RESUMEN
Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the ß-globin gene (HBB). Ex vivo ß-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.
Asunto(s)
Anemia de Células Falciformes , Compuestos Heterocíclicos , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Animales , Sistemas CRISPR-Cas/genética , Edición Génica , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas , Humanos , Ratones , Reproducibilidad de los Resultados , Globinas beta/genéticaRESUMEN
Classical nonhomologous end joining (C-NHEJ) repairs DNA double-strand breaks (DSBs) throughout interphase but predominates in G1 phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Lig4 are absolutely required for joining RAG1/2 endonuclease ("RAG")-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Lig4 also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Lig4-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA, and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially in association with G1-phase down-regulation of Lig1. Finally, we suggest that differential impacts of Ku deficiency versus Lig4 deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-deficient developing lymphocyte and neuronal cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Autoantígeno Ku/genética , Células Precursoras de Linfocitos B/metabolismo , Recombinación V(D)J , Animales , ADN Ligasa (ATP)/genética , ADN Ligasa (ATP)/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fase G1/genética , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Autoantígeno Ku/metabolismo , Ratones , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Precursoras de Linfocitos B/citologíaRESUMEN
Genome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at allelic and non-allelic target sites and report that simultaneous single-stranded DNA break formation at donor and acceptor DNA by CRISPR-Cas9 nickases (in trans paired nicking) mostly overcomes such disruptive genotype-phenotype associations. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments into essential and multiple-copy genomic sites. As shown herein by genotyping assays and high-throughput genome-wide sequencing of DNA translocations, this is achieved while circumventing most allelic and non-allelic mutations and chromosomal rearrangements characteristic of nuclease-dependent procedures. Our work demonstrates that in trans paired nicking retains target protein dosages in gene-edited cell populations and expands gene editing to chromosomal tracts previously not possible to modify seamlessly due to their recurrence in the genome or essentiality for cell function.