Gene replacement therapy for Syngap1 Syndrome

研究背景SYNGAP 1编码神经元胞质蛋白SynGAP（SYNaptic GT3 Activating Protein），是一种参与突触发育、结构、功能和可塑性的调节蛋白。SYNGAP 1基因中的杂合突变导致单倍不足，导致5型智力迟钝（MRD 5），这是一种以智力残疾（ID）、发育迟缓、癫痫、自闭症谱系障碍和其他行为异常为特征的疾病。SYNGAP 1功能丧失是ID伴癫痫的最常见原因之一。目的鉴于9型腺相关病毒（AAV9）1，2型介导的SYNGAP 1基因治疗具有显著的临床前和临床疗效，我们的目标是设计和发展AAV9基因治疗策略，以在MRD 5的临床前模型中进行概念验证。我们的具体目标是：i）设计和评估治疗性载体; ii）评估治疗性载体系统在iPSC衍生的神经元中的体外功效; iii）在MRD 5的临床前体内模型中完成概念验证功效研究。我们的Syngap1基因置换策略是一种有吸引力的解决方案，因为它靶向致病基因。syngap1基因替代疗法的研究尚未报道。实验方法AAV载体的设计和生产：目前，腺相关（AAV）为基础的系统是最精致和有效的基因传递系统之一。在罕见疾病患者中进行的2项独立I/II期临床试验报告了显著的安全性和疗效数据1，3。我们计划设计编码密码子优化的人SYNGAP 1基因的AAV。使用来自SYNGAP 1患者的iPS衍生神经元的治疗载体的体外验证。如在利用该细胞模型的最近研究中所证明的，将使用许多读数。4初步体内研究：这将在野生型小鼠中进行，以评估所产生的AAV载体的转导效率，并选择用于MRD 5小鼠模型中的临床前研究的剂量。使用体内疾病模型的临床前概念验证。然后使用充分表征的Syngap 1杂合模型（Syngap 1 +/-）在体内测试所选AAV的功效。Syngap 1 +/-模型显示出显著的表型，重现了人类疾病的许多特征。培训机会学生将加入一个由多名博士后、博士生和技术人员组成的团队。他/她将受益于他们的支持和专业知识。学生将使用/学习细胞培养，基本分子生物学技术，病毒载体设计，疾病建模，免疫染色，显微镜和体内临床前专业知识。细胞模型中的基因操作将使用慢病毒和腺相关载体来实现。该学生还将隶属于基因治疗创新和制造中心（GTIMC）。GTIMC目前是DiMen DTP准合作伙伴。新成立的中心与Cell & Gene Therapy Catapult签署了一项协议，允许技术转让用于基因治疗产品表征的分析测定。技术转让计划于2022年1月开始。学生将使用该检测方法对本项目产生的Syngap 1载体进行测序。他/她将生成数据密钥，以建立与行业的未来合作伙伴关系

Background SYNGAP1 encodes the neuronal cytoplasmic protein SynGAP (SYNaptic GTPase Activating Protein), a regulatory protein involved in synaptic development, structure, function, and plasticity. Heterozygous mutations in SYNGAP1 gene lead to haploinsufficiency, resulting in a mental retardation-type 5 (MRD5), a disease characterised by intellectual disability (ID), developmental delay, epilepsy, autism spectrum disorder and other behavioural abnormalities. SYNGAP1 loss-of-function is one of the most common causes of ID with epilepsy. ObjectivesGiven the remarkable pre-clinical and clinical efficacy mediated by adeno-associated virus serotype 9 (AAV9)1,2, our goal is to design and progress AAV9 gene therapy strategy for SYNGAP1 replacement towards proof-of-concept in pre-clinical model of MRD5. Our specific aims are: i) Design and evaluate therapeutic vectors; ii) Evaluate the in vitro efficacy of the therapeutic vector system in iPSC derived neurons; iii) complete a proof-of-concept efficacy stydy in pre-clinical in vivo model of MRD5.Novelty MRD5 patients currently have no treatment options. Our Syngap1 gene replacement strategy is an attractive solution since it is targeting the causative gene. No previous studies of syngap1 gene replacement therapy have been reported.Experimental ApproachAAV vector design and production: Currently, the adeno-associated (AAV)-based system is one of the most refined and effective gene delivery systems. Remarkable safety and efficacy data were reported from 2 separate phase I/II clinical trials in patients with rare diseases1,3. We plan to design AAV encoding codon optimised human SYNGAP1 gene. In vitro validation of the therapeutic vector using iPS-derived neurons from SYNGAP1 patients. A number of readouts will be used as demonstrated in a recent study utilising this cell model.4Pilot in vivo study: This will be performed in wild type mice to assess transduction efficiency of the generated AAV vector and select the dose for pre-clinical study in MRD5 mouse model. Pre-clinical proof-of concept using in vivo disease model. The efficacy of the selected AAV will then be tested in vivo using the well characterised Syngap1 heterozygous model (Syngap1+/-). Syngap1+/- model display significant phenotype recapitulating many features of the disease in humans. Training opportunitiesThe student will join a team of several postdocs, PhD students and technicians. He/she will benefit from their support and expertise. The student will use/learn cell culture, basic molecular biology techniques, viral vector design, disease modelling, immunostaining, microscopy and in vivo pre-clinical expertise. Gene manipulation in the cell models will be achieved using lentiviral and adeno-associated vectors. The student will also be affiliated to the Gene Therapy Innovation and Manufacturing Centre (GTIMC). GTIMC is currently a DiMeN DTP Associate Partner. The newly established centre has signed an agreement with Cell & Gene Therapy Catapult allowing Technology Transfer of analytical assays to be used for gene therapy product characterisation. The Tech transfer is scheduled to start in January 2022. The student will use the assays to characterise the Syngap1 vectors generated under this project. He/She will generate data key to establish future partnerships with industry