Age-related macular degeneration (AMD) is a common yet complex retinal degeneration that causes irreversible central blindness in the elderly. Pathology is widely believed to follow loss of retinal pigment epithelium (RPE) and photoreceptor degeneration. Here we report aberrant expression of interleukin-17A (IL17A) and the receptor IL17RC in the macula of AMD patients. In vitro, IL17A induces RPE cell death characterized by the accumulation of cytoplasmic lipids and autophagosomes with subsequent activation of pro-apoptotic Caspase-3 and Caspase-9. This pathology is reduced by siRNA knockdown of IL17RC. IL17-dependent retinal degeneration in a mouse model of focal retinal degeneration can be prevented by gene therapy with adeno-associated virus vector encoding soluble IL17 receptor. This intervention rescues RPE and photoreceptors in a MAPK-dependent process. The IL17 pathway plays a key role in RPE and photoreceptor degeneration and could hold therapeutic potential in AMD.

Production of large quantities of viral vectors is crucial for the success of gene therapy in the clinic. There is a need for higher titers of herpes simplex virus-1 (HSV-1) vectors both for therapeutic use as well as in the manufacturing of clinical grade adeno-associated virus (AAV) vectors. HSV-1 yield increased when primary human fibroblasts were treated with anti-inflammatory drugs like dexamethasone or valproic acid. In our search for compounds that would increase HSV-1 yield, we investigated another anti-inflammatory compound, aurintricarboxylic acid (ATA). Although ATA has been previously shown to have antiviral effects, we find that low (micromolar) concentrations of ATA increased HSV-1 vector production yields. Our results showing the use of ATA to increase HSV-1 titers have important implications for the production of certain HSV-1 vectors as well as recombinant AAV vectors.

The generation of clinical good manufacturing practices (GMP)-grade adeno-associated virus (AAV) vectors requires purification strategies that support the generation of vectors of high purity, and that exhibit a good safety and efficacy profile. To date, most reported purification schemas are serotype dependent, requiring method development for each AAV gene therapy product. Here, we describe a platform purification process that is compatible with the purification of multiple AAV serotypes. The method generates vector preparations of high purity that are enriched for capsids with full vector genomes, and that minimizes the fractional content of empty capsids. The two-column purification method, a combination of affinity and ion exchange chromatographies, is compatible with a range of AAV serotypes generated by either the transient triple transfection method or the more scalable producer cell line platform. In summary, the adaptable purification method described can be used for the production of a variety of high-quality AAV vectors suitable for preclinical testing in animal models of diseases.

As the most common subtype of Leber congenital amaurosis (LCA), LCA10 is a severe retinal dystrophy caused by mutations in the CEP290 gene. The most frequent mutation found in patients with LCA10 is a deep intronic mutation in CEP290 that generates a cryptic splice donor site. The large size of the CEP290 gene prevents its use in adeno-associated virus (AAV)-mediated gene augmentation therapy. Here, we show that targeted genomic deletion using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system represents a promising therapeutic approach for the treatment of patients with LCA10 bearing the CEP290 splice mutation. We generated a cellular model of LCA10 by introducing the CEP290 splice mutation into 293FT cells and we showed that guide RNA pairs coupled with SpCas9 were highly efficient at removing the intronic splice mutation and restoring the expression of wild-type CEP290. In addition, we demonstrated that a dual AAV system could effectively delete an intronic fragment of the Cep290 gene in the mouse retina. To minimize the immune response to prolonged expression of SpCas9, we developed a self-limiting CRISPR/Cas9 system that minimizes the duration of SpCas9 expression. These results support further studies to determine the therapeutic potential of CRISPR/Cas9-based strategies for the treatment of patients with LCA10.

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.

Complement dysregulation in the eye has been implicated in the pathogenesis of age-related macular degeneration (AMD), and genetic variants of complement factor H (CFH) are strongly associated with AMD risk. We therefore aimed to untangle the role of CFH and its splice variant, factor H-like 1 (FHL-1), in ocular complement regulation derived from local versus circulating sources. We assessed the therapeutic efficacy of adeno-associated viruses (AAVs) expressing human FHL-1 and a truncated version of CFH (tCFH), which retains the functional N- and C-terminal ends of the CFH protein, in restoring the alternative complement pathway in Cfh-/- mouse eyes and plasma.

Mutations in GUCY2D are associated with severe early-onset retinal dystrophy, Leber congenital amaurosis type 1 (LCA1), a leading cause of blindness in children. Despite a high degree of visual disturbance stemming from photoreceptor dysfunction, patients with LCA1 largely retain normal photoreceptor structure, suggesting that they are good candidates for gene replacement therapy. The purpose of this study was to conduct the preclinical and IND-enabling experiments required to support clinical application of AAV5-hGRK1-GUCY2D in patients harboring biallelic recessive mutations in GUCY2D. Preclinical studies were conducted in mice to evaluate the effect of vector manufacturing platforms and transgene species on the therapeutic response. Dose-ranging studies were conducted in cynomolgus monkeys to establish the minimum dose required for efficient photoreceptor transduction. Good laboratory practice (GLP) studies evaluated systemic biodistribution in rats and toxicology in non-human primates (NHPs). These results expanded our knowledge of dose response for an AAV5-vectored transgene under control of the human rhodopsin kinase (hGRK1) promoter in NHPs with respect to photoreceptor transduction and safety and, in combination with the rat biodistribution and mouse efficacy studies, informed the design of a first-in-human clinical study in patients with LCA1.