A novel gene delivery method transduces porcine pancreatic duct epithelial cells.

Gene therapy offers the possibility to treat pancreatic disease in cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene; however, gene transfer to the pancreas is untested in humans. The pancreatic disease phenotype is very similar between humans and pigs with CF; thus, CF pigs create an excellent opportunity to study gene transfer to the pancreas. There are no studies showing efficient transduction of pig pancreas with gene-transfer vectors. Our objective is to develop a safe and efficient method to transduce wild-type (WT) porcine pancreatic ducts that express CFTR. We catheterized the umbilical artery of WT newborn pigs and delivered an adeno-associated virus serotype 9 vector expressing green-fluorescent protein (AAV9CMV.sceGFP) or vehicle to the celiac artery, the vessel that supplies major branches to the pancreas. This technique resulted in stable and dose-dependent transduction of pancreatic duct epithelial cells that expressed CFTR. Intravenous (IV) injection of AAV9CMV.sceGFP did not transduce the pancreas. Our technique offers an opportunity to deliver the CFTR gene to the pancreas of CF pigs. The celiac artery can be accessed via the umbilical artery in newborns and via the femoral artery at older ages--delivery approaches that can be translated to humans.

Cystic fibrosis transmembrane conductance regulator Cl- channels with R domain deletions and translocations show phosphorylation-dependent and -independent activity.

Phosphorylation of the R domain regulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. Earlier studies suggested that the R domain controls activity via more than one mechanism; a phosphorylated R domain may stimulate activity, and an unphosphorylated R domain may prevent constitutive activity, i.e. opening with ATP alone. However, the mechanisms responsible for these two regulatory properties are not understood. In this study we asked whether the two effects are dependent on its position in the protein and whether smaller regions from the R domain mediate the effects. We found that several portions of the R domain conferred phosphorylation-stimulated activity. This was true whether the R domain sequences were present in their normal location or were translocated to the C terminus. We also found that some parts of the R domain could be deleted without inducing constitutive activity. However, when residues 760-783 were deleted, channels opened without phosphorylation. Translocation of the R domain to the C terminus did not prevent constitutive activity. These results suggest that different parts of the phosphorylated R domain can stimulate activity and that their location within the protein is not critical. In contrast, prevention of constitutive activity required a short specific sequence that could not be moved to the C terminus. These results are consistent with a recent model of an R domain composed primarily of random coil in which more than one phosphorylation site is capable of stimulating channel activity, and net activity reflects interactions between multiple sites in the R domain and the rest of the channel.

Homozygosity mapping of achromatopsia to chromosome 2 using DNA pooling.

Achromatopsia is an autosomal recessive disease of the retina, characterized clinically by an inability to distinguish colors, impaired visual acuity, nystagmus and photophobia. A genome-wide search for linkage was performed using an inbred Jewish kindred from Iran. To facilitate the genome-wide search, we utilized a DNA pooling strategy which takes advantage of the likelihood that the disease in this inbred kindred is inherited by all affected individuals from a common founder. Equal molar amounts of DNA from all affected individuals were pooled and used as the PCR template for short tandem repeat polymorphic markers (STRPs). Pooled DNA from unaffected members of the kindred was used as a control. A reduction in the number of alleles in the affected versus control pool was observed at several loci. Upon genotyping of individual family members, significant linkage was established between the disease phenotype and markers localized on chromosome 2. The highest LOD score observed was 5.4 (theta = 0). When four additional small unrelated families were genotyped, the combined peak LOD score was 8.2. Analysis of recombinant chromosomes revealed that the disease gene lies within a 30 cM interval which spans the centromere. Additional fine-mapping studies identified a region of homozygosity in all affected individuals, narrowing the region to 14 cM. A candidate gene for achromatopsia was excluded from this disease interval by radiation hybrid mapping. Linkage of achromatopsia to chromosome 2 is an essential first step in the identification of the disease-causing gene.

Fine mapping of the autosomal dominant juvenile open angle glaucoma (GLC1A) region and evaluation of candidate genes.

Juvenile Open Angle Glaucoma (GLC1A) is an autosomal optic neuropathy that has been localized previously to chromosome 1q. Here we report the fine mapping of the disease region using YACs and a high density of polymorphic microsatellite markers. This study utilized two large JOAG pedigrees genotyped at 36 loci from chromosome 1q21-q31 to refine the GLC1A locus to a approximately 3-cM region flanked by YAC-derived microsatellite markers D1S3665 and D1S3664. The candidate genes LAMC1, NPR1, and CNR2 were excluded from the region by linkage. Four other genes, SELE, SELL, TXGP1, and APT1LG1, were determined to lie within the critical region through YAC content and linkage mapping. The YAC-STS content map of the critical region provides the groundwork for the construction of a transcription map and the identification of the disease-causing gene.

Use of a DNA pooling strategy to identify a human obesity syndrome locus on chromosome 15.

Bardet-Biedl syndrome is a heterogeneous autosomal recessive disorder characterized by obesity, mental retardation, polydactyly, retinitis pigmentosa and hypogonadism. Patients with this disorder also have a high incidence of hypertension, diabetes mellitus, and renal and cardiovascular anomalies. Three independent loci causing Bardet-Biedl syndrome have previously been reported. In this study, we we utilized a DNA pooling approach using DNA samples from a highly inbred Bedouin kindred to identify a new Bardet-Biedl syndrome locus on chromosome 15. The results further demonstrate the genetic heterogeneity of this disorder. In addition, the results demonstrate the efficiency of the DNA pooling approach for identifying recessive disease loci in highly inbred human populations.

Identification of a Bardet-Biedl syndrome locus on chromosome 3 and evaluation of an efficient approach to homozygosity mapping.

Bardet-Biedl syndrome is an autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, polydactyly and hypogonadism. Individuals with this disorder also have an increased incidence of hypertension, diabetes mellitus, and renal and cardiac anomalies. We previously identified a locus on chromosome 16 causing this disorder, and provided evidence that Bardet-Biedl syndrome is heterogeneous. In this study, we identify another Bardet-Biedl syndrome locus on chromosome 3 and confirm the non-allelic heterogeneity of this disorder in Bedouin populations. In addition, we demonstrate the feasibility of using pooled DNA samples from members of large kindreds as an efficient approach to homozygosity mapping.