CX3CR1 intron 4 targeting provides a novel safe harbour for gene editing therapies strategies on haematopoietic progenitor and stem cells

I Ramos-Hernández(1,3) N Maldonado-Pérez(1,2) P Muñoz(1,2) F J Molina-Estévez(1,4) F Martín(1,2)

1:GENyO- Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia; 2:Universidad de Granada; 3:Fundacion Publica Andaluza Progreso y Salud; 4:Fundación Pública Andaluza para la Investigación Biosanitaria en Andalucía Oriental Alejandro Otero (FIBAO)

Benefit of ongoing ex-vivo gene editing clinical trials using Haematopoietic Stem Progenitor Cells (HSPCs) is undeniable. However, there are still many concerns regarding editing efficiency, safety, post-transplantation transgene regulation and Central Nervous System (CNS) repopulation that need further refinement. We identified CX3CR1 as an interesting locus with a poised myeloid regulation. Herein, we identified a candidate gRNA targeting efficiently the 4th intron of this locus with CRISPR/CAS9 system on CD34+ HSPCs (>90% indels by ICE analyses), but maintaining unaltered CX3CR1 expression pattern. Next, we ranked different AAV6 donor templates exploiting different insertional DNA repair mechanisms to set an optimized targeting protocol on HSPCs. Robust gene editing was achieved, with >50% stable GFP expression on HSPCs. Notably, despite using a strong viral promoter in our reporter cassette, GFP and CX3CR1 retained a myeloid skewed pattern. Also, this inclusion of a strong promoter in our cassette leads to increased levels of CX3CR1 protein and mRNA in HSPCs and activated macrophages differentiated from the targeted HSPCs. However, neither expansion nor phenotype were barely affected. Since CX3CR1/CX3CL1 axis is involved in migration toward inflamed and damaged organs, we proposed that CX3CR1 increase could beneficially prime migration capacity in HSPCs and their derivates. Resulting on a improve recruitment towards inflamed tissues, including the CNS. In summary, our preliminary results encourage the use CX3CR1 4th intron as a safe harbour for CRISPR/CAS9 targeted insertion.