1054. Hematopoietic and Non-Hematopoietic Engraftment after Bone Marrow Transplantation in Newborn Mice

Classical gene therapy protocols have employed different vehicles (e.g. viruses, liposomes, peptides) for tissue specific gene delivery. An alternative approach for delivery of therapeutic genes to specific tissues is to use adult stem cells as the 'vector' for a combination of cell and gene therapy. Ideally, bone marrow derived cells (BMDC) could be taken from a patient, genetically corrected in vitro via gene therapy, and then infused back to the patient to repair damaged tissue as fully functional genetically corrected cells. Recent discoveries show that, in addition to hematopoietic stem cells (HSC) that differentiate into all mature cells of the blood, there are BMDC that can engraft as epithelial cells throughout the body including the lung, liver and GI tract following transplantation into lethally irradiated recipients. Thus, autologous BM transplantation (BMT) based gene therapy could be useful for diseases associated with dysfunctions of these tissues. Treatments for several genetic diseases, to be effective, should be performed at the time of birth or in early childhood. BMT preparative regimens are well described for adult mice, but protocols for myelosuppression in newborn murine pups are few. Here, we compared different preparative regimens for newborn mice including: a) two doses of busulfan (B) in utero (15gr/kg each), b) one dose of busulfan (15gr/kg) in utero plus 400 cGy total body irradiation (TBI) on day 1 of life, c) 400 or 750cGy TBI on day 1 of life, or d) no pretreatment. For each regimen, the pups were transplanted with BM within 24h of birth (after irradiation). In order to determine which method is least toxic to the newborn mice while allowing a high level of hematopoietic engraftment, we assessed peripheral blood counts as well as donor hematopoietic and nonhematopoietic (epithelial) engraftment were evaluated at multiple times post-transplant. The level of toxicity was from highest to lowest: TBI 750 > 1B+ TBI400 > TBI400 > 2|[times]|B > no myelosuppression, and TBI 750 mice were sacrificed prior to 1 month post-BMT. The scale of hematopoietic chimerism was from best to worst: 1B +TBI400 > TBI750=TBI400 > 2|[times]|B > non- myeloablation, with peripheral blood chimerism up to 86% in 1B+ TBI400 treated mice and just 1.0% for non-myeloablated mice. BM derived (BMD) epithelial cells were found in lung and GI but not in the liver of transplanted recipients. The percentage of BMD epithelial cells ranged from 0-0.02% with the highest levels in the 2|[times]|B groups and 1B+ TBI400 (1B+ TBI400 = 2|[times]|B > 400TBI > no myeloablation). This level of BMD epithelial cells is similar to the percentage found after BM transplantation into lethally irradiated adult mice. In conclusion, our data suggest that the best myelosuppressive preparative regimens for the hematopoietic and nonhematopoietic engraftment include in utero exposure to busulfan, and that BMT in newborn mice leads to levels of epithelial engraftment that are similar to those in adult recipients.