P08

Surface-modified AAV6 vectors combined with nanoblades allow high level gene knock-in in hematopoietic stem and progenitor cells without compromising cell survival

A Gutierrez-Guerrero(1) A Leray(2) S Périan(1) C Martinello(3) M J Abrey Recalde(1) C Costa(1) M Bouzelha(4) D Alvarez-Dorta(4) S G Gouin(2) E Ayuso(5) O Adjali(5) H Büning(6) D Deniaud(2) M Mével(5) E Verhoeyen(1,3)

1:CIRI – International Center for Infectiology Research, Université de Lyon; Inserm U1111; Université Claude Bernard Lyon 1; CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Université Lyon, Lyon, F-69007, France; 2:Nantes Université, CNRS, CEISAM UMR 6230, Nantes, 44000, France; 3:C3M, Université Côte d’Azur, INSERM U1065, Nice, 06204, France; 4:Capacités, 16 rue des marchandises, Nantes, 44200, France; 5:Nantes Université, TaRGeT, Translational Research for gene Therapies, CHU de Nantes, INSERM UMR 1089, Nantes, 44200, France; 6:Laboratory for Infection Biology and Gene Transfer, Institute of Experimental Hematology, Hannover Medical School, Hannover, 30625, Germany

Nanoblades are virus-like particles (VLPs) derived from murine leukaemia virus (MLV) or human immunodeficiency virus (HIV), loaded with Cas9-gRNA ribonucleoproteins and devoid of any viral genome. They represent a new promising technology to efficiently induce DNA double-stranded breaks, proven to be remarkably efficient for entry into human T, B and hematopoietic stem and progenitor (HSPCs) cells, thanks to their surface co-pseudotyping with baboon retroviral and VSVG envelope glycoproteins.

Incubation of HSPCs with nanoblades together with rAAV6 vector containing two homologous arms to the Wiskott-Aldrich syndrome (WAS) locus flanking a GFP expression cassette, resulted in 50% stable expression cassette knock-in into the WAS locus. However, high doses of rAAV6 caused HSPC cell death, while rAAV2, carrying the same donor template, was less toxic and achieved similar transduction levels.

To improve donor template delivery, rAAV2 and rAAV6 were chemically bio-conjugated with a mannose ligand, via the lysine or tyrosine amino-acid residues exposed at the capsid surface. Our results showed higher transduction levels of HSPCs with mannose-coupled rAAV6 vectors compared to mannose-coupled rAAV2, accompanied by a remarkable lower toxicity compared to the unmodified rAAV6, which led to an increased survival of HSPCs from 10% to 80%. Additionally, we revealed that the mannose-coupled rAAV6 reduced p53-mediated DNA damage response in CD34⁺ cells, which might explain the lower cell toxicity.

Therefore, this provides an excellent tool for precise therapeutic correction in CD34⁺ HSPCs, an important feature for clinical translation of HSPC-gene editing strategies.