MOLECULAR STUDY OF MPS I--GENE THERAPY

我们建议应用分子生物学的方法来研究

We propose to apply methods of molecular biology to the study of the genetic disorders known as Mucopolysaccharidosis I (MPS I). These include the Hurler, Hurler/Scheie and Scheie syndromes, as well as a canine disease analogous to Hurler/Scheie. All forms of MPS I are caused by lack of activity of the lysosomal enzyme alpha-L-iduronidase. Our goal is two-fold: (1) to characterize precisely the mutation that give rise to human and canine MPS I and (2) to attempt therapy of the canine disease by genetic modification of bone marrow cells. The rationale for the therapeutic trial is the prior demonstration that canine MPS I responds biochemically and clinically to allogeneic bone marrow transplantation. Cloned cDNA encoding the alpha-L-iduronidase is required for both purposes. We shall purify the enzyme to homogeneity, screen lambda GT11 libraries (human and canine) with antisera and/or oligonucleotide probes, characterize and sequence the cDNA. Using biosynthetic radiolabeling in cell culture, we shall determine if cells from MPS I patients are able to synthesize and process cross-reactive protein; if not, we shall determine by Southern and Northern hybridization and nuclear run-on transcription whether the alpha-L-iduronidase gene is intact and transcribed. We plan to localize precisely to the genomic sequence those mutations deemed most informative. We will express full-length alpha-L-iduronidase cDNA in MPS I cells, using a retroviral delivery system of the type described by Mulligan and colleagues. Once expression (i.e., appearance of intralysosomal a-L-iduronidase activity) has been demonstrated in fibroblasts and hematopoietic cells, we will proceed to expression in vivo. Bone marrow will be collected from MPS I dogs, enriched in stem cells, infected with the retrovirus containing the alpha-L- iduronidase cDNA and returned to the donors. Integration and expression of the cDNA will be monitored in circulating leukocytes. After a pilot feasibility experiment, the protocol will be modeled on the recent bone marrow transplantation studies. Four to six MPS I dogs, prepared by irradiation or chemotherapeutic drugs, will be treated with their own genetically modified bone marrow. They will be compared with untreated affected and normal controls with respect to biochemical, ultrastructural and clinical parameters, in order to determine the long term consequences of such genetic therapy.