Quiet Harbor
The effectiveness, duration of action and controllability of many promising methods of gene and cell therapy of oncological, genetic and other diseases can be improved thanks to the so-called "safe genomic ports" (genomic safe harbor, GSH). These are areas in the human genome into which therapeutic genes can be safely embedded without causing undesirable changes in the cell genome that may pose a danger to the patient.
However, it is difficult to find GSH with potential for clinical application, since they must simultaneously have accessibility for genome editing technologies, have no mechanical obstacles in the form of genes and other functional sequences of nucleotides, and ensure high, stable and safe expression of the implanted therapeutic gene.
So far, only a few candidates for GSH have been studied, and all of them could be used with certain reservations. They are either located in genomic regions that are relatively densely populated with genes that may be affected during editing, or contain genes that play a role in the development of oncological diseases that may be accidentally activated. In addition, the GSHS known so far have not been evaluated for the presence of elements that, although not genes, can regulate their expression, as well as whether the inserted genes can change the expression patterns of the entire genome.
A team of researchers from the Harvard Wyss Institute, Harvard Medical School and the Zurich Graduate School of Technology has developed a computational method to identify GSH with a higher potential for the safe insertion of therapeutic genes and their long-term expression in many cell types. Two of the 2,000 such GSH sites were tested by researchers for T-cell therapy and ex vivo gene therapy of skin diseases. They inserted a reporter gene in T cells and a therapeutic gene in skin cells into the identified GSH and demonstrated safe and long-term expression of these genes.
Genome analysis for the presence of GSH
The researchers first created a computational algorithm that allowed them to find areas in the genome that are potentially suitable for use as GSH. In this step-by-step scan of the entire genome, they excluded genes encoding proteins that were involved in tumor formation and genes encoding certain types of RNA that affect expression. Regions containing enhancers, elements that activate gene expression, and regions that include the centers and ends of chromosomes were also excluded in order to avoid errors in replication and segregation of chromosomes during cell division. After such selection, there are about 2,000 candidate loci that should be further investigated for clinical and biotechnological purposes.
Out of 2,000 identified potential "safe havens" into which genes can be safely inserted, the group randomly selected five GSH and examined them on cell lines, inserting genes into each of them using a fast and efficient CRISPR-Cas9-based genome editing strategy. Two GSH showed the highest result: the genes inserted into them showed active expression significantly greater than the expression achieved by inserting genes into two previously identified GSH.
To further study the two most attractive areas of GSH, the group examined them in T cells and skin cells. T cells are used in cell therapy to treat cancer and autoimmune diseases. If the gene encoding the chimeric antigen receptor (CAR) could be neatly inserted into GSH, cell therapy would become safer. In addition, skin diseases caused by pathogenic mutations in genes controlling cell function in various layers of the skin can potentially be cured by introducing and long-term expression of a healthy copy of the mutant gene into GSH rapidly dividing skin cells.
The researchers introduced a fluorescent reporter gene into two new GSH in human T cells derived from blood, and a fully functional LAMB3 gene encoding the laminin subunit (extracellular matrix protein forming basement membranes) in human skin fibroblasts. The group managed to achieve stable expression of inserted genes in parent and daughter cells.
Sequencing of the genome of GSH-based T cells has shown that editing carries a minimal risk of effects that contribute to tumor development, which is always the main problem in the genetic modification of cells for therapeutic use. Identification of GSH in the human genome will significantly accelerate the development of more effective and safe methods of gene and cell therapy. The success achieved on skin cells can be used to rejuvenate it with the help of gene therapy.
Article by E.Aznauryan et al. Discovery and validation of human genomic safe harbor sites for gene and cell therapies is published in Cell Reports Methods. https://www.cell.com/cell-reports-methods/fulltext/S2667-2375(21)00231-9
Aminat Adzhieva, portal "Eternal Youth" http://vechnayamolodost.ru based on the materials of the Wyss Institute for Biologically Inspired Engineering at Harvard: Landing therapeutic genes safely in the human genome.