Katz Noa, An Connie, Lee Yu-Ju, Tycko Josh, Zhang Meng, Kang Jeewoo, Bintu Lacramioara, Bassik Michael C, Huang Wei-Hsiang, Gao Xiaojing J
Department of Chemical Engineering, Stanford University, Stanford, CA, USA.
Department of Neurology & Neurosurgery, Centre for Research in Neuroscience, McGill University, Montréal, QC H3G 1A3, Canada.
bioRxiv. 2024 Oct 9:2024.10.09.617463. doi: 10.1101/2024.10.09.617463.
Gene therapy holds great therapeutic potential. Yet, controlling cargo expression in single cells is limited due to the variability of delivery methods. We implement an incoherent feedforward loop based on proteolytic cleavage of CRISPR-Cas activation or inhibition systems to reduce gene expression variability against the variability of vector delivery. We demonstrate dosage control for activation and inhibition, post-delivery tuning, and RNA-based delivery, for a genome-integrated marker. We then target the gene, the haploinsufficiency and triplosensitivity of which cause two autism-related syndromes, Smith-Magenis-Syndrome (SMS) and Potocki-Lupski-Syndrome, respectively. We demonstrate dosage control for RAI1 activation in HEK293s, Neuro-2As, and mouse cortical neurons via AAVs and lentiviruses. Finally, we activate the intact copy in SMS patient-derived cells to an estimated two-copy healthy range, avoiding the harmful three-copy regime. Our circuit paves the way for viable therapy in dosage-sensitive disorders, creating precise and tunable gene regulation systems for basic and translational research.
基因治疗具有巨大的治疗潜力。然而，由于递送方法的变异性，在单细胞中控制货物表达受到限制。我们基于CRISPR-Cas激活或抑制系统的蛋白水解切割实施了一个非相干前馈回路，以减少针对载体递送变异性的基因表达变异性。我们展示了针对基因组整合标记物的激活和抑制的剂量控制、递送后调节以及基于RNA的递送。然后，我们靶向该基因，其单倍剂量不足和三倍体敏感性分别导致两种自闭症相关综合征，即史密斯-马吉尼斯综合征（SMS）和波托基-卢普斯基综合征。我们通过腺相关病毒（AAV）和慢病毒证明了在HEK293细胞、神经2A细胞和小鼠皮层神经元中对RAI1激活的剂量控制。最后，我们将SMS患者来源细胞中的完整拷贝激活到估计的双拷贝健康范围，避免有害的三拷贝状态。我们的电路为剂量敏感疾病的可行治疗铺平了道路，为基础研究和转化研究创建了精确且可调节的基因调控系统。
