Brun Mitchell J, Song Kefan, Kang Byunguk, Lueck Cooper, Chen Weitong, Thatcher Kaitlyn, Gao Erhe, Koch Walter J, Lincoln Joy, Rajan Sudarsan, Suh Junghae
Department of Chemical and Biomolecular Engineering, Rice University, 6100 Main St., Houston, TX 77005, United States.
Department of Bioengineering, Rice University, 6100 Main St., Houston, TX 77005, United States.
J Control Release. 2020 Dec 10;328:834-845. doi: 10.1016/j.jconrel.2020.10.058. Epub 2020 Nov 4.
Adeno-associated virus (AAV) is a promising vector for gene therapy, but its broad tropism can be detrimental if the transgene being delivered is harmful when expressed ubiquitously in the body, i.e. in non-target tissues. Delivering the transgene of interest to target cells at levels high enough to be therapeutically effective while maintaining safety by minimizing delivery to off-target cells is a prevalent challenge in the field of gene therapy. We have developed a protease activatable vector (provector) platform based on AAV9 that can be injected systemically to deliver therapeutic transgenes site-specifically to diseased cells by responding to extracellular proteases present at the disease site. The provector platform consists of a peptide insertion into the virus capsid which disrupts the virus' ability to bind to cell surface receptors. This peptide contains a blocking motif (aspartic acid residues) flanked on either side by cleavage sequences that are recognized by certain proteases. Exposure to proteases cleaves the peptides off the capsid, activating or "switching ON" the provector. In response to the activation, the provectors regain their ability to bind and transduce cells. Here, we have designed a provector that is activated by cysteine aspartic proteases (caspases), which have roles in inflammation and apoptosis and thus are elevated at sites of diseases such as heart failure, neurodegenerative diseases, and ischemic stroke. This provector demonstrates a 200-fold reduction in transduction ability in the OFF state compared to AAV9, reducing the virus' ability to transduce off-target healthy tissue. Following exposure to and proteolysis by caspase-3, the provector shows a 95-fold increase in transduction compared to the OFF state. The switchable transduction behavior was found to be a direct result of the peptide insertion ablating the ability of the virus to bind to cells. In vivo studies were conducted to characterize the biodistribution, blood circulation time, neutralizing antibody formation, and targeted delivery ability of the caspase-activatable provector in a model of heart failure.
腺相关病毒（AAV）是一种很有前景的基因治疗载体，但如果所递送的转基因在体内（即在非靶组织中）普遍表达时有害，其广泛的嗜性可能会产生不利影响。在基因治疗领域，将感兴趣的转基因以足够高的水平递送至靶细胞以达到治疗效果，同时通过尽量减少向非靶细胞的递送来维持安全性，是一个普遍存在的挑战。我们基于AAV9开发了一种蛋白酶可激活载体（前载体）平台，该平台可通过响应疾病部位存在的细胞外蛋白酶，经全身注射将治疗性转基因位点特异性地递送至患病细胞。前载体平台由插入病毒衣壳的一段肽组成，这会破坏病毒与细胞表面受体结合的能力。该肽包含一个阻断基序（天冬氨酸残基），其两侧为特定蛋白酶识别的切割序列。暴露于蛋白酶会将肽从衣壳上切割下来，激活或“开启”前载体。作为对激活的响应，前载体恢复其结合和转导细胞的能力。在此，我们设计了一种由半胱天冬酶激活的前载体，半胱天冬酶在炎症和细胞凋亡中起作用，因此在诸如心力衰竭、神经退行性疾病和缺血性中风等疾病部位水平会升高。与AAV9相比，这种前载体在关闭状态下的转导能力降低了200倍，降低了病毒转导非靶健康组织的能力。在暴露于半胱天冬酶-3并经其蛋白水解后，前载体与关闭状态相比，转导增加了95倍。发现这种可切换的转导行为是肽插入导致病毒结合细胞能力丧失的直接结果。进行了体内研究，以表征半胱天冬酶激活的前载体在心力衰竭模型中的生物分布、血液循环时间、中和抗体形成以及靶向递送能力。
