Schematic representation of currently available hybrid HSV-1 amplicon vector platforms. (A) The HSV/AAV hybrid amplicon contains the inverted terminal repeats (ITRs) of the adeno-associated virus (AAV), which have been designed to flank the transgene unit-of-interest. The AAV Rep open-reading frame is placed outside the ITR-flanked trangene unit and expression of Rep within the transduced cell mediates the integration of the ITR-flanked transgene into the host cell genome, preferentially at the AAVS1 locus in hChr19. (B) The HSV/EBV hybrid amplicon contains the Epstein-Barr virus (EBV) origin of replication (oriP) and EBNA-1 gene, which facilitate replication of the HSV-1 amplicon within the transduced cell. (C) Components of the Moloney-murine leukemia virus (MMLV) have been used to generate the “tribrid” HSV/EBV/RV vector. The gag-pol, and env genes are located outside the retroviral long terminal repeat (RV-LTR)-flanked trangene unit and are expressed via a cytomegalovirus promoter, which facilitates retroviral vector production in a permissive cell line. The same EBV components are included (as described in B) for episomal retention of the amplicon genome. (D) The HSV-1/Sleeping Beauty (SB) amplicon vector system requires the co-transduction of 2 amplicon vectors for stable chromosomal integration of a SB inverted/direct repeat (IR/DR)-flanked transgene unit catalyzed by the SB transposase enzyme. Panels A-C were adapted from [45].

Schematic representation of the HSV-1 virion, its genomic organization, and the basic design of the HSV-1 derived amplicon plasmid. (A) The mature wild-type HSV-1 virion is composed of 4 sub-compartments: envelope, tegument, capsid, and the 150-kb linear double-stranded DNA genome. The envelope contains glycoprotein molecules involved in the cellular binding and viral entry processes of HSV-1 infection. (B) The linear double-stranded DNA genome of wild-type HSV-1 encodes approximately 80–85 viral genes, which are located in unique long (U_L) and unique short (U_S) regions within the genome. Inverted repeat elements (ab, b’a’, a’c’, and ca) flank the unique regions and contain packaging signals required for cleavage and packaging of the replicated viral genome into virions and isomerization of the U_L and U_S genomic segments. Three viral origins of replication are located within the genome with 2 in the U_S region (oriS) and 1 in the U_L region (oriL). Several viral genes whose functions are discussed are demarcated. (C) The HSV-1 derived amplicon plasmid contains a single oriS or oriL and an “a” site and is devoid of all viral genes. A bacterial origin of replication (ColE1) and an antibiotic resistant gene (Amp^r) is included for bacterial propagation of the plasmid. A transgene unit-of-interest can be cloned into the HSV-1 amplicon using standard molecular cloning techniques and packaged into HSV-1 amplicon viral particles using helper virus-based or helper virus-free packaging methodologies. (Panel B was adapted from [17]).

Schematic representation of the latest packaging systems utilized for amplicon vector production. (A) The HSV-1LaLΔJ helper virus-based amplicon packaging technology involves initial transfection of the transgene-harbored amplicon plasmid into an ICP4-complementing cell line with subsequent superinfection with the HSV-1LaLΔJ helper virus, which contains a unique ‘floxed’ packaging signal designed to reduce helper virus contamination. The resultant viral stock from the ICP4-complementing cell line that contains both packaged amplicon particles and helper virus particles (at approximately a 1:1 ratio) is used to infect a second cell line, which expresses ICP4 and Cre recombinase. Recombination of the loxP-sites via Cre recombinase results in the deletion of the packaging signal from the helper virus, which renders its own genome packaging-defective and thereby reduces the titers of helper virus present (0.5%) in the final harvested amplicon vector stock. (B) The helper virus-free packaging technology involves the co-transfection of a permissive cell line such as Vero 2-2 cells with the transgene-harbored amplicon plasmid and a bacterial artificial chromosome (BAC) designed to harbor the HSV-1 genome deleted in the packaging signals. An accessory plasmid expressing other HSV-1 proteins, such as vhs, can be included to enhance titers. The HSV-1-BACΔpac genome has been strategically designed to preclude its packaging into viral particles, thereby eliminating the presence of helper virus contamination in the final amplicon vector stock. (Figure was adapted from [45,68]).