Intraneural GJB1 gene delivery improves nerve pathology in a model of X-linked Charcot–Marie–Tooth disease
Stavros Bashiardes PhD
Department of Molecular Virology, Cyprus Institute of Neurology and Genetics and Cyprus School of Molecular Medicine, Nicosia, Cyprus
Search for more papers by this authorJan Richter PhD
Department of Molecular Virology, Cyprus Institute of Neurology and Genetics and Cyprus School of Molecular Medicine, Nicosia, Cyprus
Search for more papers by this authorChristina Christodoulou PhD
Department of Molecular Virology, Cyprus Institute of Neurology and Genetics and Cyprus School of Molecular Medicine, Nicosia, Cyprus
Search for more papers by this authorSteven S. Scherer MD, PhD
Department of Neurology, University of Pennsylvania, Philadelphia, PA
Search for more papers by this authorCorresponding Author
Kleopas A. Kleopa MD
Neuroscience Laboratory
Neurology Clinics, Cyprus Institute of Neurology and Genetics and Cyprus School of Molecular Medicine, Nicosia, Cyprus
Address correspondence to Dr Kleopa, Neurology Clinics and Neuroscience Laboratory, Cyprus Institute of Neurology and Genetics, Cyprus School of Molecular Medicine, 6 International Airport Avenue, PO Box 23462, 1683, Nicosia, Cyprus. E-mail: [email protected]Search for more papers by this authorStavros Bashiardes PhD
Department of Molecular Virology, Cyprus Institute of Neurology and Genetics and Cyprus School of Molecular Medicine, Nicosia, Cyprus
Search for more papers by this authorJan Richter PhD
Department of Molecular Virology, Cyprus Institute of Neurology and Genetics and Cyprus School of Molecular Medicine, Nicosia, Cyprus
Search for more papers by this authorChristina Christodoulou PhD
Department of Molecular Virology, Cyprus Institute of Neurology and Genetics and Cyprus School of Molecular Medicine, Nicosia, Cyprus
Search for more papers by this authorSteven S. Scherer MD, PhD
Department of Neurology, University of Pennsylvania, Philadelphia, PA
Search for more papers by this authorCorresponding Author
Kleopas A. Kleopa MD
Neuroscience Laboratory
Neurology Clinics, Cyprus Institute of Neurology and Genetics and Cyprus School of Molecular Medicine, Nicosia, Cyprus
Address correspondence to Dr Kleopa, Neurology Clinics and Neuroscience Laboratory, Cyprus Institute of Neurology and Genetics, Cyprus School of Molecular Medicine, 6 International Airport Avenue, PO Box 23462, 1683, Nicosia, Cyprus. E-mail: [email protected]Search for more papers by this authorAbstract
Objective
X-linked Charcot–Marie–Tooth disease (CMT1X) is a common inherited neuropathy caused by mutations in the GJB1 gene encoding the gap junction protein connexin32 (Cx32). Clinical studies and disease models indicate that neuropathy mainly results from Schwann cell autonomous, loss-of-function mechanisms; therefore, CMT1X may be treatable by gene replacement.
Methods
A lentiviral vector LV.Mpz-GJB1 carrying the GJB1 gene under the Schwann cell–specific myelin protein zero (Mpz) promoter was generated and delivered into the mouse sciatic nerve by a single injection immediately distal to the sciatic notch. Enhanced green fluorescent protein (EGFP) reporter gene expression was quantified and Cx32 expression was examined on a Cx32 knockout (KO) background. A gene therapy trial was performed in a Cx32 KO model of CMT1X.
Results
EGFP was expressed throughout the length of the sciatic nerve in up to 50% of Schwann cells starting 2 weeks after injection and remaining stable for up to 16 weeks. Following LV.Mpz-GJB1 injection into Cx32 KO nerves, we detected Cx32 expression and correct localization in non–compact myelin areas where gap junctions are normally formed. Gene therapy trial by intraneural injection in groups of 2-month-old Cx32 KO mice, before demyelination onset, significantly reduced the ratio of abnormally myelinated fibers (p = 0.00148) and secondary inflammation (p = 0.0178) at 6 months of age compared to mock-treated animals.
Interpretation
Gene delivery using a lentiviral vector leads to efficient gene expression specifically in Schwann cells. Restoration of Cx32 expression ameliorates nerve pathology in a disease model and provides a promising approach for future treatments of CMT1X and other inherited neuropathies. Ann Neurol 2015;78:303–316
References
- 1Bergoffen J, Scherer SS, Wang S, et al. Connexin mutations in X-linked Charcot-Marie-Tooth disease. Science 1993; 262: 2039–2042.
- 2Kleopa KA, Scherer SS. Molecular genetics of X-linked Charcot-Marie-Tooth disease. Neuromolecular Med 2006; 8: 107–122.
- 3Scherer SS, Deschênes SM, Xu Y-T, et al. Connexin32 is a myelin-related protein in the PNS and CNS. J Neurosci 1995; 15: 8281–8294.
- 4Kleopa KA. The role of gap junctions in Charcot-Marie-Tooth disease. J Neurosci 2011; 31: 17753–17760.
- 5Hattori N, Yamamoto M, Yoshihara T, et al. Demyelinating and axonal features of Charcot-Marie-Tooth disease with mutations of myelin-related proteins (PMP22, MPZ and Cx32): a clinicopathological study of 205 Japanese patients. Brain 2003; 126: 134–151.
- 6Hahn AF, Ainsworth PJ, Naus CCG, et al. Clinical and pathological observations in men lacking the gap junction protein connexin 32. Muscle Nerve Suppl 2000; 9: S39–S48.
- 7Nakagawa M, Takashima H, Umehara F, et al. Clinical phenotype in X-linked Charcot-Marie-Tooth disease with an entire deletion of the connexin 32 coding sequence. J Neurol Sci 2001; 185: 31–36.
- 8Shy ME, Siskind C, Swan ER, et al. CMT1X phenotypes represent loss of GJB1 gene function. Neurology 2007; 68: 849–855.
- 9Omori Y, Mesnil M, Yamasaki H. Connexin 32 mutations from X-linked Charcot-Marie-Tooth disease patients: functional defects and dominant negative effects. Mol Biol Cell 1996; 7: 907–916.
- 10Yum SW, Kleopa KA, Shumas S, et al. Diverse trafficking abnormalities of connexin32 mutants causing CMTX. Neurobiol Dis 2002; 11: 43–52.
- 11Deschênes SM, Walcott JL, Wexler TL, et al. Altered trafficking of mutant connexin32. J Neurosci 1997; 17: 9077–9084.
- 12Kleopa KA, Yum SW, Scherer SS. Cellular mechanisms of connexin32 mutations associated with CNS manifestations. J Neurosci Res 2002; 68: 522–534.
- 13Anzini P, Neuberg DH-H, Schachner M, et al. Structural abnormalities and deficient maintenance of peripheral nerve myelin in mice lacking the gap junction protein connexin32. J Neurosci 1997; 17: 4545–4561.
- 14Scherer SS, Xu Y-T, Nelles E, et al. Connexin32-null mice develop a demyelinating peripheral neuropathy. Glia 1998; 24: 8–20.
10.1002/(SICI)1098-1136(199809)24:1<8::AID-GLIA2>3.0.CO;2-3 CAS PubMed Web of Science® Google Scholar
- 15Jeng LJ, Balice-Gordon RJ, Messing A, et al. The effects of a dominant connexin32 mutant in myelinating Schwann cells. Mol Cell Neurosci 2006; 32: 283–298.
- 16Sargiannidou I, Vavlitou N, Aristodemou S, et al. Connexin32 mutations cause loss of function in Schwann cells and oligodendrocytes leading to PNS and CNS myelination defects. J Neurosci 2009; 29: 4748–4761.
- 17Bruzzone R, White TW, Scherer SS, et al. Null mutations of connexin32 in patients with X-linked Charcot-Marie-Tooth disease. Neuron 1994; 13: 1253–1260.
- 18Oh S, Ri Y, Bennett MVL, et al. Changes in permeability caused by connexin 32 mutations underlie X-linked Charcot-Marie-Tooth disease. Neuron 1997; 19: 927–938.
- 19Castro C, Gomez-Hernandez JM, Silander K, et al. Altered formation of hemichannels and gap junction channels caused by C-terminal connexin-32 mutations. J Neurosci 1999; 19: 3752–3760.
- 20Liang GSL, de Miguel M, Gomez-Hernandez JM, et al. Severe neuropathy with leaky connexin32 hemichannels. Ann Neurol 2005; 57: 749–754.
- 21Scherer SS, Xu YT, Messing A, et al. Transgenic expression of human connexin32 in myelinating Schwann cells prevents demyelination in connexin32-null mice. J Neurosci 2005; 25: 1550–1559.
- 22Consiglio A, Quattrini A, Martino S, et al. In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice. Nat Med 2001; 7: 310–316.
- 23McIver SR, Lee C-S, Lee J-M, et al. Lentiviral transduction of murine oligodendrocytes in vivo. J Neurosci Res 2005; 82: 397–403.
- 24Dull T, Zufferey R, Kelly M, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998; 72: 8463–8471.
- 25Sango K, Kawakami E, Yanagisawa H, et al. Myelination in coculture of established neuronal and Schwann cell lines. Histochem Cell Biol 2012; 137: 829–839.
- 26Nelles E, Butzler C, Jung D, et al. Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice. Proc Natl Acad Sci U S A 1996; 93: 9565–9570.
- 27Ahn M, Lee J, Gustafsson A, et al. Cx29 and Cx32, two connexins expressed by myelinating glia, do not interact and are functionally distinct. J Neurosci Res 2008; 86: 992–1006.
- 28Kobsar I, Berghoff M, Samsam M, et al. Preserved myelin integrity and reduced axonopathy in connexin32-deficient mice lacking the recombination activating gene-1. Brain 2003; 126: 804–813.
- 29Vavlitou N, Sargiannidou I, Markoullis K, et al. Axonal pathology precedes demyelination in a mouse model of X-linked demyelinating/type I Charcot-Marie Tooth neuropathy. J Neuropathol Exp Neurol 2010; 69: 945–958.
- 30Hahn AF, Ainsworth PJ, Bolton CF, et al. Pathological findings in the X-linked form of Charcot-Marie-Tooth disease: a morphometric and ultrastructural analysis. Acta Neuropathol 2001; 101: 129–139.
- 31Kobsar I, Maurer M, Ott T, et al. Macrophage-related demyelination in peripheral nerves of mice deficient in the gap junction protein connexin 32. Neurosci Lett 2002; 320: 17–20.
- 32Shy ME, Tani M, Scherer SS, et al. Towards the gene therapy of Charcot-Marie-Tooth disease type 1: an adenoviral vector can transfer lacZ into Schwann cells in culture and in sciatic nerve. Ann Neurol 1995; 38: 429–436.
- 33Sørensen J, Haase G, Krarup C, et al. Gene transfer to Schwann cells after peripheral nerve injury: a delivery system for therapeutic agents. Ann Neurol 1998; 43: 205–211.
- 34Guénard V, Schweitzer B, Flechsig E, et al. Effective gene transfer of lacZ and P0 into Schwann cells of P0-deficient mice. Glia 1999; 25: 165–178.
10.1002/(SICI)1098-1136(19990115)25:2<165::AID-GLIA7>3.0.CO;2-L CAS PubMed Web of Science® Google Scholar
- 35Glatzel M, Flechsig E, Navarro B, et al. Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system. Proc Natl Acad Sci U S A 2000; 97: 442–447.
- 36Gonzalez S, Fernando R, Perrin-Tricaud C, et al. In vivo introduction of transgenes into mouse sciatic nerve cells in situ using viral vectors. Nat Protoc 2014; 9: 1160–1169.
- 37Lowenstein PR, Mandel RJ, Xiong WD, et al. Immune responses to adenovirus and adeno-associated vectors used for gene therapy of brain diseases: the role of immunological synapses in understanding the cell biology of neuroimmune interactions. Curr Gene Ther 2007; 7: 347–360.
- 38Homs J, Ariza L, Pagès G, et al. Schwann cell targeting via intrasciatic injection of AAV8 as gene therapy strategy for peripheral nerve regeneration. Gene Ther 2011; 18: 622–630.
- 39Ozçelik M, Cotter L, Jacob C, et al. Pals1 is a major regulator of the epithelial-like polarization and the extension of the myelin sheath in peripheral nerves. J Neurosci 2010; 30: 4120–4131.
- 40Cotter L, Ozçelik M, Jacob C, et al. Dlg1-PTEN interaction regulates myelin thickness to prevent damaging peripheral nerve overmyelination. Science 2010; 328: 1415–1418.
- 41Messing A, Behringer RR, Hammang JP, et al. P0 promoter directs expression of reporter and toxin genes to Schwann cells of transgenic mice. Neuron 1992; 8: 507–520.
- 42Jang SW, Svaren J. Induction of myelin protein zero by early growth response 2 through upstream and intragenic elements. J Biol Chem 2009; 284: 20111–20120.
- 43Scherer SS, Wrabetz L. Molecular mechanisms of inherited demyelinating neuropathies. Glia 2008; 56: 1578–1589.
- 44Lattanzi A, Neri M, Maderna C, et al. Widespread enzymatic correction of CNS tissues by a single intracerebral injection of therapeutic lentiviral vector in leukodystrophy mouse models. Hum Mol Genet 2010; 19: 2208–2227.
- 45Lattanzi A, Salvagno C, Maderna C, et al. Therapeutic benefit of lentiviral-mediated neonatal intracerebral gene therapy in a mouse model of globoid cell leukodystrophy. Hum Mol Genet 2014; 23: 3250–3268.
- 46Martino S, Marconi P, Tancini B, et al. A direct gene transfer strategy via brain internal capsule reverses the biochemical defect in Tay-Sachs disease. Hum Mol Genet 2005; 14: 2113–2123.
- 47Mazarakis ND, Azzouz M, Rohll JB, et al. Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery. Hum Mol Genet 2001; 10: 2109–2121.
- 48Sahenk Z, Galloway G, Clark K, et al. AAV1.NT-3 gene therapy for Charcot-Marie-Tooth neuropathy. Mol Ther 2014; 22: 511–521.
- 49Lundberg C, Björklund T, Carlsson T, et al. Applications of lentiviral vectors for biology and gene therapy of neurological disorders. Curr Gene Ther 2008; 8: 461–473.
- 50Cartier N, Hacein-Bey-Abina S, Bartholomae CC, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 2009; 326: 818–823.
- 51Biffi A, Montini E, Lorioli L, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science 2013; 341: 1233158.
- 52Kordower JH, Bloch J, Ma SY, et al. Lentiviral gene transfer to the nonhuman primate brain. Exp Neurol 1999; 160: 1–16.
- 53Abordo-Adesida E, Follenzi A, Barcia C, et al. Stability of lentiviral vector-mediated transgene expression in the brain in the presence of systemic antivector immune responses. Hum Gene Ther 2005; 16: 741–751.
- 54Biffi A, Bartolomae CC, Cesana D, et al. Lentiviral vector common integration sites in preclinical models and a clinical trial reflect a benign integration bias and not oncogenic selection. Blood 2011; 117: 5332–5339.
- 55Kleopa KA, Abrams CK, Scherer SS. How do mutations in GJB1 cause X-linked Charcot-Marie-Tooth disease? Brain Res 2012; 1487: 198–205.
Citing Literature
