Long-term in vitro correction of a-L-iduronidase deficiency ( Hurler syndrome ) in human bone marrow ( gene therapy / mucopolysaccharidosis type I / autosomal recessive )

Content Provider	Semantic Scholar
Author	Fairbairn, Leslie J. Lashford, Linda S. Spooncer, Elaine McDermott, Ruth Lebens, G. A. Bellantuono, I. Holt, R. Hatton, C. E. Cooper, Ab Gt, Nepom Wraith, James E. Dexter, T. Michael
Copyright Year	2005
Abstract	Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for a-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels ofenzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor. Mucopolysaccharidosis (MPS) type I is a lysosomal storage disorder caused by a deficiency of the enzyme a-L-iduronidase (EC 3.2.1.76; IDUA) and is characterized by accumulation of the glycosaminoglycans dermatan and heparan sulfate (1, 2). The clinical phenotype can be very variable (3). In the most severe form of Hurler syndrome, individuals show progressive neurological dysfunction, have multiple skeletal and soft tissue abnormalities, and generally die within the first decade. In the relatively mild Scheie syndrome, patients present much later in life, may have detectable but reduced (1-5% normal) levels of IDUA activity, much milder symptoms with no neurological dysfunction, and live into adulthood (4). As yet there is no specific therapy for the disorder apart from allogeneic bone marrow transplantation. This approach relies on the donor bone marrow producing and secreting functional enzyme, which is subsequently taken up by and targeted to the lysosomes of other cell types. This approach has been shown to have modest success, particularly when the transplant is undertaken before the age of 18 months. After successful transplantation, significant and progressive orthopedic problems remain and are the main problem in clinical management. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. The ultimate neurological prognosis remains guarded, but a clear slowing of neurological deterioration has been documented in patients transplanted at an early age (5-8). Unfortunately, allogeneic transplantation is limited both by the availability of donors and by significant morbidity in matched unrelated donor transplants. The cDNA and gene for IDUA have been cloned (9, 10) and expression of the cDNA in vitro in fibroblasts derived from a patient with Hurler syndrome leads to correction of the enzyme deficiency, localization of functional enzyme to the lysosomes, and reduction in the accumulation of sulfated glycosaminoglycans by corrected cells (11). Infusion of purified human IDUA in a canine model of Hurler syndrome led to normalization of lysosomal storage in some soft tissues, but not in brain, heart valves, or cornea (12). In the present report, we provide evidence that the IDUA cDNA can be transferred into primitive hemopoietic cells, that enzyme levels can be corrected, and that phenotypic correction of the deficiency occurs in vitro. MATERIALS AND METHODS Preparation ofVirus Producers. pLid (Fig. 1) was created by inserting the human IDUA cDNA into plasmid pLX, derived from the vector pLNCX (13) by deleting the cytomegalovirus and neomycin-resistance sequences. This was used to derive amphotropic retroviral producers, using GP+envAml2 cells (14) and standard methodology (15). The clone of amphotropic packaging cells that led to the highest levels of IDUA in target Hurler B cells after cocultivation was chosen for further study and named GPaLid. To estimate the titer of retrovirus produced by this clone, conditioned medium was used to transduce Hurler B cells in 24-well plates in a limiting dilution assay. This was compared with virus supernatants from a second IDUA producer that produced the LidSN virus expressing both IDUA and neomycin phosphotransferase (Fig. 1). By obtaining a comparative titer from the Lid and LidSN virus producers and obtaining a G418 titer for LidSN on 3T3 cells, it was calculated that GPaLid produced the equivalent of 1-3 x 106 Geneticin transfer units per ml. Transduction and Maintenance of Long-Term Bone Marrow Cultures (LTBMCs). After local ethics committee approval and informed consent, bone marrow was obtained from patients with Hurler syndrome and from healthy individuals. Samples were depleted of erythrocytes by gravity sedimentation through methylcellulose (0.1%, 30 min) and LTBMCs Abbreviations: LTBMC, long-term bone marrow culture; LTBMCM, LTBMC medium; MPS, mucopolysaccharidosis; IDUA, a-Liduronidase; IL, interleukin; LTCIC, long term culture initiating cell. tTo whom reprint requests should be addressed.
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