1a.
Demonstration of Specific, Non-Viral Gene Delivery Targeting Motor Neurons in-vitro
and in-vivo through the p75NTR receptor
Receptor specific, non-viral gene delivery vehicles provide a way to deliver therapeutic agents for motor neuron disease. We have constructed a highly specific non-viral gene delivery agent targeting the common neurotrophin receptor (p75NTR) in-vitro and in the SOD1G93A transgenic mouse model of motor neuron disease in-vivo. Polyethylene glycol (PEG) was covalently attached to polyethylenimine (PEI) and the construct verified by NMR. The PEI-PEG construct was then conjugated to a monoclonal antibody to p75NTR (clone MLR2) and then assessed for ability to condense GFP plasmid DNA (pGFP) electrostatically. The size, zeta potential and DNase protection ability of MLR2-PEI-PEG-pGFP was compared to PEI-PEG-pGFP. MLR2-PEI-PEG-pGFP was then tested for ability to target primary motor neurons from E12-13 SOD1G93A mice and adult SOD1G93A transgenic mice. PEI was effectively PEGylated as determined by NMR. The MLR2-PEI-PEG construct condensed pGFP and bound pGFP at a nitrogen to phosphate ratio (N/P) of 3.5 to 10 with a negative zeta potential and size less than 100 nm (n=3). PEGylated PEI-MLR2 protected pGFP from DNase digestion (n=3).
Importantly, MLR2-PEI-PEG was able to specifically target motor neurons in mixed cultures containing primary motor neurons and glia from SOD1G93A mice (n=3). Finally, MLR2-PEI-PEG-pGFP delivered into adult SOD1G93A mice by intraperitoneal injections, resulted in GFP expression in spinal motor neurons (n=4). This study shows effective non-viral gene delivery to motor neurons in vitro and in vivo. Further work is ongoing to show this agent delivers therapeutic genes to SOD1G93A mice.
1b.
Quantification of the transfection efficiency and toxicity of p75NTR non-viral
gene delivery in wild type and SOD1G93A primary motor neuron cultures
Non-viral gene delivery vehicles offer the possibility of safer therapeutic agent development for neurological conditions such as motor neuron disease (MND). Here we characterize the culture conditions, transfection efficiency and toxicity of non-viral gene delivery constructs selectively targeting p75NTR expressing motor neurons using primary embryonic motor neuron (PMN) cultures and mixed cultures (PMNm) from wild type and SOD1G93A transgenic mice. Monoclonal antibody to the neurotrophin receptor p75NTR (MLR2) was conjugated to polyethylenimine (MLR2-PEI) or pegylated polyethylenimine (MLR2-PEG-PEI) and complexed with an eGFP expression plasmid at nitrogen/phosphate (NP) ratios ranging from 2-10 to form the immunoconjugate, MLR2-PEI-pGFP/MLR2-PEG-PEI-pGFP. Transfection efficiency, toxicity and stability of various NP ratio conjugates were assessed at 72 hours in wild type and SOD1G93A PMN and PMNm cultures at day 5-10 after plating and transfected for 4, 24 and 48 hours. MLR2-PEI-pGFP and MLR2-PEG-PEI-pGFP are most stable and less toxic at NP ratios of 3.5 and 7 (n=6). Both immunogene constructs specifically transfect PMN and PMNm at a transfection efficiency of 2% and 5% respectively (n=3), however only MLR2-PEG-PEI-pGFP transfected cultures in the presence of 10% serum containing media (n=4). In PMNm cultures containing primarily astrocytes, only motor neurons were transfected (n=3). In addition MLR2-PEG-PEI-pGFP NP ratio 3.5-7 constructs were not toxic to PMN cultures as observed with MLR2-PEI-pGFP (n=4), however all immunogene constructs were 100% toxic to PMN cultures up to 4 days after plating.
Pegylation of the MLR2-PEI-pGFP construct produces a gene delivery vehicle with reduced toxicity, improved stability and transfection efficiencies in wild type and SOD1G93A PMN cultures. Reduced toxicity and stability of the immunoporter may be important in developing gene therapies that target injured or dying motor neurons in MND patients.
2.
Development of an immunogene targeting adult motor neurons
Motor neurons express the TrkC receptor, throughout life. Our collaborator Prof Uri Saragovi, University of Montreal, has produced 2B7, an antibody that internalizes and targets TrkC receptors as agonist. We have shown that labelled TrkC (2B7-Atto 488) given systemically label sensory neurons in the DRGs and motor neurons of the spinal cord. The 2B7-PEI-PEG –pGFP immunogene was shown to target motor neurons in pre-symptomatic SOD1G93A mice and GFP was expressed specifically by the motor neurons. This immunogene therefore has the potential to act at two sites to achieve significant therapeutic benefit.
3. A Biomarker for Motor Neuron Disease
There are no biochemical biomarkers for Motor Neuron Disease (MND), thus, finding a biological measure of the extent of this disease is critical. The American National Institute of Health defines a biomarker as ‘a characteristic that can be measured and evaluated as an indicator of normal biological processes, pathological processes or pharmalogical responses to therapeutic intervention’. An important step in finding effective treatments is to identify biomarkers that could aid in the detection and progression of disease, and more rapid translation of potential therapeutics from models to clinical trials. The neurotrophin receptor p75 (p75NTR) is highly expressed during development and is greatly reduced in different types of cells in adulthood. However, multiple studies have found that p75NTR is robustly induced by injury. Of particular interest, is that p75NTR has been found upregulated in the post-mortem spinal cord of persons with MND and the SOD1G93A mouse model of MND, and also in urine following sciatic nerve injury in rats. The aim of this study was to determine if p75NTR is detectable in the urine of the MND mouse model SOD1G93A and whether it could be used as a biomarker in the mouse model, allowing for more rapid translation of possible therapeutics to clinical trials. Behavioural testing showed that SOD1G93A mice (n=10) developed motor symptoms at 120 days of age reaching end-stage by 150 days. p75NTR was detectable in SOD1G93A mice urine as early as 60 days of age, increasing with age, but was not detectable in age-matched controls (n=10) until after 120 days. p75NTR has been identified as a possible biomarker for MND in the SOD1G93A mouse and on-going work is being undertaken to determine if it could be of use as a biomarker in human disease. Blood and urine samples from SOD1G93A mice and persons with MND will be tested for p75NTR by developing a more sensitive, quantitative ELISA assay.
