title: Transfer of the human glucocerebrosidase gene into hematopoietic stem cells of nonablated recipients: successful engraftment and long-term expression of the transgene.
abstract: In trying to develop methods of gene therapy for Gaucher disease that will avoid the morbidity and mortality associated with bone marrow (BM) ablation, we transplanted BM stem cells transduced with a retroviral vector containing the human glucocerebrosidase cDNA into normal, nonablated, syngeneic mice. Donor BM from untreated male mice or treated with 5-fluorouracil (5-FU) was transduced ex vivo using a standard 4-day transduction protocol. Recipient female mice were injected one time only or once daily for 5 consecutive days or once a week for 5 consecutive weeks using 2 x 10(7) (untreated BM) or 2 x 10(6) (5-FU-treated BM) cells per injection. Initial transduction efficiency into colony-forming unit-spleen (CFU-S) was 80% to 100%. Recipient analysis was performed at least 6 months after the last transplantation. The best engraftment of donor stem cells, up to 5% by secondary CFU-S analysis, was obtained with multiple injections of transduced BM not previously treated with 5-FU. Polymerase chain reaction (PCR) amplification for both the transgene and the Y chromosome identified the progeny of transduced stem cells in various hematopoietic and non-hematopoietic organs. The copy number of the transgene in stem cells was 0.13 to 2.8. Transgene expression was shown by reverse transcriptase-PCR, in situ hybridization, and immunohistochemistry. No serious side effects of the procedure were noted. We conclude that multiple transplants of retrovirally transduced BM cells into nonablated recipients may be a safe and effective therapeutic modality for a number of genetic hematopoietic disorders.
authors.affiliation: Molecular and Medical Genetics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-1260, USA.
authors.forename: R
authors.initials: R
authors.lastname: Schiffmann
authors.forename: J A
authors.initials: JA
authors.lastname: Medin
authors.forename: J M
authors.initials: JM
authors.lastname: Ward
authors.forename: S
authors.initials: S
authors.lastname: Stahl
authors.forename: M
authors.initials: M
authors.lastname: Cottler-Fox
authors.forename: S
authors.initials: S
authors.lastname: Karlsson
mesh_terms: D000818:Animals; D001483:Base Sequence; D001853:Bone Marrow / Q000201:enzymology; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D018014:Gene Transfer Techniques; D005796:Genes; D005962:Glucosylceramidase / Q000235:genetics*; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D017403:In Situ Hybridization; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D014161:Transduction, Genetic
chemical_list: D017931:DNA Primers; D005962:Glucosylceramidase