To prevent damage of healthy tissues, to decrease the risk of germ line transduction, and to design vectors that can be administered intravenously, it is necessary to achieve targeted gene therapy. Genetic modifications to the genome of HSV-1 vectors have been generated to preferentially target viral infection and/or replication to tumour cells vs
normal cells [141
Targeting viral infection to particular cells can be obtained by modifying the first steps of the virus life cycle, i.e. adsorption and penetration. Efforts for engineering the HSV-1 envelope to obtain targeted infection are currently in progress. Altering HSV-1 host range has proved a formidable task because HSV-1 infection is a complex process involving the action of several glycoproteins in cell attachment, entry, and cell-to-cell spread (Fig. ).
As a first step, to eliminate the HSV tropism, a mutant virus deleted for gC and the HS binding domain of gB, namely KgBpK-
, was generated [251
]. This virus was further engineered to redirect infection to erythropoietin (EPO)-receptor positive cells using gC-EPO fusion molecules. It was demonstrated that one of the gC-EPO fusion molecules was incorporated into a recombinant HSV-1 viral envelope and was able to stimulate proliferation of EPO growth dependent cell line FD-EPO. Otherwise, the lack of productive infection due to endocytosis of the virus resulted in an aborted infection [252
]. It was recently demonstrated that the KgBpK-
virus, further modified to express and incorporate a fusion protein between gC and the preS1 active peptide (preS1ap) of HBV, was able to bind preferentially to hepatocytes, giving a productive infection [253
]. However, this mutant still retains gD binding functions. Simultaneously, efforts are in progress to modify gD, the other glycoprotein involved in HSV-1 binding and penetration into the host cell [254
]. In a following report, R5111 mutant virus was described lacking the HS binding domains of gB and in which the HS and HVEM binding regions of gC and gD, were substituted with IL-13 coding sequences. HSV-1 R5111 can infect J13R cells containing the IL-13Rα2 receptor and lacking all other HSV-1 receptors. However, this mutant still retains the other gD binding affinities, and is not yet established whether it can productively infect the target cells, in a similar way to that obtained with wt HSV [255
]. An alternative approach reports a transiently vesicular stomatitis virus glycoprotein G (VSV-G) pseudotyped gD minus HSV-1, but to date a stable mutant has not yet been reported [254
]. A further stimulus to the search of new strategies to alter HSV cell tropism, derives from the observation, that selected mutations in gD can reduce or abolish entry/fusion activity with nectin-1 and nectin-2, the principal receptors for HSV-1 entry into neurons, without preventing activity of HVEM or 3-0-S HS, that alternatively can mediate entry into T cells and fibroblasts [256
]. It has been recently reported the construction of an HSV-1 mutant that selectively targets the HER2 (epidermal growth factor 2)-expressing tumour cells by means of a point mutation and insertion of an anti-HER2 single-chain antibody into gD that simultaneously allow the virus to be detargeted for nectin-1 and HVEM and retargeted to HER-2. The resulting recombinant, R-LM113, has been shown to enter the cells and spread to cell solely via
HER2. Moreover, such HSV-1 recombinant strongly inhibited progressive tumour growth in nude mice bearing HER-2-hyper-expressing human tumours [257
]. At the same time, other groups have demonstrated the possibility of redirecting HSV-1 tropism by antibody-binding sites incorporated on the virion surface as a gD fusion protein, by incorporating either single-chain variable fragment (scFv) anti-CD55, or anti-CD38, or anti-EGFR in place of residues 274-393 of gD, to specifically target tumour cells [259
These data suggest that strains carrying gD mutations may establish transient infections in humans, but perhaps not latent infections of neurons, and are therefore candidates for development of safe virus vaccines and vaccines vectors.
Finally, the recently discovered the PILRα gB receptor represents another possible target to modify viral tropism.
One strategy to target replication of the attenuated virus is obtained by eliminating viral functions necessary for replication in normal cells. These mutations give the virus the attenuated phenotype that leads to replication only in permissive cells such as dividing tumour cells or cells with defects in specific cancer pathways [122
]. However, a limit of this strategy is that many normal tissues also have high mitotic indices, and such viruses may not discriminate between rapidly proliferating normal and cancer cells.
A second strategy to target viral replication to tumour cells, consists in placing the expression of essential viral genes under the control of tumour or tissue-specific promoters, that are preferentially active in tumour cells [139
]. The use of tissue-specific promoters to direct viral replication to a specific tumour type have been explored more extensively in oncolytic adenoviruses and use of these promoters to regulate HSV replication could be further explored. A limitation in the use of these promoters is that viral replication is mainly targeted at a specific tumour type and often further restricted to only a subgroup. To this purpose, promoters active in most tumours [18
] have been explored [263
], or radiation-responsive promoters, but there are instances where these promoters might be active in normal cells, leading to toxicity. A further obstacle is that, in the context of a tumour, it is likely that these promoters will not be active in all tumour cells and that not all cells will be infected. As a result, a subset of tumour cells will have a selective growth advantage and survive leading to tumour recurrence.
Different reports indicate the possibility to drive expression of ICP4 or ICP34.5 with tumour specific promoters. Viruses containing ICP4 driven by either human carcinoembryonic antigen (CEA) and MUC1/DF3 tumour-associated antigens promoters were demonstrated to replicate specifically in tumour cells but regulation of ICP4 expression by the CEA promoter during HSV-1 infection overly attenuated viral replication [139
]. Conversely, regulation of ICP34.5 function by the DF3/MUC1 promoter/enhancer sequence, resulted in preferential replication into DF3/MUC1 expressing cancer cells, restricted biodistribution in vivo
, and less toxicity [139
It has also been demonstrated that viruses, based on G207 backbone, containing ICP34.5 driven by either Musashi1 (KeM34.5 vector) [265
] or rQNestin [234
] promoter can be used to drive HSV-1 virulence toward gliomas while maintaining the desirable neuro-attenuated phenotype.
The main HSV-1 targeting strategies reported to date are summarized in Table .
Summary of HSV-1 Replication-Competent Vectors Targeting Strategies