Towards gene therapy of Hurler syndrome.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler's syndrome. However, due to a lack of matched related donors and unacceptable morbidity of matched unrelated transplants, this therapy is not available to all patients. Therefore we have been developing 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 alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. A number of different 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, using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of haemopoietic colonies as around 50%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. Indeed a possible disadvantage has been identified for the use of intensive transduction procedures. The enzyme is secreted into the medium and functional localisation has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This pre-clinical work forms the basis for a clinical trial of gene therapy for Hurler syndrome.

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

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 alpha-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 of enzyme 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.

Development of DNA probes for early diagnosis and epidemiological study of cryptococcosis in AIDS patients.

We report the isolation of middle-repetitive DNA sequences from Cryptococcus neoformans that are species and variety specific. These probes were used for assessing strain relatedness among cryptococcal isolates from patients with and without AIDS who were from Zaire and the United States. Five distinct hybridization patterns were observed for the 60 isolates examined, regardless of the restriction enzyme used for digestion. The most common pattern among the isolates from the patients without AIDS was also the most common among the isolates from the patients with AIDS who were from the United States and was the only pattern observed for all isolates tested from patients with AIDS who were from Zaire. On the basis of the high specificity and sensitivity of the signals observed by hybridization, we suggest that these sequences provide a means for both biotyping and early diagnosis of C. neoformans.

Isolation of the Candida albicans histidinol dehydrogenase (HIS4) gene and characterization of a histidine auxotroph.

Genetic studies were done with Candida albicans CBS 562. Various auxotrophs were isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. SAG5 (his4C), a stable histidine auxotroph defective in histidinol dehydrogenase activity, was characterized and chosen for further molecular studies. Therefore, the C. albicans HIS4 gene was isolated. The gene was obtained from a genomic library of the wild-type strain, which was constructed in plasmid YEp24. The HIS4 gene was isolated by transformation of a Saccharomyces cerevisiae strain that carried a his4 mutation. The isolated C. albicans HIS4 gene complemented S. cerevisiae his4A, his4B, his4C, and his4ABC mutant strains, which indicates that the clone contains the entire HIS4 gene. The gene was isolated on plasmid pSTC7, whose physical map was constructed with BamHI, SalI, and EcoRV restriction endonucleases, locating the HIS4 gene on a 14-kilobase-pair DNA fragment. Hybridization experiments with HIS4 and C. albicans genomic DNA showed correspondence between the restriction patterns of the gene with that of the chromosomal DNA, indicating that the gene originates from C. albicans and appears in a single copy. Chromosomes of C. albicans CBS562 and four other strains were resolved by orthogonal-field alteration gel electrophoresis. The electrokaryotyping results showed heterogeneity in chromosomal sizes. The electrokaryotyping of CBS 562 showed a resolution of six chromosomal bands, three of which seemed to be doublets. The C. albicans HIS4 gene was located on the largest resolvable chromosome in all of the strains.

Electrophoretic karyotype of the pathogenic yeast Cryptococcus neoformans.

The electrokaryotype of the pathogenic yeast Cryptococcus neoformans is described for the first time. Three different patterns were seen: (a) serotypes B and C (variety gattii) are similar and consist of nine chromosome mobility groups of greater than 580 kb; (b) serotype A (variety neoformans) revealed eight chromosome-like groups greater than 700 kb; (c) serotype D (the second serotype of variety neoformans) not only differs from those described above, but each D isolate tested showed a different distribution of bands. The discrepancy, and the importance of electrokaryotyping as a taxonomic tool, are discussed.