Gene editing by homology-directed repair (HDR) remains the most versatile and best performing strategy for long-range editing and targeted integration of a gene-sized therapeutic sequence. Delivery of a large DNA template for such application might be challenging in cell types relevant for gene therapy, such as hematopoietic stem/progenitor cells (HSPCs), and often relies on viral transduction with recombinant adeno-associated viral vector (AAV). However, the induction of a DNA double strand break (DSB) coupled with the delivery of a viral template in the context of HDR-mediated HSPCs genome editing triggers a substantial p53-mediated DDR response which, if sustained, impacts the clonal capacity and the repopulation potential of the treated cells. Moreover, the occurrence of undesired outcomes and of possible genotoxic events upon genome editing remain poorly characterized, particularly in human HSPCs. Here, we uncover an unexpected load and prolonged persistence of AAV genome and its fragments in treated cells, which trigger sustained p53-mediated DNA damage response (DDR). Accrual of viral DNA in cell cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITR, at nuclease target sites, limiting the intended editing outcome at the former and potentially activating flanking gene transcription at the latter ones. Optimized template delivery by Integrase-Defective Lentiviral Vector (IDLV) induced lower DNA load and less persistent DDR allowing better preservation of clonogenic capacity and more efficient editing of long-term repopulating HSPCs. Because insertions of viral DNA fragments were much less frequent with IDLV and its self-inactivating long terminal repeats (SIN-LTRs) are transcriptionally silent, its choice for template delivery significantly mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate clinical translation of its application in HSPC gene therapy.
Gene editing by homology-directed repair (HDR) remains the most versatile and best performing strategy for long-range editing and targeted integration of a gene-sized therapeutic sequence. Delivery of a large DNA template for such application might be challenging in cell types relevant for gene therapy, such as hematopoietic stem/progenitor cells (HSPCs), and often relies on viral transduction with recombinant adeno-associated viral vector (AAV). However, the induction of a DNA double strand break (DSB) coupled with the delivery of a viral template in the context of HDR-mediated HSPCs genome editing triggers a substantial p53-mediated DDR response which, if sustained, impacts the clonal capacity and the repopulation potential of the treated cells. Moreover, the occurrence of undesired outcomes and of possible genotoxic events upon genome editing remain poorly characterized, particularly in human HSPCs. Here, we uncover an unexpected load and prolonged persistence of AAV genome and its fragments in treated cells, which trigger sustained p53-mediated DNA damage response (DDR). Accrual of viral DNA in cell cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITR, at nuclease target sites, limiting the intended editing outcome at the former and potentially activating flanking gene transcription at the latter ones. Optimized template delivery by Integrase-Defective Lentiviral Vector (IDLV) induced lower DNA load and less persistent DDR allowing better preservation of clonogenic capacity and more efficient editing of long-term repopulating HSPCs. Because insertions of viral DNA fragments were much less frequent with IDLV and its self-inactivating long terminal repeats (SIN-LTRs) are transcriptionally silent, its choice for template delivery significantly mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate clinical translation of its application in HSPC gene therapy.
Choice of Template Delivery Increases Efficiency and Mitigates the Genotoxic Impact of HDR-based Gene Editing in Human Hematopoietic Stem Cells
VARESI, ANGELICA
2021/2022
Abstract
Gene editing by homology-directed repair (HDR) remains the most versatile and best performing strategy for long-range editing and targeted integration of a gene-sized therapeutic sequence. Delivery of a large DNA template for such application might be challenging in cell types relevant for gene therapy, such as hematopoietic stem/progenitor cells (HSPCs), and often relies on viral transduction with recombinant adeno-associated viral vector (AAV). However, the induction of a DNA double strand break (DSB) coupled with the delivery of a viral template in the context of HDR-mediated HSPCs genome editing triggers a substantial p53-mediated DDR response which, if sustained, impacts the clonal capacity and the repopulation potential of the treated cells. Moreover, the occurrence of undesired outcomes and of possible genotoxic events upon genome editing remain poorly characterized, particularly in human HSPCs. Here, we uncover an unexpected load and prolonged persistence of AAV genome and its fragments in treated cells, which trigger sustained p53-mediated DNA damage response (DDR). Accrual of viral DNA in cell cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITR, at nuclease target sites, limiting the intended editing outcome at the former and potentially activating flanking gene transcription at the latter ones. Optimized template delivery by Integrase-Defective Lentiviral Vector (IDLV) induced lower DNA load and less persistent DDR allowing better preservation of clonogenic capacity and more efficient editing of long-term repopulating HSPCs. Because insertions of viral DNA fragments were much less frequent with IDLV and its self-inactivating long terminal repeats (SIN-LTRs) are transcriptionally silent, its choice for template delivery significantly mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate clinical translation of its application in HSPC gene therapy.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/14531