May 15, 2007 (Vol. 27, No. 10)

Growing Diversity of DNA and RNA Therapeutics Are Changing the Game

The therapeutic oligo landscape has certainly become more diverse in recent years. siRNA, siDNA, aptamers, immunostimulatory sequences, and miRNAs have joined the ranks of antisense oligos in developmental pipelines. Thus, defining synthesis, improving purity, and reducing manufacturing costs are growing concerns.

ProNAi Therapeutics ( recently reported positive preclinical findings with its DNA interference (DNAi®) therapeutic PNT2258 for treating multiple types of human cancers in xenograft mice, including evidence of a 3-log knockdown in tumor load when given in combination with rituximab to mice with human non-Hodgkin’s (Burkitt’s) lymphoma. The company plans to file an IND for PNT2258 in August.

The drug is a single-stranded, chemically unmodified phosphodiester DNA oligo directed against the BCL2 locus. It targets an untranscribed region of DNA upstream of the BCL2 promoter, a region of genomic instability. When PNT2258 binds its target, it is believed to cause a break in the chromosomal DNA, blocking transcription and initiating an apoptotic cascade resulting in cell death. ProNAi encapsulates copies of the oligo in a liposome delivery vehicle created using Novosom’s ( Smarticle® technology. The drug particle is delivered intravenously.

Safety studies have shown a good therapeutic window “with an optimal dose of about 10 mg/kg and the ability to deliver up to 75 mg/kg before seeing toxicity,” says Richard Gill, Ph.D., president and CEO of ProNAi.

“PNT-200, which targets the cMYC locus, has shown excellent knockdown of breast cancer cell lines,” says Dr. Gill, but that drug is still a year or so away from the clinic. ProNAi is also studying its DNAi-based therapeutics for the treatment of prostate cancer and melanoma.

The company also launched a cGMP clinical manufacturing campaign intended to produce a half-kilo of drug product for testing in human Phase I trials, according to Robert Forgey, COO of ProNAi. Forgey emphasizes the importance of partnering with the FDA early to lay out process development strategy, agree on analytical methods, and establish target specifications.

In the future, as the drug moves through the clinic, along with the regulatory agencies, ProNAi anticipates that it will show improvements in purity of the drug substance, according to Forgey. On entering the clinic, purity of the drug substance will be >90%, as measured by a reverse-phase HPLC. By the time the drug goes to market, purity will likely be in excess of 95–96%, with no single impurity being >1%. To achieve this, “there needs to be a breakthrough in higher purity synthesis methods,” says Forgey.

For now, though, barring a major innovation in oligonucleotide synthetic chemistry or a revolutionary advance in manufacturing technology, progress toward improved product purity will be realized through ongoing efforts to understand and define synthesis and purification processes and to experiment with modifications to those processes, implementing the ones that improve process performance.

Room for Improvement

Paul McCormac, Ph.D., director of process development at Avecia Biotechnology ( says, “There is ample scope for improvement inside the current chemistry envelope, particularly for RNA-based compounds.”

In addition to optimizing performance and product purity, tweaking the chemistry may also help bring down the cost of oligo production. Process development and optimization can affect the three main drivers of manufacturing costs (other than raw materials)—yield, cycle time, and scale. As cGMP oligo production moves into larger scale, some companies are exploring the potential of solution-phase synthesis for improving yield and reducing costs.

One of the challenges facing therapeutic oligo producers is the absence of regulatory guidance from the FDA, according to Thomas Rupp, bioprocess customer application support specialist at GE Healthcare ( Companies are working on the basis of recommendations and analytics developed by Isis Pharmaceuticals ( during the early days of its antisense oligo program.

The chemistry for synthesizing oligonucleotides on solid supports is more than two decades old, and synthesis protocols for 2´-o-TBDMS RNA chemistry are “as old and established as DNA chemistry,” says Rupp.

Isis Pharmaceuticals paved the way with the FDA for today’s developers of oligo therapeutics, in terms of how to characterize these molecules from the perspective of regulatory review, says Brian Sproat, Ph.D, CSO and general manager of Integrated DNA Technologies ( The company specializes in intermediate-scale synthesis and process transfer to cGMP manufacturing. The FDA “tends to evaluate oligos as small molecules and not really as biologicals,” says Dr. Sproat.

While synthesis technology might not have changed much, the marketplace for therapeutic oligos has undergone a major facelift. Over the past five years, direct investment in therapeutic oligo companies has exceeded $4 billion, and big pharma has inked deals valued at more than $5 billion with the oligo R&D sector, according to Gary Carter, business development and marketing manager, nucleic acids solutions division, Agilent Technologies (

This surge in funding “has created a huge clinical trial pipeline,” says Carter, with the number of therapeutic programs involving oligos now at more than 160, most still in the early stages. For siRNA alone, more than 20 companies are pursuing over 50 publicly announced therapeutic programs.

Demand Up, Prices Down

Looking back, Dr. Sproat recalls prices of RNA building blocks at $1,200/gram in the late 1980s. Since then, the cost of RNA monomers has decreased by a factor of about 40 to the current price of below $30/gram. This is still approximately 5-fold more expensive than typical DNA monomers.

At the IBC Life Sciences’“TIDES” conference, Agilent presented a novel RNA-protecting group technology intended to enhance coupling, increase yield and purity, and reduce the cost of producing RNA monomers.

Demand is another important variable in the cost equation. “There are still no large-volume products in production,” observes Dr. McCormac. When that occurs, the industry will benefit from economies of scale, shared facility space between high-volume and lower-volume compounds with improved distribution of fixed costs, and, hopefully, reduced cost of raw materials. Efforts underway to develop novel, lower-cost, higher-performance solid supports will also likely contribute to reduced cost of goods.

For contract manufacturers, one key to competitive success is to develop flexible assets with variable production capacity—from gram quantities up to multiple kilograms—to be able to support a customer’s needs from small scale through scale-up and to offer the capability to manufacture a broad range of compounds to offset some of the risk associated with drug development and clinical testing.

Avecia is increasingly relying on risk management tools, including risk assessment, process models, and DOE, to reduce risk across the product lifecycle. According to Avecia, this process-driven, increasingly analytical approach to oligo production represents a change in thinking in the industry. It combines engineering and chemistry expertise to develop batch histories and to establish and monitor process specifications.

German CMO BioSpring ( produces oligos for research, diagnostic, and therapeutic use, including both DNA and RNA oligos with various modifications of the backbones, sugars, and bases. Huseyin Ayguen, Ph.D., CSO, describes a growing emphasis among customers on the manufacturer’s analytical capabilities and methods for synthesizing and analyzing side products in order to characterize the full range of a molecule and to provide information about the potential for toxicological side products. Access to qualitative and quantitative information about existing impurities is of greatest importance to customers, says Dr. Aygun.

“We are developing methods for analyzing complex molecules such as duplexes or aptamers,” which have specific secondary structures, Aygun says. Analysis of pegylated aptamers, for example, represents a significant challenge at present.

“A 99% pure oligo, even if it might be technically feasible, would be too expensive,” says Sylvia Wojczewski, Ph.D., CEO of BioSpring.

While oligo producers have to accept a certain amount of impurity in their syntheses, they must understand and be able to characterize these impurities and show that they are not toxic.

Analytics involve primarily HPLC-based methods to characterize the product. According to Forgey of ProNAi, “the technology we need is available.”

Dr. McCormac notes the growing role LC-MS is playing as a support characterization tool to identify process-related impurities and to support process changes and scale-up. One advantage of LC-MS is its ability to detect depurinated DNA species.

The Delivery Dilemma

The synthesis and purification of double-stranded RNAi compounds presents some unique challenges. Most evident is the need to manufacture two separate strands of RNA (and the time and cost required to do this) and allow them to anneal. RNAi duplexes represent a thermodynamically unstable system. In general, RNA is less stable than DNA and at greater risk for contamination on contact with glassware and instrumentation.

Dr. McCormac points to several other obstacles: a less developed raw material supply chain; more expensive building blocks; more complicated chemistry for the isolation processes; enhanced chemical hazard management on scale-up; and more challenging purification processes that require higher pressure chromatography.

The greater complexity of RNA synthesis compared to DNA rests largely on the need for a second deprotection step to reveal the extra hydroxyl group in the sugar.

Aptamers typically range from 25–50 nucleotides in length. They behave similar to mAbs and can exert a therapeutic effect without entering the cell. Aptamers are challenging molecules to synthesize because they are highly structured, with well-defined 2-D and 3-D structures. Additionally, their lengths push the resolution limits of traditional ion exchange chromatography, yielding multiple peaks on chromatographic purification. They also tend to require longer deprotection times.

With the exception of aptamers and immunostimulatory oligos, which do not need to get inside cells to exert a therapeutic effect, delivery of oligo-based drugs to their targets remains a significant problem.

“An unresolved, important question is how to get these molecules into cells,” says Dr. Ayguen of BioSpring. “This is a bottleneck for the whole technology.” Solving this problem would dramatically change the therapeutic oligos business, as it would provide “a platform technology to develop a broad spectrum of drugs and activities.”

“Oligonucleotide therapeutics have always been a good idea,” says Dr. Sproat, “people just didn’t take the delivery issue seriously enough.”

“Nobody has yet cracked systemic delivery,” says James Powell, general manager of the nucleic acid solutions division at Agilent. This has led to a growing focus on local delivery, including topical, intraocular, and inhaled delivery mechanisms, as well as injection of anticancer oligos directly into tumors. Delivery work with nanoparticles and lipid formations is ongoing.

Innovative clinical delivery strategies are contributing to more diverse chemistry, “which requires more flexible manufacturing assets to deal with different downstream chemistry and purification,” says Dr. McCormac, citing examples such as pegylation, RNA cleavage, duplexation, and high pressure/high temperature purification.

A potential side benefit of novel delivery mechanisms might be a reduced need for modified oligos, enabling the use of more natural-looking DNA and RNA in drug formulations.

DNA Therapeutics ( is focusing its efforts on developing oligonucleotide-based therapeutics called short-inhibiting DNA, or siDNA, which interfere with DNA damage-repair mechanisms in cells. The company’s lead compound, Dbait, mimics a DNA double-strand break to “divert and disorganize” the repair system responsible for recognizing and fixing DNA damage on chromosomes, explains Jian-Sheng Sun, Ph.D., chairman and CEO of DNA Therapeutics. The intent of treatment is to compromise the ability of cancer cells to repair the chromosomal damage inflicted by traditional anticancer therapies.

Following up on MRI and cytologic analysis showing that Dbait enhances irradiation-induced necrosis and apoptosis, the company describes molecular studies demonstrating that Dbait specifically activates DNA-PK, a protein kinase that phosphorylates H2AX, an isoform of the histone H2A after exposure of a cell to ionizing radiation. Phosphorylated H2AX (or gamma-H2AX) in a cell nucleus indicates a focus of DNA repair activity to mend double-stranded breakpoints. The distribution of gamma-H2AX in cells transfected with Dbait differs substantially from that observed in irradiated cells without Dbait, leading the company to conclude that Dbait acts by “baiting” and “hijacking” enzymatic repair complexes and causing disorganization of the repair system.

Dbait is a long, single-stranded hairpin oligo measuring 64 nucleotides and, notes Dr. Sun, the main “challenge in GMP manufacturing and scale-up is process development to ensure maximal yield.” Improvements in process development, such as the use of multiple-nucleotide building blocks instead of single nucleotides, “could help increase the overall yield and thus lower manufacturing costs,” says Dr. Sun. As for any oligo therapeutic that has to penetrate cells to exert its effects, including antisense and siRNA drugs, siDNA compounds must also overcome delivery issues, which can impact formulation strategies.

DNA Therapeutics is first focusing on local delivery to serve the interventional oncology field and will then pursue systemic and targeted delivery. The company’s clinical development strategy is to first demonstrate the efficacy of its Dbait technology in indications where standard of care treatment has failed to control cancer progression, and then to focus on other segments of the oncology market.

Scaling-up to Pivotal Trials

Scale-up of oligo synthesis is basically linear. However, Dr. McCormac says, “process control during scale-up is made more of a challenge because the solid phase synthesis is an iterative multistep activity, so any inefficiencies in individual steps are amplified as the steps are repeated. A typical oligonucleotide synthesis can contain 30 different variables for each synthesis cycle and 20-plus cycles.”

Efficient scale-up requires attention to both process chemistry and process engineering. “The challenge is to ensure the equipment is performing similarly as you move through the scales,” Dr. McCormac emphasizes. Avecia constructs scalable process models for each of the unit operations with the goal of applying process development to accurately predict performance on scale-up. The use of scale-down models and DOE strategies sheds light on the potential effects of changes in process variables and aids in assessing process robustness and in optimizing process performance, according to Dr. McCormac.

“Our main challenge is not to develop new methods but to scale up the existing methods,” says Dr. Wojczewski.

“With the exception of the additional 2´ deprotection step required to synthesize RNA, scale-up is pretty much linear,” for both DNA and RNA, adds Rupp.

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