TRANSPOSON TARGETED INTEGRATION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this application is to develop novel methods for producing transgenic animals, with an eventual emphasis for the techniques learned during these experiments to be applied in gene therapy. To that effect we have developed a new active method of transgenesis that is several-fold more effective than the existing passive methods. The technique relies on a mutated hyperactive Tn5 transposase protein (*Tn5p) for the active insertion of a transgene into animal chromosomes during intracytoplasmic sperm injection (ICSI). This procedure named TN:ICSI integrates foreign DNA into the animal genome with microinjection and traditional ICSI-Transgenesis. The establishment of the active TN:ICSI technique has encouraged us to investigate methods for applying Tn5 transposase mediated transgenesis to gene therapy. We intend to accomplish this by the production of hybrid proteins with DNA recognition domains attached to the *Tn5 gene. This will be accomplished by applying principles of chimeric transposon technology (CTT) to a hybrid transposon construct and attempt to use such constructs in transgenesis and gene therapy trials in rodents. Gene therapy is defined as the means of curing genetic diseases which are inheritable via the patient's parents during fertilization. Such diseases tend to be recessive in their genetic signature and only exhibit their full effect in homozygous patients. Therefore, it is not unusual to find two heterozygous parents who are carriers for the disease with no symptoms, and a homozygous offspring who demonstrates a dramatic phenotype for the condition. The goal of gene therapy is to insert a healthy crop of a gene into a cell where it can take over for a faulty version and therefore correct such genetic diseases by using molecular biology techniques. Current gene therapy efforts have utilized deactivated Lentiviral vectors for shuttling therapeutic genes into patients cells, sometimes with disastrous consequences. The activation of oncogenes by insertional mutations caused during the vectors integration into the patients genome necessitates that alternative active gene insertion methods need to be explored. We hope that site directed *Tn5 transgene insertions during transgenesis will prove to be a good alternative to the Lentiviral technique. PI has started his collaboration with Dr. Joseph Kaminski of the Medical College of Georgia (MCG) for the synthesis of Chimeric Transposons (CTT). The constructs will target DNA regions of interest according to the DNA binding domains (DBD) attached to them, for targeted integration of transgenes contained in transposons. The eventual goal is to affect gene therapy in whole animals.