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

“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.

Tags: capture regent, iPSC, NIDA, RNAi, shRNA

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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Tags: lentivirus packaging, lentiviurs shRNA, RNAi, RNAi screening, shRNA, shRNA cloning, shRNA lentivirus packaging, shRNA library

RNAi Therapy Mediated by Linear DNA Cassettes

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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Tags: Allele RNAi, gene silencing, miRNA, non viral vector, PCR DNA manufacture, pol II promoter, pol III promtoer, RNA interference, RNAi patent, RNAi therapeutics, RNAi therapy, shRNA, siRNA, viral vector

Delivery of RNAi or Cre by Ultrasound-Guided Injection of High Titer Lentiviral Vectors

By Jiwu Wang

According to the Skin Cancer Foundation, skin cancer is the most common type of cancer in the US. Although the skin might seem to be an easy target for gene therapy or RNAi mediated functional corrections, the outer keratinized epithelial cells forms a formidable barrier to delivery of genetic material. The epidermis undergoes rapid turnover, a fact that further complicates gene therapy because gene transfer to skin stem cells would be required for sustained effects.

Before skin gene therapy can be discussed with any practical meaning, a physiologically relevant in vivo model for studying gene function in the context of tumorigenesis and epithelial biology must be established. Studies of gene functions in skin homeostasis in mouse models were mostly performed by labor-intensive knockout methods. Recently, at least two publications have shown that by using ultrasound-guided injection of lentiviruses into amniotic fluids, transgene or shRNA can be efficiently and specifically delivered to epidermis, including skin stem cells, creating a very attractive model for functional studies and therapeutic tests.

Localized injection of high titer lentiviral vectors has been widely used for studying genes in brain development and a few other areas. Instead of injection into animal tissues, Endo et al. injected tiny volume (nl) of high titer lentivirus (10e10 TU/ml) into amniotic cavities within a defined window of embryogenesis [1]. By following fluorescent protein markers (CFP, GFP, YFP, RFP), both Endo et al. and researchers from Elaine Fuchs group demonstrated high efficiency and specificity of delivery to epithelial cells, commonly resulting in multiple genomic insertions of the viral genome.

RNAi against alfa1-catenin was used by Beronja and colleagues as an example to show that loss-of-function analysis can be done rather easily using shRNA/FP bearing lentivirus [2]. nlCre was also delivered to embryos with loxP-flanked transgenes vs wildtype for conditional knockout studies. These new findings should open doors to various experiments and therapies concerning the health of the skin.

1. Endo, M., P.W. Zoltick, W.H. Peranteau, A. Radu, N. Muvarak, M. Ito, Z. Yang, G. Cotsarelis, and A.W. Flake, Efficient in vivo targeting of epidermal stem cells by early gestational intraamniotic injection of lentiviral vector driven by the keratin 5 promoter. Mol Ther, 2008. 16(1): p. 131-7.
2. Beronja, S., G. Livshits, S. Williams, and E. Fuchs, Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos. Nat Med. 16(7): p. 821-7.

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Tags: cre, Fluorescent proteins, in vivo injection, lentiviral vector, lentivirus, loxP, nlCre, RNAi, shRNA, skin cancer, skin care, skin therapy, sunscreen, transgene

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