472743 Ligand-Targeted Conjugate Systems for Delivery of siRNA to Tumors
Recently, advances in siRNA stabilization technology have made unencapsulated siRNA conjugates viable, as chemical modifications to the RNA backbone have been used to reduce susceptibility of the siRNA to endonuclease degradation. These conjugates also have desirable treatment properties, including an in-human safety profile of up to 300 mg/kg and a clear regulatory path due to a well-defined chemical structure. Furthermore, due to their small size and thus ability to diffuse relatively quickly through extracellular matrix, the conjugates can be injected subcutaneously, a home procedure familiar to many diabetics on insulin, as opposed to nanoparticles, which must be infused intravenously at a clinic or hospital.
We have developed an azide-alkyne cycloaddition based system that allows for the delivery of ligand-targeted siRNA conjugates to models of glioblastoma (a form of brain tumor) in vitro and in vivo. The ligand used is based on chlorotoxin. In vitro studies using U87 (glioblastoma) cells show a similar degree of knockdown to commercially available transfection materials relative to a scrambled siRNA control. Confocal microscopy studies using derivatives of these ligands with fluorescently-labeled siRNA demonstrate a high degree of uptake relative to unconjugated siRNA in both cases. These conjugates are also able to traverse the bloodstream, deliver a nucleic acid payload into the tumor cells, and efficiently knock down expression of a housekeeping gene. Using fluorescent derivatives of our siRNA, we are able to observe localization of the siRNA to subcutaneous or intracranial tumor xenografts. Intravital imaging experiments demonstrate the ability of these conjugates to pass the blood-brain barrier and permeate the tumor. In vivo knockdown studies show a moderate degree of tumor-specific knockdown of a housekeeping gene in an intracranial model of glioblastoma using U87 cells.
These results demonstrate that siRNA knockdown of genes in tumors using these modular conjugate systems are feasible and present a way forward for using siRNA in the treatment of cancer. Future work involves using biologically-relevant siRNA to demonstrate anti-tumor effects in this and syngeneic tumor models.