MICA: Development of gene therapy for the incurable inherited childhood epilepsy, Dravet Syndrome

Dravet综合征是一种罕见的，毁灭性的婴儿癫痫。在两岁之前，儿童会出现癫痫发作、运动障碍、认知障碍，并可能死亡。儿童往往不会说话，行为上有困难，给家庭和照料者带来巨大负担。Dravet患儿的SCN1A基因发生突变，该基因产生一种蛋白质“Nav1.1”。这种蛋白质对大脑神经元的功能至关重要，这种蛋白质的突变会破坏大脑电活动的正常平衡。不幸的是，这种疾病仍然是无法治愈的，治疗Dravet的药物效果不好。它们通常有副作用，不能完全控制癫痫发作，也不能改善疾病的其他方面，如运动和认知问题。给药也可能是一个挑战，因为Dravet儿童可能难以吞咽或拒绝食物。有一种老鼠患上了Dravet综合症。它携带着与Dravet儿童相似的突变。这种与人类疾病的相似性意味着Dravet综合征的小鼠模型非常适用于测试Dravet的新疗法。基因治疗提供了许多优于药物治疗和手术的好处。基因治疗是一种治疗方法，它不是简单地治疗症状，而是通过将SCN 1A的正确拷贝直接传递到大脑中的细胞来解决疾病的原因。基因疗法使用类似病毒的称为“载体”的粒子来传递DNA。基因疗法已经被用于治疗患有严重遗传性疾病的儿童，并且现在在神经系统疾病如遗传性帕金森症中也看到了有希望的结果。然而，Dravet综合征的基因疗法面临着一些挑战。SCN1A基因很大，这限制了最常见的基因治疗载体的整合。我们已经鉴定了能够携带全长基因的载体。这种载体已经被用于基因治疗的临床试验，以治疗帕金森氏病的成年患者。Dravet和SCN1A面临的另一个挑战是，在实验室中制备大量基因并准备交付是极其困难的。这使世界各地的基因治疗实验室受挫。我们与一家名为Touchlight Genetics的公司合作，该公司可以使用纯化的酶制造大量DNA。因此，我们希望能够制造大量的SCN1A，这将使我们能够制造基因治疗载体来治疗并可能逆转Dravet的病因。首先，Touchlight Genetics将制造大量的DNA，我们可以用这些DNA制造含有人类SCN 1A基因的慢病毒载体。由此我们将制作基因治疗载体，并测试它们在培养皿中（“体外”）的脑细胞中的作用。这些细胞将是来自具有突变的非功能性SCN1A基因的小鼠的神经元。我们将测量电流（一种称为膜片钳的技术），以检测我们的基因疗法是否成功地传递了SCN 1A基因的工作拷贝，以便细胞能够产生工作的Nav1.1蛋白。如果成功的话，我们将在突变小鼠中测试基因疗法。我们将在他们出生当天将基因治疗载体注射到他们的大脑中，并寻找疾病的症状。我们将它们与正常的未受影响的小鼠进行比较。我们将研究他们的大脑，寻找任何疾病的证据，并可能测量电流，以了解基因治疗后有多少Nav1.1蛋白质被制造出来。我们还将通过将载体注射到一些正常小鼠（不携带SCN 1A突变）的大脑中来检查基因治疗是否安全。该项目旨在开发一种药物来治疗目前无法治愈的毁灭性疾病。如果这个项目成功，我们在制造和提供这种基因疗法中使用的技术可能会使许多其他儿童疾病受益，而基因疗法以前被证明是困难的。

Dravet syndrome is a rare, devastating infantile epilepsy. Before the age of two children suffer seizures, movement problems, cognitive impairment and may die. Often children can't talk, and suffer behavioural difficulties which imposes a tremendous burden on the family and carers. Children with Dravet have mutations in the SCN1A gene, which makes a protein "Nav1.1". This protein is vital for the functioning of neurons in the brain, and the mutations in this protein disrupt the normal balance of electrical activity in the brain. Unfortunately, this disease remains is incurable and drugs to treat Dravet do not work very well. They often have side effects, fail to fully control seizures, and don't improve other aspects of the disease such as the movement and cognitive problems. Administering drugs can also be a challenge, as Dravet children may struggle to swallow or reject food. There is a strain of mice which has a very disease to Dravet syndrome. It carries a similar mutation to that in Dravet children. This similarity to the human disease means that the mouse models of Dravet syndrome are very using for testing new treatments of Dravet. Gene therapy offers many benefits over drug therapy and surgery. Gene therapy is a treatment which, instead of simply treating the symptoms, addresses the cause of the disease by delivering the corrected copies of the SCN1A directly to the cells in the brain. Gene therapy use particles called "vectors" which resemble viruses, to deliver the DNA. Gene therapy has already been used to cure children with severe genetic diseases and promising results are now being seen with neurological diseases such as inherited Parkinsonism.However, gene therapy for Dravet syndrome faces several challenges. The SCN1A gene is large, and this limits incorporation into the most common gene therapy vectors. We have identified a vector which is capable of carrying the full length gene. This vectors has already been used in gene therapy clinical trials to treat adult patients with Parkinson's Disease. Another challenge that is particular to Dravet and SCN1A, is the extreme difficulty in making large amounts of gene in the laboratory, ready for delivery. This has thwarted gene therapy laboratories around the world. We have partnered with a company called Touchlight Genetics who can make large amounts DNA, using purified enzymes. Therefore, we hope to be able to make large amounts of SCN1A which will allow us to make gene therapy vector to treat and potentially reverse the cause of Dravet. Firstly, Touchlight Genetics will make large amounts of DNA with which we can make a lentivirus vector containing the human SCN1A gene. From this we will make gene therapy vectors and test that they work in brain cells in a dish ("in vitro"). These cells will be neurons from the mice which have mutant non-functional SCN1A genes. We will measure the electrical currents (a technique called patch clamping) to detect whether our gene therapy has successfully delivered working copies of the SCN1A gene, so that the cells can make working Nav1.1 protein. If this works, we will test the gene therapy in in mutant mice. We will inject the gene therapy vector into their brain on the day of birth and look for symptoms of the diseases. We will compare them to normal, unaffected mice. We will study their brains for any evidence of disease and may measure electrical currents to see how much of the Nav1.1 protein was made after gene therapy. We will also check that the gene therapy is safe, by injecting vector into brains of some normal mice (carrying no SCN1A mutation). This project is aimed at developing a medicine to treat a devastating disease for which there is presently no cure. If this project works, the technologies we have used in making and delivering this gene therapy may benefit many other childhood diseases where gene therapy has previously proven to be difficult.