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

Abstract

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.

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

“…For library preparation, the mRNA fraction was prepared by poly(A)+ selection (Illumina). Libraries were sequenced as paired end 150 bp reads on an Illumina HiSeq 3000 with a sequencing depth of approximately [20][21][22][23][24][25][26][27][28][29][30]…”

“…However, the number of characterized sites identified following this approach have been limited and mostly characterized in established cell lines, e.g., HEK239 or K562, with few examples on primary human cells 18,19 . Other criteria based on the use of endogenous mobile elements 20 or chromatin architecture analysis 19,21 have not been particularly effective. First reported to function as GSH by Smith et al 9 , AAVS1 remains a popular site for directed transgenesis given that gene knock-in does not affect processes like cellular viability or differentiation 22,23 .…”

“…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 . Other criteria proposed for identifying GSH sites are based on using endogenous mobile elements 22 or higher-order, chromatin architecture analysis 21,23 but outcomes have not been particularly effective. Thus, AAVS1 which was developed as a GSH 10,15 , remains a popular and widely used locus for directed transgenesis given that its knock-in does not affect the cell viability or differentiation 24,25 although some reports argue that transgene silencing occurs in a cell lineage 26 or promoter-specific manner 27 .…”

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

“… 21 , 22 , 23 , 24 An ideal GSH has been defined as a region (1) that does not overlap (predicted) functional DNA elements and (2) that lacks heterochromatic marks that could impede transcription. 25 This approach was successfully used in Caenorhabditis elegans based on annotations from the ENCODE and modENCODE consortia. 26 For non-model organisms, chromatin structure annotations are often unavailable, and experiments resort to criterion (1).…”

A comprehensive catalog of 3D genome organization in diverse human genomes facilitates understanding of the impact of structural variation on chromatin structure

“…Techniques such as Hi-C have been widely used to characterize the 3D structure of the genome and uncover folding principles of chromatin, including topologically associating domains (TADs) and chromatin loops. TADs are stable genomic regions separated by insulating proteins, e.g., CCCTC-binding factor (CTCF), and provide an encapsulating domain for constraining the chromatin contacts between regulatory elements and genes 2,28,29,79 . TAD boundaries, which separate adjacent TADs, have been found to be well conserved across cell types and more evolutionarily constrained than TADs themselves 40 .…”