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Genetic Engineering & Biotechnology News | GENengnews.com | SEPTEMBER 1, 2016 | 37 a breakthrough understanding of the natu- ral role of miRNA in mammalian systems and to a new approach of using rAAV vec- tors to deliver synthetic miRNA "genes" capable of continually expressing miRNA with a sequence specificity tailored to a particular gene target. This has recently allowed for allele-specific augmentation and knockdown, meaning that a normal version of a gene can be expressed simulta- neously with a miRNA designed to knock down the offending mutant version. One example of this approach was re- cently demonstrated for alpha-1 antitryp- sin deficiency (AATD) in which liver dis- ease is caused by a toxic gain of function of the mutant protein while lung disease is caused by the lack of the normal transgene product in the circulation. Genome-Editing Tools The ability to "fix" offending genes (rath- er than augmenting expression from adding additional gene copies or blocking expres- sion of endogenous genes with the RNAi pathway) remains the holy grail of genetic approaches to therapy. While some early efforts at homologous recombination showed promise ex vivo in select circumstances, the true break- through in genome editing came with the discovery of the CRISPR/Cas9 as a host- defense pathway in bacteria such as S. pyo- genes, S. aureus, and N. meningitides. As with RNAi, the key to its success is the combination of sequence-specificity of targeting with the amplified efficiency of an enzymatic mechanism. This system has been harnessed in the form of a read- ily available technology platform in which delivery of a synthetic short-guide RNA (sgRNA) is combined with delivery of a Cas-like enzyme, allowing for the efficient creation of double-strand breaks (DSB) in the gene of interest (Figure 2). If the desire is simply to knock out the gene, host DNA repair mechanisms will generally repair the DSB with the loss or addition of two to six nucleotides resulting in a small insertion and/or deletion (indel), which will then lead to a frame-shift and termination. If the desire is to actually repair the gene of interest, a ds-DNA template for homology-dependent recombination (HDR) is also supplied along with the sgRNA and the Cas enzyme. The creation of DSBs greatly enhances the efficiency of HDR at the locus and a remarkably high number of recombination events produce intended final sequence. The application of this technology to the treatment of human disease is in its infancy. Specific therapeutic approaches have been demonstrated for genetic diseas- es of the liver such as heredi- tary tyrosinemia due to FAH deficiency. The ability to ef- ficiently deliver all needed components simultaneously and to limit the location and duration of the DNA editing activity remain challenges, but the potential of this ther- apeutic approach is clearly enormous. New Viruses, New Vistas Viruses remain the ultimate gene trans- fer machines of nature. Evolved for count- less millennia along with their hosts, par- ticularly mammals such as humans, viruses have natural means to insert genes into host cells, in most cases with the intent of allowing those host cells to survive long enough to propagate both virus and host. AAVs are undoubtedly the simplest and ar- guably the most elegant viral co-travellers that humans possess. At times described as "Almost A Virus" and "An Amazing Virus", the simplicity and ubiquity of AAVs in humans form a logical basis for understanding why this platform has been the first to achieve suc- cessful licensure in Europe and is likely to do so this year in the U.S. The explosion of applications of rAAV in human diseases has been exceeded by the explosion of new AAVs that have been discovered as potential platforms for the delivery of genes, synthetic miRNAs and CRISPR/Cas9-related systems. Beginning with the pioneering work of Gao and Wil- son, AAV has come to be understood as an incredibly diverse set of viruses in hu- mans and other mammals. There is strong evidence for ancient AAVs that have co- evolved with humans and retain the ability to transfer genes and have them persist for life, causing minimal damage to the host. The improved understanding of AAV structure, function, and evolution may prove to be a rich treasure trove of tools for gene transfer going forward for the re- mainder of the 21st century. Muse® Cell Analyzer—the simplest platform for outstanding analysis: • Mix-and-read, fully optimized assays • Flow cytometry expertise is not required • Integrated touchscreen with an 8 in x 10 in footprint Measure essential cellular parameters including count, viability, apoptosis, cell cycle and signaling—effortlessly, for every cell. Make your mark with simple, affordable flow cytometry—now at your side. Muse ® Cell Analyzer EMD Millipore, Muse and the M logo are registered trademarks of Merck KGaA, Darmstadt, Germany. BS-GEN-15-11179 03/15 ©2015 EMD Millipore Corporation, Billerica, MA USA. All rights reserved. EMD Millipore is a division of Merck KGaA, Darmstadt, Germany Make your mark. www.emdmillipore.com/muse TRANSLATIONAL MEDICINE > Alzheimer's Disease vTv Therapeutics reports that azeliragon, an orally administered small- molecule antago- nist of the receptor for advanced glycation endproducts (RAGE), given at 5mg/day, was effective in delaying the time to cognitive deterioration when compared to placebo in patients with mild Alzheimer's disease. "Azeliragon is the only treatment in ad- vanced development that works by inhibiting RAGE, which is believed to promote Aβ ac- cumulation, tau hyperphosphorylation, and chronic inflammation, the leading patholo- gies in Alzheimer's disease," commented Larry Altstiel, M.D., Ph.D., chief medical officer. > Cutaneous T-Cell Lymphoma Officials at Takeda Pharmaceutical and Se- attle Genetics announced that the Phase III ALCANZA trial evaluating ADCETRIS (bren- tuximab vedotin) in patients with cutaneous T-cell lymphoma (CTCL) met its primary end- point, demonstrating a highly statistically significant improvement in the rate of objec- tive response lasting at least four months. This randomized trial compared the use of single-agent ADCETRIS to a control arm of investigator's choice of standard therapies, methotrexate or bexarotene, in 131 patients with CD30-expressing CTCL who received prior systemic or radiation therapy. > Multidrug-Resistant Bacterial Infections Tetraphase Pharmaceuticals initiated pa- tient dosing in a Phase I trial for intravenous TP-6076, an antibiotic candidate being developed for the treatment of serious and life-threatening bacterial infections. "TP-6076 is the lead candidate selected from our second-generation discovery pro- gram because it has demonstrated potent activity against difficult-to-treat Gram- negative pathogens in preclinical studies, including carbapenem-resistant Enterobac- teriaceae and carbapenem-resistant Acineto- bacter baumannii (CRAB)," said Patrick Horn, chief medical officer. > Non-Small-Cell Lung Cancer Bristol-Myers Squibb (BMS) reported that its marketed drug Opdivo® (nivolumab) failed a Phase III trial assessing its effec- tiveness as a monotherapy in patients with previously untreated advanced non-small-cell lung cancer (NSCLC). BMS said Opdivo missed its primary endpoint in the Phase III CheckMate -026 trial, namely progression-free sur- vival in treatment-naïve NSCLC patients whose tumors expressed programmed death ligand 1 (PD-L1) at ≥5%. > Invasive Candidiasis Scynex discussed data from an interim analysis of its Phase II study evaluating the pharmacokinetics, safety, and tolerability of its glucan synthase inhibitor, SCY-078, as an oral step-down treatment in patients initially treated with intravenous echinocandin ther- apy for invasive Candida infections. A once daily oral dose of 750 mg of SCY-078 was identified as the dose that achieves the tar- get exposure with favorable safety and toler- ability in patients with invasive candidiasis. SCY-078 was safe and well tolerated. The frequency of adverse events (AEs) was similar among all treatment groups. The most com- monly reported AEs in the study were gas- trointestinal (GI) such as diarrhea, abdominal pain, nausea, and vomiting. n News Clinical Trials The ability to "fix" offending genes (rather than augmenting or block- ing expression) remains the holy grail of genetic approaches to therapy.