Liu Nan, Wang Bingyan J, Broughton Kathleen M, Alvarez Roberto, Siddiqi Sailay, Loaiza Rebeca, Nguyen Nicky, Quijada Pearl, Gude Natalie, Sussman Mark A
Biology Department, San Diego State University, San Diego, California, United States of America.
PLoS One. 2017 Mar 21;12(3):e0173963. doi: 10.1371/journal.pone.0173963. eCollection 2017.
PIM1, a pro-survival gene encoding a serine/ threonine kinase, influences cell proliferation and survival. Modification of cardiac progenitor cells (CPCs) or cardiomyocytes with PIM1 using a lentivirus-based delivery method showed long-term improved cardiac function after myocardial infarction (MI). However, lentivirus based delivery methods have stringent FDA regulation with respect to clinical trials. To provide an alternative and low risk PIM1 delivery method, this study examined the use of a non-viral modified plasmid-minicircle (MC) as a vehicle to deliver PIM1 into mouse CPCs (mCPCs) in vitro and the myocardium in vivo. MC containing a turbo gfp reporter gene (gfp-MC) was used as a transfection and injection control. PIM1 was subcloned into gfp-MC (PIM1-MC) and then transfected into mCPCs at an efficiency of 29.4±3.7%. PIM1-MC engineered mCPCs (PIM1-mCPCs) exhibit significantly (P<0.05) better survival rate under oxidative treatment. PIM1-mCPCs also exhibit 1.9±0.1 and 2.2±0.2 fold higher cell proliferation at 3 and 5 days post plating, respectively, as compared to gfp-MC transfected mCPCs control. PIM1-MC was injected directly into ten-week old adult FVB female mice hearts in the border zone immediately after MI. Delivery of PIM1 into myocardium was confirmed by GFP+ cardiomyocytes. Mice with PIM1-MC injection showed increased protection compared to gfp-MC injection groups measured by ejection fraction at 3 and 7 days post injury (P = 0.0379 and P = 0.0262 by t-test, respectively). Success of PIM1 delivery and integration into mCPCs in vitro and cardiomyocytes in vivo by MC highlights the possibility of a non-cell based therapeutic approach for treatment of ischemic heart disease and MI.
PIM1是一种编码丝氨酸/苏氨酸激酶的促生存基因，影响细胞增殖和存活。使用基于慢病毒的递送方法用PIM1修饰心脏祖细胞（CPC）或心肌细胞，在心肌梗死（MI）后显示出长期改善的心脏功能。然而，基于慢病毒的递送方法在临床试验方面受到美国食品药品监督管理局（FDA）的严格监管。为了提供一种替代的、低风险的PIM1递送方法，本研究检测了使用非病毒修饰的质粒微环（MC）作为载体，在体外将PIM1递送至小鼠CPC（mCPC）以及在体内递送至心肌的情况。含有turbo gfp报告基因的MC（gfp-MC）用作转染和注射对照。将PIM1亚克隆到gfp-MC（PIM1-MC）中，然后以29.4±3.7%的效率转染到mCPC中。经PIM1-MC工程改造的mCPC（PIM1-mCPC）在氧化处理下表现出显著更高（P<0.05）的存活率。与gfp-MC转染的mCPC对照相比，PIM1-mCPC在接种后第3天和第5天的细胞增殖分别高出1.9±0.1倍和2.2±0.2倍。在MI后，立即将PIM1-MC直接注射到10周龄成年FVB雌性小鼠心脏的边界区域。通过GFP+心肌细胞证实了PIM1在心肌中的递送。通过MC将PIM1递送至体外的mCPC和体内的心肌细胞成功，凸显了基于非细胞的治疗方法治疗缺血性心脏病和MI的可能性。
