Take Advantage of Allele’s Essential Virus Grant Program, Have High Titer Virus for Your Research on Essential Human Genes

The number of Allele products in the form of viral particles is increasing quickly. Most of these products are also listed under product groups categorized according to their respective functions (e.g. iPS production under “Induced Pluripotent Stem Cells”, shRNA under “RNA Interference”, cytokines and enzymes under “Recombinant Proteins”). Additional background information, as well as related products, may be found on the landing pages of each product group.

The increase in scientific publications reporting the conversion of “dormant” genes into “active” ones has resulted in a significant demand for cDNA clones, antibodies, expression vectors, etc., all in short order. Providing reagents for the expansion of research on such genes is scientifically important and potentially rewarding if the market catches up. Allele Biotech works diligently to supply the most up-to-date and active researchers with pre-validated viral particles for expressing many of these “hot” genes, (e.g. iPS factors, light-activated ion channels, factors that induce neuronal cells from other cell types). Allele Biotech produces custom-packaged high titer lentivirus and retrovirus using unique technologies, as described under “Services by Category”-> “Viral Packaging”.
Under the Essential Virus Grant Program, Allele Biotech accepts custom viral packaging orders, including cDNA of shRNAs against recently established critical mammalian factors. The process begins with an evaluation of your one page application explaining why the factor can potentially be in demand by other researchers. If the application is accepted, Allele Biotech will waive the charges for the project and consider it an R&D effort. Allele Biotech will also offer it as a shelf product at a much lower cost than custom projects. Interested researchers please submit your applications with your order; our accounting department will promptly forward your request to our review group.

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Tags: cDNA, essential genes, lentivirus, miRNA, promoter reporter, virus packaging

Allele Received Broad Patent on DNA-Expressed RNAi in China

Allele Biotechnology & Pharmaceuticals, a San Diego based private company with associate offices and laboratories in China and distribution channels in 30 countries, was granted a major landmark patent in China in the field of RNA interference (RNAi). The patent CN02828345.7, issued on January 20, 2010, covers compositions of DNA molecules that can be transcribed into RNAi-mediating RNA molecules, including the commonly used shRNA and miRNA-like designs. The patent also grants Allele Biotech rights to the process of introducing such DNA molecules into cells. To induce gene silencing by RNA interference, researchers often bring DNA molecules that encode interfering RNAs into cells via plasmid or viral vectors. The rights to use related technologies for the purposes of completely or partially abolishing gene functions through the mechanism of RNAi are granted to Allele Biotech.

Additional claims include methods of studying gene functions using DNA-encoded RNAi agents, or modifying gene expression profile by introducing gene expression-altering DNA molecules that will induce RNAi. The patent further protects the use of DNA-mediated RNAi in creating cell, animal models, and for curing human diseases. According to a Nov 2009 CreditSuisse analysis on the pharmaceutical market in China (and a number of other reports by JP Morgan as well as Morgan Stanley research, etc.), the drug market in China will double by 2015 and the expected revenues for major pharmaceutical companies are in the billion US dollar range each. Many large drug developers have opened research centers in China. For instance, Novartis just announced a 1.25 billion US dollar investment in Chinese R&D centers, making Shanghai one of its top three global research centers. Roche, Pfizer, JNJ, AZN, Bayer, and LLY also have substantial investments in R&D there. Some of their research teams have plans to use the virus-carried shRNA technologies in oncology and other areas, either as screening/validation tools or as therapeutic candidates. Such activities in China are now under the Allele’s recently granted RNAi patent.

The Contract Research Organization (CRO) industry in Shanghai, Suzhou, and Beijing has seen significant growth in the past few years, benefiting from R&D cost cutting in Western countries and the flow of Western-trained researchers back into China. The focus of the CRO business also shifted from chemical synthesis towards one-stop service, including functional screening and animal testing. The clarification of the RNAi patent landscape by the current granting should make the relevant CRO applications of RNAi more mature. It should also provide both the service and the customer companies with a clear route to licensing and/or collaboration.

Most major biomedical research tool and reagent companies have established themselves in the Chinese market and seen fast-growing revenues due to large funding increases to biomedical research in China. For example, Life Technologies, Promega, Millipore, Thermo Scientific, and Sigma-Aldrich all sell RNAi kits that use DNA template for expressing shRNA in mammalian cells, either by viral infection or DNA transfection. In addition, there are many local companies in China that provide reagent kits as well as services.

The Allele patent specifically states claims on reagent kits that contain shRNA-encoding DNA molecules. While being the first in China’s RNAi market, Allele Biotech manufactures in the United States and sells world-wide a set of RNAi kits in the form of retroviral or lentiviral vectors, plasmids, and linear DNA—all of which have superior design for precise shRNA production. As a matter of fact, Allele Biotech helped introduce the RNAi concept through a series of workshops in major universities in China for 3 consecutive years since 2002, at a time when most biologists had just heard of RNAi.

Allele Biotech intends to fully realize the value of this broad patent by providing opportunities to R&D centers, service providers, and reagent sellers to license at reasonable fees, so that this great technology will continue to be widely used and further developed through original research and investment. Allele Biotech intends to set licensing fees on a sliding scale in several aspects:
–the closer a drug gets to market, the higher the fees;
–the smaller the company, the lower the fees;
–the earlier the license is negotiated within an industry sector, the lower the fees.
Allele’s attorneys in China have already been contacted to start drafting plans for licensing deals and patent rights execution. “While stressing wide access, limiting the number of licenses in China is not completely out of the question. In general we want to grant all-application, non-exclusive, low-cost licenses to many companies to keep the costs affordable.” says Dr. Jiwu Wang, Allele’s CEO and the inventor of the patents. “However, if a dominant player in a particular application area is more interested in some exclusivity, a co-exclusive or conditional exclusive license may be negotiated”.

A brief background about RNAi patents:
–The original Fire and Mello patent claimed double-stranded RNAs longer than 25, eliminating use in most mammalian cells.
–The few other RNAi patents granted in the US, Europe, Japan and other markets so far mostly concern chemically synthesized siRNAs.
–The Tuschl I and II patents, with the latter being frequently mentioned in the news because it has generated hundreds of millions of dollars in licensing fees, concern siRNAs suitable for mammalian cells, but they are either chemically synthesized or processed in cell lysate.
–The Allele patent family includes 3 issued US patents on using RNA polymerase III promoter (e.g., commonly used U6 promoter) for generating RNAi. The core of the Allele patents describes making siRNAs that can be of 19 to 25 basepairs long, which are not covered by the Fire and Mello patent. Further, these transcribed siRNA are not chemically synthesized; therefore, they do not conflict with the Tuschl patents. The Allele patent in China has an even broader field of granted rights, covering any DNA-based gene silencing using double-stranded RNA as intermediates.

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Tags: Alnylam patent, Benitec, gene silencing, ISIS patents, Max Planck, miRNA, RNAi, RNAi patent, shRNA, siRNA, Tuschl patent, UMass Ownership

Choosing siRNA, shRNA, and miRNA for Gene Silencing

RNAi refers to dsRNA-induced gene silencing, a cellular process that degrades RNA homologous to one strand of the dsRNA [1, 2]. The intermediates of long dsRNA-initiated RNAi are double-stranded small interfering RNAs (siRNA), typically 21-23 nucleotide (nt) long. The siRNAs, when introduced into cells, can be used to silence genes in mammalian systems where long dsRNAs prompt protein kinase R (PKR), RNase L, and interferon activities that result in non-specific RNA degradation and general shutdown of protein synthesis [3]. siRNAs can either be chemically synthesized then directly transfected into cells or can be generated inside the cell by introducing vectors that express short-hairpin RNA (shRNA) precursors of siRNAs. The process of shRNA into functional siRNA involves cellular RNAi machinery that naturally process genome encoded microRNAs (miRNA) that are responsible for cellular regulation of gene expression by modulating mRNA stability, translation, and chromatin structures [4].

Chemically synthesized siRNA is the simplest format for RNAi. One of the biggest hurdles for achieving effective RNAi with siRNA is that many cells are difficult to transfect. An RNAi experiment is typically considered successful when the target gene expression is reduced by >70%, a threshold not reachable by many types of cells due to their low transfection efficiency. Another drawback of using synthetic siRNA is the limited duration of post-transfection effects, typically with gene silencing activities peaking around 24 hours, and diminishing within 48 hours [5]. Chemical synthesis of siRNA, which is a service Allele Biotech and Orbigen (now merged under the Allele brand) pioneered and still provides, is expensive on a per transfection basis relative to DNA vector based reagents.

shRNA can be introduced by DNA plasmid, linear template, or packaged retroviral/lentiviral vectors. Using any form of DNA construct, except the PCR template format such as Allele’s LineSilence platform, requires creating DNA constructs and sequence verification; a taxing work load if multiple genes need to be studied. However, once the constructs are made, they can be reproduced easily and inexpensively. It is difficult to directly compare the effectiveness of siRNA versus shRNA on a per molecule basis because RNA polymerase III (Pol III) promoters such as U6 or H1 commonly used to express shRNAs can make thousands of copies of shRNA from a single DNA template. However when both siRNA and shRNA are produced the same way, e.g. synthesized chemically, shRNA is reported to be somewhat more effective [6, 7]. For the goals of this research, the most important advantage using shRNA can provide over siRNA is that it can be carried on a lentiviral vector and introduced into a wide variety of cells.

Similar to the comparison between siRNA versus shRNA, it is also difficult to rank the efficiency of shRNA versus miRNA from published data, partly due to different results from different experimental systems. There have been several reports that showed shRNA can cause significant cell toxicity, especially in vivo such as after injection into mouse brain. It was originally reasoned that highly efficient expression from Pol III promoters might overwhelm the cellular machinery that is needed to execute endogenous RNAi functions such as transporting miRNA from the nucleus to the cytoplasm. It was later found out that even using Pol III promoter to create miRNA could still mitigate the toxic effects of shRNA [8]. Since shRNA and miRNA are processed by endonuclease Dicer before being incorporated into RNA induced silencing complex (RISC), the exact identity of siRNAs produced from a given shRNA or miRNA targeting the same region on the mRNA are not known in most of the earlier studies. By designing shRNA and miRNA to give exactly the same processed siRNAs, Boudreau et al. showed that shRNA is actually more potent than miRNA in various systems [9].

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1. Fire, A., S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, and C.C. Mello, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998. 391(6669): p. 806-11.
2. Hannon, G.J., RNA interference. Nature, 2002. 418(6894): p. 244-51.
3. McManus, M.T. and P.A. Sharp, Gene silencing in mammals by small interfering RNAs. Nat Rev Genet, 2002. 3(10): p. 737-47.
4. Hutvagner, G. and P.D. Zamore, A microRNA in a multiple-turnover RNAi enzyme complex. Science, 2002. 297(5589): p. 2056-60.
5. Rao, D.D., J.S. Vorhies, N. Senzer, and J. Nemunaitis, siRNA vs. shRNA: similarities and differences. Adv Drug Deliv Rev, 2009. 61(9): p. 746-59.
6. Vlassov, A.V., B. Korba, K. Farrar, S. Mukerjee, A.A. Seyhan, H. Ilves, R.L. Kaspar, D. Leake, S.A. Kazakov, and B.H. Johnston, shRNAs targeting hepatitis C: effects of sequence and structural features, and comparision with siRNA. Oligonucleotides, 2007. 17(2): p. 223-36.
7. Siolas, D., C. Lerner, J. Burchard, W. Ge, P.S. Linsley, P.J. Paddison, G.J. Hannon, and M.A. Cleary, Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol, 2005. 23(2): p. 227-31.
8. McBride, J.L., R.L. Boudreau, S.Q. Harper, P.D. Staber, A.M. Monteys, I. Martins, B.L. Gilmore, H. Burstein, R.W. Peluso, B. Polisky, B.J. Carter, and B.L. Davidson, Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proc Natl Acad Sci U S A, 2008. 105(15): p. 5868-73.
9. Boudreau, R.L., A.M. Monteys, and B.L. Davidson, Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs. Rna, 2008. 14(9): p. 1834-44.

Tags: gene knockdown, gene silencing, lentivirus packaging, miRNA, packaging systems, RNAi, shRNA, shRNA packaging, siRNA, virus packaging service

What seems to be going on with RNAi related patents in the US

Reciting Table 1 from Ref 1 and Table 3 from Ref 2:

By the end Nov 2008 it appears that Allele’s patent (US 7,294,504 and 7,422,896) are the only currently granted DNA based RNAi patents. The focus of Allele’s technology is siRNA of 21-23, either in separate sense and antisense strands, or shRNA or miRNA format, thus not covered by the Fire patent or the Kreutzer-Limmer patent. Since these RNAi inducers are not synthesized by chemical reactions, or produced with enzymes or cell lysate in vitro, they do not relate to Tuschl I or II patent groups. Allele Biotech can not guarantee that its interpretation is correct or final by any means; commercial user of any of the related technologies should perform own due diligence.

[1] Charlie Schmidt. March 2007 “Negotiating the RNAi patent thicket” Nature Biotechnology 25 (3): 273-275

[2] Dirk Haussecker. May 2008 “The Business of RNAi Therapeutics” Human Gene Therapy 19: 451-462

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Tags: gene silencing, H1, lentiviral vector, miRNA, PCR cassette, plasmid vector, pol III, retroviral vector, RNAi, shRNA, siRNA, U6