Abstract 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. Mol Cancer Ther; 17(1); 140–9. ©2017 AACR.

Title: HIF2α targeted RNAi therapeutic inhibits clear cell renal cell carcinoma Authors and affiliations: So C. Wong, Weijun Cheng, Holly Hamilton, Anthony L. Nicholas, Darren H. Wakefield, Aaron Almeida, Andrei V. Blokhin, Jeffrey Carlson, Zane C. Neal, Vladimir Subbotin, Guofeng Zhang, Julia Hegge, Stephanie Bertin, Vladimir S. Trubetskoy, David B. Rozema, David L. Lewis, Steven B. Kanner 1. Arrowhead Pharmaceuticals Inc., Madison, WI

Abstract Background: Targeted therapy including VEGF and mTOR pathway inhibitors has dramatically transformed treatment options and outcomes for patients with metastatic clear cell renal cell carcinoma (ccRCC). However, alternate treatments are needed as resistance to these initially promising agents occurs frequently. RNAi interference (RNAi), an innate gene silencing mechanism, has been explored as a new class of therapeutics where conventional treatments are lacking or have failed. The challenge in leveraging this promising approach has been efficient delivery of an RNAi trigger (siRNA) to target tissue. Over 90% of ccRCC tumors express a mutant inactive form of the von Hippel-Landau protein (pVHL), an E3 ubiquitin ligase that promotes target protein degradation. Strong evidence supports the observation that pVHL functional loss leads to the accumulation of the transcription factor hypoxia-inducible factor 2α (HIF-2α), a tumorigenic driver of ccRCC. Methods: We have developed a targeted delivery platform called Dynamic Polyconjugte™ (DPC) as an RNAi-based therapeutic targeting HIF-2α for advanced ccRCC. The ccRCC-specific DPC (ITG-DPC) comprises a membrane active polymer to promote RNAi trigger endosomal release, a ligand that binds to αV-containing integrin receptors expressed on tumor cells, reversible masking to prevent polymer activity before reaching the endosomal compartment, and a potent and specific RNAi trigger to HIF-2α. The modular nature of this delivery platform allows for flexibility to optimize each functional component independently. The ligand-dependent delivery of ITG-DPC was first evaluated in cultured tumor cells and then confirmed in ccRCC tumors established in nude mice using fluorescently-labeled ITG-DPC and confocal microscopy. To validate silencing of HIF-2α as an effective therapeutic approach, an inducible shRNA to HIF-2α was expressed in ccRCC tumors established in mice that significantly silenced HIF-2α gene expression and induced tumor regression. Results: Bi-weekly injection of ITG-DPC into nude mice with established orthotopic A498 ccRCC tumors resulted in >80% knockdown of HIF-2α mRNA and significant tumor growth inhibition. Histological examination of tumor sections showed substantial cell killing and destruction of tumor architecture. Down-regulation of HIF-2α regulated pathways genes including VEGF-A, and the corresponding reduction in tumor-associated CD31 positive neovascularization, corroborated on-target effects of HIF-2α gene silencing. Conclusion: Targeted delivery of a HIF-2α specific RNAi-based-therapeutic has the potential to radically impact the late-stage ccRCC treatment paradigm. Citation Format: So Wong, Weijun Cheng, Darren Wakefield, Aaron Almeida, Andrei Blokhin, Holly Hamilton, Vladimir Subbotin, Julia Hegge, Zane Neal, Guofeng Zhang, David Rozema, David Lewis, Steven Kanner. Novel HIF-2α targeted RNAi therapeutic for renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2064.

Evans blue dye (EBD) can be used in live mice to study muscle pathology or injury, including exercise-induced muscle damage. EBD is excluded from intact cell membranes but leaks into cells, including muscle fibers, when the cell membrane is ruptured. EBD can be visualized by its autofluorescence under a fluorescence microscope. EBD-stained myofibers can be quantified from microscope images of muscle cross-sections. These myofibers are often in clusters that lend themselves to morphometric analysis. When the damaged myofibers are interspersed among intact myofibers, however, a more suitable approach is to count individual myofibers in the field of view. A much faster approach to measure EBD in muscles from different strains of mice or between treatment groups is to extract the EBD from muscle samples and quantitate it using a spectrophotometric microplate reader. The advantages and disadvantages of using each of these approaches are discussed here. Curr. Protoc. Mouse Biol. 1:463-488 © 2011 by John Wiley & Sons, Inc.

Evans blue dye (EBD) is used to mark damaged and permeable muscle fibers in mouse models of muscular dystrophy and as an endpoint in therapeutic trials. We counted EBD‐positive muscle fibers and extracted EBD from muscles sampled throughout the hindlimbs in young adult and old mdx mice to determine if the natural variability in morphology would allow measurement of a functional improvement in one limb compared to the contralateral limb. Following one bout of rotarod or treadmill exercise that greatly increased serum creatine kinase levels, the number of EBD + muscle fibers in 12–19‐month‐old mdx mice increased 3‐fold, EBD in the muscles increased, and, importantly, contralateral pairs of muscles contained similar amounts of EBD. In contrast, the intra‐ and interlimb amounts of EBD in 2–7‐month‐old mdx mice were much too variable. A therapeutic effect can more readily be measured in old mdx mice. These results will be useful in the design of therapy protocols using the mdx mouse. Muscle Nerve, 2010

In these studies we delivered by hydrodynamic limb vein (HLV) injection plasmid DNA (pDNA) expressing the full-length mouse dystrophin gene to skeletal muscles throughout the hind limbs of the mdx mouse model for Duchenne muscular dystrophy (DMD). We evaluated the levels and stability of dystrophin expression and measured the resulting muscle protection, using Evans blue dye (EBD) to mark the damaged myofibers. Plasmid delivery was as efficient in the dystrophic mice as in wild-type mice and equally efficient in young adult and old mice, as long as the dose of pDNA was adjusted for the target muscle weight. The HLV gene delivery procedure was tolerated well by the dystrophic mice and repeat injections could be performed over an extended period of time. Multiple gene deliveries additively increased the amount of dystrophin protein and also increased the percentages of dystrophin-expressing myofibers. Plasmids expressing dystrophin from a cytomegalovirus (CMV) promoter construct containing the HMG1 intron provided stable dystrophin expression for the life of the mouse and provided significant benefit to the limbs. EBD staining showed that dystrophin gene delivery preserved myofibers in the CMV-HMGi-mDys-injected leg by 2.5- to 5-fold in large groups of muscles and by 2.5-fold throughout the injected legs, compared with the contralateral control legs injected with a nonexpressing plasmid. A similar degree of protection was measured in young adult mice evaluated soon after the last gene delivery and in aged mice injected over an extended period of time. This degree of protection resulted from 18 to 20% of the normal level of dystrophin protein, with 11-16% dystrophin-expressing myofibers. These studies show promise for the use of HLV injections to deliver therapeutic doses of full-length dystrophin-expressing plasmids for long-lasting protection of skeletal muscles in patients with DMD.

The administration route is emerging as a critical aspect of nonviral and viral vector delivery to muscle, so as to enable gene therapy for disorders such as muscular dystrophy. Although direct intramuscular routes were used initially, intravascular routes are garnering interest because of their ability to target multiple muscles at once and to increase the efficiency of delivery and expression. For the delivery of naked plasmid DNA, our group has developed a hydrodynamic, limb vein procedure that entails placing a tourniquet over the proximal part of the target limb to block all blood flow and injecting the gene vector rapidly in a large volume so as to enable the gene vector to be extravasated and to access the myofibers. The present study was conducted in part to optimize the procedure in preparation for a human clinical study. Various injection parameters such as the effect of papaverine preinjection, tourniquet inflation pressure and duration, and rate of injection were evaluated in rats and nonhuman primates. In addition, the safety of the procedure was further established by determining the effect of the procedure on the neuromuscular and vascular systems. The results from these studies provide additional evidence that the procedure is well tolerated and they provide a foundation on which to formulate the procedure for a human clinical study.

It has previously been reported that a peptide sequence of T7 phage protein p17 mediates uptake of its cargo by liver parenchymal cells. The aim of this study was to identify the phage-binding receptor. The involvement of LRP was confirmed by the observations that phage binding to Hepa 1c1c7 cells was inhibited by the LRP-binding receptor-associated protein, LRP-deficient mouse embryonic fibroblasts bound phage with lower efficiency than their wild-type counterparts, and using mouse models with ablated LRP liver expression. The identification of LRP as a cognate receptor for this sequence offers a new ligand-receptor combination for hepatocyte delivery of therapeutic agents.

In vivo phage display is a powerful source of new peptide ligands for specific organ targeting by drugs and gene therapy vectors. Since the introduction of this methodology a decade ago, a number of peptides that preferentially react with organ-specific endothelium and parenchymal markers have been selected. One organ that has been conspicuously missing from these selection studies is the liver, which possesses a multitude of acquired and hereditary disorders and represents a highly important therapeutic target. Herein, we set out to fill this gap by introducing a novel peptide display system containing cloned sequences in the tail fiber protein (p17) of phage T7. The p17 display effectively avoids the innate immune system and is well suited both for selection of new liver-specific ligands and for validation of protein sequences that have been implicated in liver targeting by the use of conventional biochemical methods.

The purpose of this work is to investigate the use of magnetic resonance imaging ( MRI) to monitor the effects of high-pressure naked plasmid DNA ( pDNA) intravascular injections in primate limbs, studying both the distribution of the injected solution in the muscle space, as well as the effects on the vascular system. The distal portion of the four limbs of each of six rhesus monkeys were hydrodynamically injected with naked pDNA, which expressed the luciferase reporter gene. Three-dimensional ( 3D) T1-weighted gradient echo and 2D multislice T2-weighted fast spin echo ( FSE) series were acquired before and immediately after the injection to confirm the volume of solution injected into the limb, and to study the distribution of the injected solution in the individual muscle groups. Time-resolved contrast-enhanced 3D magnetic resonance angiography ( MRA) was performed several days before, immediately after, and in a follow-up examination after the pDNA injection to study the effects of the procedure on the primate peripheral vascular system. T1-weighted gradient echo imaging confirmed the delivery of the majority of the solution after successful pDNA injections. T2-weighted FSE imaging demonstrated the distribution of the saline solution in individual muscles in the target limbs, with enhancement showing a weak but significant correlation with the level of gene expression. Time-resolved contrast-enhanced MRA demonstrated effects of the injection procedure on the arterial and venous vascular systems, and the intramuscular compartments; and these effects largely returned to normal on short-term follow-up.

Plasmid DNA (pDNA) is efficiently delivered to limb muscles of rodents and large animals by hydrodynamic limb vein (HLV) injection, a facile procedure that uses a tourniquet or blood pressure cuff placed distal to the injected vein to delimit the target tissue. In rhesus monkeys of various weights and ages we detected much higher reporter gene expression when the pDNA dose was above an apparent threshold level measured in pDNA dose/gram muscle of the target tissue. Studies in mice and rats were carried out to investigate these dose response effects and confirmed a threshold dose above which expression correlated directly with the dose and below which it did not. The percentages of transfected myofibers were also shown to increase as the pDNA dose increased. Co-localization of transgene expression and fiber type by immunohistochemistry showed that all types of myofibers were transfected at similar efficiencies, both in a primate and in the mdx dystrophic mouse model. Equal molar amounts of the human cytomegalovirus (CMV) promoter, the human desmin promoter and locus control region (DCR), and the mouse and human muscle creatine kinase (MCK) promoters were then compared for tissue specificity, for short-term efficiency of expression, and for stability of long-term expression in muscle. Tissue specificity was examined by use of hydrodynamic tail vein injection to deliver the plasmids to mouse liver and HLV injection to deliver the same plasmids to muscle. At lower pDNA doses, the DCR and MCK promoters had limited expression in the liver. Surprisingly, the DCR promoter was highly expressed in liver when a large dose of pDNA was injected. Short-term expression in muscle was evaluated 7 days after HLV injection of luciferase expression vectors into both mice and rats. The CMV promoter consistently gave the highest expression, two-fold higher than the mouse MCK promoter. The DCR, human MCK and mouse MCK promoters were similarly effective in rats, while the DCR and human MCK promoters were less effective in mice. A non-immunogenic mouse secreted alkaline phosphatase (mSEAP) reporter gene was then utilized to evaluate the time course and the long-term stability of expression from each promoter in mice. While high expression levels were attained by the CMV promoter within the first week, expression from both MCK promoters increased only gradually over the course of two weeks. The CMV promoter gave 2–3 fold higher long-term expression levels than the muscle promoters; however, all of these promoters gave sustained high levels of expression for at least 37 weeks. Furthermore, the amount of transgene expression could be additively increased by two to four injections into the same target area. These studies will help investigators determine effective pDNA doses and promoters for transgene expression in limb muscles.

Background The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general.Methods In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes.Results Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG) hepatocytes flooded with large amounts of IgG, appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later.Conclusions The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery. Copyright (c) 2006 John Wiley & Sons, Ltd.

Background The efficient delivery of plasmid DNA (pDNA) to hepatocytes by a hydrodynamic tail vein (HTV) procedure has greatly popularized the use of naked nucleic acids. The hydrodynamic process renders onto the tissue increased physical forces in terms of increased pressures and shear forces that could lead to transient or permanent membrane damage. It can also trigger a series of cellular events to seal or reorganize the stretched membrane. Our goal was to study the uptake mechanism by following the morphological changes in the liver and correlate these with the fate of the injected plasmid DNA.Methods We utilized both light microscopic (LM) and electron microscopic (EM) techniques to determine the effect of the HTV procedure on hepatocytes and non-parenchymal cells at various times after injection. The LM studies used paraffin-embedded livers with hematoxylin and eosin (H&E) staining. The immune-EM studies used antibodies labeled with sub-nanometer gold particles followed by silver enhancement to identify the location of injected pDNA at the subcellular level. The level of overall damage to liver cells was estimated based on alanine aminotransferase (ALT) release and clearance.Results Both the LM and EM results showed the appearance of large vesicles in hepatocytes as early as 5 min post-injection. The number of vesicles decreased by 20-60 min. Plasmid DNA molecules often appeared to be associated with or inside such vesicles. DNA could also be detected in the space of Disse, in the cytoplasm and in nuclei. Non-parenchymal cells also contained DNA, but HTV-induced vesicles could not be observed in them.Conclusions Our studies suggest an alternative or additional pathway for naked DNA into hepatocytes besides direct entry via membrane pores. It may be difficult to prove which of these pathways lead to gene expression, but the membrane pore hypothesis alone appears insufficient to explain why expression happens preferentially in hepatocytes. Further study is needed to delineate the importance of each of these putative pathways and their interrelationship in enabling oligonucleotide (siRNA) activity and pDNA expression. Copyright (c) 2006 John Wiley & Sons, Ltd.

Genetic immunization is an attractive approach to generate antibodies because native proteins are expressed in vivo with normal posttranscriptional modifications, avoiding time-consuming and costly antigen isolation or synthesis. Hydrodynamic tail or limb vein delivery of naked plasmid DNA expression vectors was used to induce antigen-specific antibodies in mice, rats, and rabbits. Both methods allowed the efficient generation of high-titer, antigen-specific antibodies with an overall success rate of Western detectable antibodies of 78% and 92%, respectively. High-titer antibodies were typically present after 3 hydrodynamic tail vein plasmid DNA deliveries, 5 weeks after the initial injection (i.e., prime). For hydrodynamic limb vein plasmid DNA delivery, two deliveries were sufficient to induce high-titer antibody levels. Tail vein delivery was less successful at generating antibodies directed against secreted proteins as compared with limb vein delivery. Material for screening was generated by,transfection of the immunization vector into mammalian cell lines. The cell line (COS-7) that produced the highest level of antigen expression performed best in Western blot analysis screens. In summary, intravenous delivery of antigen-expressing plasmid DNA vectors is an effective genetic immunization method for the induction of antigen-specific antibodies in small and large research animals.

We evaluated naked plasmid DNA (pDNA)-mediated expression of human hepatic bilirubin UDP-glucuronosyltransferase (hUGT1A1) in skeletal muscle to correct hyperbilirubinemia in the UGT1A1-deficient Gunn rat, an animal model of Crigler-Najjar syndrome type I (CN-I). After delivery of pDNA encoding hUGT1A1 via hepatic vein or femoral artery, in vitro bilirubin glucuronidation activity was detectable in Gunn rat liver and muscle extracts. Expression of hUGT1A1 in Gunn rat liver or muscle resulted in excretion of bilirubin glucuronides in bile. Total biliary bilirubin concentrations increased from a pretreatment average of 10.5 +/- 2.1 microM to 29.2 +/- 4.2 microM after gene transfer into the liver, and to 28.6 +/- 3.8 microM after gene transfer into muscle. Total serum bilirubin decreased by up to 31.2 +/- 6.9 and 29.2 +/- 3.7% and remained significantly lower for at least 1 and 2 weeks, respectively. Tissue damage associated with the procedure was minimal and reversible. Our results demonstrate that muscle can be genetically modified to glucuronidate bilirubin, leading to elimination in bile. A 30% decrease in serum bilirubin, if sustained, would provide meaningful clinical benefit for CN-I patients. However, to be clinically useful, this method needs further optimization and stable gene expression must be achieved.

Delivery is increasingly being recognized as the critical hurdle holding back the tremendous promise of nucleic acid-based therapies that include gene therapy and more recently siRNA-based therapeutics. While numerous candidate genes (and siRNA sequences) with therapeutic potential have been identified, their utility has not yet been realized because of inefficient and/or unsafe delivery technologies. We now describe an intravascular, nonviral methodology that enables efficient and repeatable delivery of nucleic acids to muscle cells (myofibers) throughout the limb muscles of mammals. The procedure involves the injection of naked plasmid DNA or siRNA into a distal vein of a limb that is transiently isolated by a tourniquet or blood pressure cuff. Nucleic acid delivery to myofibers is facilitated by its rapid injection in sufficient volume to enable extravasation of the nucleic acid solution into muscle tissue. High levels of transgene expression in skeletal muscle were achieved in both small and large animals with minimal toxicity. Evidence of siRNA delivery to limb muscle was also obtained. The simplicity, effectiveness, and safety of the procedure make this methodology well suited to limb muscle gene therapy applications.