Triplex基因编辑依赖于将稳定的肽核酸(PNA)序列与染色体靶标结合,改变DNA的螺旋结构以刺激与单链DNA(ssDNA)供体模板的位点特异性重组并引发基因校正。这里,我们评估了包裹在基于聚乳酸-共-乙醇酸(PLGA)的纳米颗粒中的PNA和供体模板共同递送是否可以纠正镰状细胞病和X-连锁严重联合免疫缺陷.然而,通过这个过程,我们已经确定了一个假阳性的PCR产物,由于PNA与ssDNA供体模板聚集的内在能力。这里,我们表明,PNA和供体模板的组合,而不是单独使用任何一种试剂,都会导致不同程度的聚集,从而导致不同但高度可重复的假阳性信号水平.我们已经在体外确定了这种现象,并证实在两种疾病模型中,在体外产生最高假定校正的PNA序列在体内都不活跃。这突出了在评估各种基因编辑技术(如PNA介导的基因编辑)中询问和消除ssDNA供体模板残留的重要性。
Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.