Abstract
Abstract: :
Purpose: To induce mitochondrial dysfunction in an animal model that resembles the optic nerve degeneration of Leber Hereditary Optic Neuropathy (LHON). While the pathologic mechanisms leading to visual failure in LHON are unknown, there is a close relationship between respiratory chain dysfunction and free radical generation. Methods: we designed hammerhead ribozymes to degrade mRNA of the NDUFA1 gene encoding the complex 1 precursor MWFE polypeptide and the manganese superoxide dismutase (MnSOD) gene. In vitro studies were used for choosing the most active ribozymes that were then cloned into recombinant adeno-associated virus (rAAV) vector and tested in cell lines. Two microliters of rAAV-ribozyme genes were injected into the right eyes of DBA/1J mice. Left eyes for control received rAAV containing the green fluorescent protein (gfp) gene. Mice were sacrificed 4 months after injection for histopathologic examination. Results: Our ribozymes showed specific cleavage in vitro against the synthetic substrates and full-length transcripts from NDUFA1 and MnSOD genes. rAAV-ribozyme infected mouse cells (NIH/3T3) exhibited a slower growth rate in a dose dependent manner. Cell death occurred at the 3rd day following infection. Relative to control infected cell, levels of MWFE mRNA decreased 64% and MnSOD mRNA decreased 81%. Assays of complex I activity in whole cells by the reduction of cytochrome c with NADH decreased by 81%. Similar infection of the human cell line (HEK293) with the mouse specific ribozyme did not result in cytopathic effects. Light microscopy exhibited optic disc edema. Quantitative analysis showed that relative to control infected eye, ganglion cell counts were reduced by 18% for Rz-MWFE and 27% for the Rz-SOD injected eyes (p < 0.01). Transmission electron microscopy of ribozyme inoculated optic nerves revealed foci of degeneration and demyelination, including cytoplasm loss and naked axons or axons enveloped by thin sheaths of myelin. Mononuclear inflammatory cells and reactive astroglia were also seen in the optic nerves. Ribozymes also caused retinal damage in some mice. These histopathologic features were not evident in AAV-gfp infected tissues. Conclusion: Mouse ocular tissues infected with ribozymes against mRNA for mitochondrial proteins appear similar histopathologic features to autopsied optic nerve tissues of LHON patients, thus suggesting we have developed an animal model of mitochondria failure to test new treatment avenues for patients blinded by LHON.
Keywords: 316 animal model • 419 gene transfer/gene therapy • 475 mitochondria