Blackmore, Murray

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Blackmore, Murray

  1. 1. HIGH CONTENT SCREENINGIDENTIFIES NOVEL REGULATORSOF CNS AXON REGENERATION Murray Blackmore The Miami Project to Cure Paralysis University of Miami
  2. 2. Why can’t axons in the CNS regenerate?
  3. 3. Why can’t axons in the CNS regenerate? Myelin-associated inhibitors Glial Scarring (CSPGs) Ephrins / Semaphorins
  4. 4. Why can’t axons in the CNS regenerate? 1. Neutralize inhibitory molecules 2. Block inhibitory signaling pathways 3. Tissue / cell transplants
  5. 5. Why can’t axons in the CNS regenerate? 1. Neutralize inhibitory molecules 2. Block inhibitory signaling pathways 3. Tissue / cell transplants ≠ robust regeneration
  6. 6. Why can’t axons in the CNS regenerate? Inadequate Cell Body Response 1. Neutralize inhibitory molecules 2. Block inhibitory signaling pathways 3. Tissue / cell transplants ≠ robust regeneration
  7. 7. Many types of neurons regenerate Zebrafish retina PNS Immature CNS
  8. 8. Many types of neurons regenerate Zebrafish retina ? PNS Immature CNS
  9. 9. In vivo, rodent corticospinal tract (CST) neurons loseregenerative capacity between postnatal day 3 and 16
  10. 10. In vivo, rodent corticospinal tract (CST) neurons loseregenerative capacity between postnatal day 3 and 16 Research strategy 1. Comparative gene profiling 2. Functional screening in culture 3. Functional gene testing in vivo
  11. 11. What CST genes change in expression between P3 and P16? Change in CST gene expression? >1000 genes change 3x or more
  12. 12. Candidate genes are available in large libraries Change in CST gene expression? >1000 genes change 3x or more Open Biosystems Library 6,200 human genes 9,800 mouse genes About 160 96-well plates
  13. 13. Workflow for in vitro screening
  14. 14. Automated quantification of neurite outgrowth~ 800 plasmids, 500 genes 1,000,000s of neurons
  15. 15. Total neurite length (normalized) 20 40 60 80 0 180 200 100 120 140 1601500001H12RIK4933407N01RIK ALDOA ATP6V1D ALDOA CHN1 DNAJB9 KLF4 CHN1 IDH3A KLF10 MAT2B PLP1 NGEF RAB24 SCG2 SLC2A3 WBP2 SLC2A3 ALDOA C1QDC2 CAMK2G CREG1 DTNA ITM2B MKKS OSBPL1A RAB24 Developmentally upregulated genes ST3GAL5 TPP1 ACTB EZH2 SBK1 Dev. ST8SIA4 (2) DCL 003 SYP DCL EEF1G (1) KLF6 DCX RPL13 Screening Identifies regulators of neurite outgrowth Genes SET KLF6
  16. 16. Total neurite length (normalized) 20 40 60 80 0 180 200 100 120 140 1601500001H12RIK4933407N01RIK ALDOA ATP6V1D ALDOA CHN1 DNAJB9 KLF4 CHN1 IDH3A KLF10 MAT2B PLP1 NGEF RAB24 SCG2 SLC2A3 WBP2 SLC2A3 ALDOA C1QDC2 CAMK2G CREG1 DTNA ITM2B MKKS OSBPL1A RAB24 Developmentally upregulated genes ST3GAL5 TPP1 ACTB EZH2 SBK1 Dev. ST8SIA4 (2) DCL 003 SYP DCL EEF1G (1) KLF6 DCX RPL13 Screening Identifies regulators of neurite outgrowth Genes SET KLF6
  17. 17. Multiple KLF family members regulate neurite outgrowth Neurite length (um) KLF9 Moore, Blackmore et al. (2009). Science, 326: 298-301.
  18. 18. Full length Domain Swap Zinc finger only VP16 (activation) Engrailed (repression) Total neurite length (um) 100 200 300 400 500 0 EGFP * KLF7 KLF7(ZF) * VP16-KLF7(ZF) Eng-KLF7(ZF) ** Total neurite length (um) KLF9-KLF7(ZF) 100 200 300 400 500 0 ** Total neurite length (µm) Total neurite length (um) (um) Total neurite length neurite length (um) Total EGFP 100 200 300 400 500 0 100 100 200 200 300 300 400 400 500 500 0 0 100 200 300 400 500 0 * KLF7 EGFP EGFP EGFP EGFP KLF7(ZF) * * * KLF7 KLF7 * KLF7 * VP16-KLF7(ZF) KLF7 KLF7(ZF) KLF7 Zinc Finger KLF7(ZF) KLF7(ZF) Eng-KLF7(ZF) ** * VP16-KLF7 * * *16-KLF7(ZF) VP16-KLF7(ZF) VP16-KLF7(ZF) KLF9-KLF7(ZF) Eng-KLF7 **ng-KLF7(ZF) Eng-KLF7(ZF) Eng-KLF7(ZF) ** ** ** **F9-KLF7(ZF) KLF9-KLF7(ZF) KLF9-KLF7(ZF) ** ** ** Structure/function studies identify critical domains
  19. 19. Testing gene function in vivoGene delivery to Dorsal hemisection CST cell bodies 1. Knock down growth suppressive genes 2. Over-express growth promoting genes 3. Overexpress/knock down in combination
  20. 20. AAV8 effectively transduces neurons Thanks to: University of Miami Viral Vector Core
  21. 21. Viral-mediated protein knockdown in vivo EGFP PTEN IHC Overlay
  22. 22. Viral-mediated protein knockdown in vivo EGFP PTEN IHC Overlay Anti-PTENAnti-luciferase control
  23. 23. Control animals have minimal axon growth below injury site Collaboration: Dr. Christopher Shields Norton Neurological Louisville, KY
  24. 24. Number of axons 100 150 200 50 0 Control anti-NGEF anti-PTENanti-SOCS3 anti-KLF2 anti-KLF5anti-KLF15 250µm anti-KLF9 Rheb1 CS 1 KLF7VP16-KLF7 Number of axons 10 20 30 40 50 60 70 0 injury in vivo Control anti-NGEF anti-PTENanti-SOCS3 anti-KLF2 anti-KLF5anti-KLF15 anti-KLF9 1000µm Rheb1 CS 1 KLF7 Quantification of axon growth after spinal cordVP16-KLF7
  25. 25. Over-expression of VP16-KLF7 in CST neuronspromotes axon growth in the injured spinal cord
  26. 26. Over-expression of VP16-KLF7 in CST neuronspromotes axon growth in the injured spinal cord ** ** ** *
  27. 27. 2. In vitro screening 3. Hypothesis driven research 4. Viral-mediated gene testing in vivo Full length Domain Swap Total neurite length (normalized) Zinc finger only 20 40 60 80 0 100 120 140 160 180 200 1500001H12RIK 4933407N01RIK ALDOA ATP6V1D VP16 (activation) CHN1 DNAJB9 IDH3A KLF10 MAT2B PLP1 RAB24 SCG2 SLC2A3 WBP2 ALDOA C1QDC2 1. Cell-cell comparisons (Microarray, RNAseq) CREG1 DTNA Engrailed (repression) ITM2B MKKS OSBPL1A RAB24 ST3GAL5 TPP1 ACTB EZH2 Total neurite length (um) SBK1 ST8SIA4 (2) 003 SYP DCL EEF1G (1) KLF6 RPL13 SET 100 200 300 400 500 0 EGFP * KLF7 KLF7(ZF) * VP16-KLF7(ZF) Eng-KLF7(ZF) ** Total neurite length (um) KLF9-KLF7(ZF) 100 200 300 400 500 0 ** Total neurite length (um) (um) Total neurite length neurite length (um) Total EGFP 100 100 200 200 300 300 400 400 500 500 0 0 100 200 300 400 500 0 * KLF7 EGFP EGFP EGFP KLF7(ZF) * * * KLF7 KLF7 KLF7 * VP16-KLF7(ZF) KLF7(ZF) KLF7(ZF) KLF7(ZF) Eng-KLF7(ZF) ** * *VP16-KLF7(ZF) VP16-KLF7(ZF) VP16-KLF7(ZF) KLF9-KLF7(ZF) ** Summary: a pipeline for therapeutic gene discovery Eng-KLF7(ZF) Eng-KLF7(ZF) Eng-KLF7(ZF) ** ** * **KLF9-KLF7(ZF) KLF9-KLF7(ZF) KLF9-KLF7(ZF) ** ** **
  28. 28. 2. In vitro screening 3. Hypothesis driven research 4. Viral-mediated gene testing in vivo Full length Domain Swap Total neurite length (normalized) Zinc finger only 20 40 60 80 0 100 120 140 160 180 200 1500001H12RIK 4933407N01RIK ALDOA ATP6V1D VP16 (activation) CHN1 DNAJB9 IDH3A KLF10 MAT2B PLP1 RAB24 SCG2 SLC2A3 WBP2 ALDOA C1QDC2 1. Cell-cell comparisons (Microarray, RNAseq) CREG1 DTNA Engrailed (repression) ITM2B MKKS OSBPL1A RAB24 ST3GAL5 TPP1 ACTB EZH2 Total neurite length (um) SBK1 ST8SIA4 (2) 003 SYP DCL EEF1G (1) KLF6 RPL13 SET 100 200 300 400 500 0 EGFP * KLF7 KLF7(ZF) * VP16-KLF7(ZF) Eng-KLF7(ZF) ** Total neurite length (um) KLF9-KLF7(ZF) 100 200 300 400 500 0 ** Total neurite length (um) (um) Total neurite length neurite length (um) Total EGFP 100 100 200 200 300 300 400 400 500 500 0 0 100 200 300 400 500 0 * KLF7 EGFP EGFP EGFP KLF7(ZF) * * * KLF7 KLF7 KLF7 * VP16-KLF7(ZF) KLF7(ZF) KLF7(ZF) KLF7(ZF) Eng-KLF7(ZF) ** * *VP16-KLF7(ZF) VP16-KLF7(ZF) VP16-KLF7(ZF) KLF9-KLF7(ZF) ** Summary: a pipeline for therapeutic gene discovery Drug discovery Eng-KLF7(ZF) Eng-KLF7(ZF) Eng-KLF7(ZF) ** ** * ** Targeted growth-KLF9-KLF7(ZF) KLF9-KLF7(ZF) KLF9-KLF7(ZF) ** ** ** promotion via virus
  29. 29. Acknowledgements Vance Lemmon Jeff GoldbergBlackmore lab Darcie Moore John BixbyZimei Wang Jessica Lerch-HanerYania Martinez Dario Motti Andrea Johnstone Christopher ShieldsOrisley Franch Tatiana Slepak Ping ZhangMathew Saccino Yan Shi University of Miami Viral Vector CoreNational Institutes of Health (NINDS, NICHD) Anthony OlivaThe Buoniconti FundCraig Neilsen Foundation Pingping JiaRalph Wilson FoundationWallace Coulter FoundationWalter G. Ross FoundationDOD

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