenovirus : dsDNA AAV: ssDNA Retrovirus incorporation: Virus is taken in then , uncoated, viral rna and goes through reverse transcription to make DNA and integrase is used to integrate it into DNA. Prevent HIV infection: receptor CCR5&CXCR4 are used to bind to the HIV, using the CRISP9 technique, cytosine is added to introduce the stop codon within the sequence or remove the start codon(the UTR 3’ and 5’ respectively). Thus the receptors are not made and HIV cannot bind. To make a therapy for SCID some safety precautions needs to be taken: remove the LT3 promoter and inactivated U3 to stop replication of Virus and integrate directly To specific site while adding a suicide gene code. Lentiviral(Lentiviral refers to a type of virus derived from the retrovirus family) gene packaging: In the virus the Viral genome is on a separate DNA strand to that of the therapeutics DNA strand. Sickle cell disease – β-thalassemia: sickle cell hemoglobin forms long chains cloating red blood cells together leading to iron overload. Treated by bone marrow transplant or blood transfusion. AAV transduction: enters in the same format at when B cells get an antigen. The virus can also espcase the lysosome and enter into the nucleus for self replication and integrating its DNA into the host cell. How is AAV produced: Plasmid Transfection: Three types of plasmids are introduced into producer cells (often HEK293 cells): Helper plasmid: Contains genes from adenovirus essential to act as a virus Rep/Cap plasmid: Encodes AAV replication (Rep) and capsid (Cap) proteins, which are necessary for viral assembly. Transgene plasmid: Contains the gene of interest flanked by AAV's. Cell Culture and Transfection: The plasmids are co-transfected into HEK293 and culture. The AAV is now separated by centrifugation in a 60% Iodixanol solution with different density solution by different iodixanol percentage. As the centrifugation occurs,the AAV supernatant will divide it self depending on there size and weight and density a AAV at the second to bottom.You can no puncture and allow the AAV to drip out for collection. In terms of coating selection to make sure the virus in engulfed, you can find naturally occuring AAV or design one or create new receptors to help in the engulfing. PRR are able to recognise the virus and the RNA inducing immune response to kill the viruses or kill the cell infected. TLR, RLR and etc. In practice, chimpanzees AAV are used to carry spike protein for the vaccines for COVID. A muscle disorder in the dystrophin causing A shortening of the intracellular protein. Becker muscle dystrophy is the shortening by a mutation in one of the exons coding for an amino acid, there are about 79 exons that match each other and if one deleted, but the chain can continue, the protein can be made but just short, DMD is caused when the chain cannot continue thus causing no protein to be formed. For the therapy we use antisense DNA to skip the mutated Exon that causes the DMD thus allowing it to create the protein but just shorter but still functional. Once produced, AAV vectors can be used for delivering genes to target cells or tissues in research and therapeutic applications. How is DNA inserted. CRIP-CAS: guide DNA binds to specific site and cas9 cleaves it. Very short time to make but could bind to the wrong effector. ZFN (Zinc Finger Nucleases): ZFNs are engineered proteins that use zinc finger domains to bind specific DNA sequences and a FokI nuclease domain to cut DNA at the target site. Once the DNA is cut, the cell repairs the break, allowing for gene editing.Takes long to make but very good TALEN (Transcription Activator-Like Effector Nucleases): TALENs use TALE proteins to recognize specific DNA sequences and a FokI nuclease to cut the DNA. This double-strand break triggers cellular repair, enabling precise gene modifications. Shorter time and quite precise. DNA Repair: Once the DNA is cut, the cell's natural repair mechanisms are activated: Non-Homologous End Joining (NHEJ): The break is repaired by directly joining the broken ends. This method is prone to errors and often results in small insertions or deletions, leading to gene disruption or knockout. Homology-Directed Repair (HDR): If a repair template (a piece of DNA with sequences similar to the broken region) is provided, the cell can use it to repair the break accurately, allowing for precise insertion, deletion, or modification of DNA. First ever CRISPR-CAS9 editing: Leber congenital amaurosis type 10 is a rare inherited blindness caused by an intronic mutation in the CEP290 gene, leading to faulty splicing and loss of primary cilia in photoreceptors. Editas and Allergen are developing EDIT-101 (AGN-151187) gene therapy using an AAV5 vector to deliver two guide RNAs and the Cas9 enzyme, aiming to restore CEP290 protein production specifically in photoreceptors. Barriers to genome editing component: Proteins, RNA and DNA are packed in nanoparticles vehicles which can be subject to phagocytosis, degradation and extraction. They can also induce an immune response. Methods of delivery: In vitro nucleus cell injection(cells in the culture are injected release the genomic information in the nucleus) or injecting the Embryo and electroporation