eukaryotic expression vectors resistant to transgene silencing

描述(申请人提供)：目前，以质粒为导向的基因表达已接近药效障碍，到目前为止，这一障碍阻碍了以质粒为基础的疗法用于人类健康应用的商业化。以前的创新，如电穿孔(EP)，通过更有效的基因传递增加了转基因表达。在第一阶段，我们开发了有效的最小化无抗生素表达载体，并证明了通过载体设计创新可以显著改善转基因表达。开发了两种平台技术。防止载体骨架介导的转基因沉默的新组合物(反沉默元件：ASE平台)在第二阶段中，我们假设将ASE和TEE载体与最先进的质粒输送相结合，将ASE和TEE载体与最先进的质粒输送相结合，将为基因治疗创造使能的载体输送平台。在特定的目标中，1和2分别确定了用于骨骼肌和皮肤基因治疗的最佳ASE/TEE无抗生素载体-EP递送平台。在具体目标3中，应用皮肤基因治疗平台，创造了一种基于缺氧诱导因子11(HIF-11)的治疗糖尿病足溃疡的基因药物。在特定的目的4中，利用角质形成细胞生长因子(KGF)载体和微磨削给药的组合，开发了一种治疗皮肤衰老的皮肤病基因疗法。具体目标3和4是与约翰·霍普金斯大学的伤口愈合基因治疗专家约翰·哈蒙博士和基因美容有限责任公司的皮肤病基因治疗专家亚伦·塔博尔博士合作进行的。这里开发的载体递送平台将进一步提高基因表达水平，使基因药物能够为未得到满足的公共卫生需求的多种应用提供许可。在第三阶段，糖尿病足部溃疡和皮肤化妆品的基因疗法将进行临床开发。 公共卫生相关性：这项建议的目标是验证一种新的不含抗生素的非病毒基因治疗平台，并因此响应NIGMS SBIR的高度优先领域，开发改进的基因转移载体。这些载体包含瞬时表达增强剂，可以提高转基因表达水平，并在基因传递到皮肤或肌肉后持续时间。该平台将被用于创造基因治疗产品，以治疗糖尿病神经性足部溃疡和皮肤老化。

DESCRIPTION (provided by applicant): Plasmid-directed gene expression is now near the efficacy barrier that to date has prevented commercialization of plasmid-based therapies for human health applications. Previous innovations such as electroporation (EP) increased transgene expression through more effective gene delivery. In Phase I we developed potent minimalized antibiotic-free expression vectors, and demonstrated dramatic improvements in transgene expression may be obtained through vector design innovations. Two platform technologies were developed. " Novel compositions that prevent vector-backbone mediated transgene silencing from plasmid vectors (Anti-Silencing Elements: ASE platform) " Novel vector backbone functionalities that improve transgene expression from plasmid vectors after transient transfection (transient expression enhancers: TEE platform) In Phase II we hypothesize that combining ASE and TEE vectors with state of the art plasmid delivery will create enabling vector-delivery platforms for gene therapy. In Specific Aims 1 and 2 optimal ASE/TEE antibiotic-free vector - EP delivery platforms for skeletal muscle and cutaneous gene therapy, respectively, are identified. In Specific Aim 3 the cutaneous gene therapy platform is applied to create a hypoxia-inducible factor 11 (HIF-11) based gene medicine for diabetic foot ulcer treatment. In Specific Aim 4 a dermatological gene therapy to treat skin aging is developed using a keratinocyte growth factor (KGF) vector- microdermabrasion delivery combination. Specific Aims 3 and 4 are performed in collaboration with wound healing gene therapy expert Dr. John Harmon at Johns Hopkins University and dermatology gene therapy expert Dr Aaron Tabor at Gene Facelift, LLC. The vector-delivery platforms developed herein will further improve gene expression to levels that will enable gene medicine licensure for multiple applications for unmet public health needs. In Phase III the gene therapies for diabetic foot ulcers and skin cosmetics will undergo clinical development. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to validate a novel antibiotic-free non-viral gene therapy platform, and as such is responsive to NIGMS SBIR high-priority area of interest in development of improved vectors for gene transfer. The vectors contain transient expression enhancers that improve transgene expression level and duration after gene delivery to skin or muscle. The platform will be applied to create gene therapy products to treat diabetic neuropathic foot ulcers and skin aging.