H-1 PV expressing RNAi effector

Abstract

The present invention relates to innovative protoparvoviruses (PV) expressing RNAi effectors, preferably shRNAs against the PD-L1 gene that exhibit improved anti-cancer activity. These new viruses are based on the ΔH-1PV silencer platform, consisting of the protoparvovirus H-1PV, which features an in-frame deletion in the NS region (ΔH-1PV) and harbors an shRNA expression cassette, where the expression of the shRNA is controlled by the H-1 polymerase III promoter. In this invention, the inventors aimed to silence the PD-L1 gene using the ΔH-1PV silencer. PD1 / PD-L1 negatively regulates T cell-mediated immune responses and serves as a mechanism for tumors to avoid antigen-specific T cell immune responses. The present invention also provides cells or organisms containing said parvoviruses.

Description

[Technical Field]

[0001] The present invention relates to innovative protoparvoviruses (PVs) that express RNAi effectors, preferably shRNAs. These new viruses are based on the ΔH-1PV silencer platform, which consists of protoparvovirus H-1PVs featuring an in-frame deletion in the NS region (ΔH-1PV) and harboring an RNA expression cassette, preferably an shRNA cassette, whose expression is controlled by the Pol III H-1 promoter. The present invention also provides cells or organisms containing the parvoviruses. [Background technology]

[0002] RNA interference, RNAi effectors and delivery RNA interference (RNAi) was first recognized in Caenorhabditis elegans by Andrew Fire and Craig Mello, who later received the Nobel Prize in 1998 for their discovery. The mechanism of RNAi is based on the sequence-specific degradation of host mRNA by double-stranded RNA complementary to the target sequence. RNAi is a naturally occurring cellular process that controls gene expression and therefore plays a central role in many cellular processes, including development. [1] It is also a crucial component of the immune response, protecting cells from pathogens such as viruses and transposons. [2] Soon after its discovery, RNAi technology was utilized to address biological questions and disease treatment options, thanks to its high specificity and ability to achieve potent knockdown of known gene sequences. [3] Therapeutically, RNAi works through the delivery of small RNA duplexes, including microRNA (miRNA) mimics, small interfering RNA (siRNA), short hairpin RNA (shRNA), and Dicer substrate small interfering RNA (dsiRNA) [4]. All four types of RNAi effectors are currently being tested in numerous phase I-III clinical trials for a variety of diseases, including multiple types of cancer [5]. In particular, RNA interference (RNAi) is the process by which RNA silences gene expression. This is initiated by the Dicer enzyme, which cleaves double-stranded RNA into smaller fragments. The passenger strand is further fragmented, and the guide strand is loaded into the RNA-induced silencing complex (RISC). The guide strand pairs with the target mRNA and Argonature-2, a protein that acts as the catalytic component of RISC, cleaving the mRNA. Typically, short hairpin RNAs (shRNAs) are delivered via a plasmid. shRNAs are processed in the nucleus and transported to the cytoplasm, where they are processed into small interfering RNAs (siRNAs) along with Dicer substrate small RNAs (dsiRNAs). shRNAs bind to specific sequences in mRNAs via RISC, leading to their degradation.

[0003] The most common problem encountered with RNAi is the efficient delivery of shRNA into cells. Although several commercially available transfection reagents exist for effective shRNA delivery, certain cell types remain difficult to efficiently transfect. Examples of viruses employed for the delivery and expression of shRNA in cancer gene therapy include adenoviruses, adeno-associated viruses, lentiviruses, and retroviruses. However, most of these viruses are replication-defective, so the silencing effect is limited to the primary infected cells. Due to safety concerns, the use of lentiviral vectors is limited in vitro and cannot be further used in in vivo settings.

[0004] Examples of genes targeted by RNAi-mediated anticancer therapy include KRAS, polo-like kinase 1, furin, ephrin type A receptor, and c-myc. Most clinical studies involve siRNA conjugated to nanoparticles (e.g., lipid nanoparticles), which have superior stability compared to naked siRNA. However, despite significant improvements, a rapid decline in siRNA is observed within the first 72 hours after injection, and most siRNA is taken up by hepatocytes (approximately 50% of the injected dose is found in the liver 30 minutes after injection). Thus, efficient delivery of siRNA remains a major obstacle in this field [5]. Because most delivery systems are transient and the intracellular concentration of siRNA is diluted during cell division, repeated administration of siRNA is often required.

[0005] Short hairpin RNAs (shRNAs) are another class of RNAi effectors [6]. shRNAs typically consist of two complementary (sense and antisense) 19–29 base pair sequences separated by a small loop of 4–11 unpaired nucleotides. Expression is typically controlled by an RNA polymerase (Pol) III promoter (e.g., U6, H1) or a modified Pol II promoter. After shRNA transcription, the sense and antisense strands, connected by the loop, pair to form a characteristic hairpin structure. This structure resembles pre-miRNAs, which are naturally used by cells to regulate gene expression and require nuclear processing [3]. The discovery of promoter-driven expression of shRNAs enabled the design of viral RNAi vectors [7]. Examples of viruses employed for the delivery and expression of shRNAs in cancer gene therapy include adenoviruses, adeno-associated viruses, lentiviruses, and retroviruses. However, most of these viruses are replication-defective, and therefore, silencing effects are limited to the primary infected cell.

[0006] Oncolytic viruses and RNAi Oncolytic viruses (OVs) selectively replicate in and kill cancer cells, possessing the ability to spread throughout tumors while sparing normal tissue. Their anticancer potential has been demonstrated preclinically in a wide variety of tumor models, and numerous OVs are currently undergoing Phase I–III clinical evaluation. In particular, talimogene laherparepvec (T-Vec; Amgen), a modified herpes simplex virus (HSV) expressing the immunostimulatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), was recently approved by the FDA and EMA for the treatment of unresectable metastatic melanoma. However, promising results obtained with OVs at the preclinical level have not always been replicated in the clinic. Tumors are often highly heterogeneous in nature, and certain tumors are likely only moderately sensitive to virus-induced oncolysis. It is also possible that a fraction of cells within a given tumor survives viral treatment, leading to tumor regrowth. Therefore, the application of RNAi technology in OV-based therapy is highly attractive because it could represent a means to enhance the efficacy of OVs by providing the virus with an additional mode of action for killing cancer cells that are less susceptible to its infection. Moreover, OV-mediated delivery of RNAi effectors could overcome a common hurdle in RNAi technology: how to achieve high levels of expression of these molecules in cancer cells, especially after intravenous administration. Furthermore, OVs can replicate and spread throughout tumors, thereby potentially amplifying shRNA production and delivery. Equipping OVs with RNAi effectors has proven to be a promising approach in the case of oncolytic adenoviruses (Ads), e.g., Ads expressing shRNAs targeting multiple tumor-associated genes, including VEGF, MYCN, SATB1, c-Met, Ki67, IL-8, hTERT, and FAK, which have superior anticancer activity compared to their parent viruses [8-15]. It has also been shown that oncolytic HSV can be engineered to efficiently express RNAi effectors (shRNAs and artificial miRNAs)

[16] .

[0007] A first-generation ΔH-1PV silencer platform has already been developed ( WO 2013 / 110464 A1 ), consisting of a ΔH-1PV viral genome featuring an in-frame deletion encompassing nucleotides (nt) 2022 to 2135. The left (LP) and right palindromic sequences (RP) serve as self-priming replication origins; the P4 promoter controls the expression of the NS gene, which encodes the nonstructural proteins NS1 and NS2, and the P38 promoter controls the expression of the VP gene, which encodes the VP1 and VP2 viral proteins. A noncoding region (NCR) is downstream of the VP region, and a target-specific nucleic acid cassette is inserted into the noncoding region at nucleotide 4687 of the wild-type H-1PV genome. However, it has been found that the target-specific nucleic acid cassette inserted at the HpaI site (positions 4686 to 4691) is not stably maintained and compromises viral fitness.

[0008] Additionally, a recombinant parvovirus containing an shRNA CDK9 expression cassette within the 3' untranslated region of the viral genome at position 4570 has been described in EP3327124A1 (WO2018 / 096148A1). The virus exhibits excellent gene silencing and maintains the ability to replicate, but the shRNA expression cassette is gradually lost during propagation of the virus in the NB324K producing cell line. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2013 / 110464A1 [Patent Document 2] EP3327124A1 [Patent Document 3] WO2018 / 096148A1 [Patent Document 4] U.S. Patent No. 8,217,149 [Patent Document 5] U.S. Patent Application Serial No. 13 / 511,538 [Patent Document 6] U.S. Patent Application Serial No. 13 / 478,511 [Patent Document 7] EP2397542A1 [Patent Document 8] U.S. Patent No. 4,873,191 [Non-patent literature]

[0010] [Non-Patent Document 1] Fingl et al., The Pharmacological Basis of Therapeutics, edited by Goodman and Gilman, Macmillan Publishing Co., New York, pp. 1-46 (1975) Summary of the Invention [Problem to be solved by the invention]

[0011] As mentioned above, when shRNAs are delivered by adenovirus, adeno-associated virus, lentivirus, and retrovirus, the viruses are often replication-defective, so the silencing effect is limited to the primary infected cells.

[0012] Therefore, an object of the present invention is to provide a means for more stably and efficiently down-regulating the expression of cancer-associated genes in cells or organisms. [Means for solving the problem]

[0013] According to the present invention, this is achieved by providing the subject matter defined in the claims. Preferred embodiments are set out in the appended claims.

[0014] The present invention relates to deletion variants of the autonomous parvovirus H-1 that express RNAi effectors, preferably shRNAs, that target cancer-associated genes or oncogenes, such as PD-L1.

[0015] H-1PV is nonpathogenic to humans and has oncolytic and oncosuppressive properties, spurring considerable interest due to its potential for anticancer. Because humans are not typically exposed to rodent parvovirus infections, pre-existing antiviral immunity is generally not a problem for these viruses. The parvovirus genome consists of approximately 5,100 bases of single-stranded DNA containing two promoters, P4 and P38, which regulate the expression of nonstructural (NS1 and NS2) and capsid (VP1 and VP2) proteins, respectively. Activation of the P4 promoter is a critical step in the PV life cycle. While the activity of the P4 promoter is later regulated by its own gene product, NS1, its initial activation is entirely dependent on host cell factors that are primarily expressed during the S phase of the cell cycle. This dependency, along with the virus's inability to stimulate quiescent cells to proliferate, contributes to the virus's oncotropism, which allows it to replicate preferentially in proliferating, transformed, or malignant cells. Parvoviral cytotoxicity is also stimulated by cellular changes associated with neoplastic transformation. NS1 is the major viral virulence protein. H-1PV has been shown to activate several death pathways in cancer cells. Although the anticancer potential of PV has been supported by numerous preclinical studies, efficacy is expected to be a limiting factor in clinical application. Some cancer cells may be resistant to viral treatment, leading to tumor recurrence.

[0016] During the experiments that led to this invention, we were able to demonstrate that insertion of an shRNA expression cassette at a specific site in the parvovirus genome is compatible with the parvovirus packaging capacity, does not interfere with viral replication, and promotes the virus's intrinsic cytotoxicity. The virus expresses high levels of shRNA and exhibits excellent gene silencing. A major advantage of H-1PV silencers compared with replication-poor vectors is their ability to replicate and grow in proliferating cells, such as cancer cells. All infected / transduced cells could theoretically generate new viral particles. Progeny virions from a second round of infection could propagate through tumors and efficiently deliver and express therapeutic shRNAs. In this setting, the silencing signal could be amplified beyond the initial inoculum. In summary, parvovirus-based vector and shRNA technologies complement each other's limitations: the natural tumor tropism of parvoviruses should enable specific and effective delivery of shRNAs to mediate shRNA transduction in proliferating cells, such as cancer cells. [Brief explanation of the drawings]

[0017] [Figure 1] Schematic diagram of ΔH-1PVRNAi genome construction. The viral genome (top) is shown as a single line terminating in a distinct hairpin telomere that serves as a self-priming replication origin. The hairpin, drawn to represent the expected structure, is drawn at approximately 20x scale relative to the rest of the genome. The viral early P4 and late P38 promoters regulate expression of the transcription units encoding the nonstructural NS1 (light gray box around the arrow) and capsid VP (dark gray box around the arrow) proteins, respectively. The transcription promoters are indicated by solid arrows. A gray triangle indicates the location of an in-frame 114-nucleotide internal deletion. The noncoding region is downstream of the VP region and is thought to be involved in regulating viral replication. An shRNA cassette (bottom) containing the H1 Pol III promoter and shRNA sequence is inserted at nt 4480 within the viral genome. [Figure 2]Figure 1 shows the testing of shRNA expression cassette stability and ΔH-1PVshPD-L1 generation. A) Scheme for generating ΔH-1PVRNAi viruses in successive rounds of infection. A virus production strategy was employed to examine the stability of the shRNA expression cassette through subsequent rounds of infection, as described in the Methods section. B) Evaluation of the stability of the shRNA expression cassette at nt4480. ΔH-1PVshEGFP, ΔH-1PVshPD-L1_1, or ΔH-1PVshPD-L1_2 viruses were obtained from passages P2, P3, P4, and P5 using the procedure shown in (A). Viral DNA was extracted, and genomic fragments containing the shRNA cassette were amplified by PCR using primers flanking the cassette. The PCR product of ΔH-1PV was loaded as a control. (C) Comparative progeny virus generation between ΔH-1PV and ΔH-1PRNAi viruses. Virus titration was performed by plaque assay (light gray). Results are shown from a representative set of two independent experiments. For quantification of encapsidated viral genomes, real-time PCR was performed (dark grey). Results are shown as the average of two independent experiments. [Figure 3] This figure shows that ΔH-1PVshPD-L1 is superior in gene silencing. (A) Western blot. U251 cells were infected with increasing MOIs (PFU / cell) of the indicated viruses, grown for 72 hours, and then lysed. Whole cell extracts were subjected to SDS-PAGE followed by immunoblot analysis of protein levels of Flag-tagged PD-L1, NS1, and vinculin (loading control) using specific antibodies. (B) qRT-PCR. U251 cells were infected as described in (A). 72 hours post-infection, cells were harvested, and total RNA was isolated and reverse-transcribed. PD-L1 mRNA levels were quantified by qRT-PCR using specific primers. PD-L1 gene expression levels were normalized to the housekeeping gene rRNA 18S. Values ​​are expressed relative to those obtained in mock-treated cells. [Figure 4]This figure shows that ΔH-1PVshPD-L1 is effective in gene silencing. (A) Western blot. AsPC-1 cells were infected with the indicated viruses at increasing MOIs (PFU / cell), grown for 72 hours, and then lysed. Western blot was performed as described in Figure 2(A). (B) qRT-PCR. AsPC-1 cells were infected as in (A). RT-PCR was performed as described in Figure 2(B). [Figure 5] Figure 1 shows activation of the NFAT-responsive luciferase reporter upon infection with ΔH-1PVshPD-L1. Either AsPC-1 (left panel) or U251 (right panel) cells were first transfected with TCR activators alone or TCR activators / PD-L1, and then infected with ΔH-1PVshPD-L1 and ΔH-1PVshEGFP at the indicated MOI (PFU / cell). Infected cells were preincubated with either anti-PD-L1 neutralizing antibody (NAb) or control antibody (CAb) at the indicated amount (ng / ml) and then co-cultured with Jurkat cells. NFAT-luciferase reporter activity was measured by a one-step luciferase assay. The relative ratios of luminescence under different treatments were calculated as described in the methods. [Figure 6] Figure 1 shows that knockdown of PD-L1 expression suppresses proliferation of the human pancreatic cancer cell line AspC-1 in a 3D spheroid highly translationally relevant model. Real-time proliferation of AsPC-1 cells was documented by Incucyte® single spheroid assay upon infection with ΔH-1PVshEGFP or ΔH-1PVshPD-L1 at an MOI of 5 PFU per cell. Cells treated with an equal volume of vehicle were used as a control. [Figure 7-1] Parvovirus H-1, complete genome (SEQ ID NO: 12). GenBank: X01457.1 [Figure 7-2] This is a continuation of Figure 7-1. [Figure 7-3] This is a continuation of Figure 7-2. DETAILED DESCRIPTION OF THE INVENTION

[0018] Thus, the present invention provides a parvovirus based on a parvovirus H-1 deletion variant for down-regulating expression of a target gene in a cell, characterized in that the parvovirus H-1 deletion variant comprises a deletion encompassing nucleotides 2022 to 2135, a target-specific nucleic acid is inserted in an untranslated region downstream of the H-1 parvovirus VP gene and is expressible under the control of a promoter or promoter region recognizable by RNA polymerase in the cell, the target-specific nucleic acid is transcribable by RNAi, and the parvovirus is capable of replicating and growing in the cell.

[0019] The parvovirus H-1 deletion variant (ΔH-1PV) features an in-frame deletion encompassing nucleotides (nt) 2022 to 2135 of wild-type parvovirus H-1 (Figure 1). The left (LP) and right palindromic sequences (RP) serve as self-priming replication origins; the P4 promoter controls the expression of the NS gene, which encodes the nonstructural proteins NS1 and NS2, and the P38 promoter controls the expression of the VP gene, which encodes the VP1 and VP2 viral proteins. The noncoding region (NCR) is downstream of the VP region and is thought to be involved in regulating viral genome replication and encapsidation.

[0020] The target-specific nucleic acid is inserted into the viral genome downstream of the parvovirus VP gene, which encodes the parvovirus capsid protein, so as not to affect viral replication and cytotoxicity. Preferably, the target-specific nucleic acid cassette is inserted at nucleotide 4480 of the ΔH-1PV genome. The inventors performed several experiments to determine the most suitable insertion site. When the shRNA expression cassette was inserted at positions 4815 or 4465, the shRNA cassette was lost at passage 1 or 2. Inserting the shRNA expression cassette at positions 4598 or 4469 improved the stability of cassette maintenance, i.e., it remained stably integrated into the viral genome even at passage 3. This is even better than insertion into the HpaI restriction enzyme site used in the first-generation ΔH-1PV silencer platform ( WO 2013 / 110464 A1 ). The best results were achieved when the shRNA expression cassette was inserted at position 4480. It is stably integrated into the viral genome at passage 4, accompanied by higher virus production and infectivity. We were able to demonstrate proof of concept that the new cassette insertion site at position 4480 is universal for all shRNAs.

[0021] In a preferred embodiment, the target gene is PD-L1. PD-1 (programmed cell death 1) and PD-L1 (programmed cell death ligand 1) play important roles in the negative regulation of T cell-mediated immune responses and serve as a mechanism for tumors to evade antigen-specific T cell immune responses. Therefore, blockade of PD-1 or PD-L1 is a suitable method for tumor treatment. Several additional immune checkpoint receptors and ligands, some of which are selectively upregulated in various types of tumor cells, are prime targets for blockade, particularly in combination with approaches that promote the activation of anti-tumor immune responses, such as vaccines. Currently, blockade is achieved through the administration of anti-PD-1 or anti-PD-L1 antibodies. Anti-PD-L1 antibodies and methods for producing them are known in the art. Such antibodies against PD-L1 can be polyclonal or monoclonal, and / or recombinant, and / or humanized. Examples of antibodies against PD-L1 are disclosed in U.S. Patent No. 8,217,149, U.S. Patent Application No. 13 / 511,538, and U.S. Patent Application No. 13 / 478,511. Currently, three antibodies against PD-L1, namely atezolizumab, avelumab, and durvalumab, have been approved by the FDA. However, there are unresolved issues associated with the administration of currently known checkpoint inhibitors: (1) Harmful immune effects: A drawback of releasing the immune brake is the induction of toxicity in healthy tissues in patients treated with immune checkpoint inhibitors (ICIs). The patients develop immune-related adverse events (irAEs), a unique range of side effects of ICIs that resemble autoimmune responses. irAEs affect almost every organ in the body, most commonly in the skin, gastrointestinal tract, lungs, and endocrine, musculoskeletal, and other systems. Although ICIs have significant clinical anticancer efficacy, they often suffer from significant systemic toxicity. Therefore, the incidence of irAEs, up to 50%, has prevented their widespread and universal use. (2) Mutations in the target protein PD-L1 result in treatment resistance. (3) For example, pancreatic ductal adenocarcinoma (PDAC) has shown disappointing results in trials of single-agent immune checkpoint blockade. A possible reason for this failure may be a combination of immune escape mechanisms and low mutation burden in PDAC.

[0022] Therefore, new strategies for blocking PD-L1 as an anti-cancer therapeutic target gene are needed, as PD1 / PD-L1 negatively regulates T cell-mediated immune responses and serves as a mechanism for tumors to evade antigen-specific T cell immune responses, which also enhances cancer cell proliferation and promotes tumorigenesis.

[0023] Specifically, we generated pΔH-1PVshPD-L1 and pΔH-1PVshEGFP infectious molecular clones by inserting shRNA expression cassettes into the noncoding region of the viral genome (Figure 1). The shRNA sequences against either PD-L1 or EGFP demonstrated efficient gene-specific silencing. To develop a new H-1PV that stably expresses shRNA cassettes, we performed several experiments to evaluate the most suitable insertion site by examining the stability of the shRNA expression cassette in the viral genome using successive rounds of infection in permissive NB324K cells (Figure 2A). When the shRNA expression cassette was inserted at position 4815 or 4465, the shRNA cassette was lost at passage 1 or 2. Inserting the shRNA expression cassette at position 4598 or 4469 improved the stability of cassette maintenance, i.e., it remained stably integrated into the viral genome even at passage 3. This is even better than insertion into the HpaI restriction enzyme site used in the first-generation ΔH-1PVshEGFP (WO 2013 / 110464 A1). The best results were achieved when the shRNA expression cassette was inserted at position 4480 (Figure 2B). This resulted in stable integration into the viral genome at passage 4, accompanied by higher virus production and infectivity (Figures 2B and 2C). This provides the first evidence that an optimal insertion site for the shRNA expression cassette is required for a parvovirus-fit intermediate and suggests that the virus has the most favorable ability to generate PV expressing an shRNA against the PD-L1 gene with good viral fitness. Virus production and infectivity were comparable across the various constructs, independent of changes in the shRNA sequence, i.e., for either PD-L1 or EGFP, indicating that the new cassette insertion site at position 4480 is universal for all shRNA sequences (Figure 2C).

[0024] To select the most effective shRNA sequence targeting PD-L1, we developed stable cell lines overexpressing Flag-tagged PD-L1 in either pancreatic ductal adenocarcinoma (PDAC) AsPC-1 or glioblastoma multiforme (GBM) U251 cells and evaluated the silencing efficiency upon infection with ΔH-1PVshPD-L1 carrying various shRNA sequences against the PD-L1 gene. Consequently, we selected ΔH-1PVshPD-L1_1, which was more capable of silencing the PD-L1 gene, for further analysis according to the evaluation of knockdown efficacy. Unless otherwise stated, the term "ΔH-1PVshPD-L1_1" hereafter refers to ΔH-1PVshPD-L1. AsPC-1 or U251 cells overexpressing Flag-tagged PD-L1 were infected with increasing amounts of either ΔH-1PVshPD-L1 or ΔH-1PVshEGFP (Figures 3 and 4). At 72 hours postinfection, cells were harvested for Western blot analysis of whole-cell lysates. Results showed that infection with H-1PVshPD-L1, but not with the ΔH-1PVshEGFP control virus, silencing the PD-L1 gene in either AsPc-1 or U251 cells was virus titer-dependent. Consistent with these results, a strong reduction in PD-L1 mRNA levels was observed in ΔH-1PVshPD-L1-infected cells, confirming that ΔH-1PVshPD-L1 has the ability to silence the PD-L1 gene.

[0025] Binding of programmed cell death protein 1 (PD-1), a receptor expressed on activated T cells, to its ligand, PD-L1, which is present in most cancers, negatively regulates immune responses. PD-1 / PD-L1 interaction inhibits T cell activity and allows cancer cells to escape immune surveillance

[35] . To investigate the possibility that blocking PD-1 / PD-L1 interaction could be achieved by silencing the PD-L1 gene, we tested whether ΔH-1PVshPD-L1 could disrupt PD-1 / PD-L1 interaction in a bioluminescent cell-based assay. As illustrated in Figure 5, either AsPC-1 or U251 cells overexpressing PD-L1 and TCR activators were treated with anti-PD-L1 neutralizing and control antibodies or infected with either ΔH-1PVshPD-L1 or ΔH-1PVshEGFP at an MOI (pfu / cell) of 12 (AsPC-1) or 2 (U251). Luminescence corresponding to nuclear factor of activated T cells (NFAT) activity was measured after co-culture with PD-1 / NFAT reporter Jurkat T cells. As expected, luminescence from cells treated with anti-PD-L1 neutralizing antibodies was 1.7 (U251) or 3.8 (AsPC-1) times higher than that from control antibodies. Interestingly, luminescence from cells infected with ΔH-1PVshPD-L1 was 1.6 (U251) or 2.8 (AsPC-1) times higher than that from the control virus ΔH-1PVshEGFP, suggesting that ΔH-1PVshPD-L1 can block and eliminate PD-1 / PD-L1 interaction. In parallel, PD-L1 gene knockdown was examined by Western blot. These results suggest that ΔH-1PVshPD-L1 may play a role in promoting T cell activation through silencing the PD-L1 gene.

[0026] This innovative virus represents the next generation of parvoviruses that not only possesses the characteristics of the original H-1PV, but also has a competitive advantage due to the important function of gene silencing that other products do not exhibit. The following results are preclinical proof of concept that ΔH-1PV pSilencer expressing shRNA against the PD-L1 gene has a unique and superior anti-cancer profile: (1) The intrinsic ability of H-1PV to induce tumor cytotoxicity coupled with eliciting a potent anti-cancer immune response that switches the immunosuppressive tumor microenvironment (TME) of cancer cells from a “cold” to a “hot” inflammatory state. (2) Silencing of the PD-L1 gene resulted in the reversal of cytotoxic T lymphocyte exhaustion, thus leading to the elimination of tumor cells via the reinduction of the “natural” function of the T cell population.

[0027] As used herein, a "PD-L1-specific nucleic acid" refers to a nucleic acid comprising at least 15, 20, 25, 50, 100, or 200 consecutive nt that has at least about 75%, particularly at least about 80%, more particularly at least about 85%, very particularly about 90%, and especially about 95% sequence identity with the complement of the transcribed nucleotide sequence of a PD-L1 target gene. The PD-L1 sequence is known and has been described in publications such as Breton et al.

[32] , Wang et al.

[36] , and Jeffrey et al.

[33] . Preferably, the following sequence was used for downregulation: 5'GATATTTGCTGTCTTTATA-3' (SEQ ID NO: 1). Using RNA consistent with this sequence, it was possible to target all known isoforms of PD-L1 (GenBank accession number NM_014143).

[0028] In the present invention, the PD-L1 gene can be downregulated in in vivo cells or in vitro cells (ex vivo). The cells may be primary cells, or cells that have been cultured for a period of time, or the cells may comprise a cultured cell line. The cells may be diseased cells, such as cancer cells or tumor cells, or virally infected cells. The cells may be all progenitor cells of the progenitor cell lineage, stem cells that give rise to more mature and fully mature cells, progenitor cells that give rise to all mature cells of the hematopoietic cell lineage, antigen-primed progenitor cells that give rise to specific hematopoietic lineages, T lymphocyte precursor cells, immature T lymphocytes, mature T lymphocytes, myeloid progenitor cells, or monocyte / macrophage cells. The cells may be omnipotent or totipotent stem cells or embryonic stem cells. The cells may be nerve cells, nervous system cells, epithelial cells, muscle cells, cardiac cells, liver cells, kidney cells, stem cells, embryonic or fetal stem cells, or fertilized egg cells.

[0029] Preferably, the parvovirus variant is formulated as a pharmaceutical composition, wherein the parvovirus is present in an effective amount and is combined with a pharmaceutically acceptable carrier.

[0030] "Pharmaceutically acceptable" is meant to encompass any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the patient to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Additional pharmaceutically compatible carriers may include gels, bioabsorbable matrix materials, implantation elements containing parvovirus (therapeutic agents), or any other suitable vehicle, delivery, or dispersion means or material. Such carriers may be formulated by conventional methods and administered to a subject in an effective amount.

[0031] An "effective amount" refers to the amount of active ingredient sufficient to effect treatment. An "effective amount" can be determined using methods known to those skilled in the art (see, for example, Fingl et al., The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds., Macmillan Publishing Co., New York, pp. 1-46 (1975)).

[0032] Administration of the parvovirus can be effected by various methods, for example, by intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. The route of administration, of course, depends on the type of therapy. Preferred routes of administration are intravenous (iv), intratumoral, or intrabronchial administration. When infectious viral particles capable of penetrating the blood-brain barrier are used, treatment is performed, or at least initiated, by intravenous injection of, for example, the H-1PV virus.

[0033] The parvovirus administration regimen can be readily determined by the attending physician within the skill of the art based on patient data, observations, and other clinical factors, including, for example, the patient's size, body surface area, age, sex, the particular modified parvovirus being administered, the time and route of administration, the type of mesenchymoma, the patient's overall health, and other medications the patient is receiving.

[0034] As another specific administration technique, the parvovirus can be administered to a patient from a source implanted in the patient. For example, a catheter made of, for example, silicone or other biocompatible material can be connected to a small subcutaneously implanted reservoir (Rickham reservoir) attached to the patient during tumor removal or by another procedure, allowing the parvovirus to be injected locally at various times without further surgical procedures. The parvovirus can also be injected into tumors by stereotactic surgical procedures or by neuronavigation targeting techniques.

[0035] Parvovirus administration can also be carried out by continuous infusion of viral particles or a fluid containing viral particles through an implanted catheter using a suitable pump system at a low flow rate, such as a peristaltic infusion pump or a convection-enhanced drug delivery (CED) pump.

[0036] Yet another method of administering parvovirus is from an implanted device constructed and arranged to distribute the parvovirus to the desired tissue. For example, wafers impregnated with parvovirus, e.g., parvovirus H1, may be employed, where the wafers are attached to the edges of the resection cavity resulting from surgical tumor removal. Multiple wafers may be employed in such interventions. Cells actively producing parvovirus H1 variants may be injected into the tumor or into the tumor cavity after tumor removal.

[0037] In a particularly preferred embodiment of the invention, the target-specific nucleic acid is inserted into the H-1PV genome at position 4480. The underlying vector is pdB del H-1PV, as described in EP2397542A1.

[0038] In a further particularly preferred embodiment of the present invention, the promoter or promoter region recognizable by an RNA polymerase is an RNA polymerase II (Pol II) promoter, such as CMV, P38, and human ubiquitin C, or an RNA polymerase III (Pol III) promoter, such as U6, H1, 7SK, and tRNA. An example of a particularly preferred RNA polymerase III (Pol III) promoter is the RNA polymerase III H1 promoter.

[0039] In a preferred embodiment of the present invention, the PD-L1-specific nucleic acid is an shRNA. shRNAs are short or short hairpin RNAs, which are RNA sequences that make sharp hairpin turns and can be used to silence gene expression via RNA interference. The shRNA hairpin structure is cleaved by the cellular machinery into siRNAs, which then bind to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the bound siRNA. However, insertion of other RNAi trigger molecules, such as microRNAs and / or antisense oligonucleotides, is also possible.

[0040] In a further particularly preferred embodiment of the invention, the PD-L1-specific nucleic acid, e.g., shRNA, has a length of at least 15 nucleotides. In a particularly preferred embodiment, the PD-L1 sequence matches the sequence 5'GATATTTGCTGTCTTTATA-3' (SEQ ID NO: 1), as previously described

[32] . The PD-L1 gene is alternatively spliced, resulting in multiple transcript variants. Of the four known variants, one is non-coding and three encode different isoforms of the PD-L1 protein.

[0041] The present invention also relates to a rodent parvovirus as characterized above for use in the treatment of cancer.

[0042] In a preferred embodiment, the parvovirus may be used to treat tumors, particularly (but not exclusively) prostate cancer, pancreatic cancer, brain cancer (preferably glioma), cervical cancer, lung cancer, head and neck cancer, breast cancer or colon cancer.

[0043] In a further preferred embodiment, the parvovirus may be used for the treatment of tumors whose cells are characterized by resistance to chemotherapy and / or radiotherapy.

[0044] Patients treatable with the parvoviruses of the present invention include humans and non-human animals, including, but not limited to, animals such as cattle, sheep, pigs, horses, dogs, and cats.

[0045] The present invention also provides an animal, fungal, or protist cell comprising a parvovirus as described hereinabove. In one embodiment, the cell is in vitro. The cell is preferably an animal cell, an isolated human cell, an in vitro human cell, a non-human vertebrate cell, a non-human mammalian cell, a fish cell, a bovine cell, a goat cell, a porcine cell, a sheep cell, a rodent cell, a hamster cell, a mouse cell, a rat cell, a guinea pig cell, a rabbit cell, a non-human primate cell, a nematode cell, a shellfish cell, a kuruma prawn cell, a crab cell, a lobster cell, an insect cell, a fruit fly cell, a Coleopteran insect cell, a Dipteran insect cell, a Lepidopteran insect cell, or a Homopteran insect cell.

[0046] Finally, the present invention also provides a transgenic non-human animal, fungus, or protist comprising a parvovirus as described hereinabove. Transgenic animals can be produced by injecting the parvovirus into the pronucleus of a fertilized oocyte, by transplanting a cell, preferably an undifferentiated cell, into a developing embryo to produce a chimeric embryo, by transplanting a nucleus from a recombinant cell into an enucleated embryo or an activated oocyte, etc. Methods for producing transgenic animals are well established in the art and are described, for example, in U.S. Pat. No. 4,873,191.

[0047] In summary, the new virus is based on the ΔH-1PV silencer platform, which consists of the protoparvovirus H-1PV, featuring an in-frame deletion in the NS region (ΔH-1PV) and harboring an RNA expression cassette, preferably an shRNA expression cassette, whose expression is controlled by the Pol III H-1 promoter. The ΔH-1PV silencer is effective for gene silencing while maintaining its replication capacity and is fully infectious. In this study, the ΔH-1PV silencer was used to silence the PD-L1 gene, which is known to cause immune system dysfunction. PD1 / PD-L1 negatively regulates T cell-mediated immune responses and serves as a mechanism for tumors to evade antigen-specific T cell immune responses. Transfection of the plasmid into HEK293T cells produced fully infectious viral particles that could be further amplified via infection in NB324K cells, following a typical parvovirus production protocol.

[0048] The following examples illustrate the invention in more detail. [Example]

[0049] Plasmid construction and virus production pΔH-1PVRNAi contains the ΔH-1PV viral genome, featuring a 114-nucleotide in-frame deletion from nucleotides 2022 to 2135 within the NS coding region according to the NCBI gene bank (reference sequence X01457.1; Figure 7)

[18] . An shRNA expression cassette was introduced into pΔH-1PV at nucleotide position 4480 by cloning an NsiI-HpaI fusion PCR DNA fragment generated with the primers shown in Table 1 below. The particularly preferred shPD-L1 in this study, containing the sequence 5'-GATATTTGCTGTCTTTATA-3' (SEQ ID NO: 1), was able to target all known PD-L1 isoforms (GenBank accession number NM_014143). All plasmid constructs carrying the shRNA expression cassette were further confirmed by sequencing (LGC Genomics, Berlin, Germany). Virus was produced, purified, titrated by plaque assay, and quantified by real-time PCR as previously described

[26] . [Example]

[0050] cell culture AsPC-1, U251, and HEK293T cell lines were grown in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, Munich, Germany) supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies, Darmstadt, Germany). PD-1 / NFAT reporter Jurkat cells (BPS Bioscience, CA, USA) were cultured in Roswell Park Memorial Institute medium 1640 (RPMI, Invitrogen) supplemented with 10% FBS. NB324K cells were grown in minimum essential medium (MEM, Sigma-Aldrich, Munich, Germany) supplemented with 5% FBS. All media contained 2 mM l -glutamic acid (Gibco), 100 U / ml penicillin, and 100 μg / ml streptomycin (Gibco). All cells were grown at 37°C in a 5% CO atmosphere and 95% humidity and routinely checked for mycoplasma contamination using a mycoplasma detection kit according to the manufacturer's instructions (Venor GeM, Minerva Biolabs, Berlin, Germany). [Example]

[0051] Establishment of stable cell lines AsPC-1 and U251 stable cell lines were generated using the pS / MARt DNA vector expressing Flag-tagged PD-L1 according to a previously described method

[37] . Positive clones were selected in medium containing 1 μg / ml puromycin according to the manufacturer's protocol (Invitrogen, C10459). The pool of selected clones was used in this study. [Example]

[0052] Assessment of the stability of shRNA expression cassettes A plasmid carrying a viral genome containing an shRNA expression cassette was transiently transfected into HEK293T cells. Three days later, cells were harvested and subjected to three freeze-thaw cycles to release viral particles. Crude cell extracts were digested with Benzonase nuclease (Merck, Germany), and viral titers were quantified by real-time PCR and expressed as encapsidated viral genomes per milliliter (Vg / ml) according to a previously described method

[26] . This period is defined as the passage 0 stage (P0). A fraction of the virus produced in HEK293T cells was used as inoculum for further amplification of the viral stock in NB324K producer cells. Cells were harvested 3–5 days postinfection and treated as described above; this period is defined as passage 1 (P1). Virus was harvested from all passages, as illustrated in Figure 2A. Viral DNA was extracted using a Qiagen viral DNA extraction kit according to the manufacturer's protocol (Qiagen, Hilden, Germany). The genomic fragment containing the shRNA cassette was amplified by PCR using primers flanking the cassette. The PCR product of ΔH-1PV was loaded as a control. [Example]

[0053] Protein extraction and Western blot analysis Cells were scraped and collected in culture medium and washed with PBS. Cell pellets were lysed on ice for 30 minutes in lysis buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate) containing protease (Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitors (Sigma-Aldrich). Cell debris was removed by centrifugation at 13,000 rpm for 15 minutes at 4°C. The supernatant was kept at -80°C for further analysis. Total cell extracts (20 μg) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond-P membranes (GE Healthcare). The following antibodies were used for analysis: mouse monoclonal anti-vinculin (sc-25336; Santa Cruz Biotechnology, Heidelberg, Germany, used at a dilution of 1:10,000), polyclonal anti-NS-1 SP8 antiserum

[31] (1:3,000), mouse monoclonal anti-Flag (AHP1074; AbD Serotec) at a dilution of 1:2,000, and rabbit anti-PD-L1 antibody (PA5-20343; Thermo Fisher Scientific, USA) at a dilution of 1:1,000. After incubation with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) at a dilution of 1:5,000, proteins were detected using ECL substrate solution with a Vilber Lourmat chemiluminescence detection system (software, Chemi-Capt 5000). [Example]

[0054] RNA isolation and real-time quantitative RT-PCR Total RNA was isolated using the TRIzol® Reagent RNA Extraction Kit (Invitrogen) according to the manufacturer's instructions. 1 μg of total cellular RNA was digested with 1 unit of DNase I (Promega) at 37°C for 20 minutes to remove genomic DNA contamination and then processed for reverse transcription (RT) with oligo(dT) primers and Moloney murine leukemia virus reverse transcriptase (Promega). For each cDNA sample, a control was generated with the RT mix without added reverse transcriptase to detect potential residual genomic DNA contamination of the cDNA sample. Quantitative PCR was performed using a fraction of the cDNA as a template with the primer pairs listed in Table 1.

[0055] [Table 1]

[0056] As previously described

[29] , rRNA 18S was chosen as the internal symmetry. The fluorescence threshold cycle (Ct) of each sample was determined by real-time PCR using the Mastercycler® ep realplex system (Eppendorf, Hamburg, Germany). Relative quantification of gene expression between groups was performed by applying the 2-ΔΔCt method

[34] . Results are expressed as fold-fold relative to transcript levels in uninfected cells (mock). [Example]

[0057] 1. Transfection and Infection AsPC-1 and GBM U251 cells were cultured at 4 × 10 5 and 2 x 10 5Cultures were seeded into 12-well plates at a density of 1000 / mL. After 24 hours, the cultures were transfected with 1 μg of a vector expressing a TCR activator (BPS Bioscience, CA, USA) and a plasmid expressing a TCR activator / PD-L1 (BPS Bioscience, CA, USA) in the presence of 3 μl of metafectene reagent (Biontex Laboratories GmbH, Munich, Germany) according to the manufacturer's instructions. After 24 hours, the cells were trypsinized and AsPC-1 cells were transfected at a density of 4.2 × 10 4 , and 2 × 10 for GBM U251 cell line. 4 For evaluation of PD-L1 gene expression upon treatment, transfected cell lines were split into 96-well plates at a density of 4 × 10 in 6 cm dishes. 5 Cells seeded in either 96-well plates or 6 cm dishes were incubated for 8 hours after splitting and then infected (or not) with either ΔH-1PVshPD-L1 or ΔH-1PVshEGFP at an MOI (pfu / cell) of 12 (AsPC-1) and 2 (U251).

[0058] 2. Co-culture with PD-1 / NFAT reporter Jurkat cells PD-1 / NFAT reporter Jurkat cells were purchased from BPS Bioscience (CA, USA). Briefly, 68 hours post-infection, infected cells in 96-well plates were pre-incubated with either anti-PD-L1 neutralizing (#71213, BPS Bioscience, CA, USA) or control antibody (PA5-20343, Thermo Fisher Scientific, USA) at a dilution of 50 ng / ml in medium according to the manufacturer's instructions, incubated at 37°C for 30 minutes, and then 6 × 10 cells per well were added. 4The cells were co-cultured with Jurkat cells in a 37°C incubator for 16 hours. After incubation, a one-step luciferase assay (BPS Bioscience, CA, USA) was performed by adding luciferase reagent to treated wells and untreated controls for 30 minutes, followed by luminescence measurement. The induction of NFAT luciferase reporter expression was calculated as the mean background-subtracted luminescence for either the various treatments or controls. Results are expressed as the relative ratio of the mean luminescence of the TCR activator / PD-L1-expressing samples to the mean luminescence of the TCR activator-alone-expressing samples for the various treatments and controls. [Example]

[0059] Spheroid generation 3D cell spheroids represent the heterogeneity of tumor models, as cells in the outer layer of the spheroid have access to nutrients and oxygen, while the core of the spheroid forms a hypoxic region due to the accumulation of cellular degradation products. Spheroids were generated from 20,000 AsPC-1 cells using the hanging drop method in the presence of a 30% methylcellulose stock solution, as previously described

[38] . After 2–3 days, formed spheroids were transfected into low-attachment round-bottom 96-well plates. 50 μl of complete medium was added per well with or without ΔH-1PVshEGFP or ΔH-1PVshPD-L1. Spheroid size was analyzed in real time using the Incucyte® 3D Single Spheroid Assay using the Spheroid Acquisition Analysis tool. Analysis was performed using Incucyte® S3 2018A software.

[0060] PD-L1 is known to be associated with tumor growth and progression. To evaluate the role of PD-L1 in the proliferation of human pancreatic cancer AsPc-1 cells, we investigated the effect of PD-L1 downregulation on cell proliferation in vitro using 3D spheroids. 3D spheroids are part of a highly translationally relevant model characterized by greater relevance to tumor biology, typified by the heterogeneity of cancer cells. Spheroid size upon treatment with ΔH-1PVshEGFP or ΔH-1PVshPD-L1 at an MOI of 5 PFU per cell was measured using the Incucyte® 3D Single Spheroid Assay using the Spheroid Acquisition Analysis Tool. The Incucyte® 3D product portfolio (system, software, and reagents) is available from Sartorious AG, Göttingen, Germany. This device allows for real-time monitoring of cell proliferation. As shown in Figure 6, on day 9 post-infection, the proliferation rate of cells infected with ΔH-1PVshPD-L1 was significantly lower than that of the control vector ΔH-1PVshEGFP, indicating that silencing of the PD-L1 gene by the ΔH-1PVshPD-L1 vector was effective in inhibiting tumor growth. Collectively, these results provide proof-of-concept that ΔH-1PVshPD-L1 has excellent anticancer activity, ensuring the clinical translation of this novel virus into cancer patients. (References) TIFF0007804694000002.tif217163TIFF0007804694000003.tif237163TIFF0007804694 000004.tif236163TIFF0007804694000005.tif238163TIFF0007804694000006.tif68163

Claims

1. A parvovirus for down-regulating expression of a target gene in a cell, the parvovirus being a parvovirus H-1 deletion variant comprising a deletion encompassing nucleotides 2022 to 2135, wherein a target-specific nucleic acid is inserted into the H-1 parvovirus VP gene in an RNA expression cassette at nucleotide 4480 of the parvovirus H-1 genome, the RNA being expressible under the control of a promoter or promoter region recognizable by an RNA polymerase in the cell, the target-specific nucleic acid being transcribable by an RNAi effector, and the parvovirus being capable of replicating and propagating in the cell, wherein the nucleic acid sequence of the parvovirus H-1 genome is set forth in SEQ ID NO:

13.

2. The parvovirus of claim 1, wherein the target gene is the PD-L1 gene.

3. The parvovirus of claim 1, wherein the promoter or promoter region recognizable by a cellular RNA polymerase is an RNA polymerase III (Pol III) promoter.

4. The parvovirus of claim 3, wherein the RNA polymerase III (Pol III) promoter is an RNA polymerase III H1 promoter.

5. The parvovirus of claim 1, wherein the target-specific nucleic acid is transcribable by shRNA.

6. The parvovirus of claim 1, wherein the target-specific nucleic acid has a length of at least 15 nucleotides.

7. The parvovirus described in claim 5, wherein the shRNA is complementary to the sequence 5'GATATTTGCTGTCTTTATA-3' (sequence number 1) of the PD-L1 gene.

8. 8. The parvovirus of any one of claims 1 to 7 for use in a method for treating a tumor.

9. 9. A parvovirus for use as defined in claim 8, characterized in that the cells of said tumor are resistant to chemotherapy and / or radiotherapy.

10. 9. The parvovirus for use as defined in claim 8, characterized in that the parvovirus is administered by intravenous (iv), intratumoral or intrabronchial administration.

11. 8. An isolated cell comprising the parvovirus of any one of claims 1 to 7.

Images