Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden.
YKI, Institute for Surface Chemistry, Stockholm, Sweden.
Mol Ther Nucleic Acids. 2014 Jan 7;2(1):e140. doi: 10.1038/mtna.2013.67.
The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, super-coiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.Molecular Therapy-Nucleic Acids (2014); doi:10.1038/mtna.2013.67.
微环（MC）仅由真核序列组成，是提高基于质粒的基因治疗载体安全性和效率的一种有趣方法。在本文中，我们研究了编码小调控 RNA 的微环（miMC）载体。我们使用了一种编码剪接校正 U7 小核 RNA 的构建体，其结果是产生了 650 个碱基对（bp）的载体，而不是携带相同表达盒的传统 3600bp 质粒。此外，我们构建了不同大小的 miMC，携带不同数量的这些盒。这使我们能够评估大小如何影响载体的生产、超螺旋、稳定性和效率。我们通过原子力显微镜描绘了卷曲形态，并测量了由注射器装置（Biojector）引起的剪切力的阻力。我们通过脂质转染和电穿孔在体外以及在小鼠体内比较了 miMC 和质粒的行为。我们在此表明，当 miMC 的大小减小时，二聚体和三聚体的形成增加。似乎存在一个有效表达的最小尺寸限制。我们证明，与质粒相比，miMC 在受到剪切力时更具韧性，并且在体内表现出延长的表达。《分子治疗-核酸》（2014 年）；doi：10.1038/mtna.2013.67。
