Ross Paul D, Gadalla Kamal K E, Thomson Sophie R, Selfridge Jim, Bahey Noha G, Benito Juliana, Burstein Suzanne R, McMinn Rachel, Bolon Brad, Hector Ralph D, Cobb Stuart R
Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh EH8 9XD, UK.
Neurogene Inc., New York, NY 10011, USA.
Sci Transl Med. 2025 Apr 2;17(792):eadq3614. doi: 10.1126/scitranslmed.adq3614.
Conventional methods of gene transfer lead to inconsistent transgene expression within cells. This variability can be problematic, particularly in conditions like Rett syndrome (RTT), a neurological disorder caused by mutations in the (methyl-CpG binding protein 2) gene, because overexpression of can also cause adverse effects. To address these challenges, we devised a gene regulation system called Expression Attenuation via Construct Tuning (EXACT), which uses a self-contained, microRNA-based feed-forward loop that not only ensures more consistent transgene expression but also protects against excessive expression. Through cell-based screening assays, we demonstrated the ability of the EXACT circuit to modulate the expression of full-length human MeCP2. Compared with a conventional construct, an EXACT- construct exhibited a narrower range of cellular protein abundance. Furthermore, the degree of regulation by the EXACT circuit increased with higher transgene doses in vitro and in wild-type mice and mice modeling RTT. On the basis of cellular and in vivo testing, we identified an optimal configuration for the adeno-associated virus serotype 9 (AAV9) construct for self-regulated gene therapy, designated NGN-401. Delivery of NGN-401 to neonatal male hemizygous mice via intracerebroventricular injection resulted in prolonged survival and amelioration of RTT-like phenotypes compared with vehicle-treated animals. NGN-401 was also well tolerated by female mice and healthy juvenile nonhuman primates, in contrast with a conventional construct, which caused toxicity. The results from these studies underpin a first-in-human pediatric trial of NGN-401 in RTT (ClinicalTrials.gov, NCT05898620).
传统的基因转移方法会导致细胞内转基因表达不一致。这种变异性可能会带来问题，尤其是在雷特综合征（RTT）等情况下，RTT是一种由甲基化CpG结合蛋白2（MeCP2）基因突变引起的神经疾病，因为MeCP2的过表达也会产生不良影响。为应对这些挑战，我们设计了一种名为“通过构建体调控实现表达衰减”（EXACT）的基因调控系统，该系统使用一个独立的、基于微小RNA的前馈环，不仅能确保更一致的转基因表达，还能防止过度表达。通过基于细胞的筛选试验，我们证明了EXACT电路调节全长人MeCP2表达的能力。与传统构建体相比，EXACT构建体的细胞蛋白丰度范围更窄。此外，在体外、野生型小鼠以及模拟RTT的小鼠中，EXACT电路的调控程度随转基因剂量的增加而增强。基于细胞和体内测试，我们确定了用于自我调节MeCP2基因治疗的腺相关病毒血清型9（AAV9）构建体的最佳配置，命名为NGN-401。与接受载体处理的动物相比，通过脑室内注射将NGN-401递送至新生雄性半合子小鼠可延长生存期并改善RTT样表型。与会导致毒性的传统构建体相比，雌性MeCP2小鼠和健康幼年非人灵长类动物对NGN-401也具有良好的耐受性。这些研究结果为NGN-401在RTT患者中的首次人体儿科试验（ClinicalTrials.gov，NCT05898620）奠定了基础。
