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Sep 1, 2014 (Vol. 34, No. 15)

Genome Editing Edges to the Clinic

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    Gene-editing technology is constantly in flux, which can prove daunting to new users. Clinical researchers, in particular, need ways to effectively access the technology regardless of their level of expertise. [Andrea Danti/shutterstock.com]

    The programmable, site-specific nuclease systems at the heart of genome editing keep evolving—even the newest system, named CRISPR (for clustered regularly interspaced short palindromic repeats) or CRISPR/Cas (for CRISPR-associated system).

    Although it was introduced just two years ago in a landmark paper by Emmanuelle Charpentier, Jennifer A. Doudna, and colleagues, CRISPR has already inspired researchers around the world to refine and elaborate the technology. The “CRISPR craze,” as the journal Science calls it, shows no sign of abating.

    At the same time, more established nuclease systems—ZFNs (for zinc finger nucleases) and TALENs (for transcription activator-like effector nucleases)—refuse to be sidelined. These technologies, along with CRISPR, occupied center stage at a recent FASEB conference. This event, entitled “Genome Engineering—Cutting-Edge Research and Applications,” not only covered the engineering of custom nucleases, it also explored emerging principles of synthetic biology, clinical translation of genome engineering, and other topics.

    ZFNs, artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain, remain central to the first gene-editing technology engineered to recognize specified targets in mammalian cells. However, these complex proteins are not trivial to engineer; screening and optimization is an iterative process.

    An expert in ZFN engineering, Sangamo Biosciences designs these enzymes to use in the development of novel therapeutics. One particular advance was cited at the FASEB event by the company’s senior director of therapeutic gene modification, Michael Holmes, Ph.D.

    “Recently, we demonstrated in mice that we could introduce ZFNs that targeted the first intron of the endogenous albumin gene,” said Dr. Holmes. “Albumin is a very highly expressed protein in the liver, so we consider the gene to be a “safe harbor” locus. Modifying a small number of these loci allows very high levels of transgene expression.”

    Using the albumin locus to convert the liver into a protein secretion factory provides flexibility for different payloads; modifying less than 1% of liver cells can produce therapeutic levels of relevant proteins.

    The human factor IX gene, a gene that occurs on the X chromosome and is mutated in hemophilia B, may be inserted into mouse albumin loci. Doing so results in secretion of 40–60% of completely functional factor IX, essentially correcting the clotting disorder. The same approach was used in a mouse model of hemophilia A, which is caused by mutations in factor VIII, resulting in secretion of up to 40% of normal factor VIII levels.

    Physiological levels of proteins from genes involved in lysosomal storage diseases, such as Hurler, Hunter, Gaucher, and Fabry, could also be expressed in mice, further validating the approach. The albumin locus may have advantages over adeno-associated virus (AAV) delivery. The AAV vector is not integrated into the liver genome; as the liver turns over, the AAV episome is likely to be lost.

  • Continual Enhancements

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    Sigma’s CRISPR-paired nickases can improve specificity and retain the design flexibility needed to target disease SNPs and other site-restricted genomic locations. Protospacer adjacent motifs (PAMs) can be spaced 30 to 150 bp apart and retain efficient editing activity.

    According to Greg Davis, Ph.D., principal R&D scientist, Sigma-Aldrich Biotechnology, key ZFN enhancements in recent years are cleavage domains, termed enhanced high-fidelity FokI domains. Because these domains work only as heterodimers, they enhance specificity by blocking an individual ZFN from homodimerizing with itself on DNA and cleaving potential off-target sites.

    Whereas ZFNs require two binding events to create the double-strand DNA break, the CRISPR technology initially required just one binding event. To improve the specificity of the CRISPR/Cas9 technology, paired nickases seemed like a tractable approach.

    If two CRISPRs bind close together and both nick an opposite strand of DNA, a double-strand break will occur. At Sigma, Cas9 mutations were undertaken to create nickases to cleave only one strand of DNA. Close nicks result in a double-strand break. Nickases allow more permissive spacing than the use of FokI, providing increased flexibility for practical research applications, such as disease SNP modeling.

    Sigma has a large lentivirus vector library, used currently for shRNA libraries, and is applying it to CRISPR. shRNA screening is limited to the exome of the genome, the protein-coding genes or roughly 1% of the genome, whereas CRISPR targets the entire chromosome. A good delivery tool, lentivirus is applicable to both arrayed and pooled high-throughput screening applications.

    RNA interference (RNAi) represses activity, but for activation and epigenetic studies, modified ZFNs, TALENs, and CRISPRs may be applicable. A natural extension to the CRISPR platform is to inactivate the nuclease activity on the Cas9 protein, turning the CRISPR into a DNA-binding protein. Then activators, such as VP16 and VP64. and repressors, such as the KRAB domain, can be fused to dead Cas9. This allows enzymatic activity to be localized to a specific part of the chromosome, permitting the study of genetic regulation at specific loci.

    Sigma has formed a partnership with Cleveland Clinic’s Molecular Screening Core to develop a CRISPR core. This new core is part of the Case Comprehensive Cancer Center at Case Western Reserve University and is open for researchers at Cleveland Clinic and other biomedical research centers in the Cleveland area.

    One of core’s first users, Paul Tesar, Ph.D., associate professor, department of genetics and genome sciences, Case Western Reserve University School of Medicine, uses gene-editing technology to study neurogenic and neurodevelopment disorders. Dr. Tesar’s group focuses on areas of the brain that impact oligodendrocytes, which make myelin.

    Specific mutations cause oligodendrocytes in the brain to produce myelin less effectively. The result: leukodystrophies, a class of pediatric congenital disorders. To better understand these diseases, the laboratory uses genome editing and aspires to create corrected oligodendrocytes from pluripotent stem cells. Once modified, the oligodendrocytes would help correct the disease in these patients.

    “Gene-editing technology continually changes and can be overwhelming to a new user,” commented Dr. Tesar. “The new CRISPR core provides a rapid and effective way to access the technology regardless of level of expertise, and builds a community of researchers. Protocols, vectors, support, and technical expertise are in place to help you get exactly what you need for your particular experiment.”


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