High value of 64Cu as a tool to evaluate the restoration of physiological copper excretion after gene therapy in Wilson's disease.

Wilson's disease (WD) is an inherited disorder of copper metabolism associated with mutations in ATP7B gene. We have shown that the administration of an adeno-associated vector (AAV) encoding a mini version of human ATP7B (VTX-801) provides long-term correction of copper metabolism in a murine WD model. In preparation of a future clinical trial, we have evaluated by positron emission tomography (PET) the value of 64Cu biodistribution, excretion pattern, and blood kinetics as pharmacodynamic biomarkers of VTX-801 effects. Six-week-old WD mice were injected intravenously with increasing doses of VTX-801 and 3 weeks or 3 months later with [64Cu]CuCl2. Untreated WD and wild-type (WT) mice were included as controls. Control WD mice showed increased hepatic 64Cu retention, reduced fecal excretion of the radiotracer, and altered 64Cu blood kinetics (BK) compared with WT mice. VTX-801 treatment in WD mice resulted in a significant reduction of hepatic 64Cu accumulation, the restoration of fecal 64Cu excretion, and the correction of 64Cu BK. This study showed that VTX-801 restores physiological copper metabolism in WD mice, confirming the mechanism of action of VTX-801, and demonstrated the translational potential of [64Cu]CuCl2-PET to explore VTX-801 pharmacodynamics in a minimally invasive and sensitive manner in WD patients.

Optimising the IgG-degrading enzyme treatment regimen for enhanced adeno-associated virus transduction in the presence of neutralising antibodies.

OBJECTIVE: Pre-existing neutralising antibodies (NAbs) to adeno-associated viruses (AAVs) remain an impediment for systemically administered AAV-mediated gene therapy treatment in many patients, and various strategies are under investigation to overcome this limitation. Here, IgG-degrading enzymes (Ides) derived from bacteria of the genus Streptococcus were tested for their ability to cleave human IgG and allow AAV-mediated transduction in individuals with pre-existing NAbs. METHODS: Cleavage activity of three different Ides was evaluated in vitro in serum from different species. Passively immunised mice or non-human primates (NHP) with naturally occurring anti-AAV NAbs were used to define the optimal IdeS dose and administration window for AAVAnc80 and AAV8 vectors in mice and AAV3B in NHPs. RESULTS: The selected candidate, IdeS, was found to be highly efficient at cleaving human IgG, less efficient against NHP IgG and inefficient against mouse IgG. In vivo, we observed differences in how IdeS affected liver transduction in the presence of NAbs depending on the AAV serotype. For AAVAnc80 and AAV3B, the best transduction levels were achieved when the vector was administered after IgG digestion products were cleared from circulation. However, for AAV8 we only observed a modest and transient inhibition of transduction by IdeS cleavage products. CONCLUSION: Preconditioning with IdeS represents a unique treatment opportunity for patients primarily excluded from participation in gene therapy clinical trials because of elevated circulating anti-AAV NAb levels. However, careful determination of the optimal IdeS dose and timing for the administration of each AAV serotype is essential for optimal transduction.

Developmentally regulated Tcf7l2 splice variants mediate transcriptional repressor functions during eye formation.

Tcf7l2 mediates Wnt/ß-Catenin signalling during development and is implicated in cancer and type-2 diabetes. The mechanisms by which Tcf7l2 and Wnt/ß-Catenin signalling elicit such a diversity of biological outcomes are poorly understood. Here, we study the function of zebrafish tcf7l2alternative splice variants and show that only variants that include exon five or an analogous human tcf7l2 variant can effectively provide compensatory repressor function to restore eye formation in embryos lacking tcf7l1a/tcf7l1b function. Knockdown of exon five specific tcf7l2 variants in tcf7l1a mutants also compromises eye formation, and these variants can effectively repress Wnt pathway activity in reporter assays using Wnt target gene promoters. We show that the repressive activities of exon5-coded variants are likely explained by their interaction with Tle co-repressors. Furthermore, phosphorylated residues in Tcf7l2 coded exon5 facilitate repressor activity. Our studies suggest that developmentally regulated splicing of tcf7l2 can influence the transcriptional output of the Wnt pathway.

Gene therapy for progressive familial intrahepatic cholestasis type 3 in a clinically relevant mouse model.

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare monogenic disease caused by mutations in the ABCB4 gene, resulting in a reduction in biliary phosphatidylcholine. Reduced biliary phosphatidylcholine cannot counteract the detergent effects of bile salts, leading to cholestasis, cholangitis, cirrhosis and ultimately liver failure. Here, we report results from treating two- or five-week-old Abcb4-/- mice with an AAV vector expressing human ABCB4, resulting in significant decreases of PFIC3 disease biomarkers. All male mice achieved a sustained therapeutic effect up through 12 weeks, but the effect was achieved in only 50% of females. However, two-week-old females receiving a second inoculation three weeks later maintained the therapeutic effect. Upon sacrifice, markers of PFIC3 disease such as, hepatosplenomegaly, biliary phosphatidylcholine and liver histology were significantly improved. Thus, AAV-mediated gene therapy successfully prevented PFIC3 symptoms in a clinically relevant mouse model, representing a step forward in improving potential therapy options for PFIC3 patients.

Liver Expression of a MiniATP7B Gene Results in Long-Term Restoration of Copper Homeostasis in a Wilson Disease Model in Mice.

Gene therapy with an adeno-associated vector (AAV) serotype 8 encoding the human ATPase copper-transporting beta polypeptide (ATP7B) complementary DNA (cDNA; AAV8-ATP7B) is able to provide long-term copper metabolism correction in 6-week-old male Wilson disease (WD) mice. However, the size of the genome (5.2 kilobases [kb]) surpasses the optimal packaging capacity of the vector, which resulted in low-yield production; in addition, further analyses in WD female mice and in animals with a more advanced disease revealed reduced therapeutic efficacy, as compared to younger males. To improve efficacy of the treatment, an optimized shorter AAV vector was generated, in which four out of six metal-binding domains (MBDs) were deleted from the ATP7B coding sequence, giving rise to the miniATP7B protein (Δ57-486-ATP7B). In contrast to AAV8-ATP7B, AAV8-miniATP7B could be produced at high titers and was able to restore copper homeostasis in 6- and 12-week-old male and female WD mice. In addition, a recently developed synthetic AAV vector, AAVAnc80, carrying the miniATP7B gene was similarly effective at preventing liver damage, restoring copper homeostasis, and improving survival 1 year after treatment. Transduction of approximately 20% of hepatocytes was sufficient to normalize copper homeostasis, suggesting that corrected hepatocytes are acting as a sink to eliminate excess of copper. Importantly, administration of AAVAnc80-miniATP7B was safe in healthy mice and did not result in copper deficiency. Conclusion: In summary, gene therapy using an optimized therapeutic cassette in different AAV systems provides long-term correction of copper metabolism regardless of sex or stage of disease in a clinically relevant WD mouse model. These results pave the way for the implementation of gene therapy in WD patients.

Erratum: Lentiviral vectors can be used for full-length dystrophin gene therapy.

This corrects the article DOI: 10.1038/srep44775.

Lentiviral vectors can be used for full-length dystrophin gene therapy.

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

Lentiviral vectors can be used for full-length dystrophin gene therapy.

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

Prevalence of lactose intolerance in Chile: a double-blind placebo study.

BACKGROUND AND STUDY AIMS: Lactase non-persistence (LNP), or primary hypolactasia, is a genetic condition that mediates lactose malabsorption and can cause lactose intolerance. Here we report the prevalence of lactose intolerance in a double-blind placebo study. METHODS: The LCT C>T-13910 variant was genotyped by RT-PCR in 121 volunteers and lactose malabsorption was assessed using the hydrogen breath test (HBT) after consuming 25 g of lactose. Lactose intolerance was assessed by scoring symptoms (SS) using a standardized questionnaire following challenge with a lactose solution or saccharose placebo. RESULTS: The LNP genotype was observed in 57% of the volunteers, among whom 87% were HBT⁺. In the HBT⁺ group the median SS was 9 and in the HBT⁻ group the median SS was 3 (p < 0.001). No difference was observed in the SS when both groups were challenged with the placebo. The most common symptoms included audible bowel sounds, abdominal pain and meteorism. In the ROC curve analysis, an SS ≥ 6 demonstrated 72% sensitivity and 81% specificity for predicting a positive HBT. To estimate prevalence, lactose intolerance was defined as the presence of an SS ≥ 6 points after subtracting the placebo effect and 34% of the study population met this definition. CONCLUSIONS: The LNP genotype was present in more than half of subjects evaluated and the observed prevalence of lactose intolerance was 34%.

Light reaches the very heart of the zebrafish clock.

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.

Red cell membrane lipid changes at 3,500 m and on return to sea level.

Previous studies have shown that acute hypobaric hypoxia, obtained in a hypobaric chamber, and subsequent reoxygenation, give rise to modifications of the erythrocyte membrane lipid dynamics, resulting in an increased lateral diffusivity of the membrane lipids, and this was interpreted as the result of a modified lipid-protein interaction. The aim of the present study was to determine the effect of the reoxygenation condition in individuals after 3 days at an altitude of 3,500 m above sea level. Reoxygenation was a consequence of returning to sea level. Resting blood samples from both conditions were obtained, and erythrocytes were separated and immediately lysed for membrane isolation. We measured the bilayer polarity in membranes with Laurdan, a fluorescent probe. We also measured malondialdehyde in membrane lipids, an indicator of oxidative damage. We found a 12% (p = 0.016, n = 7) increase in the polarity of the membrane bilayer surface, and an increase of 70% (p = 0.005, n = 7) in the formation of malondialdehyde in the membrane after the reoxygenation condition. The membrane bilayer polarity increase is due to an oxidative modification of the phospholipid backbone after reoxygenation. People working and/or recreating at moderate altitude (3,500 m) may be at risk of erythrocyte membrane oxidative damage upon returning to sea level, and therefore a better understanding of the processes occurring upon reoxygenation may lead to proposed strategies to minimize this effect.

Peroxyl-oxidized erythrocyte membrane band 3 protein with anion transport capacity is degraded by membrane-bound proteinase.

Human red blood cells anion exchange protein (band 3) exposed to peroxyl radicals produced by thermolysis of 2,2'-azo-bis(2-amidinopropane) (AAPH) is degraded by proteinases that prevent accumulation of oxidatively damaged proteins. To assess whether this degradation affects anion transport capacity we used the anionic fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-y) amino] ethanosulfonate (NBD-taurine). A decrease of band 3 function was observed after exposure to peroxyl radicals. In the presence of proteinase inhibitors the decrement of anion transport through band 3 was smaller indicating that removal achieved by proteinases includes oxidized band 3 which still retain transport ability. Proteinases recognize band 3 aggregates produced by peroxyl radicals as was evaluated by immunoblotting. It is concluded that decrease of band 3 transport capacity may result from a direct protein oxidation and from its degradation by proteinases and that band 3 aggregates removal may prevent macrophage recognition of the senescent condition which would lead to cell disposal.

Hypobaric hypoxia-reoxygenation diminishes band 3 protein functions in human erythrocytes.

We have previously shown that subjects exposed to acute hypobaric hypoxia display an erythrocyte membrane protein band 3 with an increased susceptibility to proteolytic degradation. We suggested it was due to an oxidative damage of band 3. We now report that exposure to hypobaric hypoxia followed by reoxygenation affects protein band 3 functions such as anion transport and binding of glyceraldehyde-3P-dehydrogenase. Transport capacity was assessed with the fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] ethanesulfonate (NBD-taurine). Binding capacity was evaluated from the activity of the membrane-associated enzyme. Healthy young men were exposed for 20 min to hypobaric hypoxia, simulating an altitude of 4,500 m above sea level and after recompression band 3 function was assessed. An inhibition of band 3 anion transport function and a decrease in the binding of glyceraldehyde-3P-dehydrogenase to band 3 were observed. Evidence is given supporting the hypothesis that functional alteration of band 3 is due to its oxidative modification originated as a consequence of the exposure to hypobaric hypoxia and further reoxygenation.