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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Targeted therapy against VEGF and mTOR pathways has been established as the standard-of-care for metastatic clear cell renal cell carcinoma (ccRCC); however, these treatments frequently fail and most ...patients become refractory requiring subsequent alternative therapeutic options. Therefore, development of innovative and effective treatments is imperative. About 80%-90% of ccRCC tumors express an inactive mutant form of the von Hippel-Lindau protein (pVHL), an E3 ubiquitin ligase that promotes target protein degradation. Strong genetic and experimental evidence supports the correlate that pVHL functional loss leads to the accumulation of the transcription factor hypoxia-inducible factor 2α (HIF2α) and that an overabundance of HIF2α functions as a tumorigenic driver of ccRCC. In this report, we describe an RNAi therapeutic for HIF2α that utilizes a targeting ligand that selectively binds to integrins αvβ3 and αvβ5 frequently overexpressed in ccRCC. We demonstrate that functional delivery of a HIF2α-specific RNAi trigger resulted in HIF2α gene silencing and subsequent tumor growth inhibition and degeneration in an established orthotopic ccRCC xenograft model.
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The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting ...and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites. Compared to PCR- or ELISA-based methods, this assay enables separate quantitation of non-phosphorylated full-length guide strands from 5΄-phosphorylated forms that may associate with RNA-induced silencing complexes (RISC). Biodistribution studies in mice indicated that ARC-520 guide strands predominantly accumulated in liver. 5΄-phosphorylation of guide strands was observed within 5 min after ARC-520 injection, and was detected for at least 4 weeks corresponding to the duration of HBV mRNA silencing. Guide strands detected in RISC by AGO2 immuno-isolation represented 16% of total 5΄-phosphorylated guide strands in liver, correlating with a 2.7 log10 reduction of HBsAg. The PNA method enables pharmacokinetic analysis of RNAi triggers, elucidates potential metabolic processing events and defines pharmacokinetic-pharmacodynamic relationships.
Protease-triggered siRNA delivery vehicles Rozema, David B; Blokhin, Andrei V; Wakefield, Darren H ...
Journal of controlled release,
07/2015, Volume:
209
Journal Article
Peer reviewed
Open access
The safe and efficacious delivery of membrane impermeable therapeutics requires cytoplasmic access without the toxicity of nonspecific cytoplasmic membrane lysis. We have developed a mechanism for ...control of cytoplasmic release which utilizes endogenous proteases as a trigger and results in functional delivery of small interfering RNA (siRNA). The delivery approach is based on reversible inhibition of membrane disruptive polymers with protease-sensitive substrates. Proteolytic hydrolysis upon endocytosis restores the membrane destabilizing activity of the polymers thereby allowing cytoplasmic access of the co-delivered siRNA. Protease-sensitive polymer masking reagents derived from polyethylene glycol (PEG), which inhibit membrane interactions, and N-acetylgalactosamine, which targets asialoglycoprotein receptors on hepatocytes, were synthesized and used to formulate masked polymer-siRNA delivery vehicles. The size, charge and stability of the vehicles enable functional delivery of siRNA after subcutaneous administration and, with modification of the targeting ligand, have the potential for extrahepatic targeting.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Cationic membrane disruptive peptides such as melittin would appear to have attributes necessary for DNA delivery: DNA binding via electrostatic interactions and membrane lysis to enable cytoplasmic ...delivery. However, the relatively small overall charge of membrane disruptive peptides results in weak interactions with DNA. As a model of cationic membrane disruptive peptides, amphiphilic polyvinyl ethers were synthesized. The number of positively charged groups incorporated into these polymers is substantially greater than membrane-active peptides, which enables these polymers to form stable complexes with DNA. By varying the length of the hydrophobic groups incorporated into the polymer from one to four carbons, the dependence of membrane activity on side chain length was established. The ability of these polymers to transfect DNA in tissue culture was tested, and it was found that transfection efficiency is dependent upon the membrane disruptive activity of the polymer. Comparison of melittin and synthetic polymers suggests that transfection and toxicity appear to be dependent upon their affinity for DNA. This demonstration of relationships among membrane lysis, transfection, DNA binding, and polymer side-chain composition establishes a new class of transfection reagents and may guide in the design of polymers and formulations that will enable efficient in vivo transfection.
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IJS, KILJ, NUK, PNG, UL, UM
A critical step for liver-directed gene therapy is the selective targeting of nucleic acids to hepatocytes. There are a variety of physical characteristics that affect a nucleic acid particle's ...hepatocyte-targeting ability: size, surface charge, particle stability, and targeting ligand. Using a variety of formulation methods we are able to control a particle's size, charge, and stability to maximize hepatocyte targeting. Size and stability are controlled using bifunctional crosslinking reagents to cage the nucleic acid inside a net composed of a condensing polycation. The surface charge may be reduced via acetylation, which converts cationic ammonium groups to nonbasic amide groups. Selective hepatocyte targeting is finally conferred by attachment of galactose groups, which are ligands for the asiologlycoprotein receptor.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract Background/Aims Despite the availability of effective targeted therapies, only 20-30% achieve low disease activity and even fewer attain remission. There remains a pressing need to identify ...meaningful biomarkers and disease endotypes to enable precision medicine. We aimed to identify serum protein biomarkers that associate with RA inflammation and future remission in a treatment-naïve, new-onset RA trial cohort. Methods Serum from 105 patients from the ‘VEDERA’ trial (first-line tumour necrosis factor inhibitor + methotrexate TNFi + MTX versus methotrexate treat-to-target MTX-TT) at baseline, week 24 and 48 were analysed for 92 proteins using the Olink Target 96 Inflammation panel. Clinical and MSUS assessments completed at baseline, weeks 24 and 48 were used for the analyses. Clinical remission was defined as DAS28-ESR ≤ 2.6 and joint-level remission as absence of power Doppler ultrasound synovitis (PDUS). After adjusting for age, gender and seropositive status, Bayesian mixed effects regression (posterior estimates and 95% credible intervals reported) was used to identify association of (i) proteins with clinical disease activity (DAS28-ESR) and joint-level inflammation (total power Doppler ultrasound synovitis (PDUS) scores and (ii) baseline proteins with future (week 24 or 48) clinical and joint-level remission. Results Longitudinally, out of 92 proteins, 10 and 14 were significantly associated with DAS28-ESR and total PDUS scores respectively, with 3 proteins in common: leukaemia inhibitory factor receptor (LIF-R); fractalkine (CX3CL1); interleukin-10 receptor alpha (IL-10RA). Baseline levels of 9 proteins were associated with future clinical remission (week 24 = 6 proteins; week 48 = 3 proteins) while 6 were associated with future PDUS status (week 24 = 4 proteins; week 48 = 3 proteins). Proteins that were associated with inflammation longitudinally and whose baseline levels associated with future remission included IL-17C, fibroblast growth factor 5 (FGF-5), lymphotoxin alpha and TNF. Posterior estimates and 95% credible intervals for above proteins are reported in Table 1. Conclusion Using longitudinal high-throughput proteomics in a treatment-naïve early onset RA cohort, this study sheds new light on known and novel protein associates of inflammation and future remission. With future validation, these biomarkers could provide mechanistic understanding and allow for individualised treatment strategies to improve outcomes. Disclosure R. Shukla: None. R. Wakefield: None. A. Tan: None. P. Emery: Consultancies; BristolMyersSquibb, AbbVie, Gilead, Galapagos, Eli Lilly, MSD, Pfizer, Novartis, Roche and Samsung. Grants/research support; AbbVie, Eli Lilly, Novartis, BristolMyersSquibb, Pfizer and Roche, outside the submitted work. D. Plant: None. M.H. Buch: Consultancies; AbbVie, Arxx Therapeutics, Boehringer Ingelheim, Galapagos, Gilead, and Pfizer; and speaker fees from AbbVie, CESAS Medical, Galapagos, Gilead, Medistream, and Pfizer. Grants/research support; grant support from Gilead.
A New Short Way to Furocoumarins Traven, Valery F.; Kravtchenko, Dmitrii V.; Chibisova, Tatjana A. ...
Heterocyclic communications,
1996, 1996-01-00, Volume:
2, Issue:
4
Journal Article
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