Williams Luis A, Gerber David J, Elder Amy, Tseng Wei Chou, Baru Valeriya, Delaney-Busch Nathaniel, Ambrosi Christina, Mahimkar Gauri, Joshi Vaibhav, Shah Himali, Harikrishnan Karthiayani, Upadhyay Hansini, Rajendran Sakthi H, Dhandapani Aishwarya, Meier Joshua, Ryan Steven J, Lewarch Caitlin, Black Lauren, Douville Julie, Cinquino Stefania, Legakis Helen, Nalbach Karsten, Behrends Christian, Sato Ai, Galluzzi Lorenzo, Yu Timothy W, Brown Duncan, Agrawal Sudhir, Margulies David, Kopin Alan, Dempsey Graham T
Q-State Biosciences, 179 Sidney Street, Cambridge, MA 02139, USA.
Charles River Laboratories, Montreal, QC, Canada.
Mol Ther Nucleic Acids. 2022 Jun 22;29:189-203. doi: 10.1016/j.omtn.2022.06.015. eCollection 2022 Sep 13.
Mutations in the gene are the cause of an ultra-rare neurological disorder characterized by intellectual disability, impaired speech, motor delay, and hypotonia evolving to spasticity, central sleep apnea, and premature death (SPG49 or HSAN9; OMIM: 615031). Little is known about the biological function of TECPR2, and there are currently no available disease-modifying therapies for this disease. Here we describe implementation of an antisense oligonucleotide (ASO) exon-skipping strategy targeting c.1319delT (p.Leu440Argfs∗19), a pathogenic variant that results in a premature stop codon within exon 8. We used patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived neurons homozygous for the p.Leu440Argfs∗19 mutation to model the disease . Both patient-derived fibroblasts and neurons showed lack of TECPR2 protein expression. We designed and screened ASOs targeting sequences across the exon 8 region to identify molecules that induce exon 8 skipping and thereby remove the premature stop signal. exon 8 skipping restored in-frame expression of a TECPR2 protein variant (TECPR2ΔEx8) containing 1,300 of 1,411 amino acids. Optimization of ASO sequences generated a lead candidate (ASO-005-02) with ∼27 nM potency in patient-derived fibroblasts. To examine potential functional rescue induced by ASO-005-02, we used iPSC-derived neurons to analyze the neuronal localization of TECPR2ΔEx8 and showed that this form of TECPR2 retains the distinct, punctate neuronal expression pattern of full-length TECPR2. Finally, ASO-005-02 had an acceptable tolerability profile following a single 20-mg intrathecal dose in cynomolgus monkeys, showing some transient non-adverse behavioral effects with no correlating histopathology. Broad distribution of ASO-005-02 and induction of exon 8 skipping was detected in multiple central nervous system (CNS) tissues, supporting the potential utility of this therapeutic strategy for a subset of patients suffering from this rare disease.
该基因的突变是一种极为罕见的神经疾病的病因，其特征为智力残疾、言语障碍、运动发育迟缓以及肌张力减退逐渐发展为痉挛、中枢性睡眠呼吸暂停和过早死亡（SPG49或HSAN9；OMIM：615031）。人们对TECPR2的生物学功能知之甚少，目前尚无针对该疾病的疾病修饰疗法。在此，我们描述了一种反义寡核苷酸（ASO）外显子跳跃策略的实施，该策略靶向c.1319delT（p.Leu440Argfs∗19），这一致病性变体导致第8外显子内出现过早的终止密码子。我们使用患者来源的成纤维细胞以及携带p.Leu440Argfs∗19突变的纯合诱导多能干细胞（iPSC）衍生神经元来模拟该疾病。患者来源的成纤维细胞和神经元均显示缺乏TECPR2蛋白表达。我们设计并筛选了靶向第8外显子区域序列的ASO，以鉴定能够诱导第8外显子跳跃从而去除过早终止信号的分子。第8外显子跳跃恢复了包含1411个氨基酸中1300个氨基酸的TECPR2蛋白变体（TECPR2ΔEx8）的读框内表达。ASO序列的优化产生了一个先导候选物（ASO-005-02），在患者来源的成纤维细胞中效力约为27 nM。为了检测ASO-005-02诱导的潜在功能挽救作用，我们使用iPSC衍生神经元分析TECPR2ΔEx8的神经元定位，并表明这种形式的TECPR2保留了全长TECPR2独特的点状神经元表达模式。最后，在食蟹猴中单次鞘内注射20 mg剂量后，ASO-005-02具有可接受的耐受性，显示出一些短暂的非不良行为效应，且无相关的组织病理学改变。在多个中枢神经系统（CNS）组织中检测到ASO-005-02的广泛分布以及第8外显子跳跃的诱导，支持了这种治疗策略对一部分患有这种罕见疾病患者的潜在效用。
