May 1, 2009 (Vol. 29, No. 9)

Dramatic Rise in RNA Interference Applications Has Been Boon for Sector

Despite the global economic downturn, CMOs and producers of oligonucleotides for research, therapeutic, and diagnostic applications remain optimistic that the healthy growth rate enjoyed over the past few years will continue. While some drug companies may delay development projects until the economy turns around, oligo producers appear confident that they will weather this uncertain period and maintain a steady flow of projects.

Driving this continued growth and optimism is the combination of the maturing market for DNA therapeutics—with several products in or near late-stage development—the growing demand for oligo-based molecular diagnostic products, and the dramatic rise in RNA interference (RNAi) applications in academia and industry, including small interfering RNAs (siRNA), microRNAs (miRNA), and newly emerging RNA classes.

“We have seen an increase in our sales over the same time last year,” says John Bremner, Ph.D., business development director at Link Technologies, which specializes in manufacturing products for the synthesis of modified oligonucleotides. He describes a general increase in demand for both DNA and RNA products, with a notable surge in interest in RNA.

Link is currently developing a number of RNA modifiers and, in Dr. Bremner’s view, “I think we will begin to see modifications becoming more important for RNA.” As projects in industry mature, the need for modified oligos is moving into large-scale production. Dr. Bremner notes a particular interest in amino and cholesterol modifiers, the latter being particularly attractive for cell delivery.

Additionally, as oligo therapeutics move through drug development pipelines, regulatory and safety concerns become paramount. “We are responding to an increasing demand for products that are BSE and TSE encephalopathy-free,” says Dr. Bremner. “Cholesterol is a molecule that could potentially cause problems, and therefore we have now switched to an entirely vegetable source, derived from a Mexican yam.”

Even with the economic slowdown, “we are still seeing heavy demand for oligos,” says Kathryn L. Ackley, Ph.D., director of operations at Girindus America, a manufacturer of oligo APIs for preclinical through commercial-scale applications. This demand spans oligo classes and is particularly notable for oligos with 2´ modifications, conjugated oligos, and siRNA, she notes.

Girindus has the capacity to produce hundreds of kilograms of oligos a year following completion of its $5.2 million expansion program, which doubled its oligo manufacturing space. The company also offers in-house radiolabeling of oligos.


Agilent Technologies has compiled substantial data documenting trends in oligo therapeutics programs. The data demonstrates a more than doubling of the number of programs between 2003 (103) and 2009 (231). Antisense represents the most mature sector, with the largest percentage of products in clinical development, followed by immunostimulatory oligos. Cancer and infectious disease indications account for 48% of therapeutic oligo programs, with cardiovascular, renal, respiratory, and ocular also large segments.

Agilent reports that direct investment in oligo therapeutics rose from $268 million in 2002 to $971 million in 2007, dipping to $682 million in 2008. The total potential deal value of collaborations soared from $7 million in 2003 to more than $6 billion in 2008.

RNA Takes Center Stage

siRNA programs dominate the research and preclinical arena, with antisense and miRNA molecules and aptamers also well represented. The number of clinical RNAi programs rose from 2 in 2004 to 14 at present, with the number of RNAi programs in the research and preclinical stages soaring from 17 to 100 during that same time period.

With the completed expansion of its GMP facility in Boulder, CO, Agilent’s oligo manufacturing capacity has increased to hundreds of kilograms a year, according to Gary Carter, manager of business development and marketing, nucleic acids solutions division at Agilent. “We are seeing more big pharma involvement through an increase in the number and size of collaborations,” he adds. As a result, biotech customers have access to more funding for their oligo programs.

Cost of goods remains a major market driver. Based on cost modeling, current large-scale synthesis processes for DNA are economically viable, according to James Powell, GM, Agilent nucleic acids solutions division. However, raw materials, and especially amidites, are much more expensive for RNA synthesis, and “we need a two- to three-order decrease in cost to make RNA commercially viable,” he says.

The higher costs often associated with novel therapeutics may be justified if the new drugs fulfill an unmet market need. Carter predicts that in the next wave of oligo products, which will include siRNA therapeutics, “we will definitely see more targeted to areas of unmet need.”

As GMP oligo production increases to commercial scale, the main cost driver relates to the building blocks used for nucleic acid synthesis. At the upcoming “TIDES” conference, Agilent will introduce its new ST-RNA (streamlined RNA) technology for therapeutic RNA production, which relies on a more cost-effective, large-scale process.

Applied Biosystems, a division of Life Technologies, continues to add to its portfolio of genomic assay products, which includes TaqMan® Gene Expression assays for real-time PCR, genotyping assays, and the recently launched TaqMan® Copy Number Assays.

In particular, in the gene-expression sector, “the miRNA market has exploded,” says Criss Walworth, director of Applied Biosystems’ expression assays and arrays. miRNA assays were initially being used mainly for mechanistic studies, but with the growing recognition of their potential clinical value, “we are seeing tremendous growth across the board,” notably in pharmaceutical, clinical, and life science research, as well as in biomarker discovery and validation.

“The beauty of miRNA is the manageable number of targets; less than 1,000, compared to 30,000 or more for messenger RNA,” says Walworth. But miRNAs are a challenging target for several reasons: their small size—21 nucleotides, “not much real estate to design primers or probes to”; their homologous nature—many differ by only a single base; and the fact that an miRNA will also have a longer precursor form and a primary transcript, which can result in background noise if an assay is not well designed. This month, Applied Biosystems will introduce 89 sets of mRNA TaqMan assays preplated in optical plates, covering common pathways and target classes.

Other leading providers of oligo products including Dharmacon (a division of Thermo Fisher Scientific), Biosearch Technologies, and Sigma-Aldrich have also expanded their activity in the RNAi sector.

Dharmacon offers a variety of pre-designed and custom siRNA libraries: siGenome for whole-genome screens; On-Target plus, intended to reduce off-target effects; Accell, for siRNA delivery without a transfection reagent; and siSTABLE® stability enhanced siRNA. For shRNA applications Dharmacon technology includes its Open Biosystems microRNA-adapted shRNA libraries targeting human and mouse genomes, and the SMARTvector® shRNA lentiviral platform.

Biosearch offers GMP oligo components for use in in vitro diagnostic genetic assays. The company provides oligo synthesis supports and columns for DNA synthesis and producing modified DNA, Black Hole Quencher® DNA labeling reagents, a range of fluorophore and reporter group supports for 3´ labeling of DNA, and fluorophore and reporter group amidites for 5´ and internal labeling, as well as specialty amidites and 5´ modifiers.

In late March, Sigma-Aldrich announced an exclusive custom oligo supply agreement with Gene Oracle, a gene-synthesis company. The two companies will comarket Gene Oracle’s GeneIOS system and gene synthesis services. Sigma-Aldrich’s customized oligonucleotide services encompass DNA oligos and probes, iScale Oligos™, RNA oligos, RNAtural oligos, and siRNA oligos.

Banking on Diagnostics

Late last year, Eurogentec received ISO 13485:2003 certification for the production facility it opened earlier this year, where it is making oligos for the in vitro diagnostics market. The company is in the process of obtaining ISO 13485:2003 certification for its new DNA and RNA production facility in San Diego. Further expanding its manufacturing capacity, Eurogentec has opened an IVD facility in Japan to provide GMP oligos to the Asian in vitro diagnostics market.

Eurogentec recently introduced its first cGMP Taq DNA polymerase for the molecular diagnostics market. Called Diagnostic Taq, the recombinant enzyme produced in E. coli is more than 98% pure, with low residual DNA content (typically

Avecia OligoMedicine, known for its expertise in large-scale, GMP oligo production, has responded to customer demand and improved its offering in the nonGMP preclinical market for all oligo classes. Demand is high, in particular for RNAi products, which Detlef Rethage, president of Avecia Biotechnologies, describes “as the fastest growing oligo market sector.”

Integrated DNA Technologies (IDT) also views diagnostics as an important growth market. IDT launched its GMP/ISO 1345 manufacturing group in May 2008, and it has been “working with diagnostics players to convert them from legacy methods used for research products to scalable methods appropriate for a more controlled environment,” says Trey Martin, COO.

Integrated DNA Technologies uses proprietary systems and synthesis chemistry to produce custom oligos from 2 nanomoles to 10 g of purified yield.

Emerging Markets

Since opening a 20,000-sq-ft manufacturing facility in Europe last year, and with the acquisition of Belgian oligo producer RNA-TEC, IDT has significantly expanded its global production capacity for both RNA and DNA. Martin points to the technology shift at large genome centers away from traditional Sanger sequencing methods and to high-throughput next-gen  sequencing as a key factor behind IDT’s expansion. “We have seen a lot of interest in highly modified, large-scale oligo preparations for next-gen sequencing applications; driven largely by that and by continued interest in the testing of oligos in vivo, we have doubled our large-scale synthesis and purification capacity.”

Another rapidly expanding area is the synthetic biology market and the demand for gene synthesis. “One of the biggest challenges in synthesizing genes is the assembly of complex fragments that may contain homopolymeric runs or heteropolymeric repeats,” for example, explains Martin.

“If we can push the envelope in synthesizing 400–500 base oligos, we would be able to “synthesize right through those difficult regions.” Producing a functional gene would then require combining only two or three large fragments. IDT currently offers its Ultramer oligos, which can extend up to 200 bases, and the company is working to produce oligos that are 300–400 bases in length.

As demand increases and production methods improve, the cost of synthesizing genes continues to drop and has dipped as low as $0.50/base pair in the marketplace. Martin predicts that declining costs will enable new types of projects that demand large numbers of synthetic genes. For example, a company that wants to improve the activity of a protein of interest would be able to “order the gene with a range of variants that yield amino acid substitutions at any position of interest, or a library of all possible variants, express them all, and test them functionally.”

Quality Up, Costs Down

Dr. Ackley reports that trends in the industry mirror the overall maturation of the oligo pipeline: as clinical candidates progress, GMP requirements become increasingly stringent. Dr. Ackley will speak at “TIDES” on analytical techniques the company has developed for characterizing impurities in oligo preparations. She is seeing a greater desire in the industry to understand what impurities are present in the API. Girindus is pursuing studies to characterize impurities in the raw materials used for oligo synthesis and correlating those to impurities detected in the final product.

Citing a common theme evident at the recent “BioEurope” conference, Rethage agrees that clients are seeking help in modeling the future commercial volume supply chain and cost of goods. Clients with early and mid-stage projects that are looking for funding or licensing partners want evidence—“credible numbers based on real experience”—that they will be able to bring down the cost of goods for APIs in the future.

“We are confident that our price/volume models for the future using new supply chains will be sustainable,” says Rethage. Avecia’s projections draw on its past experience producing its own amidites and leveraging its experience manufacturing a substantial project portfolio. The company has demonstrated, based on process validation experience and the use of statistical modeling in process development, that it can accelerate process validation and reduce the cost by as much as 80%, according to Rethage.

Avecia is investing in additional approaches to reduce costs associated with synthesis, primarily focusing on the installation of an Oracle enterprise resource planning system to increase standardization and automation of internal processes to achieve greater stability and predictability. Other ongoing investments include the implementation of the Pilgrim quality management system to improve and increase the reliability of regulatory compliance, of Corrective and Preventive Actions, audit, and training processes and reduced costs.

Biospring, a German CMO, also reports increased demand from the pharma sector for both DNA and RNA therapeutics, with an emphasis on intermediate- and large-scale quantities. The company is expanding its GMP production capacity by building a second facility at its existing corporate site, according to Hüseyin Aygün, Ph.D., CSO.

Commenting on overall trends in the oligo market sector, Dr. Aygün describes interest from European customers in projects focused on novel therapeutic applications for oligos. He also reports strong demand for better product characterization. In response, Biospring has initiated a program for sequencing highly modified oligos using MALDI mass spectrometry  techniques, which are based on chemical rather than enzymatic fragmentation. MALDI-MS analysis is particularly useful for RNA, including sequencing of duplex RNA.

Defying the economic slowdown, oligo manufacturers are still seeing heavy demand for their products. This phenomenon is likely the result of a myriad of factors including:

  • The number of ongoing oligo therapeutics programs more than doubled between 2003 (103) and 2009 (231).
  • Collaborations with big pharma have created more funding for biotech oligo programs.
  • The emerging synthetic biology field has created a demand for cutting-edge gene synthesis.
  • The burgeoning diagnostics market is increasingly focused on oligo-based tests.
  • As products progress through development, the need for large-scale production escalates.
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