Anterior Pituitary Transcriptome Suggests Differences in ACTH Release in Tame and Aggressive Foxes.

Domesticated species exhibit a suite of behavioral, endocrinological, and morphological changes referred to as "domestication syndrome." These changes may include a reduction in reactivity of the hypothalamic-pituitary-adrenal (HPA) axis and specifically reduced adrenocorticotropic hormone release from the anterior pituitary. To investigate the biological mechanisms targeted during domestication, we investigated gene expression in the pituitaries of experimentally domesticated foxes (Vulpes vulpes). RNA was sequenced from the anterior pituitary of six foxes selectively bred for tameness ("tame foxes") and six foxes selectively bred for aggression ("aggressive foxes"). Expression, splicing, and network differences identified between the two lines indicated the importance of genes related to regulation of exocytosis, specifically mediated by cAMP, organization of pseudopodia, and cell motility. These findings provide new insights into biological mechanisms that may have been targeted when these lines of foxes were selected for behavior and suggest new directions for research into HPA axis regulation and the biological underpinnings of domestication.

Differential genetic regulation of canine hip dysplasia and osteoarthritis.

BACKGROUND: Canine hip dysplasia (HD) is a common polygenic trait characterized by hip malformation that results in osteoarthritis (OA). The condition in dogs is very similar to developmental dysplasia of the human hip which also leads to OA. METHODOLOGY/PRINCIPAL FINDINGS: A total of 721 dogs, including both an association and linkage population, were genotyped. The association population included 8 pure breeds (Labrador retriever, Greyhounds, German Shepherd, Newfoundland, Golden retriever, Rottweiler, Border Collie and Bernese Mountain Dog). The linkage population included Labrador retrievers, Greyhounds, and their crosses. Of these, 366 dogs were genotyped at ∼22,000 single nucleotide polymorphism (SNP) loci and a targeted screen across 8 chromosomes with ∼3,300 SNPs was performed on 551 dogs (196 dogs were common to both sets). A mixed linear model approach was used to perform an association study on this combined association and linkage population. The study identified 4 susceptibility SNPs associated with HD and 2 SNPs associated with hip OA. CONCLUSION/SIGNIFICANCE: The identified SNPs included those near known genes (PTPRD, PARD3B, and COL15A1) reported to be associated with, or expressed in, OA in humans. This suggested that the canine model could provide a unique opportunity to identify genes underlying natural HD and hip OA, which are common and debilitating conditions in both dogs and humans.

COL9A2 and COL9A3 mutations in canine autosomal recessive oculoskeletal dysplasia.

Oculoskeletal dysplasia segregates as an autosomal recessive trait in the Labrador retriever and Samoyed canine breeds, in which the causative loci have been termed drd1 and drd2, respectively. Affected dogs exhibit short-limbed dwarfism and severe ocular defects. The disease phenotype resembles human hereditary arthro-ophthalmopathies such as Stickler and Marshall syndromes, although these disorders are usually dominant. Linkage studies mapped drd1 to canine chromosome 24 and drd2 to canine chromosome 15. Positional candidate gene analysis then led to the identification of a 1-base insertional mutation in exon 1 of COL9A3 that cosegregates with drd1 and a 1,267-bp deletion mutation in the 5' end of COL9A2 that cosegregates with drd2. Both mutations affect the COL3 domain of the respective gene. Northern analysis showed that RNA expression of the respective genes was reduced in affected retinas. These models offer potential for studies such as protein-protein interactions between different members of the collagen gene family, regulation and expression of these genes in retina and cartilage, and even opportunities for gene therapy.

Genome-wide association identifies a deletion in the 3' untranslated region of striatin in a canine model of arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease characterized by ventricular arrhythmias and sudden cardiac death. It is most frequently inherited as an autosomal dominant trait with incomplete and age-related penetrance and variable clinical expression. The human disease is most commonly associated with a causative mutation in one of several genes encoding desmosomal proteins. We have previously described a spontaneous canine model of ARVC in the boxer dog. We phenotyped adult boxer dogs for ARVC by performing physical examination, echocardiogram and ambulatory electrocardiogram. Genome-wide association using the canine 50k SNP array identified several regions of association, of which the strongest resided on chromosome 17. Fine mapping and direct DNA sequencing identified an 8-bp deletion in the 3' untranslated region (UTR) of the Striatin gene on chromosome 17 in association with ARVC in the boxer dog. Evaluation of the secondary structure of the 3' UTR demonstrated that the deletion affects a stem loop structure of the mRNA and expression analysis identified a reduction in Striatin mRNA. Dogs that were homozygous for the deletion had a more severe form of disease based on a significantly higher number of ventricular premature complexes. Immunofluorescence studies localized Striatin to the intercalated disc region of the cardiac myocyte and co-localized it to three desmosomal proteins, Plakophilin-2, Plakoglobin and Desmoplakin, all involved in the pathogenesis of ARVC in human beings. We suggest that Striatin may serve as a novel candidate gene for human ARVC.

Gene therapy rescues cone function in congenital achromatopsia.

The successful restoration of visual function with recombinant adeno-associated virus (rAAV)-mediated gene replacement therapy in animals and humans with an inherited disease of the retinal pigment epithelium has ushered in a new era of retinal therapeutics. For many retinal disorders, however, targeting of therapeutic vectors to mutant rods and/or cones will be required. In this study, the primary cone photoreceptor disorder achromatopsia served as the ideal translational model to develop gene therapy directed to cone photoreceptors. We demonstrate that rAAV-mediated gene replacement therapy with different forms of the human red cone opsin promoter led to the restoration of cone function and day vision in two canine models of CNGB3 achromatopsia, a neuronal channelopathy that is the most common form of achromatopsia in man. The robustness and stability of the observed treatment effect was mutation independent, but promoter and age dependent. Subretinal administration of rAAV5-hCNGB3 with a long version of the red cone opsin promoter in younger animals led to a stable therapeutic effect for at least 33 months. Our results hold promise for future clinical trials of cone-directed gene therapy in achromatopsia and other cone-specific disorders.

An expressed fgf4 retrogene is associated with breed-defining chondrodysplasia in domestic dogs.

Retrotransposition of processed mRNAs is a common source of novel sequence acquired during the evolution of genomes. Although the vast majority of retroposed gene copies, or retrogenes, rapidly accumulate debilitating mutations that disrupt the reading frame, a small percentage become new genes that encode functional proteins. By using a multibreed association analysis in the domestic dog, we demonstrate that expression of a recently acquired retrogene encoding fibroblast growth factor 4 (fgf4) is strongly associated with chondrodysplasia, a short-legged phenotype that defines at least 19 dog breeds including dachshund, corgi, and basset hound. These results illustrate the important role of a single evolutionary event in constraining and directing phenotypic diversity in the domestic dog.

Using the NAFX to measure the effectiveness over time of gene therapy in canine LCA.

PURPOSE: To use ocular motility recordings to determine the changes over time of infantile nystagmus syndrome (INS) in RPE65-deficient canines with Leber Congenital Amaurosis (LCA) and assess the time course of the recalibration of the ocular motor system (OMS). METHODS: Nine dogs were treated bilaterally with AAV-RPE65. A second cohort of four dogs was treated with AAV2.RPE65, an optimized vector. Their fixation eye movements were recorded before treatment and at 4-week intervals for 3 months, by using high-speed (500 Hz) digital videography. The dogs were suspended in a sling and encouraged to fixate on distant (57 inches) targets at gaze angles varying between +/-15 degrees horizontally and +/-10 degrees vertically. The records for each eye were examined for qualitative changes in waveform and for quantitative changes in centralisation with the expanded nystagmus acuity function (NAFX) and compared with ERG results for restoration of receptor function. RESULTS: First group: Before treatment, five of the dogs had clinically apparent INS with jerk, pendular, or both waveforms and with peak-to-peak amplitudes as great as 15 degrees . One dog had intermittent nystagmus. At the 1- and 2-month examinations, no change in nystagmus waveform or NAFX was observed in any of the initial dogs, while at 10 weeks, one dog treated bilaterally with the standard dosage showed reduced nystagmus in only one eye. The other eye did not respond to treatment, as confirmed by ERG. This result was unexpected since it was previously documented that unilateral treatment leads to bilateral reduction of INS. The other dog treated with the standard dosage showed no reduction of its small-amplitude, high-frequency pendular nystagmus despite positive ERG responses. Second group: Only one dog of the four had clinically detectable INS, similar in characteristics to that seen in the affected dogs of the first group. Unlike any previous dog studied, this one showed a damping of the nystagmus within the first 4 weeks after treatment. CONCLUSIONS: In all but one of the cases in which OMS recalibration occurred, as measured by the clinical appearance of nystagmus and by quantitative measurement using the NAFX, the improvement was apparent no sooner than 10 weeks after treatment. Longer term, dose-related studies are needed to determine the minimum necessary degree of restored receptor functionality, the duration after rescue for recalibration of the OMS, and the conditions under which recalibration information can successfully affect the contralateral eye.

Steroids do not prevent photoreceptor degeneration in the light-exposed T4R rhodopsin mutant dog retina irrespective of AP-1 inhibition.

PURPOSE: AP-1 has been proposed as a key intermediate linking exposure to light and photoreceptor cell death in rodent light-damage models. Inhibition of AP-1 associated with steroid administration also prevents light damage. In this study the role of steroids in inhibiting AP-1 activation and/or in preventing photoreceptor degeneration was examined in the rhodopsin mutant dog model. METHODS: The dogs were dark adapted overnight, eyes dilated with mydriatics; the right eye was light occluded and the fundus of the left eye photographed ( approximately 15-17 overlapping frames) with a fundus camera. For biochemical studies, the dogs remained in the dark for 1 to 3 hours after exposure. Twenty-four hours before exposure to light, some dogs were treated with systemic dexamethasone or intravitreal/subconjunctival triamcinolone. AP-1 DNA-binding activity was determined by electrophoresis mobility shift assay (EMSA) and phosphorylation of c-Fos and activation of ERK1/2 were determined by immunoblot analyses. The eyes were collected 1 hour and 2 weeks after exposure to light, for histopathology and immunocytochemistry. RESULTS: Inhibition of AP-1 activation, and phosphorylation of ERK1/2 and c-Fos were found after dexamethasone treatment in light-exposed T4R RHO mutant dog retinas. In contrast, increased AP-1 activity and phosphorylation of c-Fos and ERK1/2 were found in triamcinolone-treated mutant retinas. Similar extensive rod degeneration was found after exposure to light with or without treatment, and areas with surviving photoreceptor nuclei consisted primarily of cones. Only with systemic dexamethasone did the RPE cell layer remain. CONCLUSIONS: Intraocular or systemic steroids fail to prevent light-induced photoreceptor degeneration in the T4R RHO dog retina. Finding that systemic dexamethasone prevents AP-1 activation, yet does not prevent retinal light damage, further supports the hypothesis that AP-1 is not the critical player in the cell-death signal that occurs in rods.

Canine RD3 mutation establishes rod-cone dysplasia type 2 (rcd2) as ortholog of human and murine rd3.

Rod-cone dysplasia type 2 (rcd2) is an autosomal recessive disorder that segregates in collie dogs. Linkage disequilibrium and meiotic linkage mapping were combined to take advantage of population structure within this breed and to fine map rcd2 to a 230-kb candidate region that included the gene C1orf36 responsible for human and murine rd3, and within which all affected dogs were homozygous for one haplotype. In one of three identified canine retinal RD3 splice variants, an insertion was found that cosegregates with rcd2 and is predicted to alter the last 61 codons of the normal open reading frame and further extend the open reading frame. Thus, combined meiotic linkage and LD mapping within a single canine breed can yield critical reduction of the disease interval when appropriate advantage is taken of within-breed population structure. This should permit a similar approach to tackle other hereditary traits that segregate in single closed populations.

Operating in the dark: a night-vision system for surgery in retinas susceptible to light damage.

A standard operating microscope was modified with a bandpass infrared filter in the light path and infrared image intensifiers for each of the 2 eyepieces. We evaluated this system for subretinal injections in normal control dogs and those with a mutation in the rhodopsin gene. Rhodopsin-mutant dogs are a model for human autosomal dominant retinitis pigmentosa, and their retinas degenerate faster when exposed to modest light levels as used in routine clinical examinations. We showed that the mutant retinas developed severe generalized degeneration when exposed to the standard operating microscope light but not the infrared light. The modified operating microscope provided an excellent view of the ocular fundus under infrared illumination and allowed us to perform subretinal injections in the retinas of the rhodopsin-mutant dogs without any subsequent light-induced retinal degeneration.

Reversal of blindness in animal models of leber congenital amaurosis using optimized AAV2-mediated gene transfer.

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

Clinical light exposure, photoreceptor degeneration, and AP-1 activation: a cell death or cell survival signal in the rhodopsin mutant retina?

PURPOSE: The T4R RHO mutant dog retina shows retinal degeneration with exposures to light comparable to those used in clinical eye examinations of patients. To define the molecular mechanisms of the degeneration, AP-1 DNA-binding activity, composition, posttranslational modification of the protein complex, and modulation of ERK/MAPK signaling pathways were examined in light-exposed mutant retinas. METHODS: Dark-adapted retinas were exposed to short-duration light flashes from a retinal camera used clinically for retinal photography and were collected at different time points after exposure. Electrophoretic mobility shift assay (EMSA), supershift EMSA, Western blot analysis, and immunocytochemistry were used to examine AP-1 signaling. RESULTS: Exposure to light of mutant retinas significantly increased AP-1 DNA-binding activity by 1 hour after exposure, and levels remained elevated for 6 hours. Shielded mutant retinas had similar AP-1 levels to shielded or exposed wild-type retinas. The parallel phosphorylation of c-Fos and activation of ERK1/2 was detected only in exposed mutant retinas. Exposure to light changed the composition of the AP-1 protein complex in the mutant retina from c-Jun/Fra-1/c-Fos to JunB/c-Fos. Immunohistochemistry showed that the components of activated AP-1 (JunB, and phosphorylated c-Fos, and phosphorylated ERK1/2 isoforms) were localized in Müller cells. CONCLUSIONS: The inner nuclear layer/Müller cell localization of the key proteins induced by light exposure raises the question of the direct involvement of AP-1 in mediating photoreceptor cell death in this model of autosomal dominant retinitis pigmentosa.

A random model for mapping imprinted quantitative trait loci in a structured pedigree: an implication for mapping canine hip dysplasia.

Genetic imprinting may have played a more notable role in shaping embryonic development of plants, animals, and humans than previously appreciated. Quantitative trait loci that are imprinted (iQTL) exert monoallelic effects, depending on the parent of origin, which is an exception to the laws of Mendelian genetics. In this article, we present a modified random effect-based mapping model to use in a genome-wide scan for the distribution of iQTL that contribute to genetic variance for a complex trait in a structured pedigree. This model, implemented with the maximum likelihood method, capitalizes on a network of relatedness for maternally and paternally derived alleles through identical-by-descent sharing, thus allowing for the discrimination of the genetic variances due to alleles derived from maternal and paternal parents. The model was employed to map iQTL responsible for canine hip dysplasia in a multihierarchical canine pedigree, founded with seven greyhounds and six Labrador retrievers. Of eight significant QTL detected, three, located on CFA1, CFA8, and CF28, were found to trigger significant parent-of-origin effects on the age of femoral capital ossification measured at the left and right hips of a canine. The detected iQTL provide important candidate regions for fine-mapping of imprinted genes and for studying their structure and function in the control of complex traits.

Application of a new subretinal injection device in the dog.

The use of a new subretinal injection device (RetinaJect Subretinal Cannula, SurModics, Inc., Eden Prairie, MN) to access the subretinal space in the canine model was evaluated. Subretinal injections were performed in 33 mongrel dogs between 2 and 52 months of age (median = 9 months). In 5 normal dogs the injection of 150 microl saline or India ink occurred by using a conventional subretinal injection device (CSID) with a 30-gauge anterior chamber irrigating cannula. The sclera had to be surgically exposed and penetrated before the subretinal injection with the CSID could occur. After removing the CSID, the conjunctiva over the sclerotomy site had to be closed. In a second group of 28 dogs [16 normals, 10 RPE65 mutants, and 2 with progressive rod cone degeneration (prcd)], the 25-gauge needle of the RetinaJect was used to penetrate the conjunctiva and the sclera. Once the tip of the needle was close to the retinal surface, a 39-gauge polyimide cannula was extended and brought into apposition with the retina for the subsequent subretinal injection of 150 microl saline, India ink, or adeno-associated virus (AAV). No closure of the conjunctiva was required. The animals were clinically monitored between 1 and 59 weeks after surgery. From this second group 25 eyes were harvested for routine histological analysis either immediately after surgery or after a clinical observation time of between 1 and 40 weeks. Both devices provided equally successful access to the subretinal space. The main advantage of the RetinaJect was that no surgical dissection was required; this led to a shorter procedure time and milder postoperative conjunctival swelling. In summary, the use of the RetinaJect can be recommended as an alternative to the CSID for subretinal injections in dogs.

Modeling extent and distribution of zygotic disequilibrium: implications for a multigenerational canine pedigree.

Unlike gametic linkage disequilibrium defined for a random-mating population, zygotic disequilibrium describes the nonrandom association between different loci in a nonequilibrium population that deviates from Hardy-Weinberg equilibrium. Zygotic disequilibrium specifies five different types of disequilibria simultaneously that are (1) Hardy-Weinberg disequilibria at each locus, (2) gametic disequilibrium (including two alleles in the same gamete, each from a different locus), (3) nongametic disequilibrium (including two alleles in different gametes, each from a different locus), (4) trigenic disequilibrium (including a zygote at one locus and an allele at the other), and (5) quadrigenic disequilibrium (including two zygotes each from a different locus). However, because of the uncertainty on the phase of the double heterozygote, gametic and nongametic disequilibria need to be combined into a composite digenic disequilibrium and further define a composite quadrigenic disequilibrium together with the quadrigenic disequilibrium. To investigate the extent and distribution of zygotic disequilibrium across the canine genome, a total of 148 dogs were genotyped at 247 microsatellite markers located on 39 pairs of chromosomes for an outbred multigenerational pedigree, initiated with a limited number of unrelated founders. A major portion of zygotic disequilibrium was contributed by the composite digenic and quadrigenic disequilibrium whose values and numbers of significant marker pairs are both greater than those of trigenic disequilibrium. All types of disequilibrium are extensive in the canine genome, although their values tend to decrease with extended map distances, but with a greater slope for trigenic disequilibrium than for the other types of disequilibrium. Considerable variation in the pattern of disequilibrium reduction was observed among different chromosomes. The results from this study provide scientific guidance about the determination of the number of markers used for whole-genome association studies.

Radiation hybrid mapping of cataract genes in the dog.

PURPOSE: To facilitate the molecular characterization of naturally occurring cataracts in dogs by providing the radiation hybrid location of 21 cataract-associated genes along with their closely associated polymorphic markers. These can be used for segregation testing of the candidate genes in canine cataract pedigrees. METHODS: Twenty-one genes with known mutations causing hereditary cataracts in man and/or mouse were selected and mapped to canine chromosomes using a canine:hamster radiation hybrid RH5000 panel. Each cataract gene ortholog was mapped in relation to over 3,000 markers including microsatellites, ESTs, genes, and BAC clones. The resulting independently determined RH-map locations were compared with the corresponding gene locations from the draft sequence of the canine genome. RESULTS: Twenty-one cataract orthologs were mapped to canine chromosomes. The genetic locations and nearest polymorphic markers were determined for 20 of these orthologs. In addition, the resulting cataract gene locations, as determined experimentally by this study, were compared with those determined by the canine genome project. All genes mapped within or near chromosomal locations with previously established homology to the corresponding human gene locations based on canine:human chromosomal synteny. CONCLUSIONS: The location of selected cataract gene orthologs in the dog, along with their nearest polymorphic markers, serves as a resource for association and linkage testing in canine pedigrees segregating inherited cataracts. The recent development of canine genomic resources make canine models a practical and valuable resource for the study of human hereditary cataracts. Canine models can serve as large animal models intermediate between mouse and man for both gene discovery and the development of novel cataract therapies.

Safety of recombinant adeno-associated virus type 2-RPE65 vector delivered by ocular subretinal injection.

AAV2 delivery of the RPE65 gene to the retina of blind RPE65-deficient animals restores vision. This strategy is being considered for human trials in RPE65-associated Leber congenital amaurosis (LCA), but toxicity and dose efficacy have not been defined. We studied ocular delivery of AAV-2/2.RPE65 in RPE65-mutant dogs. There was no systemic toxicity. Ocular examinations showed mild or moderate inflammation that resolved over 3 months. Retinal histopathology indicated that traumatic lesions from the injection were common, but thinning within the injection region occurred only at the two highest vector doses. Biodistribution studies at 3 months postinjection showed no vector in optic nerve or visual centers in the brain and only isolated non-dose-related detection in other organs. We also performed biodistribution studies in normal rats at about 2 weeks and 2 months postinjection and vector was not widespread outside the injected eye. Dose-response results in RPE65-mutant dogs indicated that the highest 1.5-log unit range of vector doses proved efficacious. The efficacy and toxicity limits defined in this study lead to suggestions for the design of a subretinal AAV-2/2.RPE65 human trial of RPE65-associated LCA.

Linkage mapping of canine rod cone dysplasia type 2 (rcd2) to CFA7, the canine orthologue of human 1q32.

PURPOSE: To map the canine rcd2 retinal degeneration locus. Rod-cone dysplasia type 2 (rcd2), an early-onset autosomal recessive form of progressive retinal atrophy (PRA), is phenotypically similar to early-onset forms of retinitis pigmentosa collectively termed Leber congenital amaurosis and segregates naturally in the collie breed of dog. Multiple genes have previously been evaluated as candidates for rcd2, but all have been excluded. METHODS: A set of informative experimental pedigrees segregating the rcd2 phenotype was produced. A genome-wide scan of these pedigrees using a set of 241 markers was undertaken. To refine the localized homology between canine and human maps, an RH map of the identified rcd2 region was built using a 3000 cR panel. A positional candidate gene strategy was then undertaken to begin to evaluate potentially causative genes. RESULTS: A locus responsible for the rcd2 phenotype was mapped to CFA7 in a region corresponding to human chromosome 1, region q32.1-q32.2. Maximum linkage was observed between rcd2 and marker FH3972 (theta = 0.02; lod = 25.53), and the critical region was flanked by markers FH2226 and FH3972. As CRB1 is the closest gene on human chromosome 1q known to cause retinal degeneration, canine gene-specific markers for CRB1 were developed, and this gene was excluded as a positional candidate for rcd2. CONCLUSIONS: The rcd2 locus represents a novel retinal degeneration gene. It is anticipated that when identified, this gene will offer new insights into retinal developmental and degenerative processes, and new opportunities for exploring experimental therapies.

Quantitative trait loci for hip dysplasia in a cross-breed canine pedigree.

Canine hip dysplasia is a common developmental inherited trait characterized by hip laxity, subluxation or incongruity of the femoral head and acetabulum in affected hips. The inheritance pattern is complex and the mutations contributing to trait expression are unknown. In the study reported here, 240 microsatellite markers distributed in 38 autosomes and the X chromosome were genotyped on 152 dogs from three generations of a crossbred pedigree based on trait-free Greyhound and dysplastic Labrador Retriever founders. Interval mapping was undertaken to map the QTL underlying the quantitative dysplastic traits of maximum passive hip laxity (the distraction index), the dorsolateral subluxation score, and the Norberg angle. Permutation testing was used to derive the chromosome-wide level of significance at p<0.05 for each QTL. Chromosomes 4, 9, 10, 11 (p<0.01), 16, 20, 22, 25, 29 (p<0.01), 30, 35, and 37 harbor putative QTL for one or more traits. Successful detection of QTL was due to the cross-breed pedigree, multiple-trait measurements, control of environmental background, and marked advancement in canine mapping tools.

Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness.

The short- and long-term effects of gene therapy using AAV-mediated RPE65 transfer to canine retinal pigment epithelium were investigated in dogs affected with disease caused by RPE65 deficiency. Results with AAV 2/2, 2/1, and 2/5 vector pseudotypes, human or canine RPE65 cDNA, and constitutive or tissue-specific promoters were similar. Subretinally administered vectors restored retinal function in 23 of 26 eyes, but intravitreal injections consistently did not. Photoreceptoral and postreceptoral function in both rod and cone systems improved with therapy. In dogs followed electroretinographically for 3 years, responses remained stable. Biochemical analysis of retinal retinoids indicates that mutant dogs have no detectable 11-cis-retinal, but markedly elevated retinyl esters. Subretinal AAV-RPE65 treatment resulted in detectable 11-cis-retinal expression, limited to treated areas. RPE65 protein expression was limited to retinal pigment epithelium of treated areas. Subretinal AAV-RPE65 vector is well tolerated and does not elicit high antibody levels to the vector or the protein in ocular fluids or serum. In long-term studies, wild-type cDNA is expressed only in target cells. Successful, stable restoration of rod and cone photoreceptor function in these dogs has important implications for treatment of human patients affected with Leber congenital amaurosis caused by RPE65 mutations.