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Updated: Jan 21, 2026
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Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium
Published on: August 7, 2015
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Gene Delivery to Human Limbal Stem Cells Using Viral Vectors
Liujiang Song1, Zhenwei Song2, Nathaniel J Fry3
1Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina.
Human Gene Therapy
|August 9, 2019
Summary
Lentiviral (LV) vectors show superior stable gene delivery in human limbal stem cells (LSCs) compared to adeno-associated viral (AAV) vectors, offering potential for enhanced LSC transplantation therapies.
Area of Science:
- Ophthalmology
- Gene Therapy
- Stem Cell Biology
Background:
- Limbal stem cell (LSC) deficiency causes ocular surface diseases.
- Genetic engineering of LSCs can improve transplantation outcomes.
- Adeno-associated viral (AAV) and lentiviral (LV) vectors are key tools for gene delivery.
Purpose of the Study:
- To compare the efficacy of AAV and LV vectors for gene delivery in human LSCs.
- To evaluate transduction efficiency in fresh LSCs, cultivated colonies, and corneal tissue.
- To assess the potential for stable genetic modification of LSCs for therapeutic applications.
Main Methods:
- Transduction of human LSCs with eight AAV serotypes and LV vectors carrying a GFP cassette.
- Evaluation of transduction efficiency using flow cytometry, genome quantification, and fluorescence microscopy.
- Assessment of colony formation, size, and GFP expression in treated LSC colonies and corneal explants.
Main Results:
- AAV6 demonstrated the highest initial transduction in fresh LSCs, but LV achieved higher, stable GFP expression in cultivated LSC colonies.
- LV vectors enabled sustained gene expression in LSC colonies over serial passages, unlike AAV6 which showed restricted transduction to differentiated cells.
- Both LV and AAV6 transduced corneal stroma and endothelium; AAV6 also transduced epithelium, but neither vector effectively transduced derived LSC colonies in situ.
Conclusions:
- Lentiviral vectors are effective for stable genetic engineering of human limbal stem cells.
- Adeno-associated viral vector serotype 6 exhibits restricted transduction in multipotent limbal stem cell-derived colonies.
- LV vectors hold promise for enhancing LSC transplantation therapies through stable genetic modification.
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