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Construction and optimization of herpes simplex virus vectors for central nervous system gene delivery based on CRISPR/Cas9-mediated genome editing

Author(s):

Xinwei Huang, Xiuqing Li, Lijuan Yang, Pengfei Wang, Jingyuan Yan, Zuqing Nie, Yingzheng Gao, Zhiwei Li, Jie Wen and Xia Cao*  

Abstract:


Aims: We aim to define parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors and optimize the expression cassettes to achieve robust and sustained expression in CNS.

Background: Engineered, attenuated Herpes simplex virus (HSV) vectors are promising vehicles for gene delivery to the peripheral and central nervous systems. The virus latent promoter (LAP) is commonly used to drive exogenous gene expression; however, parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors have not been fully understood.

Objective: This study aimed to construct attenuated HSV-1 vectors using the CRISPR-Cas9 system and examine the influence of transgene cassette construction and insertion site on transgene expression and vector safety.

Method: In this study, we used a CRISPR-Cas9 system to accurately and efficiently edit attenuated HSV-1 strain 1716, and constructed two series of recombinant virus LMR and LMRx with different sets of gene cassettes insertion in Exon1(LAP2) and 2.0 kb intron downstream of LAP, respectively. The transgene expression and viral gene transcriptional kinetics were compared in in-vitro cell lines. The reporter gene expression and safety profiles of each vector were further evaluated in the mouse hippocampus gene transduction model.

Result: The in-vitro cell line analysis indicated that the insertion of a gene expression cassette would disrupt virus gene transcription. Mouse hippocampus transducing analysis suggested that complete expression cassette insertion at 2.0 kb intron could achieve robust and longtime gene expression than the other constructs. Recombinants with gene expression cassettes lacked Poly (A), which induced significant neuronal inflammation due to persistent viral antigen expression and microglia activation.

Conclusion: Our results indicated that the integrity of LAT transcripts was not necessary for the establishment of long-term latent expression. Exogenous strong promoters (like cBh promoter) could remain active during latency when placed in Exon1 or 2.0 Kb Intron of LAT locus, although their transcriptional activity declined with time. Consistent with previous research, the foreign gene expression would last much longer when the gene cassette was located downstream of Exon1, which suggested a role of LAP2 in maintaining promoter activity during latency. Besides, over-transcription of the downstream part of LAT may induce continuous activation of the attenuated vectors, suggesting an important role of LAT in maintaining viral reactivation potential.

Keywords:

HSV-1 vector, CRISPR-Cas9 genome editing, Latency-associated transcription, hippocampus gene transduction, long-term transgene expression, recombinant virus.

Affiliation:

Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101



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