Oral Nonviral Gene Delivery for Hemophilla A

描述(申请人提供)：基因治疗是治疗血友病的一种很有前途的方法。目前，由于缺乏安全有效的递送载体，它们的治疗潜力受到限制。虽然病毒仍然是实现体内高效DNA转移的首选载体，但对其长期安全性的保留意见仍在继续。免疫原性和毒性问题仍然是一个挑战。逆转录病毒的细胞有丝分裂的局限性，腺病毒的污染，以及腺相关病毒的包装限制也降低了它们的吸引力。因此，非病毒载体因其合成容易、免疫原性低和不受限制的质粒大小而受到越来越多的关注。非病毒载体在解决扩大规模、储存稳定性和质量控制等制药问题方面面临的挑战也较小。然而，非病毒基因传递所能达到的基因表达水平是低的和短暂的。因此，为了达到基因药物应用的治疗水平，经常给药是必要的。到目前为止，几乎所有的非病毒转基因注射都是静脉注射或肌肉注射，这种有侵入性的方法不具吸引力，也不适用于广泛使用。在血友病基因治疗中，这种方法没有那么有吸引力，也不比目前的蛋白质替代疗法好多少。口服给药可能会使基因治疗变得实用。本研究的目的是验证以壳聚糖和聚磷酸酯S为非病毒基因载体的FVIII基因可以通过口服途径有效传递的假说。我们已经证明，口服由质粒DNA与壳聚糖(一种天然的生物相容多糖)复合而成的DNA纳米颗粒，可导致转导基因在肠上皮细胞中表达。然而，在这些研究中，转基因表达一直很低。该方案的目的是通过优化载体设计和口服大分子制剂来改进非病毒口服基因传递技术。为了确定这一口服基因药物概念的潜力，我们将追求以下具体目标：1)设计和合成具有有利于口服给药的特性的基因载体；2)确定DNA纳米颗粒的重要特性以获得最佳摄取；以及3)在FVIII基因敲除小鼠模型中评估口服基因递送系统。通过提出一种有针对性的方法来开发有效的口服基因药物技术，我们渴望帮助实现基因治疗的潜力，即将基因应用于药物。公共卫生相关性：这项建议的目的是验证以壳聚糖和聚磷酸酯S为基因载体，非病毒基因载体可以通过口服途径有效传递的假设。如果成功，口服非病毒基因传递技术可能会改变医疗保健领域的蛋白质替代疗法，如慢性贫血、生长激素缺乏症和糖尿病。为了确定这一口服基因药物概念的潜力，我们将追求以下具体目标：1)设计和合成具有有利于口服给药的特性的基因载体；2)确定DNA纳米颗粒的重要特性以获得最佳摄取；以及3)在FVIII基因敲除小鼠模型中评估口服基因递送系统。

DESCRIPTION (provided by applicant): Gene therapy is a promising approach for treating hemophilia. Currently, their therapeutic potential is limited by the lack of safe and efficient delivery vectors. Although viruses remain the vectors of choice to achieve high efficiency of DNA transfer in vivo, reservation on their long-term safety lingers. Issues of immunogenicity and toxicity remain a challenge. Limitations of cell mitosis for retrovirus, contamination of adenovirus, and packaging constraints of adeno-associated virus also lessen their appeal. Consequently, non-viral vectors receive increasing attention on basis of ease of synthesis, low immunogenicity, and unrestricted plasmid size. Non-viral vectors also face less of a challenge in addressing pharmaceutical issues such as scale-up, storage stability, and quality control. However, the gene expression level achievable by non-viral gene delivery is low and transient. As a result, frequent administration is necessary to reach therapeutic level for gene medicine applications. To date virtually all non- viral transgene delivery has been either intravenous or intramuscular injection, which being invasive is unattractive and impractical for widespread use. In hemophilia gene therapy such an approach is less attractive and not much better than the current protein replacement therapy. Oral delivery may render gene therapy practical. The objective of this proposal is to test the hypothesis that the FVIII gene can be effectively delivered through the oral route using chitosan and poly(phosphoester)s as the non-viral gene carriers. We have demonstrated that oral administration of DNA-nanoparticles synthesized by complexing plasmid DNA with chitosan, a natural biocompatible polysaccharide, resulted in transduced gene expression in the intestinal epithelium. However, the transgene expression has been low in these studies. The objective of this proposal is to improve the nonviral oral gene delivery technology through optimization of carrier design and macroformulation for oral delivery. To define the potential of this oral gene medicine concept, we will pursue the following specific aims: 1) Design and synthesize gene carriers with characteristics favorable for oral delivery; 2) Identify the important characteristics of DNA nanoparticles for optimal uptake; and 3) Evaluate the oral gene delivery systems in a FVIII-knock-out murine model. By proposing a focused approach to develop an effective oral gene medicine technology, we aspire to help realize the potential of gene therapy, that of applying gene as a drug. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to test the hypothesis that nonviral gene vectors can be effectively delivered through the oral route using chitosan and poly(phosphoester)s as the gene carriers. If successful, the oral nonviral gene delivery technology may have the potential to transform the healthcare for protein replacement therapies such as chronic anemia, growth hormone deficiency and diabetes mellitus. To define the potential of this oral gene medicine concept, we will pursue the following specific aims: 1) Design and synthesize gene carriers with characteristics favorable for oral delivery; 2) Identify the important characteristics of DNA nanoparticles for optimal uptake; and 3) Evaluate the oral gene delivery systems in a FVIII- knock-out murine model.