NIDA Branch Chief, Jonathan D. Pollock, Ph.D., Encourages SBIR/STTR Grants on Reagent Kits Including iPSC

 From AlleleBlog:

“We’re interested in areas of genetics, in terms of smoking cessation, pharmacogenomics, treatment of substance abuse, and particularly right now, issues related to prescription substance abuse,” Jonathan D. Pollock, Ph.D., chief of the Genetics and Molecular Neurobiology Research Branch at NIDA’s Division of Basic Neuroscience and Behavioral Research, told GEN.

In addition to that solicitation, Dr. Pollock said, the branch is interested in supporting commercialization and development of products, resources, and services through SBIR/STTR relevant to brain research. They include protein capture reagents, proteomics, genomics, pharmacogenomics, molecular diagnostics, nanotechnology, gene delivery and viral vectors, identification of RNA and DNA sequences in formalin fixed nervous tissue, shRNA, microfluidics, epigenetics diagnostics, therapeutics, and tools to detect epigenetic modifications.

The branch is also looking to support commercialization and development of biomarkers, optogenetics, reagents for iPS and neural stem cells, technologies to uniquely barcode cell types, improved super resolution microscopy methods, in vivo gene expression imaging, automated sectioning, image acquisition and 3D reconstruction of electron micrographic sections, genetically encoded markers for electron microscopy, and “big” genomic and proteomic data, including data visualization, data contextualization, and data analysis.
“What we’re really looking for is products that you could basically commercialize coming out of research. These can be things that are either products or services. I think that there are opportunities, particularly for groups of individuals that have an idea, IP, and want to have a startup company.”
SBIR/STTR grants account for 2.8% of NIDA’s roughly $1 billion annual budget. NIDA spent $26.679 million on SBIR and STTR in fiscal year 2012, which ended September 30—up from $26.497 million in FY 2011. The number of SBIR/STTR research projects grants rose to 56 in FY 2012 from 44 a year earlier, according to the GEN article.

Allele Biotech’s CEO, Jiwu Wang, Ph.D., has worked with Dr. Pollock on a previous, VHH nanobody-related project under the NIDA SBIR program. He has just submitted a SBIR grant application based on Allele’s recently published mRNA-based reprogramming technology, after discussion with Dr. Pollock.

How do you make shRNA-expressing viruses for function screening?

Allele Weekly Blog: http://blog.allelebiotech.com/2011/11/how-do-you-make-shrna-expressing-viruses-for-function-screening/

Most people use standard cloning procedures when trying to insert shRNA templates into lentiviral vectors, i.e. anneal a pair of long oligos with sticky ends and ligate the dsDNA into a linearized plasmid with compatible overhangs. However, since typical lentiviral vector plasmids have terminal repeats and are relatively large, when ligated to hairpin sequence-containing shRNA templates, recombination often occurs inside bacteria that results in smaller plasmids. This problem is common for cloning shRNA or other unstable DNA pieces into viral vectors. This cloning issue is further compounded by the fact that it is difficult to sequence any shRNA template region because the hairpin may block the progress of the DNA polymerase used in sequencing, sometimes requiring several repeats under different sequencing conditions, incurring high costs charged by sequencing service providers.

To deal with these aspects of the cloning difficulties, particularly for the purpose of increasing cloning efficiency RNAi-based screening, we compared three different strategies

First, we built a smaller shRNA cloning vector to clone and sequence shRNA templates prior to transferring to lentiviral vectors. This smaller vector does not have a severe recombination problem and is easier to sequence in the hairpin-containing region. After an initial round of cloning with this new vector, we further improved it by inserting an XbaI and a NheI site between the BamHI and SpeI insertion sites, so that any plasmid preparations can be screened for recombinants by a simple XbaI or NheI digest before sequencing. After cloning into this intermediate vector, the shRNA expression cassette can be transferred into the lentivirus vectors with some flanking viral sequences so that the insert size will be around 1kb.

Second, we developed a novel DNA preparation procedure after realizing that DNA damage during miniprep of vector plasmids and gel purification of vector fragment increased recombination of these constructs, which were already less stable than usual due to hairpin structures. This procedure of DNA preparation avoids UV or guanidium exposure, which can cause nicks on double-stranded DNA and facilitate recombination. This new procedure relies on purifying DNA through surface-binding to regular reaction tubes treated with a proprietary reagent (SurfaceBind Purification). The process simply requires adding a proprietary, guanidium-free binding buffer to the DNA, which has been processed in a specially coated tube (eppendorf or thin-wall PCR tube), and purifying directly in the same tube. Vectors prepared this way indeed provide more colony counts and a higher percentage of correct constructs as shown by our test runs. The procedure also requires less time and the purified DNA can be dissolved in volumes as small as a few microliters.

Third, to enable truly high throughput shRNA screening (i.e. looking for effective RNAi reagents), we further tested and adapted a ligationless cloning protocol that can be handled by a liquid handler almost entirely. In order to increase throughput, we designed a drastically different procedure that could bypass ligation and sequencing altogether before functional tests. Briefly, DNA molecules that would provide enhanced recombination were created by one round of PCR, purified directly in the surface bind PCR reaction tubes (any template DNA would be removed with DpnI enzyme that cuts non-PCR DNA), pooled, and transformed in bacteria directly. DNA plasmids from transformed bacteria can be used for lentivirus packaging, bypassing sequencing at the initial screening stage, and choose single colonies for sequencing only after a shRNA sequence shows promise in functional assays. This is based on the fact that such cloning rarely has any background colonies, and that among all oligos (if using the correct grade of oligos from validated suppliers) inserted this way, a good portion encodes the correct sequence.

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Allele Granted Broad Patent on 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. 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.



For more views and comments by Allele Biotech and responses from others, please visit the Allele Blog on the same topic.



Website http://allelebiotech.com/blogs/

For further information: 858-587-6645, Fax 858-587-6692 RNAi@allelebiotech.com

Choosing siRNA, shRNA, and miRNA for Gene Silencing

http://allelebiotech.com/blogs/2010/02/choosing-sirna-shrna-and-mirna-for-gene-silencing/
...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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For References see original post here please.

Allele Will Receive Its 3rd US Patent On RNAi

On December 1st, 2009 Allele Biotech will be granted its 3rd US patent on using RNA polymerase III (Pol III) for creating RNAi inside mammalian cells. Previously, US patent 7,294,504 was granted to Allele Biotech that covers commercial kits with DNA template components designed for expression of shRNA, miRNA, or siRNA; US patent 7,422,896 further granted claims covering broader designs of using a Pol III promoter such as a U6 promoter for RNAi, including the use of a constitutive or inducible enhancer. The current US patent 7,625,750, protects the use of above technologies in conjunction with arrays, particularly addressable, high density DNA arrays. The RNAi encoding DNA molecules, anchored to the surface through a special peptide, can be transduced via transduction peptide into target cells grown on the array surface. The DNA will then be released from the surface after the completion of DNA transfer by a membrane protease. "This array format for RNAi using the Pol III technology should have higher efficiency and controllability than soft agar embedded siRNAs for transfecting cells, with great potential in large-scale RNAi functional screening and validation. Combined with Allele Biotech’s existing lentiviral vector-based shRNA platform, the addressable RNAi arrays provide us with the best methods available for RNAi screening”, said Dr. Jiwu Wang, CEO of Allele Biotech and the inventor of the patent.



Allele Biotech provides reagent kits and custom services from using its patented technologies in the field of RNAi. The 3 patents issued to Allele Biotech within the past 2 years are so far the only US patents on the methods and compositions of using Pol III promoter for expressing dsRNA-mediated gene silencing. Allele Biotech aims to strengthen its market position by providing superior products and services while actively protecting its intellectual properties. The current strategy includes noting providers and users of existing products that apparently fall under Allele’s patent protection in order to provide reasonable sub-licensing or co-development options.



Allele Biotech is expanding its RNAi capabilities by incorporating the Pol III promoter-driven shRNA cassettes into its popular Phoenix retroviral system and the recently added lentiviral vector system. The RNAi service will also be integrated with Allele’s viral packaging service which offers the best value in terms of per viral particle cost in the market today. High throughput and high content screenings to be conducted at Allele could be further aided by the use of low mutation oligo annealing and wall-less cell array technologies by collaborating with partner companies. In addition, RNAi target design and selection are carried out with an advanced algorism and the most effective empirical rules through Allele’s RNAi services.



Allele Biotech's RNAi technologies were developed with help from the National Institutes of Health (NIH) through several grants. The research team at Allele is currently applying for another NIH project to use these technologies in synthetic lethal screening for cancer therapy. Dr. Wang said that "It is now our goal as well as responsibility to make the RNAi technologies helpful to as many researchers as possible in their pursuit of the best results from gene function studies”.

Effective Concentrations and Effectiveness of siRNA

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.