Characterizations Of NMNAT1 Mutants,and The Construction Of NMNAT1-LCA9 Mouse Model And Gene Therapy

Leber’s Congenital amaurosis(LCA)is a severe dystrophy of the retina,typically becomes evident in the first year of life.The visual function of proband is usually poor and often accompanied by nystagmus,sluggish or near-absent pupillary responses,photophobia,high hyperopia,and keratoconus,with a worldwide incidence of 1/35,000.The nuclear nicotinamide mononucleotide adenyltransferase(NMNAT1)gene encode a six polymers protein,catalytic the biosynthesis of NAD+ in the last step,is a widely expressed in various organizations.NMNAT1 mutations can lead to LCA9.Patients with LCA9 show retinal atrophy,nystagmus and other symptoms after 2 months old.Patients with NMNAT1 truncation mutations show more severe phenotypes with macular atrophy and coloboma at birth,and there is no effective treatment for LCA9.In addition,it is not clear how those mutations of NMNAT1 gene result in LCA9.Most of the current studies have speculated that the decrease of mutant NNMNAT1 protease activity can lead to LCA9.In order to study the pathogenesis of NMNAT1 mutations,we used site-directed mutagenesis to construct the same NMNAT1 mutations as LCA9 patiens carry,then construct the mutant and wt recombined plasmid,pEGFP-NMNAT1,pCMV-NMNAT1 and pET30a-NMNAT1.The cellular localization of NMNAT1 protein was determined by cellular immunofluorescence experiment.Two nonsense mutations(Trp85*and Trp169*)changes the cellular location,and this change can affect the function of protein.In vitro enzyme activity and in vitro enzyme activity were used to test whether NMNAT1 mutation can change the enzyme activity.The results showed that Val98Gly mutant decreased the production of NAD+ significantly.NMNAT1 is a stable six polymer protein,have a long half-life,we use the bioinformatics to analyze the effect of NMNAT1 mutation on protein stability.Although no clash/contact atoms were founded in the mutant NMNAT1,the distribution of hydrogen bonds and hydrophobic bonds changed significantly.The stability of the helical structure was reduced in mutant NMNAT1 proteins(Val98Gly and Val151Phe),however Leul53Val,Glu257Lys and Asn273Asp increase the helical structure significantly.By improving the temperature,the changes of the secondary structure were observed by circular dichromatogram in real time,the results showed that the stability of the protein helical structure decreased after the mutation.In order to explore the therapeutic method for LCA9,we use CRISPR/Cas9 gene editing technique to build the NMNAT1-LCA9 mouse model.By using electroretinogram(ERG),fundus imaging and optical coherence tomography(OCT),we find needle point-like pigmentary degeneration in mice of 2-3 months,but the retinal layer was normal and the retinal layer was intact.After 32 weeks later,the symptoms of the eyes increased and the white spots were densely distributed,but the retinal structure remained intact and without defect.Compared with the missense mutation,the phenotypes of mouse models carry nonsense mutation showed earlier,and more severe retina deterioration,like the distribution of white scatter in the base of the fundus was more intensive.The results of tissue section showed that retinal structure of early NMNAT1-LCA9 mouse did not change obviously.After eight months later,outer layer,outer plexiform layer,inner nuclear layer and outer nuclear layer become thinner.The function of photoreceptors also decreased significantly after 8 months.Retinal layers brought congenital advantage for gene therapy,and the gene therapy of oculopathy ranks in the forefront of the disease gene therapy.At present,gene therapy of LCA2 in clinical phase 3 trials has completed.AAV2-CMV-NMNAT1 was used to cure LCA9 by injection.ERG was performed on NMNAT1-LCA9 mice when they were 4 months oldsubretinal.The results showed that the function of photoreceptor improved.But as a result of chosen higher injection drops of AAV2-CMV-NMNAT1,the retina sustains bigger toxicity,some severe retinal fundus atrophy in mice.In order to improve the effect of AAV2-CMV-NMNAT1 treatment,the suitable titer of AAV2-CMV-NMNAT1 used in gene therapystill needs to fumble.