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的作用。临床上，患有遗传性溶酶体贮积病的患者通常表现出生长障碍、骨骼异常和颅面畸形，以及神经变性、失明、智力迟钝、瘫痪和痴呆。目前，对这类疾病没有预防性或综合性治疗。脑、三叉神经和脊根神经节的神经元显示出储存有过量复杂大分子的肿胀空泡化胞体，导致神经元功能障碍和神经支配受损。基于这些观察结果，我们假设颅面发育需要正常的神经元功能，并且由于溶酶体储存导致的神经元功能障碍可能导致颅面发育异常。为了解决这一假设，我们将采用与由于缺乏β-氨基己糖苷酶而导致的粘多糖和GM 2神经节苷脂的过度神经元储存相关的严重颅面骨发育不全、生长迟缓和面部畸形的动物模型（hexA-/-/hexB-/-双敲除小鼠）。在严格调控的诱导型表达系统的控制下，编码人β-氨基己糖苷酶的转基因的表达将靶向hexA-/-/hexB-/-小鼠的神经元。利用该鼠模型，我们将通过在关键发育期有条件地恢复神经元中的β-氨基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，我们将开发用于治疗β-氨基己糖苷酶缺乏症的基因治疗策略，采用单纯疱疹病毒-1（HSV）扩增子和猫免疫缺陷病毒（FIV）系统。我们将在中枢（脑）和外周（受影响的颅面结构）给予β-氨基己糖苷酶治疗基因，并比较它们在体内的治疗效果。虽然这种小鼠模型本身不能复制人类疾病，但它提供了以下机会：（1）研究神经元功能在溶酶体贮积病中观察到的异常颅面发育中的作用，（2）评价嗜神经病毒用于递送治疗性基因的用途，以及（3）评估围产期基因治疗在治疗遗传性代谢紊乱中的疗效。

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.