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Archive for the ‘Oligonucleotide Therapeutics & Delivery’ Category


An Intelligent DNA Nanorobot to Fight Cancer by Targeting HER2 Expression

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

HER2 is an important prognostic biomarker for 20–30% of breast cancers, which is the most common cancer in women. Overexpression of the HER2 receptor stimulates breast cells to proliferate and differentiate uncontrollably, thereby enhancing the malignancy of breast cancer and resulting in a poor prognosis for affected individuals. Current therapies to suppress the overexpression of HER2 in breast cancer mainly involve treatment with HER2-specific monoclonal antibodies. However, these monoclonal anti-HER2 antibodies have severe side effects in clinical trials, such as diarrhea, abnormal liver function, and drug resistance. Removing HER2 from the plasma membrane or inhibiting the gene expression of HER2 is a promising alternative that could limit the malignancy of HER2-positive cancer cells.

 

DNA origami is an emerging field of DNA-based nanotechnology and intelligent DNA nanorobots show great promise in working as a drug delivery system in healthcare. Different DNA-based nanorobots have been developed as affordable and facile therapeutic drugs. In particular, many studies reported that a tetrahedral framework nucleic acid (tFNA) could serve as a promising DNA nanocarrier for many antitumor drugs, owing to its high biocompatibility and biosecurity. For example, tFNA was reported to effectively deliver paclitaxel or doxorubicin to cancer cells for reversing drug resistance, small interfering RNAs (siRNAs) have been modified into tFNA for targeted drug delivery. Moreover, the production and storage of tFNA are not complicated, and they can be quickly degraded in lysosomes by cells. Since both free HApt and tFNA can be diverted into lysosomes, so,  combining the HApt and tFNA as a novel DNA nanorobot (namely, HApt-tFNA) can be an effective strategy to improve its delivery and therapeutic efficacy in treating HER2-positive breast cancer.

 

Researchers reported that a DNA framework-based intelligent DNA nanorobot for selective lysosomal degradation of tumor-specific proteins on cancer cells. An anti-HER2 aptamer (HApt) was site-specifically anchored on a tetrahedral framework nucleic acid (tFNA). This DNA nanorobot (HApt-tFNA) could target HER2-positive breast cancer cells and specifically induce the lysosomal degradation of the membrane protein HER2. An injection of the DNA nanorobot into a mouse model revealed that the presence of tFNA enhanced the stability and prolonged the blood circulation time of HApt, and HApt-tFNA could therefore drive HER2 into lysosomal degradation with a higher efficiency. The formation of the HER2-HApt-tFNA complexes resulted in the HER2-mediated endocytosis and digestion in lysosomes, which effectively reduced the amount of HER2 on the cell surfaces. An increased HER2 digestion through HApt-tFNA further induced cell apoptosis and arrested cell growth. Hence, this novel DNA nanorobot sheds new light on targeted protein degradation for precision breast cancer therapy.

 

It was previously reported that tFNA was degraded by lysosomes and could enhance cell autophagy. Results indicated that free Cy5-HApt and Cy5-HApt-tFNA could enter the lysosomes; thus, tFNA can be regarded as an efficient nanocarrier to transmit HApt into the target organelle. The DNA nanorobot composed of HApt and tFNA showed a higher stability and a more effective performance than free HApt against HER2-positive breast cancer cells. The PI3K/AKT pathway was inhibited when membrane-bound HER2 decreased in SK-BR-3 cells under the action of HApt-tFNA. The research findings suggest that tFNA can enhance the anticancer effects of HApt on SK-BR-3 cells; while HApt-tFNA can bind to HER2 specifically, the compounded HER2-HApt-tFNA complexes can then be transferred and degraded in lysosomes. After these processes, the accumulation of HER2 in the plasma membrane would decrease, which could also influence the downstream PI3K/AKT signaling pathway that is associated with cell growth and death.

 

However, some limitations need to be noted when interpreting the findings: (i) the cytotoxicity of the nanorobot on HER2-positive cancer cells was weak, and the anticancer effects between conventional monoclonal antibodies and HApt-tFNA was not compared; (ii) the differences in delivery efficiency between tFNA and other nanocarriers need to be confirmed; and (iii) the confirmation of anticancer effects of HApt-tFNA on tumors within animals remains challenging. Despite these limitations, the present study provided novel evidence of the biological effects of tFNA when combined with HApt. Although the stability and the anticancer effects of HApt-tFNA may require further improvement before clinical application, this study initiates a promising step toward the development of nanomedicines with novel and intelligent DNA nanorobots for tumor treatment.

 

References:

 

https://pubs.acs.org/doi/10.1021/acs.nanolett.9b01320

 

https://www.ncbi.nlm.nih.gov/pubmed/27939064

 

https://www.ncbi.nlm.nih.gov/pubmed/11694782

 

https://www.ncbi.nlm.nih.gov/pubmed/27082923

 

https://www.ncbi.nlm.nih.gov/pubmed/25365825

 

https://www.ncbi.nlm.nih.gov/pubmed/26840503

 

https://www.ncbi.nlm.nih.gov/pubmed/29802035

 

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CHI’s Second Annual Oligonucleotide Discovery and Delivery conference, March 27-28, in Cambridge, MA

Reporter: Aviva Lev-Ari, PhD, RN

 

 

Oligonucleotide Discovery and Delivery

 

Recent advances in nucleic acid medicinal chemistry and delivery have led to the creation of a new generation of oligonucleotide therapies harnessing chemical modifications and conjugations to improve their stability, bioavailability, specificity and potency. These advances, along with a robust development landscape, and several late-stage clinical products poised for approval, have led to a sharp resurgence of interest in the discovery of oligonucleotide-based therapeutics.

Due to the remarkable success of the Inaugural event, Cambridge Healthtech Institute is delighted to host the Second Annual Oligonucleotide Discovery and Delivery conference, March 27-28, in Cambridge, MA. Join leading oligonucleotide developers and discovery scientists to discuss technological and scientific advances in nucleic acid synthesis, medicinal chemistry and delivery, as well as preclinical and clinical findings.

Preliminary Agenda

 

ADVANCES IN OLIGONUCLEOTIDE THERAPEUTICS

GalNAc-Conjugated siRNAs as a New Paradigm in Oligonucleotide Therapeutics

Muthiah (Mano) Manoharan, Ph.D., Senior Vice President, Drug Discovery, Alnylam Pharmaceuticals, Inc.

Talk Title to be Announced

Melissa J. Moore, Ph.D., CSO, Moderna

siRNA Therapeutics for Extrahepatic Indications: Quark’s Case Study

Elena Feinstein, M.D., Ph.D., CSO, Quark Pharmaceuticals

 

EMERGING NUCLEIC ACID DEVELOPMENT PLATFORMS

Small Molecule Nucleic Acid Hybrids (SMNH) Chemistry Platform

R.P. (Kris) Iyer, Ph.D., Co-Founder, CSO, Spring Bank Pharmaceuticals

Third-Generation Antisense (3GA) Technology: Insights into Mechanism of Action

Reina Improgo, Ph.D., Research Scientist, Idera Pharmaceuticals

 

ADVANCES IN RNA THERAPEUTICS AND DELIVERY

Development of Novel Breakthrough Cancer Therapies Based on the Unique Functions of miRNAs

Roel Schaapveld, Ph.D., CEO, InteRNA

Talk Title to be Announced

William Marshall, Ph.D., President and CEO, miRagen Therapeutics

Novel Strategies for Endogenous mRNA Upregulation

Balkrishen (Bal) Bhat, Ph.D., Vice President, Chemistry, RaNA Therapeutics

 

SYNTHESIS AND MEDICINAL CHEMISTRY

Structure Activity Relationships of Trivalent GalNAc Conjugated Antisense Oligonucleotides

Punit Seth, Ph.D., Vice President, Medicinal Chemistry, Ionis Pharmaceuticals

Solid-Phase Purification of Synthetic DNA Sequences and/or Development of 2′-Hydroxy Protecting Groups for Ribonucleosides and Oligoribonucleotides

Serge Beaucage, Ph.D., Chief, Laboratory of Biological Chemistry, CDER, FDA

 

NOVEL AND EMERGING APPROACHES FOR IN VIVO DELIVERY

Talk Title to be Announced

Anastasia Khvorova, Ph.D., Professor, Molecular Medicine, RNA Therapeutics Institute, University of Massachusetts Medical School

A Novel Nano-Medicine Platform for Oligonucleotide Discovery and Delivery

Art Levin, Ph.D., CSO, Avidity

Development of Lipid-Based Oligonucleotide Delivery Systems

Dmitry Samarsky, Ph.D., CSO, Silence Therapeutics

Self-Delivering RNAi Compounds (sd-rxRNA™) for the Treatment of Ocular Disorders

Pamela Pavco, Ph.D., Chief Development Officer, RXi Pharmaceuticals Corp‬.

Development of Three Generations of Nanoparticles for Novel siRNA Therapeutics

Patrick Y. Lu, Ph.D., President & CSO, Sirnaomics, Inc.

 


 

For more details on the conference, please contact:

Kip Harry
Senior Conference Director
Cambridge Healthtech Institute
T: (+1) 781-972-5454
E: kharry@healthtech.com

For partnering and sponsorship information, please contact:

Carolyn Benton
Manager, Business Development
Cambridge Healthtech Institute
T: (+1) 781-972-5412
E: cbenton@healthtech.com

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Gene Editing with CRISPR gets Crisper

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

 

CRISPR Moves from Butchery to Surgery   

More Genomes Are Going Under the CRISPR Knife, So Surgical Standards Are Rising

http://www.genengnews.com/gen-articles/crispr-moves-from-butchery-to-surgery/5759/

  • The Dharmacon subsidary of GE Healthcare provides the Edit-R Lentiviral Gene Engineering platform. It is based on the natural S. pyrogenes system, but unlike that system, which uses a single guide RNA (sgRNA), the platform uses two component RNAs, a gene-specific CRISPR RNA (crRNA) and a universal trans-activating crRNA (tracrRNA). Once hybridized to the universal tracrRNA (blue), the crRNA (green) directs the Cas9 nuclease to a specific genomic region to induce a double- strand break.

    Scientists recently convened at the CRISPR Precision Gene Editing Congress, held in Boston, to discuss the new technology. As with any new technique, scientists have discovered that CRISPR comes with its own set of challenges, and the Congress focused its discussion around improving specificity, efficiency, and delivery.

    In the naturally occurring system, CRISPR-Cas9 works like a self-vaccination in the bacterial immune system by targeting and cleaving viral DNA sequences stored from previous encounters with invading phages. The endogenous system uses two RNA elements, CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA), which come together and guide the Cas9 nuclease to the target DNA.

    Early publications that demonstrated CRISPR gene editing in mammalian cells combined the crRNA and tracrRNA sequences to form one long transcript called asingle-guide RNA (sgRNA). However, an alternative approach is being explored by scientists at the Dharmacon subsidiary of GE Healthcare. These scientists have a system that mimics the endogenous system through a synthetic two-component approach thatpreserves individual crRNA and tracrRNA. The tracrRNA is universal to any gene target or species; the crRNA contains the information needed to target the gene of interest.

    Predesigned Guide RNAs

    In contrast to sgRNAs, which are generated through either in vitro transcription of a DNA template or a plasmid-based expression system, synthetic crRNA and tracrRNA eliminate the need for additional cloning and purification steps. The efficacy of guide RNA (gRNA), whether delivered as a sgRNA or individual crRNA and tracrRNA, depends not only on DNA binding, but also on the generation of an indel that will deliver the coup de grâce to gene function.

    “Almost all of the gRNAs were able to create a break in genomic DNA,” said Louise Baskin, senior product manager at Dharmacon. “But there was a very wide range in efficiency and in creating functional protein knock-outs.”

    To remove the guesswork from gRNA design, Dharmacon developed an algorithm to predict gene knockout efficiency using wet-lab data. They also incorporated specificity as a component of their algorithm, using a much more comprehensive alignment tool to predict potential off-target effects caused by mismatches and bulges often missed by other alignment tools. Customers can enter their target gene to access predesigned gRNAs as either two-component RNAs or lentiviral sgRNA vectors for multiple applications.

    “We put time and effort into our algorithm to ensure that our guide RNAs are not only functional but also highly specific,” asserts Baskin. “As a result, customers don’t have to do any design work.”

    Donor DNA Formats

    http://www.genengnews.com/Media/images/Article/thumb_MilliporeSigma_CRISPR3120824917.jpg
    MilliporeSigma’s CRISPR Epigenetic Activator is based on fusion of a nuclease-deficient Cas9 (dCas9) to the catalytic histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300. This technology allows researchers to target specific DNA regions or gene sequences. Researchers can localize epigenetic changes to their target of interest and see the effects of those changes in gene expression.

    Knockout experiments are a powerful tool for analyzing gene function. However, for researchers who want to introduce DNA into the genome, guide design, donor DNA selection, and Cas9 activity are paramount to successful DNA integration.MilliporeSigma offers two formats for donor DNA: double-stranded DNA (dsDNA) plasmids and single-stranded DNA (ssDNA) oligonucleotides. The most appropriate format depends on cell type and length of the donor DNA. “There are some cell types that have immune responses to dsDNA,” said Gregory Davis, Ph.D., R&D manager, MilliporeSigma.

  • The ssDNA format can save researchers time and money, but it has a limited carrying capacity of approximately 120 base pairs.In addition to selecting an appropriate donor DNA format, controlling where, how, and when the Cas9 enzyme cuts can affect gene-editing efficiency. Scientists are playing tug-of-war, trying to pull cells toward the preferred homology-directed repair (HDR) and away from the less favored nonhomologous end joining (NHEJ) repair mechanism.One method to achieve this modifies the Cas9 enzyme to generate a nickase that cuts only one DNA strand instead of creating a double-strand break. Accordingly, MilliporeSigma has created a Cas9 paired-nickase system that promotes HDR, while also limiting off-target effects and increasing the number of sequences available for site-dependent gene modifications, such as disease-associated single nucleotide polymorphisms (SNPs).“The best thing you can do is to cut as close to the SNP as possible,” advised Dr. Davis. “As you move the double-stranded break away from the site of mutation you get an exponential drop in the frequency of recombination.”

 

  • Ribonucleo-protein Complexes

    Another strategy to improve gene-editing efficiency, developed by Thermo Fisher, involves combining purified Cas9 protein with gRNA to generate a stable ribonucleoprotein (RNP) complex. In contrast to plasmid- or mRNA-based formats, which require transcription and/or translation, the Cas9 RNP complex cuts DNA immediately after entering the cell. Rapid clearance of the complex from the cell helps to minimize off-target effects, and, unlike a viral vector, the transient complex does not introduce foreign DNA sequences into the genome.

    To deliver their Cas9 RNP complex to cells, Thermo Fisher has developed a lipofectamine transfection reagent called CRISPRMAX. “We went back to the drawing board with our delivery, screened a bunch of components, and got a brand-new, fully  optimized lipid nanoparticle formulation,” explained Jon Chesnut, Ph.D., the company’s senior director of synthetic biology R&D. “The formulation is specifically designed for delivering the RNP to cells more efficiently.”

    Besides the reagent and the formulation, Thermo Fisher has also developed a range of gene-editing tools. For example, it has introduced the Neon® transfection system for delivering DNA, RNA, or protein into cells via electroporation. Dr. Chesnut emphasized the company’s focus on simplifying complex workflows by optimizing protocols and pairing everything with the appropriate up- and downstream reagents.

From Mammalian Cells to Microbes

One of the first sources of CRISPR technology was the Feng Zhang laboratory at the Broad Institute, which counted among its first licensees a company called GenScript. This company offers a gene-editing service called GenCRISPR™ to establish mammalian cell lines with CRISPR-derived gene knockouts.

“There are a lot of challenges with mammalian cells, and each cell line has its own set of issues,” said Laura Geuss, a marketing specialist at GenScript. “We try to offer a variety of packages that can help customers who have difficult-to-work-with cells.” These packages include both viral-based and transient transfection techniques.

However, the most distinctive service offered by GenScript is its microbial genome-editing service for bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). The company’s strategy for gene editing in bacteria can enable seamless knockins, knockouts, or gene replacements by combining CRISPR with lambda red recombineering. Traditionally one of the most effective methods for gene editing in microbes, recombineering allows editing without restriction enzymes through in vivo homologous recombination mediated by a phage-based recombination system such as lambda red.

On its own, lambda red technology cannot target multiple genes, but when paired with CRISPR, it allows the editing of multiple genes with greater efficiency than is possible with CRISPR alone, as the lambda red proteins help repair double-strand breaks in E. coli. The ability to knockout different gene combinations makes Genscript’s microbial editing service particularly well suited for the optimization of metabolic pathways.

Pooled and Arrayed Library Strategies

Scientists are using CRISPR technology for applications such as metabolic engineering and drug development. Yet another application area benefitting from CRISPR technology is cancer research. Here, the use of pooled CRISPR libraries is becoming commonplace. Pooled CRISPR libraries can help detect mutations that affect drug resistance, and they can aid in patient stratification and clinical trial design.

Pooled screening uses proliferation or viability as a phenotype to assess how genetic alterations, resulting from the application of a pooled CRISPR library, affect cell growth and death in the presence of a therapeutic compound. The enrichment or depletion of different gRNA populations is quantified using deep sequencing to identify the genomic edits that result in changes to cell viability.

MilliporeSigma provides pooled CRISPR libraries ranging from the whole human genome to smaller custom pools for these gene-function experiments. For pharmaceutical and biotech companies, Horizon Discovery offers a pooled screening service, ResponderSCREEN, which provides a whole-genome pooled screen to identify genes that confer sensitivity or resistance to a compound. This service is comprehensive, taking clients from experimental design all the way through to suggestions for follow-up studies.

Horizon Discovery maintains a Research Biotech business unit that is focused on target discovery and enabling translational medicine in oncology. “Our internal backbone gives us the ability to provide expert advice demonstrated by results,” said Jon Moore, Ph.D., the company’s CSO.

In contrast to a pooled screen, where thousands of gRNA are combined in one tube, an arrayed screen applies one gRNA per well, removing the need for deep sequencing and broadening the options for different endpoint assays. To establish and distribute a whole-genome arrayed lentiviral CRISPR library, MilliporeSigma partnered with the Wellcome Trust Sanger Institute. “This is the first and only arrayed CRISPR library in the world,” declared Shawn Shafer, Ph.D., functional genomics market segment manager, MilliporeSigma. “We were really proud to partner with Sanger on this.”

Pooled and arrayed screens are powerful tools for studying gene function. The appropriate platform for an experiment, however, will be determined by the desired endpoint assay.

Detection and Quantification of Edits

 

The QX200 Droplet Digital PCR System from Bio-Rad Laboratories can provide researchers with an absolute measure of target DNA molecules for EvaGreen or probe-based digital PCR applications. The system, which can provide rapid, low-cost, ultra-sensitive quantification of both NHEJ- and HDR-editing events, consists of two instruments, the QX200 Droplet Generator and the QX200 Droplet Reader, and their associated consumables.

Finally, one last challenge for CRISPR lies in the detection and quantification of changes made to the genome post-editing. Conventional methods for detecting these alterations include gel methods and next-generation sequencing. While gel methods lack sensitivity and scalability, next-generation sequencing is costly and requires intensive bioinformatics.

To address this gap, Bio-Rad Laboratories developed a set of assay strategies to enable sensitive and precise edit detection with its Droplet Digital PCR (ddPCR) technology. The platform is designed to enable absolute quantification of nucleic acids with high sensitivity, high precision, and short turnaround time through massive droplet partitioning of samples.

Using a validated assay, a typical ddPCR experiment takes about five to six hours to complete. The ddPCR platform enables detection of rare mutations, and publications have reported detection of precise edits at a frequency of <0.05%, and of NHEJ-derived indels at a frequency as low as 0.1%. In addition to quantifying precise edits, indels, and computationally predicted off-target mutations, ddPCR can also be used to characterize the consequences of edits at the RNA level.

According to a recently published Science paper, the laboratory of Charles A. Gersbach, Ph.D., at Duke University used ddPCR in a study of muscle function in a mouse model of Duchenne muscular dystrophy. Specifically, ddPCR was used to assess the efficiency of CRISPR-Cas9 in removing the mutated exon 23 from the dystrophin gene. (Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene and significant enhancement of muscle force.)

Quantitative ddPCR showed that exon 23 was deleted in ~2% of all alleles from the whole-muscle lysate. Further ddPCR studies found that 59% of mRNA transcripts reflected the deletion.

“There’s an overarching idea that the genome-editing field is moving extremely quickly, and for good reason,” asserted Jennifer Berman, Ph.D., staff scientist, Bio-Rad Laboratories. “There’s a lot of exciting work to be done, but detection and quantification of edits can be a bottleneck for researchers.”

The gene-editing field is moving quickly, and new innovations are finding their way into the laboratory as researchers lay the foundation for precise, well-controlled gene editing with CRISPR.

 

Are Current Cancer Drug Discovery Methods Flawed?

GEN May 3, 2016   http://www.genengnews.com/gen-news-highlights/are-current-cancer-drug-discovery-methods-flawed/81252682/

 

Researchers utilized a systems biology approach to develop new methods to assess drug sensitivity in cells. [The Institute for Systems Biology]

Understanding how cells respond and proliferate in the presence of anticancer compounds has been the foundation of drug discovery ideology for decades. Now, a new study from scientists at Vanderbilt University casts significant suspicion on the primary method used to test compounds for anticancer activity in cells—instilling doubt on methods employed by the entire scientific enterprise and pharmaceutical industry to discover new cancer drugs.

“More than 90% of candidate cancer drugs fail in late-stage clinical trials, costing hundreds of millions of dollars,” explained co-senior author Vito Quaranta, M.D., director of the Quantitative Systems Biology Center at Vanderbilt. “The flawed in vitro drug discovery metric may not be the only responsible factor, but it may be worth pursuing an estimate of its impact.”

The Vanderbilt investigators have developed what they believe to be a new metric for evaluating a compound’s effect on cell proliferation—called the DIP (drug-induced proliferation) rate—that overcomes the flawed bias in the traditional method.

The findings from this study were published recently in Nature Methods in an article entitled “An Unbiased Metric of Antiproliferative Drug Effect In Vitro.”

For more than three decades, researchers have evaluated the ability of a compound to kill cells by adding the compound in vitro and counting how many cells are alive after 72 hours. Yet, proliferation assays that measure cell number at a single time point don’t take into account the bias introduced by exponential cell proliferation, even in the presence of the drug.

“Cells are not uniform, they all proliferate exponentially, but at different rates,” Dr. Quaranta noted. “At 72 hours, some cells will have doubled three times and others will not have doubled at all.”

Dr. Quaranta added that drugs don’t all behave the same way on every cell line—for example, a drug might have an immediate effect on one cell line and a delayed effect on another.

The research team decided to take a systems biology approach, a mixture of experimentation and mathematical modeling, to demonstrate the time-dependent bias in static proliferation assays and to develop the time-independent DIP rate metric.

“Systems biology is what really makes the difference here,” Dr. Quaranta remarked. “It’s about understanding cells—and life—as dynamic systems.”This new study is of particular importance in light of recent international efforts to generate data sets that include the responses of thousands of cell lines to hundreds of compounds. Using the

  • Cancer Cell Line Encyclopedia (CCLE) and
  • Genomics of Drug Sensitivity in Cancer (GDSC) databases

will allow drug discovery scientists to include drug response data along with genomic and proteomic data that detail each cell line’s molecular makeup.

“The idea is to look for statistical correlations—these particular cell lines with this particular makeup are sensitive to these types of compounds—to use these large databases as discovery tools for new therapeutic targets in cancer,” Dr. Quaranta stated. “If the metric by which you’ve evaluated the drug sensitivity of the cells is wrong, your statistical correlations are basically no good.”

The Vanderbilt team evaluated the responses from four different melanoma cell lines to the drug vemurafenib, currently used to treat melanoma, with the standard metric—used for the CCLE and GDSC databases—and with the DIP rate. In one cell line, they found a glaring disagreement between the two metrics.

“The static metric says that the cell line is very sensitive to vemurafenib. However, our analysis shows this is not the case,” said co-lead study author Leonard Harris, Ph.D., a systems biology postdoctoral fellow at Vanderbilt. “A brief period of drug sensitivity, quickly followed by rebound, fools the static metric, but not the DIP rate.”

Dr. Quaranta added that the findings “suggest we should expect melanoma tumors treated with this drug to come back, and that’s what has happened, puzzling investigators. DIP rate analyses may help solve this conundrum, leading to better treatment strategies.”

The researchers noted that using the DIP rate is possible because of advances in automation, robotics, microscopy, and image processing. Moreover, the DIP rate metric offers another advantage—it can reveal which drugs are truly cytotoxic (cell killing), rather than merely cytostatic (cell growth inhibiting). Although cytostatic drugs may initially have promising therapeutic effects, they may leave tumor cells alive that then have the potential to cause the cancer to recur.

The Vanderbilt team is currently in the process of identifying commercial entities that can further refine the software and make it widely available to the research community to inform drug discovery.

 

An unbiased metric of antiproliferative drug effect in vitro

Leonard A HarrisPeter L FrickShawn P GarbettKeisha N HardemanB Bishal PaudelCarlos F LopezVito Quaranta & Darren R Tyson
Nature Methods 2 May (2016)
                 doi:10.1038/nmeth.3852

In vitro cell proliferation assays are widely used in pharmacology, molecular biology, and drug discovery. Using theoretical modeling and experimentation, we show that current metrics of antiproliferative small molecule effect suffer from time-dependent bias, leading to inaccurate assessments of parameters such as drug potency and efficacy. We propose the drug-induced proliferation (DIP) rate, the slope of the line on a plot of cell population doublings versus time, as an alternative, time-independent metric.

  1. Zuber, J. et al. Nat. Biotechnol. 29, 7983 (2011).
  2. Berns, K. et al. Nature 428, 431437 (2004).
  3. Bonnans, C., Chou, J. & Werb, Z. Nat. Rev. Mol. Cell Biol. 15, 786801 (2014).
  4. Garnett, M.J. et al. Nature 483, 570575 (2012)

 

Mapping Traits to Genes with CRISPR

Researchers develop a technique to direct chromosome recombination with CRISPR/Cas9, allowing high-resolution genetic mapping of phenotypic traits in yeast.

By Catherine Offord | May 5, 2016

http://www.the-scientist.com/?articles.view/articleNo/46029/title/Mapping-Traits-to-Genes-with-CRISPR

 

Researchers used CRISPR/Cas9 to make a targeted double-strand break (DSB) in one arm of a yeast chromosome labeled with a green fluorescent protein (GFP) gene. A within-cell mechanism called homologous repair (HR) mends the broken arm using its homolog, resulting in a recombined region from the site of the break to the chromosome tip. When this cell divides by mitosis, each daughter cell will contain a homozygous section in an outcome known as “loss of heterozygosity” (LOH). One of the daughter cells is detectable because, due to complete loss of the GFP gene, it will no longer be fluorescent.REPRINTED WITH PERMISSION FROM M.J. SADHU ET AL., SCIENCE

When mapping phenotypic traits to specific loci, scientists typically rely on the natural recombination of chromosomes during meiotic cell division in order to infer the positions of responsible genes. But recombination events vary with species and chromosome region, giving researchers little control over which areas of the genome are shuffled. Now, a team at the University of California, Los Angeles (UCLA), has found a way around these problems by using CRISPR/Cas9 to direct targeted recombination events during mitotic cell division in yeast. The team described its technique today (May 5) in Science.

“Current methods rely on events that happen naturally during meiosis,” explained study coauthor Leonid Kruglyak of UCLA. “Whatever rate those events occur at, you’re kind of stuck with. Our idea was that using CRISPR, we can generate those events at will, exactly where we want them, in large numbers, and in a way that’s easy for us to pull out the cells in which they happened.”

Generally, researchers use coinheritance of a trait of interest with specific genetic markers—whose positions are known—to figure out what part of the genome is responsible for a given phenotype. But the procedure often requires impractically large numbers of progeny or generations to observe the few cases in which coinheritance happens to be disrupted informatively. What’s more, the resolution of mapping is limited by the length of the smallest sequence shuffled by recombination—and that sequence could include several genes or gene variants.

“Once you get down to that minimal region, you’re done,” said Kruglyak. “You need to switch to other methods to test every gene and every variant in that region, and that can be anywhere from challenging to impossible.”

But programmable, DNA-cutting champion CRISPR/Cas9 offered an alternative. During mitotic—rather than meiotic—cell division, rare, double-strand breaks in one arm of a chromosome preparing to split are sometimes repaired by a mechanism called homologous recombination. This mechanism uses the other chromosome in the homologous pair to replace the sequence from the break down to the end of the broken arm. Normally, such mitotic recombination happens so rarely as to be impractical for mapping purposes. With CRISPR/Cas9, however, the researchers found that they could direct double-strand breaks to any locus along a chromosome of interest (provided it was heterozygous—to ensure that only one of the chromosomes would be cut), thus controlling the sites of recombination.

Combining this technique with a signal of recombination success, such as a green fluorescent protein (GFP) gene at the tip of one chromosome in the pair, allowed the researchers to pick out cells in which recombination had occurred: if the technique failed, both daughter cells produced by mitotic division would be heterozygous, with one copy of the signal gene each. But if it succeeded, one cell would end up with two copies, and the other cell with none—an outcome called loss of heterozygosity.

“If we get loss of heterozygosity . . . half the cells derived after that loss of heterozygosity event won’t have GFP anymore,” study coauthor Meru Sadhu of UCLA explained. “We search for these cells that don’t have GFP out of the general population of cells.” If these non-fluorescent cells with loss of heterozygosity have the same phenotype as the parent for a trait of interest, then CRISPR/Cas9-targeted recombination missed the responsible gene. If the phenotype is affected, however, then the trait must be linked to a locus in the recombined, now-homozygous region, somewhere between the cut site and the GFP gene.

By systematically making cuts using CRISPR/Cas9 along chromosomes in a hybrid, diploid strain ofSaccharomyces cerevisiae yeast, picking out non-fluorescent cells, and then observing the phenotype, the UCLA team demonstrated that it could rapidly identify the phenotypic contribution of specific gene variants. “We can simply walk along the chromosome and at every [variant] position we can ask, does it matter for the trait we’re studying?” explained Kruglyak.

For example, the team showed that manganese sensitivity—a well-defined phenotypic trait in lab yeast—could be pinpointed using this method to a single nucleotide polymorphism (SNP) in a gene encoding the Pmr1 protein (a manganese transporter).

Jason Moffat, a molecular geneticist at the University of Toronto who was not involved in the work, toldThe Scientist that researchers had “dreamed about” exploiting these sorts of mechanisms for mapping purposes, but without CRISPR, such techniques were previously out of reach. Until now, “it hasn’t been so easy to actually make double-stranded breaks on one copy of a pair of chromosomes, and then follow loss of heterozygosity in mitosis,” he said, adding that he hopes to see the approach translated into human cell lines.

Applying the technique beyond yeast will be important, agreed cell and developmental biologist Ethan Bier of the University of California, San Diego, because chromosomal repair varies among organisms. “In yeast, they absolutely demonstrate the power of [this method],” he said. “We’ll just have to see how the technology develops in other systems that are going to be far less suited to the technology than yeast. . . . I would like to see it implemented in another system to show that they can get the same oomph out of it in, say, mammalian somatic cells.”

Kruglyak told The Scientist that work in higher organisms, though planned, is still in early stages; currently, his team is working to apply the technique to map loci responsible for trait differences between—rather than within—yeast species.

“We have a much poorer understanding of the differences across species,” Sadhu explained. “Except for a few specific examples, we’re pretty much in the dark there.”

M.J. Sadhu, “CRISPR-directed mitotic recombination enables genetic mapping without crosses,” Science, doi:10.1126/science.aaf5124, 2016.

 

CRISPR-directed mitotic recombination enables genetic mapping without crosses

Meru J Sadhu, Joshua S Bloom, Laura Day, Leonid Kruglyak

Thank you, David, for the kind words and comments. We agree that the most immediate applications of the CRISPR-based recombination mapping will be in unicellular organisms and cell culture. We also think the method holds a lot of promise for research in multicellular organisms, although we did not mean to imply that it “will be an efficient mapping method for all multicellular organisms”. Every organism will have its own set of constraints as well as experimental tools that will be relevant when adapting a new technique. To best help experts working on these organisms, here are our thoughts on your questions.

You asked about mutagenesis during recombination. We Sanger sequenced 72 of our LOH lines at the recombination site and did not observe any mutations, as described in the supplementary materials. We expect the absence of mutagenesis is because we targeted heterozygous sites where the untargeted allele did not have a usable PAM site; thus, following LOH, the targeted site is no longer present and cutting stops. In your experiments you targeted sites that were homozygous; thus, following recombination, the CRISPR target site persisted, and continued cutting ultimately led to repair by NHEJ and mutagenesis.

As to the more general question of the optimal mapping strategies in different organisms, they will depend on the ease of generating and screening for editing events, the cost and logistics of maintaining and typing many lines, and generation time, among other factors. It sounds like in Drosophila today, your related approach of generating markers with CRISPR, and then enriching for natural recombination events that separate them, is preferable. In yeast, we’ve found the opposite to be the case. As you note, even in Drosophila, our approach may be preferable for regions with low or highly non-uniform recombination rates.

Finally, mapping in sterile interspecies hybrids should be straightforward for unicellular hybrids (of which there are many examples) and for cells cultured from hybrid animals or plants. For studies in hybrid multicellular organisms, we agree that driving mitotic recombination in the early embryo may be the most promising approach. Chimeric individuals with mitotic clones will be sufficient for many traits. Depending on the system, it may in fact be possible to generate diploid individuals with uniform LOH genotype, but this is certainly beyond the scope of our paper. The calculation of the number of lines assumes that the mapping is done in a single step; as you note in your earlier comment, mapping sequentially can reduce this number dramatically.

This is a lovely method and should find wide applicability in many settings, especially for microorganisms and cell lines. However, it is not clear that this approach will be, as implied by the discussion, an efficient mapping method for all multicellular organisms. I have performed similar experiments in Drosophila, focused on meiotic recombination, on a much smaller scale, and found that CRISPR-Cas9 can indeed generate targeted recombination at gRNA target sites. In every case I tested, I found that the recombination event was associated with a deletion at the gRNA site, which is probably unimportant for most mapping efforts, but may be a concern in some specific cases, for example for clinical applications. It would be interesting to know how often mutations occurred at the targeted gRNA site in this study.

The wider issue, however, is whether CRISPR-mediated recombination will be more efficient than other methods of mapping. After careful consideration of all the costs and the time involved in each of the steps for Drosophila, we have decided that targeted meiotic recombination using flanking visible markers will be, in most cases, considerably more efficient than CRISPR-mediated recombination. This is mainly due to the large expense of injecting embryos and the extensive effort and time required to screen injected animals for appropriate events. It is both cheaper and faster to generate markers (with CRISPR) and then perform a large meiotic recombination mapping experiment than it would be to generate the lines required for CRISPR-mediated recombination mapping. It is possible to dramatically reduce costs by, for example, mapping sequentially at finer resolution. But this approach would require much more time than marker-assisted mapping. If someone develops a rapid and cheap method of reliably introducing DNA into Drosophila embryos, then this calculus might change.

However, it is possible to imagine situations where CRISPR-mediated mapping would be preferable, even for Drosophila. For example, some genomic regions display extremely low or highly non-uniform recombination rates. It is possible that CRISPR-mediated mapping could provide a reasonable approach to fine mapping genes in these regions.

The authors also propose the exciting possibility that CRISPR-mediated loss of heterozygosity could be used to map traits in sterile species hybrids. It is not entirely obvious to me how this experiment would proceed and I hope the authors can illuminate me. If we imagine driving a recombination event in the early embryo (with maternal Cas9 from one parent and gRNA from a second parent), then at best we would end up with chimeric individuals carrying mitotic clones. I don’t think one could generate diploid animals where all cells carried the same loss of heterozygosity event. Even if we could, this experiment would require construction of a substantial number of stable transgenic lines expressing gRNAs. Mapping an ~20Mbp chromosome arm to ~10kb would require on the order of two-thousand transgenic lines. Not an undertaking to be taken lightly. It is already possible to perform similar tests (hemizygosity tests) using D. melanogaster deficiency lines in crosses with D. simulans, so perhaps CRISPR-mediated LOH could complement these deficiency screens for fine mapping efforts. But, at the moment, it is not clear to me how to do the experiment.

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The late Cambridge Mayor Alfred Vellucci welcomed Life Sciences Labs to Cambridge, MA – June 1976

Reporter: Aviva Lev-Ari, PhD, RN

How Cambridge became the Life Sciences Capital

Worth watching is the video below, which captures the initial Cambridge City Council hearing on recombinant DNA research from June 1976. The first speaker is the late Cambridge mayor Alfred Vellucci.

Vellucci hoped to pass a two-year moratorium on gene splicing in Cambridge. Instead, the council passed a three-month moratorium, and created a board of nine Cambridge citizens — including a nun and a nurse — to explore whether the work should be allowed, and if so, what safeguards would be necessary. A few days after the board was created, the pro and con tables showed up at the Kendall Square marketplace.

At the time, says Phillip Sharp, an MIT professor, Cambridge felt like a manufacturing town that had seen better days. He recalls being surrounded by candy, textile, and leather factories. Sharp hosted the citizens review committee at MIT, explaining what the research scientists there planned to do. “I think we built a relationship,” he says.

By early 1977, the citizens committee had proposed a framework to ensure that any DNA-related experiments were done under fairly stringent safety controls, and Cambridge became the first city in the world to regulate research using genetic material.

 

WATCH VIDEO

How Cambridge became the life sciences capital

Scott Kirsner can be reached at kirsner@pobox.com. Follow him on Twitter@ScottKirsner and on betaboston.com.

SOURCE

How Cambridge became the life sciences capital

http://www.betaboston.com/news/2016/03/17/how-cambridge-became-the-life-sciences-capital/

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CHI’s Inaugural Oligonucleotide Therapeutics & Delivery | April 4-5, 2016 | Hyatt Regency | Cambridge, Massachusetts

Reporter: Aviva Lev-Ari, PhD, RN

Register today and join over 100 of your colleagues. See below

CHI’s Inaugural Oligonucleotide Therapeutics & Delivery | April 4-5, 2016 | Hyatt Regency | Cambridge, Massachusetts

Register | View agenda | Download final brochure

healthtech.com/oligonucleotide/

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Oligonucleotide-based therapeutics have long formed the third major drug development platform, specifically focused on modulating gene expression by targeting RNA or the genome itself. A key distinguishing attribute of nucleic acids as therapeutic agents is their ability to access the “undruggable” space left by small molecules and biologics, allowing drug developers to address a wider range of diseases, and particularly those with limited or no therapeutic options. This has generated significant interest in this field; however, first generation molecules exhibiting potency and safety issues have hindered the potential of oligonucleotide therapies dramatically impacting the drug development landscape. Recent advances in nucleic acid chemistry and delivery to improve stability, bioavailability, specificity and potency are now driving the rapid development and clinical evaluation of a new generation of therapies poised for success.

The Oligonucleotide Therapeutics and Delivery conference, April 4-5, in Cambridge, MA will gather leading drug developers and discovery scientists to discuss technological and scientific advances in oligonucleotide-based therapeutics

Topics Include

  • Advances in Oligonucleotide Therapeutics
  • Synthesis and Medicinal Chemistry
  • G-Quadruplex-Forming Oligonucleotides
  • Cancer Immunotherapy and Combinations
  • Immuno-Oncology and Oligonucleotide Intellectual Property
  • Antiviral Development
  • Advances in RNA Therapeutics and Delivery
  • Delivery to the CNS
  • Novel and Emerging Approaches for In Vivo Delivery

Featured Short Course*

Oligonucleotide Therapeutics: From Discovery to Manufacturing

Sunday, April 3rd | 5:00 – 8:00 pm

Instructor: Dmitry Samarsky, Ph.D., Senior Vice President, Technology & Global Business Development, OliX Pharmaceuticals, Inc.

*separate registration required

Keynote Speaker

Muthiah (Mano) Manoharan, Ph.D., Senior Vice President, Drug Discovery, Alnylam Pharmaceuticals, Inc.

Featured Speakers

Art Krieg, M.D., Founder and CEO, Checkmate Pharma

Dong-ki Lee, Ph.D., Professor, Sungkyunkwan University, South Korea; CEO, OliX Pharmaceuticals

Punit Seth, Ph.D., Executive Director, Medicinal Chemistry, Isis Pharmaceuticals

Here is a partial list of confirmed participants

Company

A STAR

AccuRna Inc

Advanced Cancer Therapeutics

AIC Inc

Alexion Pharmaceuticals Inc

Alexion Pharmaceuticals Inc

Alexion Pharmaceuticals Inc

Alexion Pharmaceuticals Inc

Alexion Pharmaceuticals Inc

Alexion Pharmaceuticals Inc

Alios BioPharma Inc

Alnylam Pharmaceuticals

Alnylam Pharmaceuticals

AM Biotechnologies LLC

Arcturus Therapeutics Inc

Arrowhead Research Corp

Astellas Venture Mgmt LLC

Avidity NanoMedicines

Axolabs GmbH

Bio Path Holdings Inc

Bracco Diagnostics

Brigham & Womens Hospital

Bristol Myers Squibb Co

Brown Univ

Checkmate Pharmaceuticals

Corden Pharma

CureVac GmbH

Dicerna Pharmaceuticals Inc

Dicerna Pharmaceuticals Inc

Dicerna Pharmaceuticals Inc

Dicerna Pharmaceuticals Inc

Eisai Inc

Eisai Inc

Evercyte GmbH

Exicure

George Mason Univ

Guangzhou RiboBio Co Ltd

Harvard Univ

Idera Pharmaceuticals Inc

InteRNA Technologies BV

Ionis Pharmaceuticals Inc

Johns Hopkins Univ

LGC Biosearch Technologies

Matinas Biopharma Inc

Mayo Clinic

MirImmune LLC

MiRNA Therapeutics Inc

Mitsubishi Tanabe Pharma Dev America Inc

MPEG LA

NITTO DENKO Avecia Inc

NITTO DENKO Avecia Inc

Life Therapeutics

Nubad LLC

Nutter McClennen & Fish LLP

Ono Pharma USA Inc

Ono Pharmaceutical

Pfizer Worldwide R&D

Quark Pharmaceuticals

RaNA Therapeutics Inc

RaNA Therapeutics Inc

REPLIcor Inc

Rice Univ

Roche Innovation Ctr Copenhagen

RXi Pharmaceuticals Corp

Sarepta Therapeutics

Sevident Inc

Silence Therapeutics AG

SomaLogic Inc

Sungkyunkwan Univ

The Solution Lab

Tokyo Medical & Dental Univ

Tokyo Medical & Dental Univ

Univ of California Los Angeles

Univ Of Massachusetts

Univ of Miami

Univ of Missouri Columbia

Univ of North Carolina Chapel Hill

Waters Corp

Waters Corp

Waters Corp

Wave Life Sciences

Title

Sr Principal Investigator

Dir

CSO

Dir

Assoc Dir

Assoc Dir Upstream Dev

Dev Scientist I

Dev Scientist II

Development Scientist II

Sr. Research Investigator II

Founder & CSO

Principal Research Assoc

Sr VP Drug Discovery

Dir R&D

CSO & COO

CSO

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Managing Dir

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Instructor

CEO

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CSO

Research Assoc

Scientist I

Sr Dir

Sr Research Assoc

Principal Scientist

Principal Scientist

PhD

CEO

Research Dir

Exec VP Technology & Bus Dev

PhD Candidate

President

CEO

Exec Dir Medicinal Chemistry

Asst Prof

Technical Sales Specialist

CTO

Assoc Prof & Consultant

CSO

Exec VP & CSO

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Proposal Writer

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CEO

CSO

Partner & Patent Attorney

Sr Mgr

Sr Dir

Sr Dir

CSO

CEO

VP Chemistry

CSO & VP

PostDoc

Sr Scientist

Chief Dev Officer

VP Discovery Research

President

Dir Formulation

Dir of Specialty Chemistry

Assoc Prof

Consultant

Prof

Project Asst Prof

Dir

Prof

Prof Microbiology & Immunology

Asst Research Prof

Prof

Sr Bus Dev Mgr

Sr Market Mgr

Sr Scientist

Sr VP & Head

Featured Podcast

The Resurgence of Oligonucleotide Therapeutics:
A Podcast Discussing Challenges, Advances, and an Outlook for Success

Click here to access podcast

SOURCE

From: Oligonucleotide Therapeutics and Delivery conference <richh@healthtech.com>

Date: Tuesday, March 15, 2016 at 1:11 PM

To: Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu>

Subject: You still have time to register, less than three weeks away

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