WO2009111892A1 - Microrna mediated oncolytic targeting - Google Patents

Abstract

The invention provides methods for preferentially killing target cells in a host by infecting the cells with one or more strains of a lytic virus that has been engineered to be susceptible to microRNA mediated translational repression. Methods of the invention thereby exploit differential microRNA expression in target and non-target cells of the host. For example, altered expression of microRNAs in cancer cells can be exploited to alter expression of a viral gene in cancer cells, compared to non-cancerous cells, to achieve tumour specific replication of an engineered virus, such as a vesicular stomatitis virus (VSV). In a selected embodiment, incorporation of let-7a microRNA complementary sequences within the VSV genome attenuates viral replication and associated toxicity in normal cells, but permits growth in cancer cells.

Description

MicroRNA Mediated Oncolytic Targeting

FIELD

[0001] The invention is in the field of cancer treatment, particularly oncolytic viral therapies.

BACKGROUND

[0002] Currently, there are few effective options for the treatment of many common cancer types. The course of treatment for a given individual depends on the diagnosis, the stage to which the disease has developed and factors such as age, sex and general health of the patient. The most conventional options of cancer treatment are surgery, radiation therapy and chemotherapy. Surgery plays a central role in the diagnosis and treatment of cancer. Typically, a surgical approach is required for biopsy and to remove cancerous growth. However, if the cancer has metastasized and is widespread, surgery is unlikely to result in a cure and an alternate approach must be taken. Radiation therapy, chemotherapy and immunotherapy are alternatives to surgical treatment of cancer (Mayer, 1998; Ohara, 1998; Ho et al., 1998). Radiation therapy involves a precise aiming of high energy radiation to destroy cancer cells and much like surgery, is mainly effective in the treatment of non-metastasized, localized cancer cells. Side effects of radiation therapy include skin irritation, difficulty swallowing, dry mouth, nausea, diarrhea, hair loss and loss of energy (Curran, 1998; Brizel, 1998).

[0003] Chemotherapy, the treatment of cancer with anti-cancer drugs, is another mode of cancer therapy. The effectiveness of a given anti-cancer drug therapy often is limited by the difficulty of achieving drug delivery throughout solid tumors (el-Kareh and Secomb, 1997). Chemotherapeutic strategies are based on tumor tissue growth, wherein the anti-cancer drug is targeted to the rapidly dividing cancer cells. Most chemotherapy approaches include the combination of more than one anti-cancer drug, which has proven to increase the response rate of a wide variety of cancers (U.S. Patent 5,824,348; U.S. Patent 5,633,016 and U.S. Patent 5,798,339, incorporated herein by reference). A major side effect of chemotherapy drugs is that they also affect normal tissue cells, with the cells most likely to be affected being those that divide rapidly in some cases (e.g., bone marrow, gastrointestinal tract, reproductive system and hair follicles). Other toxic side effects of chemotherapy drugs can include sores in the mouth, difficulty swallowing, dry mouth, nausea, diarrhea, vomiting, fatigue, bleeding, hair loss and infection.

[0004] Oncolytic viruses are replicating microorganisms that have been selected or engineered to grow inside tumor cells preferentially compared to normal cells. Replication-selective oncolytic viruses hold promise for the treatment of cancer (Kirn et al., 2001 ). A wide variety of oncolytic viruses have been used in preclinical and clinical cancer therapies (see Parato et al., 2005; Bell et al, 2003; Everts and van der Poel, 2005; Ries and Brandts, 2004). These viruses can cause tumor cell death through direct replication-dependent and/or viral gene expression-dependent oncolytic effects (Kirn et al., 2001 ). In addition, viruses are able to enhance the induction of cell-mediated antitumoral immunity within the host (Todo et al., 2001 ; Sinkovics et al., 2000). These viruses also can be engineered to expressed therapeutic transgenes within the tumor to enhance antitumoral efficacy (Hermiston, 2000).

[0005] A variety of mechanisms of generating tumor selectivity currently exist. Methods of targeting oncolytic viruses to cancer cells may for example be based on differential expression of receptors. An unmodified cocksackie virus (CAV21) is thought to selectively target cancer cells because the viral receptors ICAM-1 and DAF are overexpressed on malignant melanoma cells. WO 2005/087931 discloses selected Picornavirus adapted for lytically infecting a cell in the absence of intercellular adhesion molecule-1 (ICAM-1 ). Engineering to alter specificity of receptors is also possible to enhance tumor-targeting of onocolytic viruses including adenovirus (Sebestyen 2007, Carette 2007) and measles (Allen 2006, Hasegawa 2006), for example.

[0006] Other mechanisms of selectivity are rooted in the difference in biology between normal cells and cancer cells, mediated for example by mutations or altered expression of oncogenes and tumor suppressors. In normal cells, the maintenance of cell proliferation and cell death is at least partially regulated by proto-oncogenes and tumor suppressors. A proto-oncogene or tumor suppressor can encode proteins that induce cellular proliferation (e.g., sis, erbB, src, ras and myc), proteins that inhibit cellular proliferation (e.g., Rb, p16, p19, p21 , p53, NF1 and WT1 ) or proteins that regulate programmed cell death (e.g., bcl-2) (Ochi et al., 1998; Johnson and Hamdy, 1998; Liebermann et al., 1998). However, genetic rearrangements or mutations of these proto-oncogenes and tumor suppressors result in the conversion of a proto-oncogene into a potent cancer-causing oncogene, or of a tumor suppressor into an inactive polypeptide. Often, a single point mutation is enough to achieve the transformation. For example, a point mutation in the p53 tumor suppressor protein results in the complete loss of wild- type p53 function (Vogelstein and Kinzler, 1992).

[0007] Oncolytic viruses can be engineered to exploit these genetic differences in normal vs. cancer cells. For example, the adenovirus E1 B 55k protein acts to inactivate p53 function, was deleted in an oncolytic adenovirus in order to restrict its replication to cancer cells lacking p53 function (Bischoff 1996, Rogulski 2000). US 2006/18836 discloses methods for treating p53-negative human tumor cells with the Herefordshire strain of Newcastle disease virus.

[0008] Alterations in cancer cell signaling pathways lead to upregulation of genes required for cell proliferation, such as thymidine kinase (TK) and ribonucleotide reductatase (RNR) which required for the production of dNTPs used in DNA synthesis. The replication of viruses can be restricted to cancer cells with high TK or RNR activity by deleting the viral versions of TK or RNR, or other genes upgreulated in cancer cells. US 7,208,313 discloses a vaccinia virus with TK and VGF deletions and WO 2005/049845, WO 2001/053506, US

2004/120928, WO 2003/082200 , EP 1252323 and US 2004/9604 disclose herpes viruses such as HSV, which may have improved oncolytic and/or gene delivery capabilities.

[0009] The Ras/PRK pathway is commonly mutated in cancer cells. This pathway affects the interferon (IFN) response pathway, which is deficient in cancer cells with activated Ras. Some oncolytic viruses are tumor-selective based on the difference in the innate immune response pathway in normal vs. cancer cells. For example, unmodified reovirus is naturally selective to cancer cells based on the lack of innate anti-viral response in tumor cells with activations in the Ras/PRK pathway. WO 2005/002607 discloses the use of oncolytic viruses to treat neoplasms having activated PP2A-like or Ras activities, including combinations of more than one type and/or strain of oncolytic viruses, such as reovirus.

[0010] Laboratory selection or engineering can also exploit this method of targeting based on IFN response. For example, mutants of VSV which strongly induce interferon expression are attenuated in normal cells with an intact IFN response pathway, but can infect and replicate in tumor cells which are deficient in IFN response pathways. For example, EP 1218019, US 2004/208849, US 2004/115170, WO 2001/019380, WO 2002/050304, WO 2002/043647 and US 2004/170607 disclose oncolytic viruses, such as Rhabdovirus, picornavirus, and vesicular stomatitis virus (VSV), in which the virus may exhibit differential susceptibility, particularly for tumor cells having low PKR activity. Similarly, vaccinia virus engineered to lack genes that normally thwart the host IFN- response (E3L, K3L, B18R, etc.) are also attenuated in normal cells but can grow in tumor cells lacking IFN-response. WO 2005/007824 discloses oncolytic vaccinia viruses and their use for selective destruction of cancer cells, which may exhibit a reduced ability to inhibit the antiviral dsRNA dependent protein kinase (PKR) and increased sensitivity to interferon. WO 2003/008586 similarly discloses methods for engineering oncolytic viruses, which involve alteration or deletion of a viral anti-PKR activity.

[0011] Transcriptional targeting is another method of engineering selectivity into ocolytic viruses. In this method, an essential gene of the virus is under the control of tumor-specific promoters such as a the promoter for prostate-specific antigen (PSA) (Rodriguez 1997) or cyclooxygenase-2 (Cox-2) (Davydova 2004).

[0012] Although a degree of natural tumor-selectivity can be demonstrated for some virus species, and some methods exist for engineering specificity, there remains a need to enhance tumor-selectivity of oncolytic viruses. This selectivity is particularly important when intravenous administration is used, and when potentially toxic therapeutic genes are added to these viruses to enhance anti- tumoral potency.

[0013] In many instances, oncolytic viral vectors have been administered by intratumoural injection, such as vectors based on vaccinia virus, adenovirus, reovirus, newcastle disease virus, coxsackievirus and herpes simplex virus (HSV) (Shah et al., 2003; Kaufman, et al. 2005; Chiocca et al., 2004; Harrow et al., 2004; Mastrangelo et al., 1999). Particularly in metastatic disease, a systemic route of delivery for oncolytic viruses may be desirable, for example by intravenous administration (Reid et al., 2002; Lorence et al., 2003; Pecora et al., 2002;

Lorence et al., 2005; Reid et al., 2001 ; McCart et al., 2001 ). Although systemic administration of oncolytic viruses may be desireable, this exposes the virus to heightened immune surveillance, which may result in viral inactivation by serum complement components (Ikeda et al., 1999; Wakimoto et al., 2002), uptake by the reticuloendothelial system (Worgall et al, 1997; Ye et al., 2000) or neutralization by circulating antibodies (Ikeda et al., 1999; Hirasawa et al., 2003; Lang et al., 2006; Chen et al., 2000; Tsai et al., 2004).

[0014] Oncolytic viruses that have been selected or engineered to productively infect tumour cells include adenovirus (Xia et al., 2004; Wakimoto et al., 2004); reovirus; herpes simplex virus 1 (Shah, et al., 2003); Newcastle disease virus (NDV; Pecora, et al., 2002); vaccinia virus (Mastrangelo et al.,1999; US 2006/0099224); coxsackievirus; measles virus; vesicular stomatitis virus (Stojdl, et al., 2000; Stojdl, et al., 2003); Seneca Valley Virus (Reddy, et al. 2007), influenza virus; myxoma virus (Myers, R. et al., 2005).

[0015] Various strategies have been used to engineer oncolytic viruses in order to limit replication to cancer cells (Parato et al., 2005). Viral genes required for countering host cell innate immunity are deleted or mutated in a variety of oncolytics. The approach of employing deletions or mutations in viral genes that have evolved to counter the host antiviral response is a common strategy in safe oncolytic virus design (Parato et al., 2005; Muster et al., 2004; Hummel et al., 2005). This strategy has been employed with oncolytic VSV (Stojdl et al., 2000), influenza (Muster et al., 2004), and HSV-1 (Varghese & Rabkin, 2002). One function of wildtype VSV M protein in the host cell is the suppression of the host cell innate immune response and this function is eliminated by mutation in an oncolytic strain of VSV (Stojdl et al., 2003). While wildtype VSV M demonstrates toxicity in normal cells, mutant M proteins eliminate this toxicity, but may also display a reduced cytopathic effect in cancer cell lines (Stojdl et al., 2003;

Kopecky et al., 2001 ; Ahmed et al., 2004). Oncolytic VSV containing this mutant M protein induces an antiviral response in infected cells and this response may limit intratumoural replication and antitumour activity of oncolytiv viruses including VSV (Altomonte et al., 2008; Haralambieva et al., 2007; Taneja et al., 2001 ).

[0016] MicroRNAs are small (typically -21 nt), endogenous, non-coding RNAs that direct the translational repression of target mRNAs having at least a partially complementary sequence in their 3'UTR (Cerutti, 2003; Scherr & Eder, 2007; Filipowicz et al., 2005). Differential microRNA expression is a hallmark of cancer cells, and the majority of microRNAs are reportedly downregulated in cancer cells (Lu et al., 2005; Thomson et al., 2006). The general downregulation of microRNA expression in cancer is often due to a decrease in posttranscriptional processing of these RNAs, and when this processing is abrogated it promotes tumourgenesis (Thomson et al., 2006; Kumar et al., 2007). Example of microRNAs that may be abnormally expressed in cancer include: miR-9 ,miR-10b, miR-15, miR-15a, imiR- 16, miR-16-1 , miR-20a, miR-21 , miR-29, miR-31 , miR-34a, miR-96, miR-98, miR- 103, miR-107, miR-125a, miR-125b, miR-133b, miR-135b, miR-143, miR-145, miR-146, miR-181 , miR-181a, miR-181 b, miR-181c, miR-183, miR-184, miR- 196a-2, miR-221 , miR-222, miR-223, miR-301 , miR-376, let-7, let-7a, let-7a-1 , hsa-let-7a-2, let-7a-3, let-7g. The Let-7 family includes let-7, let-7a, let-7a-1 , hsa- let-7a-2, let-7a-3, let-7g. Alternative examples of differentially expressed microRNAs are miR-24 (differentially expressed in cancer vs. normal) and miR- 155 (expressed in developing dendritic cells).

[0017] One family of highly conserved microRNAs demonstrating low expression in cancer cells is let-7 (Sempere et al., 2004; Lagos-Quintana et al., 2003; Pasquinelli et al., 2000; Jannot et al., 2006; Karube et al., 2005; Kent et al., 2006; Takamizawa et al., 2004; Yanaihara et al., 2006; Yu et al., 2007; Viswanathan et al., 2008; Kumar et al., 2008). Decreased expression of let-7 is functionally linked to tumour cell biology, regulating the expression of proto- oncogenes (Johnson et al, 2005; Mayr et al., 2007), and reflecting the differentiation state of tumours (Lu et al., 2005; Yu et al., 2007; Viswanathan et al., 2008). Synthetic microRNA complementary sequences in the 3'UTR of a target gene have demonstrable let-7 specific repression (Pillai at al., 2005).

[0018] The engineered expression of siRNAs and microRNA from viruses has been suggested (see WO 2007/130604), for example in methods that are akin to gene-therapy approaches, in which an engineered siRNA sequence is used to inhibit oncogene function in the cell.

SUMMARY

[0019] In one aspect, the invention exploits the differential expression of endogenous microRNA(s) in host cells, such as cancerous cells compared to non- cancerous cells, to restrict replication of a virus, such as an oncolytic virus, the virus having been engineered to be susceptible to inhibition by the endogenous microRNA(s).

[0020] In selected embodiments, the invention exploits differential expression of a microRNA in host cells to preferentially permit expression of viral genes in selected host cells, such as cancer cells, by incorporation of recombinant microRNA target sequences in the viral genome, such as let-7 complementary sequences incorporated into an oncolytic virus genome. These sequences are selected so that they mediate inhibition of viral gene expression and replication in normal, microRNA (such as let-7) expressing cells, while low microRNA (let-7) activity in diseased (such as cancer) cells facilitates viral gene expression and subsequent lysis (such as oncolysis).

[0021] Exemplified embodiments make use of a vesicular stomatitis virus (VSV), a negative sense single-stranded RNA Rhabdovirus, that is naturally sensitive to microRNA mediated repression (Barik, 2004; Otsuka et al., 2007; Wilkins et al., 2005). Specifically, the exemplified embodiments subject the expression of the wildtype matrix protein of VSV (VSV M) to let-7 regulation. The microRNA target gene may be selected, as in the case of matrix protein, by virtue of the fact that the protein has an essential role in viral growth and replication, and thereby serves to counteract antiviral responses (Stojdl et al., 2003; Jayaker et al., 2000; Lichty et al., 2004). This strategy facilitates the use of potent viral protein targets for microRNA inhibition. Alternative viral candidates include: a Vesiculovirus (including vesicular stomatitis virus), a Rhabdovirus, a poxvirus (including myxoma and vaccinia virus), a herpes virus, an adenovirus, a Newcastle-disease virus, a measles virus, a Picornavirus (including coxsackie virus), a Seneca Valley Virus, an influenza virus, or a retrovirus. In various aspects, the invention provides methods for treating cancers with an amount of one or more strains of oncolytic virus, such as the foregoing strains. The virus will generally be selected to be effective to cause a lytic infection in cancer cells. In alternative embodiments, one or more strains of an oncolytic virus may be used in methods of the invention, simultaneously or successively.

[0022] In one aspect, the invention provides methods for selectively ablating cells in a host. The host may for example comprise: non-target cells having a microRNA response mediated by a microRNA endogenously expressed in the non-target cells; and, target cells wherein the microRNA response is attenuated compared to the non-target cells. The methods may involve administering to the host an effective amount of a recombinant lytic virus. The virus may comprise a heterologous target nucleic acid sequence incorporated into a viral gene. The methods may be carried out so that the heterologous sequence is transcribed into a target mRNA expressed from the viral gene in the target cells and in the non- target cells. The methods may further me orchestrated so that the target mRNA interacts with the microRNA in the non-target cells to mediate translational repression of the target mRNA in the non-target cells, and so that the attenuated microRNA response in the target cells permits expression of the viral gene in the target cells. Expression of the viral gene in the target cells may then mediate (directly or indirectly through any number of cellular or host mechanisms) killing of the target cells in the host.

[0023] In alternative embodiments, the oncolytic virus may be administered to the host systemically, such as intravenously, or intratumorally to infect the tumor. Alternative hosts amenable to treatments in accordance with the invention may include animals, mammals and humans.

BRIEF DESCRIPTION OF THE DRAWINGS [0024] Figure 1 : is a schematic of let-7 microRNA target elements incorporated into VSV^let"7mm, VSV^let"7 and VSV^let"7wt. The VSV genome and viral transcripts with the location of an introduced Not1 restriction site in the 3'UTR of VSV M. The mRNA of VSV M contains the original 3'UTR following the incorporated Not1 site. C. The sequence elements inserted into the Not1 site in VSV^let-^7mm, VSV^let-⁷ and VSV^le*-^7wt show partial complimentarity, complete complementarity or limited complimentarity, respectively, to the let-7 microRNA and are present in triplicate. The sequence within VSV^let"7mm has demonstrated let-7 specific translational repression, while the sequence with VSV^let"7 has demonstrated let-7 specific mRNA destabilization. The sequence within VSV^let"7wt has not demonstrated any measurable let-7 specific repression.

[0025] Figure 2: illustrate that the engineered viruses are sensitive to sequence specific let-7 activity of the host cell. (A) Vectors containing the same let-7 sequences as VSV^let"7mm in the 3'UTR of luciferase demonstrate greater repression in HeIa cells as compared to A549 cells. The repression ratio is expressed as a ratio of the VSV^let-^7mm luciferase to the VS v^let'7mut luciferase using a co-transfected renilla luciferase reporter to normalize for transfection efficiency. Quantification of mature let-7 microRNA in the indicated cell lines expressed as a ratio to U6 small RNA. (B) Titres of VSV^let-^7mm, VSV^let-^7mutand VSV^let-^7wt after 24 hours infection of HeIa and A49 cells. (C) Growth curve of the engineered viruses in A549 and GM38 at an MOI of 0.1. The dashed line indicates the limit of detection.

[0026] Figure 3: Illustrates the finding that let-7 microRNA effects replication, cytotoxicity and M mRNA levels in VSV^let'7wt infected cells. (A) HeIa cells were infected with the engineered let-7 viruses 24 hours after transfection with the indicated siRNA, Let-7, VSV M or eGFP. Titres were determined 20 hours post infection. (B) Cell survival of A549 cells, as determined by MTS assay, after transfected with the indicated siRNAs 24 hours before infection with the indicated let-7 VSV. Cell survival was determined 24 hours after infection. (C) HeIa cells were infected for 6 hours at an MOI of 1 and after 6 hours total RNA was extracted and viral mRNAs were quantified by qPCR of oligo dT primed cDNA (n=3).

[0027] Figure 4: Illustrates that let-7 target sites reduce expression of the target gene specifically and targeting M strongly attenuates VSV in the primary human fibroblast cell line GM38. (A) Luciferase expression of HeIa and A549 cells infected at an MOI of 10 for 2 hours with VSV containing luciferase containing identical let-7 target sites as depicted in Figure 1. (B) VSV titres resulting from infection of GM38 cells for 48 hours with VSV having either let-7 target sequences in the 3'UTR of M or luciferase. (C) Phase contrast pictures of infected GM38 cells 72 hours after infection at an MOI of 0.1. (D) GM38 cells mock infected or infected with VSV^let"7wt24 hours before infection with wt VSV expressing eGFP. Another 24 hours later, fluorecence microscopy was used to visualize eGFP expression.

[0028] Figure 5: (A) Balb/c mice infected intranasally with 1x10⁵pfu of the indicated virus. Body weights were expressed as a percentage of initial body weight before infection and averaged. n=5. (B) Survival of balb/c mice infected with VSV^let"7mut or VSV^let"7wt. Equal numbers of 3 week old mice were infected with 1 E5 to 1 E7 pfu of the indicated virus intranasally. Endpoints were exclusively the result of hind-limb paralysis. n=9.

[0029] Figure 6: (A) Intravenous treatment of subcutaneous tumours with VSV^let"7wt. Tumours were seeded in the hind flank of Balb/C mice by injection of 5x10⁵ CT26 cells. Mice were treated with either 1x10⁸ or 1x10⁹ pfu ofVSV^let"7wt. Control mice received an equal volume of PBS. Tumours were measures to calculate tumour volumes. (B) lmmunohistochemistry and fluorescent microsphere perfusion of subcutaneous tumours excised 24 hours after intravenous treatment with 1 x10⁹ ppfu of VSV^let-^7wt or PBS.

[0030] Figure 7: is an illustration of results from Example 2, in which HCT116 cells exposed to 100ng/ml_ doxorubicin (dox) for 48 hours were subsequently infected at an MOI of 0.1 with wt VSV or VSV carrying mir-34a mirTs (34aM) in the 3¹UTR of the matrix protein for 16 hours. Lysates were then probed with polyclonal VSV antibodies and B-actin as a loading control. The HCT116 cells are a paired cell line that contain p53 or are p53 null cells as indicated.

[0031] Figure 8: is an illustration of results from Example 2, in which HCT116 cells exposed to 100ng/ml_ doxorubicin (dox) for 48 hours were subsequently infected with wt VSV or VSV carrying mir-34a mirTs (34aM) in the 3'UTR of the matrix protein for 16 hours. Cells were infected at an MOI of 0.1 with a mir-34a sensitive VSV or an equivalent wt VSV as indicated before the phase contrast microscopy pictures were taken.

[0032] Figure 9: is an illustration of results from Example 2, in which GM38 cells were exposed to 10J/cm2 of ultraviolet radiation (UV) as indicated 24 hours before infection with a mir-34a sensitive VSV (VSV mir34a M) or a VSV carrying mock microRNA target sequences (VSV mutlet-7 M). Samples were taken from the media at 24 hours and titred.

DETAILED DESCRIPTION

[0033] In various aspects, the invention implements microRNA mediated suppression of viral gene expression to eliminate undesirable replication of a virus in non-target cells. In this way, microRNA expression may be utilized to selectively direct replication of therapeutic viruses to target tissues.

[0034] In selected embodiments, the invention utilizes a VSV virus. As an RNA virus, the VSV lifecycle is subject to interaction with endogenous microRNAs. The negative sense microRNA targets in the genome of VSV are not complementary to the endogenous microRNA. The exemplified Results indicate that in selected embodiments the positive sense genome that occurs during the viral lifecycle is not affected by the microRNA target sites, leaving only the viral mRNA transcript as the specific target using this method. This finding is supported by the data presented in Figure 3c, as viral titres are not reduced by microRNA target incorporation into the 3'UTR of the irrelevant gene, luciferase. In alternative embodiments, the invention may make use of the fact that VSV is sensitive to interferon, which may accordingly be administered to a host so as to protect the host against replication of the virus in normal tissues.

[0035] Spontaneous generation of viruses containing functional mutations in incorporated microRNA target sites is of obvious concern with this strategy. As supported by the data in Figure 4d, cells infected by VSVIet-7wt induce an antiviral state that protects all cells from a subsequent infection with an escape mutant. The escape mutant is represented in the Example by a wildtype VSV expressing eGFP. There is accordingly a particularly advantageous aspect to the use of VSV in such embodiments.

[0036] In various aspects of the invention, the microRNA target and virus may be selected so as to retain viral protein functions that serve to counteract the antiviral response in diseased (cancer) cells. This may be engineered so as to augment the selective expression of the virus in diseased target cells compared to normal non-target cells. In selected embodiments, the invention accordingly provides engineered oncolytic viruses that have particular genes under the regulatory control of incorporated microRNA target sites. The selected genes may for example be genes that are required for countering host cell innate immunity, in this way the inactivation of these genes takes place in a targeted fashion in diseased tissues. Thus, viral expression is attenuated in normal cells to minimize toxicity, while expression of these genes is maintained in cancer cells to maximize therapeutic viral efficacy. Examples of oncolytic viruses and the corresponding viral genes deleted in current oncolytics that may be selected for this approach include: HSV-1 and the gamma 34.5 gene, influenza virus and the NS-1 gene, and vaccinia virus and viral thymidine kinase. In some embodiments, for example to avoid the mutational loss of the microRNA target site, it may be desirable for the virus to carry multiple repeats of, or repeats of different, microRNA target sites.

[0037] A "substantially identical" sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, as discussed herein, or by one or more non- conservative substitutions, deletions, or insertions located at positions of the sequence that do not destroy the biological function of the amino acid or nucleic acid molecule. Such a sequence can be at least 10%, 20%, 30%, 40%, 50%, 52.5%, 55% or 60% or 75%, or more generally at least 80%, 85%, 90%, or 95%, or as much as 99% or 100% identical at the amino acid or nucleotide level to the sequence used for comparison using, for example, the Align Program (Myers and Miller, CABIOS, 1989, 4:11-17) or FASTA. For polypeptides, the length of comparison sequences may be at least 4, 5, 10, or 15 amino acids, or at least 20, 25, or 30 amino acids. In alternate embodiments, the length of comparison sequences may be at least 35, 40, or 50 amino acids, or over 60, 80, or 100 amino acids. For nucleic acid molecules, the length of comparison sequences may be at least 15, 20, or 25 nucleotides, or at least 30, 40, or 50 nucleotides. In alternate embodiments, the length of comparison sequences may be at least 60, 70, 80, or 90 nucleotides, or over 100, 200, or 500 nucleotides. Sequence identity can be readily measured using publicly available sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, or BLAST software available from the National Library of Medicine, or as described herein). Examples of useful software include the programs Pile-up and PrettyBox. Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, insertions, and other modifications.

[0038] Alternatively, or additionally, two nucleic acid sequences may be "substantially identical" if they hybridize under high stringency conditions. In some embodiments, high stringency conditions are, for example, conditions that allow hybridization comparable with the hybridization that occurs using a DNA probe of at least 500 nucleotides in length, in a buffer containing 0.5 M NaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1 % BSA (fraction V), at a temperature of 65⁰C, or a buffer containing 48% formamide, 4.8x SSC, 0.2 M Tris-CI, pH 7.6, 1x Denhardt's solution, 10% dextran sulfate, and 0.1 % SDS, at a temperature of 42⁰C. (These are typical conditions for high stringency northern or Southern hybridizations.) Hybridizations may be carried out over a period of about 20 to 30 minutes, or about 2 to 6 hours, or about 10 to 15 hours, or over 24 hours or more. High stringency hybridization is also relied upon for the success of numerous techniques routinely performed by molecular biologists, such as high stringency PCR, DNA sequencing, single strand conformational polymorphism analysis, and in situ hybridization. In contrast to northern and Southern hybridizations, these techniques are usually performed with relatively short probes (e.g., usually about 16 nucleotides or longer for PCR or sequencing and about 40 nucleotides or longer for in situ hybridization). The high stringency conditions used in these techniques are well known to those skilled in the art of molecular biology, and examples of them can be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998, which is hereby incorporated by reference.

[0039] The terms "nucleic acid" or "nucleic acid molecule" encompass both RNA (plus and minus strands) and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid may be double- stranded or single-stranded. Where single-stranded, the nucleic acid may be the sense strand or the antisense strand. A nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. By "RNA" is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. One example of a modified RNA included within this term is phosphorothioate RNA. By "DNA" is meant a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides. By "cDNA" is meant complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase). Thus a "cDNA clone" means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.

[0040] An "isolated nucleic acid" is a nucleic acid molecule that is free of the nucleic acid molecules that normally flank it in the genome or that is free of the organism in which it is normally found. Therefore, an "isolated" gene or nucleic acid molecule is in some cases intended to mean a gene or nucleic acid molecule which is not flanked by nucleic acid molecules which normally (in nature) flank the gene or nucleic acid molecule (such as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (as in a cDNA or RNA library). In some cases, an isolated nucleic acid molecule is intended to mean the genome of an organism such as a virus. An isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC. The term therefore includes, e.g., a genome; a recombinant nucleic acid incorporated into a vector, such as an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant nucleic acid which is part of a hybrid gene encoding additional polypeptide sequences. Preferably, an isolated nucleic acid comprises at least about 50, 80 or 90 percent (on a molar basis) of all macromolecular species present. Thus, an isolated gene or nucleic acid molecule can include a gene or nucleic acid molecule which is synthesized chemically or by recombinant means. Recombinant DNA contained in a vector are included in the definition of "isolated" as used herein. Also, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, as well as partially or substantially purified DNA molecules in solution. In vivo and in vitro RNA transcripts of the DNA molecules of the present invention are also encompassed by "isolated" nucleic acid molecules. Such isolated nucleic acid molecules are useful in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the nucleic acid molecule in tissue (e.g., human tissue, such as peripheral blood), such as by Northern blot analysis.

[0041] Various genes and nucleic acid sequences of the invention may be recombinant sequences. The term "recombinant" means that something has been recombined, so that when made in reference to a nucleic acid construct the term refers to a molecule that is comprised of nucleic acid sequences that are joined together or produced by means of molecular biological techniques. The term "recombinant" when made in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques. The term "recombinant" when made in reference to genetic composition refers to a gamete or progeny with new combinations of alleles that did not occur in the parental genomes. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Referring to a nucleic acid construct as '"recombinant" therefore indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and/or repair events.

[0042] As used herein, "heterologous" in reference to a nucleic acid or protein is a molecule that has been manipulated by human intervention so that it is located in a place other than the place in which it is naturally found. For example, a nucleic acid sequence from one species may be introduced into the genome of another species, or a nucleic acid sequence from one genomic locus may be moved to another genomic or extrachromasomal locus in the same species. A heterologous protein includes, for example, a protein expressed from a heterologous coding sequence or a protein expressed from a recombinant gene in a cell that would not naturally express the protein.

[0043] By "complementary" is meant that two nucleic acid molecules, e.g., DNA or RNA, contain a sufficient number of nucleotides that are capable of forming Watson-Crick base pairs to produce a region of double-strandedness between the two nucleic acids. Thus, adenine in one strand of DNA or RNA pairs with thymine in an opposing complementary DNA strand or with uracil in an opposing complementary RNA strand. It will be understood that each nucleotide in a nucleic acid molecule need not form a matched Watson-Crick base pair with a nucleotide in an opposing complementary strand to form a duplex.

Therapeutic Formulations

[0044] In one aspect, the invention involves administration (including coadministration) of therapeutic compounds or compositions, such as an oncolytic virus or agents that are effective to increase the susceptibility of a tumor cell to oncolytic viral infection in a host. In various embodiments, such agents may be used therapeutically in formulations or medicaments. Accordingly, the invention provides therapeutic compositions comprising active agents, including agents that are effective to increase the susceptibility of a tumor cell to oncolytic viral infection in a host, and pharmacologically acceptable excipients or carriers.

[0045] An effective amount of an agent of the invention will generally be a therapeutically effective amount. A "therapeutically effective amount" generally refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as increasing the susceptibility of a tumor cell to oncolytic viral infection in a host. A therapeutically effective amount a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.

[0046] In particular embodiments, a preferred range for therapeutically effective amounts may vary with the nature and/or severity of the patient's condition. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. [0047] A "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0048] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, active agents of the invention may be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

[0049] Sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0050] In accordance with another aspect of the invention, therapeutic agents of the present invention, such as agents that are effective to increase the susceptibility of a tumor or cancer cell to oncolytic viral infection in a host, may be provided in containers or kits having labels that provide instructions for use of agents of the invention, such as instructions for use in treating cancers.

[0051] Use of the present invention to treat or prevent a disease condition as disclosed herein, including prevention of further disease progression, may be conducted in subjects diagnosed or otherwise determined to be afflicted or at risk of developing the condition. In some embodiments, for oncolytic therapy, patients may be characterized as having adequate bone marrow function (for example defined as a peripheral absolute granulocyte count of >2,000/mm³ and a platelet count of 100,000/mm³), adequate liver function (for example, bilirubin<1.5 mg/dl) and adequate renal function (for example, creatinine<1.5 mg/dl).

[0052] Routes of administration for agents of the invention may vary, and may for example include intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional, percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, direct injection, and oral administration and formulation. [0053] lntratumoral injection, or injection into the tumor vasculature is contemplated for discrete, solid, accessible tumors. Local, regional or systemic administration also may be appropriate. For tumors of >4 cm, the volume to be administered may for example be about 4 to 10 ml, while for tumors of <4 cm, a volume of about 1 to 3 ml may be used. Multiple injections may be delivered as single dose, for example in about 0.1 to about 0.5 ml volumes. Viral particles may be administered in multiple injections to a tumor, for example spaced at approximately 1 cm intervals.

[0054] Methods of the present invention may be used preoperatively, for example to render an inoperable tumor subject to resection. Alternatively, the present invention may be used at the time of surgery, and/or thereafter, to treat residual or metastatic disease. For example, a resected tumor bed may be injected or perfused with a formulation comprising an oncolytic virus. The perfusion may for example be continued post-resection, for example, by leaving a catheter implanted at the site of the surgery. Periodic post-surgical treatment may also be useful.

[0055] Continuous administration of agents of the invention may be applied, where appropriate, for example, where a tumor is excised and the tumor bed is treated to eliminate residual, microscopic disease. Continuous perfusion may for example take place for a period from about 1 to 2 hours, to about 2 to 6 hours, to about 6 to 12 hours, to about 12 to 24 hours, to about 1 to 2 days, to about 1 to 2 weeks or longer following the initiation of treatment. Generally, the dose of the therapeutic agent via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs. It is further contemplated that limb perfusion may be used to administer therapeutic compositions of the present invention, particularly in the treatment of melanomas and sarcomas.

[0056] Treatments of the invention may include various "unit doses." A unit dose is defined as containing a predetermined-quantity of the therapeutic composition. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. Unit dose of the present invention may conveniently be described in terms of plaque forming units (pfu) for a viral construct. Unit doses range from 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ pfu and higher. Alternatively, depending on the kind of virus and the titer attainable, one may deliver 1 to 100, 10 to 50, 100 to 1000, or up to about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ or higher infectious viral particles (vp) to the patient or to the patient's cells.

[0057] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing. Citation of references herein is not an admission that such references are prior art to the present invention. Any priority document(s) and all publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as described herein, with reference to the examples and drawings.

[0058] The following examples illustrate the incorporation of microRNA target sites into oncolytic VSV in a manner that results in a virus that is attenuated in normal cells and has reduced in vivo toxicity while retaining anti-tumour activity.

EXAMPLE 1

[0059] This Example illustrates exploitation of differential microRNA expression to achieve selective expression of VSV M in tumour cells. The exemplified embodiment provides for let-7 sensitive expression of the VSV M gene. The result of incorporation of let-7 microRNA target elements so as to regulate VSV M expression in a wildtype toxic strain of VSV, is a virus that is attenuated specifically in normal cells and avirulent in vivo while retaining antitumour activity.

Materials and Methods

Viruses

[0060] Novel recombinant viruses were cloned as described in Figure 1 and rescued as described previously24. Viruses containing luciferase were cloned by insertion of the luciferase ORF (pGL3Basic, Promega, Madison, Wl) followed by let-7 microRNA target sites were cloned in an Nhe1 site in a VSV genome engineered to carry transgenes previously described49. Propagation of all viruses was done in A549 cells. Virions were purified from cell culture supematants by passage through a 0.2 mm Steritop filter (Millipore, Billerica, MA). For animal studies virons were concentrated by centrifugation at 30 00Og and resuspension in phosphate-buffered saline (PBS) (Hyclone, Logan, UT). Quantification of virions was done by plaque assay on Vero cells.

Cell lines

[0061] Human A549 lung carcinoma, Human HeIa cervical carcinoma, murine CT26 colon carcinoma and Human GM38 primary fibroblast (American Type Tissue Collection) were propagated in Dulbecco's modified Eagle's medium (Hyclone) supplemented with 10% fetal calf serum (Cansera, Etobicoke, Ontario, Canada).

Mice and tumour models

[0062] Female mice were obtained from Charles River Laboratories (Wilmington, MA), and injected subcutaneously with 5x105 CT26 cells to establish hind flank tumors. Tumour bearing animals were treated intravenously with 109 pfu in 10OZ]L. Tumour volume was measured using calipers and volume calculated using the formula (length/2 ^* width2). Toxicity studies were conducted with intranasal administration of 105 pfu in 51] L of VSV to 6-week-old balb/c mice or 105 to 107 pfu in 5DL to 3-week-old balb/c mice. All experiments were conducted with the approval of the University of Ottawa Animal Care and Veterinary Service. Luciferase assays

[0063] For determination of let-7 functional activity in cell lines lysates from transformations of pGL3Basic with a let-7 target sequences in the 3'UTR along with pRL (Promega, Madison, Wl) were prepared as described for the Dual Luciferase Reporter Assay System (Promega, Madison, Wl). The activity was measured using a luminometer (Lumat LB 9509, EG&G Berthold, Bad Wildbad, Germany).

qRT-PCR [0064] VSV M mRNA was reverse transcribed with Superscript III (Invitrogen, Carlsbad, CA) as per the manufacturers' instructions using oligo dT primers. qPCR was done using Platinum Taq (Invitrogen, Carlsbad, CA) as per the manufacturers instructions in the presence of SYBR? green. Expression of microRNA was quantified using mirVana qRT-PCR kit (Ambion, Austin, TX) using total RNA isolated using mirVana microRNA isolation kit (Ambion, Austin, TX) both used as per the manufacturers instructions. All PCR was quantified in real time using a Rotor-Gene GR-3000A thermocycler (Corbett Research, Sydney, Australia).

Viral infections and exogenous siRNA

[0065] Dilutions of VSV in 100 U L of DMEM were added to confluent cells in 6well plates placed in a 5%CO2 37C incubator for 40min before 2mL of DMEM containing 10%FBS was added. 50pmol of exogenous siRNA (Dharmacon) when present were transfected using oligofectamine (Invitrogen) for HeIa cells and lipofectamine 2000 (Invitrogen) for A549 cells as per the manufacturers instructions 24hrs before infection.

Results

Endogenous let-7 microRNA expression correlates with VSVIet-7wt replication [0066] VSVs engineered to carry three repeats of a sequence of differing complementarity to the mature let-7a microRNA are depicted schematically in Fig 1. Let-7a is one member of the let-7 family consisting of highly similar microRNAs. The let-7wt sequence is a perfect complement to the mature let-7a microRNA and, in a cell expressing let-7a, will be cleaved by the RISC complex resulting in transcript instability and potent translational inhibition27. The let-mm sequence is an imperfect complement to the mature let-7a microRNA, similar to sequences contained in the 3'UTR of endogenous mRNAs, and results in less potent translational repression and transcript degradation. The let-7mut sequence demonstrates no detectable inhibition in a luciferase reporter due to a high degree of noncomplimentarity to the mature let-7a microRNA and serves as a negative control (Pillai et al., 2005). These same sequences incorporated into VSVIet-7wt and VSVIet-7mm are translationally repressed when incorporated into the 3'UTR of a luciferase reporter gene in a let-7 dependant mechanism (Pillai et al., 2005).

[0067] Functional activity as assessed by luciferase reporter assay and analysis of the expression of mature let-7a microRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) shows that GM38 cells express more functional let-7 than A549 cells (Figure 2a&b). This confirms observations by others that human A549 lung carcinoma cells express low let- 713,14. The primary human fibroblast cell line, GM38, has approximately 3 times as much let-7 activity as A549 cells (Figure 2a). This let-7 microRNA functional activity correlates well with expression levels of the microRNA as measured by qPCR (Figure 2b). As such A549 and GM38 cell lines serve as models of target cells and non-target primary cells respectively, for evaluating the specificity of VSVIet-7wt. Importantly let-7 expression and activity in GM38 cells correlates with an approximate 1000-fold replication deficit of VSVIet-7wt as compared to VSVIet- 7mut after 48hours. This effect was not observed in A549 cells (Figure 2c). The growth curve also demonstrates that the greatest attenuation specific to VSVIet- 7wt occured in GM38 cells, and after 72 hours of infection VSVIet-7wt progeny were not detected. Therefore VSVIet-7wt produced the fewest progeny in a let-7 target sequence specific manner in this primary cell line.

Replication of viruses containing let-7 complementary sequences is affected by exogenous let-7

[0068] To determine if the correlation of microRNA activity and inhibition of replication of these viruses was indeed due to let-7 levels we assessed the response of the viruses to exogenous let-7a. Since both endogenous microRNA and exogenous siRNA enter the RISC complex and affect transcripts in a similar mechanism endogenous let-7 microRNA can be supplemented with exogenous siRNA of the same sequence28,29. Transfection of HeIa cells with siRNA containing the sequence of mature let-7a microRNA specifically reduced the titres produced by both viruses containing let-7a complementary sequences (Figure 3a). The results presented in Figure 3a&b using exogenous siRNA to augment endogenous let-7 and the results from Figure 2 demonstrating a correlation of let- 7 expression with VSVIet-7wt replication together suggest that the observed correlation of let-7 functional activity and VSVIet-7wt replication were due to the endogenous, sequence specific expression of let-7 microRNA.

Additional let-7 increases the survival of cells infected with viruses containing let-7 complementary sequences

[0069] In selected embodiments, the oncolytic virus will not only produce more progeny in low let-7 expressing cells, as demonstrated, but also preferentially kill these cells. To illustrate the specificity of the engineered viruses for low let-7 expressing cells we assessed the survival of cells transfected with siRNA and then infected with the let-7 complementary sequence containing viruses. Transfection of A549 cells with let-7a siRNA resulted in greater cell viability of cells infected with VSVIet-7mm and VSVIet-7wt. This protection was not seen in A549 cells infected with VSVIet-7mut suggesting that cell survival was enhanced in a let-7 sequence specific mechanism (Figure 3b). It is therefore possible to design a virus where viral replication and cell killing are sensitive to cellular microRNA levels.

VSVIet-7wt infected let-7 expressing cells contain reduced M mRNA

[0070] To illustrate that the perfectly complementary let-7 target sites were initiating degradation of the M mRNA we chose to look at the ratio of VSV gene products by qRT-PCR (Figure 3c). For these experiments we chose to use HeIa cells as these cells have measurable let-7 activityl 8,20,30. Consistent with previous observations we also detected let-7 activity in these cells by luciferase assay (as in Figure 1 ) as well as a reduced capacity to support VSVIet-7wt replication as expected (data not shown). Quantification of transcripts early in infection demonstrated the abundance of VSV transcripts as reflected by their position in the VSV genome (Figure 1 ). This supports previous findings that the nonprocessive nature of the viral RNA polymerase (VSV L) leads to this characteristic transcript abundance31 ,32. Also evident from this data was that in HeIa cells the M protein transcript is less abundant early during infection with VSVIet- 7wt as compared to VSVIet-7mut when expressed as a ratio to other genes in the VSV genome. Together this data suggests that the presence of let-7 microRNA decreases M mRNA in VSVIet-7mut infected cells, resulting in decreased viral production and oncolysis.

The sensitivity of VSVIet-7wt to let-7 expression is due to its effect on expression of VSVM

[0071] To illustrate that the attenuation of VSVIet-7wt is due to the placement of the let-7 target sequences and the decreased abundance of M mRNA specifically, new viruses were created. These viruses contained a firefly luciferase reporter gene incorporated with the let-7 target sequences, let-7mut and let-7wt in their 3'UTR. Viral luciferase expression in infected let-7 expressing HeIa cells showed a let-7 target specific reduction while A549 cells, having low let-7 activity, displayed no such reduction (Figure 4a). Titres produced after 24 hours infection of HeIa cells at an MOI of 0.1 with the Luciferase let-7mut and Luciferase let-7wt were very similar (data not shown). Infection of GM38 cells demonstrated that only VSVIet-7wt had reduced titres and the associated cytopathic effect, observed upon VSVIet-7mut and VSVIet-7mm infection, was absent from the confluent monolayer of GM38 cells infected with VSVIet-7wt (Figure 4b, c).

[0072] Prior infection of GM38 cells with VSVIet-7wt protected these cells from subsequent infection with a VSV expressing an eGFP transgene suggesting that an antiviral state was induced following by infection with VSVIet-7wt (Figure 4d). This indicates that decreased wildtype M protein specifically is responsible for the observed phenotype of VSVIet-7wt. This data indicates that, in this embodiment, incorporation into the 3'UTR of the let-7 target sequences affects only the expression of that particular viral gene and has no impact on the viral genome.

VSVIet-7wt is less pathogenic in balb/c mice

[0073] The lack of VSVIet-7wt cytotoxicity at a multiplicity of infection of 0.1 in vitro, is in keeping with evidence of reduced toxicity of this virus in vivo (Figure 5). Intranasal infections of Balb/c mice resulted in the most significant transient weight loss in VSVIet-7mut infected mice and an intermediate phenotype associated with VSVIet-7mm. No detectable transient weight loss was observed with VSVIet-7wt (Figure 5a). In young mice, prone to lethal CNS infection by VSV, no pathology was observed after intranasal infection with VSVIet-7wt. One third of young mice infected with VSVIet-7mut, however, presented with hind limb paralysis associated with CNS infection (Figure 5b).

VSVIet-7wt has antitumour activity [0074] The exemplified tumour specific viral replication in vitro is consistent with antitumour activity of VSVIet-7wt in vivo. Indeed multiple intravenous injections of VSVIet-7wt retarded CT26 tumour growth as compared to an identical schedule of PBS injections (Figure 5c). This indicates that the tumour specificity of this virus was maintained, despite containing coding sequences for wildtype viral proteins normally toxic in this tumour model.

Example 2: miR-34a regulation to limit viral replication to p53-deficient cells [0075] The microRNA miR-34a is a p53 transcriptional target, and therefore is more abundant in a cell where a functional p53 exists and is activated. Cancer cells are commonly deficient in tumor suppressors, such as p53. Certain p53- deficient cancer cells will accordingly have low levels of miR-34a.

[0076] An oncolytic virus that is subjected to miR-34a regulation will selectively replicate in p53-deficient cells, compared to host cells that are not p53-deficient. This Example illustrates an engineered oncolytic VSV in which sequences complementary to miR-34a were incorporated into the 3'UTR of the VSV M gene (Matrix protein).

[0077] In cells with functional p53, p53 is induced by various genotoxic stresses, including viral infection. Oncolytic viral infection itself may be sufficient to induce p53, and therefore mir-34a, to limit the activity of mir-34a controlled oncolytic virus in the normal cells. In some embodiments, it may also be advantageous to induce the p53 pathway in the non-cancer cells prior to oncolytic viral administration, for example by applying a genotoxic stress, which may for example be achieved using standard cancer therapies, such as radiation or chemotherapy, particularly therapies that induce DNA damage, such as treatment with cisplatin. In such embodiments, prior irradiation or chemotherapy may be carried out so as to be protective to normal cells exposed to a mir-34a controlled oncolytic virus.

Results

[0078] In HCT116 cells mir-34a is unregulated in a p53 dependant mechanism in response to doxorubicin and nutlin-3 (Braun et al., 2008; Paris et al., 2008; Kumamoto et al., 2008; Tazawa et al., 2007; He et al., 2007; Chang et al., 2007; Raver-Shapira et al., 2007). Incubation of HCT116 cells with doxorubicin for 48hours before infection with a mir-34a sensitive VSV reveals a p53 dependant delay in viral gene expression (Fig 7). This delay in viral gene expression correlates with a striking delay in apoptosis induction as seen by phase contrast microscopy of the infected monolayers (Fig 8). Together this demonstrates that VSV carrying mir-34a target sequences is sensitive to pre-treatment doxorubicin in a p53 dependant mechanism, and that p53 induction of mir-34a is inhibiting viral gene expression and apoptosis induction in these cells.

[0079] In response to ultraviolet radiation the human primary fibroblast cell line GM38 upregulates p53 (Ford & Hanawalt, 1997). The mir-34a sensitive VSV has attenuated replication in this cell line containing wt p53 and is further attenuated when these cells are exposed to 10J/cm2 ultraviolet radiation 24hours prior to infection (Fig 9).

[0080] Together these results illustrate that the mir-34a sensitive VSV is attenuated in the presence of the tumour suppressor p53.

Materials and Methods: Cell lines:

[0081] The human colon cancer cell line HCT166, the isogenic p53-/- HCT116 and the primary human fibroblast cell line were kindly provided by Dr. Bruce McKay (Ottawa Health Research Institute, ON, Canada). HCT116 cell lines were maintained in McCoy's 5A medium supplemented with 10% FBS. The GM38 cell line was maintained in Dulbecco's modified Eagle's medium supplemented with 20% FBS. All cells were incubated at 37C at 5%CO2.

Doxorubicin treatments: [0082] HCT116 cells were originally plated at 4E5 cells/well in βwell plates 24hours prior to doxorubicin treatment. Cells were exposed to 100ng/mL of doxorubicin diluted in PBS for 48hours prior to infection. Western Blot analysis of lysates were carried out using NuPAGE gradient gels (Invitrogen, Carlsbad, CA), a primary polyclonal anti-VSV rabbit antibody (Earl Brown, University of Ottawa, ON, Canada), subsequently visualized using a secondary anti-rabbit antibody conjugated to HRP and Supersignal West Pico Chemilumenescent reagents (ThermoScientific, Rockford, IL).

Ultraviolet Radiation Treatments: [0083] Media was removed from GM38 cells and the plates were exposed to 10J/cm2 UV radiation as determined by a handheld UV meter. Media was replaced and 24hours later the cells were infected with experimental VSV.

Prophetic Example 3: miR-155 regulation to limit immune response [0084] Dendritic cells are antigen presenting cells, and are therefore believed to be involved in the adaptive immune response to oncolytic viruses. An immune response against an oncolytic virus could limit the effectiveness of additional doses in immune individuals. This example accordingly illustrates embodiments that redress, at least in part, the limitations to oncolytic virotherapy imposed by the immune response.

[0085] The microRNA miR-155 is expressed in dendritic cells as they mature. To prevent viral gene expression in dendritic cells, sequences complementary to miR-155 are incorporated into the 3'UTR of a viral gene that produces and antigenic protein. The oncolytic virus may be a VSV with the N and/or G protein under miR-155 control, for example. The VSV N protein is a major antigenic determinant for CTL activity. The VSV G protein is a target for neutralizing antibodies. The oncolytic virus may also be another vesiculovirus, a rhabodvirus, a vaccinia virus or other poxvirus, an HSV-2 or other herpes virus, an adenovirus, a Newcastle-disease virus, a measles virus, an adenovirus, a coxsackievirus or other picornavirus, a Seneca Valley virus, an influenza virus, a retrovirus, etc., engineered to have sequences complementary to miR-34a incorporated into the 3'UTR of an gene encoding an antigenic protein (such as coat protein, etc).

[0086] In this way, oncolytic viruses may be engineered to not express antigenic proteins in antigen presenting cells that express miR-155. This may be carried out so as to attenuate the host adaptive immune response to the oncolytic virus, for example so as to facilitate effective multiple doses of the oncolytic virus.

REFERENCES

[0087] Citation of the following references is not an admission that such references are prior art to the present invention. The following documents are incorporated herein by reference, as if each were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein:

[0088] Ahmed, M., Cramer,S.D. & Lyles.D.S. (2004) Sensitivity of prostate tumors to wild type and M protein mutant vesicular stomatitis viruses. Virology 330, 34-49.

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[0091] Bell et al. (2003) Getting oncolytic virus therapies off the ground.

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Claims

1. A method of selectively ablating cells in a host, wherein the host comprises: non-target cells having a microRNA response mediated by a microRNA endogenously expressed in the non-target cells; and, target cells wherein the microRNA response is attenuated compared to the non-target cells; the method comprising administering to the host an effective amount of a recombinant lytic virus, wherein the virus comprises: a heterologous target nucleic acid sequence incorporated into a viral gene; so that the heterologous sequence is transcribed into a target mRNA expressed from the viral gene in the target cells and in the non-target cells, so that the target mRNA interacts with the microRNA in the non-target cells to mediate translational repression of the target mRNA in the non-target cells, and so that the attenuated microRNA response in the target cells permits expression of the viral gene in the target cells, and expression of the viral gene in the target cells mediates killing of the target cells in the host.

2. The method of claim 1 , wherein translational repression of the target mRNA mediates inhibition of viral replication in the non-target cells of the host.

3. The method of claim 1 or 2, wherein the target cells are cancer cells and the non-target cells are non-cancer cells.

4. The method of claim 1 , 2 or 3, wherein the killing of the target cells is mediated by an immune response in the host to the target cells.

5. The method of any one of claims 1 to 4, wherein the microRNA is selected from the group consisting of: miR-9 ,miR-10b, miR-15, miR-15a, miR-16, miR-16- 1 , miR-20a, miR-21 , miR-29, miR-31 , miR-34a, miR-96, miR-98, miR-103, miR- 107, miR-125a, miR-125b, miR-133b, miR-135b, miR-143, miR-145, miR-146, miR-181 , miR-181 a, miR-181 b, miR-181c, miR-183, miR-184, miR-196a-2, miR- 221 , miR-222, miR-223, miR-301 , miR-376, let-7, let-7a, let-7a-1 , hsa-let-7a-2, let- 7a-3, let-7g, miR-24 and miR-155.

6. The method of claim 5, wherein the microRNA is let-7, let-7a, let-7a-1 , hsa- let-7a-2, let-7a-3, or let-7g.

7. The method of any one of claims 1 to 6, wherein the virus is a vesiculovirus, vesicular stomatitis virus, Rhabdovirus, poxvirus, myxoma, vaccinia virus, herpes virus, adenovirus, Newcastle-disease virus, measles virus, Picornavirus, coxsackie virus, Seneca Valley Virus, influenza virus, or a retrovirus.

8. The method of claim 7, wherein the virus is a vesicular stomatitis virus (VSV).

9. The method of claim 8, wherein the gene is a VSV M protein gene.

10. The method of claim 9, wherein the microRNA is let 7a.

11. Use of a recombinant lytic virus to selectively ablate cells in a host, wherein the virus comprises a heterologous target nucleic acid sequence incorporated into a viral gene, and wherein the host comprises: non-target cells having a microRNA response mediated by a microRNA endogenously expressed in the non-target cells; and, target cells wherein the microRNA response is attenuated compared to the non-target cells; so that the heterologous sequence is transcribed into a target mRNA expressed from the viral gene in the target cells and in the non-target cells, so that the target mRNA interacts with the microRNA in the non-target cells to mediate translational repression of the target mRNA in the non-target cells, and so that the attenuated microRNA response in the target cells permits expression of the viral gene in the target cells, and expression of the viral gene in the target cells mediates killing of the target cells in the host.

12. Use of a recombinant lytic virus to formulate a medicament to selectively ablate cells in a host, wherein the virus comprises a heterologous target nucleic acid sequence incorporated into a viral gene, and wherein the host comprises: non-target cells having a microRNA response mediated by a microRNA endogenously expressed in the non-target cells; and, target cells wherein the microRNA response is attenuated compared to the non-target cells; so that the heterologous sequence is transcribed into a target mRNA expressed from the viral gene in the target cells and in the non-target cells, so that the target mRNA interacts with the microRNA in the non-target cells to mediate translational repression of the target mRNA in the non-target cells, and so that the attenuated microRNA response in the target cells permits expression of the viral gene in the target cells, and expression of the viral gene in the target cells mediates killing of the target cells in the host.

13. The use according to claim 11 or 12, wherein translational repression of the target mRNA mediates inhibition of viral replication in the non-target cells of the host.

14. The use according to claim 11 , 12 or 13, wherein the target cells are cancer cells and the non-target cells are non-cancer cells.

15. The use according to any one of claims 11 to 14, wherein the killing of the target cells is mediated by an immune response in the host to the target cells.

16. The use according to any one of claims 11 to 15, wherein the microRNA is selected from the group consisting of: miR-9 ,miR-10b, miR-15, miR-15a, miR-16, miR-16-1 , miR-20a, miR-21 , miR-29, miR-31 , miR-34a, miR-96, miR-98, miR-103, miR-107, miR-125a, miR-125b, miR-133b, miR-135b, miR-143, miR-145, miR- 146, miR-181 , miR-181 a, miR-181 b, miR-181c, miR-183, miR-184, miR-196a-2, miR-221 , miR-222, miR-223, miR-301 , miR-376, let-7, let-7a, let-7a-1 , hsa-let-7a- 2, let-7a-3, let-7g, miR-24 and miR-155.

17. The use according to claim 16, wherein the microRNA is let-7, let-7a, let- 7a-1 , hsa-let-7a-2, let-7a-3, or let-7g.

18. The use according to any one of claims 11 to 17, wherein the virus is a vesiculovirus, vesicular stomatitis virus, Rhabdovirus, poxvirus, myxoma, vaccinia virus, herpes virus, adenovirus, Newcastle-disease virus, measles virus, Picornavirus, coxsackie virus, Seneca Valley Virus, influenza virus, or a retrovirus.

19. The use according to claim 18, wherein the virus is a vesicular stomatitis virus (VSV).

20. The use according to claim 19, wherein the gene is a VSV M protein gene.

21. The use according to claim 20, wherein the microRNA is let 7a.

22. A recombinant oncolytic virus comprising a heterologous target nucleic acid sequence incorporated into a viral gene, wherein the heterologous target nucleic acid sequence is substantially complimentary to a microRNA sequence of a host cell that is permissive of infection by the virus.

23. The recombinant oncolytic virus of claim 22, wherein transcription of the heterologous sequence expressed from the viral gene in the host cell produces a target mRNA that interacts with the microRNA in the host cell to mediate translational repression of the target mRNA in the host cell.

24. The recombinant oncolytic virus of claim 23, wherein translational repression of the target mRNA mediates inhibition of viral replication in the host cell.

25. The recombinant oncolytic virus of claim 22, 23 or 24, wherein the microRNA is selected from the group consisting of: miR-9 ,miR-10b, miR-15, miR- 15a, miR-16, miR-16-1 , miR-20a, miR-21 , miR-29, miR-31 , miR-34a, miR-96, miR-98, miR-103, miR-107, miR-125a, miR-125b, miR-133b, miR-135b, miR-143, miR-145, miR-146, miR-181 , miR-181a, miR-181 b, miR-181c, miR-183, miR-184, miR-196a-2, miR-221 , miR-222, miR-223, miR-301 , miR-376, let-7, let-7a, let-7a- 1 , hsa-let-7a-2, let-7a-3, let-7g, miR-24 and miR-155.

26. The recombinant oncolytic virus of any one of claims 22 to 25, wherein the virus is a vesiculovirus, vesicular stomatitis virus, Rhabdovirus, poxvirus, myxoma, vaccinia virus, herpes virus, adenovirus, Newcastle-disease virus, measles virus, Picomavirus, coxsackie virus, Seneca Valley Virus, influenza virus, or a retrovirus.

27. The recombinant oncolytic virus of any one of claims 22 to 26, wherein the host cell is a human cell.