C4 - Targeted Gene Insertion (integrase mediated insertion -targeted or safe harbor)
1199: Innovative IDLV Design for Transgene Targeted Integration via Microhomology-Mediated End Joining Pathway
Type: Poster Session
Poster Board Number: 1199
Presentation Details
Session Title: Thursday Posters: Targeted Gene Insertion
Ex vivo gene correction of hematopoietic stem and progenitor cells (HSPCs) is a promising therapeutic approach for numerous genetic disorders. While the combination of the CRISPR-Cas9 system with various DNA donors has enable gene correction and genomic integration, several pitfalls are still present, including the limited size of the DNA payload and low editing efficiency in HSCs. Integrase-defective lentiviral vectors (IDLV) offer a notable advantage in delivering large transgenes compared to AAV6. However, their integration still relies on homology direct repair (HDR) mediated integration with low efficiency in HSCs.
Here we introduce a new class of IDLVs as DNA donor vector that leverages the microhomology end joining (MMEJ) DNA repair pathway. We designed an IDLV where the transgene expression cassette is flanked by a gRNA target site (23 bp), and a short (35 bp) sequence homologous to the intended genomic integration site (microhomology arm, MH). The underlying hypothesis is that the Cas9/gRNA cutting of IDLV will expose the MH for recombination with the genomic double strand brakes (DBS), to achieve a directional and precise target integration (TI). Compared to HDR, which requires cell cycle, MMEJ is active in all cell cycle phases and should be proficient in quiescent HSCs and the use of a small MH-arm will be an asset in terms of space for insertion of longer therapeutic cassettes. For linearizing the episomal IDLV, we used the same gRNA required to introduce DSB at the selected genomic locus, thus simplifying gRNA delivery and reducing potential off-targets associated with the use of two different gRNA.As proof of concept, a reporter gene was targeted to a safe harbor site (AAVS1) and a clinically relevant genomic locus (HBA) in K562 and T-cell lines. We showed that combining the MH-arm and the linearization of the IDLV we obtained efficient on-target, directional and seamless GFP TI (as measured by ddPCR, FACS and by Sanger sequencing of genome-IDLV junction PCR). In addition, using targeted nanopore long-read sequencing (LRS), we demonstrated the correct integration of the whole DNA expression cassette at the expected locus. To assess the contribution of the gRNA target and MH sequence alone, we generated IDLV with each one of these sequences and we demonstrated that both are required to achieve efficient and precise integration. All these data were further confirmed by molecular analysis of single cell clones. We then moved to clinically relevant human HSPC. After optimizing transduction and editing condition, we achieved and GFP integration efficiency in both genomic loci that was similar to the classical HDR based IDLV integration. Again, molecular analysis, LRS and single colony PCR confirmed the efficiency and precision of the strategy. We then challenged our IDLV to deliver and integrate a FVIII cDNA (5kb) and demonstrated once more efficient and precise integration and proficient protein expression. We are currently testing this strategy using additional therapeutic transgenes. We also have preliminary data that NHEJ inhibitors can increase IDLV integration, and we are exploring the use of different IDLV envelope to increase HSC transduction.
In conclusion, we propose an MH-IDLV system as an effective platform for cell editing, alternative to the HDR-based TI, which may have important therapeutic implications in the treatment of genetic and tumour pathologies.
Plain Language Summary
The genome editing of hematopoietic stem cells (HSCs) is a promising approach for treating various inherited blood, metabolic disorders, and cancers. Edited HSCs can express therapeutic proteins, and remain stable in patients, thereby providing a long-term treatment effect. In this context, we propose a new tool for genome editing of HSCs, namely an integrase-deficient lentiviral vector (IDLV), relying on a cellular pathway known as microhomology end joining (MMEJ). The system is based on short sequences that are identical to a specific part of the genome, in which the transgene can be integrated. Here we showed that this system enables the expression of reporter and therapeutic transgene. We also evaluated the specificity of the system, analyzing how the transgene is integrated in the genome. We are currently worked to improve the efficacy and test different therapeutic transgenes to generate a platform to the treatment of genetic, immune, and tumoral pathologies.
Giulia Scalisi1,2, Aboud Sakkal1,2, Lacombe Laurie1,2, Alexandra Tachtsidi1,2, Guillaume Corre1,2, Giulia Pavani3, Peter Lenting4, Anne Galy5, Mario Amendola1,2,6
1Genethon, Evry, France,2Université Paris-Saclay, Univ Evry, Inserm, Généthon, Integrare Research Unit UMR_S951, Evry, France,3Children's Hospital of Philadelphia, Philadelphia, PA,4UMR_S1176,Inserm, Univ. Paris-Sud, Université Paris-Saclay, Le Kremlin-Bicêtre, France,5ART-TG, Accelerator of Technological Research in Genomic Therapy - Inserm US35, EVRY, France,6Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy"
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