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Ex Vivo Rat Transected Spinal Cord Slices as a Model to Assess Lentiviral Vector Delivery of Neurotrophin-3 and Short Hairpin RNA against NG2.

ABSTRACT: The failure of the spinal cord to regenerate can be attributed both to a lack of trophic support for regenerating axons and to upregulation of inhibitory factors such as chondroitin sulphate proteoglycans including NG2 following injury. Lentiviral vector-mediated gene therapy is a possible strategy for treating spinal cord injury (SCI). This study investigated the effect of lentiviral vectors expressing Neurotrophin-3 (NT-3) and short-hairpin RNA against NG2 (NG2 sh) to enhance neurite outgrowth in in vitro and ex vivo transection injury models. Conditioned medium from cells transduced with NT-3 or shNG2 lentiviruses caused a significant increase in neurite length of primary dorsal root ganglia neurons compared to the control group in vitro. In an ex vivo organotypic slice culture (OSC) transduction with Lenti-NT-3 promoted axonal growth. Transducing OSCs with a combination of Lenti-NT-3/NG2 sh lead to a further increase in axonal growth but only in injured slices and only within the region adjacent to the site of injury. These findings suggest that the combination of lentiviral NT-3 and NG2 sh reduced NG2 levels and provided a more favourable microenvironment for neuronal regeneration after SCI. This study also shows that OSCs may be a useful platform for studying glial scarring and potential SCI treatments.

SUBMITTER: Patar A

PROVIDER: S-EPMC7150802 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Ex Vivo Rat Transected Spinal Cord Slices as a Model to Assess Lentiviral Vector Delivery of Neurotrophin-3 and Short Hairpin RNA against NG2.

Patar Azim A Dockery Peter P McMahon Siobhan S Howard Linda L

Biology 20200315 3

The failure of the spinal cord to regenerate can be attributed both to a lack of trophic support for regenerating axons and to upregulation of inhibitory factors such as chondroitin sulphate proteoglycans including NG2 following injury. Lentiviral vector-mediated gene therapy is a possible strategy for treating spinal cord injury (SCI). This study investigated the effect of lentiviral vectors expressing Neurotrophin-3 (NT-3) and short-hairpin RNA against NG2 (NG2 sh) to enhance neurite outgrowth ...[more]

PMID: 32183469

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Project description:BackgroundHIV-responsive expression vectors are all based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the use of LTR-based reporter vectors to cloned cells, where aberrantly high expressing (HIV-negative) cells can be eliminated. Enhancements in specificity would increase opportunities for expression vector use in detection of HIV as well as in experimental gene expression in HIV-infected cells.ResultsWe have constructed an expression vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites that are efficiently used in human cells. It also contains a reading frame that is removed by cellular splicing activity in the absence of HIV Rev. The vector was incorporated into a lentiviral reporter virus, permitting detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of green fluorescence protein (GFP) reporter and by PCR of reporter transcript following HIV infection. The vector displayed full HIV dependency.ConclusionAs with the earlier developed Tat-dependent expression vectors, the Rev system described here is an exploitation of an evolved HIV process. The inclusion of Rev-dependency renders the LTR-based expression vector highly dependent on the presence of replicating HIV. The application of this vector as reported here, an HIV-dependent reporter virus, offers a novel alternative approach to existing methods, in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector permits examination of living cells, can express any gene for basic or clinical experimentation, and as a pseudo-typed lentivirus has access to most cell types and tissues.

Dataset Information

Ex Vivo Rat Transected Spinal Cord Slices as a Model to Assess Lentiviral Vector Delivery of Neurotrophin-3 and Short Hairpin RNA against NG2.

Publications

Ex Vivo Rat Transected Spinal Cord Slices as a Model to Assess Lentiviral Vector Delivery of Neurotrophin-3 and Short Hairpin RNA against NG2.

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