A novel method of in vivo genome editing with the use of hybrid nucleases, such as ZFN, TALEN and the CRISPR/Cas9 system for the purpose of precise genome editing in vivo
Hybrid nucleases, including ZFN, TALEN and the CRISPR/Cas9 system, constitute indispensable tools in genome. The use of designer nucleases outperforms other technologies for genome engineering, such as viral vectors, which carry considerable risk of genetic alterations of target cells and, in a consequence, may lead to malignant transformation if inserted into inappropriate sites of the genome. Thus, development of precise genome editing technologies enabled improving safety profile of gene therapy clinical trials and holds great promise for regenerative medicine in the future.
In most instances, designer nuclease activity is needed only transiently – just long enough to cleave the target site. Prolonged expression increases the risk of so-called off-target activity, which can be genotoxic. Transient expression can be achieved ex vivo by delivering the nucleases in the forms of mRNA or protein.
For in vivo delivery, however, these platforms are not efficient. Moreover, gene delivery via liposomes or other artificial nano-vesicles seem to be inefficient and may trigger an immune response in the host, which decreases expected biological effect. Therefore, the use of EVs, which play an important role in cell-to-cell communication, constitute an attractive technological solution to deliver designer nucleases.
EVs are natural nanovesicles released by any type of cells in activated or steady-state conditions. They are composed of a cellular membrane enclosing cytosolic components. By transferring their bioactive cargo, including small RNAs, messenger RNAs, proteins and lipids, EVs may influence fate decisions of the acceptor cells.
Based on their size and origin, EVs can be divided into exosomes and microvesicles. Utility of EVs derived from various cell populations, including stem cells, in transferring biological cargo to other cells has gain considerable interest in recent years. There have been attempts to use EVs for promoting neurogenesis; treatment of neuronal diseases; in wound healing; treating cancer, pain treatment and many others. However, antigen-defined stem cell-derived EVs (in particular ectosomes) has never been considered.
The subject of the offer is a new method of in vivo genome editing with the use of hybrid nucleases, such as ZFN, TALEN and the CRISPR/Cas9 system, delivered to target cells by EVs derived from stem cells in serum-free conditions and defined as the RNA-containing vesicles expressing stem cells surface markers: CD90, CD105, CD147, CD309 (in more than 20% of RNA-positive events) and lack of expression CD45.
- novel tool for efficient in vivo genome editing for treatment of variety genetic diseases in human patients i.e. liver-associated diseases, spleen-associated diseases, lung-associated diseases, heart-associated diseases and kidney-associated diseases,
- antigen defined EVs (CD90+/CD105+/CD147+/CD309+/CD45-) can be collected from various populations of stem cells, including mesenchymal stem cells and induced pluripotent stem cells,
- surface of EVs can be either unmodified, carrying natural panel of stem cell specific antigens, or can be genetically or chemically modified to display additional receptors, ligands or other surface molecules, to increase targeting a specific tissue or a desired cell type,
- EVs are collected in a serum-free medium, which is of great significance for future clinical applications to treat human diseases,
- since the data showed enhanced gene targeting in the liver using this technology, liver-associated diseases can be of particular interest as potential targets, including i.e.: phenylketonuria, amyloidosis, cirrhosis, cancer and carcinomas, hypercholesterolemia, non-alcoholic liver disease, amyloid neuropathy, cystic fibrosis, glycogen storage diseases, polycystic kidney and hepatic disease, hepatic failure, hepatic lipase deficiency, medullary cystic kidney disease, hepatoblastoma, hepatic adenoma, and others,
- technology can be used to create gene knockouts, knock-ins and to achieve gene correction, if additional DNA donor is provided (ss/sdDNA oligos, plasmid DNA, viral vectors).
The offered in vivo delivery system of the genome DNA modifying enzymes is patent pending. Further research and development of the invention are continued at the Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University and at the Albert-Ludwigs University Freiburg in Germany. Currently the Centre for Technology Transfer CITTRU is looking for entities interested in commercial application of the invention.