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Aptamers Targeting HIV-1 Entry

Aptamers Targeting HIV-1 Entry. Hazel Mufhandu PhD student Supervisor: Dr M Khati 2 nd Regional Synthetic Biology Forum 27 July 2012. Talk Outline. What are aptamers Aptamers against HIV-1 UCLA1 aptamer against HIV-1 subtype C. Proteins. Small Molecules. Whole organisms.

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Aptamers Targeting HIV-1 Entry

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  1. Aptamers Targeting HIV-1 Entry Hazel Mufhandu PhD student Supervisor: Dr M Khati 2nd Regional Synthetic Biology Forum 27 July 2012

  2. Talk Outline • What are aptamers • Aptamers against HIV-1 • UCLA1 aptamer against HIV-1 subtype C

  3. Proteins Small Molecules Whole organisms What is an aptamer? An aptamer is an artificial nucleic acid ligand that is evolutionary engineered in vitro using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process for specific binding to a variety of targets Cells

  4. How do we make aptamers? SELEX Process

  5. The resulting enriched oligonucleotideligands are referred to as APTAMERS derived from the Latin word aptus meaning “to fit”

  6. What makes aptamers our tool of choice? • Have molecular recognition properties of antibodies • Smaller size (8-15 kDa) allow efficient entry into biological compartments • Recognize targets with high affinity and specificity • Non-immunogenic & non-toxic in therapeutic applications • Conjugation chemistries for attachment of dyes, reporter molecules or functional groups can be easily introduced during synthesis • Chemically produced and not prone to viral or bacterial contamination

  7. Applications of Aptamers

  8. What makes aptamers our tool of choice? • Limitations of aptamers • Small size makes them susceptible to renal filtration and thus shorter half-life • Unmodified aptamers are highly susceptible to serum degradation • Strategies to overcome limitations • Conjugation with PEG or cholesterol can increase circulating half-life • Chemical modifications incorporated into the sugars or internucleotidephosphodiester linkages enhance nuclease resistance

  9. Aptamers against HIV-1 entry

  10. Aptamers against HIV-1 entry • B40 stabilized with 2’-flouro-pyrimidines • UCLA1 RNA aptamer - Solid-phase synthesized • Stabilized - inverted thymidine at 3’-end and dimethoxyltrityloxy-(CH2)6-SS-(CH2)6-phospho linker at 5’-end B40t 77nt B40 117nt Dey A K et al., RNA. 2005, 11:873 Chen C et al., Virology. 2008, 381:46

  11. Rationale of current UCLA1 study To test the efficacy of UCLA1 aptamer as an entry inhibitor of HIV-1 subtype C infection

  12. Neutralization Methods • 35 EnvPseudoviruses • TZM-bl cells • Luminescent reporter gene assay • 6 Primary Isolates • PBMCs • HIV-1 Gag p24 ELISA • 5 Primary Isolates • MDMs • HIV-1 Gag p24 ELISA

  13. HIV-1 pseudovirus neutralization in TZM-bl cells

  14. UCLA1 neutralize HIV-1 subtype C pseudoviruses H T Mufhanduet al., J. Virol. May 2012, 86(9):4989

  15. UCLA1 neutralize primary isolates in PBMC

  16. Neutralization in PBMCs H T Mufhanduet al., J. Virol. May 2012, 86(9):4989

  17. UCLA1 neutralize primary isolates in MDMacrophages

  18. Neutralization in MDMs H T Mufhanduet al., J. Virol. May 2012, 86(9):4989

  19. Cytotoxicity Assays H T Mufhanduet al., J. Virol. May 2012, 86(9):4989

  20. UCLA1- gp120 binding Kinetics

  21. UCLA1- gp120 binding Kinetics H T Mufhanduet al., J. Virol. May 2012, 86(9):4989

  22. Mapping UCLA1 binding sites on gp120 H T Mufhanduet al., J. Virol. May 2012, 86(9):4989

  23. Mapping UCLA1 binding sites on gp120 H T Mufhanduet al., J. Virol. May 2012, 86(9):4989

  24. Mapping UCLA1 binding sites on gp120 ConCenvpseudovirus single point mutations H T Mufhanduet al., J. Virol. May 2012, 86(9):4989

  25. UCLA1 binding epitopes on gp120 CD4bs CoRbs L369 H330 R419 K121 L125 K305 V3 loop K305 I307 I307 R308 90° H T Mufhanduet al., J. Virol. May 2012, 86(9):4989

  26. Combination therapy • Combinations • UCLA1 + T20 • UCLA1 + IgG1b12 • Combination Index = (D)1/(Dx)1 • (D)2/(Dx)2 • 0.3 – 0.7 = Synergy • 0.7 – 0.85 = Moderate synergy • 0.9 – 1.1 = Additive • >1.1 = Antagonistic

  27. Combination therapy • Tested 6 • Env-pseudoviruses • UCLA1 + b12 • 4 synergism • CI range 0.5 – 0.7 • 1 additive • CI = 0.93 • 1 Antagonism • CI = 1.62 • Tested 6 • Env-pseudoviruses • UCLA1 + T20 • 5 synergism • CI range 0.13 – 0.46 • 1 moderate synergism • CI = 0.84 H T Mufhanduet al., J. Virol. May 2012, 86(9):4989

  28. Combination therapy H T Mufhanduet al., J. Virol. May 2012, 86(9):4989

  29. UCLA1 synergism • Dose Reduction Index • = A • A+B • 11 fold less T20 • 5 fold less b12 • 5 fold less UCLA1 H T Mufhanduet al., J. Virol. May 2012, 86(9):4989

  30. Escape Mutations • Tested 2 primary isolates in PBMC in ↑[UCLA1] • Initial [UCLA1] @ IC70 • p24 assay • 7 day intervals • No Escape mutations: • Du422 • (R5 tropic) • gp120 sequence analysis: • test and control cultures • RP1 virus propagated 12 wks • IC70 ↑ 7x: 46 – 308 nM • Du422 virus propagated 9 wks • IC70 ↑ 4x: 33 – 119 nM • 6 Escape mutations: • RP1 • (X4-tropic)

  31. UCLA1 Escape Mutations R322Q N410S P369L R202T K476R F223Y Adapted from structure 2B4C (Huang et al., 2005)

  32. Concluding remarks: UCLA RNA aptamer • HIV-1 Entry inhibitor • Broad spectrum potency against subtype C isolates • Non-cytotoxic • High binding affinity • HIV-1 ConCgp120 • Mapped binding sites • CoRbs (V3 loop base) • Bridging sheet (V1/V2 loop) • CD4bs (C3, C4, C5 regions) • Synergy: HIV-1 entry inhibitors • Escape mutations: > 5 fold IC70 of UCLA1 • Further testing of UCLA1 in clinical studies • As potential new HIV-1 entry inhibitor drug • Against subtype C viruses

  33. Aptamer Group

  34. Acknowledgements Project Supervision • Dr Makobetsa Khati (CSIR Biosciences) • Prof Lynn Morris (NICD) NICD HIV/AIDS Lab • Elin Gray • MaphutiMadiga • Nancy Tumba • Alex Kabamba • Mary Phoswa Collaborations • Lynn Morris (NICD) • William James (Oxford University) Reagents • Ian McGowan (NIH) • William James (Oxford University) • Brian Sproat (ATDBio, University of Southampton) FRC Individual Grant

  35. Thank you

  36. Can UCLA1 neutralize HIV-1 pseudoviruses?

  37. Reviewed by Zhou and Rossi, Oligonucleotides. 2011, 21(1).

  38. Larry Gold et al., Plos One. 2010, 5(12).

  39. Reviewed by Keefe A.D, et al., Nature Reviews. July 2010, 9:537.

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