Delivery of Meganucleases for Genome Engineering – DEMENAGE

The DEMENAGE partners are experts in designing and using sequence specific endonucleases (meganucleases) for gene targeting in mammalian cells. Meganucleases have a broad potential for inactivating genes or precisely inserting DNA sequences into the genome for biotechnological or therapeutic purposes.
DEMENAGE focuses on therapeutic gene transfer and will develop meganucleases, as well as specialized vectors for their delivery, in order to achieve targeted integration of therapeutic transgenes. DEMENAGE partners will develop an approach whereby therapeutic sequences will be systematically targeted to a dedicated locus (safe harbor) in the human genome. For this we will characterize a safe harbor in the human genome, where insertion of the transgene would be without unwanted toxic consequences and develop a meganuclease that allows targeting it.
In parallel, the issues of efficiency and toxicity of meganuclease delivery into the target cell will be addressed. Advanced meganuclease design will be incorporated into viral vectors derived from Lentiviruses or Parvoviruses (AAV). A central goal of the project is to deliver the nuclease transiently, preferably under the form of a protein, into the target cell. Accordingly, the nuclease will solely perform its hit-and-run function without the potential toxicity due to a sustained exposure of the genome. Lentiviral-based vectors will be tested ex-vivo in hematopoietic stem cells and in keratinocytes, whereas the AAV vectors will be used for in toto delivery to the liver. DEMENAGE should bring out advanced reagents for safe and efficient delivery of meganucleases, that will be valuable for gene therapy applications and beyond.