How I started my company and why–Inaugural Event by San Diego Entrepreneurs Exchange (SDEE)

For current graduate students, postdocs, and holders of other “in-transient” positions in bioscience-related fields today, a persistently resounding question on our minds is “What path should I follow at the end of a long and ragged journey of training?” Interestingly in our industry, like downhill skiing you see in the Winter Olympics, once you start one path it is not an easy switch to get on another.

Many of the Ph.D.s in biomed share the general view that an independent research position typically at an academic institute or non-profit organization such as San Diego’s local Salk, Scripps, or Sanford—Burnham, is the goal of the many years of training. Others soon realize that there are numerous research jobs at biotechnology and pharmaceutical companies that will make good use of their expertise, experience, and unique background knowledge in a particular field. And of course there are those who “defect” to different industries that may or may not directly relate to their extensive experience in wet labs, such as working in intellectual property laws, clinical trial management, biomedical sales, business development and management.

Research in major pharmaceutical companies (big pharma) normally focuses on a project with set goals, milestones, and layers of monitoring and management. That is how a large team can function together and get the tasks done in a timely manner. Working in smaller biotech companies can be much more flexible, researcher-initiated, and in many ways fun. On the other hand, you will be required to do much more than reading papers, designing experiments, obtaining and interpreting results. Starting a small biotech company is by no means an easy path to take, but if done correctly with some luck and a lot of determination, it can be a very rewarding career. You will get to utilize to the maximum extent of all your intelligence, knowledge, vision, and personal relations. You also have the opportunity to do real cutting-edge research in various areas, and see the fruits in journal publications, grant awards, as well as in the wild wide market.

The San Diego Entrepreneurs Exchange (SDEE) was founded by local San Diego entrepreneurs in order to provide a voice for the early stage technology startup, to encourage new entrepreneurs, and to sponsor networking and educational events that help develop the skills necessary to bring funding and business to the San Diego area.

The inaugural SDEE event to be held Wednesday March 10th at 5pm. It will help answer some of the questions you may have been thinking about regarding starting or working in a startup biotech company. Allele Biotech’s founder and CEO Dr. Jiwu Wang will be among the speakers. Ten years ago Dr, Wang was a postdoc at UCSD with an NIH fellowship, right before he started Allele with a number of NIH small business innovative research grants. He will talk about the ultimate “academic freedom”–doing any research you want but completely at your own risk– as the reason to start a technology-focused company, and the lessons he learned the hard way about running a lab vs organizing a business. Other speakers include CEOs from a number of San Diego biotech companies with great stories to share with postdocs and others. The talks will be brief yet informative, and on-site interactions are encouraged. The Sanford-Burnham building 12 is outside the main campus, with plenty of free parking. Click here for more details about the event. http://www.allelebiotech.com/allele3/SDEE-First-Event-Announcement.pdf (at AlleleNews). Let us know if you are coming by emailing to events@sdentrepreneurs.org

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Tags: biotech, biotech careers, biotech jobs, biotech startup, pharmaceutical companies, postdoc jobs, research positions., San Diego Entrepreneurs Exchange, SDEE

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.

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