Gene therapy of blood diseases with retroviral vectors

Pyruvate kinase (PK) deficiency of Basenji dogs is caused by a single base deletion in the L-gene that results in the production of a truncated protein without PK activity. A long-term bone marrow culture (LTBMC) system for gene transfer into hematopoietic progenitor cells was established for normal and PK deficient dogs. This system was useful for efficient gene transfer into myeloid and lymphoid progenitor cells. Gene transfer into hematopoietic progenitor cells of PK deficient dogs was much less efficient than that of normal dogs. Nevertheless, transfer of the R-type PK cDNA into the erythroid progenitor cells of PK deficient dogs corrected the PK phenotype as determined with an in vitro cyanide susceptibility assay. However, the PK deficient phenotype was not corrected in vivo after transplantation of hematopoietic cells that were transduced with a functional R-type PK cDNA in vitro. A chimeric human/pig FVIII retroviral vector was constructed to determine FVIIII coding sequences that negatively regulate FVIII mRNA levels and to investigate novel strategies for gene therapy of hemophilia A. The chimeric human/pig FVIII retroviral vector produced functional FVIII in both viral producer PG13 cells and viral transduced cells. In conclusion, the LTBMC system used in this study was useful for gene transfer into myeloid and lymphoid hematopoietic progenitor cells. This system is applicable to gene therapy of many genetic disorders that require gene transfer into myeloid and lymphoid progenitor cells. Correction of the PK deficient phenotype in canine erythroid progenitor cells by retroviral vector mediated gene transfer demonstrates the potential for gene therapy of PK deficiency. Characterization of the chimeric human/pig FVIII retroviral vector has provided an improved understanding of FVIII gene regulation for design of retroviral vectors that permit high levels of FVIII expression for gene therapy of hemophilia A.