Characterisation of axonal retrograde transport of rabies pseudotyped lentiviral vectors for application in gene therapy of motor neuron diseases

Abstract

Lentiviral vectors, such as those derived from Human Immunodeficiency Virus-1 (HIV-1) and Equine Infectious Anaemia Virus (EIAV), can be targeted to the neurons by replacing their natural envelope with rabies-G glycoprotein (RV-G), through a process known as pseudotyping. They are retrogradedly transported to distal projecting neurons in vivo where transgene expression occurs, an approach with significant potential for gene therapy of motor neuron diseases. However, the molecular processes that underlie retrograde transduction are unchartered and barrier(s) which result in low transduction efficiencies are thus not defined. The project aimed to characterise the processes involved in the entry and endocytic trafficking of RV-G pseudotyped lentiviral vectors in a differentiated motor neuron cell line, NSC-34, and primary rat motor neurons. For the first time, the project demonstrates the co-internalisation of RV-G pseudotyped lentiviral vectors with its 3 specific receptors, namely p75 neurotrophin receptor (p75NTR), neural cell adhesion molecule (NCAM) and nicotinic acetylcholine receptor (nAchR). Furthermore, internalised lentiviral vectors follow a sequential Rab5 to Rab7 endosomal maturation along the axonal endocytic pathway. Using the process of tetracysteine tagging, a small genetic tag was successfully introduced into the matrix of the vector capsid and labelling of the tag with the biarsenical dye FlAsH was optimised. Utilising these and other differently labelled vectors, we demonstrate through live cell imaging, that it is the RV-G pseudotype that confers on the vectors the ability to traffic retrogradedly. This trafficking pathway utilises non-acidic axonal carriers as previously demonstrated for the p75NTR and tetanus toxin trafficking in motor neurons. Additionally, the nAchR is also targeted to this retrograde trafficking pathway. Furthermore retrograde transport was studied in compartmented microfluidic motor neuron cultures and this trafficking was found to be both rapid and efficient. This suggests that the barrier(s) to efficient transduction with these vectors is post axonal transport. Additionally, studies to isolate and characterise axonal retrograde endosomes in primary motor neurons based on a system of pull-down of magnetic nano particles (MNPs) bound to a streptavidin displaying lentiviral vector were performed. The project optimised streptavidin expressing vector production and shown their ability to bind biotin coated magnetic nano particles (MNPs) and transduce motor neurons with increased efficiency. Magnetic pull-down of the vector containing endosomes and subsequent proteomics have revealed a number of potential trafficking partners of RV-G pseudotyped lentiviral vectors. Taken together, this thesis characterises the processes involved in the entry and endocytic trafficking of RV-G pseudotyped lentiviral vectors and identifies the barrier to efficient transduction of motor neurons as post axonal transport and thereby opening up avenues to improve gene therapy vectors