title: Correction of the enzyme deficiency in hematopoietic cells of Gaucher patients using a clinically acceptable retroviral supernatant transduction protocol.
abstract: Gaucher disease is a lysosomal storage disorder caused by a deficiency of the enzyme glucocerebrosidase (GC), and is an excellent candidate for gene replacement therapy. To develop a clinically acceptable protocol for this purpose, we created two amplified (A) high-titer retroviral vector-producer cell lines to efficiently transduce hematopoietic stem and progenitor cells. GP+envAm12/A-LGSN (A-LGSN), contained the GC cDNA driven by the retroviral long terminal repeat (LTR) and the neomycin phosphotransferase gene expressed from the simian virus 40 early promoter. GP+envAm12/A-LG4 (A-LG4) contained only the GC gene driven by the LTR. Both A-LGSN and A-LG4 contained multiple proviral copies and gave approximately 10-fold higher titers on 3T3 cells compared to their unamplified counterparts. These vectors were packaged in GP+envAm12 cells because vectors produced in this cell line transduced hematopoietic cells more efficiently than other packaging cells tested. Bone marrow mononuclear cells and purified CD34+ cells were infected with virus supernatants four times in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) over 96 hours in culture. Cells were then plated in semisolid cultures and colony-forming unit-granulocyte/macrophage (CFU-GM) colonies were scored for vector presence by polymerase chain reaction (PCR). Transduction efficiency of CFU-GM colonies derived from CD34+ cells was improved considerably using the amplified vectors in the GP+envAm12 packaging line. For A-LGSN, A-LG4, and unamplified LGSN, transduction efficiencies were 41, 42, and 25%, respectively. Therefore, multiple proviral copies resulting in higher titer improves retroviral transduction of human hematopoietic progenitor cells. Hematopoietic cells from Gaucher patients were transduced and placed into long-term bone marrow culture (LTBMC). Viral supernatant from the amplified producer lines transduced long-term culture initiating cells (LTCIC) efficiently (30 to 50%) using this clinically acceptable protocol. Both sustained mRNA expression and GC enzyme production are achieved in the long-term culture of LTCIC and lead to correction of the GC deficiency in their progeny cells.
authors.affiliation: Molecular and Medical Genetics Section, NINDS, Bethesda, MD 20892.
authors.forename: L
authors.initials: L
authors.lastname: Xu
authors.forename: S K
authors.initials: SK
authors.lastname: Stahl
authors.forename: H P
authors.initials: HP
authors.lastname: Dave
authors.forename: R
authors.initials: R
authors.lastname: Schiffmann
authors.forename: P H
authors.initials: PH
authors.lastname: Correll
authors.forename: S
authors.initials: S
authors.lastname: Kessler
authors.forename: S
authors.initials: S
authors.lastname: Karlsson
mesh_terms: D001854:Bone Marrow Cells; D002478:Cells, Cultured; D005776:Gaucher Disease / Q000201:enzymology / Q000628:therapy*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D005962:Glucosylceramidase / Q000172:deficiency / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000201:enzymology*; D006801:Humans; D019868:Kanamycin Kinase; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D012190:Retroviridae / Q000235:genetics*; D013539:Simian virus 40 / Q000235:genetics
chemical_list: D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D005962:Glucosylceramidase