High Transgene Expression by Lentiviral Vectors Causes Maldevelopment of Purkinje Cells In Vivo

Abstract

Lentiviral vectors are promising as gene-transfer vehicles for gene therapy targeted to intractable brain diseases. Although lentiviral vectors are thought to exert little toxicity on infected cells, the adverse influence of viral infection on vulnerable developing neurons has not been well studied. Here, we examined whether lentiviral vector infection and subsequent transgene expression affected the morphological and functional maturation of vigorously developing cerebellar Purkinje cells in vivo. Lentiviral vectors expressing GFP under the control of the murine stem cell virus (MSCV) promoter were injected into the cerebellar cortex of neonatal rat pups. Three weeks after treatment, GFP-expressing Purkinje cells were compared with control Purkinje cells from phosphate-buffered saline-injected rats. Analysis of the dendritic tree showed that total dendrite length in GFP-expressing Purkinje cells was almost 80% that in control Purkinje cells. Electrophysiological examination showed that short-term synaptic plasticity at parallel fiber-Purkinje cell synapses and climbing fiber-Purkinje cell synapses was significantly altered in GFP-expressing Purkinje cells. In contrast, maldevelopment of infected Purkinje cells was substantially attenuated when lentiviral vectors with much weaker promoter activity were used. These results suggest that the maldevelopment of Purkinje cells was mainly caused by subsequent expression of a high amount of GFP driven by the strong MSCV promoter. Thus, the use of lentiviral vectors carrying a strong promoter may require particular precautions when applying them to neurological disorders of infants.

Recent Developments in Gene Therapy Research Targeted to Cerebellar Disorders

“…We have also verified that this injection method is applicable to neonatal pups and mature mice (Fig. 2c) (Sawada et al, 2010;Torashima et al, 2006a;Torashima et al, 2006b).…”

Role of post-translational modifications in modulating the structure, function and toxicity of α-synuclein

“…However, a recent study by Hyman and colleagues reported a similar toxicity for the three variants (WT, S129D and S129A) (McFarland et al, 2009). As mentioned above, the results from this study might be biased by co-expression of GFP and a-syn (Baens et al, 2006;Sawada et al, 2010;Ulusoy et al, 2009). …”

“…The discrepancy with the previous two models might be due to the fact that McFarland et al (2009) used a bi-cistronic mRNA to co express a-syn and green fluorescent protein (GFP), which may lead to lower expression of a syn compared to classical monocistronic RNA. Recent reports suggest neuronal toxicity associated with AAV-mediated over-expression of GFP in the substantia nigra, which could mask the differences in terms of neuronal loss caused by a-syn and the two S129 mutants (Baens et al, 2006;Sawada et al, 2010;Ulusoy et al, 2009). An additional contribut ing factor might be the fact the immunohistological analysis was carried out 6 weeks after virus injec tion, compared to 8 weeks in the studies by Azeredo da Silveira et al (2009) and Gorbatyuk et al (2008).…”

Activity-Dependent Neurotrophin Signaling Underlies Developmental Switch of Ca2+Channel Subtypes Mediating Neurotransmitter Release

“…Lentiviral vectors were designed to express EGFP under the control of a PC-specific promoter, L7-4N. The L7-4N promoter is a fusion promoter consisting of a minimal CMV sequence and a truncated (1 kb) PC-specific L7 promoter (Sawada et al, 2010). It exhibits much greater transcriptional activity than the original L7 promoter without compromising PC specificity (H. H., unpublished observation).…”