Optimization of Capillary-Based Western Blotting for MYO7A.

Myosin VIIA (MYO7A)-associated Usher syndrome type 1B (USH1B) is a severe disorder that impacts the auditory, vestibular, and visual systems of affected patients. Due to the large size (~7.5 kb) of the MYO7A coding sequence, we have designed a dual adeno-associated virus (AAV) vector-based approach for the treatment of USH1B-related vision loss. Due to the added complexity of dual-AAV gene therapy, careful attention must be paid to the protein products expressed following vector recombination. In order to improve the sensitivity and quantifiability of our immunoassays, we adapted our traditional western blot protocol for use with the Jess™ Simple Western System. Following several rounds of testing, we optimized our protocol for the detection of MYO7A in two of our most frequently used sample types, mouse eyes, and infected HEK293 cell lysates.

Novel AAV44.9-Based Vectors Display Exceptional Characteristics for Retinal Gene Therapy.

The majority of inherited retinal diseases (IRDs) are caused by mutations in genes expressed in photoreceptors (PRs). The ideal vector to address these conditions is one that transduces PRs in large areas of retina with the smallest volume/lowest titer possible, and efficiently transduces foveal cones, the cells responsible for acute, daylight vision that are often the only remaining area of functional retina in IRDs. The purpose of our study was to evaluate the retinal tropism and potency of a novel capsid, AAV44.9, and rationally designed derivatives thereof. We found that AAV44.9 and AAV44.9(E531D) transduced retinas of subretinally injected (SRI) mice with higher efficiency than did benchmark AAV5- and AAV8-based vectors. In macaques, highly efficient cone and rod transduction was observed following submacular and peripheral SRI. AAV44.9- and AAV44.9(E531D)-mediated GFP fluorescence extended laterally well beyond SRI bleb margins. Notably, extrafoveal injection (i.e., fovea not detached during surgery) led to transduction of up to 98% of foveal cones. AAV44.9(E531D) efficiently transduced parafoveal and perifoveal cones, whereas AAV44.9 did not. AAV44.9(E531D) was also capable of restoring retinal function to a mouse model of IRD. These novel capsids will be useful for addressing IRDs that would benefit from an expansive treatment area.

Development of an AAV-CRISPR-Cas9-based treatment for dominant cone-rod dystrophy 6.

Cone-rod dystrophy 6 (CORD6) is caused by gain-of-function mutations in the GUCY2D gene, which encodes retinal guanylate cyclase-1 (RetGC1). There are currently no treatments available for this autosomal dominant disease, which is characterized by severe, early-onset visual impairment. The purpose of our study was to develop an adeno-associated virus (AAV)-CRISPR-Cas9-based approach referred to as "ablate and replace" and evaluate its therapeutic potential in mouse models of CORD6. This two-vector system delivers (1) CRISPR-Cas9 targeted to the early coding sequence of the wild-type and mutant GUCY2D alleles and (2) a CRISPR-Cas9-resistant cDNA copy of GUCY2D ("hardened" GUCY2D). Together, these vectors knock out ("ablate") expression of endogenous RetGC1 in photoreceptors and supplement ("replace") a healthy copy of exogenous GUCY2D. First, we confirmed that ablation of mutant R838S GUCY2D was therapeutic in a transgenic mouse model of CORD6. Next, we established a proof of concept for "ablate and replace" and optimized vector doses in Gucy2e+/-:Gucy2f-/- and Gucy2f-/- mice, respectively. Finally, we confirmed that the "ablate and replace" approach stably preserved retinal structure and function in a novel knockin mouse model of CORD6, the RetGC1 (hR838S, hWT) mouse. Taken together, our results support further development of the "ablate and replace" approach for treatment of CORD6.

Dual-AAV vector-mediated expression of MYO7A improves vestibular function in a mouse model of Usher syndrome 1B.

Usher syndrome is the most common cause of deafness-blindness in the world. Usher syndrome type 1B (USH1B) is associated with mutations in MYO7A. Patients with USH1B experience deafness, blindness, and vestibular dysfunction. In this study, we applied adeno-associated virus (AAV)-mediated gene therapy to the shaker-1 (Myo7a4626SB/4626SB) mouse, a model of USH1B. The shaker-1 mouse has a nonsense mutation in Myo7a, is profoundly deaf throughout life, and has significant vestibular dysfunction. Because of the ∼6.7-kb size of the MYO7A cDNA, a dual-AAV approach was used for gene delivery, which involves splitting human MYO7A cDNA into 5' and 3' halves and cloning them into two separate AAV8(Y733F) vectors. When MYO7A cDNA was delivered to shaker-1 inner ears using the dual-AAV approach, cochlear hair cell survival was improved. However, stereocilium organization and auditory function were not improved. In contrast, in the vestibular system, dual-AAV-mediated MYO7A delivery significantly rescued hair cell stereocilium morphology and improved vestibular function, as reflected in a reduction of circling behavior and improved vestibular sensory-evoked potential (VsEP) thresholds. Our data indicate that dual-AAV-mediated MYO7A expression improves vestibular function in shaker-1 mice and supports further development of this approach for the treatment of disabling dizziness from vestibular dysfunction in USH1B patients.

A Novel Mouse Model of MYO7A USH1B Reveals Auditory and Visual System Haploinsufficiencies.

Usher's syndrome is the most common combined blindness-deafness disorder with USH1B, caused by mutations in MYO7A, resulting in the most severe phenotype. The existence of numerous, naturally occurring shaker1 mice harboring variable MYO7A mutations on different genetic backgrounds has complicated the characterization of MYO7A knockout (KO) and heterozygote mice. We generated a novel MYO7A KO mouse (Myo7a - / -) that is easily genotyped, maintained, and confirmed to be null for MYO7A in both the eye and inner ear. Like USH1B patients, Myo7a - / - mice are profoundly deaf, and display near complete loss of inner and outer cochlear hair cells (HCs). No gross structural changes were observed in vestibular HCs. Myo7a - / - mice exhibited modest declines in retinal function but, unlike patients, no loss of retinal structure. We attribute the latter to differential expression of MYO7A in mouse vs. primate retina. Interestingly, heterozygous Myo7a + / - mice had reduced numbers of cochlear HCs and concomitant reductions in auditory function relative to Myo7a +/+ controls. Notably, this is the first report that loss of a single Myo7a allele significantly alters auditory structure and function and suggests that audiological characterization of USH1B carriers is warranted. Maintenance of vestibular HCs in Myo7a - / - mice suggests that gene replacement could be used to correct the vestibular dysfunction in USH1B patients. While Myo7a - / - mice do not exhibit sufficiently robust retinal phenotypes to be used as a therapeutic outcome measure, they can be used to assess expression of vectored MYO7A on a null background and generate valuable pre-clinical data toward the treatment of USH1B.

Reliable Detection of Herpes Simplex Virus Sequence Variation by High-Throughput Resequencing.

High-throughput sequencing (HTS) has resulted in data for a number of herpes simplex virus (HSV) laboratory strains and clinical isolates. The knowledge of these sequences has been critical for investigating viral pathogenicity. However, the assembly of complete herpesviral genomes, including HSV, is complicated due to the existence of large repeat regions and arrays of smaller reiterated sequences that are commonly found in these genomes. In addition, the inherent genetic variation in populations of isolates for viruses and other microorganisms presents an additional challenge to many existing HTS sequence assembly pipelines. Here, we evaluate two approaches for the identification of genetic variants in HSV1 strains using Illumina short read sequencing data. The first, a reference-based approach, identifies variants from reads aligned to a reference sequence and the second, a de novo assembly approach, identifies variants from reads aligned to de novo assembled consensus sequences. Of critical importance for both approaches is the reduction in the number of low complexity regions through the construction of a non-redundant reference genome. We compared variants identified in the two methods. Our results indicate that approximately 85% of variants are identified regardless of the approach. The reference-based approach to variant discovery captures an additional 15% representing variants divergent from the HSV1 reference possibly due to viral passage. Reference-based approaches are significantly less labor-intensive and identify variants across the genome where de novo assembly-based approaches are limited to regions where contigs have been successfully assembled. In addition, regions of poor quality assembly can lead to false variant identification in de novo consensus sequences. For viruses with a well-assembled reference genome, a reference-based approach is recommended.