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ALACHUA, FL, United States

MacLachlan T.K.,Genzyme | Lukason M.,Genzyme | Collins M.,Charles River Laboratories Preclinical Services Nevada | Munger R.,Animal Ophthalmology Clinic | And 10 more authors.
Molecular Therapy | Year: 2011

AAV2-sFLT01 is a vector that expresses a modified soluble Flt1 receptor designed to neutralize the proangiogenic activities of vascular endothelial growth factor (VEGF) for treatment of age-related macular degeneration (AMD) via an intravitreal injection. Owing to minimal data available for the intravitreal route of administration for adeno-associated virus (AAV), we initiated a 12-month safety study of AAV2-sFLT01 administered intravitreally at doses of 2.4 × 10 9 vector genomes (vg) and 2.4 × 10 10 vg to cynomolgus monkeys. Expression of sFlt01 protein peaked at ∼1-month postadministration and remained relatively constant for the remainder of the study. Electroretinograms, fluorescein angiograms, and tonometry were assessed every 3 months, with no test article-related findings observed in any group. Indirect ophthalmoscopy and slit lamp exams performed monthly revealed a mild to moderate but self-resolving vitreal inflammation in the high-dose group only, which follow-up studies suggest was directed against the AAV2 capsid. Histological evaluation revealed no structural changes in any part of the eye and occasional inflammatory cells in the trabecular meshwork, vitreous and retina in the high-dose group. Biodistribution analysis in rats and monkeys found only trace amounts of vector outside the injected eye. In summary, these studies found AAV2-sFLT01 to be well-tolerated, localized, and capable of long-term expression. © The American Society of Gene & Cell Therapy. Source


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 143.08K | Year: 2010

DESCRIPTION (provided by applicant): Complete achromatopsia is an inherited retinal disorder characterized by severely reduced visual acuity, nystagmus, severe photophobia, a small central scotoma, eccentric fixation, and complete loss of color discrimination. In 50% of patients with achromatopsia the disease is caused by mutations in the cyclic nucleotide gated channel beta subunit (CNGB3) gene. Preliminary studies indicate that gene therapy using a recombinant adeno-associated virus serotype 5 (rAAV5) vector expressing a human CNGB3 gene can restore cone photoreceptor function in a dog model of achromatopsia caused by mutations in the CNGB3 gene. The objectives of the studies proposed in this application are to confirm and extend these findings using a rAAV5-CNGB3 vector produced using a commercially relevant manufacturing method. This will be accomplished by producing and purifying a rAAV5-hCNGB3 vector and evaluating the safety and efficacy of subretinal administration of a range of vector concentrations (1 x 1010, 1 x 1011, and 1 x 1012 vg/mL) of the rAAV5-CNGB3 vector in a dog model of achromatopsia caused by mutations in the CNGB3 gene. Results of these studies will be important for future advanced development of rAAV-CNGB3 gene therapy for evaluation in patients with CNGB3-related achromatopsia. PUBLIC HEALTH RELEVANCE: Complete achromatopsia is an inherited retinal disease characterized by severely reduced visual acuity and complete loss of color discrimination. In 50% of patients, the disease is caused by mutations in the CNGB3 gene. No treatment for achromatopsia is currently available. This project will evaluate a novel, CNGB3 gene therapy product for treatment of achromatopsia in a dog model.


Grant
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 657.70K | Year: 2013

Project Summary/Abstract 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 acuityunder 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 humansbut 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 codon340, 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 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 onhow to evaluate the safety of gene therapy vectors in an animal model of the disease.


Patent
Applied Genetic Technologies Corporation | Date: 2015-04-15

This invention relates generally to a codon optimized nucleic acid encoding a retinitis pigmentosa GTPase regulator (RPGR) protein. The nucleic acid has enhanced stability during plasmid production relative to a wildtype cDNA encoding the RPGR protein. The invention also relates to expression cassettes, vectors, and host cells comprising the codon optimized nucleic acid. Methods for preparing a recombinant adeno-associated (rAAV) expression vector comprising the codon optimized nucleic acid sequence are also provided. The nucleic acids, expression cassettes, vectors, and host cells provided may be useful in the large scale production of rAAV expression vectors for gene therapy applications.


Patent
Applied Genetic Technologies Corporation | Date: 2010-07-13

The invention generally provides methods for producing recombinant AAV viral particles using cells grown in suspension. The invention provides recombinant AAV particles for use in methods for delivering genes encoding therapeutic proteins, and methods for using the recombinant AAV particles in gene therapy.

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