title: Design and optimization of lentiviral vectors for transfer of GALC expression in Twitcher brain.
abstract: BACKGROUND
  Demyelination in globoid cell leukodystrophy (GLD) is due to a deficiency of galactocerebrosidase (GALC) activity. Up to now, in vivo brain viral gene transfer of GALC showed modest impact on disease development in Twitcher mice, an animal model for GLD. Lentiviral vectors, which are highly efficient to transfer the expression of therapeutic genes in neurons and glial cells, have not been evaluated for direct cerebral therapy in GLD mice.
  
  
  METHODS
  Lentiviral vectors containing the untagged cDNA or the hemagglutinin (HA)-tagged cDNA for the full-length mouse GALC sequence were generated and validated in vitro. In vivo therapeutic efficacy of these vectors was evaluated by histology, biochemistry and electrophysiology after transduction of ependymal or subependymal layers in young Twitcher pups.
  
  
  RESULTS
  Both GALC lentiviral vectors transduced neurons, oligodendrocytes and astrocytes with efficiencies above 75% and conferred high levels of enzyme activity. GALC accumulated in lysosomes of transduced cells and was also secreted to the extracellular medium. Conditioned GALC medium was able to correct the enzyme deficiency when added to non-transduced Twitcher glial cultures. Mice that received intraventricular injections of GALC vector showed accumulation of GALC in ependymal cells but no diffusion of the enzyme from the ependymal ventricular tree into the cerebral parenchyma. Significant expression of GALC-HA was detected in neuroglioblasts when GALC-HA lentiviral vectors were injected in the subventricular zone of Twitcher mice. Life span and motor conduction in both groups of treated Twitcher mice were not significantly ameliorated.
  
  
  CONCLUSIONS
  Lentiviral vectors showed to be efficient for reconstitution of the GALC expression in Twitcher neural cells. GALC was able to accumulate in lysosomes as well as to enter the secretory pathway of lysosomal enzymes, two fundamental aspects for gene therapy of lysosomal storage diseases. Our in vivo results, while showing the capacity of lentiviral vectors to transfer expression of therapeutic GALC in the Twitcher brain, did not limit progression of disease in Twitchers and highlight the need to evaluate other routes of administration.
authors.affiliation: Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy. dolcetta.diego@hsr.it
authors.forename: D
authors.initials: D
authors.lastname: Dolcetta
authors.forename: L
authors.initials: L
authors.lastname: Perani
authors.forename: M I
authors.initials: MI
authors.lastname: Givogri
authors.forename: F
authors.initials: F
authors.lastname: Galbiati
authors.forename: S
authors.initials: S
authors.lastname: Amadio
authors.forename: U
authors.initials: U
authors.lastname: Del Carro
authors.forename: G
authors.initials: G
authors.lastname: Finocchiaro
authors.forename: A
authors.initials: A
authors.lastname: Fanzani
authors.forename: S
authors.initials: S
authors.lastname: Marchesini
authors.forename: L
authors.initials: L
authors.lastname: Naldini
authors.forename: M G
authors.initials: MG
authors.lastname: Roncarolo
authors.forename: E
authors.initials: E
authors.lastname: Bongarzone
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000378:metabolism; D001681:Biological Assay; D001921:Brain / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D018076:DNA, Complementary; D004195:Disease Models, Animal; D005698:Galactosylceramidase / Q000032:analysis / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005823:Genetics; D006367:HeLa Cells; D006388:Hemagglutinins / Q000737:chemistry; D006720:Homozygote; D006801:Humans; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics*; D007965:Leukodystrophy, Globoid Cell / Q000235:genetics / Q000473:pathology / Q000628:therapy; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D009474:Neurons / Q000378:metabolism; D009836:Oligodendroglia / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D006388:Hemagglutinins; D005698:Galactosylceramidase