RNA interference (RNAi) has been demonstrated to be a powerful tool to silence gene expression. Therapies based on RNAi are being developed in numerous application areas at fast paces. Although in basic research both expressed and synthetic double-stranded RNA molecules are broadly used to induce gene silencing, synthetic small interfering RNAs (siRNAs) are deemed easier to deliver in preclinical and clinical studies. Compared to synthetic siRNAs, DNA cassettes that express small hairpin RNA (shRNA), microRNA (miRNA), or strands of siRNAs have advantages of prolonged effects.
RNAi-expressing DNA cassettes have been incorporated into viral and non-viral vectors for delivery. Viral vectors for RNAi carry the same risks as those for gene therapies, and are currently not the method of choice for human therapies. Non-viral DNA molecules, often in the form of plasmids, can be easily created and reproduced, but their efficacy is hindered by delivery barriers at the tissue, cell, and the nucleus levels. These difficulties are in part due to the plasmids’ large size, presence of antibiotic resistance genes, and immuresponse-generating CpG islands created in bacteria during propagation.
One way to alleviate these difficulties with non-viral DNA vectors for RNAi is to use linear DNA cassettes. Linear DNAs traverse nucleopores efficiently. The DNA molecules can be conveniently produced by PCR reactions without going through production in bacteria, avoiding DNA modifications such as CpG motifs and the need for replication origin or drug-resistance genes. Linear DNA encompassing a promoter, coding region, and poly(A) signals has been used for protein production. Similarly, by incorporating a miRNA cassette into linear transcription unit driven by a Pol II promoter was used to express RNAi for inhibiting HBV (Chattopadhyay et al. (2009). There are now available technologies and commercial services (e.g. Vandalia Research, Inc.) to produce therapeutic grade linear DNA by specialized PCR reactions.
Allele Biotech’s patents on DNA-expressed RNAi provide a platform for highly express shRNA or siRNA from a DNA molecule as short as fewer than 200 basepairs, potentially more suitable for large scale production, and even more efficient transduction trough tissue, cell membrane, and nuclear pores than the large linear cassettes used by Chattopadhyay et al. A set of experiments similar to the cited HBV studies could quickly lead to the validation of a possibly the most effect way yet for RNAi therapeutics.
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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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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 . 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 .
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 . 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 . 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 .
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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.
Since Tuschl et al. published the first empirical guidelines on how to design effective siRNA , the most significant advancement (based on the understanding of biochemical mechanisms of RNAi such as how RISC is assembled) is the recognition of asymmetric thermostability of the 5’ end of the antisense strand (AS) relative to that of the sense strand (SS) [2, 3]. siRNAs with an A/T-rich AS 5’ end can be more easily integrated into RISC. By biasing against the sense strand for RISC loading, the off-target effects due to the presence of the SS (as one of the sources of off-targets effects) can also be minimized. In recent years datasets of increased number of siRNAs and shRNAs became available and statistical analysis suggested additional rules for RNAi design. These newer rules in general define the siRNA prediction parameters in more detail, for instance, the number of bases of the 5’ ends that should be included when calculating asymmetric thermostability, base preferences at each particular position, and the identity of the 2 nt 3’ overhang [4, 5]. Computer programs and websites are developed based on these features also resulting from NIH funded research through universities and organizations. Among the well-known ones, Design of SIRna (DSIR at biodev.extra.cea.fr/DSIR/DSIR.html) and the shRNA search program at the Broad Institute (broadinstitute.org/genome_bio/trc/publicSearchForHairpinsForm.php) are freely available.
Several companies such as Open Biosystems, System Biosciences, Dharmacon/ThermoFisher, Sigma-Aldrich, Invitrogen/LifeTech, provide premade RNAi reagents against various numbers of human and rodent genes. Although some product lines from these suppliers are labeled as validated RNAi reagents, apparently only one reveals clone sequences and only a few hundred among the claimed 4,500 shRNA clones. It is not possible to find what shRNAs are used against any target gene from most companies even though many of them claim to have a few hundred pre-validated constructs. Some of them may provide additional information upon purchase.
Even with the recent advancement of RNAi design technologies, prediction of effective RNAi is still far from accurate. Depending on the datasets used to score the success rates of the programs at DSIR, Broad or any other software, the general consensus is that about 50% of predicted RNAi target sequences will be effective, resulting in better than 70% gene knockdown. Allele Biotech uses a software that was trained with known RNAi results to predict siRNA target candidates on a given mRNA, and then applies an additional set of rules to pick the most promising candidates. Off-target effects caused by partial-matching between AS strand and untended targets are reduced by searching the chosen site against the NCBI gene base. The basic rules Allele Biotech uses include most currently known ones and are similar to what are listed by The RNAi Consortium (TRC) program at the Broad Institute.
Criteria for RNAi design:
(1) Overall GC content is between 30-55%
(2) The 4 bases at the 5’ of AS is more AT-rich than those of the SS
(3) The first base of AS and SS 5’ is preferably A/T and G/C, respectively
(4) “U” is preferred at the 10th position of the antisense from the 5’ end
(5) “C” is to be avoided as the last base of an overhang
(6) Avoid 4-nt mono-nucleotide regions
(7) Avoid 6-nt GC-rich regions
(8) If possible, do not include those with apparent secondary structures
These selected rules are based on a number of publications (for example, [4-6]), but it is impossible to include all known rules, many of which conflict with each other. In case of conflicting rules we rely more on recent discoveries and our own experience from providing RNAi service during the past 8 years.
Allele Biotech provides RNAi validation and screening services to customers using synthetic siRNA, linear DNA cassettes with engineered Pol III promoter, and shRNA expressing lentiviral vectors in high throughput formats. In a unique design, all RNAi target candidate sequences of a gene transcript are fused consecutively to a bright green fluorescent protein, mWasabi, on a lentiviral vector. Instead of analyzing gene silencing by QPCR, the initial selection of effective RNAi can be performed by measuring fluorescence.
RNAi screening has been conducted to identify correlations between gene functions and cellular phenotypes such as synthetic lethality among DNA damage signaling and repair pathway factors. Successfully performing high throughput screenings requires capabilities of efficient RNAi design, viral packaging, fluorescent proteins, and advanced cell culture and analysis techniques. In addition to these capabilities, Allele’s RNAi services are provided with access to commercial use of Allele’s own patents on Pol III promoter driven shRNA expression, and licensed patents on lentiviral vector, packaging, and fluorescent proteins.
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1. Tuschl, T., P.D. Zamore, R. Lehmann, D.P. Bartel, and P.A. Sharp, Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev, 1999. 13(24): p. 3191-7.
2. Khvorova, A., A. Reynolds, and S.D. Jayasena, Functional siRNAs and miRNAs exhibit strand bias. Cell, 2003. 115(2): p. 209-16.
3. Schwarz, D.S., G. Hutvagner, T. Du, Z. Xu, N. Aronin, and P.D. Zamore, Asymmetry in the assembly of the RNAi enzyme complex. Cell, 2003. 115(2): p. 199-208.
4. Vert, J.P., N. Foveau, C. Lajaunie, and Y. Vandenbrouck, An accurate and interpretable model for siRNA efficacy prediction. BMC Bioinformatics, 2006. 7: p. 520.
5. Zhou, H. and X. Zeng, Energy profile and secondary structure impact shRNA efficacy. BMC Genomics, 2009. 10 Suppl 1: p. S9.
6. Ui-Tei, K., Y. Naito, F. Takahashi, T. Haraguchi, H. Ohki-Hamazaki, A. Juni, R. Ueda, and K. Saigo, Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res, 2004. 32(3): p. 936-48.
RNA oligo is significantly more difficult to synthesize than DNA oligos, mainly because the efficiency of coupling each new ribonucleotide during RNA synthesis is a few fold lower than deoxyribonucleotide during DNA synthesis. Typically, there is an ~10% chance a DNA oligo of 21 bases will have a mutation (most frequently a deletion mutation); for an RNA oligo of 21 bases, as in an siRNA pair, such chance is much higher. Furthermore, after combining the sense and antisense siRNA strands, some RNA molecules will remain as single-stranded thereby not fitting for the RNAi apparatus.
RNA interference is a dose-sensitive process — specificity of gene silencing is meaningful only relative to the active concentration of siRNA used. When the concentration is too low, even the most effective siRNAs would fail to cause gene expression knockdown; when too high, non-specific effects will be duly observed. Therefore, it is essential that the concentrations of siRNAs are measured correctly. When doing so, one must consider not only what the apparent concentrations are by OD260 reading, but also whether the RNA strands are of full-length and whether only dsRNA molecules are counted. This issue might not affect data interpretation if appropriate controls are included in one set of RNAi experiments, but it could have significant influence on conclusions if data from different experiment sets or labs are compared or combined.
HPLC purification currently provides the best means to remove RNA molecules with deletions or remain single-stranded, however, the price tag added by most reagent providers for such treatment has been prohibiting because manufacturers either need to start synthesis at a much bigger scale to obtain promised amount, or they do not promise the delivery quantity at all. The phosphoramidites (oligo building blocks) for RNA synthesis can be 10 times or more expensive than for DNA. Some companies offer alternative purification methods such as a cartridge type device, but they can only remove salt and small impurities, not RNA oligos of shorter lengths accumulated at each cycle of amide coupling. The AllHPLC siRNAs within Allele’s RNAi product line, pre-validated or custom made, are uniformly HPLC purified with 5 OD or 12.5 nmol of double-stranded, annealed siRNA delivered. Allele passes to customers the cost savings from manufacturing our own RNA amidites and other reagents for oligo synthesis. The pre-validated HPLC purified double-stranded siRNA is offered today at $149/12.5 nmol.
Before purchasing siRNAs, even at a low cost of $29 per pair of HPLC purified control siRNA from Allele, researchers still need to consider how well their cells can be transfected. For hard-to-transfect cells, lentiviral vectors carrying a shRNA expressing cassette is often a better choice. To establish stable cell lines, plasmid vectors should be considered. For low cost target screening, the PCR format linear siRNA expression cassettes have advantages.
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