Oral Presentation ASSCR, AGCTS, ISCT ANZ and Friends Joint Scientific Conference 2019

Generation of novel cardiac specific AAV vectors by directed evolution in human iPSC derived cardiomyocytes (#24)

Cindy Kok 1 2 , Sindhu Igoor 1 , Rhys Skelton 1 , James Chong 1 2 3 , Kimberley Dilworth 4 , Marti Cabanes Creus 5 , Ian Alexander 6 7 , Leszek Lisowski 4 5 8 , Eddy Kizana 1 2 3 , Samantha Ginn 6
  1. Centre for Heart Research, Westmead Institute for Medical Research, Westmead, NSW, Australia
  2. Westmead Clinical School, The Faculty of Medicine and Health, The University of Sydney, Westmead, NSW , Australia
  3. Department of Cardiology, Westmead Hospital, Westmead, NSW
  4. Vector and Genome Engineering Facility, Children’s Medical Research Institute, The University of Sydney, Westmead, NSW, Australia
  5. Translational Vectorology Group, Children’s Medical Research Institute, The University of Sydney, Westmead, NSW, Australia
  6. Gene Therapy Research Unit, Children’s Medical Research Institute, The University of Sydney, Westmead, NSW , Australia
  7. Sydney Medical School, Discipline of Child and Adolescent Health, The University of Sydney, Westmead, NSW, Australia
  8. Military Institute of Hygiene and Epidemiology, The Biological Threats Identification and Countermeasure Centre, Pulawy, Poland

Recombinant adeno-associated viral (rAAV) vectors have emerged as one of the most promising gene therapy vectors. However, recent evidence has indicated that successful rAAV-mediated gene therapy in animal models may not translate to the same therapeutic benefit in humans. This is mainly due to the species difference in rAAV transduction efficiency, thus leading to the search for new capsids which exhibit high efficiency in human cells. This may be achieved through capsid shuffling, in which rAAV capsid gene sequences can be reconstituted from fragments of naturally occurring rAAV capsids to generate a library comprised of novel rAAVs with unique capsid protein sequences. This library could be screened using Directed Evolution (DE) in human cardiomyoctyes to select for cardiotropic rAAVs. In the development of strategies for personalised medicine, patient-specific iPSC-CMs could be used to evolve patent specific rAAVs. To test this strategy, we have used an rAAV library (rAAV1-12) to perform DE in human iPSC-CMs. The resulting rAAVs were then recovered from cell lysates and used for subsequent rounds of selection in fresh iPSC-CMs. After six rounds of selection, the recovered rAAVs were analysed for enrichment of cardiotropic candidates. Five candidates emerged. Functional analysis using rAAV-GFP indicated that while rAAV6 was the most efficient at transducing iPSC-CM among natural rAAVs, two of our novel variants led to higher GFP expression at the same vector dose. Our results validate the use of DE to develop novel, highly functional rAAVs for use in clinical studies.

  • Have you presented your abstract at another international meeting?: Yes