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The potential use of induced pluripotent stem cells (iPSCs) in personalized regenerative medicine applications may be augmented by transgenics, including the expression of constitutive cell labels, differentiation reporters, or modulators of disease phenotypes. Thus, there is precedence for reproducible transgene expression amongst iPSC sub-clones with isogenic or diverse genetic backgrounds. Using virus or transposon vectors, transgene integration sites and copy numbers are difficult to control, and nearly impossible to reproduce across multiple cell lines. Moreover, randomly integrated transgenes are often subject to pleiotropic position effects as a consequence of epigenetic changes inherent in differentiation, undermining applications in iPSCs. To address this, we have adapted popular TALEN and CRISPR/Cas9 nuclease technologies in order to introduce transgenes into pre-defined loci and overcome random position effects. AAVS1 is an exemplary locus within the PPP1R12C gene that permits robust expression of CAG promoter-driven transgenes. Gene targeting controls transgene copy number such that reporter expression patterns are reproducible and scalable by ∼2-fold. Furthermore, gene expression is maintained during long-term human iPSC culture and in vitro differentiation along multiple lineages. Here, we outline our AAVS1 targeting protocol using standardized donor vectors and construction methods, as well as provide practical considerations for iPSC culture, drug selection, and genotyping.

Background Emicizumab is an anti-activated factor IX/FX bispecific antibody that mimics activated FVIII cofactor function. Emicizumab does not require activation by thrombin, and its effect on shortening the activated partial thromboplastin time (APTT) is much greater than that of FVIII. Therefore, the APTT has limited utility in hemophilia A (HA) patients treated with emicizumab. Aim To evaluate the global coagulation potential of emicizumab. Methods Clot waveform analysis (CWA) with prothrombin time (PT)/APTT mixed reagents was used to define hemostatic monitoring protocols in HA patients. A modified parameter, adjusted-|min1| (Ad|min1|), was developed. Maximum and minimum percentage transmittance were defined as 100% and 0% in the precoagulation and postcoagulation phases, respectively. Ad|min1| was calculated as an index of the maximum velocity of the coagulation process. Results Ad|min1| obtained with mixed-trigger reagent (PT/APTT/buffer, 1 : 15 : 135) in the presence of emicizumab optimally corresponded to the conversion rate estimated in animals; 0.2-0.4 IU dL equivalent FVIII per 1 μg mL emicizumab). Ex vivo addition of emicizumab to HA plasma with or without inhibitors resulted in concentration-dependent increases in Ad|min1|, with some individual variations. The addition of various concentrations of FVIII to HA plasma mixed with emicizumab resulted in dose-dependent increases in Ad|min1|. Similarly, mixtures of activated prothrombin complex concentrate and emicizumab added to HA plasma resulted in dose-dependent increases in Ad|min1|. In contrast, enhanced coagulation potential appeared to be better defined by the clot time than by Ad|min1| in experiments using recombinant activated FVII. Conclusion The PT/APTT reagent-triggered adjusted CWA could provide a useful means of assessing global coagulation potential in emicizumab-treated HA patients, with enhanced activity neither masking nor being masked by FVIII or bypassing agents.

The mechanistic interconnectivity between circadian regulation and the genotoxic stress response remains poorly understood. Here we show that the expression of Period 2 (Per2), a circadian regulator, is directly regulated by p53 binding to a response element in the Per2 promoter. This p53 response element is evolutionarily conserved and overlaps with the E-Box element critical for BMAL1/CLOCK binding and its transcriptional activation of Per2 expression. Our studies reveal that p53 blocks BMAL1/CLOCK binding to the Per2 promoter leading to repression of Per2 expression. In the suprachiasmatic nucleus (SCN), p53 expression and its binding to the Per2 promoter are under circadian control. Per2 expression in the SCN is altered by p53 deficiency or stabilization of p53 by Nutlin-3. Behaviorally, p53−/− mice have a shorter period length that lacks stability and they exhibit impaired photo-entrainment to a light pulse under a free-running state. Our studies demonstrate that p53 modulates mouse circadian behavior.