Oligonucleotide therapeutics aim to treat disease by altering gene expression. They work by modulating gene expression through antisense or antigene technology to complementarily bind to RNA or DNA, respectively. Successful oligonucleotide therapy requires overcoming challenges such as nuclease degradation and poor cellular uptake. Various delivery strategies including liposomes, dendrimers, polymers, and conjugation to carrier peptides are being explored to protect oligonucleotides and improve cellular delivery, though none have achieved full success yet. Overall, oligonucleotide therapeutics show promise if these delivery challenges can be addressed.

1. Oligonucleotide Therapeutics: Basic Principles and Delivery Strategies Ashish Sarode February 17, 2006

2. Outline Introduction Basic Principles Delivery Strategies Summary Conclusion

3. Introduction

4. Definitions Oligonucleotide (ON) is a molecule composed of 25 or fewer nucleotides ON strategies designed to treat disease by altering gene expression of an affected individual

5. Past and Present 1,2 First synthetic oligonucleotide – Zamecnik and Stephenson (1978) – RS virus First oligonucleotide drug – Fomivirsen (Vitravene) – FDA approval (1998/99) – Cytomegalovirus (CMV) retinitis – AIDS Approximately 70 to 80 oligonucleotides are currently in clinical trials

6. Current Research 3 Isis Pharmaceuticals 2003 annual report

7. Clinical Trials 4 Isis Pharmaceuticals 2004 annual report

8. Basic Principles

9. Gene Therapy vs. ON Therapy 5 Gene Therapy Missing or defective genes are added or replaced with functional versions ON Therapy Existing but abnormally expressed genes are modulated

10. Modulation of gene expression 2,6 Antisense technology ONs are synthesized that are complementary to the RNA of interest (Control of translation) Antigene technology ONs are synthesized for direct binding to DNA (Control of transcription)

11. Antisense Technology 6 Specificity is mediated through Watson-Crick base pairing J. Dagle, D Weeks 2001

12. Antisense Technology 7 E. Devor 2005

13. Antigene Technology 6 J. Dagle, D Weeks 2001

14. Antigene Technology 8 Intramolecular triplex formation Intermolecular triplex formation R. Guntaka, B. Varma, K. Webber2003

15. Antigene Technology 2 S. Buchini, C. Leumann 2003

16. Antisense vs. Antigene 2 Potency Gene expression Selective modification Chemical modification p H sensitivity of C-GC K + sensitivity of GA or GT Low target accessibility and affinity No antigene ON in clinical trials S. Buchini, C. Leumann 2003

17. Steps in successful ON therapy 9 Design and chemistry Stability Cell association and entry Net accumulation (influx > efflux) Avoid compartmentalization Localization at active sites Exert activity

18. Designing ONs 9 Gene-walking RNaseH mapping Scanning combinatorial ON arrays S. Akhtar 2000

19. Stability 2 Nuclease digestion S. Buchini, C. Leumann 2003

20. Delivery Strategies

21. Delivery Strategies 9 Liposomes Dendrimers Carrier peptide-mediated Receptor-mediated Polymers (microsphere formulations)

22. Liposomes 10 Anionic liposomes pH sensitive liposomes Immunoliposomes Fusogenic liposomes Cationic liposomes

23. Anionic Liposomes Low encapsulation efficiency Phosphatidylserine and calcium Cardiolipin (MVE) technique Dipalmitoyl-phosphatidylglycerol (DPPG) O. Zelphati, F. Szoka1996

24. pH-sensitive Liposomes Principle of action – enveloped viruses Dioleylphosphatidylethanol-amine (DOPE) Non-specific electrostatic adsorption 90% of the contents is degraded in lysosomes Plasma and serum instability – incorporation of cholesterol / ganglioside / cholesterol hemisuccinate (CHEMS) – loss of encapsulation capacity O. Zelphati, F. Szoka1996

25. Immunoliposomes Double specificity – antibody Amount of binding depends on density of targeted cell membrane molecules Endocytic pathway Poor encapsulation capacity – ONs coupled to cholesterol via disulfide linkage Lysosomal destruction Immunogenic O. Zelphati, F. Szoka1996

26. Fusogenic Liposomes Liposomes merge with cell membranes Fusogenic agents – PEG, glycerol, Poly-vinyl alcohol, reconstituted viral membranes Immunogenicity Poor cellular specificity Instability in plasma and serum O. Zelphati, F. Szoka1996

27. Cationic Liposomes No encapsulation step – electrostatic interaction between ONs and cationic lipids Charge ratio is critical for efficiency Fusion of cationic lipids with anionic cell membranes Enocytosis but uncoated vesicles – acidification is not required O. Zelphati, F. Szoka1996

28. Cationic Liposomes O. Zelphati, F. Szoka1996

29. Dendrimers 9 Supermolecular delivery systems – Polymerization – monodisperse, reproducible product Several functional groups – versatile Polyamidoamine (PAMAM) starburst dendrimers – hydrocarbon core – charged surface amino groups Stable complex – plasma and serum Reduce degradation of ONs in serum and lysosomes

30. Carrier peptide-mediated 5,9 Poly-L-lysine (PLL) – polycationic drug carrier Non-specific adsorptive endocytosis PLL interacts nonspecifically with negatively charged molecules on the cell membrane Cytotoxicity and nonspecificity Specific peptides can be conjugated to PLL

31. Receptor-mediated 5 Affinity of the receptor target may increase ON-cellular association Combination approach – endosome disrupting agents and labile linkages between ON and targeting moiety Y. Rojanasakul 1996

32. Polymers 9 ONs are encapsulated in biodegradable polymers – copolymers of lactic acid and glycolic acid (PLA and PLGA) Entrapment provides nuclease protection Controlled release – size of microspheres, length of ONs, and M w of the polymer Triphasic profiles – initial ‘burst effect’ (48 hrs) – sustained release – increased release (due to bulk degradation of microspheres)

33. Summary ON therapeutics has a potential to specifically alter gene expression However ON activity is restricted by lack of target cell recognition, low cellular uptake, and nuclease degradation Chemical modification of ONs and delivery strategies explored to overcome these drawbacks show promising results at some level

34. Conclusion ON therapeutics can be used to treat diseases such as cancer, viral infections, inflammatory and genetic disorders There is a wide scope to improve the available delivery strategies as well as to invent new strategies for successful application of ON therapeutics

35. References J. Rossi. A society of our own. Oligonucleotides. 2005; 15:71-71 S. Buchini, C. Leumann. Recent improvements in antigene technology. Current opinion in chemical biology. 2003; 7:717-726 Isis Pharmaceuticals 2003 annual report Isis Pharmaceuticals 2004 annual report Y. Rojanasakul. Antisense oligonucleotide therapeutics: drug delivery and targeting. ADDR. 1996; 18:115-131 J. Dagle, D Weeks. Oligonucleotide-based strategies to reduce gene expression. Differentiation. 2001; 69:75-82 E. Devor. ID Tutorial: Antisense Technologies. Integrated DNA technologies. 2005 R. Guntaka, B. Varma, K. Webber. Triplex forming oligonucleotides as modulators of gene expression. IJBCB. 2003; 35:22-31 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 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

36. Acknowledgement Dr. S. Kislalioglu Dr. H. Zia