Increased prime edit rates in KCNQ2 and SCN1A via single nicking all-in-one plasmids.

BACKGROUND: Prime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models. RESULTS: pAIO-EF1α-PE2 and pAIO-EF1α-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1α-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1α-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors. CONCLUSION: Taken together, our study shows an improved efficacy of EF-1α driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important.

Force-time characteristics during sustained maximal handgrip effort according to age and clinical condition.

BACKGROUND: Muscle fatigue, a prominent symptom in older patients, can be assessed by sustained maximal handgrip testing. The force decline during sustained maximal contraction is described for young adults, but data for elderly persons are scarce. The aim of this study was to investigate force-time characteristics during a sustained maximal handgrip effort according to age and clinical condition. METHODS AND MATERIALS: Force-time data were continuously recorded during sustained maximal grip effort in 91 elderly patients (aged 83±5years), 100 elderly controls (aged 74±5years) and 100 young controls (aged 23±3years). The force-time curve was divided in 4 parts per 25% strength drop observed. Time (representing fatigue resistance (FR)) was measured during which grip strength (GS) dropped to 75% (FR75), 50% (FR50), 25% (FR25) of its maximum and to exhaustion (FRexhaustion). Grip work ((GW), the area under the force-time curve) was measured for the 4 parts as well as for the first 20 and 30s of the fatigue protocol test. Strength decay (GWdecay), defined as the difference between the area under the curve (% GW) and a theoretical maximal area under the curve (assuming there's no strength drop), was also studied. In the elderly participants, relationships (controlling for age and sex) of GS, FR and GW with circulating IL-6 and TNF-α were analyzed. RESULTS: FRexhaustion was similar for all groups, whereas the duration of each of the 4 parts was significantly different between the 3 groups. FR75 was shortest in old patients (p=0.004), FR75-50 was almost twice as long in old community-dwelling compared to old patients and young controls (p<0.001). This contrast was inverted for FR50-25 which was significantly shorter in old community-dwelling compared to the other groups (p=0.013). FR25-exhaustionwas significantly longer in young controls compared to the groups of older participants (p=0.017). Old patients showed lower GW for the first 2 parts compared to old community-dwelling and young controls. Also, GWdecay values during the first 20 and 30s were significantly higher in old patients compared to old community-dwelling and young controls (both p<0.001). IL-6 was significantly related to lower GSmax, FR75, FR50, FR25, FRexhaustion, GW75, GW50 and GW75-50. CONCLUSION: This is the first study reporting differences in strength decay during a sustained maximal handgrip effort according to age and clinical condition. Old patients showed a particularly rapid decline in GW during the first part of sustained handgrip. GW was also significantly related to circulating IL-6. Future studies should confirm whether a shorter FR test protocol (i.e. until FR75) but using a continuous registration of the strength decay could be more informative in a clinical setting compared to the classical FR test (measuring only FR50).