Altering Energy Balance by Systemic Delivery of RNAi to the Neuroendocrine Brain

描述（由申请人提供）：迄今为止，操纵下丘脑功能主要依赖于使用条件敲除小鼠或转基因过表达。这些方法的主要局限性在于，它们没有考虑到神经内分泌神经元及其投射发育的复杂性，以及这些神经元在生命早期被操纵时发生的代偿性适应。另外，递送sirna的腺相关病毒（AAV）微注射已被用于改变成年期下丘脑功能。该技术的最大限制是侵入性和相对低效率的过程。本应用旨在通过开发一种新颖的微创方法，以时间定义和细胞特异性的方式来操纵下丘脑神经元功能，从而绕过这些限制。在可以作为这些研究原型的下丘脑系统中，弓状核（ARC）的黑素皮质素系统是一个理想的候选者。它已被广泛研究，并证明在调节食物摄入、体重和葡萄糖稳态中调节能量平衡方面发挥关键作用。它由两个主要的神经元群组成，它们的功能相反；含有POMC的神经元抑制进食并刺激能量消耗，含有Y/ agouti相关肽（NPY/AgRP）的神经元刺激摄食行为并抑制能量消耗。通过测量食物摄入量和体重，可以无创地可靠地评估改变任一神经元亚群功能的后果。从人类健康的角度来看，开发新的工具来研究这一系统具有巨大的价值；由于早期营养改变导致儿童和成人肥胖的急剧增加，因此迫切需要开发新的方法来更好地理解控制摄食行为和能量稳态的核心机制。这是一个特别重要的问题，因为在人类妊娠后期和啮齿类动物产后早期的“发育规划”关键时期发生的营养挑战可能永久性地影响能量平衡。我们拟开发的技术的一个主要优点是，在完成能量平衡的开发规划后，可以对ARC功能进行修改。我们建议通过在血管内给药修饰的AAV2颗粒转导下丘脑细胞，将RNA干扰（RNAi）传递到ARC，从而沉默POMC和AgRP基因。我们期望这些研究的成功实施将为最终应用类似的方法来治疗神经内分泌脑疾病铺平道路。我们还预计这些研究将为基础研究目的和新兴疗法提供新的大脑递送策略的基础。

DESCRIPTION (provided by applicant): To date, manipulating hypothalamic function mostly relies on the use of conditional knockout mice or transgenic overexpression. The major limitation to these approaches is that they do not take into account the complexities in the development of neuroendocrine neurons and their projections, and the compensatory adaptations that occur when these neurons are manipulated during early life. Alternatively, microinjections of adeno-associated viruses (AAV) delivering siRNAs have been used to modify hypothalamic function in adulthood. The greatest limitation of this technique is the invasiveness and relative inefficiency of the procedure. The present application intends to circumvent these limitations by developing a novel, minimally invasive method to manipulate hypothalamic neuronal function in a temporally defined and cell-specific manner. Among the hypothalamic systems that can be used as a prototype for these studies, the melanocortin system of the arcuate nucleus (ARC) stands out as an ideal candidate. It has been extensively studied and shown to play a critical role in regulating energy balance through modulation of food intake, body weight and glucose homeostasis. It is composed of two major populations of neurons with opposite functions; neurons containing pro-opiomelanocortin (POMC) inhibit the drive to eat and stimulate energy expenditure, neurons containing neuropeptide Y/Agouti-related peptide (NPY/AgRP) stimulate feeding behavior and inhibit energy expenditure. The consequences of altering the functions of either neuronal subset can be reliably assessed non-invasively, by measuring food intake and body weight. From the human health standpoint, developing new tools to study this system has an enormous value; the dramatic increase in childhood and adult obesity resulting from nutritional alterations during early life makes it urgent to develop novel methods to better understand the central mechanisms underlying the control of feeding behavior and energy homeostasis. This is a particularly important issue because energy balance can be permanently affected by nutritional challenges taking place during the critical period of "developmental programming" that in humans occurs during late gestation and in rodents, during the early postnatal period. A major advantage of the technology we propose to develop is that it can be used to modify ARC function after the developmental programming of energy balance is complete. We propose to silence the POMC and AgRP genes by delivering RNA interference (RNAi) to the ARC via the intravascular administration of modified AAV2 particles engineered to transduce hypothalamic cells. We anticipate that the successful execution of these studies will pave the way to the eventual application of similar approaches to treat disorders of the neuroendocrine brain. We also anticipate that these studies will provide the basis for new delivery strategies to the brain for basic research purposes and emerging therapies.