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Functional mitochondrial genomics II

Functional mitochondrial genomics II. The antigenomic approach to developing therapies for mtDNA disorders. Dimitra Smaragda Kyriakouli. International Symposium on Mitochondrial Physiology and Pathology Bari 22-26 June 2008. Mitochondria – Mitochondrial genome/defects – Diseases.

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Functional mitochondrial genomics II

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  1. Functional mitochondrial genomics II The antigenomic approach to developing therapies for mtDNA disorders Dimitra Smaragda Kyriakouli International Symposium on Mitochondrial Physiology and Pathology Bari 22-26 June 2008

  2. Mitochondria – Mitochondrial genome/defects – Diseases Defects of the mt genome cause wide variety of clinical disorders BUT no effective treatment for the majority of patients

  3. No effective treatment … consider genetic-based therapies

  4. No effective treatment … consider genetic-based therapies Approaches: • Rescue of a defect by expression of an engineered gene product from the nucleus (allotopic or xenotopic expression)

  5. No effective treatment … consider genetic-based therapies Approaches: • Rescue of a defect by expression of an engineered gene product from the nucleus (allotopic or xenotopic expression) • Import of normal copies or relevant sections of mtDNA into mitochondria

  6. No effective treatment … consider genetic-based therapies Approaches: • Rescue of a defect by expression of an engineered gene product from the nucleus (allotopic or xenotopic expression) • Import of normal copies or relevant sections of mtDNA into mitochondria • Manipulation of the mitochondrial genetic content

  7. No effective treatment … consider genetic-based therapies Approaches: • Rescue of a defect by expression of an engineered gene product from the nucleus (allotopic or xenotopic expression) • Import of normal copies or relevant sections of mtDNA into mitochondria • Manipulation of the mitochondrial genetic content Great potential BUT still require the delivery of foreign genes to affected cells (similar problem to many conventional gene therapy approaches) Impressive BUT still has no direct relevance to treatment unless the transfected material can be autonomously maintained Particularly attractive idea, especially if the intervention is non-invasive or is mediated by a pharmaceutical agent

  8. Re-introduce the possibility of antigenomics by developing novel nucleic acid derivatives that will follow the 4 ‘golden rules’ …

  9. Re-introduce the possibility of antigenomics by developing novel nucleic acid derivatives that will follow the 4 ‘golden rules’ … 1-2. Hydrophilic/Lipophilic

  10. Re-introduce the possibility of antigenomics by developing novel nucleic acid derivatives that will follow the 4 ‘golden rules’ … 1-2. Hydrophilic/Lipophilic 3. Bind specifically and strongly …to mutated mtDNA 4. Inhibit mtDNA polymerase γ

  11. Antigenomics • Manipulation of the heteroplasmy levels via an antigenomic approach - Antigenomic molecule selectively inhibit propagation of mutated mtDNA species • - Replication of the WT species only

  12. Antigenomics • Manipulation of the heteroplasmy levels via an antigenomic approach - Antigenomic molecule selectively inhibit propagation of mutated mtDNA species • - Replication of the WT species only • Overtime • - reduce mutated mtDNA to SUBTHRESHOLD levels REDUCTION OF MUTANT mtDNA

  13. Antigenomics • Manipulation of the heteroplasmy levels via an antigenomic approach - Antigenomic molecule selectively inhibit propagation of mutated mtDNA species • - Replication of the WT species only • Overtime • - reduce mutated mtDNA to SUBTHRESHOLD levels • - rescue the cellular defect and REDUCTION OF MUTANT mtDNA CLINICAL DEFECTS NO CLINICAL DEFECTS

  14. Antigenomics • Manipulation of the heteroplasmy levels via an antigenomic approach - Antigenomic molecule selectively inhibit propagation of mutated mtDNA species • - Replication of the WT species only • Overtime • - reduce mutated mtDNA to SUBTHRESHOLD levels • - rescue the cellular defect and • - reverse disease progression REDUCTION OF MUTANT mtDNA CLINICAL DEFECTS NO CLINICAL DEFECTS

  15. BUILDING BLOCK / UNIT ESTER LINKER 2 MAIN BODY LINKER 1 B Ugichem monomer (U)

  16. BUILDING BLOCK / UNIT ESTER LINKER 2 vs. MAIN BODY MAIN BODY LINKER 1 LINKER 1 B B Ugichem monomer (U) Nielsen monomer (N) Un Un Un Un Un Un Nn Nn Nn Cell Membrane Crossing Oligomer (CMCO) Cell Membrane Crossing Oligomer (CMCO) Cell Membrane Crossing Oligomer (CMCO) Cell Membrane Crossing Oligomer (CMCO) Cell Membrane Crossing Oligomer (CMCO) Peptide Nucleic Acid (PNA) Peptide Nucleic Acid (PNA)

  17. MAIN BODY: amino-ethyl-glycine BUILDING BLOCK / UNIT ESTER LINKER 2 vs. 2 2 2 2 LINKER 1 LINKER 1 B B Ugichem monomer (U) Nielsen monomer (N) Un Nn Cell Membrane Crossing Oligomer (CMCO) Peptide Nucleic Acid (PNA)

  18. MAIN BODY: amino-ethyl-glycine Linker 1=Linker to nucleobase= methyl carbonyl/acetyl BUILDING BLOCK / UNIT ESTER LINKER 2 vs. 2 2 2 2 B B Ugichem monomer (U) Nielsen monomer (N) Un Nn Cell Membrane Crossing Oligomer (CMCO) Peptide Nucleic Acid (PNA)

  19. MAIN BODY: amino-ethyl-glycine Linker 1=Linker to nucleobase= methyl carbonyl/acetyl Linker 2=Side chain linker= ethyl Ester= diethoxy phosphonic ester 2’-ethyl-(diethoxy)-phosphonic ester BUILDING BLOCK / UNIT vs. 2 2 2 2 B B Ugichem monomer (U) Nielsen monomer (N) Un Nn Cell Membrane Crossing Oligomer (CMCO) Peptide Nucleic Acid (PNA)

  20. MAIN BODY: amino-ethyl-glycine Linker 1=Linker to nucleobase= methyl carbonyl/acetyl Linker 2=Side chain linker= ethyl Ester= diethoxy phosphonic ester 2’-ethyl-(diethoxy)-phosphonic ester BUILDING BLOCK / UNIT R vs. 2 2 2 2 B B Ugichem monomer (U) Nielsen monomer (N) More polar More hydrophobic

  21. Concept of antigenomics is pretty old … Novel molecules “Cell Membrane Crossing Oligomers” … unknown behaviour ! Novel monomer → Novel polymers → Behaviour ? n

  22. Concept of antigenomics is pretty old … Novel molecules “Cell Membrane Crossing Oligomers” … unknown behaviour ! Novel monomer → Novel polymers → Behaviour ? n Establish fidelity of CMCOs Test hypothesis

  23. Novel molecules “Cell Membrane Crossing Oligomers” … unknown behaviour ! IN VITRO studies IN VITRO studies Studies in Cultured Cells Studies in Cultured Cells Establish fidelity of CMCOs Test hypothesis

  24. SPR analysis Thermal melts EMSA Novel molecules “Cell Membrane Crossing Oligomers” … unknown behaviour ! IN VITRO studies IN VITRO studies Studies in Cultured Cells Binding Ability and Specificity Inhibition of mtDNA Replication (Collaboration with Dr. M. Falkenberg) Mini-replisome technique Establish fidelity of CMCOs Test hypothesis

  25. Novel molecules “Cell Membrane Crossing Oligomers” … unknown behaviour ! IN VITRO studies IN VITRO studies Studies in Cultured Cells Cellular Uptake and Mitochondrial targeting Cytotoxicity Effect / Functionality Binding Ability and Specificity Inhibition of mtDNA Replication (Collaboration with Dr. M. Falkenberg) Establish fidelity of CMCOs Test hypothesis

  26. Novel molecules “Cell Membrane Crossing Oligomers” … unknown behaviour ! IN VITRO studies IN VITRO studies Studies in Cultured Cells Cellular Uptake and Mitochondrial targeting Cytotoxicity Effect / Functionality Binding Ability and Specificity Inhibition of mtDNA Replication (Collaboration with Dr. M. Falkenberg) Establish fidelity of CMCOs Test hypothesis

  27. Novel molecules “Cell Membrane Crossing Oligomers” … unknown behaviour ! IN VITRO studies IN VITRO studies Studies in Cultured Cells Cellular Uptake and Mitochondrial targeting Cytotoxicity Effect / Functionality Binding Ability and Specificity Inhibition of mtDNA Replication (Collaboration with Dr. M. Falkenberg) Establish fidelity of CMCOs Test hypothesis “The A2 molecule study”

  28. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen

  29. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties Strong binding (KD<10-8 M & Tm>65°C) Specificity (30-fold weaker with singly mismatched template) Potential in inhibiting replication of pathogenic mtDNA in vitro (Ki ~ 2.5μM)

  30. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties • Accessory group (Nitrophenol moiety) → mt targeting/matrix entrapment

  31. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties • Accessory group (Nitrophenol moiety) → mt targeting/matrix entrapment Punctate co-localisation pattern

  32. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties • Accessory group (Nitrophenol moiety) → mt targeting/matrix entrapment Punctate co-localisation pattern

  33. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties • Accessory group (Nitrophenol moiety) → mt targeting/matrix entrapment Punctate co-localisation pattern Taken up by cells but problems in localising Diffused cytoplasmic

  34. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties • Accessory group (Nitrophenol moiety) → mt targeting/matrix entrapment Punctate co-localisation pattern UNTREATED Hela

  35. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties • Accessory group (Nitrophenol moiety) → mt targeting/matrix entrapment Punctate co-localisation pattern

  36. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties • Accessory group (Nitrophenol moiety) → mt targeting/matrix entrapment Punctate co-localisation pattern Association (a) Significant (as determined by r2 G/R fluor-signal; 0.6 vs. 0.004) (b) Not artifactual (c) Time-dependent (4hrs 50%, 24hrs saturation)

  37. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties • Accessory group (Nitrophenol moiety) → mt targeting/matrix entrapment • Target sequence → Antisense (part of 5’ terminus of WT hMTCOIgene & transcript)

  38. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties • Accessory group (Nitrophenol moiety) → mt targeting/matrix entrapment • Target sequence → Antisense (part of 5’ terminus of WT hMTCOIgene & transcript) Could potentially 1. have an antisense effect by binding to MTCOI mRNA 2. inhibit transcription by binding to ssDNA template or ideally 3. inhibit mtDNA replication by preventing extension of DNA synthesis 4. combination ?!

  39. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties • Accessory group (Nitrophenol moiety) → mt targeting/matrix entrapment • Target sequence → Antisense (part of 5’ terminus of WT hMTCOIgene & transcript) • Non-cytotoxic (Galactose growth assessment 17 days) …

  40. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties • Accessory group (Nitrophenol moiety) → mt targeting/matrix entrapment • Target sequence → Antisense (part of 5’ terminus of WT hMTCOIgene & transcript) • Non-cytotoxic (Galactose growth assessment 17 days) … … retarded growth

  41. Concept of antigenomics is pretty old … EXPERIMENTAL PROCEDURES Nitrophenol moiety Getting to the A2 molecule … = Ugichem = Nielsen • Backbone composition → desired kinetic properties • Accessory group (Nitrophenol moiety) → mt targeting/matrix entrapment • Target sequence → Antisense (part of 5’ terminus of WT hMTCOIgene & transcript) • Non-cytotoxic (Galactose growth assessment 17 days) … … retarded growth Possible functionality in mitochondria

  42. Concept of antigenomics is pretty old … The A2 study Steady state level COX1 NO ARTIFICIAL MEANS OF TRANSFECTION – Supplement media with cmco Ensure equal loading – 4 protein amounts & Porin as the loading marker 3 days exposure

  43. Concept of antigenomics is pretty old … The A2 study Steady state level COX1 NO ARTIFICIAL MEANS OF TRANSFECTION – Supplement media with cmco Ensure equal loading – 4 protein amounts & Porin as the loading marker

  44. Concept of antigenomics is pretty old … The A2 study Steady state level COX1 NO ARTIFICIAL MEANS OF TRANSFECTION – Supplement media with cmco Ensure equal loading – 4 protein amounts & Porin as the loading marker 70 70 RATIO COX1:PORIN

  45. Concept of antigenomics is pretty old … The A2 study Steady state level COX1 NO ARTIFICIAL MEANS OF TRANSFECTION – Supplement media with cmco Ensure equal loading – 4 protein amounts & Porin as the loading marker 70 70 RATIO COX1:PORIN 9days exposure

  46. Concept of antigenomics is pretty old … The A2 study Steady state level COX1 NO ARTIFICIAL MEANS OF TRANSFECTION – Supplement media with cmco Ensure equal loading – 4 protein amounts & Porin as the loading marker 70 70 RATIO COX1:PORIN

  47. Concept of antigenomics is pretty old … The A2 study Steady state level COX1 NO ARTIFICIAL MEANS OF TRANSFECTION – Supplement media with cmco Ensure equal loading – 4 protein amounts & Porin as the loading marker 70 70 RATIO COX1:PORIN RATIO COX1:PORIN 20 20 RATIO COX1:PORIN

  48. Concept of antigenomics is pretty old … The A2 study Steady state level COX1 NO ARTIFICIAL MEANS OF TRANSFECTION – Supplement media with cmco Concentration Series Concentration Series 20% 20% 48% 55% 75% 48% 55% 75% 48% 55% 75% 80% 88% 100% 80% 88% 100% 80% 88% 100% RATIO COX1:PORIN RATIO COX1:PORIN

  49. Concept of antigenomics is pretty old … The A2 study Steady state level COX1 …………COX2 Steady state level COX1 …………COX2 Steady state level COX1 …………COX2 NO ARTIFICIAL MEANS OF TRANSFECTION – Supplement media with cmco Concentration Series 20% 20% 20% 48% 55% 75% 48% 55% 75% 48% 55% 75% 80% 88% 100% 80% 88% 100% 80% 88% 100% RATIO COX1:PORIN 30% 30% 40% 59% 100% 40% 59% 100% 40% 59% 100% RATIO COX2:PORIN

  50. Concept of antigenomics is pretty old … The A2 study Steady state level COX1 …………COX2 Steady state level COX1 …………COX2 Steady state level COX1 …………COX2 Is it sequence specific ? Or has to do with nitrophenol moiety ? NO ARTIFICIAL MEANS OF TRANSFECTION – Supplement media with cmco Concentration Series 20% 20% 20% 48% 55% 75% 48% 55% 75% 48% 55% 75% 80% 88% 100% 80% 88% 100% 80% 88% 100% RATIO COX1:PORIN 30% 30% 40% 59% 100% 40% 59% 100% 40% 59% 100% RATIO COX2:PORIN

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