Forces Shaping the Blastocyst.

The blastocyst forms during the first days of mammalian development. The structure of the blastocyst is conserved among placental mammals and is paramount to the establishment of the first mammalian lineages. The blastocyst is composed of an extraembryonic epithelium, the trophectoderm (TE), that envelopes a fluid-filled lumen and the inner cell mass (ICM). To shape the blastocyst, embryos transit through three stages driven by forces that have been characterized in the mouse embryo over the past decade. The morphogenetically quiescent cleavage stages mask dynamic cytoskeletal remodeling. Then, during the formation of the morula, cells pull themselves together and the strongest ones internalize. Finally, the blastocyst forms after the pressurized lumen breaks the radial symmetry of the embryo before expanding in cycles of collapses and regrowth. In this review, we delineate the force patterns sculpting the blastocyst, based on our knowledge on the mouse and, to some extent, human embryos.

Ectoderm to mesoderm transition by down-regulation of actomyosin contractility.

Collective migration of cohesive tissues is a fundamental process in morphogenesis and is particularly well illustrated during gastrulation by the rapid and massive internalization of the mesoderm, which contrasts with the much more modest movements of the ectoderm. In the Xenopus embryo, the differences in morphogenetic capabilities of ectoderm and mesoderm can be connected to the intrinsic motility of individual cells, very low for ectoderm, high for mesoderm. Surprisingly, we find that these seemingly deep differences can be accounted for simply by differences in Rho-kinases (Rock)-dependent actomyosin contractility. We show that Rock inhibition is sufficient to rapidly unleash motility in the ectoderm and confer it with mesoderm-like properties. In the mesoderm, this motility is dependent on two negative regulators of RhoA, the small GTPase Rnd1 and the RhoGAP Shirin/Dlc2/ArhGAP37. Both are absolutely essential for gastrulation. At the cellular and tissue level, the two regulators show overlapping yet distinct functions. They both contribute to decrease cortical tension and confer motility, but Shirin tends to increase tissue fluidity and stimulate dispersion, while Rnd1 tends to favor more compact collective migration. Thus, each is able to contribute to a specific property of the migratory behavior of the mesoderm. We propose that the "ectoderm to mesoderm transition" is a prototypic case of collective migration driven by a down-regulation of cellular tension, without the need for the complex changes traditionally associated with the epithelial-to-mesenchymal transition.

HIF2α-Targeted RNAi Therapeutic Inhibits Clear Cell Renal Cell Carcinoma.

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 αvß3 and αvß5 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.

Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses.

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.

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.

Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection.

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.

Co-injection of a targeted, reversibly masked endosomolytic polymer dramatically improves the efficacy of cholesterol-conjugated small interfering RNAs in vivo.

Effective in vivo delivery of small interfering (siRNA) has been a major obstacle in the development of RNA interference therapeutics. One of the first attempts to overcome this obstacle utilized intravenous injection of cholesterol-conjugated siRNA (chol-siRNA). Although studies in mice revealed target gene knockdown in the liver, delivery was relatively inefficient, requiring 3 daily injections of 50 mg/kg of chol-siRNA to obtain measurable reduction in gene expression. Here we present a new delivery approach that increases the efficacy of the chol-siRNA over 500-fold and allows over 90% reduction in target gene expression in mice and, for the first time, high levels of gene knockdown in non-human primates. This improved efficacy is achieved by the co-injection of a hepatocyte-targeted and reversibly masked endosomolytic polymer. We show that knockdown is absolutely dependent on the presence of hepatocyte-targeting ligand on the polymer, the cognate hepatocyte receptor, and the cholesterol moiety of the siRNA. Importantly, we provide evidence that this increase in efficacy is not dependent on interactions between the chol-siRNA with the polymer prior to injection or in the bloodstream. The simplicity of the formulation and efficacy of this mode of siRNA delivery should prove beneficial in the use of siRNA as a therapeutic.

Breaking the bonds: non-viral vectors become chemically dynamic.

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.

Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes.

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.

Hepatocyte targeting of nucleic acid complexes and liposomes by a T7 phage p17 peptide.

A critical step for liver-directed gene therapy is the selective targeting of nucleic acids to hepatocytes. We have previously discovered that the proximal half of the T7 phage tail fiber protein (p17) targeted intact T7 phage and recombinant proteins to hepatocytes in vivo. In the present study, we have localized the targeting activities to a 33 amino acid sequence within the p17 coiled-coil rod domain. Given that the tail fiber domain from which the peptide was derived may form alpha and triple helical structures, biophysical studies (CD spectra and analytical ultracentrifugation) were conducted to determine the secondary and tertiary structures of the peptide. This peptide is able to target proteins, polymers, and siRNA and also particles such as DNA polyplexes and liposomes to hepatocytes. A variety of coupling strategies and chemistries were employed, thus demonstrating that this peptide is a versatile system for delivering cargo. The ability of this hepatocyte-targeting peptide to target DNA-containing particles suggests that it should be useful in the development of both nonviral and viral vectors. However, biological function of delivered cargo has not been demonstrated. This was primarily due to failure of delivered cargo to escape the endosomes. Further studies are in progress to provide functional activity of delivered nucleic acids by enabling their endosomal escape.

Membrane activity and transfection ability of amphipathic polycations as a function of alkyl group size.

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.

Effects of side chain configuration and backbone spacing on the gene delivery properties of lysine-derived cationic polymers.

A series of lysine-based oligomers (18 residues) that differ in side chain configuration or side chain spacing along the backbone was tested for DNA transfection activity. Although materials constructed from lysine are not the most effective polymeric transfection agents, we have chosen L-lysine-based molecules as a starting point because this system allows us to examine the functional effects of incremental changes in polycation structure. The oligomer constructed from beta(3)-homolysine (beta(3)-hLys) and that from alpha-D-lysine were superior to an alpha-L-lysine 18-mer in gene delivery assays. This improved activity is attributed to the fact that the alpha-L-peptide is a protease substrate while the other 18-mers are not. This conclusion is supported by the effects of chloroquine on transfection activity, based on the protease inhibition activity of chloroquine. To our knowledge, these results represent the first direct comparison of a D-lysine oligomer with an L-lysine oligomer in the context of gene delivery. Poly(beta(3)-hLys) was synthesized from the ring opening polymerization of the corresponding lactam. The DNA transfection ability of this polymer was compared with that of commercially available poly(L-lysine) (PLL). In each case the polymer was more active than the corresponding oligomer.

Endosomolysis by masking of a membrane-active agent (EMMA) for cytoplasmic release of macromolecules.

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).