Cationic Derivatives of Polyisoprenoids and Commercial Lipid‐Based DNA Carriers for Transfection in vivo in Spontaneously Hypertensive Rats
Authors: Gawrys Olga , Baranowska Iwona , Rak Monika , Bobis‐Wozowicz Sylwia , Szaro Karolina , Madeja Zbigniew , Swiezewska Ewa , Kompanowska‐Jezierska Elzbieta 
Published: June 2021

Development of efficient vectors for transfection constitutes a major challenge in modern gene therapy. Viral vectors are considered more effective, but plagued by safety concerns. Research shows that derivatives of polyisoprenoid alcohols (trimethylpolyprenylammonium iodides, called PTAI), may serve as lipofectants. In previous research PTAI were proved to be effective as DNA carriers in vitro and in vivo as carriers of DNA vaccines against influenza virus.

In the present study the effectiveness of two compositions (PTAI‐6–8 and PTAI‐10–14, containing a mixture of polyprenols with chain lengths from 6 to 8 subunits and from 10 to 14 subunits, respectively and helper lipids: DOPE (1,2‐dioleoyl‐sn‐glycero‐3‐phosphatidylethanolamine) and DC‐cholesterol (3β‐[N‐(N′,N′‐dimethylaminoethane)‐carbamoyl]cholesterol hydrochloride) was investigated for DNA delivery into rats' cells and compared to the commercially available reagents for transfection: in vivo‐jetPEI® (Polyplus‐transfection) and Avalanche®‐in vivo Transfection Reagent (EZ Biosystems LLC). Selected mixtures were used to enhance the expression of vascular endothelial growth factor type A (VEGF‐A) in spontaneously hypertensive rats (SHR). VEGF deficiency is considered one of the factors in the development of hypertension and related kidney diseases.

In preliminary experiments, the mixtures containing lipid‐carriers and plasmid DNA (encoding reporter gene and VEGF‐A) were administered intravenously and observed for 24, 48 or 72 h, to establish the optimal conditions for transfection. Subsequently rats were anesthetized, perfused with PBS, and tissues (liver, spleen, kidney, lung) were collected for analysis. Transfection efficiency was evaluated by flow cytometry (BD FACSCanto™ II system) in single cell suspensions prepared from collected tissues.

In almost all tested cell suspensions the level of fluorescence of reporter gene (eGFP or mCherry) was undetectable. Only in the liver higher expression of mCherry was observed with commercial reagents (the % of mCherry positive liver cells with Avalanche® was 42 versus 12% in control group receiving 5% glucose; p<0.05). Due to low efficiency of intravenously applied carriers, we tested if selected mixtures are able to provide stable expression of VEGF‐A gene after direct administration to the kidney. SHR were implanted with osmotic pumps (model 2001, ALZET®) connected to catheters allowing the infusion into the renal medulla.

The fluorescence of mCherry in the kidneys was undetectable and there were no changes in tissue, plasma and urine levels of VEGF‐A (ELISA). Unfortunately we were not able to prove the usefulness of PTAI for transfection, however all selected mixtures, including commercially available reagents failed to deliver DNA to renal cells

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NCN 2015/17/N/NZ4/02831

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.