Targeted Chemoprevention for Melanoma

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。尽管它具有致命性，但目前还没有靶向的局部化学预防剂来抑制这种癌症的早期形式，或者当细胞存在于早期原发病变附近的淋巴系统中时。其中一个可能的化学预防靶点是Akt3，它的活性在约70%的肿瘤中增加，从而促进黑色素瘤的发展。使用基于siRNA的药物靶向早期黑素细胞病变细胞来抑制Akt3和其他关键激酶对于靶向黑色素瘤化学预防可能是重要的，但siRNA的递送仍然是一个挑战。目前，没有技术或方法利用siRNA作为化学预防剂来抑制黑色素瘤的早期发展。该应用侧重于黑色素瘤化学预防，通过使用基于sirna的药物靶向早期黑色素细胞病变，抑制Akt3和突变体V600EB-Raf， GSK31或Wee1激酶，这些激酶被证明可以协同抑制黑色素细胞病变细胞。因此，本研究的核心假设是，靶向Akt3和突变型V600EB-Raf、GSK31或Wee1激酶的siRNA可以通过新型超声纳米脂体技术递送到含有早期黑色素细胞病变的皮肤中，以防止黑色素瘤的发展和这些细胞入侵淋巴系统。其基本原理是靶向这些基因的siRNA将作为途径特异性靶向化学预防剂来预防黑色素瘤的发展。我们基于初步发现的主要证明，确定了可以与Akt3抑制协同作用的激酶。此外，我们发现针对Akt3和V600EB-Raf的siRNA可以被加载到纳米脂体中，并在超声治疗后放置在含有黑素细胞病变的皮肤上，有效地抑制黑色素瘤细胞的存活，如果一起递送，可以协同预防疾病的发展。因此，本应用的目的是首先评估含有sirna靶向Akt3和突变体V600EB-Raf、GSK31或Wee1激酶的新型纳米脂质体在转基因动物模型中的化学预防效果，其中Akt3和V600EB-Raf都被解除调控以促进自发黑色素细胞病变的发展。这将通过将靶向Akt3和V600EB-Raf、GSK31或Wee1的siRNA加载到抗体靶向纳米脂质体中，并经过超声治疗，有效地将药物输送到动物皮肤中自发形成的早期黑色素细胞病变中来实现。其次，我们将确定这些药物是否可以预防或减少早期黑素细胞对淋巴结的侵袭，以减少疾病的发展和帮助生存。这将通过在黑素细胞病变细胞侵入淋巴结池之前或之后用纳米脂质体siRNA治疗小鼠来完成，以确定它是否会阻止疾病进展。在超声介导的皮肤渗透后，使用抗体靶向纳米脂质体制剂递送siRNA对抗关键激酶是一种新的方法，对于更有效地化学预防黑色素瘤和抑制其进入和通过淋巴系统的扩散至关重要。

DESCRIPTION (provided by applicant): Malignant melanoma is the most deadly of all skin cancers. Despite its lethality, no targeted topical chemopreventive agents exist to inhibit this cancer in its earliest forms or when cells are present in the lymphatic system in the proximity of the early primary lesion. Among possible chemopreventive targets is Akt3 whose activity increases in ~70% of tumors to promote melanoma development. Targeting early melanocytic lesion cells using siRNA-based agents to inhibit Akt3 and other key kinases could be important for targeted melanoma chemoprevention, but delivery of siRNA remains a challenge. Currently, no technology or approach utilizes siRNA as a chemopreventive agent to inhibit early melanoma development. This application focuses on melanoma chemoprevention by targeting early melanocytic lesions using siRNA-based agents to inhibit Akt3 and mutant V600EB-Raf, GSK31 or Wee1 kinases, which are shown to synergistically inhibit melanocytic lesion cells. Thus, the central hypothesis for the proposed research is that siRNA targeting Akt3 and mutant V600EB-Raf, GSK31 or Wee1 kinases can be delivered via novel ultrasound-nanoliposomal technology into skin containing early melanocytic lesions to prevent melanoma development and invasion of these cells into or through the lymphatic system. The rationale is that siRNA targeting these genes would serve as pathway specific targeted chemopreventive agents to prevent melanoma development. We formulated this hypothesis based on proof-of-principal preliminary discoveries identifying kinases to target that can synergise with Akt3 inhibition. Furthermore, we show that siRNA against Akt3 and V600EB-Raf can be loaded into nanoliposomes and placed on skin containing melanocytic lesions following ultrasound treatment, effectively inhibiting melanoma cell survival, and if delivered together it can cooperatively prevent disease development. Thus, the objectives of this application are to first, evaluate the chemopreventive efficacy of novel nanoliposomes containing siRNA-targeting Akt3 and mutant V600EB-Raf, GSK31 or Wee1 kinases in a transgenic animal model of the disease in which both Akt3 and V600EB-Raf are deregulated to promote spontaneous melanocytic lesion development. This will be accomplished by loading siRNA targeting Akt3 and V600EB-Raf, GSK31 or Wee1 into antibody-targeted nanoliposomes and following ultrasound treatment, effectively deliver agents into spontaneously developing early melanocytic lesions in skin of animals. Second, we will determine whether these agents can prevent or decrease lymph node invasion by early melanocytic cells to decreased disease development and aid survival. This will be accomplished by treating mice with nanoliposomal siRNA prior to or following invasion of the lymph node basin by melanocytic lesion cells to establish whether it will prevent disease progression. Delivery of siRNA against key kinases using an antibody-targeted nanoliposomal formulation following ultrasound-mediated skin permeabalization is a novel approach critically needed for more effective chemoprevention of melanoma and inhibition of its spread into and through the lymphatic system.