机构：[1]Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA[2]Li Weibo Institute for Rare Diseases Research, University ofMassachusetts Medical School, Worcester, MA 01605, USA[3]Department of Microbiology and Physiological Systems, University of Massachusetts Medical School,Worcester, MA 01605, USA[4]Viral Vector Core, University of Massachusetts Medical School, Worcester, MA 01605, USA[5]Pacific Biosciences Inc., Menlo Park, CA94025, USA[6]Program in Molecular Medicine and Center for AIDS Research, University of Massachusetts Medical School, Worcester, MA 01605, USA[7]Institute ofUrology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China四川大学华西医院[8]Department of Thoracic Oncology, West China Hospital, Sichuan University,Chengdu, Sichuan 610041, China四川大学华西医院[9]Cancer Center and National Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China四川大学华西医院
Recombinant adeno-associated virus (rAAV)-based gene therapy has entered a phase of clinical translation and commercialization. Despite this progress, vector integrity following production is often overlooked. Compromised vectors may negatively impact therapeutic efficacy and safety. Using single molecule, real-time (SMRT) sequencing, we can comprehensively profile packaged genomes as a single intact molecule and directly assess vector integrity without extensive preparation. We have exploited this methodology to profile all heterogeneic populations of self-complementary AAV genomes via bioinformatics pipelines and have coined this approach AAV-genome population sequencing (AAV-GPseq). The approach can reveal the relative distribution of truncated genomes versus full-length genomes in vector preparations. Preparations that seemingly show high genome homogeneity by gel electrophoresis are revealed to consist of less than 50% full-length species. With AAV-GPseq, we can also detect many reverse-packaged genomes that encompass sequences originating from plasmid backbone, as well as sequences from packaging and helper plasmids. Finally, we detect host-cell genomic sequences that are chimeric with inverted terminal repeat (ITR)-containing vector sequences. We show that vector populations can contain between 1.3% and 2.3% of this type of undesirable genome. These discoveries redefine quality control standards for viral vector preparations and highlight the degree of foreign products in rAAV-based therapeutic vectors.
基金：
This work was supported by Public Health Service grants
1R01NS076991-05, R01 HL097088, 1P01AI100263-05, and
4P01HL131471-01 from the NIH and an internal grant from University
of Massachusetts Medical School to G.G.
语种：
外文
PubmedID：
中科院(CAS)分区：
出版当年[2018]版：
大类|3 区医学
小类|3 区医学：研究与实验
最新[2023]版：
大类|2 区医学
小类|2 区医学：研究与实验
第一作者：
第一作者机构：[1]Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA[2]Li Weibo Institute for Rare Diseases Research, University ofMassachusetts Medical School, Worcester, MA 01605, USA[3]Department of Microbiology and Physiological Systems, University of Massachusetts Medical School,Worcester, MA 01605, USA
共同第一作者：
通讯作者：
通讯机构：[1]Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA[2]Li Weibo Institute for Rare Diseases Research, University ofMassachusetts Medical School, Worcester, MA 01605, USA[3]Department of Microbiology and Physiological Systems, University of Massachusetts Medical School,Worcester, MA 01605, USA[7]Institute ofUrology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China[8]Department of Thoracic Oncology, West China Hospital, Sichuan University,Chengdu, Sichuan 610041, China[9]Cancer Center and National Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China[*1]Horae Gene Therapy Center, University of Massachusetts Medical School, 386 Plantation Street, Worcester, MA 01605, USA
推荐引用方式(GB/T 7714)：
Tai Phillip W L,Xie Jun,Fong Kaiyuen,et al.Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras.[J].Molecular therapy. Methods & clinical development.2018,9:130-141.doi:10.1016/j.omtm.2018.02.002.
APA：
Tai Phillip W L,Xie Jun,Fong Kaiyuen,Seetin Matthew,Heiner Cheryl...&Gao Guangping.(2018).Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras..Molecular therapy. Methods & clinical development,9,
MLA：
Tai Phillip W L,et al."Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras.".Molecular therapy. Methods & clinical development 9.(2018):130-141
