A ribonucleoprotein-based decaplex CRISPR/Cas9 knockout strategy for CHO host engineering.

Chinese hamster ovary (CHO) cell engineering based on CRISPR/Cas9 knockout (KO) technology requires the delivery of guide RNA (gRNA) and Cas9 enzyme for efficient gene targeting. With an ever-increasing list of promising gene targets, developing, and optimizing a multiplex gene KO protocol is crucial for rapid CHO cell engineering. Here, we describe a method that can support efficient targeting and KO of up to 10 genes through sequential transfections. This method utilizes Cas9 protein to first screen multiple synthetic gRNAs per gene, followed by Sanger sequencing indel analysis, to identify effective gRNA sequences. Using sequential transfections of these potent gRNAs led to the isolation of single cell clones with the targeted deletion of all 10 genes (as confirmed by Sanger sequencing at the DNA level and mass spectrometry at the protein level). Screening 704 single cell clones yielded 6 clones in which all 10 genes were deleted through sequential transfections, demonstrating the success of this decaplex gene editing strategy. This pragmatic approach substantially reduces the time and effort required to generate multiple gene knockouts in CHO cells.

Bax and Bak knockout apoptosis-resistant Chinese hamster ovary cell lines significantly improve culture viability and titer in intensified fed-batch culture process.

In the field of therapeutic protein production, process intensification strategies entailing higher starting cell seeding densities, can potentially increase culture productivity, lower cost of goods and improve facility utilization. However, increased cell densities often trigger apoptotic cell death at the end of the cell culture process and thus reduce total viable cell count. Apoptosis-resistant Chinese hamster ovary cell lines may offer the possibility to diminish this undesired outcome of the intensified production process. In this study, we have generated and tested Bax/Bak double-knock-out (DKO) apoptosis resistant hosts to express standard and bispecific antibodies, as well as complex molecules in intensified production processes both as pools and single cell clones, and at different scales. In all cases, therapeutic proteins expressed from clones or pools generated from the Bax/Bak DKO hosts showed not only better viability but also enabled extended productivity in the later stages of the 14-day intensified production process. The product qualities of the produced molecules were comparable between Bax/Bak DKO and wild type cells. Overall, we showed that Bax/Bak DKO apoptosis-resistant host cell lines significantly improve viability and volumetric productivity of the intensified production cultures without altering product qualities.

Down-regulated MicroRNAs in Gastric Carcinoma May Be Targets for Therapeutic Intervention and Replacement Therapy.

Gastric cancer is one of the leading types of cancer with an annual death toll of 700,000 worldwide. Despite the fact that several agents are approved for its treatment, high percentage of recurrence and intractability of metastatic disease remain a major problem. The identification of new targets and modalities for treatment are therefore of high priority. We have searched the literature for microRNAs down-regulated in gastric cancer with efficacy in gastric cancer-related murine xenograft models after reconstitution therapy. Among the identified miRs were 25 miRs targeting transcription factors, seven of them regulating cell-cycle and apotosis-related targets, and five of them regulating GTPase-related targets such as GAPs and GEFs. According to criteria such as prognostic impact, functional data, and tractability, miR-133 b/a (MCL1) and miR-518 (MDM2) are suggested as potentially valuable targets for further evaluation and possible treatment of gastric cancer.

Gastric Cancer: Identification of microRNAs Inhibiting Druggable Targets and Mediating Efficacy in Preclinical In Vivo Models.

In addition to chemotherapy, targeted therapies have been approved for treatment of locally advanced and metastatic gastric cancer. The therapeutic benefit is significant but more durable responses and improvement of survival should be achieved. Therefore, the identification of new targets and new approaches for clinical treatment are of paramount importance. In this review, we searched the literature for down-regulated microRNAs which interfere with druggable targets and exhibit efficacy in preclinical in vivo efficacy models. As druggable targets, we selected transmembrane receptors, secreted factors and enzymes. We identified 38 microRNAs corresponding to the criteria as outlined. A total of 13 miRs target transmembrane receptors, nine inhibit secreted proteins and 16 attenuate enzymes. These microRNAs are targets for reconstitution therapy of gastric cancer. Further target validation experiments are mandatory for all of the identified microRNAs.

microRNAs and Corresponding Targets Involved in Metastasis of Colorectal Cancer in Preclinical In Vivo Models.

The high death toll of colorectal cancer patients is due to metastatic disease which is difficult to treat. The liver is the preferred site of metastasis, followed by the lungs and peritoneum. In order to identify new targets and new modalities of intervention we surveyed the literature for microRNAs (miRs) which modulate metastasis of colorectal cancer in preclinical in vivo models. We identified 12 up-regulated and 19 down-regulated miRs corresponding to the latter criterium. The vast majority (n=16) of identified miRs are involved in modulation of epithelial-mesenchymal transition (EMT). Other categories of metastasis-related miRs exhibit tumor- and metastasis-suppressing functions, modulation of signaling pathways, transmembrane receptors and a class of miRs, which interfere with targets which do not fit into these categories. Finally, we discuss the principles of miR inhibition and reconstitution of function, prospective clinical evaluation of with miR-related agents in the context of clinical evaluation in metastasis relevant settings.