Project Details
Projekt
AAV-based gene replacement therapy in a murine model of Hereditary Spastic Paraplegia Type 5 (SPG5)
Subject Area
Molecular and Cellular Neurology and Neuropathology
Gastroenterology
Gastroenterology
Term
since 2024
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 541715925
Hereditary spastic paraplegia type 5 (SPG) is a rare neurodegenerative disease that leads to progressive spastic gait disorder due to degeneration of the corticospinal tract. SPG5 is caused by mutations in CYP7B1 resulting in loss of function of 7 alpha-hydroxylase, particularly in the liver. Lack of CYP7B1 leads to increased concentrations of its substrates 25-hydroxycholesterol and 27-hydroxycholesterol in blood and, since these oxysterols can cross the blood-brain barrier, also in cerebrospinal fluid of SPG5 patients. In neuronal cell cultures, 25- and 27-hydroxycholesterol were neurotoxic at concentrations similar to those in SPG5 patients. Oxysterols are incorporated into lipid membranes and likely disrupt membrane function because of their hydrophilic properties. Therefore, elevated oxysterol concentrations are considered the primary factor causing degeneration of long fiber tracts in the spinal cord in SPG5. As a first step in the development of a gene therapy for SPG5, we strive for a gene supplementation in cyp7b1-/- knockout mice, which show very similar metabolic alterations as SPG5 patients. Since most of the oxysterols in the brain are of hepatic origin, we aimed to replace CYP7B1 in the liver of cyp7b1-/- knockout mice. We used AAV8 as a serotype with high expression in the liver, which has been used already successfully for gene therapy in humans. Intravenous injection of AAV8 carrying human CYP7B1 under control of the TTR promoter resulted in normalization of oxysterol levels in liver and blood of cyp7b1-/- mice, but only limited effects in brain after 6 weeks. This preliminary study suggests that either longer treatment periods or simultaneous treatment of liver and brain are needed. Both will be investigated in the proposed study, which includes (i) a long-term study with AAV8-TTR-hCYP7B1 and (ii) novel AAV vectors (AAV-PHP.eB and AAV.CAP-B22) that cross the blood-brain barrier in mice and in nonhuman primates. For ubiquitous transgene expression including the brain, we will use the elongation factor 1 alpha short (EFS) promoter. We will investigate therapeutic efficacy and potential toxicity of these new vectors with increasing vector doses. Oxysterol levels in blood, liver, and brain will be determined as the primary readout parameter by isotope dilution mass spectrometry using deuterium-labelled internal standards. Vectors will be evaluated for liver toxicity by transaminase levels in blood and by liver histology. Potential neurotoxicity will be assessed as neurofilament light chain concentration using single molecule array technology and histological examination of brain, spinal cord, and dorsal root ganglia focused on inflammatory changes. In addition, neural tropism and potential neural toxicity of AAV-PHP.eB-EFS-CYP7B1 and AAV.CAP-B22-EFS-CYP7B1 will be tested in vitro in iPSC-derived human neural cell cultures and brain organoids.
DFG Programme
Research Grants
