Next Generation Gene-Gun Delivered DNA and RNA Immunotherapeutic Vaccines for Melanoma.

项目总结 Orlance已经开发出一种下一代基因枪(Mach-1 GG)，它可以有效地将DNA和RNA输送到 表皮细胞，导致强大的免疫反应。对个别患者的肿瘤进行测序已经导致了 识别可作为癌症疫苗靶点的个性化新抗原。然而， 能够有效地传递这些癌症新抗原并促进局部肿瘤诱导的技术 仍然需要特定的T细胞反应。使用特定疫苗的核酸(NA；DNA和RNA)疫苗 实现细胞内递送的制剂或递送技术提供了实现的巨大希望 这个目标。这些方法包括电穿孔(EP)或喷射注射，用于即时传递DNA或脂质纳米粒(LNPs) 用于即时消息传递RNA。然而，这些交付方式有不同的缺点，包括要求 适用于高剂量(1-5毫克DNA)、因稳定性有限(RNA/LNPs)、反应性或疼痛而超低温储存 给药后，针对特定组织的免疫反应的能力有限。GG需要 以金微粒为载体输送室温稳定的冻干DNA或RNA疫苗。它实现了无痛 并以极低的剂量(1-4微克)直接进入皮肤细胞内，从而导致全身、粘膜和 局部皮肤免疫反应，可以为癌症的治疗提供好处，特别是， 黑色素瘤。Mach-1 GG是基于之前成功的GG，该GG诱导了强大的抗体和T细胞 I期人体临床试验的反应。马赫-1在早期的基础上提供了显著的改进 设备。在这里，我们将研究使用Mach-1在小鼠体内传递DNA或RNA癌症疫苗的可行性 并验证Mach-1将在免疫原性和有效性方面优于其他DNA/RNA的假设 治疗黑色素瘤的递送技术。我们将首先确定共同交付一组新的基因佐剂是否会 提高Mach-1 DNA和RNA疫苗诱导黑色素瘤特异性T细胞反应的能力。接下来，我们 将确定在相同剂量或最佳增强方案中结合DNA和RNA是否提供协同效应。 然后我们将比较马赫-1递送的DNA和/或RNA黑色素瘤疫苗与通过EP或RNA递送的DNA LNPs对小鼠免疫原性和保护作用的研究。这项工作将通过两个目标完成： 目的1：探讨基因佐剂对GG介导的DNA疫苗免疫原性和免疫效果的影响 RNA黑色素瘤疫苗。目的2：确定是否将优化的佐剂DNA和RNA疫苗结合在 与EP递送相比，相同剂量或在主要增强方案中可增强免疫原性和疗效 DNA和LNP在黑色素瘤小鼠模型中的RNA传递。这些目标的成功实现将 使马赫-1成为运送癌症疫苗的有效设备。

PROJECT SUMMARY Orlance has developed a next-generation Gene Gun (MACH-1 GG) that efficiently delivers DNA and RNA into epidermal cells, leading to robust immune responses. Sequencing of tumors from individual patients has led to the identification of personalized neoantigens that could be targeted with cancer vaccines. However, technologies that can effectively deliver these cancer neoantigens and promote the induction of localized tumor specific T cell responses are still needed. Nucleic acid (NA; DNA and RNA) vaccines administered using specific formulations or delivery technologies that achieve intracellular delivery offer considerable promise to achieve this goal. These include electroporation (EP) or jet injection for IM delivery of DNA or lipid nanoparticles (LNPs) for IM delivery of RNA. These delivery modalities, however, have different drawbacks including a requirement for high doses (1-5 mg of DNA), ultra-cold storage due to limited stability (RNA/LNPs), reactogenicity or pain post-administration, and a limited ability to target immune responses to specific tissues. The GG entails the delivery of room temperature stable lyophilized DNA or RNA vaccines on gold microparticles. It achieves painless and direct intracellular delivery into skin cells with very low doses (1-4 µg) that results in systemic, mucosal and localized skin immune responses that could provide a benefit for treatment of cancers and, in particular, melanoma. The MACH-1 GG is based on a previous successful GG that induced strong antibody and T cell responses in phase I human clinical trials. The MACH-1 provides significant improvements over this earlier device. Here, we will investigate the feasibility of using MACH-1 to deliver DNA or RNA cancer vaccines in mice and test the hypothesis that MACH-1 will offer advantages in immunogenicity and efficacy over other DNA/RNA delivery technologies for melanoma. We will first determine if co-delivering a novel set of genetic adjuvants will increase the ability of MACH-1 DNA and RNA vaccines to induce melanoma-specific T cell responses. Next, we will determine if combining DNA and RNA in the same dose or in a prime-boost regimen offers synergistic effects. We will then compare MACH-1 delivery of DNA and/or RNA melanoma vaccines to DNA delivery by EP or RNA delivery by LNPs for immunogenicity and protective efficacy in mice. This work will be accomplished in two Aims: Aim 1: Investigate the impact of genetic adjuvants on the immunogenicity and efficacy of GG delivered DNA and RNA melanoma vaccines. Aim 2: Determine if combining the optimized adjuvanted DNA and RNA vaccines in the same dose or in a prime-boost regimen enhances immunogenicity and efficacy compared to EP delivery of DNA and LNP delivery of RNA in a mouse model of melanoma. Successful completion of these Aims will establish MACH-1 as an effective device to deliver cancer vaccines.