Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305.
Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2021 Nov 9;118(45). doi: 10.1073/pnas.2109175118.
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.
三重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) 供体模板的特异性重组，并引发基因校正。在这里，我们评估了将 PNA 和包裹在聚乳酸-共-羟基乙酸 (PLGA) 纳米颗粒中的供体模板共递送至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，我们已经确定了一种由于 PNA 与 ssDNA 供体模板固有聚集能力而产生的假阳性 PCR 假象。在这里，我们表明 PNA 和供体模板的组合，但不是单独的任何一种试剂，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。我们已经在体外鉴定了这种现象，并证实了在两种疾病模型中，体外产生最高校正的 PNA 序列在体内均不活跃，这突出了在评估各种基因编辑技术（如 PNA 介导的基因编辑）时，检测和消除 ssDNA 供体模板残留的重要性。
