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Nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) is a ubiquitously expressed enzyme involved in nuclear NAD+production throughout the body. However, mutations in theNMNAT1gene lead to retina-specific disease with few reports of systemic effects. We have previously demonstrated that AAV-mediated gene therapy using self-complimentary AAV (scAAV) to ubiquitously express NMNAT1 throughout the retina prevents retinal degeneration in a mouse model ofNMNAT1-associated disease. We aimed to develop a better understanding of the cell types in the retina that contribute to disease pathogenesis inNMNAT1-associated disease, and to identify the cell types that require NMNAT1 expression for therapeutic benefit. To achieve this goal, we treatedNmnat1V9M/V9Mmice with scAAV using cell type-specific promoters to restrict NMNAT1 expression to distinct retinal cell types. We hypothesized that photoreceptors are uniquely vulnerable to NAD+depletion due to mutations inNMNAT1. Consistent with this hypothesis, we identified that treatments that drove NMNAT1 expression in the photoreceptors led to preservation of retinal morphology. These findings suggest that gene therapies forNMNAT1-associated disease should aim to express NMNAT1 in the photoreceptor cells.

Nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) is required for nuclear nicotinamide adenine mononucleotide (NAD+) biosynthesis in all nucleated cells, and despite its functional ubiquity, mutations in this gene lead to an isolated retinal degeneration. The mechanisms underlying how mutant NMNAT1 causes disease are not well understood, nor is the reason why the pathology is confined to the retina. Using a mouse model of NMNAT1-associated retinal degeneration that harbors the p.Val9Met mutation, we tested the hypothesis that decreased function of mutant NMNAT1 has a greater effect on the levels of NAD+ in the retina than elsewhere in the body. Measurements by liquid chromatography with tandem mass spectrometry showed an early and sustained decrease of NAD+ in mutant retinas that was not observed in other tissues. To understand how consumers of nuclear NAD+ are affected by the reduced availability of NAD+ in mutant retinas, poly(ADP-ribose) polymerase (PARP) and nuclear sirtuin activity were evaluated. PARP activity was elevated during disease progression, as evidenced by overproduction of poly(ADP-ribose) (PAR) in photoreceptors, whereas histone deacetylation activity of nuclear sirtuins was not altered. We hypothesized that PARP could be activated because of elevated levels of oxidative stress; however, we did not observe oxidative DNA damage, lipid peroxidation, or a low glutathione to oxidized glutathione ratio. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed that photoreceptors appear to ultimately die by apoptosis, although the low NAD+ levels and overproduction of PAR suggest that cell death may include aspects of the parthanatos cell death pathway.

No treatment is available for nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1)-associated retinal degeneration, an inherited disease that leads to severe vision loss early in life. Although the causative gene, NMNAT1, plays an essential role in nuclear nicotinamide adenine dinucleotide (NAD)+ metabolism in tissues throughout the body, NMNAT1-associated disease is isolated to the retina. Since this condition is recessive, supplementing the retina with a normal copy of NMNAT1 should protect vulnerable cells from disease progression. We tested this hypothesis in a mouse model that harbors the p.Val9Met mutation in Nmnat1 and consequently develops a retinal degenerative phenotype that recapitulates key features of the human disease. Gene augmentation therapy, delivered by subretinal injection of adeno-associated virus (AAV) carrying a normal human copy of NMNAT1, rescued retinal structure and function. Due to the early-onset profile of the phenotype, a rapidly activating self-complementary AAV was required to initiate transgene expression during the narrow therapeutic window. These data represent the first proof of concept for a therapy to treat patients with NMNAT1-associated disease., Graphical Abstract, This study demonstrates that gene therapy rescues retinal structure and function in a mouse model of NMNAT1-associated retinal degeneration, a disease that causes severe, early-onset vision loss and has no treatment available to patients. A self-complementary AAV delivery vector, rather than a single-stranded vector, proved necessary because of the rapid disease progression.