Author: Kevin Smith
Smith, Kevin, 2015 Neurotrophin Receptor p75 as a target for gene delivery to motor neurons, Flinders University, School of Medicine
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Motor neuron disease (MND) is a relentlessly progressive neurodegenerative disease, which is typically fatal within 3 years. The only FDA approved treatment for MND is Riluzole, which increases life by approximately 3 months, with little to no increase in quality of life. Due to the heterogeneity of the disease and the blood brain barrier, developing treatments for MND is difficult. This study used a novel non-viral gene delivery agent (called the ‘immunogene’) to bypass the blood brain barrier and deliver pDNA to neonatal mouse motor neurons, which has potential to be used as a therapeutic for MND. An immunogene consists of a monoclonal antibody that targets a receptor that is conjugated to a PEGylated polycation allowing it to carry a cargo of pDNA or siRNA. In this study we used a monoclonal antibody (MLR2) that targets the neurotrophin receptor p75 (p75NTR) that is able to be retrogradely transported in motor neurons with the receptor. We also used polyethylenamine (PEI) as the cation that electrostatically binds and condenses pDNA. PEGylation (polyethylene glycol) of the PEI was used to stealth and shield the complex from the immune system and blood components (full complex; MLR2-PEI-PEG12). The common neurotrophin receptor p75 (p75NTR) binds all neurotrophins and is highly expressed in motor neurons during development and down-regulated following birth. However, p75NTR is re-expressed in motor neurons of the SOD1G93A MND mouse model and motor neurons of MND patients. The aim of this study was to use p75NTR expression on motor neurons in neonatal mice and re-expression of p75NTR on symptomatic SOD1G93A mice that model MND to deliver pDNA via the immunoporter MLR2-PEI-PEG12. Motor neurons re-expressing p75NTR in SOD1G93A mice were characterised for expression time, apoptotic markers and morphology. This was to determine if the MLR2 immunogene would be feasible as a gene therapy agent in SOD1G93A mice. The study first addressed the ability for the MLR2-immunogene to specifically transfect p75NTR expressing cells and primary motor neurons. Motor neuron-like NSC34 cells and primary motor neurons from E12.5 control C57BL/6J mice were able to be transfected with the MLR2-immunogene (~23 and ~8%, respectively), while p75NTR-ve cell lines (including glial cells) were unable to be transfected. Removing the MLR2 from the immunogene (i.e. PEI-PEG12) abolishes the p75NTR+ve cell specificity. p75NTR was found to be highly expressed on ~92% of all motor neurons in PND1 C57BL/6J mice. This was reduced to ~50% of motor neurons at PND7, and was completely absent at PND14. PND1 mice were injected intraperitoneally with the MLR2-immunogene which resulted in the transfection of ~20% of all motor neurons at all levels of the spinal cord, assessed by the presence of the pDNA expressing green fluorescent protein (GFP). Transfection levels peaked 10 days after injection. Targeting of p75NTR is integral for the immunogene to properly transfect motor neurons as injection of PEI-PEG12 carrying pDNA (MLR2 antibody) resulted in no transfection. Only a small number (0-5%) of motor neurons in SOD1G93A adult mice (60-140 days of age) expressed p75NTR at any one time. These p75NTR+ve motor neurons were larger than p75NTR-ve neurons, and the majority (~ 80%) co-expressed the apoptotic marker cleaved caspase 3. The results from this study show that the MLR2-immunogene is able to deliver pDNA to p75NTR expressing motor neurons in neonatal mice after systemic (intraperitoneal) injection. Targeting motor neurons in adult SOD1G93A mice via p75NTR may be difficult as the percentage of neurons expressing the receptor at any specific time is low (<5%) and appear destined to die due to the presence of cleaved caspase 3.
Keywords: p75NTR, p75 neurotorphin receptor, MND, motor neuron disease, ALS, amytrophic lateral sclerosis, gene delivery, motor neuron, SOD1G93A
Subject: Medicine thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Mary-Louise Rogers