Nonviral Gene Therapy

Plasmid vectors show potential in future of gene therapy for inherited retinal diseases. Roibeárd O’hÉineacháin reports. Plasmids are showing promising results in preclinical studies as an alternative to adeno-associated virus vectors (AAVs) in treating hereditary retinal diseases, says Professor Mariya Moosajee BSc, MBBS, PhD, FRCOphth. Prof Moosajee noted AAV vectors have good transduction efficiency and are generally well tolerated. However, AAV vectors have some limitations. For example, they are restricted to transfection of genes of a certain size, no more than 4.7 kB. In addition, in 10–20% of cases, they will cause inflammatory reactions, which can result in sight loss. Moreover, gene therapy with AAV vectors has had variable results. While LUXTURNA® (voretigene neparvovec-rzyl) has been extremely successful in treating Leber congenital amaurosis, the gene therapy trials for choroideremia with an AAV viral vector have failed. She pointed out DNA plasmids have the advantage of having unlimited cloning capacity and freedom from toxic viral components. Researchers have manufactured the plasmids with scaffold/matrix attachment regions (S/MARs). An S/MAR is a 300 to 3000 AT-rich base pair nucleotide motif found naturally in the nuclear matrix of cells. S/MARs help with the binding formation of chromatin structure for DNA transcription and replication. When placed into the DNA plasmids, they confer episomal maintenance, prevent epigenetic silencing, and provide mitotic stability. CHOROIDEREMIA Prof Moosajee’s team is developing S/MAR plasmid gene therapies for choroideremia and Usher syndrome with many encouraging preclinical findings. She noted choroideremia (CHM) is an X-linked chorioretinal dystrophy with a prevalence of 1 in 50,000–100,000. The condition is caused by a mutation in the CHM gene that has a molecular weight of 1.9 kB and encodes for the REP1 protein. She and her associates have successfully cloned an S/MAR vector for the CHM gene. In laboratory experiments, fibroblasts from choroideremia patients transfected with the S/MAR vector have shown persistent expression of the transfected gene. In a zebrafish animal model for choroideremia, embryo transfection resulted in human gene expression in the photoreceptors of the adult fish. Future work on gene therapy for choroideremia will involve testing S/MAR vectors in retinal pigment epithelium (RPE) cells derived from choroideremia patient induced pluripotent stem cells and evaluation of functional vision in a mouse model of the disease. USHER SYNDROME Prof Moosajee noted Usher syndrome has a prevalence of 4–17 in 100,000 and causes hearing and sight loss with variable vestibular dysfunction. A mutation in the large USH2A gene (19 kB cDNA), which codes the Usherin protein, causes the most common form (type 2). Prof Moosajee and her team have successfully cloned the entire USH2A gene into an S/MAR vector. In laboratory experiments, there was a persistent expression of USH2A in transfected human fibroblasts and transfected zebrafish embryos. The team is now undertaking studies using intravitreal and subretinal injections in animal experiments. “Nonviral gene therapy holds a great deal of promise, but future work now needs to be focused on improving the transfection efficiency in the retina in a safe and efficient manner. It may involve chemical or physical approaches, such as the use of nanoparticles or electroporation,” she concluded. The study was presented at the Retina 2021 Meeting in Dublin, Ireland. Prof Mariya Moosajee BSc, MBBS, PhD, FRCOphth is a Professor of Molecular Ophthalmology at the UCL and the Francis Crick Institute and a Consultant Ophthalmologist in Genetic Eye Disease Moorfields Eye Hospital and Great Ormond Street Hospital for Children, London, UK. m.moosajee@ucl.ac.uk