Complete achromatopsia is an autosomal recessive inherited congenital disorder of retinal cone photoreceptors. Patients with complete achromatopsia experience extreme light sensitivity and daytime blindness, and best visual acuity under non-bright light conditions is usually 20/200 or worse and generally stable over time. In addition to poor acuity, hypersensitivity to light is an extremely troubling symptom. No specific therapy is currently available. Previous studies in a dog model of achromatopsia caused by mutations in the CNGB3 gene showed that subretinal injection of a recombinant adeno-associated virus (rAAV) vector expressing human CNGB3 rescued cone photoreceptor function but at high doses was associated with findings consistent with immune-mediated toxicity that may be due to the low amino acid identity between human and canine CNGB3. The objectives of the studies proposed in this Fast-Track Phase I/II STTR application are to confirm and extend these findings by comparing rAAV vectors expressing human or canine CNGB3. This will be accomplished by constructing an AAV proviral plasmid or rHSV helper virus containing canine CNGB3 cDNA driven by a cone-specific promoter and using the construct to produce rAAV vectors expressing canine or human CNGB3 that will be tested for safety and efficacy in the dog model of CNGB3-related achromatopsia. Previous attempts to clone a stable, full-length dog CNGB3 (dCNGB3) coding region into a plasmid to generate an AAV expression cassette that could be used to packaging a rAAV-CNGB3 vector have been uniformly unsuccessful. To overcome this problem, we will use two innovative approaches. In one approach we will modify the codons of the dCNGB3 cDNA in a way that they favor gene expression in humans but are rarely utilized in E. coli. To overcome toxicity related to a presumed cryptic promoter, we will also introduce a mutation at codon 340 (from methionine to leucine), and additional silent mutations (i.e. using synonymous codons) upstream of codon 340, in order to suppress possible internal promoter functions. In a second approach we will directly introduce a synthesized dCNGB3 expression cassette into a recombinant herpes simplex virus (HSV) helper virus that can be used for rAAV production using AGTC's HSV-based rAAV production system, thereby bypassing the potential of toxicity in E. coli meditated by plasmids containing a dCNGB3 expression cassette. These studies will also support development of several assays critical to support of human clinical studies of a product to treat Achromatopsia. A better understanding of the effects of rAAV-CNGB3 vectors in animals, especially with respect to toxicity seen at higher doses, will help to guide future development of rAAV-CNGB3 gene therapy for human patients.
Public Health Relevance Statement: Public Health Relevance: Complete achromatopsia is an inherited retinal disorder characterized by severely reduced visual acuity, daytime blindness and complete loss of color discrimination. It can be caused by mutations in any one of four different genes. Gene therapy using a modified virus containing a normal copy of one of these genes can improve vision and correct daytime blindness in animal models of achromatopsia. The current research project will provide information on how to evaluate the safety of gene therapy vectors in an animal model of the disease.
NIH Spending Category: Biotechnology; Eye Disease and Disorders of Vision; Gene Therapy; Genetics; Neurosciences
Project Terms: achromatopsia; Address; adeno-associated viral vector; Aftercare; Amino Acid Sequence; Amino Acids; Animal Disease Models; Animal Model; Animals; Antibodies; aqueous; base; Biological Assay; Blindness; Bypass; Canis familiaris; Capsid; Capsid Proteins; Clinical Research; Code; Codon Nucleotides; Color; Complementary DNA; Complete Blindness; Congenital Disorders; Detection; Development; Discrimination (Psychology); Dose; Endotoxins; enzyme linked immunospot assay; Enzyme-Linked Immunosorbent Assay; Escherichia coli; experience; Future; Gene Expression; gene therapy; Gene Transduction Agent; Genes; Helper Viruses; Human; Hypersensitivity; Immune; Immune response; improved; Inherited; innovation; Interferons; Laboratories; large scale production; Length; Leucine; Light; Measures; Mediating; Methionine; Methods; Modeling; Mutation; neutralizing antibody; Patients; Peptides; Peripheral Blood Mononuclear Cell; Phase; Photophobia; Plasmids; Procedures; Production; Promotor (Genetics); protein expression; Proteins; public health relevance; Recombinant adeno-associated virus (rAAV); Recombinants; Research Project Grants; Research Proposals; response; Retina; Retinal; Retinal Cone; Retinal Diseases; Safety; safety testing; Sampling; Serum; Silent Mutation; Simplexvirus; Small Business Technology Transfer Research; Specific qualifier value; Sterility; subretinal injection; Symptoms; System; T cell response; Testing; Time; Tissues; Toxic effect; Transfection; vector; Virus; Vision; Visual Acuity
