Recovery of Retroviral Transduction via DOTAP

June 09, 2015

University of Michigan
Pharmaceutical Sciences
Research Fellow

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Company:

Avanti Polar Lipids Inc

Product Name:

18:1 TAP (DOTAP, MS Salt)

Catalog Number:

890895P

Retroviruses are commonly used gene therapy vectors but can be extremely unstable, inefficient and expensive due to sensitivities in performance and targeting associated with their envelope protein. We used retroviruses without the envelope protein (to eliminate their intracellular uptake and transduction) and in turn used synthetic agents to revive the original retroviral transduction performance. In this study, we used DOTAP - a commonly used synthetic transfection lipid. Overall, while it was an easy to use lipid, its performance was limited and had to be supplemented with other lipids like cholesterol and DOPE to provide drastically improved performance. See papers for more information - http://www.ncbi.nlm.nih.gov/pubmed/23485145 & http://www.ncbi.nlm.nih.gov/pubmed/25883029

Experimental Design and Results Summary

Application

Viral Transduction

Starting Material

Cellular derived retroviruses (See http://www.clontech.com/US/Products/Viral_Transduction/Retroviral_Packaging/High-Titer_Retrovirus)

Protocol Overview

A relatively simple protocol of mixing was used to obtain hybrid synthetic viral vectors. Predetermined volumes of virus solutions and lipids formulated as liposomes were mixed to obtain required stoichiometry of the synthetic virus. See paper for more details on the different protocols used to obtain these vectors - http://www.ncbi.nlm.nih.gov/pubmed/23485145

Tips

Did not use if it was more than 6 months post purchasing. If dissolved in chloroform, we used up the solution within a month of preparation.

Results Summary

The cellular uptake was remarkably high for the vectors formulated with DOTAP but the transduction was rather abysmal. Only when the vector was co-formulated with DOPE and Cholesterol in varying ratios did the performance increase and optimal performance was achieved using a composition of DOTAP:DOPE:Cholesterol = 5:8:7 (w/w/w). A representative electron microscopy image of the vector co-formulation is shown. (Left) - only liposome (without virus) in white arrows (Right) is the hybrid vector comprising of the liposome formulated with DOTAP, DOPE and Cholesterol (in white arrows) while in black arrows, is the virus particle without the envelope protein

Additional Notes

None

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Summary

The Good

Easy to use. Ready to use and no need of any more purification once bought from Avanti.

The Bad

Be wary of shelf life. needs -20 deg freezer.

The Bottom Line

Excellent lipid for transduction performance provided is co-formulated with other lipids like DOPE and Cholesterol. Regular non-viral gene therapy vectors like plasmid DNA or siRNA or shRNA may do without the co-formulation with DOTAP alone providing very good gene delivery.

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