Oral Nonviral Gene Delivery for Hemophilla A

描述（由申请人提供）：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转移的载体，但对其长期安全性的质疑仍然存在。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、腺病毒的污染和腺相关病毒的包装限制也降低了它们的吸引力。因此，非病毒载体因其易于合成、免疫原性低和质粒大小不受限制而受到越来越多的关注。非病毒载体在解决诸如扩大规模、储存稳定性和质量控制等制药问题方面也面临较少的挑战。然而，通过非病毒基因传递可实现的基因表达水平低且短暂。因此，频繁给药是达到基因医学应用的治疗水平所必需的。迄今为止，几乎所有的非病毒转基因传递都是静脉注射或肌肉注射，这是侵入性的，不具有吸引力，也不适合广泛使用。在血友病基因治疗中，这种方法不那么有吸引力，也不比目前的蛋白质替代疗法好多少。口服给药可能使基因治疗变得可行。本研究的目的是验证壳聚糖和聚磷酸酯作为非病毒基因载体可通过口服途径有效递送FVIII基因的假设。我们已经证明，口服由质粒DNA与壳聚糖（一种天然生物相容性多糖）复合物合成的DNA纳米颗粒可导致肠上皮中基因的转导表达。然而，在这些研究中，转基因表达一直很低。本课题旨在通过优化载体设计和口服给药大剂配方来改进非病毒口服基因给药技术。为了确定这种口服基因医学概念的潜力，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特性的基因载体；2)确定DNA纳米颗粒的重要特征，以获得最佳的吸收；3)在fviii敲除小鼠模型中评估口服基因传递系统。通过提出一种有效的口服基因药物技术，我们希望帮助实现基因治疗的潜力，即将基因作为药物应用。公共卫生相关性：本提案的目的是验证非病毒基因载体可以通过口服途径以壳聚糖和聚磷酸酯作为基因载体有效递送的假设。如果成功，口服非病毒基因传递技术可能有潜力改变蛋白质替代疗法的医疗保健，如慢性贫血、生长激素缺乏症和糖尿病。为了确定这种口服基因医学概念的潜力，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特性的基因载体；2)确定DNA纳米颗粒的重要特征，以获得最佳的吸收；3)在FVIII基因敲除小鼠模型中评估口服基因传递系统。

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.