Generation Of The SCN1A Epilepsy Mutation In HiPS Cells Using The TALEN Technique

Severe mycological epilepsy of infancy (SMEI) also called Dravet’s Syndrome is an intractable epilepsy which has many complicated symptoms include severe, intractable epilepsy co-morbidities of ataxia and cognitive impairment. SMEI is typically resistant to standard anticonvulsant pharmacotherapy. Many years’endeavor demonstrated that the genetic etiology of this epilepsy was the mutations in sodium channels and the al subunit of the voltage-gated Na+(NaV1.1) channel encoded by the SCN1A gene, which is the most frequent target of mutations. Different types of SCN1A mutations such as nonsense, frameshift, and missense mutations are located at different sites of the SCN1A gene, which have been identified in patients with SMEI. Some groups proposed a hypothesis for the spectrum of epilepsy syndromes caused by genetic mutations in the SCN1A gene. However, the correlations between phenotypes and genotypes have remained inconclusive so far. Some researches about the pathogenesis indicated that SMEI mutations leaded the loss-of-function of the mutant channels and showed remarkably attenuated or absence of inward sodium currents. Then they proposed that the decrease of the sodium currents might underlie the neuronal hyperexcitability and lead to epileptic seizures. Also the researches on the animal models which were in accordance with this propose indicated those loss-of-function mutant Navl.1channels have severely impaired sodium currents in GABAergic inhibitory interneurons may cause the hyperexcitability in SMEI. Further studies would be necessary to understand the molecular pathology of SMEI.TALEN technology is a powerful tool for genome engineering, which can cleave unique genomic sequences in living cells. TALEN is mainly composed of two parts. One is the Transcription activator-like (TAL) effector, which is a virulence factor of plant pathogenic bacteria in the genus Xanthomonas. The native function of TAL effectors is to subvert host genome regulatory networks after translocation into host cells via the bacterial type III secretion system and bind to effector-specific sequences. The other one is the FokI nuclease, which can efficiently cleave DNA to create targeted DNA double-strand breaks (DSBs) in vivo for genome editing. As FokI cleaves as a dimmer, these TAL effector nucleases (TALENs) function in pairs to create the break. These DSBs are repaired by cell itself through non-homologous end joining (NHEJ) or homologous recombination (HR) to drive targeted gene disruption including small insertions or deletions (InDel). However, homologous recombination (HR) requires a homologous DNA segment as a template to copy the information across the break, which can be used for gene insertion or replacement. Hence TALEN technology provides a robust and rapid designable DNA targeting platform for the interrogation and engineering of biological systems.So far, the scientists challenged so many difficulties on the research of the neurodegenerative diseases because of the limited experimental access to disease-affected human nervous system tissue. Human induced pluripotent stem cell (hiPSC) technology enables the epigenetic reprogramming of human somatic cells into a pluripotent state. The hiPSC can differentiate into any disease-relevant cells type and tissues. So they provide an access to virtually unlimited numbers of patient-specific and disease-specific adult cells for modeling neurological disorders in vitro. The iPSCs can bypass the ethical concerns related to the embryonic stem (ES) cell derivation and potentially issues of allogeneic immune rejection. In principle, patient-specific iPSCs that carry all disease-relevant genetic alterations represents a significant progress for basic biomedical research and drug development in this field.Commonly, we compared the cells from between patient-derived disease-relevant and the normal people but it was a significant defect to do this kind of compare. On one hand, neurodegenerative diseases are usually late age-onset diseases with long latency and slow progression of cellular and pathological changes in vivo. Even more the phenotypes of these diseases are very subtle in vitro. On the other hand, the differences in genetic background of the two cell lines presents a particularly significant impediment to in vitro research. Any difference between the two cell lines could not be concluded of the disease-relevant lesion or the variable genetic background. It’s hard to distinguish these disease-relevant phonotypical changes from unpredictable background-related variations.In order to develop a genetically defined experimental system to study the pathology of epilepsy, we work on:1、The normal person’s induced pluripotent stem cells (iPSCs) was reprogrammed from the skin flbroblast. TALENs technology was performed to target SCN1A gene and genetically engineer endogenous loci in iPSCs. Combining TALEN and iPSC methods, we introduced the point mutations (A5768G) in the SCN1A gene of the iPSCs from the normal people, and then made an "artificial patient" cell line.2、Correspondingly, disease iPSCs were reprogrammed from patient’s skin fibroblasts, repaired the disease-related mutation by TALEN, and then generated a "mutation corrected" cell line.3、The pluripotency of these cell lines was confirmed that they maintained the pluripotent state after the gene manipulation.4、These engineering iPSCs were successfully differentiated into the neurons with functional characters.Based on all the work above, we will do research between the normal cell line and the artificial patient one, which the disease-causing genetic lesion of interest is the sole modified variable. The robust capability to genetically introduce disease-causing point mutations in normal hiPS cell lines may produce a human epileptic cell model for studying epilepsy mechanisms and drug screening. Our study may give a more accurate and credible exposition of the pathogenesis and even some possible suggestions of therapies of epilepsy.