Melanoma Chemoprevention by Targeting Mutant (V600E) B-Raf

描述（由申请人提供）：恶性黑色素瘤是所有皮肤癌中最致命的。一种称为B-Raf的蛋白质在大约90%的痣和癌前早期黑色素瘤细胞中突变为组成型活性形式（V600 E-B-Raf）。可以制备特异性降低突变体V600 E-B-Raf蛋白而非正常野生型蛋白表达的SiRNA。使用这种siRNA靶向V600 E-B-Raf抑制早期黑素瘤发展，使其成为理想的化学预防剂。挑战在于开发一种策略，将这种针对V600 E-B-Raf的化学预防性siRNA递送到皮肤细胞中，这是该项目的目标。目前，没有技术或方法利用siRNA作为化学预防剂来抑制早期黑色素瘤发展。所提出的研究的中心化学预防假设是，通过新的超声脂质体技术将针对突变V600 E-B-Raf的siRNA递送到含有早期黑色素细胞病变的皮肤中，将是逆转或延缓痣或早期病变发展的有效化学预防剂。我们根据初步发现制定了这一假设，使用一个模型，其中皮肤是在组织培养皿中制成的，其中含有来自早期黑色素瘤的细胞，这些细胞发展成组织学上类似于早期癌前黑色素瘤的肿瘤。作为原理证明，我们可以证明针对突变体V600 E-B-Raf的siRNA延缓这些早期黑素细胞病变的发展。此外，我们正在开发一种新颖的创新技术，用于将siRNA递送到皮肤中的癌细胞中，该技术将皮肤超声处理与脂质体递送载体结合在一起。我们准备进行拟议的研究，已确定突变体V600 E-B-Raf作为潜在的化学预防目标。此外，我们有初步的数据，使用含有早期黑色素瘤的皮肤重建，这已经被超声处理，并暴露于脂质体装载siRNA显示这种技术的潜在效用。此外，我们还组建了一个研究团队，独特地结合了黑色素瘤，皮肤重建，siRNA靶向以及超声和脂质体技术的多学科专家，每个人都对黑色素瘤和化学预防剂的开发具有长期的研究兴趣。这种化学预防剂的开发将通过减少黑色素瘤的发展而显著影响人类健康，从而直接降低死亡率。

DESCRIPTION (provided by applicant): Malignant melanoma is the most deadly of all skin cancers. A protein called B-Raf is mutated to a constitutively active form (V600E-B-Raf) in approximately 90% of moles and premalignant early melanoma cells. SiRNA can be made that specifically reduces expression of mutant V600E-B-Raf protein but not normal wild-type protein. Targeting V600E-B-Raf using this siRNA inhibits early melanoma development making it an ideal chemopreventive agent. The challenge is to develop a strategy to deliver this chemopreventive siRNA against V600E-B-Raf into cells in the skin, which is the goal of this project. Currently, no technology or approach utilizes siRNA as a chemopreventive agent to inhibit early melanoma development. The central chemoprevention hypothesis for the proposed research is that siRNA against mutant V600E-B-Raf delivered via novel ultrasound-liposome technology into skin containing early melanocytic lesions will be an effective chemopreventive agent for reversing or retarding development of moles or early lesions. We formulated this hypothesis based on preliminary findings using a model in which skin is made in a tissue culture dish that contains cells derived from early-stage melanomas, which develop into tumors histologically resembling early premalignant melanomas. As proof-of-principle, we can show that siRNA against mutant V600E-B-Raf retards development of these early melanocytic lesions. Furthermore, we are developing a novel and innovative technology for delivering siRNA into cancer cells in skin that combines skin ultrasonication together with liposomal delivery vehicles. We are prepared to undertake the proposed research, having identified mutant V600E-B-Raf as a potential chemopreventive target. Furthermore, we have preliminary data using skin reconstructs containing early melanomas, which have been ultrasonicated and exposed to liposomes loaded with siRNA showing the potential utility of this technology. In addition, we have assembled a research team that uniquely combines multidisciplinary experts in melanoma, skin reconstruction, siRNA targeting, as well as ultrasound and liposome technology, each of who has long-standing research interests in melanoma and development of chemopreventive agents. Development of this chemopreventive agent would significantly impact human health by reducing melanoma development, thereby directly decreasing mortality rates.