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

M13噬菌体衣壳与双链AAV(腺相关病毒)基因组杂交载体的特性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的发生有关，传统的治疗方法不能有效地靶向转移。因此，寻找新的、安全的、系统的治疗转移性癌症的方法势在必行。为此目的使用病毒是一个有吸引力的选择，腺相关病毒(AAV)是首选候选之一。尽管AAV衍生载体具有良好的表型，但它仍然存在一些缺点，如在人类群体中普遍存在的预先存在的免疫力、其有限的转基因编码能力或其复杂的生产方案。噬菌体/腺相关病毒混合载体已被提出作为一种替代方案来克服AAV相关的一些主要缺点。这些混合载体具有极好的安全性，与其噬菌体来源有关，并被设计为在全身递送后专门针对肿瘤和肿瘤血管。它们的转基因包装能力比AAVs高得多，而且它们的生产更简单，更容易扩展。它们还被证明能够穿越血脑屏障，扩大了它们的应用范围，适用于发生在大脑中的肿瘤或转移。这些载体的最后一代是基于噬菌体系统，该系统经过进一步修改，以增强内体逃逸，减少与细胞外分子的非特异性结合。此外，他们的基因组被设计成产生一个自我互补的转基因，以克服单链到双链DNA合成步骤，这是AAV及其衍生系统中公认的主要瓶颈。在这里，我将首先对上一代PAAV杂交载体的最后一代进行体外鉴定。然后，我将评估这些编码三种不同治疗性转基因候选基因的载体在体外环境中的效率：白介素IL12、IL15和肿瘤坏死因子α。接下来，将从4T1小鼠乳腺肿瘤细胞和B16F10小鼠黑色素瘤细胞中选择最合适和最有效的载体来靶向在免疫活性小鼠中建立的转移癌。

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.