Our Areas of Focus
Developing a Treatment for GUCY2D Leber Congenital Amaurosis (LCA1)-
The Boye Lab has demonstrated the ability to restore retinal function and visually-guided behavior and preserve retinal structure in several animal models of this devastating early-onset retinal dystrophy. Dr. Boye is now engaged in IND-enabling safety studies and is hopeful that this treatment will be applied to patients within the next year. In 2014, Dr. Boye partnered with Genzyme/Sanofi to conduct the final pre-clinical studies necessary to file an IND and bring a gene therapy for LCA1 into the clinic. Phase I/II clinical trials began in November 2019 and are ongoing. This is one of the first gene therapies targeted to retinal photoreceptors. A press release from Genzyme and a story on ABC News highlight this work.
Engineering AAV for Safe and Efficient Gene Delivery to the Human Retina
FDA approval of an Adeno associated virus (AAV)- based gene therapy for RPE65-Leber congenital amaurosis (LCA2) solidified gene therapy’s place in current medical practice. However, injection of vector under the fovea of some patients led to central retinal thinning and loss of visual acuity. Similar decreases in retinal thickness were also observed in Choroideremia clinical trials. In more severe conditions, like X-linked Retinoschisis (XLRS), there is concern that subretinal injection (SRI) will further damage patient retinas. Since most inherited retinal diseases (IRDs) are caused by mutations in photoreceptor (PR)- and retinal pigment epithelial (RPE)- specific genes, development of gene therapies that more safely and efficiently target these cells remains a significant, unmet need. Targeting foveal cones is especially important, as they are responsible for acute, daylight vision. We have developed AAV capsids capable of efficient retinal transduction following intravitreal injection (IVI) in primate. The inner limiting membrane (ILM) is the major barrier to AAV transduction via this this route.
However, results from clinical trials utilizing IVI AAVs that show dose-limiting inflammation, and neutralization of the AAV capsid by pre-existing antibodies (NAbs) implicate the host immune system as a more immediate ‘barrier’ to clinical translation. The eye’s ‘immune-privilege’ has perhaps led to an under appreciation of the immune system’s role in shaping the outcome of intra-ocularly delivered AAVs. Naturally occurring antibodies to capsids capable of transducing retina via the vitreous (i.e. AAV2) are prevalent in up to 70% of humans. As such, a large percentage of patients will not meet inclusion criteria for emerging therapies. We are actively working to overcome these barriers by 1) enhancing transduction and safety of intravitreally delivered AAVs by engineering the capsid and genome to avoid immune recognition, 2) enhancing retinal transduction by subILM delivery of AAVs to enable efficient and specific transduction of inner and outer retina, and 3) enhancing transduction by subretinally delivered AAVs that spread laterally beyond the injection site. These novel vectors and methods will have an immediate impact on planned clinical trials to address inherited retinal diseases as well as non-orphan indications such as AMD. Development of these tools by academia (rather than industry) will ensure the availability of shared resources with the broader scientific community.
Developing Treatments for Inherited Retinal Disease Associated with Mutations in Large Genes
A significant hurdle in the retinal gene therapy field is how to efficiently deliver large therapeutic genes to photoreceptors. A limitation of AAV is its relatively modest genetic payload capacity. Despite this, it is to date the only viral (and non-viral) vector capable of efficiently transducing photoreceptors. To overcome the size limitation, the Boye Lab has developed dual AAV vector platforms wherein the therapeutic gene is split in half and delivered via two different, matched vectors. Once inside the cell, the matching halves recombine to form full-length gene. We have confirmed the sequence fidelity of the recombined gene, that it encodes full-length protein, and that it is well tolerated following subretinal injection in a clinically relevant species. We are currently funded to use dual AAV vectors to develop a gene therapy for a severe deaf-blinding condition, MYO7A– associated Ushers syndrome 1B.
Developing AAV-CRISPR/Cas9-Based Therapies for Inherited Retinal Disease
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing system can efficiently disrupt genes at desired loci, enabling either complete gene knockout or homology directed repair. The Boye lab is harnessing this state-of-the-art technology to address multiple forms of inherited retinal disease, assessing its utility in both small and large animal models.
Mutations in GUCY2D, the gene encoding retinal guanylate cyclase-1 (retGC1), are the leading cause of autosomal dominant cone-rod dystrophy. GUCY2D-CORD6 patients present with loss of visual acuity, abnormal color vision, photophobia, visual field loss and macular atrophy within the first decade. Rod degeneration and peripheral visual field loss follow. Significant progress towards clinical application of gene replacement therapy for LCA due to recessive mutations in GUCY2D (LCA1) has been made, but a different approach is needed to treat CORD6 where gain of function mutations cause dysfunction and dystrophy. Our preliminary data show that 1) selective and efficient somatic knock-out of GUCY2D and Gucy2e (murine homologue) with AAV-CRISPR/Cas9 results in a subsequent loss of retinal structure/function that manifests from reduced retGC1 expression in macaque and mouse, respectively, 2) a ‘knock-out + complementation in trans’ approach (wherein complementation is performed with ‘hardened’ Gucy2e not recognized by Gucy2e gRNA) preserves retinal function in mice, and 3) AAV-CRISPR/Cas9- based editing of GUCY2D is therapeutic in a R838S transgenic (Tg) mouse model of CORD6. Ongoing work aims to establish the optimal parameters for AAV-CRISPR/Cas9-based gene editing in two R838S CORD6 Tg mouse lines, and the optimal AAV capsid/dose, durability of therapy, treatment window, and feasibility of transient Cas9 expression systems. We are also evaluating safety/efficacy of AAV-CRISPR/Cas9-based gene editing in a clinically relevant species by looking for off-target editing and assessing the potential impact of AAV vector insertions and long-term Cas9 expression. Finally, we are comparing ‘knock out + complementation in trans’ vs. ‘allele-targeted’ approaches for treating CORD6. Our findings will identify the optimal capsid/dose, and treatment age for therapeutic AAV-CRISPR/Cas9-based disruption of R838S GUCY2D in vivo. We will establish the safety profile, and regional efficiencies of gene editing by AAV-CRISPR/Cas9 in a species with both genomic and clinical relevance. In addition, we will identify materials and approaches that will allow clinical application of AAV-CRISPR/Cas9 therapies for CORD6 as well as other dominantly inherited retinal diseases.