New Research Identifies Safe Harbors for Gene Editing Therapies

Gene therapies can replace defective genes, enhance cell functions and improve the safety of engineered cells. Thanks to the rapid development of genome engineering technologies, Gene therapies have the potential to treat once-incurable diseases. However, transgenes are delivered with lentivirus/retrovirus vectors and integrated into the genome in a random or semi-random manner, leading to unpredictable gene expression patterns, disruption of endogenous transcription, and malignancy. A large amount of effort has been spent to establish complicate experiment systems to identify and prevent these potential deleterious effects.

One approach to improve the safety of gene therapy is to deliver transgenes into predefined genomic loci called genomic safe harbors (GSHs). Transgenes integrated into GSHs can be stalely expressed and have minimal effects on normal cellular functions. So far, only a few human GSHs have been defined, including AAVS1, CCR5, and Rosa26 loci. However, none of the current GSH sites show adequate evidence for therapeutic safety. In addition, since the functional consequences of genomic alternation may only be detected in a specific cell type during a specific development stage and/or under specific conditions, it is challenging to comprehensively evaluate all these factors.

In this study, we developed a novel GSH discovery approach. First, we selected GSH candidate regions based on polymorphic mobile elements insertions (pMEIs) that are identified in more than 10% healthy human populations. Because these pMEIs have been subjected topurifying selection and remained common in human populations, they marked genomic regions that are selectively neutral with little or no impact on genomic functions. These large DNA fragments integrated in these candidate GSHs for thousands of years provides extra information that is not available from any cell or animal models. 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.

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Second, unlike most current GSH mapping approaches that mask genome with arbitrary defined linear windows near important DNA elements, 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. Thus, it is crucial that the GSHs are outside of the repressive/heterochromatin regions. To this end, we use tissue-specific epigenetic signatures to identify genomic regions that are open for transcription in the tissue of interest. This step is crucial for GSH selection, as we found that 90% of the pMEIs overlaps repressive chromatin marks.We also developed a computer program, Genomics and Epigenetic Guided Safe Harbor mapper (GEG-SH mapper), to facilitate this knowledge-based tissue-specific GSH selection process. 

One potential application is to generate safer and more efficient Chimeric Antigen Receptor (CAR) T-cells. CAR T-cell therapy is a breakthrough in the field of cancer treatment. During CAR T-cell manufacturing, viral vectors are commonly used to deliver the chimer receptor sequences into ex vivo expanded T-cells. Due to the semi-random integration pattern of lentiviral vectors, there are potential risks for magnificent transformation of engineered CAR-T cells. In addition, the CAR expression can also be silenced by the chromatin of host cell and lead to sub-optimal antitumor efficiency. By collaborating with the Experimental Cellular Therapeutics Laboratory at St. Jude Children’s Research Hospital, we have generated different epigenomic and chromatin organization profiles in T-cells. Using our GEG-SH mapper, we further identified 11 candidate T-cell specific GSHs. We are now testing the efficiency and safety of those loci. If successful, this will lead to more uniform CAR expression and less cellular toxicity. 

In summary, combining with the fast-growing genome engineering technologies, this new approach has the potential to improve the overall safety and efficiency of gene and cell-based therapy in the near future.