Gustavo Aguirre - US grants

Abnormalities in outer segment renewal and/or cyclic nucleotide metabolism exist in rodents with hereditary retinal degeneration. Defects in these basic processes have been hypothesized to exist in other species, including man, affected with retinal degeneration. Developmental and degenerative forms of hereditary retinal degeneration are present in different breeds of dogs. In affected animals, the P.I. will determine if defective outer segment renewal and/or cyclic nucleotide metabolism are associated with the degeneration of the visual cells. Moreover, the P.I. will determine if these changes are the result of photoreceptor degeneration or cause the disease. Breeding experiments with affected dogs of different breeds will determine if single or multiple gene defects exist in dogs with hereditary retinal degeneration.

Cats are affected with mucopolysaccharidosis VI and I (MPS VI, MPS I), two recessively inherited but distinct deficiencies of lysosomal hydrolases [MPS VI=arylsulfatase B (ASB); MPS I=Alpha L-iduronidase (Alpha L-i)]. In MPS VI, the disease in the RPE has been characterized and found to correlate with temporal, spatial and pigment density factors. A similar characterization is proposed for MPS I. In vitro studies in MPS I will determine if the RPE retains the essential features of the in vivo disease. Biochemical studies will characterize the relationship between age and pigmentation factors on the residual enzyme (Alpha L-i) activity. The glycosaminoglycans (GAGs) present in normal RPE and accumulating in diseased (MPS I and VI) RPE will be determined. Synthesis and secretion of GAGs will be measured in cultured cells and correlated with age, pigmentation, spatial position and enzyme activity levels. The effect of the disease on the RPE's ability to synthesize extracellular matrix material and/or type IV collagen will be studied. Experimental modification or "therapy" of the diseases will be investigated in vivo and in vitro. The relationship between outer segment phagocytosis and/or GAG turnover and disease in the RPE will be determined in vitro. Bone marrow transplantation or enzyme activation (MPS VI-dimerization of ASB with cystamine) will be investigated as potential therapeutic modalities. In vitro studies will be used to screen potential therapies prior to their in vivo use.

Specific strains of dogs, affected with well-characterized hereditary retinal degenerations (Irish setter: Rod-Cone Dysplasia, miniature poodle: Progressive Rod-Cone Degeneration, Norwegian elkhound: Early Retinal Degeneration, Alaskan malamute: Hemeralopial, together with appropriate nonaffected control dogs, will be bred and maintained. The progeny of these dogs will be distributed to approved research investigators, either directly or by collection, processing and distribution of requested tissues. The primary aims in breeding setters, poodles and elkhounds are to ensure the continued availability of their respective mutations and to promote their utilization by research investigators. The primary aim in breeding malamutes is to preserve this unique mutation and ensure its future availability. The utilization of these mutants will be promoted by solicitation of requests from investigators. Responses will be reviewed for scientific merit and prioritized by an independent Animal Distribution Committee (ADC). The allocation and distribution of dogs and/or tissues will be according to the priorities set by the ADC. Assistance will be offered to investigators in development and implementation of specific protocols for optimal utilization of these mutants.

Abnormalities in outer segment renewal and/or cyclic nucleotide metabolism exist in rodents with hereditary retinal degeneration. Defects in these basic processes have been hypothesized to exist in other species, including man, affected with retinal degeneration. Developmental and degenerative forms of hereditary retinal degeneration are present in different breeds of dogs. In affected animals, the P.I. will determine if defective outer segment renewal and/or cyclic nucleotide metabolism are associated with the degeneration of the visual cells. Moreover, the P.I. will determine if these changes are the result of photoreceptor degeneration or cause the disease. Breeding experiments with affected dogs of different breeds will determine if single or multiple gene defects exist in dogs with hereditary retinal degeneration.

The retinal pigment epithelium (RPE) plays a number of vital roles in maintaining visual cell function; one of these is the metabolism of extracellular matrix (ECM) components. The studies outlined in this proposal will answer specific questions concerning the mechanisms involved in the degradation of one of the components of the ECMs surrounding the RPE: glycosaminoglycans (GAGs). The proposed studies will identify and characterize GAG degradative pathways in both the intracellular and extracellular compartments of the RPE in vitro. The experimental studies utilize RPE cultures initiated from defined regions of the eye to measure the turnover of GAGs within individual compartments of the RPE cell layer (intracellular compartment, trypsin-soluble glycocalyx, basal extracellular matrix). Exogenously applied compounds as well as inherited enzyme deficiencies will be used to experimentally modulate (stimulate, inhibit) in a selective manner either intracellular or extracellular GAG turnover. Biosynthetically radiolabeled basement membrane preparation will be used to determine whether ECM components are reinternalized during degradation or remain extracellular, and what factors affect this process. Radiolabeled basement membrane preparations also will be used to distinguish degradation by cell surface enzymes (e.g. requiring direct cell contact) from that of secreted enzymes. These studies will provide valuable information concerning the normal functioning of the RPE, and its interactions with extracellular matrices. In addition, these studies may provide insights into aging or disease related changes in the RPE and/or the ECMs surrounding the RPE.

This application proposes continuance of the project "Canine Models of Hereditary Retinal Degeneration", as a national research resource for studies of hereditary retinal degenerations. The need for these canine mutants in research studies is addressed, as is the importance of maintaining them in a centralized breeding and distribution facility. Examples of research studies that will utilize the colony, and their significance, are summarized. Specific and comprehensive details of such research are not included, however, as these form part of the peer reviewed research programs (funded by ROI or other support) of those individual investigators utilizing the colony's resources. Four specific mutant strains of dog, each transmitting a different, unique gene for a well-characterized, autosomal recessive hereditary retinal degeneration (rcd1: Rod-Cone Dysplasia Type 1; prcd: Progressive Rod-Cone Degeneration; erd: Early Retinal Degeneration; cd: Cone Degeneration) together with appropriate nonaffected control dogs, will be bred, maintained and made available for approved research investigations. The progeny of these dogs will be distributed to approved research investigators either directly or by collection, processing and distribution of requested tissues. DNA from representative individuals and from informative pedigrees of each mutant strain will be preserved and made available to all approved investigators. The primary aims of this project are to ensure continued availability of the respective mutations and to promote their utilization by research investigators. Utilization will be promoted by solicitation of requests from investigators. Responses will be reviewed for scientific merit and priorized by an independent Retinal Research Resources Committee (RRRC), appointed and chaired by appropriate NEI/NIH staff. Allocation of distributable resources (such as dogs, tissues, DNA) from the colony will be according to priorities set by the RRRC. Investigators will be assisted in the development and implementation of protocols for optimal utilization of these mutants.

The progressive rod-cone degeneration (prcd) dog mutant has a progressive retinal degenerative disorder that serves as a model for retinitis pigmentosa in man. The disease is topographically defined and is characterized by a decreased rod outer segment (ROS) renewal rate. The studies proposed in this application examine the association between retinal protein synthesis and/or transport, renewal rate, disease and degeneration. Work with collaborators will correlate regional retinal lipid and protein synthesis as well as examining the composition of the different ROS phospholipid classes. The studies will test the hypothesis that perturbations wither in synthesis, transport or renewal compromise the visual cell and result in disease and degeneration. The protein and lipid studies will utilize both in vivo and in vitro approaches. Using single or double label combinations of various radioactively tagged precursors, it will be possible to examine the synthesis, transport or turnover of newly synthesized proteins or lipids. These studies are based on our ability to maintain the retina structurally intact and metabolically active under organ culture conditions. By using a sampling method that preserves disease, quadrant and area relationships, the biochemical, morphologic and/or autoradiographic studies will establish the causal relationship between disease and the observed abnormalities. Studies are also proposed to examine the variation in retinal degeneration phenotype that exists at the prcd locus. The prcd- slow mutant will be used to establish the temporal association between the renewal rate abnormality and disease. Since this mutant has a milder and slowly progressive disease, the proposed experiments will determine if this results from a renewal rate defect that is expressed at a later age and/ or to a lesser extent. On the other hand, the prcd mutant will be used to examine whether local factors modulate disease an can be used to experimentally manipulate disease progression. Based on our recent findings that the tissue distribution of vitamin E parallels the disease topography in the prcd retina, nutritional experiments will examine the effect of vitamin E (marginal, adequate, excess) levels on disease severity and progression. These studies will determine the association between tissue vitamin E levels, disease and degeneration and establish if the disease phenotype can be modulated (accelerated, retarded) nutritionally.

The progressive rod-cone degeneration (prcd) dog mutant has a progressive retinal degenerative disorder that serves as a model for retinitis pigmentosa in man. The disease is topographically defined and is characterized by a decreased rod outer segment (ROS) renewal rate. The studies proposed in this application examine the association between retinal protein synthesis and/or transport, renewal rate, disease and degeneration. Work with collaborators will correlate regional retinal lipid and protein synthesis as well as examining the composition of the different ROS phospholipid classes. The studies will test the hypothesis that perturbations wither in synthesis, transport or renewal compromise the visual cell and result in disease and degeneration. The protein and lipid studies will utilize both in vivo and in vitro approaches. Using single or double label combinations of various radioactively tagged precursors, it will be possible to examine the synthesis, transport or turnover of newly synthesized proteins or lipids. These studies are based on our ability to maintain the retina structurally intact and metabolically active under organ culture conditions. By using a sampling method that preserves disease, quadrant and area relationships, the biochemical, morphologic and/or autoradiographic studies will establish the causal relationship between disease and the observed abnormalities. Studies are also proposed to examine the variation in retinal degeneration phenotype that exists at the prcd locus. The prcd- slow mutant will be used to establish the temporal association between the renewal rate abnormality and disease. Since this mutant has a milder and slowly progressive disease, the proposed experiments will determine if this results from a renewal rate defect that is expressed at a later age and/ or to a lesser extent. On the other hand, the prcd mutant will be used to examine whether local factors modulate disease an can be used to experimentally manipulate disease progression. Based on our recent findings that the tissue distribution of vitamin E parallels the disease topography in the prcd retina, nutritional experiments will examine the effect of vitamin E (marginal, adequate, excess) levels on disease severity and progression. These studies will determine the association between tissue vitamin E levels, disease and degeneration and establish if the disease phenotype can be modulated (accelerated, retarded) nutritionally.

DESCRIPTION (Adapted from applicant's abstract): Inherited disorders affecting the retinal pigment epithelium (RPE) and photoreceptor layers cause blindness in both humans and animals. The research will address the issue of whether gene therapy of a potentially proliferating cell population (RPE) can effect a cure for an inherited disease affecting the RPE and, secondarily, the photoreceptors. Autologous RPE cells will deliver gene products to the retina to treat primary disorders of the RPE. This research is now feasible because of recently-developed surgical techniques that provide access to the subretinal space, and because viral vectors can be used for high efficiency gene transfer to RPE cells. The canine mucopolysaccaridoses (MPS) type VII model offers a unique target tissue for therapy and evaluation, where cone photoreceptor pathology is a secondary result of marked alterations in the chondroitin sulfate turnover in the IPM. Two approaches will be used: (1) ex vivo correction of the RPE followed by cell transplantation into the fellow eye, and (2) direct in situ transduction. Both approaches will be assessed for efficacy by clinical, functional, biochemical, morphological and gene expression techniques. The proposed studies will serve as an experimental model for the development of the technology necessary to use genetically modified RPE to treat primary disorders of photoreceptor cells and/or the RPE.

XLPRA, an X-linked retinal degeneration, is the canine equivalent of human RP3. RP3 maps to a 520 kb interval between OTC and DXS1110. RPGR is the only gene for which RP3-causal mutations are known, yet most RP3 patients do not appear to have RPGR mutations. RPGR mutations yet to be identified might account for the balance of RP3 patients. If not, then other genes located in this interval must be responsible. Two distinct models of XLPRA, XLPRA1 and XLPRA2, cosegregate with RPGR and, in both, the RPGR cDNA sequence is normal. Our objective is to use XLPRA dogs to determine 1) do RPGR mutations cause the disease(s), or 2) can novel disease genes in this region be incriminated for either retinal degeneration phenotype? Identification of the gene(s) and mutation(s) responsible for XLPRA will allow for studies of the molecular mechanisms of these diseases, and provide new large animal models for pre-clinical studies and therapy. We will first refine the XLPRA1 and XLPRA2 minimal regions by linkage, recombination and physical mapping. If these regions overlap, we will also define their minimum common zero- recombination interval under the hypothesis that XLPRA1 and XLPRA2 represent diseases caused by different mutations in the same gene. Under this hypothesis, the combined pedigrees yields much greater power to reduce the search area to significantly less than 1 Mb. If RPGR falls within the zero recombination region for either or both diseases, we will examine RPGR genomic sequence in detail for deletions or rearrangements in the appropriate disease(s). In the absence of such RPGR genomic alterations, full genomic sequencing of wild type canine RPGR will be done from RPGR-specific canine BACs, followed by genomic sequencing of the gene for both XLPRA1 and XLPRA2. If RPGR is excluded by these analyses for either disease, we will make a minimal tiling path of canine BACs and cosmids from the XLPRA zero-recombination interval, and evaluate other genes, ESTs and conserved sequences identified therein.

DESCRIPTION (provided by applicant): Mutations in the RP GTPase regulator gene (RPGR) cause the RP3 form of X-linked RP, one of the most prevalent and severe forms of retinitis pigmentosa. Over half of these mutations occur in ORF15, a purine rich repetitive domain of unknown function. During the current grant we fine mapped two X-linked canine retinal degenerations (XLPRA1 and 2), and found 2 different causative rnicrodeletions in ORF15. These are the only animal models for ORF15-associated disease. Because the 2 disorders are phenotypically very distinct, the major differences between them reflect the nature of the RPGRORF15 microdeletions. Within XLPRA1 however, although all affected dogs share a common stable mutation originating from a single founder, significant variation in disease severity exists. This clearly results not from environmental factors or heterogeneity at the primary locus, but as a semi-dominant trait segregating independently of the primary disease locus. SNP-based haplotypes will be developed to test genes known to interact with RPGR, as candidate disease modifiers. Informative pedigrees will be examined for cosegregation with disease severity. If no such association is found, a genome scan will then be undertaken to define genomic regions with potential positional candidates. We will exploit differences in expression, at both the mRNA and protein level, to identify disease-associated transcripts and gene products. RNase protection assays will be used to identify ORF15 containing transcripts and evaluate expression in retinal vs. nonretinal tissues. Because both mutations alter the length and charge of the ORF15 proteins, 2D-gels and peptide sequencing will be used to identify disease relevant isoforms. Specific antibodies will be developed for immunochemical studies to evaluate expression and localization of each isoform during development and degeneration. Using our custom canine retinal cDNA microarray we will identify changes in gene expression that are either mutation specific or common to both disorders. This will reveal insights into the molecular processes underlying the disease and degeneration of visual cells. Further, it will help to indicate when irreversible metabolic changes occur that could limit therapeutic intervention, and establish time points for implementation and evaluation of our planned gene therapy using AAV 2/5 pseudotyped vectors. Two hypotheses will be tested experimentally with gene therapy: a) -that disease from the stop mutation (XLPRA1) is caused by loss of function requiring gene replacement; and b) -that disease from the frameshift mutation (XLPRA2) arises from a deleterious gain of function, necessitating early intervention with combined ribozyme knockdown and gene replacement.

Project summary . A multi-investigator, multi-center plan is proposed to develop gene-based retinal therapies for LCA- ciliopathies using the dog NPHP5 model. A subgroup of these human ciliopathies show early onset and profound congenital retinal and visual malfunction that results from abnormally developed photoreceptors (PR) that subsequently degenerate. The proposal builds on success achieved in the current grant period in moving RPGR-XLRP to a clinical trial, and recent studies in the NPHP5 model showing that a scAAV2/8- based viral vector, together with the human GRK1 promoter and human full-length NPHP5 cDNA, rescues ERG rod and cone function, and vision, for at least 1 year, but that PRs continue to degenerate, albeit at a much slower rate. This vector now serves as the benchmark test vector to assess treatment paradigms to optimize PR targeting, infectivity and therapeutic transgene expression that will result in permanent disease correction. The proposal will evaluate gene therapy in dogs having this aggressive and severe LCA- ciliopathy, and is divided into three aims that will: 1- establish the benchmark dose and disease stage treatment efficacy of the test vector; 2- select the lead vector pseudotype and promoter with optimized transduction efficiency, and efficacy in targeting different PR disease stages; 3- facilitate translational studies by defining the natural history of the disease in dogs and patients, the effect of treatment in dogs, and determining the degree of PR/retinal disease still amenable to treatment. While the test platform is the NPHP5-LCA dog model, the therapeutic questions addressed apply broadly to other LCA-ciliopathies. Four coordinated groups [a.k.a. modules (M)], are described that take advantage of the special expertise of each group to create a complementary and focused approach to the proposed translational studies. M1 (Large Animal Experimental) will produce the dog models, and provide infrastructure resources for the work; M2 (Large Animal Therapy) will carry out therapy studies and develop measures for outcome assessment; M3 (Non-invasive Patient and Dog Studies) will establish functional and structural disease features in the patients and model, and evaluate success of therapies in dogs using non-invasive outcome measures that correlate with ex vivo morphologic studies; M4 (Molecular Therapeutic Development) will provide therapeutic vectors. The research studies described in this proposal represents a continuation of a longstanding collaboration between the module scientists that already has brought retinal gene therapy for the treatment of patients with RPE65-LCA (Phase I clinical trial), and CNGB3-achromatopsia and RPGR-XLRP (both in final preparations for clinical trials). The University of Pennsylvania leads this collaboration with the University of Florida.

Project Summary/Abstract This project is focused on accelerating the development and pre-clinical testing of new and effective approaches to therapy of hereditary retinal degenerations. These diseases are a major cause of blindness in people, affecting over 100,000 Americans, and are caused by a large number of different gene mutations, not all of which have yet been identified. Similar diseases also affect dogs, in many cases caused by identical or essentially similar gene mutations to those affecting people. In this project, studies will be undertaken in a research colony of dogs affected by such hereditary retinal diseases to better understand the genetic and pathogenetic mechanisms of these diseases, and evaluate potential methods of disease prevention, therapy or amelioration. Specific canine strains with well characterized retinal disorders will be maintained, bred, and made available to research investigators for collaborative studies aimed at a) increasing our understanding of the molecular mechanisms involved in these diseases and b) preclinical evaluation of potential therapies. Collaborations to effectively utilize these mutants will be initiated by the Principal Investigators interacting with independently funded investigators, to develop, implement and conduct specific protocols for optimal utilization of these mutants. Special emphasis will be placed on collaborative studies that: i) develop vectors for gene therapy that primarily target rod and/or cone photoreceptors, and test these vectors in appropriate canine models. For example, cone-specific vectors will be tested in canine models of achromatopsia, and rod-specific vectors will be tested in a canine model of autosomal dominant retinitis pigmentosa. ii) identify the causative mutations in new canine hereditary retinal degenerations, and investigate the cell biologic mechanisms critical to the pathogenesis of such diseases. For example, the mutations responsible for 3 canine cone-rod dystrophies will be identified. This will then allow these models to be used for gene-specific therapy studies. iii) Identify molecular signals favoring either the death or survival of photoreceptors during the onset of disease, and attempt to modulate such processes as either an adjunct or alternative to gene-specific therapies.

A multi-investigator, multi-center research plan is proposed to develop and test gene-based retinal therapy in animal models (mouse and dog) for translation to patients with autosomal dominant RP caused by mutations in the rhodopsin gene (RHO). RHO mutations constitute one of the most common molecularly-identified causes of human RP, and more than 100 of them account for > 12 % of RP. The proposal has been divided into 4 aims that will: (Aim#1) develop viral vectors, promoters, knockdown constructs and replacement cDNAs, and compare the efficacy of a RHO cDNA augmentation approach, to that of an allele-independent knockdown and replacement strategy in two mouse models; (Aim #2) evaluate in a large animal model (dog) which of these strategies provides optimal rescue of rods, (Aim #3) develop outcome measures for clinical trials of gene therapy in RHO-ADRP patients, and (Aim #4) evaluate the optimal strategy and vector construct (based on results of Aims #1 and 2) in pre-clinical safety studies. Six coordinated modules (M) are described, each with a specific set of aims that contributes in a unique but complementary way to the translational studies. M1 (Vector Development) will provide AAVs carrying knockdown (siRNA, ribozymes) reagents, and resistant (hardened) RHO cDNAs. M2 (Small Animal-mouse- Therapy Studies) will test the 2 gene therapy approaches in two mouse models. M3 (Large Animal Experiemntal Support) will produce the dogs, and provide infrastructure resources for this work). M4 (Large anima I- dog - Therapy Studies) will test the 2 approaches in a naturally -occurring canine model of RHO-ADRP. M5 (Human RHO-ADRP) will identify retinal regions that can be targeted for focal retinal therapy in patients. M6 (Vector safety studies in Animals) will conduct GLP-based preclinical toxicology and biodistribution studies in small and large animals to test the safety of the optimal (lead) therapeutic vector as the essential first step fro FDA consideration of an IND for a future Phase I Clinical Trial. The research studies described in this proposal represent a continuation of a longstanding collaboration between the module scientists that already has brought retinal gene therapy for RPE65-LCA patients to a Phase I clinical trial.

DESCRIPTION (provided by applicant): The overarching goal of this project is to accelerate the development and pre-clinical evaluation of new and effective approaches to therapy of hereditary retinal degenerations in man. These diseases are a major cause of human blindness, affecting ~200,000 Americans, and are caused by a large number mutations in over 200 retinally expressed genes, not all of which have yet been identified. Similar diseases also affect dogs, in many cases caused by similar gene mutations. In this project, studies will be undertaken using research colonies of dogs affected by such hereditary retinal diseases to better understand the genetic and pathogenetic mechanisms of the diseases, and evaluate potential methods of disease prevention, therapy or amelioration. Specific canine strains with well characterized retinal disorders will be maintained and bred at a centralized resource facility Mutant and age-matched control dogs will be made available to research investigators either for independent or collaborative studies aimed at understanding the molecular mechanisms involved in the diseases, and developing potential therapies that can be evaluated on a short- or long-term basis. In parallel, this centralized resource will be used by multiple investigators to accomplish the research goals of their own NIH funded grants. Lastly, hypothesis driven studies by the PI and collaborators will identify new patient-relevant retinal disease models, examine the cellular/molecular mechanisms resulting in photoreceptor cell death, survival or proliferation, and develop interventions that will modulate the diseases, and develop therapies targeted for late-stage retinal disease with an emphasis on promoter selection and combinatorial therapies adjunctive to gene augmentation. The proposed studies provide support for the principal hypothesis that collaborative research using these canine models, from a centralized well maintained resource colony, and with an experienced team of investigators will lead to critical proof of principle studies directed at developing safe and effective novel therapies for human retinal degenerations.