November 1, 2012 (Vol. 32, No. 19)

Over the years, researchers have demonstrated that RNAi compounds can be designed to specifically reduce the expression of target genes before their mRNA is translated into protein. By leveraging this knowledge, RNAi compounds can be developed into drugs to prevent overexpression of deleterious proteins in target cells, thus providing therapeutic benefit.

At GTC’s recent “RNAi Research and Therapeutics” conference, speakers presented their solutions to the challenges faced by clinical researchers looking to develop RNAi therapeutics. How do I deliver RNAi compounds to target cells? Do I need a delivery vehicle to facilitate cellular uptake? Can I demonstrate sufficient knockdown, either transiently or permanently, in my target cells?

RNAi screens provide an essential tool for gene discovery and validation of gene function. One of the preferred approaches for RNAi screening recommended by Thermo Fisher Scientific employs the use of pooled shRNAs. This approach is supported by protocols and a large portfolio of products, including shRNA libraries.

Although RNAi research has experienced some ups and downs over the past few years, ample evidence exists that this technology can be used to develop new therapeutics. [iStock photos/Pgiam]

“The screening strategy of using pooled shRNAs provides a large collection of lentivirus-expressed shRNAs that target thousands of genes in a single well,” indicated Devin Leake, Ph.D., global director of R&D, molecular biology at Thermo.

“Cells with shRNAs targeting certain genes respond differently to phenotypic selection and become enriched or depleted during the screen. Identifying the exact shRNA producing the phenotype is then determined using next-gen sequencing (NGS) of genomic DNA isolated from the cells.”

Success depends on three factors: first, the use of high-performance shRNAs that effectively silence (knockdown) target gene expression; second, a high representation of shRNAs in the pool; and third, maintenance of the relative abundance of shRNA molecules during analysis.

Experimentally, Dr. Leake’s team has determined that providing the shRNAs in 500-fold excess yields the best, most consistent results. It is important to remember that in the context of the screen, there will be both high-abundant targets and low-abundant targets.

The shRNAs are packaged in lentiviral particles and delivered to the cells via the normal retroviral process. That means that the transduced shRNA can integrate into the cells’ genome, providing a permanent knockdown of the targeted gene.

Cells transduced with pools of shRNA can be treated with a particular therapeutic agent of interest and screened for specific phenotypes. Cells lacking a response indicate a wild-type phenotype suggesting that shRNAs in those cells target genes that are not involved in the disease state.

In contrast, cells that respond indicate that shRNAs target relevant genes to the disease state. In negative selection screens, shRNAs from responders will either be overrepresented in the final NGS analysis because of cell proliferation, or underrepresented because of cell death. Both outcomes help identify genes involved in the disease-state pathway and the role that they play during therapeutic intervention.

“The power of RNAi is enabling,” said Dr. Leake. “It is much faster and easier to make a knockdown than to create a gene knockout in either a cell line or in an animal model.”

According to Thermo Fisher Scientific, using pooled shRNAs provides a large collection of lentivirus-expressed shRNAs that target thousands of genes in a single well.

Low-Dose Delivery

Xavier de Mollerat du Jeu, Ph.D., senior scientist in the R&D transfection group at Life Technologies, is directing the effort to improve the current set of delivery vehicles used in life science research to deliver siRNA molecules in vivo. The ultimate goal is to develop a delivery vehicle that will enable therapeutic drug delivery.

While the company cut its teeth on transfection reagents that enabled nucleic acid delivery into primary cells and established cell cultures, a completely new formulation is required for efficient siRNA delivery in vivo. The optimal reagent, called Invivofectamine® 2.0, is derived from different lipids and has been shown to deliver siRNA molecules into hepatocytes in the liver following tail vein injections in mice.

Since the launch, the team has discovered new lipids and has been working on optimizing the ratios of the different components as well as mixing parameters. These improvements have resulted in a greater than 50-fold improvement in efficacy; ED50 levels have been reduced to 0.02 mg/kg. This has been a big step in moving from a great tool for research to an effective tool for therapeutics.

These liponanoparticles (LNPs) are not toxic based on liver tox and interferon testing. They are resistant to blood nucleases and significantly smaller than in vitro transfection reagents, so that they can pass through capillaries and into the cellular matrix.

“As a tool provider, we provide the clinical researcher with our Invivofectamine 2.0 reagent as an empty delivery vehicle. We also provide researchers with a protocol to encapsulate their siRNA molecule of interest. This delivery reagent works best for encapsulation of siRNA, miRNA, miRNA mimics, and miRNA antimirs,” indicated Dr. de Mollerat du Jeu.

“With intravenous injection in the mouse tail vein, these loaded particles deliver to the liver and facilitate uptake in hepatocytes. We recommend the use of Factor VII as a positive control for this system. Factor VII is synthesized in the liver and secreted into the bloodstream. Efficacy of delivery and gene expression knockdown is easily monitored by the measurement of Factor VII protein levels from a drop of blood taken from treated vs. untreated mice.

“To reach other organ systems, alternative routes of injection may be required. Preliminary results indicate that intraperitoneal injections enable delivery to the pancreas and the spleen, intracranial injections enable access to the brain, and intraorbital injections enable access to the eye. We have not yet tried it, but we believe that inhalation delivery should enable access to targets in the lung.”

The team is also working on alternative formulations that might facilitate delivery to organs outside the liver following IV injection. For this effort, different lipid formulations are being rationally developed based on all the design elements, including particle size, overall charge, and the ratio of each of the different components in the LNP. Analytical methods are used in vitro to screen for the best candidates. A subset of candidates are then tested in vivo using fluorescent-labeled siRNAs as probes to monitor sites of accumulation and cellular uptake.

Polymer-Based Delivery Technology

Safe and efficient delivery of siRNA to the appropriate target cells is the critical advance needed to enable RNAi therapeutics. The R&D team at Arrowhead Research has been working to achieve this goal with their polymer-based delivery technology, Dynamic Polyconjugates (DPC).

What makes the DPC technology so unique is that these membrane-active polymers can be reversibly masked so that their ability to disrupt the integrity of membranes is inhibited until they reach the acidic environment of endosomes. Within this environment the acid-labile mask is removed and the polymer reveals its net cationic charge that disrupts the endosomal membrane, effectively dumping the siRNA molecules into the cytoplasm where they can interact with the cells’ RNAi machinery—thereby effecting knockdown of gene expression.

“While the DPC delivery system is truly dynamic, upon IV injection, the polymer carries a net-negative charged mask to protect cellular membranes it encounters en route to the liver. But it is kind of a misnomer to call the current DPC structure a conjugate. We find that we no longer need to tether the siRNA to the polymer in order to deliver both to the liver hepatocyte target cells,” shared David Rozema, Ph.D., vp of chemistry.

“This new formulation is much simpler now, and not limited by consideration of the ‘where’ and ‘how many’ siRNA molecules to attach to the polymer to maximize efficacy. This also makes the analytics in CMC manufacturing more straightforward. Current protocols involve simply co-injecting the polymer and the siRNA.”

The elegance of the DPC technology is in its simplicity. By attaching targeting ligands for binding to cell surface receptors separately to the polymer and the siRNA, both the masked polymer and the siRNA molecule accumulate on hepatocytes in the liver. Cellular uptake of both molecules is facilitated by receptor-mediated endocytosis. Then, within the acidic environment of the endosome, the unmasked polymer displays its membrane lytic activity, thereby dumping the siRNA payload into the cytoplasm where it exerts target gene knockdown. Using siRNA directed against Factor VII in proof-of-concept studies, Dr. Rozema said the team has demonstrated that the polymer and the siRNA can be co-injected separately with the same outcome of specific, high-level gene knockdown.

“We will be taking this DPC technology to the clinic. The therapeutic siRNA ARC-520 is directed against hepatitis B virus, a small virus that produces RNAs that all overlap. By virtue of this overlap, the therapeutic siRNA acts to cleave all the viral RNA preventing viral protein production. Further, because HBV replicates through an RNA intermediate, treating infected cells with siRNA directed against that RNA blocks formation of new viral particles,” said David Lewis, Ph.D., vp of biology at Arrowhead.

“We are confident our approach will result in knockdown of viral proteins and block replication. Our goal is to enable robust HbsAg sero conversion in patients. This only occurs in approximately 10% of the cases using current HBV therapies.”

While there is plenty of work to be done focusing on disease states in the liver, including viral infectious disease, metabolic disorders, and cancer, the DPC technology could conceivably be targeted anywhere in the body based on the attachment of cell-specific ligands on the polymer and therapeutic siRNA. Arrowhead Research is investing in this approach as indicated by the acquisition of Alvos Therapeutics, which has developed a clinically validated technology platform that discovers tissue-specific receptor targets and homing peptides.

Arrowhead Research designed its Dynamic Polyconjugates (DPC) technology to deliver siRNA safely and efficiently to target cells.

Self-Delivery RNAi Compounds

At RXi Pharmaceuticals the R&D effort has led to the development of novel RNAi compounds that have drug-like properties. What makes these small hydrophobically modified, asymmetric RNAs truly unique is that they don’t require an additional delivery vehicle for cellular uptake. Hence their name, self-delivering RNAi compounds, or sd-rxRNA.

The sd-rxRNA compounds are hybrids between conventional antisense RNA molecules and RNAi compounds with the best features of both. Like antisense RNA they have good PK/PD profiles while maintaining the highly potent intracellular activity typical of RNAi compounds.

The modifications that have been made to the molecule not only promote cellular uptake (such as hydrophobic modifications) but are also beneficial to protect the molecule from nuclease attack and prevent an immune response. Research has shown that cellular uptake is mediated by endosomal uptake and the RNAi compounds don’t require a receptor-mediated internalization mechanism.

“In the design of the sd-rxRNA molecules, there is a critical balance between the size of the RNA (

“Our results from testing cellular uptake in over 15 different cell lines in vitro showed that regardless of the cell type, whether the cells are primary or established cell lines, adherent or in suspension, all cells tested readily take up the sd-rxRNA based on their structure and show significant gene silencing, greater than 70%.”

RXI-109 was developed based on the novel chemistry of the sd-rxRNA platform. RXI-109 targets and reduces connective tissue growth factor (CTGF), a key regulator of fibrosis, through an RNAi mechanism to prevent overexpression of the protein in response to injury.

“Because CTGF promotes dermal scarring, silencing its overexpression is predicted to minimize scar formation at the site of trauma or surgery,” shared Pamela Pavco, Ph.D., chief development officer. “Our nonclinical toxicology data to date indicates that RXI-109 is tolerated systemically and that reduction of CTGF is not deleterious in the context of wound healing.”

RXI-109 is RXi’s first sd-rxRNA to be advanced into clinical trials and is currently involved in a Phase I trial, a dose-escalation study designed to evaluate safety and tolerability of RXI-109 in humans following intradermal administration. Following proof of concept studies in dermal scarring, the use of RXI-109 as a treatment for other fibrotic indications will be investigated.

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