Genetic Engineering & Biotechnology News

NOV15 2017

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12 | NOVEMBER 15, 2017 | | Genetic Engineering & Biotechnology News Kenneth Albrecht, Ph.D. Pressure to accelerate drug discovery is high, and can be impacted by how mouse models are generated and bred. This tutorial reviews the use of two techniques that, when com- bined, reduce the time needed to reach the experimental cohort stage when CRISPR/ Cas9-mediated gene editing in embryos is not the best option. Mouse models are often customized to suit study objectives and improve translat- ability of results. Yet the process to generate a new model and produce a suitably sized co- hort can be lengthy, especially when complex genetic modifications are required. While the fastest gene-editing protocols—such as CRIS- PR/Cas9 in embryos—are efficient when used for simple modifications, complex modifica- tions are typically generated more efficiently using embryonic stem (ES) cell–mediated techniques. The timeline for this approach depends on factors such as the number of animals generated in Phase I, cohort size, and genotype. However, the average time for ge- nome modification, generation of chimeras, and production of a few heterozygous mice is 42 weeks. An additional 28 weeks of breed- ing usually is needed to produce a study-size cohort, for a total of 70 weeks. For research projects that are best accom- plished using an ES cell-mediated approach, molecular analysis of sperm to directly deter- mine germline chimerism can be combined with in vitro fertilization (IVF) to speed cohort production. This strategy reduces the model generation timeline by 12–16 weeks, without sacrificing quality control (see Figure 1). Employing Molecular Analysis of Sperm to Directly Determine Germline Chimerism Identifying the male founder mice that are most likely to quickly transmit the modified allele accelerates the initial breeding steps. Molecular analysis of sperm from male chi- meras can be used to make this identification. When using ES cell-based methods for genome modification, the ES cells are first manipulated in culture and then injected into blastocyst-stage embryos. The intent is that they will integrate into the developing embryo and become germ cells, enabling transmission of the genetic modification to the next generation. The injected embryos become chimeras—mice with multiple, ge- netically distinct cell lineages. While there are techniques to generate founder mice completely derived from ES cells, they re- quire specialized skills and/or equipment, or specific mutant mouse lines to produce blastocyst-stage embryos. This complexity may be unnecessary if ES cells with strong germline potential are employed, and the process described here is used to speed co- hort generation. Coat color of the resulting chimeras typi- cally is used as an easy but indirect marker of the percentage of germ cells derived from the ES cells. For example, if embryos from a white mouse are injected with ES cells from a black mouse, resulting in a black-and-white coat, a preponderance of black patches is viewed as indicative of a high uptake of ES cells. In turn, chimeras with the most black fur would be selected as breeders. Yet, there is not always a strong correlation between coat color and the percentage of germ cells with the modified allele, which may take multiple rounds of breeding to discover. Molecular analysis of sperm (genotyping) can help avoid this potential delay by ascer- taining directly the ES cell contribution to the germline, providing a more accurate method for selecting male chimeras for breeding. Sperm is harvested from all sexually mature chimeras and cryopreserved using standard protocols. Genomic DNA is extracted from a sperm aliquot and quantitative PCR is used to assess the percentage of cells carrying the wild type and modified gene. Rather than rely on coat color as an indirect measure, this A Strategy for Projects Requiring Complex Genetic Modifications Expediting the Creation of Mouse Models Drug Discovery Tutorial Insights Discovery & Development An antibody-drug conjugate (ADC) represents, as its name suggests, molecular teamwork. It brings together a monoclonal antibody, a linker, and a therapeutic payload (a cytotoxic agent). Also, ADCs often repre- sent teamwork at the drug-development level, where biotechs and pharmas collaborate to assemble novel ADCs. Collaboration also occurs at the preclinical- and clinical-trial levels of development. All these kinds of collaboration are evident in recent ADC news. For example, Seattle Genetics, a biotech that specializes in ADCs—and that already has one approved ADC (Adcetris) to its credit—announced that it is pursuing collaborations with GenMab and Astellas Pharma. The work with GenMab involves a Phase II study of tisotumab vedotin as a monotherapy for patients with cervical cancer. The work with Astellas involves a Phase II study of enforumab vedotin as a monotherapy for patients with urothelial cancer. Seattle is also taking part in clinical collaborations to evaluate SGN-LIV1A, another ADC, in combination therapies against breast cancer. One trial will evaluate the ADC, which targets the LIV-1 protein expressed by most metastatic breast cancers, in combination with Keytruda, an anti-PD-1 therapy marketed by Merck. Another trial will evaluate the ADC followed by standard chemotherapy (doxorubicin and cyclophosphamide) as a neoadjuvant treatment. Other recent ADC developments include an IND application coming into effect for ImmunoGen's IMGN632, an ADC for patients with hematological malignancies. NanoValent Pharmaceuticals has licensed its nanoparticle-based ACD platform to Chil- dren's Hospital of Los Angeles, and ADC Therapeutics has secured $200 million to finance clinical development of several ADCs against subtypes of lymphoma and leukemia. Such developments are helping to drive the growth of the global ADC market, which according to BCC Research, should reach $4.2 billion by 2021, $2.0 billion of which represents the North American share. The global figure reflects 25.5% annual growth from a 2016 base of $1.3 billion. n Reports of ADC Progress from Seattle Genetics, Others Figure 1. Using an embryonic stem (ES) cell-mediated approach to mouse model generation reduces the timeline by 12–16 weeks, without sacrificing quality control. Figure 2. Investigators use molecular analysis to select the mice that are the most suitable candidates for breeding.

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