Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes

Abstract

Achieving efficient in vivo delivery of siRNA to the appropriate target cell would be a major advance in the use of RNAi in gene function studies and as a therapeutic modality. Hepatocytes, the key parenchymal cells of the liver, are a particularly attractive target cell type for siRNA delivery given their central role in several infectious and metabolic disorders. We have developed a vehicle for the delivery of siRNA to hepatocytes both in vitro and in vivo, which we have named siRNA Dynamic PolyConjugates. Key features of the Dynamic PolyConjugate technology include a membrane-active polymer, the ability to reversibly mask the activity of this polymer until it reaches the acidic environment of endosomes, and the ability to target this modified polymer and its siRNA cargo specifically to hepatocytes in vivo after simple, low-pressure i.v. injection. Using this delivery technology, we demonstrate effective knockdown of two endogenous genes in mouse liver: apolipoprotein B (apoB) and peroxisome proliferator-activated receptor alpha (ppara). Knockdown of apoB resulted in clear phenotypic changes that included a significant reduction in serum cholesterol and increased fat accumulation in the liver, consistent with the known functions of apoB. Knockdown of ppara also resulted in a phenotype consistent with its known function, although with less penetrance than observed in apoB knockdown mice. Analyses of serum liver enzyme and cytokine levels in treated mice indicated that the siRNA Dynamic PolyConjugate was nontoxic and well tolerated.pH labile bonds ͉ nonviral siRNA delivery ͉ siRNA-polymer conjugates ͉ endosomolytic polymers

Gene therapy progress and prospects: synthetic polymer-based systems

“…The synthetic nature of siRNA opened new options, such as stabilizing modifications and covalent conjugation to carrier molecules. 36 Also, the different requirements and polyplex characteristics have become evident, with the smaller size of siRNA and the aim to deliver and release into the cytosol as opposed to the required nuclear delivery of pDNA. For example, some PEI formulations that effectively work for pDNA delivery show only moderate siRNA delivery capacity.…”

“…This is consistent with the notion that receptor-mediated cellular uptake into tumor cells by the transferrin receptor is essential for effective siRNA delivery. 45 Other examples of siRNA polyplexes for receptor-targeted in vivo delivery were recently reported, such as hepatocyte targeting utilizing the asialoglycoprotein receptor, 36 or brain targeting with a rabies virus-derived peptide ligand for the acetyl-choline receptor. 46 Multifunctional polymers can be designed with defined topology and dynamic transport domains…”

“…57 pH-responsive polyplexes were also developed for siRNA delivery. 3,36 An elegant strategy was developed for hepatocyte-targeted in vivo siRNA delivery by Rozema et al 36 A membrane-destabilizing poly(vinyl ether) polymer was covalently conjugated with siRNA through a disulfide bond and reversibly masked by pH-labile ligation of PEG as shielding domain and N-acetylgalactosamine as targeting ligand. The applied linkers are either cleaved at acidic pH in endosomes or the reductive intracellular environment.…”

Special delivery: targeted therapy with small RNAs

“…Another example of this approach is 'siRNA dynamic polyconjugates' . 43 In this intelligently designed delivery system, siRNAs are conjugated to membrane-penetrating polymers whose activity is unmasked only after reaching the acidic environment of the endosome. Therefore, in principle, cell membrane toxicity by the polymer is limited.…”

Conjugation of siRNA with Comb‐Type PEG Enhances Serum Stability and Gene Silencing Efficiency

“…[1,2] However, the therapeutic potency of siRNA is severely limited by delivery problems. [2][3][4][5] PEGylation, a technique commonly used to improve the in vivo pharmacokinetics of protein therapeutics, [6,7] has also been investigated for siRNA delivery. [8][9][10][11][12][13][14][15][16] Covalent conjugation of a PEG chain to siRNA has shown to increase serum stability of siRNA with sufficient gene silencing efficiency.…”