Herpes simplex virus type 1/adeno-associated virus hybrid vectors

Abstract

Herpes simplex virus type 1 (HSV-1) amplicons can accommodate foreign DNA of any size up to 150 kbp and, therefore, allow extensive combinations of genetic elements. Genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell type-specific expression, multiple transgenes, and genetic elements from other viruses to create hybrid vectors may be inserted in a modular fashion. Hybrid amplicons use genetic elements from HSV-1 that allow replication and packaging of the vector DNA into HSV-1 virions, and genetic elements from other viruses that either direct integration of transgene sequences into the host genome or allow episomal maintenance of the vector. Thus, the advantages of the HSV-1 amplicon system, including large transgene capacity, broad host range, strong nuclear localization, and availability of helper virus-free packaging systems are retained and combined with those of heterologous viral elements that confer genetic stability to the vector DNA. Adeno-associated virus (AAV) has the unique capability of integrating its genome into a specific site, designated AAVS1, on human chromosome 19. The AAV rep gene and the inverted terminal repeats (ITRs) that flank the AAV genome are sufficient for this process. HSV-1 amplicons have thus been designed that contain the rep gene and a transgene cassette flanked by AAV ITRs. These HSV/AAV hybrid vectors direct site-specific integration of transgene sequences into AAVS1 and support long-term transgene expression.

Keywords: HSV-1 amplicon vectors; adeno-associated virus; herpes simplex virus type 1; hybrid vectors..

Fig. (1)

Schematic representation of the HSV-1 genome. The HSV-1 genome is a linear double stranded DNA of approximately 152 kb in size, composed of two unique segments, U_L and U_S, which are flanked by inverted repeats, TR_L /IR_L and IR_S/TR_S. The minimal cis elements required for HSV-1 DNA replication and packaging include the origin of DNA replication, ori, and the cleavage/packaging, pac, signals.

Fig. (2)

Viral vectors. A) HSV-1 amplicon. The HSV-1 amplicon contains three types of genetic elements: i) sequences from bacteria (colE1 and ampR) that allow plasmid propagation in E. coli; ii) sequences from HSV-1, in particular an origin of DNA replication (ori) and a DNA packaging/cleavage signal (pac), which allow replication and packaging of the amplicon DNA into HSV-1 particles in the presence of HSV-1 helper functions in mammalian cells; and iii) a transgene cassette with the gene of interest. B) Recombinant AAV vector. Recombinant AAV vectors are bacterial plasmids that contain the AAV ITRs flanking a transgene of interest. Replication of the ITR cassette and packaging into AAV particles is achieved by supplying helpervirus functions and the rep and cap genes in cis or trans but outside the ITR cassette. C) HSV/AAV hybrid amplicon. In addition to the HSV-1 amplicon elements, HSV/AAV hybrid amplicon vectors contain the AAV rep gene and a transgene of interest flanked by AAV ITRs. D) HSV/EBV hybrid amplicon. In addition to the HSV-1 amplicon elements, HSV/EBV hybrid amplicon vectors contain the EBV origin of DNA replication (oriP) and the gene encoding EBNA-1 which together can support episomal retention and segregation of the vector in dividing cells. E) HSV/RV hybrid amplicon. In addition to the HSV-1 amplicon elements, HSV/RV hybrid amplicon vectors contain the retrovirus (MoMLV) gag, pol, and env genes, and the RV LTRs flanking a transgene of interest.

Fig. (3)

Schematic map of the wild type AAV genome. A) Secondary structure formed by the inverted terminal repeat, ITR. Depicted are the Rep binding sites, RBEs, and the terminal resolution site, TRS. B) The AAV genome expresses two clusters of genes, rep and cap, from three different promoters, p5, p19, p40, by alternative splicing.

Fig. (4)

The life cycle of AAV. Co-infection of AAV and helpervirus, adenovirus or HSV-1, leads to viral gene expression, viral DNA replication, and production of progeny virus. In the absence of helpervirus, the genome of AAV can integrate into a specific site on human chromosome 19. In the presence of helpervirus, integrated AAV genomes are rescued and enter the lytic replication cycle.

Fig. (5)

Comparison between self-complementary AAV (scAAV) and rAAV vectors. scAAV delivers a dimeric inverted repeated DNA molecule thereby bypassing the rate-limiting second-strand DNA synthesis of rAAV.