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• W4284882005 abstract "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. 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. IntroductionWilson’s disease (WD) is a rare autosomal recessive, debilitating, and life-threatening disorder of copper homeostasis that affects approximately one in 30,000 individuals, with wide geographical variations.1Członkowska A. Litwin T. Dusek P. Ferenci P. Lutsenko S. Medici V. Rybakowski J.K. Weiss K.H. Schilsky M.L. Wilson disease.Nat. Rev. Dis. Primers. 2018; 4: 21https://doi.org/10.1038/s41572-018-0018-3Crossref PubMed Scopus (254) Google Scholar In WD, mutations of the copper transporter ATP7B lead to decreased biliary copper excretion and a reduction in circulating holoceruloplasmin levels.1Członkowska A. Litwin T. Dusek P. Ferenci P. Lutsenko S. Medici V. Rybakowski J.K. Weiss K.H. Schilsky M.L. Wilson disease.Nat. Rev. Dis. Primers. 2018; 4: 21https://doi.org/10.1038/s41572-018-0018-3Crossref PubMed Scopus (254) Google Scholar, 2Saroli Palumbo C. Schilsky M.L. Clinical practice guidelines in Wilson disease.Ann. Transl. Med. 2019; 7: S65https://doi.org/10.21037/atm.2018.12.53Crossref PubMed Google Scholar, 3Ferenci P. Diagnosis of Wilson disease.Handb. Clin. Neurol. 2017; 142: 171-180https://doi.org/10.1016/B978-0-444-63625-6.00014-8Crossref PubMed Scopus (39) Google Scholar As a result, toxic levels of copper accumulate, primarily in the liver but also in the central nervous system; left untreated, WD is considered uniformly fatal. Current medical WD management includes copper chelators and/or zinc salt treatment, together with low-copper diet.2Saroli Palumbo C. Schilsky M.L. Clinical practice guidelines in Wilson disease.Ann. Transl. Med. 2019; 7: S65https://doi.org/10.21037/atm.2018.12.53Crossref PubMed Google Scholar, 3Ferenci P. Diagnosis of Wilson disease.Handb. Clin. Neurol. 2017; 142: 171-180https://doi.org/10.1016/B978-0-444-63625-6.00014-8Crossref PubMed Scopus (39) Google Scholar, 4European Association for Study of LiverEASL clinical practice guidelines: wilson’s disease.J. Hepatol. 2012; 56: 671-685https://doi.org/10.1016/j.jhep.2011.11.007Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar, 5Roberts E.A. Schilsky M.L. American Association for Study of Liver Diseases (AASLD)Diagnosis and treatment of Wilson disease: an update.Hepatology. 2008; 47: 2089-2111https://doi.org/10.1002/hep.22261Crossref PubMed Scopus (910) Google Scholar Despite recognized benefits, current life-long management options have important limitations, including poor compliance, incomplete resolution of symptoms, and various side effects, among them severe and often not fully reversible neurological deterioration.2Saroli Palumbo C. Schilsky M.L. Clinical practice guidelines in Wilson disease.Ann. Transl. Med. 2019; 7: S65https://doi.org/10.21037/atm.2018.12.53Crossref PubMed Google Scholar,3Ferenci P. Diagnosis of Wilson disease.Handb. Clin. Neurol. 2017; 142: 171-180https://doi.org/10.1016/B978-0-444-63625-6.00014-8Crossref PubMed Scopus (39) Google Scholar As of today, liver transplantation, together with life-long immunosuppression, remains the only therapeutic option that can permanently restore physiological copper metabolism and is mostly limited to patients with acute liver failure or end-stage liver disease.2Saroli Palumbo C. Schilsky M.L. Clinical practice guidelines in Wilson disease.Ann. Transl. Med. 2019; 7: S65https://doi.org/10.21037/atm.2018.12.53Crossref PubMed Google Scholar, 3Ferenci P. Diagnosis of Wilson disease.Handb. Clin. Neurol. 2017; 142: 171-180https://doi.org/10.1016/B978-0-444-63625-6.00014-8Crossref PubMed Scopus (39) Google Scholar, 4European Association for Study of LiverEASL clinical practice guidelines: wilson’s disease.J. Hepatol. 2012; 56: 671-685https://doi.org/10.1016/j.jhep.2011.11.007Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar, 5Roberts E.A. Schilsky M.L. American Association for Study of Liver Diseases (AASLD)Diagnosis and treatment of Wilson disease: an update.Hepatology. 2008; 47: 2089-2111https://doi.org/10.1002/hep.22261Crossref PubMed Scopus (910) Google Scholar However, recent data suggest the potential for added benefit of liver transplantation to newly diagnosed neurological WD patients unresponsive to medical treatment.6Poujois A. Sobesky R. Meissner W.G. Brunet A.S. Broussolle E. Laurencin C. Lion-François L. Guillaud O. Lachaux A. Maillot F. et al.Liver transplantation as a rescue therapy for severe neurologic forms of Wilson disease.Neurology. 2020; 94: e2189-e2202https://doi.org/10.1212/WNL.0000000000009474Crossref PubMed Scopus (21) Google ScholarLiver-directed adeno-associated vector (AAV)-based gene therapy has been recently shown to offer a non-surgical, permanent correction of copper metabolism in WD mice.7Murillo O. Luqui D.M. Gazquez C. Martinez-Espartosa D. Navarro-Blasco I. Monreal J.I. Guembe L. Moreno-Cermeño A. Corrales F.J. Prieto J. et al.Long-term metabolic correction of Wilson’s disease in a murine model by gene therapy.J. Hepatol. 2016; 64: 419-426https://doi.org/10.1016/j.jhep.2015.09.014Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 8Murillo O. Moreno D. Gazquez C. Barberia M. Cenzano I. Navarro I. Uriarte I. Sebastian V. Arruebo M. Ferrer V. et al.Liver expression of a MiniATP7B gene results in long-term restoration of copper homeostasis in a Wilson disease model in mice.Hepatology. 2019; 70: 108-126https://doi.org/10.1002/hep.30535Crossref PubMed Scopus (13) Google Scholar, 9Moreno D. Murillo O. Gazquez C. Hernandez-Alcoceba R. Uerlings R. Gonzalez-Aseguinolaza G. Weiskirchen R. Visualization of the therapeutic efficacy of a gene correction approach in Wilson’s disease by laser-ablation inductively coupled mass spectrometry.J. Hepatol. 2018; 68: 1088-1090https://doi.org/10.1016/j.jhep.2017.12.022Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar The administration of VTX-801, a hepatotropic recombinant AAV carrying an ATP7B-minigene under the transcriptional control of a liver-specific promoter, to WD mice resulted in the reduction of copper concentration in liver and urine, restoration of fecal copper excretion, and normalization of ceruloplasmin activity and transaminase levels in circulation.8Murillo O. Moreno D. Gazquez C. Barberia M. Cenzano I. Navarro I. Uriarte I. Sebastian V. Arruebo M. Ferrer V. et al.Liver expression of a MiniATP7B gene results in long-term restoration of copper homeostasis in a Wilson disease model in mice.Hepatology. 2019; 70: 108-126https://doi.org/10.1002/hep.30535Crossref PubMed Scopus (13) Google Scholar,9Moreno D. Murillo O. Gazquez C. Hernandez-Alcoceba R. Uerlings R. Gonzalez-Aseguinolaza G. Weiskirchen R. Visualization of the therapeutic efficacy of a gene correction approach in Wilson’s disease by laser-ablation inductively coupled mass spectrometry.J. Hepatol. 2018; 68: 1088-1090https://doi.org/10.1016/j.jhep.2017.12.022Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar Furthermore, sustained therapeutic efficacy was demonstrated for at least 1 year with preserved liver histology and increased animal survival.8Murillo O. Moreno D. Gazquez C. Barberia M. Cenzano I. Navarro I. Uriarte I. Sebastian V. Arruebo M. Ferrer V. et al.Liver expression of a MiniATP7B gene results in long-term restoration of copper homeostasis in a Wilson disease model in mice.Hepatology. 2019; 70: 108-126https://doi.org/10.1002/hep.30535Crossref PubMed Scopus (13) Google ScholarIn anticipation of a first-in-human clinical trial, we have explored the possibility of using a non-invasive technique to demonstrate the restoration of physiological copper metabolism in VTX-801-treated patients. Pioneering studies performed in the 50s–60s showed abnormal copper metabolism (reduced fecal excretion and abnormal plasma kinetics) in WD patients using radioactive copper isotopes, including short-lived 64Cu.10Bearn A.G. Kunkel H.G. Localization of Cu64 in serum fractions following oral administration: an alteration in Wilson’s disease.Proc. Soc. Exp. Biol. Med. 1954; 85: 44-48https://doi.org/10.3181/00379727-85-20780Crossref PubMed Scopus (91) Google Scholar, 11Osborn S.B. Walshe J.M. Studies with radioactive copper (64Cu and 67Cu) in relation to the natural history of Wilson’s disease.Lancet. 1967; 1: 346-350https://doi.org/10.1016/s0140-6736(67)92893-0Abstract PubMed Scopus (22) Google Scholar, 12O’Reilly S. Weber P. Pollycove M. Shipley L. Detection of the heterozygote of Wilson’s disease.J. Nucl. Med. 1969; 10: 143-144PubMed Google Scholar The excellent WD diagnostic accuracy of the 48-h-to-1-to-2-h 64Cu plasma ratio following intravenous [64Cu]CuCl2 administration showed initially by Sternlieb and Scheinberg13Sternlieb I. Scheinberg I.H. Radiocopper in diagnosing liver disease.Semin Nucl. Med. 1972; 2: 176-188https://doi.org/10.1016/s0001-2998(72)80071-0Crossref PubMed Google Scholar was recently corroborated by Czlonkowska et al.14Członkowska A. Rodo M. Wierzchowska-Ciok A. Smolinski L. Litwin T. Accuracy of the radioactive copper incorporation test in the diagnosis of Wilson disease.Liver Int. 2018; 38: 1860-1866https://doi.org/10.1111/liv.13715Crossref PubMed Scopus (16) Google ScholarHere, we have investigated the biodistribution and excretion and secretion pattern of Cu after VTX-801 treatment in a murine model of WD using 64Cu. Its use makes possible to perform non-invasive in vivo imaging studies for assessment of copper metabolism imbalance with positron emission tomography (PET) technology. In addition, the presence of 64Cu in biological samples and tissues was measured in a gamma counter, adding valuable information about copper biodistribution and routes of elimination.ResultsDistinctive biodistribution pattern of radiocopper in 9-week-old Atp7b−/− and WT miceTo set up optimal experimental conditions for the evaluation of VTX-801’s effect on copper metabolism, we first studied the biodistribution of copper-64 in untreated 9-week-old Atp7b−/− (WD), Atp7b+/− (heterozygous), and WT (Atp7b+/+) male (n = 3) and female (n = 3) mice (total n = 6). Blood samples were harvested at different time points, weighted, and measured in gamma counter. All animal groups showed a rapid decrease of the radioactive signal in blood shortly after injection, followed by a slight signal increase (starting 8 h post-injection) in WT and Atp7b+/− animals, and a distinctive continuous decrease in Atp7b−/− mice (Figure 1A ). The average 48-h-to-1-h ratio of radiocopper signal in blood was 0.87 for WT animals, 0.79 for Atp7b+/−, and 0.4 for Atp7b−/−, being similar for WT and Atp7b+/−, although statistically different between Atp7b−/− versus WT (Figure 1B). These data are consistent with data reported in humans.13Sternlieb I. Scheinberg I.H. Radiocopper in diagnosing liver disease.Semin Nucl. Med. 1972; 2: 176-188https://doi.org/10.1016/s0001-2998(72)80071-0Crossref PubMed Google Scholar During the experiment, all animals were placed in individual metabolic cages. Twenty-four-hour cumulative feces and urine were harvested for 2 consecutive days, and the presence of radioactive signal was determined (Figure 1C). Significant differences in the concentration of copper-64 in feces were observed between Atp7b−/− and WT or Atp7b+/− mice. While fecal excretion of radiocopper was observed in WT and Atp7b+/− mice, the signal was nearly undetectable in Atp7b−/− mice. Slightly lower levels of 64Cu excretion were observed in Atp7b+/− mice during the first 24 h in comparison to WT, but no differences were observed at 48 h. Regarding urinary radiocopper excretion, levels were very low and highly variable in all three groups, and no significant differences were observed among them; surprisingly, Atp7b−/− showed the lowest levels (Figure S1). Thus, radiocopper blood kinetics (BK) and fecal excretion, but not urinary excretion, represent useful biomarkers to determine copper metabolism.Radiocopper biodistribution was analyzed by full-body in vivo PET, 24 and 48 h after 64Cu injection. PET images in WT and Atp7b+/− animals showed the presence of radioactive signal in liver as well as in the gut region. In untreated Atp7b−/− animals, a strong signal was detected in the liver area, but no signal was detected in the gut area (Figure 2A ). The quantification of the liver standardized uptake value (SUV), i.e., the radiocopper uptake in liver, showed a significantly higher hepatic retention of radiocopper in Atp7b−/− animals in comparison to WT and Atp7b+/− mice (Figure 2B). Seventy-two hours after radiocopper injection, animals were sacrificed, and the radioactive signal was quantified in liver, brain, kidneys, lungs, and spleen (Figure 2C). As observed with PET data, significantly higher radioactive signal was detected in liver of Atp7b−/− mice in comparison to livers of WT and Atp7b+/− mice. Interestingly, radiocopper levels were significantly lower in all other organs evaluated, brain, kidneys, lungs, and spleen in Atp7b−/− mice in comparison to WT or Atp7b+/− mice.Figure 2In vivo and ex vivo biodistribution analysis of 64Cu by whole-body PET imaging and gamma counter measurements in WT, Atp7b+/−, and Atp7b−/− mice at 9 weeks of ageShow full captionPET analysis was performed 24 and 48 h post-64Cu injection. Ex vivo biodistribution analysis was performed at sacrifice, 48 h after 64Cu administration. (A) Representative PET coronal slices obtained 24 and 48 h after intravenous (i.v.) injection of the radiotracer co-registered with a CT 3D image of other animal used as anatomical reference. The volumes of interest (VOIs) containing the entire liver were used to obtain quantitative data. (B) Graphical representation of the quantitative measures obtained by PET data analysis is shown. (C) Ex vivo biodistribution data of selected organs are shown. All data were presented as mean ± SD. The statistical analysis was performed using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. lv, liver; i, intestine; f, feces.View Large Image Figure ViewerDownload Hi-res image Download (PPT)VTX-801 administration to 6-week-old Atp7b−/− mice significantly modifies physiological radiocopper biodistributionSix-week-old male and female Atp7b−/− mice (n = 6) received intravenously three different doses of VTX-801: 5 × 1011, 1.5 × 1012, and 5 × 1012 viral genome copies (VG)/kg. The highest dose administered to animals corresponds to one previously shown to restore copper metabolism and improve survival in Atp7b−/− mice.8Murillo O. Moreno D. Gazquez C. Barberia M. Cenzano I. Navarro I. Uriarte I. Sebastian V. Arruebo M. Ferrer V. et al.Liver expression of a MiniATP7B gene results in long-term restoration of copper homeostasis in a Wilson disease model in mice.Hepatology. 2019; 70: 108-126https://doi.org/10.1002/hep.30535Crossref PubMed Scopus (13) Google Scholar Three weeks post-injection, VTX-801-treated animals received an intravenous injection of 64Cu. Blood samples were harvested at different time points, and radiocopper BK was analyzed (Figure 3A ). As controls, we used untreated WT and Atp7b−/− mice from Figures 1 and 2. In Atp7b−/− animals, VTX-801 treatment prevented the continuous decrease of the radiocopper signal observed in untreated WD mice. Moreover, all three doses of VTX-801 administered showed an initial decrease in the 64Cu signal followed by stabilization after 24 h. A direct dose relationship was also observed for radiocopper 48 h/1 h ratio with average values of 0.61, 0.67, and 0.79 in WD animals receiving 5 × 1011 VG/kg, 1.5 × 1012 VG/kg, and 5 × 1012 VG/kg, respectively (Figure 3B). No differences were observed depending on the gender.Figure 3Copper-64 kinetics in blood and feces and in vivo and ex vivo biodistribution analysis of the radiotracer by whole-body PET imaging and gamma counter measurements in Atp7b−/−, WT, and VTX-801-treated Atp7b−/− at 9 weeks of ageShow full captionAtp7b−/− and WT mice data are the ones shown in Figures 1 and 2. Blood samples were collected 1, 4, 8, 24, and 48 h post-[64Cu]CuCl2 administration. Feces were harvested 24 and 48 h after 64Cu injection. PET analysis was performed 24 and 48 h post-administration of the radiotracer. Ex vivo biodistribution analysis was performed at sacrifice, 72 h after 64Cu administration. (A) Radiocopper plasma kinetics (BK), fold-change ratios of 64Cu in blood at different time points relative to 1 h have been represented. (B) Forty-eight-hours-to-1-hour ratio of 64Cu in the serum 1 h is shown. (C) Cumulative fecal excretion of 64Cu is shown. (D) Representative PET coronal slices obtained 24 h after 64Cu administration are shown. (E) Ex vivo biodistribution data of selected organs are shown. All data were presented as mean ± SD. The statistical analysis was performed using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Presence of radiocopper in feces also revealed a direct dose response to VTX-801 (Figures 3C and 3D), indicating that gene therapy and the expression of functional ATP7B protein restores physiological excretion of copper through the biliary and fecal route.Radiocopper biodistribution was analyzed by PET at 24 and 48 h post [64Cu]CuCl2 injection. As previously described for untreated Atp7b−/− mice, copper-64 was retained in the liver and no signal was detected in the gut area; however, in animals treated with the higher dose of VTX-801, the presence of radiocopper was clearly detected in the gut region, same as in WT animals (Figure 3D). Furthermore, when 64Cu activity was measured in whole organs at sacrifice (Figure 3E), a dose-dependent reduction of radiocopper activity in liver was observed for WD mice treated with VTX-801. Levels of 64Cu in brain, kidneys, and lungs of untreated Atp7b−/− mice were lower than in WT animals; VTX-801 treatment at the highest dose resulted in a significant increase of 64Cu in brain and lungs (Figure 3E).Long-lasting VTX-801 restoration of fecal excretion of Cu and BK in WD miceSix-week-old Atp7b−/− male mice (n = 3) were treated with either of four doses (5E11, 1.5E12, 5E12, and 1.5E13 VG/kg) of VTX-801 and 3 months later received an intravenous injection of [64Cu]CuCl2. Blood samples were harvested at different time points as previously described. The radiocopper BK of untreated WT and WD animals of matching age (18 weeks old) was different from that observed at 9 weeks of age. In WT mice, radiocopper levels remained stable from 1 to 4 h post-injection and slightly increased thereafter up to 48 h. In WD mice, an initial drop of signal was observed during the first 8 h, followed by a sharp increase in activity levels, higher than those observed in WT animals at 48 h post-injection (Figure 4A ). The average 48 h/1 h ratio of radiocopper concentration in serum was 1.32 in WT and 1.59 in WD mice (Figure 4B). Interestingly, Atp7b−/− mice treated with the two higher doses of VTX-801 (5E12 and 1.5E13 VG/kg) showed a profile similar to that observed in WT mice. At lower doses, copper-64 levels in blood either remained stable (1.5E12 VG/kg) or exhibited a continuous decrease (5E11 VG/kg) (Figure 4A), however, not as dramatic to that observed in untreated 9-week-old Atp7b−/− mice (Figure 1A). The radiocopper 48 h/1 h ratio in blood increased in a dose-dependent manner with average values of 0.6, 0.86, 1.05, and 1.06 after treatment with 5E11, 1.5E12, 5E12, and 1.5E13 VG/kg, respectively (Figure 4B).Figure 4Copper-64 kinetics in blood and feces of Atp7b−/−, WT, and VTX-801-treated Atp7b−/− mice at 18 weeks of ageShow full captionBlood samples were collected 1, 4, 8, 24, 48, and 72 h post-64Cu administration. Feces were harvested 24, 48, and 72 h after 64Cu injection. (A) Fold change ratio of 64Cu in blood relative to 1 h is shown. (B) Forty-eight-hours-to-1-hour ratio of 64Cu in the serum is shown. (C) Cumulative fecal excretion of 64Cu is shown. All data were presented as mean ± SD. The statistical analysis was performed using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc. ∗p < 0.05; ∗∗p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Twenty-four-hour feces were harvested for 3 consecutive days, and the presence of radioactivity was represented as cumulative values (Figure 4C). WD animals treated with VTX-801 showed a direct dose dependency for levels of copper excreted in feces, with those that received the highest dose of VTX-801 reaching levels not significantly different from WT animals. The analysis of urinary copper-64 excretion revealed no significant differences among the different groups, although once again, levels in WT animal were slightly higher that in WD mice and WD animals treated with the highest dose of VTX-801 showed the highest levels (Figure S2).PET images revealed retention of radiocopper in liver of untreated Atp7b−/− mice. VTX-801 treatment induced a dose-dependent reduction of 64Cu signal in the liver area as well as a dose-dependent increase of radiocopper signal in the gut area (Figure 5A ). Furthermore, radiocopper signal was also quantified by drawing volumes of interest (VOIs) in liver at 90 min (min), 24 h, and 48 h after 64Cu injection and 24 h/90 min or 48 h/90 min ratios were calculated (Figure 5B). In WT animals, both ratios were below 1, indicating a reduction of radiocopper in liver over time. In contrast, untreated Atp7b−/− mice exhibited ratios above 1, suggesting retention of the radiotracer in the organ. WD mice treated with VTX-801 showed a dose-dependent reduction of 64Cu signal in liver as well as a reduction in the 24 h/90 min or 48 h/90 min ratios in comparison to WD untreated mice.Figure 5In vivo and ex vivo biodistribution analysis of 64Cu by whole-body PET imaging and gamma counter measurements in Atp7b−/−, WT, and VTX-801-treated Atp7b−/− mice at 18 weeks of ageShow full captionPET analysis was performed 90 min and 24 and 48 h post-64Cu injection. Ex vivo biodistribution analysis was performed at sacrifice, 72 h after 64Cu administration. (A) Representative PET coronal slices obtained 48 h after the injection of the radiotracer are shown. The VOIs)containing the entire liver (dashed line) were used to obtain quantitative data. (B) Graphical representation of hepatic concentration of 64Cu (SUV) at 90 min and 24 and 48 h post-injection and ratio of 24 h/90 min and 48 h/90 min hepatic SUV values derived from PET data analysis is shown. (C) Ex vivo biodistribution data of selected organs are shown. All data were presented as mean ± SD. The statistical analysis was performed using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc. ∗p < 0.05; ∗∗p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)At sacrifice, radioactive signal was quantified in liver, brain, kidneys, lungs, and spleen. A VTX-801 dose-dependent reduction in hepatic 64Cu concentration was observed (Figure 5C). The radioactive signal was increased in kidneys and lungs at higher doses of vector at similar levels to WT (no significant differences). Interestingly, high levels of radiocopper were found in the spleen of 18-week-old untreated Atp7b−/− animals (not observed in 9-week-old animal) that were significantly reduced by VTX-801 treatment (Figure 5C).DiscussionThe Atp7b knockout (Atp7b−/−) mouse is a good model of WD, allowing proof-of-concept and pharmacology studies of AAV-mediated gene therapy. It exhibits the typical biochemical and pathophysiological alterations found in WD patients, including progressive liver involvement and lack of biliary elimination of copper. The advantages of the Atp7b−/− mouse model to test the therapeutic efficacy of gene therapy approaches include a well-defined genetic background, good survival with disease progression, good AAV-transduction efficiency, and availability of the original strain to serve as a control in comparative analyses.15Buiakova O.I. Xu J. Lutsenko S. Zeitlin S. Das K. Das S. Ross B.M. Mekios C. Scheinberg I.H. Gilliam T.C. Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation.Hum. Mol. Genet. 1999; 8: 1665-1671https://doi.org/10.1093/hmg/8.9.1665Crossref PubMed Scopus (162) Google Scholar We demonstrated that an AAV vector carrying an ATP7B-minigene (named VTX-801) was able to restore copper metabolism and physiological copper excretion in WD mice in a safe and very efficient manner.8Murillo O. Moreno D. Gazquez C. Barberia M. Cenzano I. Navarro I. Uriarte I. Sebastian V. Arruebo M. Ferrer V. et al.Liver expression of a MiniATP7B gene results in long-term restoration of copper homeostasis in a Wilson disease model in mice.Hepatology. 2019; 70: 108-126https://doi.org/10.1002/hep.30535Crossref PubMed Scopus (13) Google Scholar Next, and in anticipation of a future clinical study, we worked on the development of a clinically compatible and minimally invasive method to demonstrate the efficacy of gene therapy in restoring copper metabolism in WD patients.Copper-64 is a radionuclide that exhibits physical properties that are complementary for diagnosis, therapeutic purposes, and biodistribution studies. In different types of cancers, copper-64 is being used both as a theranostic agent and to monitor the efficacy of antitumoral agents.16Keinänen O. Fung K. Brennan J.M. Zia N. Harris M. van Dam E. Biggin C. Hedt A. Stoner J. Donnelly P.S. et al.Harnessing 64Cu/67Cu for a theranostic approach to pretargeted radioimmunotherapy.Proc. Natl. Acad. Sci. USA. 2020; 117: 28316-28327https://doi.org/10.1073/pnas.2009960117Crossref PubMed Scopus (29) Google Scholar,17Mirzaei S. Mohammed F. Zandieh S. Theranostics of metastatic prostate cancer applying 64Cu/18F/68Ga PSMA PET-CT and 177Lu radiopharmaceuticals.Curr Radiopharm. 2020; https://doi.org/10.2174/1874471013666200908122845Crossref Scopus (4) Google Scholar Furthermore, copper-64 labeling of antibodies, small molecules, nanoparticles, or cells followed by PET imaging represent a highly sensitive and non-invasive method for biodistribution and pharmacokinetic studies.18Krishnan A. Adhikarla V. Poku E.K. Palmer J. Chaudhry A. Biglang-Awa V.E. Bowles N. Nathwani N. Rosenzweig M. Sahebi F. et al.Identifying CD38+ cells in patients with multiple myeloma: first-in-human imaging using copper-64-labeled daratumumab.Blood Adv. 2020; 4: 5194-5202https://doi.org/10.1182/bloodadvances.2020002603Crossref PubMed Google Scholar, 19Caserta E. Chea J. Minnix M. Poku E.K." @default.
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• W4284882005 title "High value of 64Cu as a tool to evaluate the restoration of physiological copper excretion after gene therapy in Wilson’s disease" @default.
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