Strategy for the consistent preparation of sufficient non-viral large chromosomal vectors for biopharmaceutical applications

现代药物和疗法正变得越来越复杂，针对特定疾病或患者群体的针对性也越来越强。一种非常特殊的疗法是基因疗法。基因治疗是利用基因作为药物。这些基因可以通过使用携带基因的转基因病毒或使用从细菌中分离出来的称为质粒的环状DNA分子的非病毒方法传递给患者。这两种技术仍处于起步阶段，但已经有望在疫苗接种、癌症治疗和遗传疾病纠正方面取得巨大的医学进步。然而，病毒有几个缺点，包括安全方面的考虑和限制携带大量的遗传信息。另一方面，被称为BACs（细菌人工染色体）的大质粒DNA分子可以被修改，以纳入广泛的重要控制区域，从而允许基因在正确的组织和正确的时间表达。这种更大的尺寸给科学和工业带来了挑战，因为这种尺寸限制了单个细菌可以制造的数量，此外，这种尺寸还需要特殊的处理考虑，因为bac是脆弱的。面临的挑战是能够制备出所需数量的BAC分子，以治疗可能从这些药物中受益的人数。我们需要在生物制造业中研究并开发大规模制造DNA分子的方法。DNA分子的质量也是至关重要的。它们需要以最适合递送给人类（和动物）的形式存在，并且不含污染物质。我们提出的研究将使工业界能够制造出大规模生产的DNA分子。拟议的研究成果将使其他参与基因治疗的人能够为治疗癌症、疫苗和纠正遗传疾病准备大的DNA分子。拟议的科学还将使相关学科的其他研究人员能够从能够制造和操纵大的DNA结构中受益。我们还将探索如何使DNA以正确的三维形式有效地进入细胞，从而使DNA成为一种有效的药物。

Modern medicines and therapies are becoming increasingly complex and specific for a particular disease or group of patients. One very specific type of therapy is Gene Therapy. Gene therapy is the use of genes as medicines. These genes can be delivered to patients either by the use of genetically modified viruses to carry the genes or by using non-viral methods which employ circular DNA molecules called plasmids isolated from bacteria. Both techniques are still in their infancy but are already promising huge medical advances in vaccination, cancer therapy and the correction of genetic disorders. However, viruses suffer from several drawbacks including safety considerations and limited to carry large genetic information. On the other hand large plasmid DNA molecules called BACs ( Bacterial Artificial Chromosomes) can be modified to incorporate a wide range of important control regions which allow expression of the gene in the correct tissue and at the correct time. This larger size poses a scientific and industrial challenge because the size limits the amount that can be made in a single bacterium, in addition, the size also calls for special processing considerations because BACs are fragile. The challenge is to be able to prepare the BAC molecules in the amounts needed to treat the numbers of people who could benefit from these medicines. We need to investigate then develop methods of making the DNA molecules at large scales in the biomanufacturing industry. The quality of the DNA molecules is also paramount. They need to be in the form that is most appropriate for delivering to humans (and animals) and free of contaminating material. The research we propose will enable industry to make DNA molecules that can be made at the large scales. The proposed research outcomes will allow others involved in gene therapy to prepare large DNA molecules for the treatment of cancers, for vaccines and to correct genetic disorders The science proposed will also allow other researchers in related disciplines to benefit from being able to make and manipulate large DNA constructs .We will also explore ways of making the DNA in the correct three dimensional form needed for efficient uptake into cells so that the DNA is an effective medicine.