Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity
Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity
复制标题
DOI：
10.1016/j.ymthe.2017.03.028
复制
发表时间：
2017-06-07
影响因子：
12.4
通讯作者：
Gao, Guangping
中科院分区：
文献类型：
--
作者：
Xie, Jun;Mao, Qin;Gao, Guangping
由重组腺相关病毒（recombinant adeno-associated viruses，rAAVs）介导的短发夹状RNA（Short hairpin RNA，sh RNA）是研究体内基因功能的重要工具，也是一种很有前途的基因治疗平台。我们的数据表明，将shRNA转基因掺入rAAV构建体中降低了载体产量，并产生了一群截短和有缺陷的基因组。我们证明了具有发夹或发夹样结构的序列在病毒基因组复制期间通过聚合酶重定向机制驱动修剪的AAV基因组的产生。我们的研究结果揭示了基因组二级结构优化病毒载体设计时的重要性。我们还发现，shDNAs可以适应作为替代突变体反向末端重复序列（mTRs），以前被认为是功能性自身互补的AAV载体所需的序列。使用shDNA作为人工mTR为工程化新一代AAV载体打开了大门，其具有改善的效力、遗传稳定性和安全性，用于临床前研究和人类基因治疗。
Short hairpin (sh)RNAs delivered by recombinant adeno-associated viruses (rAAVs) are valuable tools to study gene function in vivo and a promising gene therapy platform. Our data show that incorporation of shRNA transgenes into rAAV constructs reduces vector yield and produces a population of truncated and defective genomes. We demonstrate that sequences with hairpins or hairpin-like structures drive the generation of trimcated AAV genomes through a polymerase redirection mechanism during viral genome replication. Our findings reveal the importance of genomic secondary structure when optimizing viral vector designs. We also discovered that shDNAs could be adapted to act as surrogate mutant inverted terminal repeats (mTRs), sequences that were previously thought to be required for functional self-complementary AAV vectors. The use of shDNAs as artificial mTRs opens the door to engineering a new generation of AAV vectors with improved potency, genetic stability, and safety for both preclinical studies and human gene therapy.