ORAL GENE THERAPY WITH NiMOS FOR INFLAMMATORY BOWEL DISEASE

描述(申请人提供)：炎症性肠病(IBD)影响多达100,000个30岁以下的美国人。最近的研究表明，IBD可能是由胃肠道(GI)促炎和抗炎细胞因子之间微妙平衡的改变引起的。肿瘤坏死因子-α(TNF)和白介素10(IL-10)分别作为促炎和抗炎细胞因子的作用已在临床前和临床研究中得到验证。在这项拟议的研究中，一个跨学科的研究小组将评估在急性结肠炎诱导的Balb/c小鼠中使用可溶性肿瘤坏死因子受体-1(sTNF-R1)和IL-10表达质粒DNA的口服基因治疗。质粒DNA将被包裹在明胶纳米颗粒中，再由聚己内酯(PCL)基质保护，形成直径1-5 mm的纳米颗粒微球口腔系统(NIMOS)。我们使用非病毒基因递送载体的创新策略依赖于这样一个事实，即明胶纳米颗粒可以有效地包裹质粒DNA，通过内吞作用内化到细胞内，防止细胞中的DNA降解，并允许有效的转染。PCL基质有望在口服时保护含有DNA的纳米颗粒，并允许由于脂肪酶的酶降解作用而在小肠和大肠内释放。我们的初步研究表明，优化的尼莫斯制剂在禁食大鼠的小肠和大肠中的滞留时间更长，并且可以提供报告质粒在小肠和大肠中的转染。本R01应用的具体目的是：(1)制备、表征和优化包含编码sTNF-R1和IL-10的质粒DNA的nimos配方；(2)评估nimos口服给药后在NAVE和急性结肠炎诱导的Balb/c小鼠中的生物分布模式和滞留情况；(3)测定含DNA的Nimos在NAVE和结肠炎诱导的小鼠中的定性和定量转染效率；以及(4)评价表达的sTNF-R1和IL-10对口服和直肠给药的急性结肠炎小鼠的治疗效果。这项研究将对口服治疗性基因传递的障碍和NIMOS作为安全有效的非病毒载体系统的潜力提供重要的理解。使用这种临床可翻译的策略进行口服基因治疗IBD的潜力在有效治疗这种疾病方面具有巨大的前景。 项目简介：炎症性肠病(IBD)是一个重要的临床问题，每10万名30岁以下的美国人中就有100人受到影响。最近的研究表明，IBD可能是由胃肠道(GI)中促炎症和抗炎蛋白分子(称为细胞因子)之间微妙平衡的变化引起的。由于蛋白质降解酶的存在，口服蛋白质药物并不能提供有效的治疗方法。在这项拟议的研究中，一个跨学科的研究小组将评估用编码可溶性受体(可溶性肿瘤坏死因子受体-1，sTNF-R1)和抗炎细胞因子白细胞介素10(IL-10)的质粒DNA进行口服基因治疗是否可以作为治疗IBD的临床可翻译替代方案。我们研制了纳米微球口服系统(NIMOS)，以提供安全和有效的基因传递机制，通过口服给药到小肠和大肠。编码sTNF-R1和IL-10的质粒DNA将被包裹在明胶纳米颗粒中，并由聚己内酯(PCL)基质进一步保护，形成1-5微米的颗粒，以实现有效的输送。一旦NIMO到达小肠和大肠，PCL外壳就被降解，含有DNA的明胶纳米颗粒被释放出来，供细胞摄取和有效的基因表达。我们的初步研究表明，针对口服基因传递进行优化的NIMOS可以将包裹的纳米颗粒定位在禁食大鼠的小肠和大肠中，并导致包裹的报告质粒转染。在这项研究中，我们将开发带有编码sTNF-R1和IL-10的质粒DNA的尼莫斯，对其进行表征，并优化其口服给药。在优化了含有DNA的NIMOS之后，我们将研究NIMOS在NAVE和结肠炎诱导的Balb/c小鼠中的分布和滞留。接下来将检测sTNF-R1和IL-10的定性和定量转染效率。最后，我们将仔细评估口服含有编码sTNF-R1和IL-10的质粒DNA的nimos对结肠炎小鼠的治疗效果。这项研究的结果将是非常有益的，因为表达的治疗性蛋白可以在疾病部位局部产生很长一段时间。此外，NIMOS将用于其他口服基因治疗策略，用于治疗疾病或口服DNA疫苗。

DESCRIPTION (provided by applicant): Inflammatory bowel disease (IBD) affects as many as 100 per 100,000 Americans under the age of 30. Recent studies suggest that IBD may be caused by alterations in the delicate balance between pro- and anti- inflammatory cytokines in the gastro-intestinal (GI) tract. The role of tumor necrosis factor-alpha (TNF) and interleukin-10 (IL-10) as pro- and anti-inflammatory cytokines, respectively, has been examined in preclinical and clinical studies. In the proposed study, an interdisciplinary team of investigators will evaluate oral gene therapy using soluble TNF receptor-1 (sTNF-R1) and IL-10 expressing plasmid DNA in acute colitis-induced Balb/c mice. The plasmid DNA will be encapsulated in gelatin nanoparticles, which are further protected by a poly(epsilon- caprolactone (PCL) matrix to form Nanoparticles-in-Microsphere Oral System (NiMOS) of 1-5 mm in diameter. Our innovative strategy using non-viral gene delivery vector relies on the fact that gelatin nanoparticles can efficiently encapsulate plasmid DNA, are internalized in cells by endocytosis, protects against DNA degradation in cells, and allows for efficient transfection. The PCL matrix is expected to protect the DNA- containing nanoparticles upon oral administration and allow the release to occur in the small and large intestine due to enzymatic degradation with lipases. Our preliminary studies show that optimized NiMOS formulation have longer residence time in the small and large intestine upon administration in fasted rats and can afford transfection of reporter plasmids in the small and large intestine. The specific aims of this R01 application are: (1) to prepare, characterize, and optimize NiMOS formulations containing plasmid DNA encoding for sTNF-R1 and IL-10, (2) evaluate the biodistribution pattern and residence profile of NiMOS upon oral administration in naove and acute colitis-induced Balb/c mice, (3) determine qualitative and quantitative transfection efficiency upon and oral and rectal administration of DNA- containing NiMOS in naove and colitis-induced mice, and (4) evaluate the therapeutic efficacy of expressed sTNF-R1 and IL-10 in acute colitis-induced mice upon oral and rectal administration. This study will provide important understanding of the barriers to oral therapeutic gene delivery and the potential of NiMOS as safe and effective non-viral vector system. The potential of oral gene therapy for the treatment of IBD using this clinically-translatable strategy has tremendous promise in the effective management of this disease. Project Narrative: Inflammatory bowel disease (IBD) is a significant clinical problem affecting as many as 100 per 100,000 Americans under the age of 30. Recent studies suggest that IBD may be caused by the alternations in the delicate balance between pro- and anti-inflammatory protein molecules, called cytokines, in the gastro- intestinal (GI) tract. Due to the presence of proteolytic degrading enzymes, oral administration of protein drugs does not provide an effective therapy. In the proposed study, an interdisciplinary group of investigators will evaluate if oral gene therapy with plasmid DNA that encodes for soluble receptor (soluble tumor necrosis factor receptor-1, sTNF-R1) and anti-inflammatory cytokine interleukin-10 (IL-10) can be used as a clinically translatable alternative for treatment of IBD. We have formulated Nanoparticles-in-Microsphere Oral System (NiMOS) to provide a safe and effective gene delivery mechanism to the small and large intestine upon oral administration. The sTNF- R1- and IL-10-encoding plasmid DNA will be encapsulated in gelatin nanoparticles, which are further protected with a poly(epsilon-caprolactone) (PCL) matrix to form 1-5 micron particles for effective delivery. Once the NiMOS reach small and large intestine, the PCL shell is degraded and the DNA-containing gelatin nanoparticles are released for cellular uptake and efficient gene expression. Our preliminary studies show that NiMOS, optimized for oral gene delivery, can localize the encapsulated nanoparticles in the small and large intestine in fasted rats and lead to transfection of the encapsulated reporter plasmid. In this study we will develop NiMOS with plasmid DNA encoding for sTNF-R1 and IL-10, characterize, and optimize them for oral delivery. Following the optimization of DNA-containing NiMOS, we will examine distribution and residence of NiMOS upon oral administration in naove and colitis-induced Balb/c mice. The qualitative and quantitative transfection efficiency of sTNF-R1 and IL-10 will be examined next. Lastly, we will carefully evaluate the therapeutic effectiveness of orally-administered NiMOS containing plasmid DNA that encodes for sTNF-R1 and IL-10 in colitis-induced mice. The results of this study will be extremely beneficial, since the expressed therapeutic protein can be produced locally at the disease site for a long period of time. In addition, NiMOS would be useful for other oral gene therapy strategies for treatment of diseases or oral DNA vaccination.