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

Abstract

Existing approaches for the integration and expression of genes of interest in a desired human cellular context are marred by the safety concerns related to either the random nature of viral-mediated integration or unpredictable pattern of gene expression in currently employed targeted genomic integration sites. Disadvantages of these methods lead to their limited use in clinical practice, thus encouraging future research in identifying novel human genomic sites that allow for predictable and safe expression of genes of interest. We conducted a bioinformatic search followed by experimental validation of novel genomic sites and identified two that demonstrated stable expression of integrated reporter and therapeutic genes without detrimental changes to cellular transcriptome. The cell-type agnostic criteria used in our bioinformatic search suggest wide-scale applicability of our sites for engineering of a diverse range of tissues for therapeutic as well as enhancement purposes, including modified T-cells for cancer therapy and engineered skin to ameliorate inherited diseases and aging. Additionally, the stable and robust levels of gene expression from identified sites allow for their use in industry-scale biomanufacturing of desired proteins in human cells.

Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells

“…However, in contrast to the candidate sites we present (Fig. 1 A-B), the previously reported sites do not utilise criteria to avoid potential regulatory elements or criteria for universally stable and active genomic regions (Aznauryan et al, 2021;Costa et al, 2005;Eyquem et al, 2013;Papapetrou et al, 2011;Pellenz et al, 2019;Rodriguez-Fornes et al, 2020). We utilised directed differentiation of our Pansio-1, Olônne-18 and Keppel-19 targeted hESC to show consistent expression in hESC and cells from all three germ lineages (Fig.…”

“…We utilised directed differentiation of our Pansio-1, Olônne-18 and Keppel-19 targeted hESC to show consistent expression in hESC and cells from all three germ lineages (Fig. 3 C-H), whereas majority of the previously reported sites remain studied in only a limited number of cell types (Aznauryan et al, 2021;Eyquem et al, 2013;Pellenz et al, 2019;Rodriguez-Fornes et al, 2020).…”

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

“…The cell lines SKBR3 and MCF-7 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 gRNA was specific for CCR5 and taken from [44]; 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.…”