Cyclin-dependent kinase-like 5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene. It predominantly affects females who typically present with severe early epileptic encephalopathy, global developmental delay, motor dysfunction, autistic features and sleep disturbances. To develop a gene replacement therapy, we initially characterized the human CDKL5 transcript isoforms expressed in the brain, neuroblastoma cell lines, primary astrocytes and embryonic stem cell-derived cortical interneurons. We found that the isoform 1 and to a lesser extent the isoform 2 were expressed in human brain, and both neuronal and glial cell types. These isoforms were subsequently cloned into recombinant adeno-associated viral (AAV) vector genome and high-titre viral vectors were produced. Intrajugular delivery of green fluorescence protein via AAV vector serotype PHP.B in adult wild-type male mice transduced neurons and astrocytes throughout the brain more efficiently than serotype 9. Cdkl5 knockout male mice treated with isoform 1 via intrajugular injection at age 28-30 days exhibited significant behavioural improvements compared to green fluorescence protein-treated controls (1012 vg per animal, n = 10 per group) with PHP.B vectors. Brain expression of the isoform 1 transgene was more abundant in hindbrain than forebrain and midbrain. Transgene brain expression was sporadic at the cellular level and most prominent in hippocampal neurons and cerebellar Purkinje cells. Correction of postsynaptic density protein 95 cerebellar misexpression, a major fine cerebellar structural abnormality in Cdkl5 knockout mice, was found in regions of high transgene expression within the cerebellum. AAV vector serotype DJ efficiently transduced CDKL5-mutant human induced pluripotent stem cell-derived neural progenitors, which were subsequently differentiated into mature neurons. When treating CDKL5-mutant neurons, isoform 1 expression led to an increased density of synaptic puncta, while isoform 2 ameliorated the calcium signalling defect compared to green fluorescence protein control, implying distinct functions of these isoforms in neurons. This study provides the first evidence that gene therapy mediated by AAV vectors can be used for treating CDKL5 disorder.

中文翻译：

---

基因替换改善了细胞周期蛋白依赖性激酶样5紊乱的小鼠和人类模型中的缺陷。

细胞周期蛋白依赖性激酶样5障碍是一种严重的神经发育障碍，由X连锁的细胞周期蛋白依赖性激酶样5（CDKL5）基因突变引起。它主要影响通常患有严重的早期癫痫性脑病，整体发育迟缓，运动功能障碍，自闭症和睡眠障碍的女性。为了开发基因替代疗法，我们最初表征了在大脑，神经母细胞瘤细胞系，原代星形胶质细胞和胚胎干细胞衍生的皮质中间神经元中表达的人类CDKL5转录亚型。我们发现同工型1和较少的同工型2在人脑中以及神经元和神经胶质细胞类型中表达。随后将这些同工型克隆到重组腺相关病毒（AAV）载体基因组中，并生产出高滴度病毒载体。在成年野生型雄性小鼠中，通过AAV载体血清型PHP.B进行颈内绿色荧光蛋白的传递比在血清型9中更有效地转导了整个大脑的神经元和星形胶质细胞。Cdkl5基因敲除的雄性小鼠在28-30天时通过颈内注射用亚型1处理。与用PHP.B载体处理的绿色荧光蛋白处理的对照（每只动物1012 vg，每组n = 10）相比，它们表现出明显的行为改善。同种型1转基因的脑表达在后脑中比前脑和中脑更为丰富。转基因脑表达在细胞水平上是偶发性的，在海马神经元和小脑浦肯野细胞中最为突出。校正突触后密度蛋白95小脑的错误表达，这是Cdkl5基因敲除小鼠的主要细小脑结构异常，在小脑内高转基因表达的区域中发现了这种蛋白。AAV载体血清型DJ有效转导了CDKL5突变型人诱导的多能干细胞来源的神经祖细胞，随后将其分化为成熟的神经元。当治疗CDKL5突变神经元时，与绿色荧光蛋白对照相比，同工型1表达导致突触点的密度增加，而同工型2改善了钙信号传导缺陷，这暗示了这些同工型在神经元中的独特功能。这项研究提供了第一个证据，证明由AAV载体介导的基因治疗可用于治疗CDKL5疾病。随后被分化为成熟的神经元。当治疗CDKL5突变神经元时，与绿色荧光蛋白对照相比，同工型1表达导致突触点的密度增加，而同工型2改善了钙信号传导缺陷，这暗示了这些同工型在神经元中的独特功能。这项研究提供了第一个证据，证明由AAV载体介导的基因治疗可用于治疗CDKL5疾病。随后被分化为成熟的神经元。当治疗CDKL5突变型神经元时，与绿色荧光蛋白对照相比，同工型1表达导致突触点的密度增加，而同工型2改善了钙信号传导缺陷，这暗示了这些同工型在神经元中的独特功能。这项研究提供了第一个证据，证明由AAV载体介导的基因治疗可用于治疗CDKL5疾病。