Intravitreal Implantation of Genetically Modified Autologous Bone Marrow-Derived Stem Cells for Treating Retinal Disorders.

A number of retinal degenerative diseases may be amenable to treatment with continuous intraocular delivery of therapeutic agents that cannot be delivered effectively to the retina via systemic or topical administration. Among these disorders are lysosomal storage diseases resulting from deficiencies in soluble lysosomal enzymes. Most cells, including those of the retina, are able to take up these enzymes and incorporate them in active form into their lysosomes. In theory, therefore, continuous intraocular administration of a normal form of a soluble lysosomal enzyme should be able to cure the molecular defect in the retinas of subjects lacking this enzyme. Experiments were conducted to determine whether genetically modified bone marrow-derived stem cells implanted into the vitreous could be used as -vehicles for continuous delivery of such enzymes to the retina. Bone marrow-derived mesenchymal stem cells (MSCs) from normal mice were implanted into the vitreous of mice undergoing retinal degeneration as a result of a mutation in the PPT1 gene. The implanted cells appeared to survive indefinitely in the vitreous without proliferating or invading the retina. This indicates that intravitreal implantation of MSCs is likely a safe means of long-term delivery of proteins synthesized by the implanted cells. Experiments have been initiated to test the efficacy of using genetically modified autologous MSCs to inhibit retinal degeneration in a canine model of neuronal ceroid lipofuscinosis.

GM2 gangliosidosis associated with a HEXA missense mutation in Japanese Chin dogs: a potential model for Tay Sachs disease.

GM2 gangliosidosis is a fatal lysosomal storage disease caused by a deficiency of ß-hexosaminidase (EC 3.2.1.52). There are two major isoforms of the enzyme: hexosaminidase A composed of an α and a ß subunit (encoded by HEXA and HEXB genes, respectively); and, hexosaminidase B composed of two ß subunits. Hexosaminidase A requires an activator protein encoded by GM2A to catabolize GM2 ganglioside, but even in the absence of the activator protein, it can hydrolyze the synthetic substrates commonly used to assess enzyme activity. GM2 gangliosidosis has been reported in Japanese Chin dogs, and we identified the disease in two related Japanese Chin dogs based on clinical signs, histopathology and elevated brain GM2 gangliosides. As in previous reports, we found normal or elevated hexosaminidase activity when measured with the synthetic substrates. This suggested that the canine disease is analogous to human AB variant of G(M2) gangliosidosis, which results from mutations in GM2A. However, only common neutral single nucleotide polymorphisms were found upon sequence analysis of the canine ortholog of GM2A from the affected Japanese Chins. When the same DNA samples were used to sequence HEXA, we identified a homozygous HEXA:c967G>A transition which predicts a p.E323K substitution. The glutamyl moiety at 323 is known to make an essential contribution to the active site of hexosaminidase A, and none of the 128 normal Japanese Chins and 92 normal dogs of other breeds that we tested was homozygous for HEXA:c967A. Thus it appears that the HEXA:c967G>A transition is responsible for the GM2 gangliosidosis in Japanese Chins.

A truncating mutation in ATP13A2 is responsible for adult-onset neuronal ceroid lipofuscinosis in Tibetan terriers.

A recessive, adult-onset neuronal ceroid-lipofuscinosis (NCL) occurs in Tibetan terriers. A genome-wide association study restricted this NCL locus to a 1.3Mb region of canine chromosome 2 which contains canine ATP13A2. NCL-affected dogs were homozygous for a single-base deletion in ATP13A2, predicted to produce a frameshift and premature termination codon. Homozygous truncating mutations in human ATP13A2 have been shown by others to cause Kufor-Rakeb syndrome (KRS), a rare neurodegenerative disease. These findings suggest that KRS is also an NCL, although analysis of KRS brain tissue will be needed to confirm this prediction. Generalized brain atrophy, behavioral changes, and cognitive decline occur in both people and dogs with ATP13A2 mutations; however, other clinical features differ between the species. For example, Tibetan terriers with NCL develop cerebellar ataxia not reported in KRS patients and KRS patients exhibit parkinsonism and pyramidal dysfunction not observed in affected Tibetan terriers. To see if ATP13A2 mutations could be responsible for some cases of human adult-onset NCL (Kufs disease), we resequenced ATP13A2 from 28 Kufs disease patients. None of these patients had ATP13A2 sequence variants likely to be causal for their disease, suggesting that mutations in this gene are not common causes of Kufs disease.

A missense mutation in canine CLN6 in an Australian shepherd with neuronal ceroid lipofuscinosis.

The childhood neuronal ceroid lipofuscinoses (NCLs) are inherited neurodegenerative diseases that are progressive and ultimately fatal. An Australian Shepherd that exhibited a progressive neurological disorder with signs similar to human NCL was evaluated. The cerebral cortex, cerebellum, and retina were found to contain massive accumulations of autofluorescent inclusions characteristic of the NCLs. Nucleotide sequence analysis of DNA from the affected dog identified a T to C variant (c.829T>C) in exon 7 of CLN6. Mutations in the human ortholog underlie a late-infantile form of NCL in humans. The T-to-C transition results in a tryptophan to arginine amino acid change in the predicted protein sequence. Tryptophans occur at homologous positions in the CLN6 proteins from all 13 other vertebrates evaluated. The c.829T>C transition is a strong candidate for the causative mutation in this NCL-affected dog. Dogs with this mutation could serve as a model for the analogous human disorder.

A mutation in canine PPT1 causes early onset neuronal ceroid lipofuscinosis in a Dachshund.

The neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage diseases characterized by progressive neurodegeneration and accumulation of autofluorescent storage granules. A 9-month-old Miniature Dachshund presented with NCL-like signs that included disorientation, ataxia, weakness, visual impairment, and behavioral changes. Neurons throughout the CNS contained autofluorescent lysosomal inclusions with granular osmiophilic deposit (GROD) ultrastructure characteristic of classical infantile NCL (INCL). Human INCL is an autosomal recessive disorder that results from mutations in PPT1, a gene that encodes the enzyme palmitoyl protein thioesterase 1 (PPT1; EC 3.1.22). Resequencing of PPT1 from the affected dog revealed that the dog was homozygous for a single nucleotide insertion in exon 8 (PPT1 c.736_737insC), upstream from the His289 active site. Brain tissue from this dog lacked PPT1 activity. The sire and dam of the propositus were heterozygous for the c.736_737insC mutation; whereas, 127 unrelated Dachshunds were homozygous for the wild-type allele. This is the first reported instance of canine NCL caused by a mutation in PPT1.

RPE65 gene mutation prevents development of autofluorescence in retinal pigment epithelial phagosomes.

During senescence, autofluorescent lysosomal storage bodies known as lipofusin or age pigment accumulate in many post-mitotic types of cells. Among these cell types is the retinal pigment epithelium (RPE) of the mammalian eye. The mechanisms of lipofuscin formation and accumulation have been studied more extensively in the RPE than in any other cell type. Substantial evidence indicates that Vitamin A derivatives (retinoids) are required for RPE lipofuscin formation. The RPE and adjacent retina contain retinoids in the forms of retinol, retinyl esters, and retinaldehyde. Previous research has demonstrated that retinaldehydes are directly involved in the formation of one RPE lipofuscin fluorophore. However, RPE lipofuscin contains many other fluorophores. It has not been determined which retinoids are involved in the formation of these fluorescent compounds. Mice with a mutation in the Rpe65 gene contain substantial levels of retinol and retinyl esters in the RPE, but little if any retinaldehydes in either the RPE or retina. Therefore, these mice could be used to determine whether retinaldehydes are required for formation of all of the RPE lipofuscin fluorophores. Normal mice were given intraocular injections of a protease inhibitor, which resulted in the rapid accumulation in the RPE of lipofuscin-like inclusions. These inclusions exhibited fluorescence properties typical of RPE lipofuscin. Rpe65-/- mice treated with the protease inhibitor also accumulated inclusions similar to those observed in the normal mice. However, these inclusions did not fluoresce under the conditions used to visualize lipofuscin fluorescence. These findings indicate that the aldehyde form of Vitamin A is required for the formation of not only one, but all of the RPE lipofuscin fluorophores.

Retinal capillary basement membrane thickening in a porcine model of diabetes mellitus.

PURPOSE: Current animal models of diabetic microangiopathy, including diabetic retinopathy (DR), have substantial limitations. To determine whether the diabetic pig would provide an improved large animal model of DR, a 20-week porcine model of diabetic dyslipidemia that manifests appreciable macrovasculopathy was evaluated for development of retinal microvascular changes associated with diabetes mellitus (hereafter referred to as diabetes) in humans. METHODS: The effect of diabetes alone or in combination with high dietary fat intake on retinal capillary morphology was assessed. Cell density in the retinal capillaries was evaluated by use of elastase digestion isolation of the retinal vascular bed, and retinal capillary basement membrane thickness was determined by use of electron microscopic analyses. RESULTS: Diabetic pigs fed a normal diet had significantly (P < 0.001) thicker retinal capillary basement membranes than did nondiabetic animals. High fat intake did not contribute to basement membrane thickening. Significant effects of diabetes or diet on retinal capillary cell densities were not observed, nor did diabetes induce formation of microaneurysms typical of advanced diabetic retinopathy. CONCLUSIONS: The rapid development of retinal capillary basement membrane thickening in diabetic pigs makes this animal model useful for early intervention and mechanistic studies for this diabetes-associated microvascular change.

Assessment of plasma carnitine concentrations in relation to ceroid lipofuscinosis in Tibetan Terriers.

OBJECTIVE: To determine whether the late onset form of inherited ceroid lipofuscinosis (CL) in Tibetan Terriers is accompanied by low plasma carnitine concentrations prior to the appearance of clinical signs. ANIMALS: 129 healthy Tibetan Terriers, 12 Tibetan Terriers with CL, and 95 healthy purebred dogs of other breeds. PROCEDURE: After withholding food, blood samples were collected from all dogs into tubes containing EDTA. Blood samples were analyzed for plasma-free carnitine and acyl-carnitines concentrations. RESULTS: Neither the mean plasma total carnitine concentration nor the mean fraction of carnitine in the free form differed significantly between Tibetan Terriers with CL and healthy Tibetan Terriers. Among Tibetan Terriers and the general dog population, plasma carnitine concentration increased with age. Castrated males had an overall increase in plasma carnitine concentrations and variability, compared with sexually intact males. By comparison, plasma carnitine concentrations were not significantly different between spayed and sexually intact females. The mean plasma carnitine concentration in the Tibetan Terriers was approximately 22% higher than in the general population of healthy dogs of other breeds. CONCLUSIONS AND CLINICAL RELEVANCE: Contrary to what is seen in early onset CL in English Setters and in humans with some forms of CL, plasma carnitine concentrations are not decreased in the late-onset disorder in Tibetan Terriers. Our large-scale study establishes reference range values for plasma carnitine concentrations in dogs as functions of age and sex that will be useful in evaluating potential carnitine deficiencies in other disorders in dogs.

An ADAMTS17 splice donor site mutation in dogs with primary lens luxation.

PURPOSE: To identify the genetic cause of isolated canine ectopia lentis, a well-characterized veterinary disease commonly referred to as primary lens luxation (PLL) and to compare the canine disease with a newly described human Weill-Marchesani syndrome (WMS)-like disease of similar genetic etiology. METHODS: Genomewide association analysis and fine mapping by homozygosity were used to identify the chromosomal segment harboring the PLL locus. The resequencing of a regional candidate gene was used to discover a mutation in a splice donor site predicted to cause exon skipping. Exon skipping was confirmed by reverse transcription-polymerase chain reaction amplification of RNA isolated from PLL-affected eyes and from skin fibroblast cultures from PLL-affected dogs. An allelic discrimination assay was used to genotype individual dogs at the splice donor site mutation. RESULTS: The PLL locus was mapped to a 664-kb region of canine chromosome 3 containing regional candidate gene ADAMTS17. Resequencing ADAMTS17 revealed a GT-->AT splice-donor-site mutation at the 5' end of intron 10. The predicted exon 10 skipping and resultant frame shift were confirmed with RNA derived from PLL-affected dogs. The ADAMTS17 mutation was significantly associated with clinical PLL in three different dog breeds. CONCLUSIONS: A truncating mutation in canine ADAMTS17 causes PLL, a well-characterized veterinary disease, which can now be compared to a recently described rare WMS-like disease caused by truncating mutations of the human ADAMTS17 ortholog.

Export of the siderophore enterobactin in Escherichia coli: involvement of a 43 kDa membrane exporter.

The enterobactin system for iron transport in Escherichia coli is well characterized with the exception of the mechanism of enterobactin secretion to the extracellular environment. Escherichia coli membrane protein P43, encoded by ybdA in the chromosomal region of genes involved in enterobactin synthesis, shows strong homology to the 12-transmembrane segment major facilitator superfamily of export pumps. A P43-null mutation was created and siderophore nutrition assays, performed with a panel of E. coli strains expressing one or more outer membrane receptors for enterobactin-related compounds, demonstrated that the P43 mutant was unable to secrete enterobactin efficiently. Products released from the mutant strain were identified with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), revealing that the P43 mutant secretes little, if any, enterobactin, but elevated levels of enterobactin breakdown products 2,3- dihydroxybenzoylserine (DHBS) monomer, dimer, and trimer. These data establish that P43 is a critical component of the E. coli enterobactin secretion machinery and provides a rationale for the designation of the previous genetic locus ybdA as entS to reflect its relevant biological function.