Avirulent oncolytic herpes simplex virus strains engineered to counter the innate host response

#The present invention relates to an oncolytic avirulent modified herpes simplex virus having a high level of replication in neoplastic cells, and uses thereof for immunization against and treatment of disease conditions.

#One hundred years ago, the first anecdotal observations correlating viral infection with tumor regression were reported, which lead to several investigations, over the course of the last century, evaluating the potential of various natural human and animal viruses to treat cancer. (Sinkovics et al., “New Developments in the Virus Therapy of Cancer: A Historical Review,” Intervirology 36:193-214 (1993)). These observations suggest that malignant lesions could regress in response to viral infection. To be effective in treatments of malignancies, such isolates should only grow productively and exhibit virulence in neoplastic cells, and should not be capable of propagating a productive infection through surrounding normal, terminally differentiated tissue. As virulence is governed by specific viral genes, one approach would be to genetically alter the virulence of viruses to obtain viruses which selectively destroy neoplastic cells. However, it was the achievements of the last two decades, most notably technical innovation in the area of molecular biology coupled with a heightened understanding of viral replication and pathogenesis at the genetic level, which ushered in the possibility of creating viruses in the laboratory which were selectively pathogenic for neoplastic cells. The advent of genetic engineering techniques has made it possible to selectively ablate viral virulence genes in the hope of creating a safe, avirulent virus that can selectively replicate in and destroy tumor cells. Such a virus would be able to propagate an infection throughout a tumor mass and directly kill the cancer cells, but be unable to inflict substantial damage to normal terminal differentiated cells. Although safe viruses have been created by such methodology, the attenuation process often has an overall deleterious effect on viral replication. This replication defect prevents the virus from completely destroying the tumor mass, and the surviving cancer cells can simply repopulate.

#HSV-1 is a double-stranded DNA virus which is replicated and transcribed in the nucleus of the cell. HSV-1 consists of at least three groups of genes, α, β, and γ, whose expression is coordinately regulated and sequentially ordered in a cascade fashion. (Honess et al., “Regulation of Herpesvirus Macromolecular Synthesis, I. Cascade Regulation of the Synthesis of Three Groups of Viral Proteins,” J Vir 14:8-19 (1974)). Five immediate early (IE or α) genes are the first genes expressed during productive infection. While one of these genes encodes the immunomodulatory protein ICP47, the remaining four a genes encode infected cell proteins (ICPs) 0, 4, 22, and 27, the major regulatory proteins of the virus. These immediate early proteins then activate the expression of viral genes of the early (E or β) and late (L or γ) classes. Once the viral lifecycle advances beyond the IE stage, the ICP4 protein also autoregulates IE gene expression by reducing transcription from IE promoters (Preston, “Control of Herpes Simplex Virus Type 1 mRNA Synthesis in Cells Infected with Wild-Type Virus or the Temperature-Sensitive Mutant tsK,” J Vir 29: 275-284 (1979); Roberts et al., “Direct Correlation Between a Negative Autoregulatory Response Element at the Cap Site of the Herpes Simplex Virus Type 1 IE175(α4) Promoter and a Specific Binding Site for the IE175 (α4) Protein,” J Vir 62: 4307-4320 (1988); Lium et al., “Repression of the alpha0 gene by ICP4 During a Productive Herpes Simplex Virus Infection,”J Vir 70: 3488-3496 (1996)). Proteins of the E class are responsible for viral DNA replication. The late (L) genes are induced after DNA replication and encode the structural components and enzymes required for assembly of virus particles.

#FIG. 1 is a line diagram of the wild-type HSV-1 genome. Boxed regions designate inverted terminal repeat (TR) regions that flank the unique short (Us) and unique long (UL) components, represented by solid lines, Dotted lines indicate an expanded view of a region of the genome. The Us-TRs junction region containing the Us10, Us11, and Us12 open reading frames (ORFs), designated by open rectangles, appears expanded. Stars represent the respective cis-acting promoter elements. The arrow above each box extending from the promoter element denotes the mRNA transcript that encodes each gene product. All of these mRNAs are polyadenylated at a common polyadenylation signal (not depicted) downstream from the Us10 ORF. The Us12 mRNA is spliced, as indicated by the dip in the arrow joining two non-contiguous regions to form the mRNA.

#FIGS. 2A and 2B are detailed maps of the genome of wild-type HSV-1 and the Δ34.5 modified (deletion mutant) virus, respectively. FIG. 2A shows that HSV-1 possesses a linear dsDNA genome composed of two sections: the Unique Long (UL) and Unique Short (Us), each represented by solid black lines flanked by open boxes on the top of each page. The boxes represent the inverted terminal repeats that flank the UL and Us (TRL and TRs). The letters above the representation of the genome identify the BamHI restriction enzyme digestion fragments which contain the γ34.5 and Us11 loci. The thick black lines between and below selected restriction sites indicate the probes used in southern blotting. The horizontal arrows indicate various transcription units and the stars represent their respective promoters. The boxes below the horizontal lines denote open reading frames (ORF), the names of which are printed within the boxes. The γ34.5 locus is diploid because it is contained within the TRL repeats, while the Us11 locus is haploid because it is located in the Us region. Both γ34.5 loci can be distinguished by size after BamHI digestion and southern blotting, because the first γ34.5 locus is wholly contained within the BamHI S fragment, whereas the second, larger locus is comprised of a fusion between the terminal BamHI S and P fragments. This fusion fragment is termed Bam SP. The γ34.5 ORF is situated between the NcoI and SacI sites and is transcribed by a promoter (indicated by star) located between the DraI and NcoI sites. FIG. 2B is a diagram of the Δ34.5 modified virus, in which both copies of the γ134.5 gene are replaced by sequences encoding β-glucuronidase.

#FIGS. 3A-B show the Us11 gene product is required for translation of viral proteins in primary human fibroblasts treated with IFNα. FS4 cells, untreated or treated overnight with human INFα, were mock infected or infected (MOI=5) with either a γ134.5 deletion mutant (Δ34.5), a Δ34.5 strain that expressed Us11 as an IE protein (Δ34.5-(IE)Us11), a Us11 null mutant (pAUs11), a virus in which the Us11 mutant allele was repaired (pAUs11-Rep), or wild type HSV-1 (WT). At 18 hours post-infection, the cultures were metabolically labeled with 35S amino acids for 1 hour. Total protein was isolated and fractionated by SDS-PAGE. FIG. 3A is an exposure of the fixed, dried gel. FIG. 3B, bottom panel, shows an immunoblot of the samples probed with a polyclonal antibody raised against the γ134.5 protein. The arrowhead to the right of the blot denotes the position of the full-length γ134.5 polypeptide encoded by the HSV-1 Patton strain. The migration of molecular weight standards (in kilodaltons) appears to the left of each panel.

#The present invention relates to an avirulent, oncolytic herpes simplex virus that is modified from a wild-type herpes simplex virus so that both γ134.5 genes of the virus have been deleted and each is replaced with an interferon-resistance gene that is expressed as an immediate-early gene. It also retains the ability to express an anti-interferon gene, exemplified by Us11, as a late gene and can counteract the acquired immune response through the action of the Us12 gene product. The modified virus is non-neurovirulent and replicates in and destroys neoplastic cells.

#The optimal intracellular environment for virus replication develops through events that begin to take place with attachment of virus to the cell membrane. Binding of the herpes simplex virus to the cell membrane receptor(s) is followed by a cascade of events that are associated with biochemical, physiological, and morphological changes in the cells. Following infection in susceptible cells, lytic replication is regulated by a temporally coordinated sequence of gene transcription. Binding of the virus to a host cell membrane activates the immediate-early (IE or a) genes (ICP0, ICP4, ICP22, ICP27, and ICP47), which are transactivating factors allowing the production of next group of genes to be transcribed, the early (β) genes. Expression of immediate-early gene products is followed by the expression of proteins encoded by the early and then, the late (γ) genes. The entire cascade of gene activation and viral replication in the WT virus takes about 18 hours and invariably results in cell death. The modified HSV mutant of the present invention circumvents the protein synthesis shutoff phenotype of γ134.5 deletion viruses, creating a more robust γ134.5 deletion virus. In WT HSV, the US11 gene product binds to RNA and is expressed with true late (γ2) kinetics. However, the modified virus of the present invention has two Us11 genes placed under the control of an IE gene promoter. Us11 expressed an IE gene compensates for deletions in γ134.5, in that eIF-2α is not phosphorylated and protein translation is not inhibited. Thus, the modified HSV of the present invention overcomes PKR-mediated protein synthesis shutoff. As a result, the modified virus of the present invention, having both γ134.5 genes replaced with Us11 having IE gene kinetics, replicates to a higher titer compared to other γ134.5 null virus mutants, and has greater efficacy as an avirulent oncolytic agent.

#Exemplary viral interferon resistance genes with which both HSV γ134.5 genes are replaced in the modified oncolytic virus of the present invention include, without limitation, the HSV Us11 gene, which, as described in greater detail herein, is an interferon antagonist. Interferon antagonists encoded by other viruses can also be utilized, as it is likely that many viruses can counteract host interferons (He et al., “Regulation of mRNA Translation and Cellular Signaling by Hepatitis C Nonstructural Protein NS5A,”J Virol 75: 5090-5098 (2001); Basler et. al., “The Ebola virus VP35 protein functions as a type I IFN antagonist,” Proc. Natl Acad Sci USA 97: 12289-12294 (2000); Hakki et al., “Double-Stranded RNA Binding by Human Cytomegalovirus pTRS1,” J Virol 79: 7311-7318 (2005), which are hereby incorporated by reference in their entirety). As more antagonists are identified in other viruses, they can be incorporated into the present invention as well. Significantly, it is well established that interferon antagonists from one virus can function to provide an anti-interferon function when incorporated into the genome of a heterologous virus (He et al., “Regulation of mRNA Translation and Cellular Signaling by Hepatitis C Nonstructural Protein NS5A,” J Virol 75: 5090-5098 (2001); Basler et al., “The Ebola virus VP35 Protein Functions as a Type I IFN Antagonist,” Proc Natl Acad Sci USA 97: 12289-12294 (2000); Beattie et al., “Reversal of the Interferon-Sensitive Phenotype of a Vaccinia Virus Lacking E3L by Expression of the Reovirus S4 Gene,” J Virol 69: 499-505 (1995); Child et al., “Complementation of Vaccinia Virus Lacking the Double-Stranded RNA-Binding Protein Gene E3L by Human Cytomegalovirus,” J Virol 76: 4912-4918 (2002), which are hereby incorporated by reference in their entirety). Other suitable interferon resistance genes include, without limitation, the influenza NS1 gene (Bergmann et al., “Influenza Virus NS1 Protein Counteracts PICA-Mediated Inhibition of Replication,” J Virol 74:6203-6 (2000); Garcia-Sastre et al., “Influenza A Virus Lacking the NS1 Gene Replicates in Interferon-Deficient Systems,” Virology 252:324-30 (1998); Hatada et al., “Mutant Influenza Viruses with a Defective NS1 Protein Cannot Block the Activation of PKR in Infected Cells,” J Virol 73:2425-33 (1999), which are hereby incorporated by reference in their entirety), the vaccinia E3L gene (Davies et al., “The E3L and K3L Vaccinia Virus Gene Products Stimulate Translation Through Inhibition of the Double-Stranded RNA-Dependent Protein Kinase by Different Mechanisms,” J Virol 67:1688-92 (1993); Beattie et al., “Distinct Patterns of IFN Sensitivity Observed in Cells Infected with Vaccinia K3L- and E3L-Mutant Viruses,” Virology 210:254-63 (1995); Beattie et al., “Host-Range Restriction of Vaccinia Virus E3L-Specific Deletion Mutants,” Virus Genes (12:89-94 (1996); Beattie et al., “Reversal of the Interferon-Sensitive Phenotype of a Vaccinia Virus Lacking E3L by Expression of the Reovirus S4 Gene,” J Virol 69:499-505 (1995), which are hereby incorporated by reference in their entirety), the reovirus sigma 3 gene (Imani et al., “Inhibitory Activity for the Interferon-Induced Protein Kinase is Associated with the Reovirus Serotype 1 sigma 3 Protein,” Proc Natl Acad Sci USA 85:7887-91 (1988), which is hereby incorporated by reference in its entirety), and the adenovirus virus associated RNAs (Ad VA RNAs) (Mori et al., “Anti-Interferon Activity of Adenovirus-2-Encoded VAI and VAII RNAs in Translation in Cultured Human Cells,” Virus Res 42:53-63 (1996), which is hereby incorporated by reference in its entirety).