Gene Delivery to Human Limbal Stem Cells Using Viral Vectors.

Human Gene Therapy
Liujiang SongMatthew L Hirsch

Abstract

Limbal stem cell (LSC) transplantation is a promising treatment for ocular surface diseases especially LSC deficiency. Genetic engineering represents an attractive strategy to increase the potential for success in LSC transplantations either by correcting autologous diseased LSCs or by decreasing the immunogenicity of allogeneic LSCs. Therefore, two popular viral vectors, adeno-associated viral (AAV) vector and lentiviral (LV) vector, were compared for gene delivery in human LSCs. Transduction efficiency was evaluated by flow cytometry, quantitation of viral genomes, and fluorescence microscopy after introducing eight self-complementary AAV serotypes or LV carrying a green fluorescent protein (GFP) cassette to fresh limbal epithelial cells, cultivated LSC colonies, or after corneal intrastromal injection into human explant tissue. For fresh limbal epithelial cells, AAV6 showed the highest transduction efficiency, followed by LV and AAV4 at 24 h after vector incubation, which did not directly correlate with internalized genome copy number. The colony formation efficiency, as well as colony size over time, showed no significant differences among AAV serotypes, LV, and nontreated controls. The percentage of GFP+ colonies at 14 day...Continue Reading

References

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Citations

Jun 17, 2020·Molecular Therapy. Methods & Clinical Development·Liujiang SongBrian C Gilger
Apr 25, 2020·Molecular Therapy : the Journal of the American Society of Gene Therapy·Keiko MiyaderaMatthew L Hirsch

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Methods Mentioned

BETA
genetic modification
transgenic
flow cytometry
dissecting
electrophoresis
transfection
reverse transcription PCR
fluorescence-activated cell sorting
biopsy

Software Mentioned

GraphPad Prism
cellSens
cellSens Dimension
GraphPad

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