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

RNA interference (RNAi)-based therapeutics have the potential to treat chronic hepatitis B virus (HBV) infection in a fundamentally different manner than current therapies. Using RNAi, it is possible to knock down expression of viral RNAs including the pregenomic RNA from which the replicative intermediates are derived, thus reducing viral load, and the viral proteins that result in disease and impact the immune system's ability to eliminate the virus. We previously described the use of polymer-based Dynamic PolyConjugate (DPC) for the targeted delivery of siRNAs to hepatocytes. Here, we first show in proof-of-concept studies that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines. Using transient and transgenic mouse models of HBV infection, we show that a single coinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBV sequences resulted in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect. These results suggest that coinjection of NAG-MLP and chol-siHBVs holds great promise as a new therapeutic for patients chronically infected with HBV.

No abstract

Conditions that promote renaturation of an unfolded protein also promote protein aggregation, in many cases, because these competing intramolecular and intermolecular processes are driven by similar networks of noncovalent interactions. The GroEL/GroES system and related biological chaperones facilitate the renaturation of substrate proteins by minimizing the aggregation pathway. We have devised a two-step method in which small molecules, "artificial chaperones," facilitate protein refolding from a chemically denatured state. In the first step, the protein is captured by a detergent as guanidinium chloride is diluted to a non-denaturing concentration; formation of a protein-detergent complex prevents both protein aggregation and proper refolding. In the second step, a cyclodextrin strips detergent from the protein, allowing the protein to refold. Here we describe the first application of this method to a protein that must form disulfides in the native state. Lysozyme (hen egg white) can be refolded from the Gdm-denatured, DTT-reduced state in good yields at final protein concentrations as high as 1 mg/mL with the artificial chaperone method. Several mechanistic aspects of artificial chaperone-assisted refolding have been probed, and a detailed mechanism for the kinetically controlled stripping step is proposed.

We recently reported a new approach to protein refolding that utilizes a pair of low molecular weight folding assistants, a detergent and a cyclodextrin (Rozema, D., and Gellman, S. H. (1995) J. Am. Chem. Soc. 117, 2373-2374). Here, we provide a detailed study of carbonic anhydrase B (CAB) refolding assisted by these "artificial chaperones." When CAB is heated in the presence of a competent detergent, or when guanidiniumdenatured CAB is diluted to nondenaturing guanidinium concentration in the presence of such a detergent, the detergent forms a complex with the nonnative protein, thereby preventing aggregation. CAB is unable to refold from the detergent-complexed state, but folding can be induced by introduction of a cyclodextrin, which strips the detergent away from the protein.Use of artificial chaperones provides excellent yields of reactivated CAB under conditions that lead to little or no reactivation in the absence of the refolding assistants. Our studies show that the detergent can capture the unfolded protein even at submicellar concentrations, but that not all CAB-detergent complexes lead efficiently to refolded enzyme upon introduction of the stripping agent. Effective refolding appears to require that detergent stripping occur as rapidly as possible; intrinsically slow methods of detergent removal (dialysis or use of macroscopic adsorbents) are less effective than cyclodextrin at inducing renaturation upon detergent removal. The detailed characterization of artificial chaperone-assisted CAB refolding reported here should guide the application of this strategy to other proteins.The process by which protein molecules achieve their native conformations is a subject of fundamental and practical importance. Fundamental interest in the "protein folding problem" arises because we do not yet understand how a complex network of noncovalent interactions can specify one particular compact conformation for an intrinsically flexible polypeptide (1), how the polypeptide rapidly finds that compact conformation (2, 3), or why such a purely noncovalent process involves relatively large kinetic barriers (4). Practical interest in the "protein refolding problem" stems from the fact that proteins overproduced by genetically engineered cells are often obtained in non-native forms (e.g. inclusion bodies), and the use of such proteins for basic research or biotechnological applications requires that the native conformation be achieved (5, 6).Revolutionary advances in genetic manipulation techniques have made protein refolding an increasingly pressing problem, but there have been relatively few efforts to devise general renaturation strategies. In a common scenario, an overproduced protein is purified in inclusion body form, the inclusion bodies are solubilized with a chemical denaturant (typically guanidinium or urea), and refolding is attempted by removing the denaturant, via dilution or dialysis. For many proteins, however, denaturant removal leads predominantly or completely to protein aggregation rather than refolding (...

Endosomolysis, a critical barrier to efficient delivery of macromolecules such as nucleic acids, has been breached using a novel approach: endosomolysis by masking of a membrane-active agent (EMMA). To demonstrate the concept of EMMA, a cationic membrane-active peptide, melittin, was reversibly inhibited using a maleic anhydride derivative. At neutral pH, the lysines of melittin are covalently acylated with the anhydride, thereby inhibiting melittin's membrane disruption activity. Under acidic conditions such as those present within endosomes, the amide bond of the maleamate is cleaved, thus unmasking melittin. The active melittin can then disrupt the endosomal membrane resulting in release of biologically active molecules into the cytoplasm. This approach avoids cellular toxicity by restricting melittin's activity until it reaches the endosomal compartment. The utility of this approach was demonstrated by delivery phosphorodiamidate morpholino oligonucleotides (PMOs).

Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a 1 , the a 2 , or the X position of the a-factor Ca 1 a 2 X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a 1 position, V, L, I, C, or M at the a 2 position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a 1 substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

The delivery of a variety of nucleic acids such as plasmid DNA (pDNA) and small interfering RNA (siRNA) to mammalian cells is both an important research tool and potential therapeutic approach. Synthetic vehicles (SVs) that include lipoplexes and polyplexes, are widely used for non-viral delivery. A promising method of improving the efficacy of this approach is to create SVs that are chemically dynamic, so that delivery is enabled by the cleavage of chemical bonds upon exposure to various physiological environments or external stimuli. An example of this approach is the use of masked endosomolytic agents (MEAs) that improve the release of nucleic acids from endosomes, a key step during transport. When the MEA enters the acidic environment of the endosome, a pH-labile bond is broken, releasing the agent';s endosomolytic capability. Another challenge has been to develop SVs that enable in vivo delivery. Recently, an MEA that was used within dynamic polyconjugates (DPCs) enabled the efficient delivery of siRNA into hepatocytes in vivo. The use of labile bonds to mask endosomolytic agents, provides a critical design feature, because it enables efficient in vivo delivery without sacrificing endosomolytic function for release into the cytoplasm.