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Gene therapy of Wilson disease: A “golden” opportunity using rAAV on the 50th anniversary of the discovery of the virus

2015; Elsevier BV; Volume: 64; Issue: 2 Linguagem: Inglês

10.1016/j.jhep.2015.11.017

1600-0641

Jayanta Roy‐Chowdhury, Michael L. Schilsky,

Infectious Encephalopathies and Encephalitis

Long-term metabolic correction of Wilson’s disease in a murine model by gene therapyJournal of HepatologyVol. 64Issue 2PreviewWilson’s disease (WD) is a rare autosomal recessive inborn error of copper metabolism caused by mutations in the gene that encodes the ATPase copper transporting beta polypeptide, ATP7B. Copper is a potentially toxic metal but it is essential for a wide number of physiological functions acting as a co-factor of a variety of enzymes [1]. After its intestinal absorption, copper is transported to hepatocytes where it binds to ATP7B located in the membrane of the trans-Golgi network (TGN). This large transmembrane protein is in charge of transferring the metal to copper-dependent enzymes. Full-Text PDF Wilson disease (WD) was a once progressive and uniformly fatal inherited disorder of copper metabolism. Medical therapy to arrest progression or prevent complications of WD was developed in the 1950s with the introduction of parenterally administered BAL [[1]Roberts E. Schilsky M.L. A practice guideline on Wilson disease.Hepatology. 2008; 47: 2089-2111Crossref PubMed Scopus (936) Google Scholar], and over the next two decades by oral therapy with d-penicillamine, trientine, and zinc [[2]Schilsky M.L. Liver Transplantation for Wilson Disease.Ann N Y Acad Sci. 2014; 1315: 45-49Crossref PubMed Scopus (41) Google Scholar]. Effective therapy for WD requires life-long administration of daily medication. At least 30–50% of patients on medication for WD have periods of non-adherence, some suffering liver failure, others developing potentially irreversible neurologic or psychiatric symptoms. Herein lies the justification for developing therapies that would provide correction instead of serial treatment of WD. Liver transplantation (LT) is curative for WD. Biliary copper excretion is restored, ceruloplasmin and copper levels in the circulation normalize, neurologic and psychiatric disease stabilizes or improves and Kayser Fleischer rings disappear over time [[2]Schilsky M.L. Liver Transplantation for Wilson Disease.Ann N Y Acad Sci. 2014; 1315: 45-49Crossref PubMed Scopus (41) Google Scholar]. Following LT, patients do not require WD-specific therapy, but rather life-long immunosuppression. Thus LT proved that the defect for WD lay within the liver, consistent with physiologic studies showing reduced biliary copper excretion in WD patients [[3]Sternlieb I. Van den Hamer C.J. Morell A.G. Alpert S. Gregoriadis G. Scheinberg I.H. Lysosomal defect of hepatic copper excretion in Wilson’s disease (hepatolenticular degeneration).Gastroenterology. 1973; 64: 99-105Abstract Full Text PDF PubMed Scopus (96) Google Scholar]. Therefore targeting the liver to correct the defect in biliary copper excretion would provide a cure for WD. The WD gene was identified as the trans-membrane copper transporter ATP7B in 1993, and studies showed that the ATP7B protein was preferentially expressed in hepatocytes [[6]Roybal J.L. Endo M. Radu A. Gray L. Todorow C.A. Zoltick P.W. et al.Early gestational gene transfer with targeted ATP7B expression in the liver improves phenotype in a murine model of Wilson’s disease.Gene Ther. 2012; 19: 1085-1094Crossref PubMed Scopus (31) Google Scholar]. ATP7B facilitates copper transport into bile and incorporation of copper into ceruloplasmin. Animal models of WD, the LEC rat and the toxic milk mouse, lack ATP7B and manifest hepatic copper accumulation due to impaired biliary copper excretion and low circulating ceruloplasmin levels [[4]Tanzi R.E. Petrukhin K. Chernov I. Pellequer J.L. Wasco W. Ross B. et al.The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene.Nat Genet. 1993; 5: 344-350Crossref PubMed Scopus (1181) Google Scholar]. Atp7b knockout mice develop the same phenotype. Therefore, introduction of ATP7B into hepatocytes should correct the phenotype. Proof of principle for gene therapy came from the expression of ATP7B in the liver of LEC rats and murine models of WD using adenoviral and lentiviral vectors, which achieved transient correction of copper excretion and incorporation of copper into ceruloplasmin [5Meng Y. Miyoshi I. Hirabayashi M. Su M. Mototani Y. Okamura T. et al.Restoration of copper metabolism and rescue of hepatic abnormalities in LEC rats, an animal model of Wilson disease, by expression of human ATP7B gene.Biochim Biophys Acta. 2004; 1690: 208-219Crossref PubMed Scopus (33) Google Scholar, 6Roybal J.L. Endo M. Radu A. Gray L. Todorow C.A. Zoltick P.W. et al.Early gestational gene transfer with targeted ATP7B expression in the liver improves phenotype in a murine model of Wilson’s disease.Gene Ther. 2012; 19: 1085-1094Crossref PubMed Scopus (31) Google Scholar]. What was missing was longer term ATP7B expression and robust transduction without oncogenesis. How many hepatocytes must be transduced with ATP7B to improve the phenotype? This was answered in part from studies where hepatocytes with normal ATP7B expression were transplanted into congeneic LEC rats with hepatic copper accumulation [[7]Irani A.N. Malhi H. Slehria S. Gorla G.R. Volenberg I. Schilsky M.L. et al.Correction of liver disease following transplantation of normal rat hepatocytes into Long-Evans Cinnamon rats modeling Wilson’s disease.Mol Ther. 2001; 3: 302-309Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]. With ∼40% hepatocyte repopulation, liver damage from copper accumulation was prevented and circulating ceruloplasmin was increased. This proved that a fraction of hepatocytes with normal copper transport in an affected liver was sufficient to provide a conduit for copper into bile, and suggested that transduction of only a fraction of hepatocytes with ATP7B by gene therapy should protect against copper toxicity. In this issue of the Journal of Hepatology, gene therapy by systemic administration of a viral vector into a murine model of WD was reported by Murillo et al. [[8]Murillo O. Luqui D.M. Gazquez C. Martinez-Espartosa D. Navarro-Blasco I. Monreal J.I. et al.Long-term metabolic correction of Wilson’s disease in a murine model by gene therapy.J Hepatol. 2016; 64: 419-426Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. Initially, liver directed gene therapy involved ex vivo transduction of hepatocytes isolated from a resected liver segment using recombinant oncoretroviral vectors that integrate permanently in the host genome [[9]Roy-Chowdhury J. Grossman M. Gupta S. Roy-Chowdhury N. Baker Jr., J.R. Wilson J.M. Long term improvement of hypercholesterolemia after ex vivo gene therapy in LDL-receptor deficient rabbits.Science. 1991; 254: 1802-1805Crossref PubMed Scopus (325) Google Scholar]. Inefficient retroviral transduction of primary hepatocytes and inefficiency of hepatocyte engraftment restricted the success of these efforts. The risk of activation of protooncogenes by randomly integrated transgenes was highlighted by the occurrence of leukemia in some children undergoing bone marrow-directed ex vivo gene therapy for severe combined immunodeficiency. Subsequently, systemic gene therapy utilized episomal recombinant adenovirus that can infect hepatocytes and express the transgene with high efficiency without integrating into the host genome [[10]Takahashi M. Ilan Y. Roy-Chowdhury N. Guida J. Horwitz M.S. Roy-Chowdhury J. Long-term correction of bilirubin UDP-glucuronosyltransferase deficiency in Gunn rats by administration of a recombinant adenovirus during the neonatal period.J Biol Chem. 1996; 271: 26536-26542Crossref PubMed Scopus (108) Google Scholar]. However, intrinsic host immunity and adaptive immune response shortened the duration of transgene expression and precluded repeated administration of the vector. The death of a recipient of adenoviral gene therapy was a major setback to gene therapy efforts [[11]Wilson J.M. Lessons learned from the gene therapy trial for ornithine transcarbamylase deficiency.Mol Genet Metab. 2009; 96: 151-157Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar]. Although, viral gene-deleted adenoviral vectors permitted prolonged transgene expression, the problem of adaptive immune response remains. Partly due to setbacks with retroviral and adenoviral vectors, attention was focused on a small (20 nm) single stranded non-enveloped parvovirus, termed adeno-associated virus (rAAV), which was employed by Murillo et al. [[8]Murillo O. Luqui D.M. Gazquez C. Martinez-Espartosa D. Navarro-Blasco I. Monreal J.I. et al.Long-term metabolic correction of Wilson’s disease in a murine model by gene therapy.J Hepatol. 2016; 64: 419-426Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar] for the treatment of the Atp7b−/− mouse model of WD (WD mice). This year marks the 50th anniversary of the discovery of AAV [[12]Atchison R.W. Casto B.C. Hammon W.M. Adenovirus-associated defective virus particles.Science. 1965; 149: 754-756Crossref PubMed Scopus (556) Google Scholar], which has taken the center stage in gene therapy because of many desirable features as a gene delivery vector (Table 1). Up to 90% of the population has been exposed to wild-type AAV serotype 2 (AAV2), without any deleterious effect, indicating safety of the virus. All viral genes can be deleted in rAAV vectors, enhancing safety and reducing immunogenicity. Eleven AAV serotypes have been characterized that can infect both dividing and quiescent cells in liver, brain, retina, heart, muscle, lung, and pancreas, making rAAV a versatile gene transfer platform.Table 1Desirable features of recombinant adeno-associated virus for gene therapy and goals for genetic correction of Wilson disease. Open table in a new tab AAV belongs to the genus Dependoparvovirus because its replication is dependent on a “helper virus” like adenovirus or herpes simplex virus. Adenoviral gene products providing this help were identified [[13]Matsushita T. Elliger S. Elliger C. Podsakoff G. Villarreal L. Kurtzman G.J. et al.Adeno-associated virus vectors can be efficiently produced without helper virus.Gene Ther. 1998; 5: 938-945Crossref PubMed Scopus (414) Google Scholar], and rAAV is now generated by co-transfection with helper plasmids into the packaging cell. Specific AAV serotypes differ in their cellular tropism [[14]Wu Z.J. Asokan A. Samulski R.J. Adeno-associated virus serotypes: Vector toolkit for human gene therapy.Mol Ther. 2006; 14: 316-327Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar]. rAAV lacks the Rep gene products required for site-specific integration into the human chromosome 19. After the rAAV genome is converted to double stranded DNA, the inverted terminal repeats (ITR) of the rAAV genome mediate the formation of circular concatemers that persist episomally in non-dividing cells, and integrate into the host genome very infrequently and in a semi-random manner [[15]Nakai H. Wu X. Fuess S. Storm T.A. Munroe D. Montini E. et al.Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver.J Virol. 2005; 79: 3606-3614Crossref PubMed Scopus (143) Google Scholar]. The 4.7 kb single stranded DNA genome of AAV contains two open reading frames (ORF), rep and cap, flanked on both ends by ITRs required for packaging the genome into the viral capsid [[16]Yang Q.C. Kadam A. Trempe J.P. Mutational analysis of the adenoassociated virus rep gene.J Virol. 1992; 66: 6058-6069PubMed Google Scholar]. rAAVs are generated by replacing the rep and cap ORFs with the gene of interest and providing rep and cap as helper genes in trans to package the transgene inside the capsid. The capsid proteins from different serotypes determine their cell specificity and immunogenicity. Taking advantage of this, the well-characterized AAV2 genome is packaged with capsids of different serotypes to generate pseudotyped rAAVs that can efficiently transduce cells of interest in vivo. Murillo et al. [[8]Murillo O. Luqui D.M. Gazquez C. Martinez-Espartosa D. Navarro-Blasco I. Monreal J.I. et al.Long-term metabolic correction of Wilson’s disease in a murine model by gene therapy.J Hepatol. 2016; 64: 419-426Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar] inserted their genes of interest (ATP7B, eGFP or luciferase) with hepatocyte-specific promoters between the 5′ and 3′ ITRs of the AAV2. To achieve liver specificity they used the capsid of the AAV8 serotype to package the rAAV vector. Using such pseudotyped vectors has become a standard procedure to achieve cell type-preferred gene delivery [[17]Grieger J.C. Choi V.W. Samulski J. Production and characterization of adeno-associated viral vectors.Nat Protoc. 2006; 1: 1412-1428Crossref PubMed Scopus (400) Google Scholar]. Different approaches for engineering capsids to achieve enhanced cell specificity and reduce or circumvent an immune response have been reviewed recently [[18]Grimm D. Zolotukhin S. E Pluribus Unum: 50 Years of research, millions of viruses, and one goal-tailored acceleration of AAV evolution.Mol Ther. 2015; ([Epub ahead of print])https://doi.org/10.1038/mt.2015.173Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar]. In the paper by Murillo et al. [[8]Murillo O. Luqui D.M. Gazquez C. Martinez-Espartosa D. Navarro-Blasco I. Monreal J.I. et al.Long-term metabolic correction of Wilson’s disease in a murine model by gene therapy.J Hepatol. 2016; 64: 419-426Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar], a single injection of rAAV at doses (viral genomes/kg body weight) similar to those used in patients with hemophilia B (Factor IX deficiency) resulted in normalization of plasma holoceruloplasmin levels, increased biliary copper excretion as evidenced by increased fecal copper, and reduced hepatic and urinary copper in a dose related manner 6 months after the treatment. Many, but not most host hepatocytes stained positive for ATP7B, confirming the predictions derived from hepatocyte transplantation experiments that copper excretion by a fraction of hepatocytes can deplete the excessive copper in the remaining ATP7B deficient cells (7). In patients with hemophilia B, Nathwani et al. reported only 1–6% of normal plasma Factor IX levels for a median of 3.2 years [[19]Nathwani A.C. Reiss U.M. Tuddenham E.G.D. Rosales C. Chowdary P. McIntosh J. et al.Long-term safety and efficacy of factor ix gene therapy in hemophilia B.N Engl J Med. 2014; 371: 1994-2004Crossref PubMed Scopus (879) Google Scholar]. Although this level was therapeutically significant, similar levels of ATP7B expression in WD mice would be inadequate to achieve normalization of copper stores as observed by Murillo et al. [[8]Murillo O. Luqui D.M. Gazquez C. Martinez-Espartosa D. Navarro-Blasco I. Monreal J.I. et al.Long-term metabolic correction of Wilson’s disease in a murine model by gene therapy.J Hepatol. 2016; 64: 419-426Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. While there may be a species difference, preexisting antibodies against wild-type AAV or Factor IX, and/or adaptive immune response might have reduced plasma levels of this protein. Intriguingly, rAAV transduced hepatocytes of WD mice more efficiently than hepatocytes of wild-type mice [[8]Murillo O. Luqui D.M. Gazquez C. Martinez-Espartosa D. Navarro-Blasco I. Monreal J.I. et al.Long-term metabolic correction of Wilson’s disease in a murine model by gene therapy.J Hepatol. 2016; 64: 419-426Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar], suggesting a higher transduction efficiency of rAAV in livers with genotoxic hepatic injury, which occurs in WD mice due to excess copper, but not in patients with hemophilia B or inherited metabolic liver diseases without liver injury. In the human hemophilia B trial, a high proportion of patients exhibited increased serum alanine aminotransferase (ALT) levels 7–10 weeks after the rAAV injection, which was ameliorated by prednisolone therapy. This indicated the loss of some rAAV-transduced hepatocytes through an adaptive immune response. Such ALT spikes were not reported in treated WD mice [[8]Murillo O. Luqui D.M. Gazquez C. Martinez-Espartosa D. Navarro-Blasco I. Monreal J.I. et al.Long-term metabolic correction of Wilson’s disease in a murine model by gene therapy.J Hepatol. 2016; 64: 419-426Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar], but were perhaps masked by their baseline elevated ALT levels. What is the prospect of rAAV-mediated gene therapy of WD? Because it required up to 5 weeks for transgene expression to reach peak levels after rAAV injection in canine models of hemophilia B [[20]Snyder R.O. Miao C. Meuse L. Tubb J. Donahue B.A. Lin H.-F. et al.Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors.Nat Med. 1999; 5: 64-70Crossref PubMed Scopus (315) Google Scholar], this vector is unlikely to be useful in the treatment of acute liver failure due to WD. Prolonged ATP7B expression after a single rAAV injection would be attractive for treating WD patients as an alternative to life-long daily maintenance therapy. Although the hemophilia B clinical trial showed therapeutic levels of Factor IX persisting for a median period of 3.2 years after one rAAV injection, episomal rAAV will likely be lost eventually through cell division, and re-administration of the vector may be hindered by humoral and cell-mediated immune responses resulting from prior treatment. This may be addressed by packaging the rAAV genome with the capsid from alternative serotypes, although antibodies to one AAV serotype can sometimes cross-react with the capsid of a different serotype. However, extensive research on directed evolution of AAV capsids by multiple laboratories is likely to yield rAAVs with capsids engineered to evade preexisting immunity against the originally used rAAV capsid [[18]Grimm D. Zolotukhin S. E Pluribus Unum: 50 Years of research, millions of viruses, and one goal-tailored acceleration of AAV evolution.Mol Ther. 2015; ([Epub ahead of print])https://doi.org/10.1038/mt.2015.173Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar]. Thus, on the “golden” anniversary year of the discovery of AAV [[12]Atchison R.W. Casto B.C. Hammon W.M. Adenovirus-associated defective virus particles.Science. 1965; 149: 754-756Crossref PubMed Scopus (556) Google Scholar], there is reason for optimism about the application of rAAV vectors in liver directed gene therapy for WD and other monogenic liver-based disorders. National Institutes of Health – United States grants 1RO1 DK092469, 1PO1 DK 096990, 5 P30 DK 41296-24. The authors declare no conflict of interest in relation to this manuscript.