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J Biol Chem. 2014 Jun 6;289(23):16148-63. doi: 10.1074/jbc.M114.549980. Epub 2014 Apr 21.

Rabies virus envelope glycoprotein targets lentiviral vectors to the axonal retrograde pathway in motor neurons.

Author information

1
From Gene Therapy, Centre for Neuroinflammation and Neurodegeneration, Division of Brain Sciences, Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom.
2
Molecular NeuroPathoBiology Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom, and.
3
Molecular NeuroPathoBiology Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom, and Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, University College London, Queen Square, London WC1N 3BG, United Kingdom.
4
From Gene Therapy, Centre for Neuroinflammation and Neurodegeneration, Division of Brain Sciences, Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom, n.mazarakis@imperial.ac.uk.

Abstract

Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.

KEYWORDS:

Axon; Endosome; Glycoprotein; Lentivirus; Rab Proteins; Trafficking

PMID:
24753246
PMCID:
PMC4047386
DOI:
10.1074/jbc.M114.549980
[Indexed for MEDLINE]
Free PMC Article
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