Improved cell metabolism prolongs photoreceptor survival upon retinal-pigmented epithelium loss in the sodium iodate induced model of geographic atrophy

Age-related macular degeneration (AMD) is characterized by malfunction and loss of retinal-pigmented epithelium (RPE) cells. Because the RPE transfers nutrients from the choriocapillaris to photoreceptor (PR), PRs are affected as well. Geographic atrophy (GA) is an advanced form of AMD characterized by severe vision impairment due to RPE loss over large areas. Currently there is no treatment to delay the degeneration of nutrient deprived PRs once RPE cells die. Here we show that cell-autonomous activation of the key regulator of cell metabolism, the kinase mammalian target of rapamycin complex 1 (mTORC1), delays PR death in the sodium iodate induced model of RPE atrophy. Consistent with this finding loss of mTORC1 in cones accelerates cone death as cones fail to balance demand with supply. Interestingly, promoting rod survival does not promote cone survival in this model of RPE atrophy as both, rods and cones suffer from a sick and dying RPE. The findings suggest that activation of metabolic genes downstream of mTORC1 can serve as a strategy to prolong PR survival when RPE cells malfunction or die

Muscle-Directed Delivery of an AAV1 Vector Leads to Capsid-Specific T Cell Exhaustion in Nonhuman Primates and Humans

With the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) approvals for Zolgensma, Luxturna, and Glybera, recombinant adeno-associated viruses (rAAVs) are considered efficient tools for gene transfer. However, studies in animals and humans demonstrate that intramuscular (IM) AAV delivery can trigger immune responses to AAV capsids and/or transgenes. IM delivery of rAAV1 in humans has also been described to induce tolerance to rAAV characterized by the presence of capsid-specific regulatory T cells (Tregs) in periphery. To understand mechanisms responsible for tolerance and parameters involved, we tested 3 muscle-directed administration routes in rhesus monkeys: IM delivery, venous limb perfusion, and the intra-arterial push and dwell method. These 3 methods were well tolerated and led to transgene expression. Interestingly, gene transfer in muscle led to Tregs and exhausted T cell infiltrates in situ at both day 21 and day 60 post-injection. In human samples, an in-depth analysis of the functionality of these cells demonstrates that capsid-specific exhausted T cells are detected after at least 5 years post-vector delivery and that the exhaustion can be reversed by blocking the checkpoint pathway. Overall, our study shows that persisting transgene expression after gene transfer in muscle is mediated by Tregs and exhausted T cells

Intralingual and Intrapleural AAV Gene Therapy Prolongs Survival in a SOD1 ALS Mouse Model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results in death from respiratory failure. No cure exists for this devastating disease, but therapy that directly targets the respiratory system has the potential to prolong survival and improve quality of life in some cases of ALS. The objective of this study was to enhance breathing and prolong survival by suppressing superoxide dismutase 1 (SOD1) expression in respiratory motor neurons using adeno-associated virus (AAV) expressing an artificial microRNA targeting the SOD1 gene. AAV-miR(SOD1) was injected in the tongue and intrapleural space of SOD1(G93A) mice, and repetitive respiratory and behavioral measurements were performed until the end stage. Robust silencing of SOD1 was observed in the diaphragm and tongue as well as systemically. Silencing of SOD1 prolonged survival by approximately 50 days, and it delayed weight loss and limb weakness in treated animals compared to untreated controls. Histologically, there was preservation of the neuromuscular junctions in the diaphragm as well as the number of axons in the phrenic and hypoglossal nerves. Although SOD1 suppression improved breathing and prolonged survival, it did not ameliorate the restrictive lung phenotype. Suppression of SOD1 expression in motor neurons that underlie respiratory function prolongs survival and enhances breathing until the end stage in SOD1(G93A) ALS mice

Editing out five Serpina1 paralogs to create a mouse model of genetic emphysema

Chronic obstructive pulmonary disease affects 10% of the worldwide population, and the leading genetic cause is alpha-1 antitrypsin (AAT) deficiency. Due to the complexity of the murine locus, which includes up to six Serpina1 paralogs, no genetic animal model of the disease has been successfully generated until now. Here we create a quintuple Serpina1a-e knockout using CRISPR/Cas9-mediated genome editing. The phenotype recapitulates the human disease phenotype, i.e., absence of hepatic and circulating AAT translates functionally to a reduced capacity to inhibit neutrophil elastase. With age, Serpina1 null mice develop emphysema spontaneously, which can be induced in younger mice by a lipopolysaccharide challenge. This mouse models not only AAT deficiency but also emphysema and is a relevant genetic model and not one based on developmental impairment of alveolarization or elastase administration. We anticipate that this unique model will be highly relevant not only to the preclinical development of therapeutics for AAT deficiency, but also to emphysema and smoking research

CX3CL1 (fractalkine) protein expression in normal and degenerating mouse retina: in vivo studies

We aimed to investigate fractalkine (CX3CL1) protein expression in wild type (wt) retina and its alterations during retinal degeneration in mouse model (rd10) of retinitis pigmentosa. Forms of retinal protein CX3CL1, total protein and mRNA levels of CX3CL1 were analyzed at postnatal days (P) 5, 10, 14, 22, 30, 45, and 60 by Western blotting and real-time PCR. Cellular sources of CX3CL1 were investigated by in situ hybridization histochemistry (ISH) and using transgenic (CX3CL1cherry) mice. The immunoblots revealed that in both, wt and rd10 retinas, a membrane integrated approximately 100 kDa CX3CL1 form and a cleaved approximately 85 kDa CX3CL1 form were present at P5. At P10, accumulation of another presumably intra-neuronal approximately 95 kDa form and a decrease in the approximately 85-kDa form were observed. From P14, a approximately 95 kDa form became principal in wt retina, while in rd10 retinas a soluble approximately 85 kDa form increased at P45 and P60. In comparison, retinas of rd10 mice had significantly lower levels of total CX3CL1 protein (from P10 onwards) and lower CX3CL1 mRNA levels (from P14), even before the onset of primary rod degeneration. ISH and mCherry reporter fluorescence showed neurons in the inner retina layers as principal sites of CX3CL1 synthesis both in wt and rd10 retinas. In conclusion, our results demonstrate that CX3CL1 has a distinctive course of expression and functional regulation in rd10 retina starting at P10. The biological activity of CX3CL1 is regulated by conversion of a membrane integrated to a soluble form during neurogenesis and in response to pathologic changes in the adult retinal milieu. Viable mature neurons in the inner retina likely exhibit a dynamic intracellular storage depot of CX3CL1

Genetic Deficiency of Flavin-Containing Monooxygenase 3 ( Fmo3) Protects Against Thrombosis but Has Only a Minor Effect on Plasma Lipid Levels-Brief Report

Objective- FMO (flavin-containing monooxygenase) 3 converts bacterial-derived trimethylamine to trimethylamine N-oxide (TMAO), an independent risk factor for cardiovascular disease. We generated FMO3 knockout (FMO3KO) mouse to study its effects on plasma TMAO, lipids, glucose/insulin metabolism, thrombosis, and atherosclerosis. Approach and Results- Previous studies with an antisense oligonucleotide (ASO) knockdown strategy targeting FMO3 in LDLRKO (low-density lipoprotein receptor knockout) mice resulted in major reductions in TMAO levels and atherosclerosis, but also showed effects on plasma lipids, insulin, and glucose. Although FMO3KO mice generated via CRISPR/Cas9 technology bred onto the LDLRKO background did exhibit similar effects on TMAO levels, the effects on lipid metabolism were not as pronounced as with the ASO knockdown model. These differences could result from either off-target effects of the ASO or from a developmental adaptation to the FMO3 deficiency. To distinguish these possibilities, we treated wild-type and FMO3KO mice with control or FMO3 ASOs. FMO3-ASO treatment led to the same extent of lipid-lowering effects in the FMO3KO mice as the wild-type mice, indicating off-target effects. The levels of TMAO in LDLRKO mice fed an atherogenic diet are very low in both wild-type and FMO3KO mice, and no significant effect was observed on atherosclerosis. When FMO3KO and wild-type mice were maintained on a 0.5% choline diet, FMO3KO showed a marked reduction in both TMAO and in vivo thrombosis potential. Conclusions- FMO3KO markedly reduces systemic TMAO levels and thrombosis potential. However, the previously observed large effects of an FMO3 ASO on plasma lipid levels appear to be due partly to off-target effects

AAV9 gene replacement therapy for respiratory insufficiency in very-long chain acyl-CoA dehydrogenase deficiency

Very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is an autosomal recessive disorder of fatty acid oxidation. Fatty acids are a major source of energy during catabolic stress, so the absence of VLCAD can result in a metabolic crises and respiratory insufficiency. The etiology of this respiratory insufficiency is unclear. Thus, our aims were: (1) to characterize respiratory pathophysiology in VLCADD mice (VLCAD(-/-) ), and (2) to determine if AAV9-mediated gene therapy improves respiratory function. For the first aim, VLCAD(-/-) and wild-type (WT) mice underwent an exercise/fast stress protocol and awake spontaneous breathing was evaluated using whole-body plethysmography (WBP) both at baseline and during a hypercapnic respiratory challenge (FiO2 : 0.21; FiCO2 : 0.07; nitrogen balance). During hypercapnia, VLCAD (-/-) mice had a significantly lower frequency, tidal volume, minute ventilation, and peak inspiratory and expiratory flow, all of which indicate respiratory insufficiency. Histologically, the cardiac and respiratory muscles of stressed VLCAD (-/-) animals had an accumulation of intramyocellular lipids. For the second aim, a single systemic injection of AAV9-VLCAD gene therapy improved this respiratory pathology by normalizing breathing frequency and enhancing peak inspiratory flow. In addition, following gene therapy, there was a moderate reduction of lipid accumulation in the respiratory muscles. Furthermore, VLCAD protein expression was robust in cardiac and respiratory muscle. This was confirmed by immuno-staining with anti-human VLCAD antibody. In summary, stress with exercise and fasting induces respiratory insufficiency in VLCAD(-/-) mice and a single injection with AAV9-VLCAD gene therapy ameliorates breathing

Western blot analysis of fractalkine protein expression in mouse neural retina at P5 until P60 being representative of five independent experiments.

<p>Blots were re-probed with anti-GAPDH antibody. (A). Fractalkine membrane integrated (100-kDa) and soluble (85-kDa) protein forms are present in both control (wt) and rd10 retina lysates during development at P5. (B). Accumulation of presumably intracellular (95-kDa) protein form is seen in both wt (B) and rd10 (C) developing neural retina at P10. Remarkably, no cleaved (85-kDa) protein form was associated with membrane integrated (100-kDa) form in both rd10 and wt retina lysates at P10. Both wt and rd10 retinas show lower to beneath detection level of 100-kDa form and higher level of 95-kDa form at P14 through P60 (D-G). Increased level of 85-kDa form is clearly detectable in rd10 retinas at P45 and P60 (G). Histogram showing relative percent levels of each of the three CX3CL1 protein forms in wt retina (H) and degenerating rd10 retina (I). Data are expressed as percent of densitometric arbitrary units. Values are mean ± SEM, (n = 5). In rd10 retinas, positive correlation between the relative levels of a ∼100-kDa and corresponding 85-kDa bands was found at P45 and P60 (Pearson product-moment correlation coefficient r = 0.683, n = 12, p = 0.014 in RD10 retina at P45 and r = 0.882, n = 9, p = 0.0017 at P60) as well as for the relative levels of a 100-kDa and 95-kDa bands (r = 0.928, n = 11, p<0.0001 at P14, r = 0.861, n = 9, p = 0.0029 at P22, r = 0.892, n = 9, p = 0.0012 at P30, r = 0.966, n = 12, p<0.0001 at P45, and r = 0.628, n = 12, p = 0.0288 at P60 in rd10 retina). The degree of association between the levels of 95 kDa and 85 kDa protein band was r = 0.712, n = 12, p = 0.0094 at P45 and r = 0.907, n = 9, p = 0.0007 at P60. It was not, however, possible, to make similar analysis for the wild type retina samples, as the level of the ∼95-kDa protein form was the highest and the levels of cleaved and full-length forms were far below the limit of detection.</p

Localization of CX3CL1 mRNA expressing cells in the retina and brain of wt and rd10 mice.

<p>(A-D). Light micrographs of radial cryosections through mouse central retina probed with a DIG-labeled anti-sense CX3CL1 riboprobe. In both, wt (A) and rd10 (B) mouse retinas, CX3CL1 mRNA signal is present exclusively in the neurons of the ganglion cell layer (GCL) and the inner- and outermost areas of the inner nuclear layer (INL). The outer nuclear (photoreceptor) layer is devoid of CX3CL1 signal at the time points examined (P22 and P30). Enlargements showing juxtanuclear localization of mRNA in the cells of inner nuclear (C) and ganglion (D) cell layers in wt mouse retina. (E-G). Distribution of CX3CL1 mRNA expressing neurons in wt mouse hippocampus. Light micrographs of a paraffin section used as positive control. (E). Numerous CX3CL1 mRNA-positive neurons are located in all hippocampal subfields of the pyramidal (PCL) and in the dentate granule cell layers (GCL). (F). Higher magnification of the dentate gyrus tips and of hilus. (G). Enlargement showing high levels of CX3CL1 mRNA expression within the grey matter of the cerebral cortex. PCL, pyramidal cell layer; GCL, layer of ganglion cells in dentate granule cell layer; H, hilus; T, dentate gyrus tips. Cells expressing CX3CL1 mRNA are visualized as of brown product accumulations in cryosections and red-brown in paraffin sections. The different color of nuclei staining (blue in cryosections and purple in paraffin sections) is due to a hemalaun counterstaining. Scale bars represent: 50 µm (A, B), 35 µm (C, D), 250 µm (E), 125 µm (F) and 25 µm (G).</p