Development and Application of Replication-Incompetent HSV-1

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Gene Therapy (2005) 12, S98–S102 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt CONFERENCE PAPER Development and application of replication- incompetent HSV-1-based vectors

E Berto, A Bozac and P Marconi Department of Experimental and Diagnostic Medicine, Section of Microbiology, University of Ferrara, Ferrara, Italy

The replication-incompetent HSV-1-based vectors are of different HSV-1-based nonreplicative vectors for speciﬁc herpesviruses in which genes that are ‘essential’ for viral gene therapy applications have been developed so far. They replication have been either mutated or deleted. These have been tested in different gene therapy animal models of deletions have substantially reduced their cytotoxicity by neuropathies (Parkinson’s disease, chronic pain, spinal cord preventing early and late viral gene expression and, together injury pain) and lysosomal storage disorders. Many replication- with other deletions involving ‘nonessential’ genes, have incompetent HSV-1-based vectors have also been used either also created space to introduce distinct and independently as potential anti-herpes vaccines, as well as vaccine vectors regulated expression cassettes for different transgenes. for other pathogens in murine and simian models. Anticancer Therapeutic effects in gene therapy applications requiring gene therapy approaches have also been successfully set up; simultaneous and synergic expression of multiple gene gene therapy to other targets by using these vectors is feasible. products are easily achievable with these vectors. A number Gene Therapy (2005) 12, S98–S102. doi:10.1038/sj.gt.3302623

Keywords: Herpes simplex virus; replication-incompetent viral vectors; neuronal gene delivery; cancer gene therapy; vaccination

Introduction genes in one vector, to achieve a synergistic therapeutic effect in gene therapy applications of multifactorial Many diseases are the consequence of single or multi- diseases. factorial gene defects. A greater understanding of the The replication-defective HSVs are viral vectors, molecular, biochemical and genetic factors that entail where ‘essential’ genes for in vitro viral replication are the progression of a specific disease state has led to the either mutated or deleted. Immediate-early (IE) genes, development of gene therapy-based strategies using which encode infected cell proteins (ICP) ICP0, ICP4, direct gene transfer either to ameliorate the disease ICP22, ICP27 and ICP47, are expressed shortly after viral condition or to correct a genetic defect. Several gene entry into the host cell and are required for initiation delivery approaches have been explored, each of which of the cascade of early (E) and late (L) viral gene has both advantages and disadvantages but natural gene transcription. ICP4 and ICP27 are essential for replication transfer agents such as viruses remain by far the most and the deletion of one or both of them requires adequate efficient and proven vectors. complementing cell lines capable of providing in trans Recombinant nonreplicative Herpes simplex viruses the deleted viral proteins.1,2 Besides its transcriptional (HSV) are potential vectors for several applications in functions, ICP27 also affects the splicing, polyadenyla- human health (Figure 1). These include: (i) delivery and tion and stability of mRNAs and inhibits the transcrip- expression of human genes to central nervous system tion of many host genes.3–5 ICP0 is a multifunctional (CNS) cells, (ii) selective destruction of cancer cells, (iii) protein acting as a promiscuous transactivator, able to prophylaxis and immunotherapy against tumors and (iv) modify host functions in order to promote viral gene prophylaxis against HSV-mediated and other infectious expression and viral productive infection.6,7 ICP22 is a diseases. Each application requires a different kind of nonessential regulatory protein required for optimal vector and, in principle, different kinds of genetic expression of the ICP0 protein; deletion of the ICP22 IE engineering. Some gene therapy applications will require gene can be responsible for a disregulated expression of short-term transgene expression, while others will ICP0. ICP47 inhibits MHC class I antigen presentation demand a long-term expression of the exogenous gene. contributing to the virus escape from the immune Although delivery of a single gene should be adequate in surveillance.8 The ‘first generation’ of replication-defec- many cases, other applications will likely require the tive HSV-1-based vectors consisted of mutants deleted in delivery of multiple transgenes. One of the major the single essential IE gene encoding ICP4, namely d120 advantages of nonreplicative vectors is the possibility, and S4TK vectors.9,10 Although these vectors show due to their capacity to accommodate multiple trans- reduced pathogenicity and can be used to efficiently transfer and transiently express reporter genes in brain, Correspondence: Dr P Marconi, Department of Experimental and they are nonetheless cytotoxic for neurons in culture. Diagnostic Medicine, University of Ferrara, Via Luigi Borsari 46, 44100 It is presumed that residual cytotoxicity results from the Ferrara, Italy expression of the other four IE genes. ICP4, ICP27, ICP0 Replication-incompetent HSV-1-based vectors E Berto et al S99 frequency of recombination has been described. Efficient insertion of transgenes into specific sites within the viral genome has been achieved in vitro using a recombination system derived from phage P1.19 Among various strategies for targeted deletion of genes within a specific viral locus, a rapid two-step method has been successfully used to facilitate the insertion of foreign sequences into the viral genome. The first step requires the insertion of a reporter gene cassette (Escherichia coli b-galactosi- dase, lacZ) flanked either by PacI or PmeI restriction recognition sites that are absent in the viral genome and surrounded by HSV sequences necessary for homologous recombination with the viral genome.10,20 The recombinant viral progeny is then selected by a ‘blue plaque’ phenotype after X-gal staining. The second step consists in the removal of the lacZ reporter gene from the recombinant virus, now containing the PacI sites, that results in the generation of two large fragments unable to produce infectious viral particles. The PacI-deleted Figure 1 Schematic representation of a second generation replication- fragment is then replaced by the heterologous gene, incompetent HSV-1-based gene transfer vector prototype, and its potential following cotransfection of the complementing cell line fields of application. These vectors have been obtained by deletion of ICP27, with the deleted HSV-1 viral genome and the plasmid ICP4 (both copies) and ICP22 IE genes, as indicated by red boxes; yellow containing the transgene. Potential recombinants are boxes represent loci that can be deleted and substituted by exogenous DNA sequences, to obtain vectors expressing multiple gene products simulta- identified by the ‘clear plaque’ phenotype after X-gal neously. staining. The insertion of the transgene construct into the viral genome eliminates the PacI sites, and allows the use and ICP22 have all been shown to be toxic in stable of this method repeatedly to insert multiple transgenes transfection assays,2 so deletion of these genes in in different loci of HSV-1 genome. This methodology combination is required to eliminate toxicity.9,11–14 Cell increases the frequency (ranging from 40 to 90%, instead lines that complement ICP4 and ICP27 have permitted of from 4 to 5%) of the homologous recombination the construction of a ‘second generation’ of highly between the viral genome and the HSV sequences defective mutants.14–16 These multiple-deleted mutants present in the plasmid. show substantially reduced cytotoxicity for neurons in A different procedure involves transfection of cells culture and unusually prolonged transgene expression with overlapping cosmids containing appropriate inser- from the ICP0 IE promoter or the HCMV IE promoter in tion or deletions. Expression of genes contained in neurons in vitro. To date, several replication-defective cosmids leads through recombination to the construction 21 vectors have been constructed in which ICP0, ICP4, of full-length viral genome. To select recombinant ICP27, ICP22 and ICP47 genes have been deleted in vectors it is critical to have a system by which to identify various combinations.12,17 The second generation of successful recombinants containing the deletions and the replication-defective vectors are characterized by transgenes. absence of early and late viral gene expression and provide enough space to introduce distinct and inde- Viral stocks production and purification pendently regulated expression cassettes for different transgenes.12 Deletion of all the five IE genes (ICP0, ICP4, The large-scale HSV-1 recombinant vectors production is ICP22, ICP27 and ICP47) prevents virus toxicity for cells currently performed by purifying the viral particles from at high multiplicity of infection, allowing the vector cell debris by low-speed centrifugation followed by high- genome to persist in cells for long periods of time.18 speed centrifugation on density gradient.22,23 This prac- However, these vectors grow poorly in culture and tice is, however, both time- and labor-consuming and express transgenes at very low levels in the absence of is thus not suitable for the manufacturing processes at ICP0 transactivator.17 the industrial scale. Efforts have been made recently to substitute the density gradient purification method with a more efficient and cheaper purification practice represented by column chromatography. Successful use Construction of nonreplicative HSV-1-based of immobilized metal affinity chromatography (IMAC) vectors: engineering techniques in the viral particles purification has been reported in a recent publication.24 Furthermore, optimal culture Modifications of the HSV genome can be achieved in a conditions to facilitate large-scale preparation of non- number of ways. They usually require a two-step process replicative vector stocks have also been described.25 in which portions of the herpes genome, that have been cloned into plasmid vectors, are first modified in vitro. The plasmid DNA is then cotransfected into cultured Gene transfer in the nervous system cells with infectious wild-type viral DNA and recombinant viruses are selected. Several methods have been HSV has evolved to persist in neurons as an episomal described to insert exogenous DNA sequences into element in a potentially life-long latent state and without the viral genome. Technique designs to enhance the interference with host cell function; these characteristics

Gene Therapy Replication-incompetent HSV-1-based vectors E Berto et al S100 make replication-defective viruses an attractive vehicle Therapy of lysosomal storage disorders with neurolo- for gene therapy of neural tissue.26 However, the genetic gical involvement such as Tay–Sachs (TS) disease modifications of these viruses introduced to reduce their requires production and distribution of the missing pathogenicity and increase their safety profile often enzyme into the CNS. Several therapeutic approaches result also in the loss of viral activities required for allow restoring the defective enzymatic activity in many efficient gene delivery and life-long persistence within key tissues (kidney, liver, spleen, etc) but the reduction of the host. As for the long-term transgene expression, it is the GM2 ganglioside deposits in the CNS is difficult to still controversial which promoter would better suit this achieve since CNS is kept in a privileged environment, purpose in vivo. Based on previous studies, it seems separated from the blood system by the blood–brain likely that the latency-associated transcript (LAT) pro- barrier (BBB), representing an obstacle to therapy. moters, namely latency-active promoters LAP1 and S Martino and co-workers (manuscript in preparation) LAP2, may be the most effective in the long-term have demonstrated that a nonreplicating Herpes simplex expression of transgenes in neurons.27,28 Hybrid variants vector encoding the hexosaminidase (Hex) A a-subunit of these promoters constituted by LAP elements linked (HSV-T0aHex) when delivered into the internal capsule to strong viral IE promoters and able to sustain long- of the TS brain animal model was able to reestablish the term expression of a transgene have been reported.29,30 Hex A activity resulting in the removal of the GM2 Nonreplicative HSV vectors have been tested in many ganglioside storage in both injected and controlateral different gene therapy animal models of various neuro- hemisphere and in the cerebellum 1 month after pathies (Parkinson’s disease,31,32 chronic pain,33 spinal treatment. cord injury pain,34 cisplatin neuropathy35) or lysosomal storage disorders with neurological involvement (manuscript in preparation). Among them, vectors expressing Vaccination multiple trophic factors seem to be very promising as a side treatment for neurodegenerative diseases. Motor The use of HSV vectors for prophylaxis against viral neuron disease (MND) is a group of neurological infections and tumors requires genetically stable mu- diseases characterized by the degenerative process of tants, incapable of replicating in CNS and of spreading in the upper and lower motor neurons36 in different parts of immunocompromised individuals, unable to reactivate, the motor system including the spinal cord, brain stem not transmissible from immunized vaccinated indivi- and motor cortex. Experimental evidence indicates that duals to contacts; however, the recombinant viral vectors treatment with multiple neurotrophic factors can sig- have to retain their immunogenicity in order to elicit nificantly increase motor neuron survival in comparison protective immunity against diseases in their hosts. A with the delivery of a single factor alone.15,37 HSV vectors large number of defective viruses have been proposed have been engineered to express multiple neurotrophic for immunization on the ground that although they factors and used to deliver these molecules to specific cannot produce infectious progeny and fundamentally motor neuron populations. Nonreplicative vectors con- make proteins only in the set of initially infected cells, the taining bFGF, CNTF and EGFP (as a reporter gene) have presentation of viral antigens and/or transgene, synthe- already been shown to induce proliferation and differ- sized ex novo in these cells, to the host’s immune system entiation in O-2A oligospheres obtained from newborn is able to elicit long-lived and potent cellular and rat brains in vitro and also in the rat hippocampus in vivo humoral immune responses. Various nonreplicative (Figure 2).38 HSV-1-based vectors have been successfully used either as potential anti-herpes vaccines in murine model39–41 or as vaccine vectors in murine and simian models. Murphy et al42 have demonstrated that two of the seven rhesus monkeys vaccinated with recombinant HSV expressing HIV antigenes were solidly protected, and another showed a sustained reduction in viral load following rectal challenge with pathogenic SIVmac239. A previous study performed in mice by T Brocker and our group has shown the efficacy of recombinant HSV-1 vaccines by using a vector expressing chicken ovalbumin.43 A single vaccination, with a strong antigen as ovalbumine, was sufficient to elicit a protective immune response characterized by high frequency of both primary and memory antigene-specific CTL response. Poor induction of CD4 helper T-cell responses and a similarly weak induction of T-dependent antibodies were observed. Apparently, the lack of antibodies did not negatively affect the ability of HSV-1-derived vaccines to give long- Figure 2 Biological activity of the TH-FGF2/0-CNTF/EGFP vector: term protection against L. monocytogenes expressing Ova. differentiation.38 Nonreplicative HSV-1-based vectors containing bFGF The results obtained with Ova viral vector are in and CNTF induce differentiation in the rat hippocampus in vivo. The accordance with the data we collected by immunizing images represent TH-FGF2/0-CNTF/EGFP-induced proliferating cells double-stained with BrdU and markers for astrocytes (GFAP), neurons mice with an HIV-1 Tat expressing nonreplicative vector (MAP2) and oligodendrocytes (CNPase). Sections were prepared at rat (manuscript in preparation). Notably, herpes simplex dentate gyrus level, 4 weeks after vector inoculation. The relative viral recombinants have been reported to elicit antigen- representation of the different cell types is indicated under each panel. specific immune response, despite pre-existing immu-

Gene Therapy Replication-incompetent HSV-1-based vectors E Berto et al S101 nity against viral antigens in host, which is very developed so far. The challenge for scientists is to important in prospective of their potential therapeutic develop the means to render these vectors harmless yet use in human population where the virus is widely effective for targeted gene transfer and appropriate gene distributed.44,45 expression. Recent advances and present applications of existing vectors, are focused on improving the characteristics of these vectors. Efforts are being made, in Cancer gene therapy particular, to reduce their toxicity, to modulate the Different replication-defective HSV vectors have been breadth and the time course of transgene expression, to produced that deliver anticancer transgenes to tumors precisely target specific cell populations and to transfer such as melanoma,12 glioma,46,47 gliosarcoma13 or glio- multiple genes by a unique vector. These recombinant blastoma.48 These mutant vectors express, in association viruses with reduced cytotoxicity can represent a very with the autologous HSV-1 thymidine kinase (tk) gene efficient delivery reagent for gene transfer in vitro and acting as a suicide gene when accompanied by its pro- in vivo. Multiple exogenous genes delivery, whether they drug ganciclovir (GCV), further transgenes chosen for are synergistically acting or not, is easily achievable with their potential to synergize in tumor cell killing and these HSV mutants and thus suggests their possible induction of antitumor immunity. Among immunosti- clinical applications in multifactorial diseases such as mulatory molecules, soluble human cytokines Interleu- tumors and neurodegenerative diseases. kin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), Interferon-g (IFN-g), human tumor necrosis factor-a (TNFa)49 and also the costimulatory Acknowledgements surface antigen CD80-encoding genes have been stu- Part of the research works cited in this review have been 12 died. Furthermore, rat connexin 43 gene has been supported by the Italian Ministry for the University and successfully used to improve the HSV-1 TK/GCV killing Scientific Research (FISR), the Italian National Institute of of glioma cells by increasing the TK/GCV proximal Health (Program Stem Cells, CS 126.1 and ICAV, Italian 50 bystander effect. Recently, an HSV-1-derived replica- Concerted Action on HIV-AIDS Vaccine Development) tion-defective vector (T0-IFI16) has been developed, and the Italian Association for Cancer Research (AIRC). which has been shown to efficiently transduce an interferon-inducible gene (IFI16), into primary human umbilical vein endothelial cells (HUVEC), which are References usually poorly transfectable.51 We have also been able to infect HUVEC cells with similar HSV-1-based vectors 1 Marconi P et al. Replication-defective herpes simplex virus expressing antiangiogenic fusion proteins endostatin::an- vectors for gene transfer in vivo. Proc Natl Acad Sci USA 1996; 93: giostatin and endostatin::kringle 5. The expression of 11319–11320. antiangiogenic proteins by directly infected HUVEC cells 2 Wu N, Watkins SC, Schaffer PA, DeLuca NA. Prolonged gene has been shown to induce cytostatic effects in prolifera- expression and cell survival after infection by a herpes simplex tion assays in vitro. Also, by addition of ganciclovir to the virus mutant defective in the immediate-early genes encoding cell culture media, we have obtained major cell killing ICP4, ICP27, and ICP22. J Virol 1996; 70: 6358–6369. effect too (manuscript in preparation). 3 Hardwicke MA, Sandri-Goldin RM. The herpes simplex virus regulatory protein ICP27 contributes to the decrease in cellular mRNA levels during infection. J Virol 1994; 68: 4797–4810. New targets for future gene therapy 4 Hardy WR, Sandri-Goldin RM. Herpes simplex virus inhibits host cell splicing, and regulatory protein ICP27 is required for applications this effect. J Virol 1994; 68: 7790–7799. In a recent publication, a replication-defective HSV-1 5 Spencer CA, Dahmus ME, Rice SA. Repression of host RNA vector has been used to deliver and express nerve polymerase II transcription by herpes simplex virus type 1. growth factor (NGF) in a rat model of diabetic J Virol 1997; 71: 2031–2040. cystopathy. 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