Therapeutic benefit after intracranial gene therapy delivered during the symptomatic stage in a feline model of Sandhoff disease.

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by defects in the ß-subunit of ß-N-acetylhexosaminidase (Hex), the enzyme that catabolizes GM2 ganglioside. Hex deficiency causes neuronal storage of GM2 and related glycoconjugates, resulting in progressive neurodegeneration and death, typically in infancy. No effective treatment exists for human patients. Adeno-associated virus (AAV) gene therapy led to improved clinical outcome and survival of SD cats treated before the onset of disease symptoms. Most human patients are diagnosed after clinical disease onset, so it is imperative to test AAV-gene therapy in symptomatic SD cats to provide a realistic indication of therapeutic benefits that can be expected in humans. In this study, AAVrh8 vectors injected into the thalamus and deep cerebellar nuclei of symptomatic SD cats resulted in widespread central nervous system enzyme distribution, although a substantial burden of storage material remained. Cats treated in the early symptomatic phase showed delayed disease progression and a significant survival increase versus untreated cats. Treatment was less effective when administered later in the disease course, although therapeutic benefit was still possible. Results are encouraging for the treatment of human patients and provide support for the development AAV-gene therapy for human SD.

Therapeutic response in feline sandhoff disease despite immunity to intracranial gene therapy.

Salutary responses to adeno-associated viral (AAV) gene therapy have been reported in the mouse model of Sandhoff disease (SD), a neurodegenerative lysosomal storage disease caused by deficiency of ß-N-acetylhexosaminidase (Hex). While untreated mice reach the humane endpoint by 4.1 months of age, mice treated by a single intracranial injection of vectors expressing human hexosaminidase may live a normal life span of 2 years. When treated with the same therapeutic vectors used in mice, two cats with SD lived to 7.0 and 8.2 months of age, compared with an untreated life span of 4.5 ± 0.5 months (n = 11). Because a pronounced humoral immune response to both the AAV1 vectors and human hexosaminidase was documented, feline cDNAs for the hexosaminidase α- and ß-subunits were cloned into AAVrh8 vectors. Cats treated with vectors expressing feline hexosaminidase produced enzymatic activity >75-fold normal at the brain injection site with little evidence of an immune infiltrate. Affected cats treated with feline-specific vectors by bilateral injection of the thalamus lived to 10.4 ± 3.7 months of age (n = 3), or 2.3 times as long as untreated cats. These studies support the therapeutic potential of AAV vectors for SD and underscore the importance of species-specific cDNAs for translational research.

Neurodegenerative lysosomal storage disease in European Burmese cats with hexosaminidase beta-subunit deficiency.

GM2 gangliosidosis is a fatal, progressive neuronopathic lysosomal storage disease resulting from a deficiency of beta-N-acetylhexosaminidase (EC 3.2.1.52) activity. GM2 gangliosidosis occurs with varying degrees of severity in humans and in a variety of animals, including cats. In the current research, European Burmese cats presented with clinical neurological signs and histopathological features typical of a lysosomal storage disease. Thin layer chromatography revealed substantial storage of GM2 ganglioside in brain tissue of affected cats, and assays with a synthetic fluorogenic substrate confirmed the absence of hexosaminidase activity. When the hexosaminidase beta-subunit cDNA was sequenced from affected cats, a 91 base pair deletion constituting the entirety of exon 12 was documented. Subsequent sequencing of introns 11 and 12 revealed a 15 base pair deletion at the 3' end of intron 11 that included the preferred splice acceptor site, generating two minor transcripts from cryptic splice acceptor sites in affected Burmese cats. In the cerebral cortex of affected cats, hexosaminidase beta-subunit mRNA levels were approximately 1.5 times higher than normal (P<0.001), while beta-subunit protein levels were substantially reduced on Western blots.

Phage probes for malignant glial cells.

Early diagnosis and effective treatment of malignant gliomas, which are heterogeneous brain tumors with variable expression of cell surface markers, are inhibited by the lack of means to characterize and target tumor-selective molecules. To create molecular profiles for RG2 rat glioma cells, we used phage display technology, an approach capable of producing valuable ligands to unknown cell surface targets. The ligands were selected from libraries of peptides displayed as fusion molecules on phage particles. Modifications of the selection conditions resulted in identification of three distinctive families of peptide ligands for malignant glioma cells. The first family with V (D)/(G) L P (E)/(T) H(3) binding motif appeared to target a marker that is common for glioma cells, normal brain cells, and cells of non-brain origin. The second group of peptide-presented phage displayed D (T)/S/(L) T K consensus sequence and contained peptides with pronounced glioma-selective properties. Phage clones expressing peptides with E (L)/V/(S) R G D S motif were found in cell lysates and represented the third family of glioma-specific ligands. All peptides within this family contain the RGD amino acid sequence, which is known to bind to a number of integrins. Phage clones that belong to this family were internalized by RG2 glioma cells about 63-fold more efficiently than by astrocytes. The approach described could be applicable for accurate detection of glioma expression patterns in individual tumors. Such patterns could be beneficial in the design of effective combinations of drugs for anti-glioma treatments.

Molecular cloning, sequencing, and distribution of feline GnRH receptor (GnRHR) and resequencing of canine GnRHR.

GnRH receptors play vital roles in mammalian reproduction via regulation of gonadotropin secretion, which is essential for gametogenesis and production of gonadal steroids. GnRH receptors for more than 20 mammalian species have been sequenced, including human, mouse, and dog. This study reports the molecular cloning and sequencing of GnRH receptor (GnRHR) cDNA from the pituitary gland of the domestic cat, an important species in biomedical research. Feline GnRHR cDNA is composed of 981 nucleotides and encodes a 327 amino acid protein. Unlike the majority of mammalian species sequenced so far, but similar to canine GnRHR, feline GnRHR protein lacks asparagine in position three of the extracellular domain of the protein. At the amino acid level, feline GnRHR exhibits 95.1% identity with canine, 93.8% with human, and 88.9% with mouse GnRHR. Comparative sequence analysis of GnRHRs for multiple mammalian species led to resequencing of canine GnRHR, which differed from that previously published by a single base change that translates to a different amino acid in position 193. This single base change was confirmed in dogs of multiple breeds. Reverse transcriptase PCR analysis of GnRHR messenger RNA in different tissues from four normal cats indicated the presence of amplicons of varying lengths, including full-length as well as shortened GnRHR amplicons, pointing to the existence of truncated GnRHR transcripts in the domestic cat. This study is the first insight into molecular composition and expression of feline GnRHR and promotes better understanding of receptor organization, and distribution in various tissues of this species.

Cell targeted phagemid rescued by preselected landscape phage.

We have developed a gene delivery system that utilizes a cell-binding helper phage preselected from a landscape phage display library, and a phagemid harboring a marker gene and all regulatory elements (origins of replication and promoter-enhancer cassettes) necessary for replication of the phagemid and expression of the marker gene in the targeted cell. All the proteins required for encapsulation of the phagemid DNA and cell targeting are provided by the phage helper and are separate from the phagemid. Therefore, the resultant Phagemid Infective Particles (PIPs) are able to bind and infect target cells and express the marker gene from within the cell. Our approach, shown here for glioma cells, differs from others in that a phagemid expressing a model marker or particular therapeutic gene can be easily exchanged for a phagemid expressing a different therapeutic gene. Also, a different helper phage, selected from a phage display library, such as the f8-8-mer landscape library used here, can target any cell type and direct the encapsulation of any therapeutic gene encoding phagemid. Because of its versatility, the PIPs system may be readily used for optimization of the gene-delivery strategies applied to specific cell and tissue targets.

Targeting peptides for microglia identified via phage display.

Screening with a 7-mer phage display peptide library, a panel of cell-targeting peptides for the murine microglial cell line, EOC 20, was recognized. A number of similar, but not identical, sets of sequences representing more than 75% of all the cell line-binding clones were identified. Comparative analysis indicated that motif S/(T) F T/(X) Y W is present in the vast majority of the binding sequences. The selectivity and specificity of the dominant peptide sequence identified for microglia was confirmed using both phage displaying the peptide and the synthetic peptide alone.

Phage matrix for isolation of glioma cell membrane proteins.

Cell-binding ligands for RG2 rat glioma were identified in our recent study from a library of peptides that are displayed as fusion molecules on phage particles. Here, one of the phage clones was used to affinity purify those cell membrane components to which the displayed peptides bind. This phage clone, displaying the ELRGDSLP peptide, was shown to recognize glioma cells specifically in comparison to control phage-expressing peptides of either similar or irrelevant sequences. Blocking experiments with synthetic RGDS peptide demonstrated that the phage-glioma cell recognition occurs via the RGD motif known to be present in many integrin-binding proteins. To form an affinity matrix that would bind to glioma cell membrane molecules, ELRGDSLP phage particles were cross-linked using dextran polymer. Whole cell lysate from RG2 rat glioma cells was passed through the matrix, resulting in the isolation of cell membrane components having strong affinity to the peptides on phage and molecules associated with those components. One of the isolated proteins was found to be CD44s, a cell surface adhesion molecule involved in glioma cell invasion and migration, which likely formed a complex with an RGD-binding integrin. Cell membrane proteins isolated with this innovative approach could be used for the design of cell-specific anticancer treatments.

Influence of dietary calcium and phosphorus content in a fixed ratio on growth and development in Great Danes.

OBJECTIVE: To study the musculoskeletal development of Great Dane puppies fed various dietary concentrations of calcium (Ca) and phosphorus (P) in fixed ratio by use of dual energy x-ray absorptiometry (DEXA), determination of serum insulin-like growth factor 1 and parathyroid hormone concentrations, radiography, and blood chemistry analysis results. ANIMALS: 32 purebred Great Dane puppies from 4 litters. PROCEDURE: At weaning, puppies were assigned randomly to 1 of 3 diets. Blood was collected for biochemical analyses and hormone assays, and radiography and DEXA were performed through 18 months of age. Changes in body weight, bone mineral content, fat tissue weight, lean mass, result of serum biochemical analyses, hormonal concentrations, and radius lengths were analyzed through 18 months of age. RESULTS: Bone mineral content of puppies correlated positively with Ca and P content of the diets fed. Significant differences between groups in bone mineral content, lean mass, and body fat were apparent early. The disparity among groups increased until 6 months of age and then declined until body composition was no longer different at 12 months of age. Accretion rates for skeletal mineral content, fat, and lean tissue differed from each other and by diet group. CONCLUSIONS AND CLINICAL RELEVANCE: Ca and P concentrations in the diet of young Great Dane puppies are rapidly reflected in the bone mineral content of the puppies until 5 to 6 months of age, after which hormonal regulation adjusts absorption and excretion of these minerals. Appropriate Ca and P concentrations in diets are important in young puppies < 6 months of age.

Sustained normalization of neurological disease after intracranial gene therapy in a feline model.

Progressive debilitating neurological defects characterize feline G(M1) gangliosidosis, a lysosomal storage disease caused by deficiency of lysosomal ß-galactosidase. No effective therapy exists for affected children, who often die before age 5 years. An adeno-associated viral vector carrying the therapeutic gene was injected bilaterally into two brain targets (thalamus and deep cerebellar nuclei) of a feline model of G(M1) gangliosidosis. Gene therapy normalized ß-galactosidase activity and storage throughout the brain and spinal cord. The mean survival of 12 treated G(M1) animals was >38 months, compared to 8 months for untreated animals. Seven of the eight treated animals remaining alive demonstrated normalization of disease, with abrogation of many symptoms including gait deficits and postural imbalance. Sustained correction of the G(M1) gangliosidosis disease phenotype after limited intracranial targeting by gene therapy in a large animal model suggests that this approach may be useful for treating the human version of this lysosomal storage disorder.

Generation and characterization of recombinant feline beta-galactosidase for preclinical enzyme replacement therapy studies in GM1 gangliosidosis.

Lysosomal beta-galactosidase is required for the degradation of GM1 ganglioside and other glycolipids and glycoproteins with a terminal galactose moiety. Deficiency of this enzyme leads to the lysosomal storage disorder, GM1 gangliosidosis, marked by severe neurodegeneration resulting in premature death. As a step towards preclinical studies for enzyme replacement therapy in an animal model of GM1 gangliosidosis, a feline beta-galactosidase cDNA was cloned into a mammalian expression vector and subsequently expressed in Chinese hamster ovary (CHO-K1) cells. The enzyme secreted into culture medium exhibited specific activity on two synthetic substrates as well as on the native beta-galactosidase substrate, GM1 ganglioside. The enzyme was purified from transfected CHO-K1 cell culture medium by chromatography on PATG-agarose. The affinity-purified enzyme preparation consisted mainly of the protein with approximate molecular weight of 94 kDa and displayed immunoreactivity with antibodies raised against a 16-mer synthetic peptide corresponding to C-terminal amino acid sequence deduced from the feline beta-galactosidase cDNA.

Mutation of the GM2 activator protein in a feline model of GM2 gangliosidosis.

The G(M2) activator protein is required for successful degradation of G(M2) ganglioside by the A isozyme of lysosomal beta-N-acetylhexosaminidase (EC 3.2.1.52). Deficiency of the G(M2) activator protein leads to a relentlessly progressive accumulation of G(M2) ganglioside in neuronal lysosomes and subsequent fatal deterioration of central nervous system function. G(M2) activator deficiency has been described in humans, dogs and mice. This manuscript reports the discovery and characterization of a feline model of G(M2) activator deficiency that exhibits many disease traits typical of the disorder in other species. Cats deficient in the G(M2) activator protein develop clinical signs at approximately 14 months of age, including motor incoordination and exaggerated startle response to sharp sounds. Affected cats exhibit central nervous system abnormalities such as swollen neurons, membranous cytoplasmic bodies, increased sialic acid content and elevated levels of G(M2) ganglioside. As is typical of G(M2) activator deficiency, hexosaminidase A activity in tissue homogenates appears normal when assayed with a commonly used synthetic substrate. When the G(M2) activator cDNA was sequenced from normal and affected cats, a deletion of 4 base pairs was identified as the causative mutation, resulting in alteration of 21 amino acids at the C terminus of the G(M2) activator protein.

Novel metal clusters isolated from blood are lethal to cancer cells.

Unfolding and subsequent aggregation of proteins is a common phenomenon that is linked to many human disorders. Misfolded hemoglobin is generally manifested in various autoimmune, infectious and inherited diseases. We isolated micrometer and submicrometer particles, termed proteons, from human and animal blood. Proteons lack nucleic acids but contain two major polypeptide populations with homology to the hemoglobin alpha-chain. Proteons form by reversible seeded aggregation of proteins around proteon nucleating centers (PNCs). PNCs are comprised of 1- to 2-nm metallic nanoclusters containing 40-300 atoms. Each milliliter of human blood contained approximately 7 x 10(13) PNCs and approximately 3 x 10(8) proteons. Exposure of isolated blood plasma to elevated temperatures increased the number of proteons. When an aliquot of this heated plasma was introduced into untreated plasma that was subsequently heated, the number of proteons further increased, reaching a maximum after a total of three such iterations. Small concentrations of PNCs were lethal to cultured cancer cells, whereas noncancerous cells were much less affected.