Genetic Engineering & Biotechnology News

MAY1 2015

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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18 | MAY 1, 2015 | | Genetic Engineering & Biotechnology News See CRISPR on page 20 that appeared in Nature Biotechnology. "The method robustly detects DNA DSBs generated by engineered nucleases across the human genome based on their transloca- tion to other endogenous or ectopic DSBs," the article read. "HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot numbers for given nucleas- es that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously character- ized nucleases more than 10-fold." When HTGTS was used to compare Cas9 nuclease and Cas9 paired nickases, paired nickases showed reduced off-target activity. Paired nickases were also assessed by scien- tists at Sigma-Aldrich. "We compared paired nickases to Cas9- FokI nucleases. Paired nickases have about a 10-fold increase in design density, the number of nucleases that target a specifc sequence in the selected area," commented Gregory Da- vis, R&D manager, molecular biotechnology. "The more nuclease options, the better the chances of fnding an active one near site-re- stricted locations such as disease single-nucle- otide polymorphisms (SNPs)." Like other companies, Sigma-Aldrich is evaluating methods to boost homologous recombination (HR) rates and inhibit NHEJ. Small molecules are being investigated, along with components of the DNA repair ma- chinery such as mRNAs for RAD proteins. Enhancement techniques offer some improve- ment, but those improvements are not univer- sally applicable to all cell types. The company recently introduced a nu- clease-based kinase knockout lentiviral li- brary, but the challenge is increasing library screening effectiveness for cancer cell lines, which typically demonstrate some level of polyploidy. When the Cas9 nuclease library on the A459 lung cancer cell line was evalu- ated, a target diploid gene responded with a robust knockout, yet an expected knockout response for another gene was not seen. That particular gene turned out to be tetraploid. Epigenetically based activators and inhibi- tors may be another approach, and the com- pany is considering CRISPR-based gene regu- lation for inhibition or activation, CRISPRi or CRISPRa. Gene regulation may better simu- late drugs that suppress activity and prove more effective than the nuclease-knockout method in lentiviral screening applications. Measurement Systems HDR and NHEJ editing events generally occur at low frequencies, necessitating ultra- sensitive techniques for detection and quan- tifcation of edited alleles. While some stud- ies have relied on NGS, a next-generation PCR technology called droplet digital PCR (ddPCR) is providing researchers with rapid, low-cost, ultrasensitive quantifcation of both NHEJ and HDR editing events. ddPCR has already been widely used for high-sensitivity and high-precision applica- tions such as rare cancer mutation detection and copy number analysis, noted Jennifer Berman, Ph.D., staff scientist, Digital Biol- ogy Center, Bio-Rad Laboratories. Since HDR and NHEJ editing events can occur at very low frequency (<1%), especially HDR in primary or induced pluripotent stem (iPS) cells, ddPCR appears to be a ft for re- searchers wanting a rapid, sensitive, quantita- tive readout of editing in cells and tissues. The technique also enables empirical validation of guide RNA effciency and measurement of the ratio of HDR:NHEJ at a targeted locus. "ddPCR is one of the frst sophisticated measurement systems for genome editing. The other option is sequencing, which is time-consuming, expensive and out of reach for most people," explained Bruce Conklin, M.D., a senior investigator at the Gladstone Institute of Cardiovascular Disease and a professor of medicine at the University of California, San Francisco. The Conklin laboratory works with iPS cells and is primarily interested in HDR, which is typically less than 1% of total alleles. A recent Nature Methods article by the group was the frst demonstration that the genome could be changed one base at a time without any mark of an antibiotic reselection marker, a scarless replacement. Populations of cells that have a very rare cell with a single-base change are isolated using ddPCR as a measurement tool, then enriched sequentially, until a pure clone results, in a method termed sib-selection. "With our method, you can see if the mutation you want is there from the start," asserted Dr. Conklin. "Single base changes cause many human genetic diseases. To fg- ure out the problem, you want to be able to change one thing and see what happens." "We are also looking at ddPCR to quan- tify HDR and NHEJ simultaneously to iso- late conditions where there is more HDR than NHEJ," he added. "Conditions are different in every cell type, for each location, and we do not understand the rules." Application to Animal Models Mouse models have the potential to quickly screen and build a causal relationship between sequence variations in humans and their phe- notypes. Historically, either pronuclear injec- tion of a transgene into a mouse embryo or conventional gene targeting using embryonic stem (ES) cells produced new models. In 2013, a study led by Rudolph Jaenisch, M.D., a professor of biology at MIT and a founding member of the Whitehead Institute for Biomedical Research, culminated in a pub- lished work that was the frst to describe a CRISPR/Cas-engineered animal species. CRIS- PR's ability to engineer targeted mutagenesis in the genome directly on the zygotes circumvents the need for germline-competent ES cells, and appears to result in more predictable models in a fraction of the previous time and cost. CRISPR Continued from page 1 > NanoString, MD Anderson Enter Gene and Protein Expression Profling Partnership The University of Texas MD Anderson Cancer Center and NanoString Technologies entered a multiyear col- laboration to accelerate the development and adop- tion of a new type of assay based on NanoString's nCounter®Analysis System. The collaboration will involve the development of "multi-omic" assays, which simulta- neously profle both gene and protein expression, with a primary focus on identifying important biomarkers in the feld of immuno-oncology as well as extending pro- grams for targeting therapeutics. nCounter is an automated platform that utilizes a novel digital barcoding chemistry to deliver high-preci- sion multiplexed assays across a number of research ap- plications. It uses color-coded molecular barcodes that can hybridize directly to many diferent types of target molecules. "Our current PanCancer Pathway and Immune Pro- fling gene-expression panels ofer researchers power- ful assays for understanding the tug-of-war between the drivers of tumor growth and the immune sys- tem's response," said Brad Gray, president and CEO of NanoString. "In collaboration with experts at MD Ander- son, we will be adding a new proteomic dimension to these assays, and aiming to demonstrate their potential to inform drug development and selection." Key objectives of the collaboration include devel- opment of new multi-omic assays and signatures that profle key oncology disease pathways and immune re- sponse from tumor tissue; incorporation of these multi- omic assays into select clinical studies being run at MD Anderson to predict response and monitor response to cancer immuno-therapies and targeted therapies, both as single agents and combinations; and identifcation of clinically actionable proteomic markers across multiple tumor types. > BMS, uniQure Launch Up-to-$1B+ Gene Therapy Collaboration Bristol-Myers Squibb (BMS) will gain exclusive access to uniQure's gene therapy technology platform for multiple targets in cardiovascular diseases, under a collaboration that could net the Dutch gene therapy developer more than $1 billion, the companies said. The companies agreed to col- laborate on up to 10 targets—including uniQure's lead gene therapy program for congestive heart failure. The program is intended to improve clinical outcomes for patients with reduced ejection fraction by restoring the heart's ability to synthesize S100A1, a calcium sensor and master regulator of heart function. uniQure will lead discovery eforts and be responsible for manufacturing of clinical and commercial supplies using its vector technologies and its industrial, insect-cell based manu- facturing platform. The parties have also inked a supply con- tract, under which uniQure will undertake manufacturing of all gene therapy products under the collaboration. BMS will lead development and regulatory activities across all programs, shoulder all R&D costs, and be solely responsible for commercialization of all products from the collaboration. In return, BMS will comply with an agreed-upon payment structure, providing uniQure about $100 million short-term; payments tied to research, development, and regulatory mile- stones equaling up to $254 million for S100A1; and up to $217 million for each other gene therapy product to be developed through the collaboration. n OMICS News GENOMICS & PROTEOMICS Sigma-Aldrich research scientists Greg Davis, Ph.D., and Fuqiang Chen, Ph.D., discuss genome- editing technologies and best practices. Droplet digital PCR (ddPCR), a next-generation polymerase chain reaction technology from Bio -Rad Laboratories, can provide rapid, low-cost, ultra-sensitive quantifcation of both NHEJ- and HDR-editing events.

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