Discovery and validation of human genomic safe harbor sites for gene and cell therapies

Aznauryan, Erik; Yermanos, Alexander; Kinzina, Elvira; Devaux, Anna; Kapetanovic, Edo; Milanova, Denitsa; Church, George M.; Reddy, Sai T.

doi:10.1016/j.crmeth.2021.100154

Cited by 32 publications

(38 citation statements)

References 71 publications

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“…We undertook a computational search to predict intergenic genome safe harbor (GSH) sites in S. mansoni based on accepted criteria, newly introduced principles 13 , and genome sequence resources available for this schistosome, that could satisfy benign and stable gene expression.…”

Section: Resultsmentioning

confidence: 99%

Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke,Schistosoma mansoni

Tanno

Moscheid

Chaparro³

et al. 2022

Preprint

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We identified genomic safe harbor sites (GSH) in the human blood fluke, Schistosoma mansoni and developed a CRISPR-focused protocol for insertion of a reporter transgene into a representative GSH. The protocol employed ribonuclear protein complexes of Cas9 nuclease, three overlapping guide RNAs, and phosphorothioate-modified, double stranded donor DNAs encoding green fluorescent protein driven by a strong endogenous promoter. Gene-editing efficiencies of >20% and reporter transgene fluorescence of >50% of gene-edited eggs were obtained by five days after CRISPR transfection. These methods and results advance functional genomics for multicellular parasites, and represent a tractable path towards transgenic schistosomes using homology directed repair catalyzed transgene insertion. Identification and characterization of GSH is expected to facilitate consistent transgene activity with neutral influence on the host cell genome and, concurrently, provide a privileged locus for transgene activity. This approach should be adaptable to platyhelminths generally.

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Section: Resultsmentioning

confidence: 99%

Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke,Schistosoma mansoni

Tanno

Moscheid

Chaparro³

et al. 2022

Preprint

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“…Among common pMEIs, we then excluded loci that are associated with tissue-specific expression of nearby genes to further increase the likelihood of selecting region with no functional impacts. Second, unlike most current GSH mapping approaches that mask genome with arbitrary defined linear windows near important DNA elements [ 14 , 15 ], our approach is knowledge-based and considers 3D chromatin organizations of the genome and the 3D spatial distance between genomic loci. Third, stable expression of the transgene is essential for an effective gene therapy.…”

Section: Discussionmentioning

confidence: 99%

“…With the increasing availability of genomics and epigenomics data, different criteria have been applied to genome-wide searches for GSHs in the human genome [ 14 , 15 ]. Generally, those criteria require a minimal linear distance from functional DNA elements such as promoters, enhancers, and coding sequences.…”

Section: Introductionmentioning

confidence: 99%

Genomics and epigenetics guided identification of tissue-specific genomic safe harbors

Shrestha

Bag

et al. 2022

Genome Biol

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Background Genomic safe harbors are regions of the genome that can maintain transgene expression without disrupting the function of host cells. Genomic safe harbors play an increasingly important role in improving the efficiency and safety of genome engineering. However, limited safe harbors have been identified. Results Here, we develop a framework to facilitate searches for genomic safe harbors by integrating information from polymorphic mobile element insertions that naturally occur in human populations, epigenomic signatures, and 3D chromatin organization. By applying our framework to polymorphic mobile element insertions identified in the 1000 Genomes project and the Genotype-Tissue Expression (GTEx) project, we identify 19 candidate safe harbors in blood cells and 5 in brain cells. For three candidate sites in blood, we demonstrate the stable expression of transgene without disrupting nearby genes in host erythroid cells. We also develop a computer program, Genomics and Epigenetic Guided Safe Harbor mapper (GEG-SH mapper), for knowledge-based tissue-specific genomic safe harbor selection. Conclusions Our study provides a new knowledge-based framework to identify tissue-specific genomic safe harbors. In combination with the fast-growing genome engineering technologies, our approach has the potential to improve the overall safety and efficiency of gene and cell-based therapy in the near future.

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“…Cancer cell line culture and genome editing HER2 expressing cell lines SKBR3 and MCF-7 27 were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco) and 50 mg/mL Normocin (Invivogen), thereafter referred to as cell line growth medium. CRISPR-Cas9 genome editing in these cell lines was performed with RNP particles as described above with the following differences: the crRNA was specific for CCR5 56 (Supplementary Table 1); the nucleofection buffer was Dulbecco's phosphate-buffered saline (DPBS) (Gibco); the nucleofector protocol was EO-117 for SKBR3 and EN-130 for MCF-7; and the cells were diluted in cell line growth medium.…”

Section: Primary Human T Cell Genome Editingmentioning

confidence: 99%

speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing

et al. 2022

Self Cite

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Chimeric antigen receptors (CARs) consist of an antigen-binding region fused to intracellular signaling domains, enabling customized T cell responses against targets. Despite their major role in T cell activation, effector function and persistence, only a small set of immune signaling domains have been explored. Here we present speedingCARs, an integrated method for engineering CAR T cells via signaling domain shuffling and pooled functional screening. Leveraging the inherent modularity of natural signaling domains, we generate a library of 180 unique CAR variants genomically integrated into primary human T cells by CRISPR-Cas9. In vitro tumor cell co-culture, followed by single-cell RNA sequencing (scRNA-seq) and single-cell CAR sequencing (scCAR-seq), enables high-throughput screening for identifying several variants with tumor killing properties and T cell phenotypes markedly different from standard CARs. Mapping of the CAR scRNA-seq data onto that of tumor infiltrating lymphocytes further helps guide the selection of variants. These results thus help expand the CAR signaling domain combination space, and supports speedingCARs as a tool for the engineering of CARs for potential therapeutic development.

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Discovery and validation of human genomic safe harbor sites for gene and cell therapies

Cited by 32 publications

References 71 publications

Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke,Schistosoma mansoni

Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke,Schistosoma mansoni

Genomics and epigenetics guided identification of tissue-specific genomic safe harbors

speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing

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