Di Pasquale Giovanni, Perez Riveros Paola, Tora Muhibullah, Sheikh Tayyab, Son Aran, Teos Leyla, Grewe Brigitte, Swaim William D, Afione Sandra, Zheng Changyu, Jang Shyh-Ing, Shitara Akiko, Alevizos Ilias, Weigert Roberto, Chiorini John A
Adeno-Associated Virus Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.
Salivary Gland Biology and Disorder Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.
Mol Ther Methods Clin Dev. 2020 Oct 14;19:459-466. doi: 10.1016/j.omtm.2020.10.006. eCollection 2020 Dec 11.
The loss of salivary gland function caused by radiation therapy of the head and neck or autoimmune disease such as Sjögren's syndrome is a serious condition that affects a patient's quality of life. Due to the combined exocrine and endocrine functions of the salivary gland, gene transfer to the salivary glands holds the potential for developing therapies for disorders of the salivary gland and the expression of therapeutic proteins via the exocrine pathway to the mouth, upper gastrointestinal tract, or endocrine pathway, systemically, into the blood. Recent clinical success with viral vector-mediated gene transfer for the treatment of irradiation-induced damage to the salivary glands has highlighted the need for the development of novel vectors with acinar cell tropism able to result in stable long-term transduction. Previous studies with adeno-associated virus (AAV) focused on the submandibular gland and reported mostly ductal cell transduction. In this study, we have screened AAV vectors for acinar cell tropism in the parotid gland utilizing membrane-tomato floxed membrane-GFP transgenic mice to screen CRE recombinase encoding AAV vectors of different clades to rapidly identify capsid isolates able to transduce salivary gland acinar cells. We determined that AAVRh10 and a novel isolate found as a contaminant of a laboratory stock of simian adenovirus SV15, AAV44.9, are both able to transduce parotid and sublingual acinar cells. Persistence and localization of transduction of these AAVs were tested using vectors encoding firefly luciferase, which was detected 6 months after vector administration. Most luciferase expression was localized to the salivary gland compared to that of distal organs. Transduction resulted in robust secretion of recombinant protein in both blood and saliva. Transduction was species specific, with AAVRh10 having stronger transduction activity in rats compared with AAV44.9 or AAV2 but weaker in human primary salivary gland cells. This work demonstrates efficient transduction of parotid acinar cells by AAV that resulted in secretion of recombinant protein in both serum and saliva.
头颈部放射治疗或自身免疫性疾病（如干燥综合征）导致的唾液腺功能丧失是一种严重影响患者生活质量的疾病。由于唾液腺兼具外分泌和内分泌功能，将基因导入唾液腺具有开发唾液腺疾病治疗方法以及通过外分泌途径向口腔、上消化道输送治疗性蛋白质，或通过内分泌途径将其系统性地输送到血液中的潜力。近期，病毒载体介导的基因转移用于治疗放射性唾液腺损伤的临床成功突出了开发具有腺泡细胞嗜性、能够实现稳定长期转导的新型载体的必要性。先前对腺相关病毒（AAV）的研究主要集中在下颌下腺，且大多报道的是导管细胞转导。在本研究中，我们利用膜番茄floxed膜绿色荧光蛋白转基因小鼠，在腮腺中筛选具有腺泡细胞嗜性的AAV载体，以筛选编码不同进化枝的CRE重组酶的AAV载体，从而快速鉴定能够转导唾液腺腺泡细胞的衣壳分离株。我们确定AAVRh10和一种作为猿猴腺病毒SV15实验室储备污染物发现的新型分离株AAV44.9，均能够转导腮腺和舌下腺腺泡细胞。使用编码萤火虫荧光素酶的载体测试了这些AAV转导的持久性和定位情况，在载体给药6个月后检测到荧光素酶。与远端器官相比，大多数荧光素酶表达定位于唾液腺。转导导致重组蛋白在血液和唾液中大量分泌。转导具有物种特异性，与AAV44.9或AAV2相比，AAVRh10在大鼠中的转导活性更强，但在人原代唾液腺细胞中较弱。这项工作证明了AAV对腮腺腺泡细胞的有效转导，导致重组蛋白在血清和唾液中分泌。
