Comparing Genetic Transduction between VSV-G and Rabies-G Lentiviral Pseudotypes Open Access

Allen, Dexter Chase (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/d791sg366?locale=en
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Abstract

In an effort to optimize gene therapy to the spinal cord, this study compares the neuronal tropism of the VSV-G (vesicular stomatitis virus) lentiviral pseudotype with that of the rabies-G lentiviral pseudotype. To this end, these pseudotyped vectors, which will express Green Fluorescent Protein (GFP), will be injected into the lumbar spinal cord of the Sprague Dawley rat. Three weeks post-injection, rats will be sacrificed and their lumbar spinal cords will be sectioned. Immunohistochemistry will be performed to determine where the spread of GFP expression occurs in the spinal cord. Co-staining will be performed with Choline Acetyltransferase (ChAT) to determine what cell types are transduced by the two lentiviral pseudotypes. We found that direct injection of VSV-G lentiviral pseudotype exhibited significantly greater longitudinal spread in the lumbar spinal cord than direct injection of Rab-G lentiviral pseudotype. Additionally, we found transduction from the Rab-G lentiviral pseudotype to be localized within cells and transduction from the VSV-G lentiviral pseudotype to be localized within axons. These results suggest a lentiviral pseudotype that would optimally deliver therapeutic genetic material in the treatment of motor neuron related diseases, such as Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy (SMA), and spasticity.

Table of Contents

Introduction.................................................................................1

Methods......................................................................................5

Vector Construction...............................................................5

Animals...............................................................................6

Gene Delivery.......................................................................6

Perfusion.............................................................................7

Immunohistochemistry...........................................................8

Motor Neuron Counting...........................................................9

Green Fluorescent Protein (GFP) Expression Analysis...................9

Results.......................................................................................10

Statistical Analysis...............................................................10

Surgeries Performed.............................................................10

Motor Neuron Density............................................................11

Green Fluorescent Protein (GFP) Expression Analysis..................14

Colocalization......................................................................19

Figures and Tables........................................................................10

Figure 1...............................................................................7

Figure 2..............................................................................11

Figure 3..............................................................................12

Figure 4..............................................................................13

Table 1...............................................................................14

Figure 5..............................................................................14

Figure 6..............................................................................15

Figure 7..............................................................................17

Discussion...................................................................................18

Conclusion...................................................................................22

References..................................................................................23

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