NEURONAL FUNCTION IN CRANIOFACIAL DEVELOPMENT

越来越多的证据表明，神经系统在颅面发育中起着重要作用。临床上，遗传性溶酶体储存疾病患者常表现为生长发育障碍、骨骼异常和颅面畸形，以及神经变性、失明、智力低下、瘫痪和痴呆。目前，还没有针对这类疾病的预防或综合治疗。大脑、三叉神经节和脊神经节的神经元表现出肿胀的空泡化核膜，储存了过量的复杂大分子，导致神经元功能障碍和神经支配受损。基于这些观察，我们假设正常的神经元功能是颅面发育所必需的，而溶酶体储存引起的神经元功能障碍可能是导致颅面发育异常的原因之一。为了解决这一假设，我们将使用一个严重的颅面部发育不良、生长迟缓和面部畸形的动物模型，该模型与由于缺乏β-己糖胺酶而导致的粘多糖和GM2神经节苷脂的过度神经元存储相关(六基因敲除小鼠)。在一个严格调控的可诱导表达系统的控制下，编码人β-己糖氨酸酶的转基因将被靶向于6-/-/6-B-/-小鼠的神经元。利用这一小鼠模型，我们将通过在关键发育时期有条件地恢复神经元中的β-己糖苷酶活性，来确定正常颅面发育是否需要神经元功能。此外，我们将利用单纯疱疹病毒1型(HSV)扩增子和猫咪免疫缺陷病毒(FIV)系统，开发治疗β-己糖氨酸酶缺乏症的基因治疗策略。我们将在中枢(脑)和外周(受影响的颅面结构)应用β-己糖苷酶治疗基因，并在体内比较它们的治疗效果。尽管该小鼠模型本身不能复制人类疾病，但它提供了机会(1)研究神经功能在溶酶体储存疾病中出现的异常头面部发育中的作用，(2)评估嗜神经病毒用于治疗基因的使用，以及(3)评估围产期基因治疗在遗传性代谢疾病治疗中的效果。

An increasing body of evidence suggests that the nervous system plays an important role in craniofacial development. Clinically, patients suffering from inherited lysosomal storage diseases often exhibit growth impairment, skeletal abnormalities and craniofacial malformations, as well as neurodegeneration, blindness, mental retardation, paralysis and dementia. Currently, there is no preventive or comprehensive treatment for this class of disorders. The neurons of the brain, trigeminal and spinal root ganglia display swollen vacuolated perikarya stored with excessive amounts of complex macromolecules, leading to neuronal dysfunction and impaired innervation. Based on these observations, we hypothesize that normal neuronal function is required for craniofacial development, and that neuronal dysfunction due to lysosomal storage may contribute to aberrant craniofacial development. To address this hypothesis, we will employ an animal model with severe craniofacial dysostosis, growth retardation and facial dysmorphism associated with excessive neuronal storage of mucopolysaccharides and GM2 gangliosides due to lack of beta-hexosaminidase (hexA-/-/hexB-/- double knockout mice). The expression of a transgene encoding for human beta-hexosaminidase under the control of a tightly regulated inducible expression system will be targeted to the neurons of hexA-/-/hexB-/- mice. Utilizing this murine model we will determine whether neuronal function is required for normal craniofacial development by conditionally restoring beta- hexosaminidase activity in neurons during critical developmental periods. Moreover, we will develop gene therapy strategies for the treatment of beta-hexosaminidase deficiency, employing the Herpes Simplex Virus-1 (HSV) Amplicon and the Feline Immunodeficiency Virus (FIV) systems. We will administer the beta-hexosaminidase therapeutic gene centrally (brain) as well as peripherally (affected craniofacial structures), and compare their treatment efficacy in vivo. Although this mouse model does not replicate a human disease per se, it provides the opportunity to (1) investigate the role of neuronal function in aberrant craniofacial development seen in lysosomal storage diseases, (2) evaluate the use of neurotropic viruses for the delivery of therapeutic genes, and (3) assess the efficacy of perinatal gene therapy in the treatment of inherited metabolic disorders.