Oligonucleotide Therapeutics


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Oligonucleotide Therapeutics

  1. 1. Oligonucleotide Therapeutics: Basic Principles and Delivery Strategies Ashish Sarode February 17, 2006
  2. 2. Outline <ul><li>Introduction </li></ul><ul><li>Basic Principles </li></ul><ul><li>Delivery Strategies </li></ul><ul><li>Summary </li></ul><ul><li>Conclusion </li></ul>
  3. 3. <ul><li>Introduction </li></ul>
  4. 4. Definitions <ul><li>Oligonucleotide (ON) is a molecule composed of 25 or fewer nucleotides </li></ul><ul><li>ON strategies designed to treat disease by altering gene expression of an affected individual </li></ul>
  5. 5. Past and Present 1,2 <ul><li>First synthetic oligonucleotide – Zamecnik and Stephenson (1978) – RS virus </li></ul><ul><li>First oligonucleotide drug – Fomivirsen (Vitravene) – FDA approval (1998/99) – Cytomegalovirus (CMV) retinitis – AIDS </li></ul><ul><li>Approximately 70 to 80 oligonucleotides are currently in clinical trials </li></ul>
  6. 6. Current Research 3 Isis Pharmaceuticals 2003 annual report
  7. 7. Clinical Trials 4 Isis Pharmaceuticals 2004 annual report
  8. 8. <ul><li>Basic Principles </li></ul>
  9. 9. Gene Therapy vs. ON Therapy 5 <ul><li>Gene Therapy </li></ul><ul><ul><li>Missing or defective </li></ul></ul><ul><ul><li>genes are added or </li></ul></ul><ul><ul><li>replaced with functional </li></ul></ul><ul><ul><li>versions </li></ul></ul><ul><li>ON Therapy </li></ul><ul><ul><li>Existing but abnormally </li></ul></ul><ul><ul><li>expressed genes are </li></ul></ul><ul><ul><li>modulated </li></ul></ul>
  10. 10. Modulation of gene expression 2,6 <ul><li>Antisense technology </li></ul><ul><ul><li>ONs are synthesized that </li></ul></ul><ul><ul><li>are complementary to </li></ul></ul><ul><ul><li>the RNA of interest </li></ul></ul><ul><ul><li>(Control of translation) </li></ul></ul><ul><li>Antigene technology </li></ul><ul><ul><li>ONs are synthesized for </li></ul></ul><ul><ul><li>direct binding to DNA </li></ul></ul><ul><ul><li>(Control of transcription) </li></ul></ul>
  11. 11. Antisense Technology 6 <ul><li>Specificity is mediated through Watson-Crick base pairing </li></ul>J. Dagle, D Weeks 2001
  12. 12. Antisense Technology 7 E. Devor 2005
  13. 13. Antigene Technology 6 J. Dagle, D Weeks 2001
  14. 14. Antigene Technology 8 <ul><li>Intramolecular triplex formation </li></ul><ul><li>Intermolecular triplex formation </li></ul>R. Guntaka, B. Varma, K. Webber2003
  15. 15. Antigene Technology 2 S. Buchini, C. Leumann 2003
  16. 16. Antisense vs. Antigene 2 <ul><li>Potency </li></ul><ul><li>Gene expression </li></ul><ul><li>Selective modification </li></ul><ul><li>Chemical modification </li></ul><ul><li>p H sensitivity of C-GC </li></ul><ul><li>K + sensitivity of GA or GT </li></ul><ul><li>Low target accessibility and affinity </li></ul><ul><li>No antigene ON in clinical trials </li></ul>S. Buchini, C. Leumann 2003
  17. 17. Steps in successful ON therapy 9 <ul><li>Design and chemistry </li></ul><ul><li>Stability </li></ul><ul><li>Cell association and entry </li></ul><ul><li>Net accumulation (influx > efflux) </li></ul><ul><li>Avoid compartmentalization </li></ul><ul><li>Localization at active sites </li></ul><ul><li>Exert activity </li></ul>
  18. 18. Designing ONs 9 <ul><li>Gene-walking </li></ul><ul><li>RNaseH mapping </li></ul><ul><li>Scanning combinatorial ON arrays </li></ul>S. Akhtar 2000
  19. 19. Stability 2 <ul><li>Nuclease digestion </li></ul>S. Buchini, C. Leumann 2003
  20. 20. <ul><li>Delivery Strategies </li></ul>
  21. 21. Delivery Strategies 9 <ul><li>Liposomes </li></ul><ul><li>Dendrimers </li></ul><ul><li>Carrier peptide-mediated </li></ul><ul><li>Receptor-mediated </li></ul><ul><li>Polymers (microsphere formulations) </li></ul>
  22. 22. Liposomes 10 <ul><li>Anionic liposomes </li></ul><ul><li>pH sensitive liposomes </li></ul><ul><li>Immunoliposomes </li></ul><ul><li>Fusogenic liposomes </li></ul><ul><li>Cationic liposomes </li></ul>
  23. 23. Anionic Liposomes <ul><li>Low encapsulation efficiency </li></ul><ul><li>Phosphatidylserine and calcium </li></ul><ul><li>Cardiolipin (MVE) technique </li></ul><ul><li>Dipalmitoyl-phosphatidylglycerol (DPPG) </li></ul>O. Zelphati, F. Szoka1996
  24. 24. pH-sensitive Liposomes <ul><li>Principle of action – enveloped viruses </li></ul><ul><li>Dioleylphosphatidylethanol-amine (DOPE) </li></ul><ul><li>Non-specific electrostatic adsorption </li></ul><ul><li>90% of the contents is degraded in lysosomes </li></ul><ul><li>Plasma and serum instability – incorporation of cholesterol / ganglioside / cholesterol hemisuccinate (CHEMS) – loss of encapsulation capacity </li></ul>O. Zelphati, F. Szoka1996
  25. 25. Immunoliposomes <ul><li>Double specificity – antibody </li></ul><ul><li>Amount of binding depends on density of targeted cell membrane molecules </li></ul><ul><li>Endocytic pathway </li></ul><ul><li>Poor encapsulation capacity – ONs coupled to cholesterol via disulfide linkage </li></ul><ul><li>Lysosomal destruction </li></ul><ul><li>Immunogenic </li></ul>O. Zelphati, F. Szoka1996
  26. 26. Fusogenic Liposomes <ul><li>Liposomes merge with cell membranes </li></ul><ul><li>Fusogenic agents – PEG, glycerol, Poly-vinyl alcohol, reconstituted viral membranes </li></ul><ul><li>Immunogenicity </li></ul><ul><li>Poor cellular specificity </li></ul><ul><li>Instability in plasma and serum </li></ul>O. Zelphati, F. Szoka1996
  27. 27. Cationic Liposomes <ul><li>No encapsulation step – electrostatic interaction between ONs and cationic lipids </li></ul><ul><li>Charge ratio is critical for efficiency </li></ul><ul><li>Fusion of cationic lipids with anionic cell membranes </li></ul><ul><li>Enocytosis but uncoated vesicles – acidification is not required </li></ul>O. Zelphati, F. Szoka1996
  28. 28. Cationic Liposomes O. Zelphati, F. Szoka1996
  29. 29. Dendrimers 9 <ul><li>Supermolecular delivery systems – Polymerization – monodisperse, reproducible product </li></ul><ul><li>Several functional groups – versatile </li></ul><ul><li>Polyamidoamine (PAMAM) starburst dendrimers – hydrocarbon core – charged surface amino groups </li></ul><ul><li>Stable complex – plasma and serum </li></ul><ul><li>Reduce degradation of ONs in serum and lysosomes </li></ul>
  30. 30. Carrier peptide-mediated 5,9 <ul><li>Poly-L-lysine (PLL) – polycationic drug carrier </li></ul><ul><li>Non-specific adsorptive endocytosis </li></ul><ul><li>PLL interacts nonspecifically with negatively charged molecules on the cell membrane </li></ul><ul><li>Cytotoxicity and nonspecificity </li></ul><ul><li>Specific peptides can be conjugated to PLL </li></ul>
  31. 31. Receptor-mediated 5 <ul><li>Affinity of the receptor target may increase ON-cellular association </li></ul><ul><li>Combination approach – endosome disrupting agents and labile linkages between ON and targeting moiety </li></ul>Y. Rojanasakul 1996
  32. 32. Polymers 9 <ul><li>ONs are encapsulated in biodegradable polymers – copolymers of lactic acid and glycolic acid (PLA and PLGA) </li></ul><ul><li>Entrapment provides nuclease protection </li></ul><ul><li>Controlled release – size of microspheres, length of ONs, and M w of the polymer </li></ul><ul><li>Triphasic profiles – initial ‘burst effect’ (48 hrs) – sustained release – increased release (due to bulk degradation of microspheres) </li></ul>
  33. 33. Summary <ul><li>ON therapeutics has a potential to specifically alter gene expression </li></ul><ul><li>However ON activity is restricted by lack of target cell recognition, low cellular uptake, and nuclease degradation </li></ul><ul><li>Chemical modification of ONs and delivery strategies explored to overcome these drawbacks show promising results at some level </li></ul>
  34. 34. Conclusion <ul><li>ON therapeutics can be used to treat diseases such as cancer, viral infections, inflammatory and genetic disorders </li></ul><ul><li>There is a wide scope to improve the available delivery strategies as well as to invent new strategies for successful application of ON therapeutics </li></ul>
  35. 35. References <ul><li>J. Rossi. A society of our own. Oligonucleotides. 2005; 15:71-71 </li></ul><ul><li>S. Buchini, C. Leumann. Recent improvements in antigene technology. Current opinion in chemical biology. 2003; 7:717-726 </li></ul><ul><li>Isis Pharmaceuticals 2003 annual report </li></ul><ul><li>Isis Pharmaceuticals 2004 annual report </li></ul><ul><li>Y. Rojanasakul. Antisense oligonucleotide therapeutics: drug delivery and targeting. ADDR. 1996; 18:115-131 </li></ul><ul><li>J. Dagle, D Weeks. Oligonucleotide-based strategies to reduce gene expression. Differentiation. 2001; 69:75-82 </li></ul><ul><li>E. Devor. ID Tutorial: Antisense Technologies. Integrated DNA technologies. 2005 </li></ul><ul><li>R. Guntaka, B. Varma, K. Webber. Triplex forming oligonucleotides as modulators of gene expression. IJBCB. 2003; 35:22-31 </li></ul><ul><li>S. Akhtar, M. Hughes, A. Khan, M. Bibby, M. Hussain, Q. Nawaz, J Double, P. Sayyed. The delivery of antisense therapeutics. ADDR. 2000; 44:3-21 </li></ul><ul><li>O. Zelphati, F. Szoka. Liposomes as a carrier for intracellular delivery of antisense oligonucleotides: a real or magic bullet? Journal of controlled release. 1996; 41:99-119 </li></ul>
  36. 36. Acknowledgement <ul><li>Dr. S. Kislalioglu </li></ul><ul><li>Dr. H. Zia </li></ul>
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