Login Register Cart 0
Research Article

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*

Volume 22, Issue 1, 2022

Published on: 29 November, 2021

Page: [66 - 77] Pages: 12

DOI: 10.2174/1566523219666210618154326

Price: $65

Abstract

Aims: We aim to define parameters that affect 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 longterm transgene expression of attenuated HSV-1 vectors have not been fully understood.

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

Methods: In this study, we used a CRISPR-Cas9 system to accurately and efficiently edit attenuated HSV-1 strain 1716, and construct 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 mouse hippocampus gene transduction model.

Results: 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 lacking Poly (A) 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 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, which suggests 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.

« Previous
Graphical Abstract

[1]
Nesburn AJTCMC, St. Louis Mo. Report of the corneal disease panel: vision research: a national plan 1983–1987 1983.
[2]
Hjalmarsson A, Blomqvist P. Sköldenberg BJCid Herpes simplex encephalitis in Sweden 1990–2001: incidence, morbidity, and mortality 2007; 45(7): 875-80.
[http://dx.doi.org/10.1086/521262] [PMID: 17806053]
[3]
Rock DL, Nesburn A, Ghiasi H, Ong J, Lewis T, Lokensgard J, et al. Detection of latency-related viral RNAs in trigeminal ganglia of rabbits latently infected with herpes simplex virus type 1 1987; 61(12): 3820-6.
[PMID: 2824816]
[4]
Stevens J, Wagner E, Devi-Rao G, Cook M, Feldman LJS. RNA complementary to a herpesvirus alpha gene mRNA is prominent in latently infected neurons 1987; 235(4792): 1056-9.
[http://dx.doi.org/10.1126/science.2434993]
[5]
Wechsler S, Nesburn A, Watson R, Slanina S. Ghiasi HJJov Fine mapping of the latency-related gene of herpes simplex virus type 1: Alternative splicing produces distinct latency-related RNAs containing open reading frames 1988; 62(11): 4051-8.
[PMID: 9696825]
[6]
Farrell MJ, Dobson AT. Feldman LTJPotNAoS Herpes simplex virus latency-associated transcript is a stable intron 1991; 88(3): 790-4.
[http://dx.doi.org/10.1073/pnas.88.3.790] [PMID: 1846963]
[7]
Umbach JL, Kramer MF, Jurak I, Karnowski HW, Coen DM, Cullen BR. MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs. Nature 2008; 454(7205): 780-3.
[http://dx.doi.org/10.1038/nature07103] [PMID: 18596690]
[8]
Palmer JA, Branston RH, Lilley CE, et al. Development and optimization of herpes simplex virus vectors for multiple long-term gene delivery to the peripheral nervous system. J Virol 2000; 74(12): 5604-18.
[http://dx.doi.org/10.1128/JVI.74.12.5604-5618.2000] [PMID: 10823868]
[9]
Margolis TP, Bloom DC, Dobson AT, Feldman LT, Stevens JGJV. Decreased reporter gene expression during latent infection with HSV LAT promoter constructs 1993; 197(2): 585-92.
[http://dx.doi.org/10.1006/viro.1993.1632]
[10]
Goins WF, Sternberg LR, Croen KD, et al. A novel latency-active promoter is contained within the herpes simplex virus type 1 UL flanking repeats. J Virol 1994; 68(4): 2239-52.
[http://dx.doi.org/10.1128/JVI.68.4.2239-2252.1994] [PMID: 8139009]
[11]
Puskovic V, Wolfe D, Goss J, et al. Prolonged biologically active transgene expression driven by HSV LAP2 in brain in vivo. Mol Ther 2004; 10(1): 67-75.
[http://dx.doi.org/10.1016/j.ymthe.2004.04.004] [PMID: 15233943]
[12]
Berthomme H, Lokensgard J, Yang L, Margolis T, Feldman LT. Evidence for a bidirectional element located downstream from the herpes simplex virus type 1 latency-associated promoter that increases its activity during latency. J Virol 2000; 74(8): 3613-22.
[http://dx.doi.org/10.1128/JVI.74.8.3613-3622.2000] [PMID: 10729137]
[13]
Amelio AL, McAnany PK, Bloom DC. A chromatin insulator-like element in the herpes simplex virus type 1 latency-associated transcript region binds CCCTC-binding factor and displays enhancer-blocking and silencing activities. J Virol 2006; 80(5): 2358-68.
[http://dx.doi.org/10.1128/JVI.80.5.2358-2368.2006] [PMID: 16474142]
[14]
Lee JS, Raja P, Pan D, Pesola JM, Coen DM, Knipe DM. CCCTC-binding factor acts as a heterochromatin barrier on herpes simplex viral latent chromatin and contributes to poised latent infection. MBio 2018; 9(1): e02372-17.
[http://dx.doi.org/10.1128/mBio.02372-17] [PMID: 29437926]
[15]
Miyagawa Y, Marino P, Verlengia G, et al. Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity. Proc Natl Acad Sci USA 2015; 112(13): E1632-41.
[http://dx.doi.org/10.1073/pnas.1423556112] [PMID: 25775541]
[16]
Han F, Miyagawa Y, Verlengia G, et al. Cellular antisilencing elements support transgene expression from herpes simplex virus vectors in the absence of immediate early gene expression. J Virol 2018; 92(17): e00536-18.
[http://dx.doi.org/10.1128/JVI.00536-18] [PMID: 29950408]
[17]
Umbach JL, Nagel MA, Cohrs RJ, Gilden DH. Cullen BRJJov Analysis of human alphaherpesvirus microRNA expression in latently infected human trigeminal ganglia 2009; 83(20): 10677-83.
[http://dx.doi.org/10.1128/JVI.01185-09] [PMID: 19656888]
[18]
Pan D, Flores O, Umbach JL, et al. A neuron-specific host microRNA targets herpes simplex virus-1 ICP0 expression and promotes latency. Cell Host Microbe 2014; 15(4): 446-56.
[http://dx.doi.org/10.1016/j.chom.2014.03.004] [PMID: 24721573]
[19]
Jiang X, Brown D, Osorio N, Hsiang C, BenMohamed L, Wechsler SL. Increased neurovirulence and reactivation of the herpes simplex virus type 1 latency-associated transcript (LAT)-negative mutant dLAT2903 with a disrupted LAT miR-H2. J Neurovirol 2016; 22(1): 38-49.
[http://dx.doi.org/10.1007/s13365-015-0362-y] [PMID: 26069184]
[20]
Perng G-C, Jones C, Ciacci-Zanella J, et al. Virus-induced neuronal apoptosis blocked by the herpes simplex virus latency-associated transcript. Science 2000; 287(5457): 1500-3.
[http://dx.doi.org/10.1126/science.287.5457.1500] [PMID: 10688801]
[21]
Tormanen K, Allen S, Mott KR, Ghiasi H. LAT inhibits apoptosis via downregulation of components of type I IFN pathway during latent HSV-1 ocular infection. Journal of Virology 2019. JVI. 00103-.
[22]
Lin C, Li H, Hao M, et al. Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing of HSV-1 virus in human cells. Sci Rep 2016; 6: 34531.
[http://dx.doi.org/10.1038/srep34531] [PMID: 27713537]
[23]
Conrady CD, Zheng M, van Rooijen N, et al. Microglia and a functional type I IFN pathway are required to counter HSV-1-driven brain lateral ventricle enlargement and encephalitis. J Immunol 2013; 190(6): 2807-17.
[http://dx.doi.org/10.4049/jimmunol.1203265] [PMID: 23382563]
[24]
Bi Y, Sun L, Gao D, et al. High-efficiency targeted editing of large viral genomes by RNA-guided nucleases. PLoS Pathog 2014; 10(5)e1004090
[http://dx.doi.org/10.1371/journal.ppat.1004090] [PMID: 24788700]
[25]
Yuen K-S, Chan C-P, Wong NM, et al. CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells. J Gen Virol 2015; 96(Pt 3): 626-36.
[http://dx.doi.org/10.1099/jgv.0.000012] [PMID: 25502645]
[26]
Howes R, Schofield C. Genome engineering using adeno-associated virus (AAV)Chromosomal Mutagenesis. Springer 2015; pp. 75-103.
[http://dx.doi.org/10.1007/978-1-4939-1862-1_5]
[27]
Berges BK, Wolfe JH, Fraser NW. Stable levels of long-term transgene expression driven by the latency-associated transcript promoter in a herpes simplex virus type 1 vector. Mol Ther 2005; 12(6): 1111-9.
[http://dx.doi.org/10.1016/j.ymthe.2005.06.478] [PMID: 16122987]
[28]
Liu W, Griffin G, Clarke T, et al. Bilateral single-site intracerebral injection of a nonpathogenic herpes simplex virus-1 vector decreases anxiogenic behavior in MPS VII mice. Mol Ther Methods Clin Dev 2015; 2: 14059.
[http://dx.doi.org/10.1038/mtm.2014.59] [PMID: 26052529]
[29]
Lokensgard JR, Berthomme H, Feldman LT. The latency-associated promoter of herpes simplex virus type 1 requires a region downstream of the transcription start site for long-term expression during latency. J Virol 1997; 71(9): 6714-9.
[http://dx.doi.org/10.1128/JVI.71.9.6714-6719.1997] [PMID: 9261395]
[30]
Lachmann RH, Efstathiou S. Utilization of the herpes simplex virus type 1 latency-associated regulatory region to drive stable reporter gene expression in the nervous system. J Virol 1997; 71(4): 3197-207.
[http://dx.doi.org/10.1128/JVI.71.4.3197-3207.1997] [PMID: 9060683]
[31]
Srivastava R, Dervillez X, Khan AA, Chentoufi AA, Chilukuri S, Shukr N, et al. The herpes simplex virus LAT gene is associated with a broader repertoire of virus-specific exhausted CD8+ T cells retained within the trigeminal ganglia of latently infected HLA transgenic rabbits. J Virol 2021; 90(8): 3913-28. Available from: https://jvi.asm.org/content/jvi/90/8/3913.full.pdf
[32]
Chentoufi AA, Dervillez X, Dasgupta G, et al. The herpes simplex virus type 1 latency-associated transcript inhibits phenotypic and functional maturation of dendritic cells. Viral Immunol 2012; 25(3): 204-15.
[http://dx.doi.org/10.1089/vim.2011.0091] [PMID: 22512280]
[33]
Leib DA, Bogard CL, Kosz-Vnenchak M, et al. A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with reduced frequency. J Virol 1989; 63(7): 2893-900.
[http://dx.doi.org/10.1128/JVI.63.7.2893-2900.1989] [PMID: 2542601]
[34]
Hill JM, Sedarati F, Javier RT, Wagner EK, Stevens JG. Herpes simplex virus latent phase transcription facilitates in vivo reactivation. Virology 1990; 174(1): 117-25.
[http://dx.doi.org/10.1016/0042-6822(90)90060-5] [PMID: 2152989]
[35]
Block TM, Deshmane S, Masonis J, Maggioncalda J, Valyi-Nagi T, Fraser NW. An HSV LAT null mutant reactivates slowly from latent infection and makes small plaques on CV-1 monolayers. Virology 1993; 192(2): 618-30.
[http://dx.doi.org/10.1006/viro.1993.1078] [PMID: 8380666]
[36]
Perng G-C, Dunkel EC, Geary PA, et al. The latency-associated transcript gene of herpes simplex virus type 1 (HSV-1) is required for efficient in vivo spontaneous reactivation of HSV-1 from latency. J Virol 1994; 68(12): 8045-55.
[http://dx.doi.org/10.1128/JVI.68.12.8045-8055.1994] [PMID: 7966594]
[37]
Nicoll MP, Hann W, Shivkumar M, et al. The HSV-1 latency-associated transcript functions to repress latent phase lytic gene expression and suppress virus reactivation from latently infected neurons. PLoS Pathog 2016; 12(4)e1005539
[http://dx.doi.org/10.1371/journal.ppat.1005539] [PMID: 27055281]
[38]
Kesari S, Lasner TM, Balsara KR, et al. A neuroattenuated ICP34.5-deficient herpes simplex virus type 1 replicates in ependymal cells of the murine central nervous system. J Gen Virol 1998; 79(Pt 3): 525-36.
[http://dx.doi.org/10.1099/0022-1317-79-3-525] [PMID: 9519831]
[39]
Nicoll MP, Efstathiou S. Expression of the herpes simplex virus type 1 latency-associated transcripts does not influence latency establishment of virus mutants deficient for neuronal replication. J Gen Virol 2013; 94(Pt 11): 2489-94.
[http://dx.doi.org/10.1099/vir.0.056176-0] [PMID: 23907392]
[40]
Ousman SS, Kubes P. Immune surveillance in the central nervous system. Nat Neurosci 2012; 15(8): 1096-101.
[http://dx.doi.org/10.1038/nn.3161] [PMID: 22837040]

Rights & Permissions Print Cite
Article Metrics
37
2
© 2024 Bentham Science Publishers | Privacy Policy