Multiplex HDR for disease and correction modeling of SCID by CRISPR genome editing in human HSPCs

Advances in CRISPR/Cas gene therapy for inborn errors of immunity

“…By employing Cas9mediated HDR strategy, Chang et al achieved correction of JAK3 mutations in SCID patient-specific induced pluripotent stem cells (iPSCs) and observed restoration of T cell development (214). Recently, Iancu et al reported a CRISPR-mediated multiplex HDR strategy for disease modeling and correction in SCID patient-derived HSPCs; biallelic knockout of genes was utilized to generate a disease model while knockin/knockout strategy was used for single-allelic gene correction (215). The most common form of SCID, X-SCID, is caused by mutations in the genes encoding interleukin 2 receptor gamma (IL2RG).…”

Rare immune diseases paving the road for genome editing-based precision medicine

“…By designing a chemically modified 20-nucleotide RNA guide ( Hendel et al, 2015 ) (single guide RNA; sgRNA) complementary to a pre-selected DNA sequence in the human genome ( Mali et al, 2013 ), CRISPR/Cas9-sgRNA can now be programmed to recognize and induce DSBs, at most genomic locations, an event that recruits DNA repair machinery and prompts DNA sequence modifications ( Yeh et al, 2019 ). The versatility ( Gaudelli et al, 2017 ; Kim et al, 2017 ; Komor et al, 2017 ; Bak et al, 2018 ; Koblan et al, 2018 ; Rees and Liu, 2018 ; Anzalone et al, 2019 ; Jeong et al, 2020 ; Sakata et al, 2020 ; Sato et al, 2020 ; Zeng et al, 2020 ; Anzalone et al, 2022 ), efficacy ( Genovese et al, 2014 ; Hoban et al, 2015 ; Dever et al, 2016 ; Schiroli et al, 2017 ; Kuo et al, 2018 ; Pavel-Dinu et al, 2019 ; Roman-Rodriguez et al, 2019 ; Goodwin et al, 2020 ; Rai et al, 2020 ; Cromer et al, 2021 ; De Ravin et al, 2021 ; Sweeney et al, 2021 ; Iancu et al, 2023 ) and specificity ( Vakulskas et al, 2018 ) of the CRISPR/Cas9-sgRNA-based genome editing platforms have revolutionized our ability to recognize and correct disease-causing variants directly in the genome of primary human cells.…”

“…This corrective cassette is often delivered by an adeno-associated virus (AAV) with serotypes specific to the cell type of interest (e.g., AAV6 for HSPCs and T cells). The CRISPR/Cas9-AAV6 strategy was used to restore the endogenous expression of a mutated gene by inserting a therapeutic cassette that delivers a copy of a sequence (up to 4.7 kb) coding for a functional protein ( Genovese et al, 2014 ; Hubbard et al, 2016 ; Schiroli et al, 2017 ; Pavel-Dinu et al, 2019 ; Goodwin et al, 2020 ; Cromer et al, 2021 ; Sweeney et al, 2021 ; Pavel-Dinu et al, 2022 ; Iancu et al, 2023 ).…”

“…In a more recent publication ( Iancu et al, 2023 ), another in vitro RAG2- SCID disease model was established. In this model, the RAG2 gene was KO in human CD34 + cells using CRISPR/Cas9-AAV6 platform.…”

“…The decades-long effort to identify and modify pathogenic nucleotide variants in genomic DNA ( Smithies et al, 1985 ; Rouet et al, 1994 ; Choulika et al, 1995 ; Jasin, 1996 ; Bibikova et al, 2003 ; Porteus and Baltimore, 2003 ; Porteus et al, 2003 ; Porteus and Carroll, 2005 ; Urnov et al, 2005 ; Grizot et al, 2009 ; Miller et al, 2011 ; Voit et al, 2014 ) has yielded a powerful technology—genome editing - based on inducing DNA double-stranded breaks (DSBs) or nicks to achieve defined genetic changes and restore normal gene function. The safety and efficacy of genome editing in human hematopoietic stem and progenitor cells (HSPCs) and T cells have been tested preclinically ( Dever et al, 2016 ; Hubbard et al, 2016 ; Liang et al, 2017 ; Pavel-Dinu et al, 2019 ; Goodwin et al, 2020 ; Cromer et al, 2021 ; Gray et al, 2021 ; Sweeney et al, 2021 ; Vavassori et al, 2021 ; Wilkinson et al, 2021 ; Iancu et al, 2023 ) with some approved by Food and Drug Administration (FDA) for Phase 1 clinical trials in indications such as hemoglobinopathies ( Porteus et al, 2022 ), congenital blindness ( Maeder et al, 2019 ), and hearing loss ( Botto et al, 2021 ). The therapeutic application of genome editing technology to correct gene dysfunction in IEIs has gained momentum, mainly due to the ability to preserve critical endogenous expression and regulatory features, which are indispensable for proper immune development and function.…”