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Genome editing has the potential to treat genetic diseases in a variety of tissues, including the lung. We have previously developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing in the airways of mice. To validate this delivery vehicle in a large animal model, we have shown that intratracheal instillation of CRISPR/Cas9 in AAV5 can edit a housekeeping gene or a disease-related gene in the lungs of young rhesus monkeys. We observed up to 8% editing of angiotensin-converting enzyme 2 (ACE2) in lung lobes after single-dose administration. Single-nuclear RNA sequencing revealed that AAV5 transduces multiple cell types in the caudal lung lobes, including alveolar cells, macrophages, fibroblasts, endothelial cells, and B cells. These results demonstrate that AAV5 is efficient in the delivery of CRISPR/Cas9 in the lung lobes of young rhesus monkeys.

In this study, we built upon our previous work to demonstrate the distribution and transport of AAV5-green fluorescent protein (GFP) following a single convection-enhanced delivery infusion into the nonhuman primate cerebellum, with no untoward side effects noted. Dosing under magnetic resonance imaging guidance revealed a sixfold larger volume of distribution compared with the volume of infusion, with no evidence of reflux underscoring the convective properties of the cerebellum and step design of the cannula. Postmortem tissue analysis, 4 weeks post-adeno-associated viral (AAV) delivery, revealed the robust presence of the transgene in situ , with GFP detection in secondary regions not directly targeted by the infusion, denoting distal transport of the vector. Irrespective of tropism, a twofold larger area of transgene expression was found and was corroborated against the presence of contrast on T1-weighted images. Different levels of transduction were detected between animals, which were negatively correlated with the level of antibody titer against the GFP construct, whereby the higher the antibody titer, the lower the level of transgene expression. These findings support the use of the posterior fossa as a potential target site for direct delivery of gene-based therapeutics for cerebellar diseases.

Achromatopsia causes severely reduced visual acuity, photoaversion, and inability to discern colors due to cone photoreceptor dysfunction. In 2010, we reported on day-blindness in sheep caused by a stop-codon mutation of the ovine CNGA3 gene and began gene augmentation therapy trials in this naturally occurring large animal model of CNGA3 achromatopsia. The purpose of this study was to evaluate long-term efficacy and safety results of treatment, findings that hold great relevance for clinical trials that started recently in CNGA3 achromatopsia patients. Nine day-blind sheep were available for long-term follow up. The right eye of each sheep was treated with a single subretinal injection of an Adeno-Associated Virus Type 5 (AAV5) vector carrying either a mouse (n = 4) or a human (n = 5) CNGA3 transgene under control of the 2.1-Kb red/green opsin promoter. The efficacy of treatment was assessed periodically with photopic maze tests and electroretinographic (ERG) recordings for as long as 74 months postoperatively. Safety was assessed by repeated ophthalmic examinations and scotopic ERG recordings. The retinas of three animals that died of unrelated causes >5 years post-treatment were studied histologically and immunohistochemically using anti-hCNGA3 and anti-red/green cone opsin antibodies. Passage time and number of collisions of treated sheep in the photopic maze test were significantly lower at all follow-up examinations as compared with pretreatment values ( p  = 0.0025 and p  < 0.001, respectively). ERG Critical Flicker Fusion Frequency and flicker amplitudes at 30 and 40 Hz showed significant improvement following treatment ( p  < 0.0001) throughout the study. Ophthalmic examinations and rod ERG recordings showed no abnormalities in the treated eyes. Immunohistochemistry revealed the presence of CNGA3 protein in red/green opsin-positive cells (cones) of the treated eyes. Our results show significant, long-term improvement in cone function, demonstrating a robust rescue effect up to six years following a single treatment with a viral vector that provides episomal delivery of the transgene. This unique follow-up duration confirms the safe and stable nature of AAV5 gene therapy in the ovine achromatopsia model.