Developing a protocol in vivo for recombinant adeno-associated virus-mediated gene therapy of hemoglobinopathies

Towards gene therapy for thalassemias and sickle cell disease this laboratory has constructed recombinant adeno-associated viruses (rAAVs) that harbored human α- and β-globin cDNAs/ genes, which efficiently expressed globin mRNAs and polypeptides in erythroid cells (Ohi, S. et al., Gene 89: 279, 1990; Dixit, M. et al., Gene 104: 253, 1991; Ohi, S. and Kim, B.C., Blood 84: 263a, 1994, J. Pharm. Sei., in press, 1995). As the test system, we are now developing a protocol(s) for gene therapy of a mouse model of β-thalassemia (C57BL/6Hbb.th/Hbb.th) using the rAAVs with a possible application to humans in the future. Mouse bone marrow hematopoietic stem/progenitor cells (BMHSCs) were purified/ enriched by Histopaque 1077 (Sigma, density 1.077 g/c.c.) density gradient centrifugation and immunomagnetic purging (Ohi, S. et al. FASEB J. 7: A1304, 1993). The L3T4-, Lyt2-, Thy 1.2+ cells had a capability to differentiate into BFU-E, CFU-GM, and CFU-GEMM in the methyl-cellulose clonogenic assay. In addition, when the host marrow cells were depleted by 70% by cyclophosphamidetreatment, about 10,000 purified BMHSCs restored the cell number of the host marrow in about a week. The rAAV vectors effected synthesis of human hemoglobin in the BMHSCs in culture. (Supported by grants from The American Heart Association-NCA and The Armstead-Barnhill Foundation for Sickle Cell Anemia)

Stress neuropeptide levels in adults with chest pain due to coronary artery disease: potential implications for clinical assessment

: Substance P (SP) and neuropeptide Y (NPY) are neuropeptides involved in nociception. The study of biochemical markers of pain in communicating critically ill coronary patients may provide insight for pain assessment and management in critical care. Purpose of the study was to to explore potential associations between plasma neuropeptide levels and reported pain intensity in coronary critical care adults, in order to test the reliability of SP measurements for objective pain assessment in critical care