Characterization of a novel hybrid vector between M13 phage capsid and double double-stranded AAV (adeno-associated virus) genome, and its application

M13噬菌体衣壳与双双链AAV（腺相关病毒）基因组新型杂交载体的表征及其应用

• 批准号: 2288808
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2288808
• 关键词: Characterization; novel; hybrid; vector; between

Characterization of a novel hybrid vector between M13 phage capsid and double double-stranded AAV (adeno-associated virus) genome, and its application in cancer gene therapyMost of cancer related deaths are linked to the development of metastases, against which traditional therapies are not efficient at targeting. Therefore, it is imperative to find new, safe and systemic approaches able to treat metastatic cancer. The use of viruses for this purpose is an attractive option, with the adeno-associated virus (AAV) being one of the preferred candidates. Despite its good profile, AAV derived vectors still have several drawbacks, such as the generalized pre-existing immunity in the human population, its limited transgene encoding capacity or its complex production protocols. Phage/adeno-associated virus hybrid vectors have been proposed as an alternative option to overcome some major AAV-associated disadvantages. These hybrid vectors have an excellent safety profile linked to their bacteriophage origin and were engineered to specifically target tumours and tumour vasculature after systemic delivery. They have a substantially higher transgene packaging capacity than AAVs and their production is simpler and easily scalable. They also have been proven to be able to cross the blood brain barrier, expanding their application to tumours or metastases occurring in the brain. The last generation of these vectors is based on a phagemid system, that was further modified to enhance the endosomal escape and reduce the unspecific binding to extracellular molecules. Also, their genome has been designed to generate a self-complementary transgene to overcome the single stranded to double stranded DNA synthesis step, a recognized major bottleneck in AAV and derived systems. Here, I will first characterize the last generation of the last generation of PAAV hybrid vectors in vitro. Then, I will evaluate the efficiency of these vectors encoding three different therapeutic transgene candidates: interleukins IL12, IL15 and tumor necrosis factor alpha (TNF) in an ex vivo setting. Next, the most suitable and efficient vector will be selected to target metastatic cancer established in immunocompetent mice from 4T1 mouse mammary tumour cells and B16F10 mouse melanoma cells.

M13噬菌体衣壳蛋白与双链腺相关病毒基因组杂交载体的构建及其在肿瘤基因治疗中的应用大多数癌症相关死亡与转移有关，而传统的治疗方法对其靶向效果不佳。因此，迫切需要找到新的，安全的和系统的方法来治疗转移性癌症。为此目的使用病毒是一种有吸引力的选择，腺相关病毒（AAV）是优选的候选者之一。尽管AAV衍生载体具有良好的特性，但其仍具有几个缺点，例如人类群体中普遍存在的免疫性、其有限的转基因编码能力或其复杂的生产方案。已经提出噬菌体/腺相关病毒杂合载体作为克服一些主要的AAV相关缺点的替代选择。这些杂交载体具有与其噬菌体来源相关的优异的安全性，并且被工程化以在全身递送后特异性靶向肿瘤和肿瘤脉管系统。它们具有比AAV高得多的转基因包装能力，并且它们的生产更简单且易于扩展。它们还被证明能够穿过血脑屏障，将其应用扩展到脑中发生的肿瘤或转移。这些载体的最后一代是基于噬菌粒系统，其被进一步修饰以增强内体逃逸并减少与细胞外分子的非特异性结合。此外，它们的基因组已被设计成产生自身互补的转基因，以克服单链到双链DNA合成步骤，这是AAV和衍生系统中公认的主要瓶颈。在这里，我将首先在体外表征最后一代PAAV杂交载体的最后一代。然后，我将评估这些载体的效率编码三种不同的治疗转基因候选人：白细胞介素IL 12，IL 15和肿瘤坏死因子α（TNF）在离体设置。接下来，将选择最合适和有效的载体来靶向在免疫活性小鼠中从4 T1小鼠乳腺肿瘤细胞和B16 F10小鼠黑素瘤细胞建立的转移性癌症。