CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells

Vlassis, Arsenios; Jensen, Tanja L.; Mohr, Marina; Jedrzejczyk, Dominika J.; Meng, Xiangyou; Kovacs, Gergo; Morera-Gómez, Martí; Barghetti, Andrea; Muyo Abad, Sergi; Baumgartner, Roland F.; Natarajan, Kedar N.; Nielsen, Lars K.; Warnecke, Tanya; Gill, Ryan T.

doi:10.1016/j.isci.2023.108287

Cited by 2 publications

(2 citation statements)

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“…1a, 1b, and Supplementary table 1); (ii) predicted editability, and (iii) predicted transcriptional activity in human cells. In contrast to alternative approaches for identifying GSH candidate loci that rely on arbitrary assumptions considered to reduce risks of insertional mutagenesis 16,19,21 , the methodology applied in the current study was intended to identify and characterize hematopoietic-compatible GSH loci. The criteria for establishing an ontogenic GSH (e.g., as in germline editing for transgenic mouse generation) are much different than the criteria used to establish a cell-, or lineage-, specific safe harbor, and as such, the "global" GSH is likely to be suboptimal for adult stem cells.…”

Section: Discussionmentioning

confidence: 99%

“…To date, efforts are ongoing to find new human GSH sites beyond the widely applied AAVS1 [8][9][10] , CCR5 11,12 , and hRosa26 [13][14][15] loci. Criteria have been proposed for selecting potential GSH sites that are intergenic and preferentially 50-300 kb distant from known or predicted transcriptional units [16][17][18][19] . However, the number of characterized sites following this approach have been limited and mostly evaluated in established cell lines with few examples using primary human cells 20,21 .…”

Section: Introductionmentioning

confidence: 99%

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Development of evolutionarily conserved viral integration sites as safe harbors for human gene therapy

Quezada-Ramírez,

Loncar,

Campbell

et al. 2023

Preprint

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Gene transfer into hematopoietic stem and progenitor cells (HSPCs) involving indiscriminately integrating viral vectors has unpredictable outcomes including potential adverse events such as leukemogenesis, resulting from insertional mutagenesis. Therefore, identifying and characterizing genomic safe harbor (GSH) sites where exogenous genetic information can be integrated safely into adult progenitor and stem cells is critically important for therapeutic gene addition. Here, we present a novel approach to identify new GSH sites based on a proven system of stable transgene insertion: the evolutionarily conserved integration of parvovirus DNA into the germlines of host species. From a dataset of 199 unique endogenous parvovirus element (EPV) integration events identified in host genomes, 102 loci were mapped to the human genome with 17 being evaluated for potential use as GSHs in primary human CD34+ HSPCs. Six of the edited loci resulted in sustained transgene expression in both erythroid and myeloid phenotypes while three exhibited myeloid specific-expression and the remaining four loci resulted in a subset of myeloid and erythroid lineages. Following this approach, a minimum of nine promising GSH sites suitable for gene therapy of blood disorders were identified and additional GSH sites are likely to emerge from the remaining mapped loci which may be suitable for gene addition in stem and progenitor cells other than hematopoietic tissues. This novel approach extends the options for gene knock-ins while reducing the risks of insertional mutagenesis, unpredictable expression profiles, and increasing the safety and therapeutic effects.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development of evolutionarily conserved viral integration sites as safe harbors for human gene therapy

Quezada-Ramírez,

Loncar,

Campbell

et al. 2023

Preprint

View full text Add to dashboard Cite

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Engineered yeast cells simulating CD19+ cancers to control CAR T cell activation

Jensen,

Deichmann,

Schiesaro

et al. 2023

Preprint

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Chimeric antigen receptor (CAR) T cells have become an established immunotherapy and show promising results for the treatment of hematological cancers in most patients. However, modulation of the surface levels of the targeted antigen in cancer cells affects the quality and safety of CAR T cell therapy. Here we present the successful engineering of yeast to simulate cancer cells with controllable surface antigen-densities for synthetic cell-cell communication with CAR T cells. Hence, we establish a novel tool for controlled activation of CAR T cell responses and the assessment of antigen-density thresholds. Specifically, we demonstrate i) controllable antigen-densities of CD19 on yeast using G protein-coupled receptors (GPCRs), ii) a customizable system applying heterologous GPCRs that define signal input types and signal pathway engineering for tuning the output intensity, and iii) efficient and robust activational control of clinically-derived CAR T cells using CD19-displaying yeast cells compared to the activation elicited by a NALM6 cancer cell line. Based on this yeast-based antigen-presenting cell system, we envision efficient assessment of how varying antigen densities in cancer cells affect CAR T cell responses and ultimately support development of safer and better quality of personalized cancer therapies.

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CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells

Cited by 2 publications

References 62 publications

Development of evolutionarily conserved viral integration sites as safe harbors for human gene therapy

Development of evolutionarily conserved viral integration sites as safe harbors for human gene therapy

Engineered yeast cells simulating CD19+ cancers to control CAR T cell activation

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