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GEN News Highlights : Sep 15, 2011
Scientists Use HDL to Efficiently Deliver siRNAS into Brain Capillary Endothelial Cells
Team showed that delivered siRNAs effectively silenced target gene but didn’t cross BBB into neurons or glial cells.!--h2>
Scientists have developed a method for delivering short interfering RNAs (siRNAs) directly and specifically to brain capillary endothelial cells (BCECs) within the blood brain barrier by attaching them to endogenous lipoproteins. The Tokyo Medical and Dental University team conjugated an siRNA designed to silence organic transporter 3 (OAT3) to cholesterol, and then incorporated the construct into endogenous HDL lipoproteins.
When injected into experimental mice, the lipoprotein-formulated Chol-siOAT3 construct was taken up by receptor mediated transfer, and resulted in a significant reduction of OAT3 mRNA. However, the construct was not taken up by neurons or glial cells. Reporting in Molecular Therapy, Takanori Yokota, Ph.D., and colleagues claim the technique could be used for gene silencing therapy in diseases involving BCECs. Their paper is titled “Efficient In Vivo Delivery of siRNA into Brain Capillary Endothelial Cells along with Endogenous Lipoprotein.”
The team had previously developed a hydrodynamic technique for delivering siRNAs into BCECs, but admit this strategy isn’t really applicable clinically because of volume overload and the extremely high hydrostatic pressure involved. The ideal in vivo carrier of siRNA into BCECs would be a molecule that is taken up into BCECs but cannot pass through the blood brain barrier. In their search for an alternative approach, they considered independent work demonstrating that extracted endogenous lipoproteins represent effective vectors for the delivery of siRNA to the liver by conjugation of cholesterol (Chol-siRNA).
To test this strategy for delivering siRNAs to BCECs, they developed a Chol-siOAT3 construct incorporated into HDL, which they injected intravenously into experimental mice. OAT3 is exclusively expressed by endothelial cells in the brain, they note. For comparison, different animals were administered with "naked" siOAT3, Chol-siOAT3 without the HDL fraction, or Chol-siOAT3 incorporated into LDL. Subsequent analysis of brain tissue confirmed that only Chol-siOAT3 incorporated into HDL was efficiently taken up by BCECs. Moreover, the formulation didn’t actually cross the blood brain barrier and reach glial cells or neurons.
Importantly, administration of Chol-siOAT3/HDL led to significant reductions in OAT3 mRNA in relevant cells in a dose-dependent manner, but had no effect on levels of OAT3 mRNA in the epithelial cells of renal proximal tubules, which also produce OAT3.
Subsequent studies using Chol-SiOAT3/HDL generated using HDL taken from the sera of ApoE-deficient mice, and recipient mice that were also deficient in ApoE, showed that delivery of Chol-SiOAT3/HDL was mostly ApoE-dependent. Similarly, administering unmodified Chol-SiOAT3/HDL to mice that were deficient in LDLR showed that the receptor for the Chol-SiOAT3/HDL was LDLR.
This was particularly surprising given that Chol-siOAT2/LDL wasn’t efficiently taken up by the BCECs. However, the authors point out, this may relate to the unique lipoprotein profile in rodents, which display an LDL clearance rate that is 40 times greater than that in humans; the observation that Chol-siOAT2/LDL was not taken up efficiently into BCECs may simply be because it was rapidly eliminated from the blood circulation into the liver.
“This is the first report to demonstrate the concept of an endogenous lipoprotein vector for the efficient delivery of siRNA into BCECs in mice via an intravenous injection,” the authors conclude. “Our results indicate that HDL works as an efficient vector for transporting Chol-siOAT3 into the BCECs, being mainly mediated by ApoE as the ligand and LDLR as the receptor...As a therapeutic approach in humans, LDL as well as HDL has a potential to work as another effective vector of Chol-siRNA.”
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