Landing therapeutic genes safely in the human genome

Researchers at Harvard’s Wyss Institute and ETH Zurich’s Department of Biosystems Science and Engineering predict and validate genomic safe harbor sites for therapeutic genes, enabling safer, more efficient, and predictable gene and cell therapies.

Aznauryan-Erik_genomic-safe-harbor-sites_2022_Cell-Reports-Method
A collaborative research team at Harvard’s Wyss Institute and the ETH Zurich in Switzerland has identified genomic safe harbors (GSHs) in the tumultuous see of human genome sequence to land therapeutic genes in. As part of their validation, they inserted a fluorescent GFP reporter gene into candidate GSHs and followed its expression over time. The GSHs could enable safer and longer-lasting expression of genes in future gene and cellular therapies. This illustration won the team the cover of the Cell Reports Methods issue the study is published in. (Image credit: Erik Aznauryan, Wyss Institute at Harvard University).

Many future gene and cell therapies to treat diseases like cancer, rare genetic and other conditions could be enhanced in their efficacy, persistence, and predictability by so-called genomic safe harbors (GSHs). These are landing sites in the human genome able to safely accommodate new therapeutic genes without causing other, unintended changes in a cell’s genome that could pose a risk to patients.

However, finding GSHs with potential for clinical translation has been as difficult as finding a lunar landing site for a spacecraft — which has to be in smooth and approachable territory, not too steep and surrounded by large hills or cliffs, provide good visibility, and enable a safe return. A GSH, similarly, needs to be accessible by genome editing technologies, free of physical obstacles like genes and other functional sequences, and allow high, stable, and safe expression of a “landed” therapeutic gene.

Thus far, only few candidate GSHs have been explored and they all come with certain caveats. Either they are located in genomic regions that are relatively dense with genes, which means that one or several of them could be compromised in their function by a therapeutic gene inserted in their vicinity, or they contain genes with roles in cancer development that could be inadvertently activated. In addition, candidate GSHs have not been analyzed for the presence of regulatory elements that, although not being genes themselves, can regulate the expression of genes from afar, nor whether inserted genes change global gene expression patterns in cells across the entire genome.

Now, a collaboration of researchers at external pageHarvard’s Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, and ETH Zurich’s Department of Biosystems Science and Engineering has developed a computational approach to identify GSH sites with significantly higher potential for the safe insertion of therapeutic genes and their durable expression across many cell types. For two out of 2,000 predicted GSH sites, the team provided an in-depth validation with adoptive T cell therapies and in vivo gene therapies for skin diseases in mind. By engineering the identified GSH sites to carry a reporter gene in T cells, and a therapeutic gene in skin cells, respectively, they demonstrated safe and long-lasting expression of the newly introduced genes. The study is published in external pageCell Reports Methods.
 

The identification of multiple GSH sites is especially relevant in T cell engineering for cancer immunotherapy.  Sai Reddy, D-BSSE Laboratory for Systems and Synthetic Immunology

The researchers first set up a computational pipeline that allowed them to predict regions in the genome with potential for use as GSHs by harnessing the wealth of available sequencing data from human cell lines and tissues. "In this step-by-step whole-genome scan we computationally excluded regions encoding proteins, including proteins that have been involved in the formation of tumors, and regions encoding certain types of RNAs with functions in gene expression and other cellular processes. We also eliminated regions that contain so-called enhancer elements, which activate the expression of genes, often from afar, and regions that comprise the centers and ends of chromosomes to avoid mistakes in the replication and segregation of chromosomes during cell division,” said first-author Erik Aznauryan, Ph.D. “This left us with around 2,000 candidate loci all to be further investigated for clinical and biotechnological purposes.”

Aznauryan started the project as a graduate student with other members of Sai Reddy’s Systems and Synthetic Immunology lab before he teamed up with Wyss Fellow Denitsa Milanova, co-author of the study. He since has joined the group of Wyss Core Faculty member George Church as a Postdoctoral Fellow.

Out of the 2,000 identified GSH sites, the team randomly selected five and investigated them in common human cell lines by inserting reporter genes into each of them using a rapid and efficient CRISPR-Cas9-based genome editing strategy. “Two of the GSH sites allowed particularly high expression of the inserted reporter gene — in fact, significantly higher than expression levels achieved by the team with the same reporter gene engineered into two earlier-generation GSHs. Importantly, the reporter genes harbored by the two GSH sites did not upregulate any cancer-related genes,” said Aznauryan. This also can become possible because regions in the genome distant from one another in the linear DNA sequence of chromosomes, but near in the three-dimensional genome, in which different regions of folded chromosomes touch each other, can become jointly affected when an additional gene is inserted.

To evaluate the two most compelling GSH sites in human cell types with interest for cell and gene therapies, the team investigated them in immune T cells and skin cells, respectively. T cells are used in a number of adoptive cell therapies for the treatment of cancer and autoimmune diseases that could be safer if the receptor-encoding gene was stably inserted into a GSH. “An extensive sequencing analysis that we undertook in GSH-engineered primary human T cells clearly demonstrated that the insertion has minimal potential for causing tumor-promoting effects, which always is a main concern when genetically modifying cells for therapeutic use,” said Reddy. “The identification of multiple GSH sites, as we have done here, also supports the potential to build more advanced cellular therapies that use multiple transgenes to program sophisticated cellular responses, this is especially relevant in T cell engineering for cancer immunotherapy.”

This text is a shortened version of a external pagepress release issued on 24 January 2022 by the external pageWyss Institute for Biologically Inspired Engineering at Harvard University.

Publication details:

Aznauryan, E, A Yermano, E Kinzina, A Devaux, E Kapetanovic, D Milanova, G M Church, and S T Reddy (2022) Discovery and validation of human genomic safe harbor sites for gene and cell therapies. Cell Reports Methods, external pagehttps://doi.org/10.1016/j.crmeth.2021.100154

Learn about the Systems and Synthetic Immunology lab led by Sai Reddy.

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