1:Inserm; 2:CIRI; Inserm U1111

Chimeric Antigen Receptor (CAR)-T cell immunotherapy has modified the concept of treatment in hematological malignancies. In contrast to T cells, cord blood derived-NK cells (CB-NK) and CAR-NK cells derived from CB-NK can be used “off-the-shelf” as immune cells with anti-tumor properties for the treatment of cancer patients. We have recently show that NK cells can be gene-modified by CAR-CD22 expressing LVs pseudotyped with baboon retroviral envelopes (BAEVs) at high efficiency and that these CAR+NKs show stronger anti-tumor activity than the original NKs. Moreover, in collaboration, we showed that cord blood derived NK enhanced the fitness of both CAR positive and CAR negative T cells, promoting lower levels of exhaustion and senescence. 
 
We developed ‘nanoblades’ which are MLV- or HIV-derived virus like particle (VLP), in which the viral structural protein Gag has been fused to the Cas9, which are thus loaded with Cas9 protein together with gRNAs.  To assure efficient nanoblade delivery of Cas9/sgRNA cargo into human T, B and HSCs we pseudotyped them with baboon retroviral, measles virus or/and VSV-G envelopes. We obtained efficient gene editing in T, B and HSCs. In addition, we inserted an expression cassette in a specific genomic locus of HSCs by combining nanoblades with AAV6 vectors carrying the donor cassette. Moreover, we have recently through bio-conjugation of AAV6 vectors with ligands increased AAV6 transduction of HSCs at high vector doses, while strongly reducing CD34+ cell toxicity. The introduction of a specific ligand on rAAV6 increased donor mediated gene knock-in and significantly increased HSC survival, an important feature for clinical translation of HSC gene editing strategies.
 We will also report on the use of nanoblades for generation of CRISPR/Cas9 mediated knock-out of organoids.