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CCDS Symposium 2018: Recognizing Gene Sequencing and Gene Therapy
- 1. Recognizing Gene Sequencing and Gene Therapy Matthew R. Skelton, Ph.D.
- 2. Gene Therapy
- 3. Conflicts of interest/disclosures • I do not have any personal financial stake in any treatments under development for CTD • Sponsored research agreements (expired and ongoing):
- 4. Overview • Gene therapy • What is gene therapy? • Are CCDSs good candidates for gene therapy? • How is gene therapy performed? • Gene replacement • Gene repair • What is standing in the way of gene therapy for CCDS?
- 5. What is gene therapy? • Gene therapy is a way to use genes to combat disease by: • Restoring gene expression • Eliminating gene function • Fixing gene mutations
- 6. Gene therapy in humans-first trials in 2017
- 7. How is gene therapy performed? Source:FDA.gov
- 8. Did you say virus? Is that safe? Normal virus that makes you sick Virus used to deliver genes Remove gene that makes more virus No new viruses!
- 9. Are CCDSs a good candidate for gene therapy? • YES! • They are caused by mutations in a single gene • They have a clear phenotype • They can be identified using gene sequencing • Restoring CRT/SLC6A8 function in human cells increases Cr uptake • When Crt function is restored in mice, they gain weight • Suggests increase Cr uptake • No changes in behavior • Further testing needed 1W 2W 4W 8W 12W 14 16 18 20 22 Weight(g) AAV9-hCrT Control (KO) KO 0 5000 10000 15000 Cr(mMol/mgprotein)
- 10. AAV treatment increases Cr 0 1 2 3 4 5 FoldChange KO-hCRT KO-CON * K O 2W 4W 14W W T 0 5000 10000 15000 Cr(mMol/mgprotein) AAV9-hCRT Udobi, K.C. and Skelton M.R., unpublished
- 11. Small cognitive improvement in AAV treatment D ay 1 D ay 2 D ay 3 D ay 4 0 2 4 6 8 PathLength(m) AAV9-hCrT KO AAV9-hCrT cKO 0 10 20 30 40 50 %DistanceinTargetQuadrant * AAV9-hCrT KO 0.0 0.2 0.4 0.6 0.8 DiscriminationIndex D ay 1 D ay 2 D ay 3 D ay 4 0 2 4 6 8 10 PathLength(m) AAV9-hCrT KO AAV9-hCrT cKO 0 10 20 30 40 %DistanceinTargetQuadrant D ay 1 D ay 2 D ay 3 0 20 40 60 80 Latency AAV9-hCrT KO Udobi, K.C. and Skelton M.R., unpublished
- 13. What is Crispr? • A protein (Cas9) “breaks” DNA • Using a template from a healthy donor, the cell repairs the “break” and replaces the mutated DNA with the healthy DNA • Can be used to repair DNA or introduce modifications
- 14. How would it be used to treat CCDS? • In theory, it could be packaged into viral vectors directed to repair AGAT, GAMT, or SLC6A8 • Must express guide-RNA and Cas9 enzyme
- 15. Limitations • Crispr is not as precise as once thought • Using Crispr/Cas9 to make genetically modified mice has run into problems • Would need to be directed to every cell • Would need to be turned off after repair
- 16. Sounds great, what’s standing in the way? • There’s still a lot we don’t know • How long does it last? • Can it be inherited? • Will it interfere with the function of other genes? • The virus doesn’t really get into the brain well • For SMA1 patients it may be enough-uptake via motor neurons • Cognitive delays are caused by loss in large, deep regions of the brain • AAV9 is not effective in restoring expression at a high level • We need to control when and where the gene is expressed • Still more work required to understand when the gene turns on and off • The delivery of a new gene is beneficial, but it would be better to mimic the real-life expression of the gene
- 18. What works in the mouse… ***Gives us important information that can be used for other therapeutics!
- 19. Questions? Thanks to: • CCRF Animal Behavior Core • Charles Vorhees, Ph.D. • Michael Williams, Ph.D. • Cincinnati Children’s Research Foundation • Skelton Lab – Kenea Udobi – Marla Perna, Ph.D. – Amanda Kokenge – Keila Miles – Zuhair Abdulla