Making in utero gene therapy safer and more efficient: A first step towards clinical realization

Gene therapy harbors the potential to treat and cure some of the most devastating diseases affecting humans. Monogenic disorders are the ideal target for developing and testing gene therapy procedures that will allow correction of genetically-based diseases. In this study gene therapy tools were developed and evaluated in an effort to improve the efficacy and safety of viral vectors. A two-vector delivery system was constructed to perform cell-specific in utero gene therapy, and I showed proof-of-principle that the system functions in vitro in human and sheep cells. An Ad5 serotype adenoviral vector (Ad5) acts as the first delivery-vehicle, which carries a hepatocyte-specific promoter (pHep) that drives transcription of the murine cationic amino acid transporter type 1 (mCAT-1) receptor gene which is found only in mouse and rat. The murine stem cell virus (MSCV) gamma-retroviral vector (MR4IN) acts as the second delivery-vehicle, which carries the therapeutic protein and is packaged with the murine ecotropic envelope which restricts binding of the MR4IN vector to the mCAT-1 receptor, thus limiting its transduction to human or sheep cells that have been transduced by the first vector and express the vector-encoded mCAT-1. The pHep promoter activated GFP-expression primarily in human hepatocarcinoma cells (HepG2) in vitro, indicating that this system could be used to achieve targeted transduction of human hepatocytes. Also, upon transfection with the plasmid construct for the first vector, sheep bone marrow mesenchymal stem cells (sheep BM MSC) demonstrated the ability to produce functional mCAT-1 receptor proteins at their cell surface, which specifically allowed transduction by the MR4IN viral vector.;These studies have thus established a foundation from which the Two-Vector System will be completed for potential use in treating or curing genetic disorders in utero before disease onset. Also, sheep were evaluated as a potential pre-clinical experimental animal model for testing strategies for circumventing pre-existing adeno-associated virus (AAV) neutralizing antibodies. An indirect ELISA was performed using the AAV serotypes as antigen and sheep serum as the primary antibody, and it was discovered that sheep are the only large animal model (to our knowledge) that makes anti-AAV antibodies to AAV serotypes 1, 2, 9 and possibly 8. These results suggest that sheep may be employed for testing gene therapy strategies for circumventing pre-existing neutralizing antibodies that could permit the use of AAV-based gene therapy procedures in prenatal and postnatal human subjects. Finally, linker-mediated polymerase chain reaction (LM-PCR) was employed to pinpoint MSCV gamma-retroviral vector integration sites and thereby analyze the safety of these sites in sheep hematopoietic stem cells. The results revealed that MSCV integrated near cancer-related genes in over 59% of the retrievable integration sites. This result demonstrates the need to fully evaluate viral vectors to determine their safety for use in human therapies and to undertake efforts to re-engineer these tools to make them safe for treatments in human. These studies represent a step forward in improving the efficacy and safety of gene therapy viral vectors so that they may be used as therapeutics for genetically-based human disorders.