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Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques

机译：使用非侵入性生物发光成像技术评估体内的弹射基因转移方法

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Background Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun") delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI) methods. Results Plasmid DNA carrying the firefly luciferase (LUC) reporter gene under the control of the human Cytomegalovirus (CMV) promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter) using different DNA Loading Ratios (DLRs), and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50) at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. Conclusions The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results demonstrate that different tissues show different expression kinetics following gene transfer of the same reporter plasmid to different mouse tissues in vivo. We evaluated superficial (skin) and abdominal organ (liver) targets, and found that reporter gene expression peaked within the first two days post-transfer in each case, but declined most rapidly in the skin (3-4 days) compared to liver (10-14 days). This information is essential for designing effective gene therapy strategies in different target tissues.
机译：背景技术基因疗法继续具有治疗许多不同类型的疾病和功能障碍的巨大潜力。需要安全有效的体内基因转移和表达技术，以使基因治疗策略对患者有效。当前，最常用的方法使用复制缺陷型病毒载体进行基因转移，而诸如基因枪介导的“基因枪”的物理基因转移方法尚未广泛研究。在本研究中，我们评估了使用非侵入性生物发光成像（BLI）方法在体内的生物射弹基因转移技术的功效。结果在人巨细胞病毒（CMV）启动子/增强子的控制下，携带萤火虫荧光素酶（LUC）报告基因的质粒DNA被转染到小鼠皮肤和肝脏中。使用不同的DNA负载率（DLR）将质粒偶联至金微球（直径1μm），并使用氦驱动的基因枪将其“射入”靶组织。发现最佳的DLR在4-10的范围内。在转移后的各个时间点，使用体内成像系统（IVIS-50）测量生物发光。转移到小鼠皮肤的基因枪基因转移在转移后一天产生峰值报告基因表达。表达在四天内保持可检测，但是在基因转移后六天下降至不可检测的水平。生物射弹转移至腹部皮肤后组织穿透的最大深度为200-300μm。类似地，生物射弹基因在体内转移至小鼠肝脏也产生了高峰早期表达，随后随时间下降。然而，与皮肤相反，报告基因的肝脏表达在生物基因转移后4-8天相对稳定，并且在近两周内仍可检测到。结论使用生物发光成像技术能够有效评估报告基因在体内的表达。我们的结果表明，不同的组织在体内将同一报告质粒基因转移到不同的小鼠组织后显示出不同的表达动力学。我们评估了浅表（皮肤）和腹部器官（肝脏）的靶标，发现在每种情况下，报告基因的表达在每种情况下均在转移后的前两天内达到峰值，但与肝脏相比，在皮肤中（3-4天）下降最快（ 10-14天）。此信息对于在不同目标组织中设计有效的基因治疗策略至关重要。

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