Rational engineering of a functional CpG-free ITR for AAV gene therapy

被引:26
作者
Pan, Xiufang [1 ]
Yue, Yongping [1 ]
Boftsi, Maria [2 ,3 ]
Wasala, Lakmini P. [1 ,2 ]
Tran, Ngoc Tam [4 ,5 ]
Zhang, Keqing [1 ]
Pintel, David J. [1 ,3 ]
Tai, Phillip W. L. [4 ,5 ]
Duan, Dongsheng [1 ,6 ,7 ,8 ]
机构
[1] Univ Missouri, Sch Med, Dept Mol Microbiol & Immunol, Columbia, MO 65212 USA
[2] Univ Missouri, Pathobiol Area Grad Program, Columbia, MO 65212 USA
[3] Univ Missouri, Christopher S Bond Life Sci Ctr, Columbia, MO 65212 USA
[4] UMass Chan Med Sch, Horae Gene Therapy Ctr, Worcester, MA 01605 USA
[5] UMass Chan Med Sch, Dept Microbiol & Physiol Syst, Worcester, MA 01605 USA
[6] Univ Missouri, Coll Vet Med, Dept Biomed Sci, Columbia, MO 65212 USA
[7] Univ Missouri, Sch Med, Dept Neurol, Columbia, MO 65212 USA
[8] Univ Missouri, Coll Engn, Dept Biomed Biol & Chem Engn, Columbia, MO 65212 USA
基金
美国国家卫生研究院;
关键词
ADENOASSOCIATED VIRUS VECTORS; HIGH-EFFICIENCY TRANSDUCTION; IN-VITRO; TRANSGENE EXPRESSION; NUCLEOTIDE-SEQUENCE; INFECTIOUS CLONES; DNA; GENERATION; GENOME; CLONING;
D O I
10.1038/s41434-021-00296-0
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Inverted terminal repeats (ITRs) are the only wild-type components retained in the genome of adeno-associated virus (AAV) vectors. To determine whether ITR modification is a viable approach for AAV vector engineering, we rationally deleted all CpG motifs in the ITR and examined whether CpG elimination compromises AAV-vector production and transduction. Modified ITRs were stable in the plasmid and maintained the CpG-free nature in purified vectors. Replacing the wild-type ITR with the CpG-free ITR did not affect vector genome encapsidation. However, the vector yield was decreased by approximately 3-fold due to reduced vector genome replication. To study the biological potency, we made micro-dystrophin (mu Dys) AAV vectors carrying either the wild-type ITR or the CpG-free ITR. We delivered the CpG-free mu Dys vector to one side of the tibialis anterior muscle of dystrophin-null mdx mice and the wild-type mu Dys vector to the contralateral side. Evaluation at four months after injection showed no difference in the vector genome copy number, microdystrophin expression, and muscle histology and force. Our results suggest that the complete elimination of the CpG motif in the ITR does not affect the biological activity of the AAV vector. CpG-free ITRs could be useful in engineering therapeutic AAV vectors.
引用
收藏
页码:333 / 345
页数:13
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