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有前途的方法。目前，它们的治疗潜力受到缺乏安全有效的递送载体的限制。尽管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限制、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，非病毒载体基于易于合成、低免疫原性和不受限制的质粒大小而受到越来越多的关注。非病毒载体在解决制药问题（如规模扩大、储存稳定性和质量控制）方面面临的挑战也较少。然而，通过非病毒基因递送可实现的基因表达水平是低的和瞬时的。因此，频繁给药是必要的，以达到基因药物应用的治疗水平。迄今为止，几乎所有的非病毒转基因递送都是静脉内或肌内注射，这是侵入性的，对于广泛使用来说是没有吸引力和不切实际的。在血友病基因治疗中，这种方法的吸引力较小，并且不比当前的蛋白质替代疗法好多少。口服给药可能使基因治疗实用化。本提案的目的是检验使用壳聚糖和聚（磷酸酯）作为非病毒基因载体可以通过口服途径有效递送FVIII基因的假设。我们已经证明，口服给药的DNA-纳米颗粒复合质粒DNA与壳聚糖，一种天然的生物相容性多糖，导致在肠上皮细胞中的转导基因表达。然而，在这些研究中，转基因表达一直很低。本研究的目的是通过优化载体设计和口服给药的宏观处方来改进非病毒口服基因传递技术。为了确定这种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递送特征的基因载体; 2）确定DNA纳米颗粒的重要特征以实现最佳摄取; 3）在FVIII敲除小鼠模型中评估口服基因递送系统。通过提出一个有针对性的方法来开发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假设，非病毒基因载体可以有效地通过口服途径使用壳聚糖和聚（磷酸酯）作为基因载体。如果成功，口服非病毒基因递送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递送的特征的基因载体; 2）鉴定DNA纳米颗粒的重要特征以获得最佳摄取;以及3）在FVIII敲除小鼠模型中评价口服基因递送系统。

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.