RNAi Therapy
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The document discusses RNA interference (RNAi) based therapeutics and purification methods. It provides an overview of RNAi, including the biogenesis of small interfering RNAs (siRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and long non-coding RNAs (lncRNAs). It then examines the advantages and challenges of RNAi therapies. Several RNA purification methods are analyzed, including boronate affinity chromatography, RNA affinity tags, and amino acid-based affinity chromatography. The author concludes that a high-throughput, robust, and cost-effective RNA purification technique is needed to translate lab-scale methods to product-scale while maintaining RNA stability.
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RNAi Therapy
- 1. Ahmed Abouelnour RNAi Based Therapeutics Purifications
- 2. Ahmed Abouelnour • RNAi Overview. • Biogenesis of regulatory non-coding RNAs. • Advantages and Challenges of RNAi Therapies. • RNAi affinity-chromatography . – Boronate affinity chromatography – RNA affinity tags – Amino acid-based affinity chromatography • Discussion. Overview
- 3. Ahmed Abouelnour RNAi Overview DNA mRNA Protein (Peptide,Hormone,Enzyme, Receptor,..etc) ncRNA Intracellular mechanism, involving the recognition and post-transcriptional control of specific messenger RNA (mRNA), mediated by non-coding RNAs (ncRNAs), that can result in the silencing gene
- 4. Ahmed Abouelnour RNAi Overview Allergen mRNA Protein (Pro-inflammatory IL) Inflammation ncRNA
- 5. Ahmed Abouelnour RNAi Overview ncRNA Small <200 nt Regulatory RNAs siRNAs piRNAs miRNAInfrastructural RNAsLarge 200nt-100kb
- 6. Ahmed Abouelnour FBIO731 :Molecular Pharmacology & Pharmacogenomics RNAi Biogenesis ncRNA Small <200 nt Regulatory RNAs siRNAs piRNAs miRNAInfrastructural RNAsLarge 200nt-100kb
- 7. Ahmed Abouelnour FBIO731 :Molecular Pharmacology & Pharmacogenomics RNAi Biogenesis 1-Small Interferening RNA Identification in Cytoplasm. RISC Incorporated. Non Sense Degradation and Expel mRNA degradation P. Pereira, et al 2016
- 8. Ahmed Abouelnour FBIO731 :Molecular Pharmacology & Pharmacogenomics RNAi Biogenesis ncRNA Small <200 nt Regulatory RNAs siRNAs piRNAs miRNAInfrastructural RNAsLarge 200nt-100kb
- 9. Ahmed Abouelnour RNAi Biogenesis 2-Micro RNA RNA polymerase II produces long primary miRNAs transcripts Pre-miRNA Mature miRNAs duplexes RISC and translation repression or mRNA degradation P. Pereira, et al 2016
- 10. Ahmed Abouelnour FBIO731 :Molecular Pharmacology & Pharmacogenomics RNAi Biogenesis ncRNA Small <200 nt Regulatory RNAs siRNAs piRNAs miRNAInfrastructural RNAsLarge 200nt-100kb
- 11. Ahmed Abouelnour RNAi Biogenesis 3-PIWI-interacting RNAs From long and single- stranded RNA precursors In Cytoplasm , RNA– protein complexes (piRNP complexes) -miRNA target RNA degradation- Methylation Ping-pong cycle Amplification P. Pereira, et al 2016
- 12. Ahmed Abouelnour FBIO731 :Molecular Pharmacology & Pharmacogenomics RNAi Biogenesis ncRNA Small <200 nt Regulatory RNAs siRNAs piRNAs miRNAInfrastructural RNAsLarge 200nt-100kb
- 13. Ahmed Abouelnour RNAi Biogenesis 4-Long non-coding RNAs • Found in nucleus, cytoplasm, or into specific sub-cellular compartments (p.e. mitochondria). • Structural features as mRNA, Including 5’ capping, polyadenylated 3’ tails and undergo alternative splicing to give rise to the final product • Recently, lncRNAs can be spliced at their 5’and 3’-ends to give stable circular RNAs (circRNAs) .Binds with: – circRNAs + miRNAs= Positive Regulators. – circRNAs + RNA Binding Protiens= Negative Regulators. • Induce translation repression or mRNA degradation .
- 14. Ahmed Abouelnour FBIO731 :Molecular Pharmacology & Pharmacogenomics Advantages and Challenges of RNAi Therapies. • Under clinical trials in cancers, viral infections, metabolic diseases, cardiovascular disease, hypertension , stroke and many others. Purity -Specific ,Selective, targeted therapy -Low dosage Intracellular Stability Long lasting therapy
- 15. Ahmed Abouelnour FBIO731 :Molecular Pharmacology & Pharmacogenomics Why Technology needed? Whatever Synthesis is chemical ,Enzymatic or Recombinant, You MUST Consider: • RNA stability. (RNases every where) • Selectivity of specific RNA molecule and yield Purity. (Avoid off target) • Time Factor (Avoid Denaturation). • HTP application. (Cost) Gel Electrophoresis, Ion Exchange, Size Exclusion RNAi affinity-chromatography • Time-consuming. • Expensive. • Tedious. • Difficult to scale up. • Degradation of the RNA molecules (toxic solvents and denaturing conditions) • Highly efficient , very rapid. • Cost Effective. • High-throughput applications • Easily adapted to any molecular weight RNA • RNA stability .
- 16. Ahmed Abouelnour FBIO731 :Molecular Pharmacology & Pharmacogenomics Affinity Chromatography? • combination of multiple intermolecular forces (Selective interaction ;Affinity) • Selective Ligands bind RNA (See types later). (Tőzsér et al., 2011) • Specific Elusion (Biospesefic ; competing agent ) or Non Specific (polarity or buffer manipulation).
- 17. Ahmed Abouelnour Affinity Chromatography 1-Boronate affinity chromatography • Columns packed with Boronate. • The presence of cis-diol groups of ribose sugar at RNA.(Not in DNA ; Selective). • Less specific but requires gradient elusion to increase selectivity (Time). • Aminoacylated tRNAs only. (Fluckiger, R 1988)
- 18. Ahmed Abouelnour FBIO731 :Molecular Pharmacology & Pharmacogenomics Affinity Chromatography 2-RNA affinity tags • Based on RNA–protein interactions found in nature. • Tags inserted in the target RNAs molecules, during in vitro transcription. • Fast and more robust for native (most types) RNA purification of long constructs produced. • Limitations =Elusion after prolonged incubations= structural modifications =Maybe degradation. • Modifications?? competitive elution (with biotin or dextran) or cleavage by a protease with a recognition site that is incorporated along with the affinity tag (P. Pereira, et al 2016)
- 19. Ahmed Abouelnour FBIO731 :Molecular Pharmacology & Pharmacogenomics Affinity Chromatography 2-RNA affinity tags • Activatable Ribozyme (the glmS ribozyme) and the BoxB RNA from bacteriophage λ. • The RNA is first transcribed as an ARiBo-fusion RNA. • Captured on Glutathione- Sepharose (GSH-Sepharose) resin via a Glutathione-S- Transferase (GST) fusion with the λN peptide. • Eluted by self-cleavage of the glmS ribozyme upon activation by glucosamine-6-phosphate (GlcN6P). Geneviève Di Tomasso et al. Nucl. Acids Res. 2011;39:e18
- 20. Ahmed Abouelnour FBIO731 :Molecular Pharmacology & Pharmacogenomics Affinity Chromatography 3-Amino acid-based affinity chromatography • Exploits natural affinity interactions occurring between amino acids (immobilized agents). • Negative charge of the RNA backbone and single, stranded nature of RNA and high base exposure on RNA species. • Control Factors are :Amino Acid, RNA backbone and RNA conformational rearrangement. • Selective & Single Step, high efficiency, selectivity and integrity. • Limitations: – Stability of RNA. – Conventional columns restrictions ; low capacity & poor pore diffusion .
- 21. Ahmed Abouelnour Affinity Chromatography 3-Amino acid-based affinity chromatography Monolithic Column: • Higher flow rates ; very fast separation. • High reproducibility both at small and large scales. • Decreased degradation. • High-throughput separations.
- 22. Ahmed Abouelnour Conclusion • Great need for HTP reliable robust ,selective ,cost effective, fast RNA purification technique keeping RNA stability to transfer from Lab scale to product. • RNA affinity tags : Selective but time consuming . • Amino acid –based seems promising. • Can oligonucleotides be an affinity binding ncRNA on a colums? Multi step?
Editor's Notes
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- Competes with RNA
- Competes with RNA
- Competes with RNA
- Types of affinity differs according to ligand
- Selective separation of RNA from DNA matrix
- There are 2 modifications ,what is the recent one?
- This RNA tag is preceded by the glmS ribozyme sequence which isactivated by glucosamine-6-phosphate A bridge of glmS ribozyme sequence which isactivated by glucosamine-6-phosphate, removing the tag used at the end of the RNA transcript,
- This RNA tag is preceded by the glmS ribozyme sequence which isactivated by glucosamine-6-phosphate A bridge of glmS ribozyme sequence which isactivated by glucosamine-6-phosphate, removing the tag used at the end of the RNA transcript,
- This RNA tag is preceded by the glmS ribozyme sequence which isactivated by glucosamine-6-phosphate A bridge of glmS ribozyme sequence which isactivated by glucosamine-6-phosphate, removing the tag used at the end of the RNA transcript,
- This RNA tag is preceded by the glmS ribozyme sequence which isactivated by glucosamine-6-phosphate A bridge of glmS ribozyme sequence which isactivated by glucosamine-6-phosphate, removing the tag used at the end of the RNA transcript,
- This RNA tag is preceded by the glmS ribozyme sequence which isactivated by glucosamine-6-phosphate A bridge of glmS ribozyme sequence which isactivated by glucosamine-6-phosphate, removing the tag used at the end of the RNA transcript,