Directed evolution of a sequence-specific targeting technology for therapeutic gene delivery to the human genome.

PROJECT SUMMARY Unnecessary risk by random insertional mutagenesis and uncontrolled expression of inserted transgenes represent critical issues with current non-targeted gene therapy approaches. Homology directed repair (HDR) can target inserts but is inefficient, especially in non-dividing cells or if the donor DNA is large. Currently, a

system for efficiently inserting a therapeutic gene to a known sequence is critically needed. To address these challenges, the applicants have developed a novel system for DNA integration by evolving integrase enzymes to insert DNA of flexible size at a desired sequence in the human genome. The long-term goal is to develop

clinical therapies that use insertional vectors to treat genetic disease. The central hypothesis is that directed evolution will produce an integrase capable of targeting a single attP site in the genome without off-target insertion. To demonstrate translational applicability, the evolved integrase will be assayed for delivery of the

therapeutic Factor IX gene to the liver of hemophilic mice. This hypothesis has been formulated on the basis of preliminary data produced in the applicant's laboratories clearly demonstrating that their directed evolution approach successfully improves integrase specificity. The rationale is that, development of this new tool will allow

researchers and clinicians to deliver therapeutic transgenes to a single, known sequence. This would overcome risks of insertional mutagenesis and facilitate predictable transgene expression. In Aim 1, directed evolution will be used to repeatedly evolve and select for variants with improved targeting specificity in order to generate

integrases active on a single sequence in the genome. In Aim 2, evolved integrases will be screened for activity in human cell lines and a genome-wide analysis of possible off-targets will be performed. In Aim 3, the site- specific integrase will be delivered to the liver of a mouse model of Hemophilia B. The therapeutic potential of

treating Hemophilia B with this technology will be assessed. The project is highly innovative because it uses an advanced continuous directed evolution system that is orders of magnitude faster than traditional approaches and has never been applied to improving integrase vectors. These sequence targeting vectors will be combined

with a non-invasive, tissue-specific delivery approach for the first time. The proposed research is significant because it develops a tool capable of safely and efficiently directing therapeutic genes to a desired sequence in the genome without negative off-target consequences. Patients suffering from genetic diseases frequently have

a variety of distinct mutations. This technology could be used to insert a corrected gene copy to treat disease, irrespective of an individual's mutation. Complex disorders could be treated by delivering multiple genes or whole biosynthetic pathways. In order to demonstrate a therapeutic application, Factor IX will be delivered to mice to

model a treatment for Hemophilia B. Significantly, because the size and sequence of the inserted DNA is flexible, this platform technology is adaptable to preclinical research applications as well as potential treatments of any disease requiring gene replacement.