Rt-sir2双基因表达自噬系统及其逆转细胞衰老的分子机制研究

基因治疗的有效性已被广泛认可，但其安全性尚未解决，构建安全可控的新型基因治疗载体是基因治疗领域的迫切需求！哺乳类细胞特有的分子伴侣介导的自噬（CMA）取决于底物蛋白的靶向基序“KFERQ”，前期研究发现这一靶向基序和DNA结合域的合成肽nap可将质粒DNA分子介导进入CMA过程。前期也证明粘红酵母sir2家族基因Rt-sirt2和Rt-sirt4分别对哺乳细胞线粒体的裂殖和自噬起重要作用。本课题拟构建双基因表达自噬质粒pIRES-Rt-sirt2/4-Tet-nap，使nap在四环素调控下表达，诱导时质粒DNA被细胞自噬清除，通过单细胞测序和小角散射谱仪等法研究此双基因自噬质粒DNA在靶细胞自噬的分子机制，进一步观察其对衰老细胞和小鼠的表观逆转及诱导后消除效果。此项双基因共时可控表达研究不仅为Rt-sir2基因的抗衰老提供依据，也为众多外源基因实现有效可控的基因治疗拓展新思路。

The effectiveness of gene therapy has been widely recognized, but its safety has not yet been resolved. Constructing a safe and controllable new gene therapy vector is an urgent need in the field of gene therapy! Mammalian cell-specific molecular chaperone-mediated autophagy (CMA) depends on the targeting motif "KFERQ" of the substrate protein. Previous studies have found that this synthetic peptide nap, which targets the motif and DNA binding domain, can mediate plasmid DNA molecules into the CMA process. It has also been proved earlier that Rhodosporidium toruloides sir2 family genes Rt-sirt2 and Rt-sirt4 play important roles in mitochondria and autophagy of mammalian cells, respectively. This project intends to construct a dual-gene expression autophagy plasmid pIRES-Rt-sirt2/4-Tet-nap, so that nap is expressed under the control of tetracycline, and the plasmid DNA is cleared by cell autophagy during induction. Single-cell sequencing and small-angle scattering spectrometry were used to study the molecular mechanism of this dual-gene autophagy plasmid DNA in target cells, and further observe its apparent reversal and elimination effects after induction on senescent cells and mice. This dual-gene simultaneous controllable expression study not only provides the basis for the anti-aging of the Rt-sir2 gene, but also expands new ideas for effective and controllable gene therapy for many foreign genes.