Developing siRNA Therapies: A Technical Forum

Small interfering RNA (siRNA) therapies are a type of RNA-interfering drugs (RNAi) that have shown to be effective at silencing genes in preclinical and clinical studies. Unlike biologically based gene therapy treatments, synthetic siRNA therapeutics can be manufactured under completely controlled conditions. They are considered to be large small-molecule therapies and, therefore, follow the established regulatory pathway for those types of drugs. As clinical trials for siRNA therapies continue to advance toward approvals for this class of drugs, Pharmaceutical Technology asked leading companies about their work in this emerging field.

Representatives from Alnylam Pharmaceuticals (Cambridge, MA), MDRNA (Bothell, WA), and Quark Pharmaceuticals (Fremont, CA) discuss their approaches and techniques for developing and delivering siRNA therapeutics, specifically, liposomal and naked siRNA delivery. Participants in the forum are Mark Tracy, PhD, senior director of pharmaceutical operations at Alnylam Pharmaceuticals; Michael Houston, vice-president of chemistry and formulations at MDRNA; and James Thompson, vice-president of pharmaceutical development at Quark Pharmaceuticals.

PharmTech: What novel approaches is your company taking to deliver siRNA therapies?

Tracy (Alnylam): Dosage forms for systemic delivery of siRNA must meet several important criteria. In particular, they must maintain drug stability, enable transport of the siRNA through the body to the desired organ and cell type(s) within that organ, and, then, allow transport of the siRNA to the cytoplasm within the desired target cells.

Novel lipid nanoparticle (LNP) formulations containing specially designed lipids are one of the few siRNA delivery systems to date that have demonstrated highly potent silencing in vivo of multiple targets in multiple species, including non-human primates. These formulations are able to overcome the delivery challenges described above.

LNP formulations can be designed to achieve silencing of target genes in a variety of cell types in the liver, tumors, and immune cells, among other cell types and tissues. LNP formulations that have advanced to the clinic to date are specifically designed for delivery to cell types in the liver, including hepatocytes and tumor cells.

Houston (MDRNA): MDRNA is developing liposome formulations based on its DiLA² (di-alkylated amino acid) platform. This platform allows MDRNA to create a library of DiLA² molecules, and subsequent formulations for delivery of siRNA, where key characteristics such as charge, fusogenicity, and pH responsiveness can be tailored to meet the delivery requirements associated with specific therapeutic indications. MDRNA has formulations for systemic and local delivery.

Formulations currently in development do not use active targeting moieties, but this is a focused area of our research. The DiLA² platform readily enables the incorporation of peptide, protein, and other classes of targeting ligands. MDRNA has developed a proprietary phage display library based on the [self-folding mini proteins called a Trp cage peptide] as a source of novel, highly specific targeting agents.

Thompson (Quark): We have not devoted our initial efforts toward liposomal delivery. Quark's strategy has been, with synthetic siRNAs, to evaluate what cell types take up the siRNAs and where they distribute after various routes of administration. For example, when you administer our type of siRNAs intravenously, they are rapidly cleared by the kidneys and distribute almost exclusively to the kidney. Because of this, we are focusing one program on kidney indications. We're basically going where the siRNAs go. Formulations allow you to change the distribution and to lower the dose of the siRNA to gain more efficient delivery with certain delivery vehicles. There's always a tradeoff, however, when developing formulations in terms of the cost:benefit ratio.

Manufacturing and administration

PharmTech: How are siRNA therapies manufactured? How stable is the finished dosage form?

Tracy (Alnylam): siRNAs are manufactured as the active pharmaceutical ingredient with standardized chemical synthetic procedures. LNP formulations are produced by mixing defined components of specific lipids and siRNAs to achieve a defined drug product. The final product is stable at refrigerated conditions (2 to 8 °C) for up to two years or longer.

Houston (MDRNA): The drug product, which includes the delivery agents and the siRNA, is manufactured using an impinging stream process whereby the siRNA and delivery agents (DiLA² and other components) are combined forming the initial particles. The intermediate product is incubated for a prescribed time under conditions that favor formation of highly active and physically stable liposomes. The formulations undergo filtration and concentration steps prior to sterile fill–finish steps. The formulation allows the drug product to be frozen and stored as a concentrate. Under these conditions, greater than one year stability at –20 °C has been demonstrated.

Thompson (Quark): Quark's systemically administered drug is formulated at 25 mg/ml in phosphate-buffered saline. In this formulation the drug has exhibited excellent stability for three years at room temperature. Our siRNAs are very well behaved drugs in terms of manufacturing and stability. We're now extending our current stability studies beyond three years, because we haven't had an instance yet where we've fallen out of specification.

PharmTech: How often are the therapies administered? Can a patient take the drug at home or is it administered as an outpatient procedure?

Tracy (Alnylam): The administration frequency depends on the clinical indication and dosing requirements. This could range from once-monthly to bimonthly administration regimens.

Houston (MDRNA): The exact frequency for administration will depend on the indication. However, it is expected the administration schedules for oncology drug products will be similar to established regimens such as once every three weeks. Preclinical studies in models of liver and bladder cancer have confirmed that the inhibition of messenger RNA (mRNA) persists for at least two to three weeks after dose administration. For oncology indications we assume the dosing would be conducted within a clinical setting as expertise in intravenous (i.v.) (in the treatment of liver cancer, for example) or intravesicle (in the treatment of bladder cancer, for example) administration are needed, and drug administration will be a part of overall therapy and monitoring. It is possible that administration of this drug could take place in an outpatient situation because the patient would not be required to remain in the doctor's care after administration of the drug. Future drug products that allow for self-administration to treat indications other than oncology or maintenance therapies in which patients can be adequately trained are also under consideration.

Thompson (Quark): At the moment, all of our programs that are being evaluated at the clinic for siRNAs involve some sort of injection. We have two programs that target the eye because siRNAs can be administered locally through intravitreal administration, which is the standard of care for the diseases we're following such as macular degeneration. The local administration means we can put a large amount of siRNA in a small space that works for days and weeks, resulting in efficient uptake in the target cells. In our systemic program, because siRNAs naturally go to the kidney following i.v. administration, our initial clinical programs are focused on that region. When a nonformulated siRNA is administered into the bloodstream, the molecules do not bind to any plasma proteins. Because of that, they are very rapidly filtered by the kidney, and subsequently the drug is taken up by the kidney's proximal tubule cells.

Liposomal delivery

PharmTech: When the liposomally packaged siRNA drug enters the cell, how is it activated at the appropriate time to perform the appropriate function? What features of the liposome enable the drug to travel to the appropriate place in the body?

Tracy (Alnylam): Once LNPs cross into the cell, they are at first contained within an endosomal vesicle. The cationic or ionizeable lipid component of the LNP is designed to promote disruption of the endosomal membrane to promote release of the siRNA into the cytoplasm of the cell. Once the siRNA is in the cytoplasm, it is able to associate with the cellular RNAi machinery to enable RNA interference for the desired target.

Houston (MDRNA): DiLA²-based liposomes are designed to carefully balance several properties to enable successful delivery of the siRNA in the cytoplasm. Key properties include: the ability to interact with a cell to initiate internalization, or endosomal uptake; fusion with the endosome to enable endosomal escape; and release of the siRNA in the cytoplasm. These features can be designed into a specific delivery component(s), or they can be engineered into the liposome structure. Interaction with a cell surface leading to endosomal internalization is characteristically driven by nonspecific properties and is primarily manifested by a slight cationic charge on the liposome surface. Endosomal release is typically achieved by fusion of the liposome with the endosomal membrane, and requires pH-responsive characteristics. The interaction of the liposome and the endosomal membrane, in response to the pH gradient in the endosome, is usually based on the pKa of the liposome components and can be designed into the DiLA² molecule. Ultimate release within the cytoplasm is also achieved by charge properties, again the pKa, that allows for neutrality and loss of the electrostatic attraction between a positively charged component and the negatively charged siRNA.

On the horizon

PharmTech: Looking forward, do you foresee having the ability to target various tissues and areas of the body?

Tracy (Alnylam): Yes. We and others have made significant progress over the last few years in developing delivery systems for siRNA and in understanding mechanisms of siRNA delivery. The progress made in the field in a short time has given us great optimism that we will continue to significantly broaden the target cells and tissues addressable by RNAi delivery technology.

Houston (MDRNA): The ability to target organs besides the liver via systemic delivery is a central goal for MDRNA. Given the nature of the liver to filter out particles from the systemic circulation, it will be a matter of biasing delivery away from the liver and into other organs. Physical/chemical characteristics such as size and apparent charge, and careful choice of targeting ligand may allow for this biasing and effective distribution to other tissue.

For our bladder program, dosing occurs via direct instillation into the bladder; this is more of a topical delivery approach with the intent to localize and maintain the siRNA within the bladder tissue. The goal for local delivery to other sites such as lung or skin, would focus on maximal uptake at the site of administration, and the formulation would need to be tailored to provide the characteristics most appropriate for the target cell type.

Thompson (Quark): Our near-term, newer programs focus on opportunities where we can obtain effective delivery of the siRNAs without complex formulations. We are concentrating on disease indications such as the lung, eye, and kidney where we can get effective delivery and activity of these molecules without any sort of complicated formulation. We are, however, looking at formulations, and we will consider those further downstream, but at the moment it is more cost-effective (i.e., from a cost-of-goods perspective) to use nonformulated forms of the drug. Looking forward, we at Quark are working on noninvasive routes of administration (e.g., inhalation or topical) so that patients can self-administer the drug without involving injection.