The applications of genomic medicines are currently limited in the field of ophthalmology. However, owning to the future of genomic medicine, it is the responsibility of ophthalmologists, who have access to clinical samples, to lead cutting-edge research based on genome information. We focus on translational research on the genomic diagnosis of ocular infection and genome editing-based gene therapies for retinal dystrophies with the aim of developing treatment methods for such diseases. This article reveals the outcomes of our most recent research, which aims to establish new genome medicine platforms for ocular diseases and apply these platforms clinically.
I. Application of Nanopore long-read sequencing for the diagnosis of ocular infections
Recently, metagenomic analysis, an approach to comprehensively sequence DNA and RNA of microorganisms purified directly from specimens, bypassing the conventional culturing process, is being widely explored in the research of infectious diseases. We are currently working on the development of a metagenomic analysis platform for the diagnosis of ocular infectious diseases using a long-read Nanopore sequencer equipped with artificial intelligence, which allows easy and quick genome testing. When the metagenomic analysis was applied to aqueous or vitreous samples from diagnosed patients with conventional multiplex polymerase chain reaction (mPCR), the pathogen, including herpesvirus, was detected in many samples in agreement with the diagnosis. Nevertheless, pathogens were not detected in some samples, and considerable contamination with microorganisms was observed in all samples. To overcome these problems and comprehensively search for causative pathogens, we carried out a metagenomic association study in order to compare mPCR results between negative cases and controls. Despite successful detection of genome fragments that were considered as causative pathogens, we found that the sequence was shared between multiple microorganisms, including Chlamydia Trachomatis. Unfortunately, we have not yet obtained convincing results that reveal the source of the detected genome fragments associated with mPCR-negative uveitis cases. We applied a multifaceted genomic analysis to identify the pathogen from which the DNA sequence in question is derived.
Nanopore analysis revealed Torque teno virus in some cases of uveitis. This virus has been implicated in immunosuppressive conditions; However, little has been reported on it in the field of ophthalmology. We studied the association between Torque teno virus and a history of systemic immunosuppression by performing quantitative PCR (qPCR) and testing for the presence or absence of the virus in aqueous samples from uveitis cases. There was high detection rate of the virus in patients with a history of immunosuppression, which may indicate an association between intraocular Torque teno infection and immunosuppression.
II. Genome editing-based gene therapy strategy for retinal disorders
Most gene therapies currently used for ocular diseases utilize a gene supplementation approach based on adeno-associated viruses (AAVs). The basic concept behind this approach is to supplement loss-of-function mutations with wild-type copies of mutated genes in the diseased cells. However, due to the limited cargo capacity of AAVs, it is only possible to supplement mutations in small pathogenic genes. This is a critical problem, as only a small fraction of Japanese patients with retinitis pigmentosa (RP) can be treated with this method. In contrast, genome editing-based gene therapy targets the local genome; therefore, in theory, nearly all mutations are treatable regardless of the size of the gene. Unfortunately, the clinical application of this approach is hampered by its low treatment efficacy.
In order to improve treatment efficacy, we developed three different genome editing methods for AAV-mediated genome editing-based gene therapy, all of which may be clinically applicable in the future. First, we developed a versatile single-AAV genome editing-based gene therapy platform that allows the replacement of a mutation with a wild-type sequence. The utility of this approach was demonstrated in a mouse model. Second, by targeting insertions or deletions that result in frameshift mutations, we devised a simple and highly effective genomic reframing strategy aimed at reversing the shift in downstream codons caused by the mutation. The utility of this approach was shown in a patient-derived cell line harboring the S1653Kfs mutation in the eyes shut homolog (EYS) gene, which is carried by approximately 12% of all Japanese RP patients. Third, we were able to conveniently combine two different genome repair methods, i.e., microhomology-mediated end joining (MMEJ) and homology-independent targeted integration, to generate a compact MMEJ-H approach. We are using this to develop a therapy that mainly targets non-coding areas of the genome, including introns.
We plan to continue our efforts to pursue translational research focusing on the clinical application of genome-related technology for a wide range of ocular diseases and to contribute to advancements in genomic medicine in ophthalmology.
Nippon Ganka Gakkai Zasshi (J Jpn Ophthalmol Soc) 127: 402-422, 2023.