Engineered histones as DNA carriers with application in therapeutic gene delivery

我们打算应用我们对蛋白质转运到细胞核的知识来增强DNA向靶细胞的输送。这涉及使用基因疗法来治疗遗传缺陷，例如先天的新陈代谢错误，在这种情况下，致病的功能缺失突变可以通过在生物体内改造细胞来克服，这些细胞在必要的数量和对适当的调节信号做出反应时，表达缺乏的特定成分。基因治疗方法中的一个限制因素是导入的DNA的核摄取效率低，据估计，实际摄取的DNA中有1%是表达的。我们的建议旨在开发增强非病毒介导的基因传递的方法，特别是通过优化这一关键的、限制外源DNA传递到细胞核的步骤。我们打算应用核靶向和染色质组装研究中的知识来改进基因转移技术。我们将在我们工作的基础上表明，核进口的特定信号-核靶向信号(NTSS)-可以用于增强核基因的传递和表达。由于正常细胞环境中的DNA是染色质的形式--一种与组蛋白等蛋白质的特定复合体--我们打算使用重组的染色质作为转基因DNA，由此将使用经改造包括NTSS和其他模块化序列元件的组蛋白。染色质不仅应该使NTSS和其他序列模块与DNA相连，而且还应该防止核酶在核进入之前的降解，使DNA浓缩以实现更有效的细胞-核进入，并通过在生理环境中将报告基因呈现给细胞来确保报告基因的表达。我们的方法应该有助于使基因治疗更接近临床现实。

We intend to apply our knowledge of protein transport to the nucleus to enhance the delivery of DNA to target cells. This relates to the use of gene therapy to treat genetic defects such as inborn errors of metabolism, where a disease-causing lack-of-function mutation can be overcome by engineering cells within the organism which express, in the necessary quantities and in response to the appropriate regulatory signals, the particular component which is lacking. A limiting factor in gene therapy approaches is the low efficiency of nuclear uptake of introduced DNA, where it has been estimated that < 1% of the DNA taken up is actually expressed. Our proposal seeks to develop approaches to enhance non-viral-mediated gene delivery, in particular by optimising this critical, limiting step of the delivery of exogenous DNA to the nucleus. We intend to apply knowledge from studies of nuclear targeting and chromatin assembly to improve gene transfer technologies. We will build on our work showing that specific signals for nuclear import - nuclear targeting signals (NTSs) - can be used to enhance nuclear gene delivery and expression. Since DNA in the normal cellular context is in the form of chromatin - a specific complex with proteins such as histones - we intend to use reconstituted chromatin as the transfecting DNA, whereby histones engineered to include NTSs and other modular sequence elements will be used. Chromatin should not only enable NTSs and other sequence modules to be linked to the DNA but also protect against nuclease-mediated degradation prior to nuclear entry, condense the DNA to enable more efficient cellular-nuclear entry, and ensure expression of the transfected reporter gene by presenting it to the cell in a physiological context. Our approaches should contribute to bringing gene therapy closer to reality in the clinic.