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Non-viral vectors for gene therapy may accommodate large genes, such as USH2A for Usher syndrome patients.

Researchers at the UCL Institute of Ophthalmology, London, UK have reported early pre-clinical research using non-viral gene therapy treatments for certain inherited retinal degenerations (IRDs), including Usher syndrome, affecting vision, hearing and balance.  While many gene therapy strategies use adeno-associated viral (AAV) vectors with a 4.7kb cDNA capacity, genes such as USH2A, with a coding sequence of 15.6kb, will not be able to accommodate these vectors. In addition, inflammatory issues may arise from some viral vectors, potentially providing a further challenge, and therefore an alternative application using non-viral options may be valuable.  One of the non-viral approaches are engineered plasmids providing stable episomal maintenance showing persistent gene expression in vitro and in vivo, reported in Molecular Therapy (vol. 31, no. 8, Aug. 2023).

Usher syndrome is a rare disease arising from mutations in at least 9 genes that can cause different types of the disorder, including: MY07A, USH1C, CDH23, PCHD15, USH1G, USH2A, GPR98, WHRN and CLRN1.  Three subtypes of Usher (I, II and III) can be classified by the severity of the phenotype and genotype. Patients with Usher II, causing from USH2A, may present a combination of retinitis pigmentosa (RP) and congenital hearing impairment, with a delayed onset and slowly progressive pathology. USH2A gene translates a protein called usherin found in basement membranes in the inner ear and in the retina that plays an important role in cell development and maintenance.  The USH2A gene spans an estimated 800kb of DNA on chromosome 1q41 and the usherin coding sequence comprises 72 exons with 15.6kb.  This size of the coding sequence is challenging in the context of attempting to deliver a gene augmentation strategy with current viral gene capacity.

In the current study, the alternative strategy used DNA plasmid vectors containing the full-length of the human USH2A coding sequence, including a green fluorescent protein (GFP) reporter gene and a ubiquitous promoter, totalling  approximately 23 kb. The DNA plasmid vectors were transfected in cells, including USH2A patient-derived dermal fibroblasts and an experimental model using zebrafish, microinjected with the vector at the one-cell stage. Results showed that the vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Furthermore, after 1 year, GFP expression was found in the photoreceptor cells and with rescue of the sequence in the photoreceptors of zebrafish retinas. The researchers commented that, “this is the first reported vector that can be used to express full-length usherin with functional rescue” and “although further investigation showing efficiency in the mammalian retina is necessary, developing a safe and effective non-viral large gene therapy will be hugely beneficial to a significant proportion of retinal disease patients requiring functional genes that exceed the AAV limit”.