(F1227) IN VIVO SINGLE AND DUAL TRANSDUCTION OF AAV6 IN EXPANDED NON-MOBILIZED HAEMATOPOIETIC STEM CELLS FROM TRANSFUSION-DEPENDENT THALASSEMICS IN HUMANIZED MICE
Clinician-Scientist National University of Singapore, Singapore
Abstract: Hematopoietic stem cells (HSC) are an ideal target for gene modification therapies (GMT) as they reconstitute the entire haemopoietic and immune system, and HSC genetic diseases often cause severe lifelong morbidity. Ex-vivo GMT is established for major β-hemoglobinopathies, but limitations include accessibility, affordability and myeloablation and immunosuppression-associated morbidity. These barriers may be circumvented by in-vivo HSC GMT. Adeno- associated viral vector (AAV) hold numerous benefits for in-vivo GMT, but its small payload poses packaging challenges for large gene editors, preferred for their ability to correct mutations in situ. A dual-AAV strategy delivering split-intein editors directly addresses this limitation. We produced a humanized mouse (Humice) engrafted with in-vitro expanded human adult non-mobilised CD45+CD34+ HSC from transfusion-dependent β-thalassaemics to demonstrate the feasibility of dual-AAV serotype 6 (AAV6) targeting of human HSC in vivo. Humice (4-10% human cells, CD45+) were injected intravenously with 5E+12 vector genome (vg)/kg of ssAAV6 expressing GFP or mCherry (1:1 mixture). Peak transduction was 56.82±11.59%, achieved at one-week post-injection, with 31.77±26.21% dual transgene expression in peripheral circulating hCD45+. In Thal humice, single or dual transduction was observed in 0.2-98.2% of nested hCD45 in bone marrow, liver and spleen at four weeks, with AAV6 concentration ranging from 0.8-306017.3 copies per cell. Compared to non-thal humice (0.1–91.5% transduction and 0.0-9276.0 copies per cell), an increase in transduction efficiency and VCN was seen. Secondary transplantation of hCD45 initially transduced at 0.6% resulted in engraftment of hCD45 with 4.1-60.7% transduction, confirming AAV6 transduction of long-term repopulating HSC, with minimal activation of pro-inflammatory cytokines compared to non-AAV6 humice. This model demonstrates the feasibility of in-vivo targeting of circulating and nested HSC with AAV6, lending this strategy useful to gene or base editors amenable to split-intein designs packaged as dual AAV6, while benefiting from the promising safety profile of in-vivo AAV therapy and avoiding complications of ex-vivo HSC GMT.
Funding Source: This study was funded by the Singapore Ministry of Health National Medical Research Council NMRC/CSA INV/0012/2016. CM is supported by grants from the NMRC/TA/0003/2012, NMRC/CSA-INV/0012/2016, and NMRC/CSAINV20nov-0016.
