Gene delivery using a pH-sensitive molecular tweezer to catch and release nucleic acids

经过长期的潜伏期，基因治疗已经成为现实。自2015年以来，已有四种基于基因的产品获得批准，其中几种正在进行中。批准的加速表明，研究人员已经积累了足够的基础知识来支持基因治疗，并认可其可行性和临床相关性。目前针对罕见病的基因治疗产品，未来将涉及更多常见疾病，如癌症，并将为更多患者提供服务。然而，这种疗法的生产和价格仍然是一个巨大的挑战，最近已经导致Glybera（100万美元/治疗）的撤回。我们相信这种改善可能来自于治疗基因的传递策略。病毒载体已经商业化，但其高昂的生产成本、生物安全问题、潜在的免疫原性限制了其大规模生产。到目前为止，合成载体在临床试验中失败，原因是功效不足和非特异性毒性，通常归因于它们的正电荷。* 我们最近开发了pH敏感的分子镊子，灵感来自仿生主客体化学。这些结构的灵感来自于2016年诺贝尔奖授予的分子机器领域。我们已经证明，这些镊子能够结合药物在生理pH值和释放它在酸性pH值。我们还纳入了镊子在脂质结构，以获得pH敏感的脂质体。这些纳米载体能够在体外和体内显著改善亲水性药物和核酸的胞质递送。然而，它们的阳离子电荷仍然限制了它们的体内功效。* 在这个项目中，我们希望开发分子镊子来捕获和释放DNA。在第一步中，它们将被设计用于核酸的非特异性结合，并将用作基因治疗产品的非阳离子递送系统。它们将被合成，表征，并在体外和体内的最新阳离子系统进行比较。在第二步中，我们将设计DNA镊子，用于序列，错配或三核苷酸重复的特异性识别。基因组DNA中的这些缺陷与许多遗传疾病如亨廷顿病或肌营养不良症相关。这种镊子的特异性将在体外进行优化。然后，将开发基于芯片的生物传感器，并将其用作DNA缺陷和遗传疾病的诊断工具。

After a long period of latency, gene therapy has become a reality. Four gene-based products have been approved since 2015 and several are on the way. This acceleration of approvals shows that researchers have accumulated enough fundamental knowledge to support gene therapy and endorse its feasibility and clinical relevance. Currently targeting rare diseases, gene therapy products will, in the future, concern more common disease, such as cancer, and will be accessible for a larger number of patients. Nevertheless, the production and the price of this therapy is still a great challenge, and has recently conducted to the withdrawn of Glybera (1 million $/treatment). We believe that improvement could come from the delivery strategy of the therapeutic gene. Viral vectors have been commercialized, but their exorbitant production cost, biosafety issues, potential immunogenicity, limit their large-scale production. Up to now, synthetic vectors have failed in clinical trials due to insufficient efficacy and non-specific toxicity, often attributed to their positive charge. ***We have recently developed pH-sensitive molecular tweezers, inspired from the biomimetic host-guest chemistry. These structures are inspired from the molecular machine field awarded by the Nobel Price in 2016. We have shown that these tweezers are able to bind a drug at physiological pH and release it at acidic pH. We also incorporated the tweezers in a lipid structure to get pH-sensitive liposomes. These nanocarriers were able to drastically improve the cytosolic delivery of hydrophilic drugs and nucleic acids in vitro and in vivo. Nevertheless, their cationic charge still limited their in vivo efficacy. ***In this project, we want to develop molecular tweezers to catch and release DNA. In a first step, they will be designed for non-specific binding of nucleic acids, and will be used as a non-cationic delivery system for gene therapy products. They will be synthesized, characterized, and compared in vitro and in vivo to the latest cationic systems. In a second step, we will design DNA tweezers for the specific recognition of sequences, mismatches or trinucleotide repeats. These defects in genomic DNA are associated with a number of genetic disease such as Huntington disease or muscular dystrophy. The specificity of such tweezers will be optimized in vitro. Then, a chip-base biosensor will be developed and used as a diagnostic tool for DNA defects and genetic diseases.**