Safe landing of therapeutic genes in humans

Image: A collaborative research team at Harvard University’s Wyss Institute and the ETH Institute in Zurich in Switzerland has identified genomic safe ports (GSHs) in a disturbed landscape of human genome sequencing for the landing of therapeutic genes. As part of their validation, they inserted a fluorescent GFP reporter gene into the candidate GSHs and followed its expression over time. GSHs could enable safer and more persistent expression of genes in future genetic and cellular therapies. This team illustration won the cover of the Cell Reporting Methods issue in which the study was published.
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Credit: Eric Aznorian

(Boston) — Many of the future gene and cell therapies for diseases such as cancer, rare genetic conditions and others could be improved in terms of their efficacy, persistence and predictability through so-called “genomic safe harbors” (GSHs). These are landing sites in the human genome that are able to safely accommodate new therapeutic genes without causing other unintended changes to the cell’s genome that could pose a risk to patients.

However, finding GSHs with clinical translation potential was as difficult as finding a lunar landing site for a spacecraft – which had to be in a smooth and easily accessible area, not too steep and surrounded by large hills or slopes, provide good visibility, and enable a return Safe. Likewise, GSH needs to be accessible by genome-editing technologies, free from physical barriers such as genes and other functional sequences, and permitting high, stable and safe expression of a ‘stable’ therapeutic gene.

So far, only a few candidate GSHs have been explored and all come with some caveats. Either they are located in genomic regions that are relatively dense with genes, which means that one or more of them could be compromised in their function by a therapeutic gene inserted in their vicinity, or they contain genes that have a role in the development of cancer that could be inadvertently Enabled. In addition, candidate GSHs were not analyzed for the presence of regulatory elements that, although not genes per se, could regulate the expression of genes from afar, nor whether the inserted genes alter global gene expression patterns in cells across the entire genome.

Now, a collaboration of researchers at Harvard University’s Wyss Institute for Bioinspired Engineering, Harvard Medical School (HMS), and ETH Zurich in Switzerland has developed a computational approach to identify high-potential GSH sites for the safe introduction and durable expression of therapeutic genes across many cell types. For two of the 2,000 predicted GSH sites, the team provided in-depth validation of adoptive T-cell therapies and in vivo Genetic therapies for skin diseases. By engineering specific 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 was published in Cell Reporting Methods.

“While GSHs can be used as universal landing platforms for targeting genes, thus accelerating the clinical development of gene and cell therapies, to date no site in the human genome has been fully validated and all are only acceptable for research applications,” said Wyss primary faculty member George Church, Ph. , lead author of the study. “This makes our collaborative approach to highly validated GSHs an important step forward. Together with the more effective target gene integration tools we develop in vitro, these GSHs can enable a variety of future clinical translation efforts.” Church is the leader of the synthetic biology platform. at the Wyss Institute, as well as the Robert Winthrop Professor of Genetics at HMS and Professor of Health Sciences and Technology at Harvard University and the Massachusetts Institute of Technology (MIT).

Genome screening for GSHs

The researchers initially created a computational pipeline that allowed them to predict regions in the genome with potential for use as natural gas buffers by harnessing the wealth of available sequencing data from human cell lines and tissues. “In this step-by-step whole-genome scan, we have computationally excluded regions that encode proteins, including those involved in tumorigenesis, and regions that encode specific types of RNA with functions in gene expression and other cellular processes. We also excluded regions that contain on so-called enhancer elements, which activate gene expression, often from afar, and regions comprising the centers and ends of chromosomes to avoid errors in replication and segregation of chromosomes during cell division,” said first-author Eric Aznorian, PhD. “We left this with about 2,000 candidate sites for further investigation for clinical and biotechnological purposes.”

Aznauryan started the project as a graduate student with other members of Sai Reddy’s lab at ETH’s Department of Biosystems Science and Engineering in Zurich before visiting Church’s lab as part of his graduation work, collaborating with fellow Wyss Technology Development, Denitsa Milanova, Ph. .Dr. He has since joined the Church Group as a Postdoctoral Fellow. Reddy, lead author and lead author of the collaborative study, is Associate Professor of Systems and Artificial Immunology at ETH Zurich and is focused on developing new methods in systems and synthetic biology to engineer immune cells for diverse research and clinical applications.

Of the 2,000 GSH loci identified, the team randomly selected five and screened them in common human cell lines by inserting reporter genes into each of them using a fast and efficient CRISPR-Cas9-based genome editing strategy. “Two of the GSH sites allowed for particularly high expression of the inserted reporter gene—in fact, much higher than the expression levels achieved by the team with the same reporter gene engineered in two generations of the previous generation of GSH. Importantly, the reporter genes harbored by the two GSH sites It did not regulate any genes associated with cancer,” Aznorian said. This could also become possible because regions in the genome that are far from each other in the linear DNA sequence of chromosomes, but near the 3D genome, where different regions of the folded chromosomes come into contact with each other, can be jointly affected when an additional gene is inserted .

Looking forward to clinical translation

To evaluate the most pressing GSH sites in human cell types with interest in cellular and genetic therapies, the team examined them in immune T cells and skin cells, respectively. T cells are used in a number of adoptive cell therapies to treat cancer and autoimmune diseases that could be much safer if a receptor-encoding gene was stably inserted into GSH. Also, skin diseases caused by harmful mutations in the genes that control the function of cells in the different layers of the skin can be treated by the introduction and long-term expression of a healthy copy of the mutated GSH gene of the dividing skin cells that nourish those layers.

“We have introduced a fluorescent reporter gene into two new sets of GSHs in primary human T cells obtained from blood, and a fully functional organ LAMB3 The gene, an extracellular protein in the skin, is in the same GSHs in human primary dermal fibroblasts, and observed long-lasting activity,” Milanova said. “While GSHs are uniquely positioned to improve levels and persistence of gene expression in parental and daughter cells for treatment, I am particularly excited about the treatment. Concerning emerging cellular ‘gain of function’ enhancements that can increase the normal function of cells and organs. Hence the safety aspect is of paramount importance.” With a leading team at Wyss, Milanova is developing a platform for genetic regeneration and enhancements with a focus on skin rejuvenation.

“Our comprehensive sequencing analysis in GSH-engineered primary human T cells clearly showed that the insertion has the lowest potential to cause tumor-promoting effects, which is always a major concern when cells are genetically modified for therapeutic use,” Reddy said. “Determining multiple GSH loci, as we did here, also supports the possibility of building more advanced cellular therapies that use modifier genes to program complex cellular responses, and this is particularly important in engineering T cells for cancer immunotherapy.”

This collaborative, interdisciplinary effort demonstrates the power of integrating computational methods with genome engineering while maintaining a focus on clinical translation. Identification of GSHs in the human genome will greatly augment future developmental therapeutic efforts focused on engineering more effective and safer gene and cellular therapies,” said Wyss founding director Donald Ingber, PhD, who is also the founding director. Yehuda Volkman Professor of Vascular Biology at HMS and Boston Children’s Hospital, and Professor of Bioengineering at Harvard’s John A. Paulson School of Engineering and Applied Science.

Additional authors on the study are Alexander Yermanos, Ph.D., and Ido Kapetanovic, members of the Reddy group; Anna Defoe from University of Basel, Switzerland; and Elvira Kinsina of the McGovern Institute for Brain Research at the Massachusetts Institute of Technology. The study was supported by ETH Research Grants, the Helmut Horten Stiftung Fund and Research on Aging and Longevity at HMS, as well as a 2019 Genome Engineer Innovation Grant from Synthego to Aznauryan.

Media contact

Wyss Institute for Biologically Inspired Engineering at Harvard University
Benjamin Buettner,, +1 617-432-8232

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Wyss Institute for Biologically Inspired Engineering at Harvard University ( uses Nature’s design principles to develop bio-inspired technologies that will transform medicine and create a more sustainable world. Wyss researchers develop new and innovative solutions for healthcare, energy, architecture, robotics, and manufacturing that translate into commercial products and treatments through collaborations with clinical investigators, corporate alliances, and the formation of new start-ups. The Wyss Institute creates transformative technology breakthroughs by engaging in high-risk research, transcending disciplinary and institutional barriers, working as an alliance of Harvard Medical, Engineering, Art, Science and Design, and in partnership with the Beth Israel Deaconess Medical Center. Brigham and Women’s Hospital, Boston Children’s Hospital, Dana-Farber Cancer Institute, Massachusetts General Hospital, University of Massachusetts Medical School, Spaulding Rehabilitation Hospital, Boston University, Tufts University, Charity-University Medicine Berlin, University of Zurich, and MIT.

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