RNA trans-splicing molecules

Abstract

The present invention relates to an RNA trans-splicing molecule (RTM) that targets human endogenous retrovirus (HERV) pre-mRNA. The RTM comprises (i) a binding region specific for HERV pre-mRNA, (ii) a trans-splicing splice domain, and (iii) a coding sequence for a suicide protein. The binding region of the RTM binds to HERV pre-mRNA in a cell, and the coding sequence is trans-spliced ​​through the trans-splicing domain with the HERV pre-mRNA, resulting in a chimeric mRNA that expresses the suicide protein in the cell. The RTM of the present invention may be useful for selectively killing cells expressing HERV genes, such as cancer cells. The RTM, encoding nucleic acid, therapeutic methods and related methods and uses are provided.

Description

[Technical field]

[0001] The present invention relates to RNA trans-splicing molecules (RTMs), in particular RTMs that mediate trans-splicing of suicide genes, and their use in the treatment of cancer. [Background technology]

[0002] RNA trans-splicing is a spliceosome-mediated process in which two different RNA molecules are spliced ​​together to generate a chimeric mRNA molecule in the nucleus, which, after nuclear export, is translated in the cytoplasm to generate a chimeric protein.

[0003] RNA trans-splicing is used to exchange defective RNA transcripts with corrective mRNA molecules delivered in trans (Hong et al (2020) Br Med Bull. 2020 Dec 15;136(1):4-20).

[0004] RNA trans-splicing has also been used to deliver the two-step herpes simplex virus thymidine kinase-ganciclovir (HSV-tk / GCV) cell death system as a potential cancer therapy (Kim et al. (2016). Theranostics 6 357-368; Kwon et al. (2005). Mol. Ther 12, 5 824-834; Jung et al. (2006). Biochem. Biophys. Res. Commun., 349, 556-563; Song et al. (2009). Cancer Gene Ther 16, 113-125; Song et al. (2006). FEBS Letters 580, 5033-5043; Won et al. (2007). J. Biotechnol. 129, 614-619; Won et al. (2012). J. Biotechnol. 158, 44-49). Herpes simplex virus thymidine kinase catalyzes the conversion of the prodrug ganciclovir to the active compound by phosphorylation, causing chain termination during DNA replication and cell death (Duarte, S et al (2012). Cancer Letters 324, 160-170).

[0005] RNA trans-splicing has also been used to target HIV (Ingemarsdotter, C. K. et al (2017) Mol. Ther. Nucleic Acids, 7, 140-154) and cancer-specific RNAs (Poddar et al (2018). Mol. Ther. Nucleic Acids, 11, 41-56; WO2017171654A1, US2015079678A1, WO2014068063A1). Summary of the Invention

[0006] We have developed an RNA trans-splicing molecule (RTM) that targets human endogenous retrovirus (HERV) pre-mRNA, which may be useful for selectively killing cells expressing HERV genes, such as cancer cells.

[0007] The first aspect of the present invention is (i) a binding region specific to HERV pre-mRNA; (ii) a trans-splicing splice domain; and (iii) providing an RNA trans-splicing molecule (RTM) comprising a coding sequence for a suicide protein;

[0008] The binding region of the RTM binds to the HERV pre-mRNA in the cell, and the coding sequence is trans-spliced ​​through the trans-splicing domain to the HERV pre-mRNA, resulting in a chimeric mRNA that expresses the suicide protein in the cell.

[0009] A preferred RTM may comprise the nucleic acid sequence of SEQ ID NO: 13, SEQ ID NO: 15, or a variant of either of these.

[0010] A second aspect of the invention provides a nucleic acid encoding the RTM of the first aspect. Preferred nucleic acids may comprise the nucleotide sequence of SEQ ID NO: 14, SEQ ID NO: 16, or a variant of any of these.

[0011] A third aspect of the invention provides an expression vector comprising a nucleic acid of the second aspect.

[0012] A fourth aspect of the invention provides a viral particle comprising an RTM of the first aspect, a nucleic acid of the second aspect, or an expression vector of the third aspect.

[0013] A fifth aspect of the invention provides an isolated cell comprising an RTM of the first aspect, a nucleic acid of the second aspect, and / or an expression vector of the third aspect.

[0014] A sixth aspect of the invention provides a pharmaceutical composition comprising an RTM according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect and / or a viral particle according to the fourth aspect.

[0015] A seventh aspect of the present invention is The present invention provides a method for treating cancer, comprising administering to an individual in need thereof an RTM according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect, a viral particle according to the fourth aspect and / or a pharmaceutical formulation according to the sixth aspect.

[0016] The method of the seventh aspect may further comprise administering to the individual a cytotoxic compound that is activated by the suicide protein.For example, an inactive pro-form can be administered to the individual, and in this case, the suicide protein can convert the inactive pro-form into the active form of the cytotoxic compound in the cell in which the suicide protein is expressed.The cytotoxic compound can be administered to the individual in a first treatment, and one or more additional treatments can be administered after the first treatment.

[0017] An eighth aspect of the invention provides an RTM of the first aspect, a nucleic acid of the second aspect, an expression vector of the third aspect, a viral particle of the fourth aspect and / or a pharmaceutical formulation of the sixth aspect for use in a method of treating cancer, such as the method of the seventh aspect; and the use of an RTM of the first aspect, a nucleic acid of the second aspect, an expression vector of the third aspect, a viral particle of the fourth aspect and / or a pharmaceutical formulation of the sixth aspect in the manufacture of a medicament for use in a method of treating cancer, such as the method of the seventh aspect.

[0018] The RTM, nucleic acid, expression vector, viral particle, and / or pharmaceutical formulation may be provided in combination with a cytotoxic compound that is activated by the suicide protein.

[0019] A ninth aspect of the invention relates to a method for preventing the occurrence or recurrence of cancer in an individual undergoing cell therapy, comprising administering to an individual in need thereof a population of cells according to the fifth aspect.

[0020] The method may further include administering to the individual an inactive pro-form of the cytotoxic compound that is activated by the suicide protein, such that the cytotoxic compound is activated in cells of a cancerous or cancerous population in the individual.

[0021] A tenth aspect of the invention provides a method of killing a cell in vitro, comprising the steps of: contacting a cell with an RTM of the first aspect, a nucleic acid of the second aspect, an expression vector of the third aspect, a viral particle of the fourth aspect and / or a pharmaceutical formulation of the sixth aspect, such that said cell expresses a suicide protein; contacting the cell with a cytotoxic compound that is activated by a suicide protein, whereby the suicide protein activates the cytotoxic compound to kill the cell.

[0022] An eleventh aspect of the invention provides a method of depleting HERV gene expressing cells in a population, comprising the steps of: contacting a cell population with an RTM according to the first aspect, a nucleic acid according to the second aspect, an expression vector according to the third aspect, a viral particle according to the fourth aspect and / or a pharmaceutical formulation according to the sixth aspect, whereby HERV gene expressing cells in said cell population express a suicide protein; contacting the population with a cytotoxic compound that is activated by the suicide protein, such that the suicide protein activates the cytotoxic compound in HERV gene-expressing cells in the population, thereby depleting HERV gene-expressing cells in the population.

[0023] In preferred embodiments of the first to eleventh aspects, the suicide protein may be HSV thymidine kinase and the cytotoxic compound may be ganciclovir.

[0024] Other aspects and embodiments of the invention are described in more detail below. [Brief description of the drawings]

[0025] [Figure 1]The genomic organization of HERV-K class I and class II is shown (top panel) with the gag, pro, pol, and env open reading frames (ORFs) shown. Pro and Pol are expressed via a -1 ribosomal frameshift event. HERV-K class I has a 292 base pair deletion in the env gene that results in an alternative splicing event in HERV-K class I, generating the Np9 RNA transcript, whereas Rec is generated from HERV-K class II (middle panel). HERV-K env can be targeted for RNA trans-splicing at the RTM, which contains a binding domain complementary to the Rec / Np9 intron in env (bottom panel). [Diagram 2] Sequences and alignments of Np9 RNA trans-splicing target and binding domains are shown. 2A. Target sequence of RNA trans-splicing binding domain. The target sequence corresponds to nucleotides 6513-6556 of HERV-K HML-2-22q11.21, GenBank sequence number JN675087.1 within the env open reading frame. 2B. Nucleotide alignment of selected target sequence with HERV-K HML-2-22q11.21, GenBank sequence number JN675087.1 confirming nucleotide positions 6513-6556. 2C. Reverse complementary nucleotide sequence of target selected for optimization of binding domain design (top). Optimized binding domain DNA sequence (middle). Optimized binding domain RNA sequence (bottom). Arrowed nucleotides indicate C to U wobble base substitution, A to U mismatch, and A to G wobble base substitution for improved RNA structure. 2D. Nucleotide alignment of the binding domain sequence with target sequence HERV-K HML-2-22q11.21, Genbank sequence number JN675087.1 shows sequence complementarity. [Diagram 3]RNA secondary structure prediction of the binding domain sequence is shown. 3A. Minimum free energy (MFE) (left) and centroid (right) RNA secondary structure prediction using the RNA fold web server. 3B. RNA secondary structure prediction of the Np9 target RNA binding domain folded within an RNA trans-splicing cassette. RNA centroid fold (left) and MFE fold (right). The insert indicates the RNA binding domain region. [Figure 4A] 1 shows a lentiviral RNA trans-splicing delivery system. Diagram showing a third generation lentiviral gene delivery system, including packaging plasmids (Gag / Pol, VSV-G, and Rev) and a gene transfer construct containing an RNA trans-splicing cassette shown between XbaI and XhoI restriction enzyme sites. The RNA trans-splicing cassette is driven by an internal promoter, such as the thyroxine-binding globulin promoter (TBG) or the cytomegalovirus promoter (CMV) or other tissue-specific or non-specific or system-specific (e.g., hypoxia-responsive) promoter. [Figure 4B] Figure 2 shows a lentiviral RNA trans-splicing delivery system. A detailed view of the RNA trans-splicing cassette shown in A. The TBG promoter is followed by an RNA binding domain sequence targeting HERV-K Np9. A spacer is included downstream of the binding domain to separate it from the trans-splicing domain, followed by a P2A cleavage site. The transgene Herpes Simplex Virus Thymidine Kinase (HSV-tk) lacks the first ATG translation initiation codon, but the second and third ATGs (ATG46 and ATG60) are intact. A mini-intron is placed further downstream. [Figure 4C] 1 shows the lentiviral RNA trans-splicing delivery system. Optimized RNA trans-splicing cassette. The BbvCI restriction enzyme site and downstream nucleotides are mutated to destroy potential RNA splice acceptor sites, potentially leading to RNA splicing in cis. [ka] (The BbvCI restriction enzyme site is highlighted.) [ka] The second and third ATGs encoding the AUG translation initiation sites were mutated to ATG 46 and A.T.G. 60 was mutated from ATG to CTG and ATC. [Figure 5A] Figure 4 shows the expression of HSV-tk protein in HEK293T cells transfected with RNA trans-splicing constructs targeting Np9. Schematic diagram of RNA trans-splicing constructs, HSV-tk-ts and HSV-tk-ts-opt, for directing RNA trans-splicing of HSV-tk pre-mRNA onto Np9 pre-mRNA. Both constructs are derived from the CMV promoter. HSV-tk-ts and its optimized version HSV-tk-ts-opt are described in Figure 4C. [Figure 5B] Figure 1 shows the expression of HSV-tk protein in HEK293T cells transfected with an RNA trans-splicing construct targeting Np9. Graph showing HSV-tk mRNA. The arrow and green block indicate the HSV-tk translation initiation site. Four isoforms can be produced from the wild-type HSV-tk mRNA transcript: P1 from ATG1, P2 from a non-canonical ATG translation initiation site located between ATG1 and ATG46, P3 from ATG46, and P4 from ATG60. Among the various HSV-tk isoforms, it was found that P1 and P3 are catalytically active, P2 is inactive, and P4 has very low activity (Ellison, AR & Bishop, JO (1996) Nucleic acids research 24 2073-2079). [Figure 5C]Figure 1 shows HSV-tk protein expression in HEK293T cells transfected with RNA trans-splicing constructs targeting Np9. Predictions of HSV-tk isoform protein expression for HSV-tk wild-type transcript, HSV-tk-ts, and HSV-tk-ts-opt in both Np9-expressing and non-Np9-expressing cells. Wild-type HSV-tk transcript is expected to express P1, P2, P3, and P4 to some extent. Both HSV-tk-ts and HSV-tk-ts-opt are expected to express P1 only in Np9-expressing cells. However, expression of P3 and P4 is expected to be suppressed in HSVtk-ts-opt transfected cells. [Figure 5D] Figure 1 shows HSV-tk protein expression in HEK293T cells transfected with RNA trans-splicing constructs targeting Np9. Protein lysates from HEK 293T cells analyzed by Western blot 72 hours after transfection with either the RNA trans-splicing constructs, HSV-tk-ts or HSV-tk-ts opt, or the controls, CMV-GFP or CMV-HSV-tk. HSV-tk antibody staining detected at both high and low doses. [Figure 5E] Figure 1 shows the expression of HSV-tk protein in HEK293T cells transfected with an RNA trans-splicing construct targeting Np9. Quantification of P1 expression in HSV-tk-ts or HSV-tk-ts-opt transfected cells. P1 protein was normalized to vinculin protein in each lane (n=3). [Figure 5F] Figure 1 shows the expression of HSV-tk protein in HEK293T cells transfected with an RNA trans-splicing construct targeting Np9. Quantification of P3 expression in HSV-tk-ts or HSV-tk-ts-opt transfected cells. P1 protein was normalized to vinculin protein in each lane (n=3). For all graphs, error bars indicate SD; ns=not significant; **P<0.01; unpaired t-test. [Figure 6] Figure 5 shows the loss of cell viability in hepatocellular carcinoma cell lines (HCC) Hep3B and Huh7-mRFP after transduction with RNA trans-splicing lentiviral vectors targeting Np9 pre mRNA. A. Hep3B cells (1x10^4 cells / well in 96-well plates) were untreated (ctrl) or transduced at an MOI (multiplicity of infection) of 1 with the negative control vector pSico, the trans-splicing vector, 3'ER-HSVtk, 3'ER-HSVtk, or HSVtk (positive control). 3'ER-HSVtk and 3'ER-HSVtk opt are identical to HSV-tk-ts and HSV-tk-ts-opt shown in Figure 5A, respectively, except that they are driven from the TBG promoter. One day after transduction, cells were treated with a dose of 300 μM ganciclovir (GCV) and a second dose the next day. Cell viability 4 days after GCV treatment (5 days after transduction) was analyzed by MTT assay (Pannecouque, C. et al (2008) Nature protocols, 3, 427-434). B. HuH7-mRFP cells were transduced and treated with GCV as described in A, and cell viability was analyzed 4 days after treatment. C. Hep3B cells were transduced as in A, and treated with GCV using two consecutive doses of 10 μM or 100 μM GCV on the 2nd and 3rd days after transduction, and then cell viability analysis was performed 3 days later by MTT assay. Error bars indicate standard deviation (SD). Statistical analysis was performed by Student's t-test (two-tailed, assuming unequal variances) with *P<0.05; **P<0.01; ***P<0.001, comparing each sample + GCV to each sample without GCV. [Figure 7A]We show that RNA trans-splicing lentiviral vectors induce cell death in HEK293T cells and pancreatic cancer cell lines Panc-1, Aspc-1, and MIA PaCa-2. HEK293T cells were transduced with Lv-CMV-GFP, Lv-HSV-tk-ts, Lv-HSV-tk-ts-opt, or Lv-CMV-HSV-tk at an MOI of 1 and treated with 1000 μM GCV. A significant decrease in cell viability was observed in almost all cell lines in cells treated with RNA trans-splicing lentiviral vectors carrying the CMV promoter. Cell viability was quantified using the MTT assay. (n=3); error bars represent SD; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; two-way ANOVA followed by Tukey's post hoc multiple comparison test. [Figure 7B] We show that RNA trans-splicing lentiviral vectors induce cell death in HEK293T cells and pancreatic cancer cell lines Panc-1, Aspc-1, and MIA PaCa-2. HEK293T cells were transduced with Lv-CMV-GFP, Lv-HSV-tk-ts, Lv-HSV-tk-ts-opt, or Lv-CMV-HSV-tk at an MOI of 2 and treated with 1000 μM GCV. A significant decrease in cell viability was observed in almost all cell lines in cells treated with RNA trans-splicing lentiviral vectors carrying the CMV promoter. Cell viability was quantified using the MTT assay. (n=3); error bars represent SD; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; two-way ANOVA followed by Tukey's post hoc multiple comparison test. [Figure 7C]We show that RNA trans-splicing lentiviral vectors induce cell death in HEK293T cells and pancreatic cancer cell lines Panc-1, Aspc-1, and MIA PaCa-2. Panc-1 cells were transduced with Lv-CMV-GFP, Lv-HSV-tk-ts, Lv-HSV-tk-ts-opt, or Lv-CMV-HSV-tk at an MOI of 1 and treated with 1000 μM GCV. A significant decrease in cell viability was observed in almost all cell lines in cells treated with RNA trans-splicing lentiviral vectors carrying the CMV promoter. Cell viability was quantified using the MTT assay. (n=3); error bars represent SD; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; two-way ANOVA followed by Tukey's post hoc multiple comparison test. [Figure 7D] We show that RNA trans-splicing lentiviral vectors induce cell death in HEK293T cells and pancreatic cancer cell lines Panc-1, Aspc-1, and MIA PaCa-2. Panc-1 cells were transduced with Lv-CMV-GFP, Lv-HSV-tk-ts, Lv-HSV-tk-ts-opt, or Lv-CMV-HSV-tk at an MOI of 2 and treated with 1000 μM GCV. A significant decrease in cell viability was observed in almost all cell lines in cells treated with RNA trans-splicing lentiviral vectors carrying the CMV promoter. Cell viability was quantified using the MTT assay. (n=3); error bars represent SD; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; two-way ANOVA followed by Tukey's post hoc multiple comparison test. [Figure 7E]We show that RNA trans-splicing lentiviral vectors induce cell death in HEK293T cells and pancreatic cancer cell lines Panc-1, Aspc-1, and MIA PaCa-2. Aspc-1 cells were transduced with Lv-CMV-GFP, Lv-HSV-tk-ts, Lv-HSV-tk-ts-opt, or Lv-CMV-HSV-tk at an MOI of 1 and treated with 1000 μM GCV. A significant decrease in cell viability was observed in almost all cell lines in cells treated with RNA trans-splicing lentiviral vectors carrying the CMV promoter. Cell viability was quantified using the MTT assay. (n=3); error bars represent SD; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; two-way ANOVA followed by Tukey's post hoc multiple comparison test. [Figure 7F] We show that RNA trans-splicing lentiviral vectors induce cell death in HEK293T cells and pancreatic cancer cell lines Panc-1, Aspc-1, and MIA PaCa-2. Aspc-1 cells were transduced with Lv-CMV-GFP, Lv-HSV-tk-ts, Lv-HSV-tk-ts-opt, or Lv-CMV-HSV-tk at an MOI of 2 and treated with 1000 μM GCV. A significant decrease in cell viability was observed in almost all cell lines in cells treated with RNA trans-splicing lentiviral vectors carrying the CMV promoter. Cell viability was quantified using the MTT assay. (n=3); error bars represent SD; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; two-way ANOVA followed by Tukey's post hoc multiple comparison test. [Figure 7G]We show that RNA trans-splicing lentiviral vectors induce cell death in HEK293T cells and pancreatic cancer cell lines Panc-1, Aspc-1, and MIA PaCa-2. MIA PaCa-2 cells were transduced with Lv-CMV-GFP, Lv-HSV-tk-ts, Lv-HSV-tk-ts-opt, or Lv-CMV-HSV-tk at an MOI of 1 and treated with 1000 μM GCV. A significant decrease in cell viability was observed in almost all cell lines in cells treated with RNA trans-splicing lentiviral vectors carrying the CMV promoter. Cell viability was quantified using the MTT assay. (n=3); error bars represent SD; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; two-way ANOVA followed by Tukey's post hoc multiple comparison test. [Figure 7H] We show that RNA trans-splicing lentiviral vectors induce cell death in HEK293T cells and pancreatic cancer cell lines Panc-1, Aspc-1, and MIA PaCa-2. MIA PaCa-2 cells were transduced with Lv-CMV-GFP, Lv-HSV-tk-ts, Lv-HSV-tk-ts-opt, or Lv-CMV-HSV-tk at an MOI of 2 and treated with 1000 μM GCV. A significant decrease in cell viability was observed in almost all cell lines in cells treated with RNA trans-splicing lentiviral vectors carrying the CMV promoter. Cell viability was quantified using the MTT assay. (n=3); error bars represent SD; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; two-way ANOVA followed by Tukey's post hoc multiple comparison test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention relates to a ribonucleic acid (RNA) trans-splicing molecule (RTM) comprising (i) a binding region specific for HERV pre-mRNA, (ii) a trans-splicing domain, and (iii) a sequence encoding a suicide protein. The RTM mediates trans-splicing of HERV pre-mRNA by a sequence encoding the suicide protein, e.g., a 3' exon substitution (3'ER) or a 5' exon substitution (5'ER). This results in the expression of the suicide protein in cells expressing the HERV pre-mRNA. Cells expressing the suicide protein can then be selectively killed by exposure to a cytotoxic compound that is activated by the suicide protein.

[0027] Ribonucleic acid (RNA) trans-splicing molecules (RTMs) are heterologous RNA molecules that can induce trans-splicing events between endogenous target pre-mRNAs and RTMs to generate chimeric mRNAs that contain nucleotide sequences from both the target pre-RNA and the RTM. RTMs typically contain a binding region that defines specificity for the target pre-mRNA, a splicing element that mediates trans-splicing, and a coding sequence that replaces a portion of the target pre-mRNA. The design and use of RTMs for use in gene therapy has been reported (see, for example, Wally et al. J Invest Dermatol 2012;132: 1959-1966. Pttaraju et al Nat. Biotechnol. 1999;17: 246-252). The RTMs described herein target HERV pre-mRNAs. The RTM can be contacted with a HERV pre-mRNA under conditions where the coding sequence of the RTM is trans-spliced ​​with the HERV pre-mRNA to form a chimeric mRNA molecule that can be further processed and expressed in a cell.

[0028] RTM features (i)-(iii) may be arranged sequentially in the 5' to 3' direction, e.g., in the case of a 3' exon substitution, in the order (i), (ii)(iii), or, e.g., in the case of a 5' exon exchange, in the order (iii), (ii), (i) (see, e.g., Poddar et al (2018) Molecular therapy. Nucleic Acids 11, 41-56).

[0029] The RTM described herein mediates trans-splicing with HERV precursor mRNA (pre-mRNA). Pre-mRNA is RNA transcribed from a gene in the nucleus of a cell that has not yet been processed into mRNA. Thus, pre-mRNA contains introns and other features not present in mRNA. Pre-mRNA may also be referred to as primary transcript, or heterogeneous nuclear RNA (hnRNA).

[0030] HERV pre-mRNAs may comprise unlinked or partially spliced ​​transcripts of genes derived from a HERV provirus (HERV genes). Suitable HERV pre-mRNAs include HERV-K pre-mRNAs.

[0031] Human endogenous retroviruses (HERVs) are endogenous viral elements or proviruses present in the human genome. HERVs display a similar genomic organization to exogenous retroviruses and are vertically transmitted through successive generations in the germline. Expression of genes from HERV proviruses is tightly regulated in normal cells, but HERV-K genes can be dysregulated and overexpressed in cancer (see, e.g., Hohn et al Frontiers in Oncology, 3, 246; Kassiotis, G. (2014) J. Immunol. 192, 1343-1349; Attig, J., et al Genome Res, 29, 1578-1590).

[0032] The HERVs can be of any class or group having a complete or incomplete genome. For example, the HERVs can be a class I HERV of any one of groups 1-6, a class II HERV of any one of groups 1-10, or a class III HERV. In some preferred embodiments, the HERVs can be a class II HERV, such as HERV-K. HERV-K is a class of human endogenous retroviruses (HERVs) in the human genome (see, e.g., Bannert et al (2018) Front Microbiol 9,178). HERV-2 can include HERV-K HML-2 proviruses, such as HERV-K HML-2 proviruses of groups 1 and 2. Suitable HERV pre-mRNAs may be transcribed from a HERV proviral gene, such as the gag, pro, pol or env gene, for example the HERV-K HML-2 gag, pro, pol or env gene.

[0033] In some preferred embodiments, the HERV proviral gene can be an env gene. For example, a suitable HERV pre-mRNA can be transcribed from a HERV env gene, such as a HERV-K env gene. A suitable HERV pre-mRNA is transcribed from a HERV-K HML-2 env gene. HERV-K HML-2 proviruses can be classified into two groups (group 1 and group 2). The env gene of the HERV-K HML-2 group 1 provirus has a 291 base pair deletion relative to the env gene of the HERV-K HML-2 group 2 provirus. This deletion causes alternative splicing in group 1 and group 2 proviruses, producing the Np9 protein in group 1 proviruses and the Rec protein in group 2 proviruses.

[0034] The RTM described herein can target both types of HERV-K HML-2 env gene transcripts. For example, the HERV-K pre-mRNA can be (i) a HERV-K pre-mRNA encoding Np9 and / or (ii) a HERV-K pre-mRNA encoding Rec. For example, the RTM described herein can target both the Np9 pre-mRNA expressed by the HERV-K HML-2 group 1 provirus and the Rec pre-mRNA expressed by the HERV-K HML-2 group 2 provirus.

[0035] The binding region of the RTM specifically binds to the HERV pre-mRNA. For example, the binding region can specifically bind to the HERV pre-mRNA transcribed from the env gene. The binding of the binding region targets the RTM to the HERV pre-mRNA and allows the coding sequence encoding the suicide protein to be trans-spliced ​​with the HERV pre-mRNA. In some preferred embodiments, the binding region can specifically bind to the HERV-K pre-mRNA transcribed from the env gene, such as HERV-K Np9 or HERV-K Rec pre-mRNA. The binding of the binding region targets the RTM to the HERV-K pre-mRNA and allows the coding sequence encoding the suicide protein to be trans-spliced ​​with the HERV-K pre-mRNA.

[0036] The RTMs described herein may contain one or more binding regions, i.e., one continuous sequence or multiple continuous regions that hybridize to HERV pre-mRNA. Multiple binding regions may be useful, for example, to enhance cell death (e.g., Poddar et al (2018) supra). Preferably, the RTMs described herein contain a single binding region, i.e., only one continuous sequence that hybridizes to HERV pre-mRNA.

[0037] The binding region may have an open structure, e.g., it may lack self-complementary sequences and may contain a stretch of unstructured nucleotides that are not paired or bound to other nucleotides in the RTM. The binding region may, for example, contain less than 25 consecutive unstructured nucleotides, e.g., 12-25 consecutive unstructured nucleotides.

[0038] Preferably, the binding region of the RTM specifically binds to an intron sequence of a HERV pre-mRNA, for example, a HERV-K pre-mRNA. The binding region can specifically bind to an intron sequence close to a splice site in the HERV pre-mRNA. Suitable splice sites can be identified using standard sequence analysis tools (e.g., Neural Network Server within the Berkeley Drosophila Genome Project; CrypSkip software, Bioinformatics HUSAR server, German Cancer Research Centre). In the case of a 3' exon substitution, the binding region can specifically bind to an intron sequence downstream (3') of a splice site in the HERV pre-mRNA. In the case of a 5' exon substitution, the binding region can specifically bind to an intron sequence upstream (5') of a splice site in the HERV pre-mRNA.

[0039] The binding region can bind to a region that is identified as having a high minimum free energy and a high proportion of unstructured nucleotides compared to other intron sequences in pre-mRNA. Suitable regions can be identified using standard sequence analysis tools (e.g., Foldanalyse, Bioinformics HUSAR server, German Cancer Research Center). For example, the binding region of RTM targeting HERV-K Env gene can bind within the region of HERV-K Np9 or HERV-K Rec pre-mRNA corresponding to SEQ ID NO: 1 (nucleotides 6513-6556 of HERV-K HML-2-22q11.21; Genbank ID: JN675087.1).

[0040] A suitable binding region may comprise a sequence in HERV pre-mRNA or a reverse complement of a variant thereof. For example, a suitable binding region targeting HERV-K gene may comprise a sequence in HERV-K pre-mRNA or a reverse complement of a variant thereof. The binding region may comprise the nucleotide sequence of SEQ ID NO:3 (reverse complement of SEQ ID NO:1) or SEQ ID NO:4 (reverse complement of SEQ ID NO:2), or may be a variant thereof. A suitable binding region may be less than 200 nucleotides in length, preferably less than 100 nucleotides in length.

[0041] In some preferred embodiments, the nucleotide sequence of the binding region can be modified to remove potential splice sites, inhibit RNA editing, and / or increase trans-splicing efficiency. For example, a binding region targeting HERV-K env pre-mRNA may comprise the nucleotide sequence of SEQ ID NO: 3 or a variant thereof, which has modifications relative to SEQ ID NO: 3 at one or more, preferably all, of positions 4, 19, 20, 32, and 34. For example, the nucleotide sequence may have U at positions 4, 19, and 20, and G at positions 32 and 34. A suitable binding region may comprise a nucleotide sequence corresponding to SEQ ID NO: 3, with a C to U substitution at position 4; an A to U substitution at position 19; an A to U substitution at position 20; an A to G substitution at position 32; and an A to G substitution at position 34. For example, the binding region may comprise the nucleotide sequence of SEQ ID NO: 5 or a variant thereof.

[0042] A variant of a reference amino acid sequence or a reference nucleotide sequence described herein may comprise an amino acid sequence or a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence. A particular amino acid sequence variant may differ from the reference sequence by the insertion, addition, substitution, or deletion of one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids. A particular nucleotide sequence variant may differ from the reference sequence by the insertion, addition, substitution, or deletion of one, two, three, four, five, six, seven, eight, nine, or ten or more nucleotides.

[0043] Sequence similarity and identity are generally defined with reference to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences in a way that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with gap creation penalty=12 and gap extension penalty=4. Although the use of GAP may be preferred, other algorithms may also be used, such as BLAST (using the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (using the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), or the TBLASTN program of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.Computerized implementations of these algorithms are available (GAP, BESTFIT, PASTA, and FASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), and publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)), Genomequest™ software (Gene-IT, Worcester MA USA), and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780 software) can be used. When using such software, it is advisable to use default parameters, such as, for example, gap penalties and extension penalties. Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Sequence comparisons can be performed over the entire length of the relevant sequences described herein.

[0044] Amino acid residues in the reference amino acid sequence may be altered or mutated by insertion, deletion or substitution, preferably substitution with a different amino acid residue, to produce a variant of the reference amino acid sequence. Nucleotides in the reference nucleotide sequence may be altered or mutated by insertion, deletion or substitution, preferably substitution with a different nucleotide, to produce a variant of the reference nucleotide sequence.

[0045] In some embodiments, a spacer may be placed between the binding domain and the trans-splicing domain of the RTM described herein. The spacer may reduce or prevent the interaction between the binding domain and the trans-splicing domain or the coding sequence. The spacer may comprise any nucleotide sequence that is not complementary to the binding domain region and does not hybridize or otherwise interact with the binding region, the trans-splicing domain or the coding sequence. In some embodiments, the spacer may further comprise a stop codon to block the translation of any unconjugated RTM. A suitable spacer may comprise the nucleotide sequence of SEQ ID NO: 10 or a variant thereof.

[0046] In other embodiments, the RTMs described herein may lack a spacer between the binding domain and the trans-splicing domain of the RTM.

[0047] After binding to HERV-K pre-mRNA via the binding region, the trans-splicing domain in the RTM is spliceosome-mediated trans-spliced ​​with the HERV pre-mRNA. Trans-splicing links a nucleotide sequence encoding a suicide protein to trans-splice the HERV pre-mRNA. In 3' exon substitution, the coding sequence is located downstream (3') of the trans-splicing domain and is spliced ​​with a nucleotide sequence upstream (5') of the donor splice site in the HERV pre-mRNA. In 5' exon substitution, the coding sequence is located upstream (5') of the trans-splicing domain of the RTM and is spliced ​​with a nucleotide sequence downstream (3') of the acceptor splice site in the HERV pre-mRNA. The technology of trans-splicing by 3' and 5' exon substitution has been established in the art (see, for example, Poddar et al. (2018) supra).

[0048] The trans-splicing between RTM and HERV pre-mRNA is mediated by trans-splicing domain.Trans-splicing domain is a nucleotide sequence that contains the motif required for recruiting spliceosome and mediating trans-splicing with endogenous pre-mRNA.For example, trans-splicing domain can contain the splice site where RTM is connected to HERV pre-mRNA by trans-splicing.

[0049] The trans-splicing domain of the RTM suitable for use in 3' exon replacement can include a splice acceptor site. The splice acceptor site can include an AG dinucleotide sequence. For example, the splice acceptor site can include the sequence CAGG (e.g., AG). Suitable splice acceptor sites are well known in the art.

[0050] The trans-splicing domain of the RTM suitable for use in 3' exon replacement may further comprise a polypyrimidine tract (PPT). The polypyrimidine tract may be located upstream of the splice site, for example 5-40 nucleotides upstream of the splice acceptor site in the 3' exon replacement RTM. The polypyrimidine tract may comprise a sequence of 15-20 nucleotides rich in pyrimidines (C and U). Suitable PPTs include 5'-UUUUUUUCCCUUUUUUUCC-3' and variants thereof. Other suitable PPTs are known in the art (see, for example, Wagner et al 2001 Mol Cell Biol21(10):3281-3288; WO2017171654A1).

[0051] The trans-splicing domain of the RTM suitable for use in 3' exon replacement may further comprise a branchpoint sequence. The branchpoint sequence may be located upstream of the PPT, for example 20-50 nucleotides upstream of the splice acceptor site. The branchpoint sequence may comprise the sequence YURAC or YNURAC, where R=purine, Y=pyrimidine, and N=any nucleotide. Suitable branchpoint sequences include 5'-UACUAACA-3' and are known in the art. (See, e.g., Gao et al Nucl Acid Res 2008 36(7)2257-2267; US20060094675)

[0052] The trans-splicing domain of the RTM suitable for use in 3' exon replacement may further comprise an intronic splice enhancer (ISE). The ISE may be located upstream of the branch point sequence. A suitable ISE may be: [ka] and are known in the art (see, e.g., Wang et al Nat Struct. Mol. Biol. (2012) 19(10) 1044-1052; McCarthy et al (1998) Hum Mol Genet 7 1491-1496; Yeo et al (2004) PNAS USA 101 15700-15705).

[0053] Suitable trans-splicing domains for use in 3' exon replacement are well known in the art (see, e.g., Poddar et al. 2018, supra). For example, a suitable trans-splicing domain may comprise the nucleotide sequence of SEQ ID NO: 12 or may be a variant thereof.

[0054] The trans-splicing domain of the RTM suitable for use in 5' exon replacement can include a splice donor site. The splice donor site can include a GU dinucleotide sequence. For example, the splice donor site can include the sequence 5'-CAG / GUAAGTAT-3'. Other suitable splice donor acceptor sites are well known in the art.

[0055] The trans-splicing domain of RTM suitable for use in 5' exon replacement can further comprise an intronic splice enhancer (ISE). ISE can be located downstream of splice donor sequence. Suitable ISE is well known in the art (see, for example, Wang et al Nat Struct.Mol.Biol.(2012)19(10)1044-1052; McCarthy et al(1998)Hum Mol Genet 7 1491-1496; Yeo et al(2004)PNAS USA 101 15700-15705).

[0056] Trans-splicing domains suitable for use in 5' exon replacement are well known in the art (see, e.g., Poddar et al. 2018, supra).

[0057] Preferably, the RTM described herein does not contain a splice site that interferes with the trans-splicing of the trans-splicing domain, for example by mediating cis-splicing. For example, the 3' exon-substituted RTM may lack a splice site upstream of the trans-splicing domain, i.e., the trans-splicing domain is not downstream (3') of the 5' splice site of the 3'ER RTM. The 5' exon-substituted RTM may lack a splice site downstream of the trans-splicing domain, i.e., the trans-splicing domain is not upstream (5') of the 3' splice site of the 5'ER RTM.

[0058] The RTMs described herein may further comprise a separation element that allows for the production of a suicide protein that does not contain the amino acids encoded by the HERV sequence in the chimeric mRNA. Suitable separation elements are well known in the art (Poddar et al., 2018, supra) and include a termination codon and a sequence that encodes a self-cleaving peptide, such as a 2A peptide. The coding region for the suicide protein may be followed by a polyA tail, such as SV40 polyA (Poddar et al., 2018).

[0059] For example, the 5'ER RTM described herein may further include a stop codon, such as UAG, UAA or UGA, at the 3' end of the sequence encoding the suicide protein. Translation of the chimeric mRNA after trans-splicing terminates at the stop codon, producing the suicide protein without the additional amino acids encoded by the HERV sequence in the chimeric mRNA.

[0060] The 3'ER RTM described herein may further comprise a self-cleaving peptide coding sequence, such as a 2A peptide coding sequence. The self-cleaving peptide coding sequence may be located between the trans-splicing domain and the suicide protein coding sequence, for example at the 5' end of the coding sequence. The self-cleaving peptide cleaves the nascent peptide chain during translation, separating the suicide protein from the HERV amino acid sequence. Suitable 2A peptides may include T2A, P2A, E2A and F2A peptides (Poddar et al (2018); Kim et al (2011) PLoS ONE 6, e18556, supra), and may comprise the amino acid sequence of SEQ ID NO: 11 or a variant thereof. The self-cleaving peptide coding sequence may comprise the nucleotide sequence of SEQ ID NO: 17 or a variant thereof.

[0061] The 5'ER RTM described herein may further comprise a ribozyme, such as a hammerhead ribozyme, either catalytically active or inactive, or a stabilized RNA element. The ribozyme may be located downstream of the binding domain. The ribozyme or stabilized RNA element may be followed by a spacer and a polyA tail. The ribozyme may remove a nucleotide sequence downstream (3') of the binding domain, such as a polyA tail, from the RTM. Suitable ribozyme sequences and stabilized RNA elements are available in the art (Poddar et al (2018) supra).

[0062] The RTM described herein further comprises the coding sequence of suicide protein.In HERV gene expressing cells such as cancer cells, the coding sequence is trans-spliced ​​into HERV pre-mRNA via the trans-splicing domain of RTM, and the suicide protein is expressed in HERV gene expressing cells.

[0063] A suicide protein is a protein expressed in a cell that interacts with at least one other molecule to cause cell induction or cell death. The coding sequence that encodes the suicide protein can be called a suicide gene. The coding sequence can include a complete coding sequence for the suicide protein. In a 3'ER RTM, the coding sequence can lack a 5' translation initiation codon (start codon: AUG). This prevents expression of the coding sequence in the absence of trans-splicing. In a 5'ER RTM, the coding sequence can include a 5' translation initiation codon (start codon: AUG).

[0064] Preferably, the suicide protein is a prodrug-activating enzyme that converts an inactive pro-form of a cytotoxic compound into an active form, such that cells expressing the suicide protein are sensitive to exposure to the pro-form, since the suicide protein generates the active cytotoxic compound from the inactive pro-form. Suicide proteins suitable for use as described herein are available in the art (see, e.g., Malekshah et al (2016) Curr Pharmacol Reps 2 299-308) and include cytosine deaminase, which activates 5-fluorocytosine (5-FC); cytochrome P450, which activates ifosfamide (IFO) or cyclophosphamide; nitroreductase, which activates 5-[aziridin-1-yl]-2,4-dinitrobenzamide; purine nucleoside phosphorylase, which activates fludarabine (ePNP; Secrist et al Nucleos. Nucleot. 1999, 18, 745-757; Krohne et al Hepatology (2001) 34(3):511-8) and thymidine kinase, which activates ganciclovir (GCV).

[0065] In some preferred embodiments, the suicide protein is herpes simplex virus (HSV) thymidine kinase (tk). HSV-tk phosphorylates ganciclovir to produce the cytotoxic ganciclovir triphosphate. HSV thymidine kinase can comprise or consist of the sequence shown in SEQ ID NO: 6 (database accession number AF057310.1), or a variant thereof. A suitable coding sequence encoding HSV thymidine kinase can comprise the nucleotide sequence of SEQ ID NO: 7, or a variant thereof.

[0066] In an RTM suitable for 3' exon replacement, the HSV thymidine kinase may lack an N-terminal M residue at a position corresponding to position 1 of SEQ ID NO:6. This may prevent expression of the suicide protein in the absence of a trans-splicing event. The coding sequence may lack a 5' translation initiation site (AUG). In an RTM suitable for 5' exon replacement, the HSV thymidine kinase may include an N-terminal M residue at a position corresponding to position 1 of SEQ ID NO:6.

[0067] In some embodiments, the coding sequence of the HSV thymidine kinase may include an exonic splice enhancer (ESE). Advantageously, alternative degenerate codons may be used, so that the encoded amino acid sequence is not altered by the presence of the ESE. In some embodiments, the coding sequence may further include an intron, such as a β-globin mini-intron. Suitable sequences are known in the art (Poddar et al. 2018, supra).

[0068] Preferably, the coding sequence encoding HSV thymidine kinase may be modified to reduce the expression of aberrant isoforms of thymidine kinase. This may be useful to reduce the production of active HSV thymidine kinase from HERV-K pre-mRNA and non-trans-spliced ​​sequences. For example, HSV thymidine kinase may be modified to delete or replace M residues encoded by the translation initiation codon. A suitable variant of the HSV thymidine kinase sequence of SEQ ID NO:6 may lack one or more M residues encoded by the translation initiation codon. For example, the M residues of HSV thymidine kinase at positions corresponding to positions 1 and 46 of SEQ ID NO:6, preferably positions 1, 46 and 60 of SEQ ID NO:6, may be deleted or replaced by other residues. For example, the M residue of HSV thymidine kinase at the position corresponding to position 1 of SEQ ID NO:6 may be deleted. The M residue of HSV thymidine kinase at the position corresponding to position 46 of SEQ ID NO:6 may be replaced by a different residue, preferably an L residue. The M residue of the HSV thymidine kinase at the position corresponding to position 60 of SEQ ID NO:6 may be replaced with a different residue, preferably an I residue. Preferably, the HSV thymidine kinase lacks a functional translation initiation site. A preferred HSV thymidine kinase encoded by the coding sequence may comprise the amino acid sequence of SEQ ID NO:8 or a variant thereof.

[0069] The coding sequence of the modified HSV thymidine kinase may comprise an amino acid sequence that is a variant of SEQ ID NO:7, which includes one or more codons that initiate translation modification. For example, one or more nucleotides of the translation initiation codon (ATG / AUG) at positions corresponding to positions 136-138 of SEQ ID NO:7 may be replaced with other nucleotides such that the codon ATG / AUG is destroyed or abolished. In some embodiments, the A at position 136 may be replaced with a different nucleotide. For example, the nucleotide sequence may include an A>C substitution at position 136. A suitable nucleotide sequence may lack the translation initiation codon at positions 136-138 and not support translation initiation from these positions. One or more nucleotides of the translation initiation codon (ATG / AUG) at positions corresponding to positions 178-180 of SEQ ID NO:7 may be replaced with other nucleotides such that the translation initiation codon (ATG / AUG) is destroyed or abolished. In some embodiments, the G at position 180 may be replaced with a different nucleotide. For example, a nucleotide sequence may contain a G>C substitution at position 180. A suitable nucleotide sequence may lack a translation initiation codon at positions 178-180 and not support translation initiation from these positions.

[0070] In some preferred embodiments, the nucleotide sequence may lack translation initiation codons at positions 136-138 and 178-180. For example, the nucleotide sequence may include an A>C substitution at position 136 and a G>C substitution at position 180.

[0071] A preferred coding sequence for HSV thymidine kinase may comprise the nucleotide sequence of SEQ ID NO:9 or a variant thereof.

[0072] The nucleic acid sequence encoding the modified HSV thymidine kinase as described above may generally be useful as a suicide gene for RNA trans-splicing and is provided as an aspect of the present invention. As described above, the modified HSV thymidine kinase may include a deletion or substitution of residues M1 and M46, preferably M1, M46 and M60. For example, the modified HSV thymidine kinase may include an M46L mutation, preferably an M46L and an M60I mutation, as described above. The modified HSV thymidine kinase may include the sequence of SEQ ID NO:8 or a variant thereof. The nucleic acid sequence may include the sequence of SEQ ID NO:9 or a variant thereof. Nucleic acid constructs, such as RTMs and expression vectors, that include the nucleic acid sequence, and the use of such nucleic acids in the manufacture of nucleic acid constructs, such as RTMs or expression vectors, are also provided as aspects of the present invention. The constructs and nucleic acids may be useful for a range of trans-splicing applications, including 3' exon replacement trans-splicing and ribozyme-mediated trans-splicing.

[0073] A preferred RTM as described herein may comprise the nucleotide sequence of SEQ ID NO: 13 or a variant thereof, or the nucleotide sequence of SEQ ID NO: 15 or a variant thereof.

[0074] The RTMs described above may be encoded by a nucleic acid, such as a DNA molecule. The nucleic acid molecule may be isolated. The nucleic acid may be partially or wholly synthetic.

[0075] A preferred RTM as described herein may be encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 14 or a variant thereof, or the nucleotide sequence of SEQ ID NO: 16 or a variant thereof.

[0076] The nucleic acid encoding the RTM may be operably linked to one or more control elements or regulatory sequences capable of directing expression of the RTM. Control elements or regulatory sequences suitable for promoting expression of heterologous nucleic acid coding sequences in mammalian cells, preferably human cells, are well known in the art and include constitutive promoters, e.g., viral promoters such as CMV or SV40; and tissue-specific promoters, e.g., promoters such as the human thyroxine-binding globulin (TBG) promoter, or system-specific promoters, such as hypoxia-responsive promoters.

[0077] Tissue-specific promoters include cancer-specific promoters (i.e., promoters that have specific activity in cancer cells) or promoters that are specific to the tissue in which cancer occurs in an individual.Suitable promoters can include, for example, Cox-2 or Muc-1 promoters for pancreatic cancer.

[0078] Further provided are constructs in the form of plasmids, vectors (e.g., expression vectors), transcription or expression cassettes, or other delivery systems, that contain the RTMs described herein or nucleic acids encoding the RTMs described herein. For example, the nucleic acids encoding the RTMs can be included in an expression vector. Suitable expression vectors can be selected or constructed with appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as necessary. The vectors may also contain sequences such as origins of replication, promoter regions, and selectable markers, which allow their selection, expression, and replication in bacterial hosts, such as E. coli.

[0079] Preferred vectors may be directed to the cell type in which trans-splicing is required and may contain appropriate control and regulatory elements to enhance specific expression in that cell type, in some embodiments, the vectors may be non-oncolytic to avoid endogenous deleterious cis-acting effects on splicing.

[0080] Vectors can be plasmids, viruses such as phages, or phagemids, as appropriate. For further details, see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulating nucleic acids, such as preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells, and expressing genes, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds. John Wiley & Sons, 1992.

[0081] In some preferred embodiments, the expression vector may be a viral vector, such as a lentivirus or an adeno-associated virus (AAV) vector.

[0082] A viral expression vector is a recombinant nucleic acid that contains a heterologous nucleic acid encoding an RTM and a viral sequence that is expressed in a target cell. A viral expression vector can be packaged into a viral particle. A viral particle can contain a viral expression vector encapsulated in a viral capsid. Suitable methods for packaging a viral vector into a viral particle are well established in the art.

[0083] It is possible to use a single viral expression vector that encodes all the viral components necessary for viral particle formation and function, however, most often multiple plasmid expression vectors or individual expression cassettes stably integrated into host cells such as human embryonic kidney (HEK) 293 cells are utilized to separate the various genetic components that generate the viral vector particles.

[0084] Expression cassettes encoding one or more viral packaging and envelope proteins are stably integrated into mammalian cells, and transduction of these cells with the viral expression vectors described herein is sufficient to produce viral particles without the addition of additional expression vectors.

[0085] Alternatively, multiple expression vectors may be used. In some embodiments, mammalian cells may be transduced with one or more expression vectors encoding viral packaging and envelope proteins necessary for viral packaging and particle formation. For example, recombinant AAV vectors may be prepared by co-transfecting a plasmid containing heterologous nucleic acid flanked by two AAV inverted terminal repeat (ITR) regions and a plasmid carrying the AAV encapsidase gene (rep and cap genes) into a cell line infected with a human helper virus (e.g., adenovirus) or expressing its isolated essential genes. Recombinant lentiviral vectors may be prepared by transfecting a packaging cell line, such as HEK293, with a transfer vector plasmid and two or more helper plasmids. The transfer plasmid contains heterologous nucleic acid encoding the RTM, which is flanked by long terminal repeat (LTR) sequences, which facilitates integration of the transfer plasmid sequences into the host cell. The two or more helper plasmids may include one or more packaging plasmids encoding virion proteins such as Gag, Pol, Tat and Rev, and an envelope plasmid encoding an envelope protein such as VSV-G. In some embodiments, two packaging plasmids may be employed, a first packaging plasmid encoding Gag and Pol, and a second packaging plasmid encoding Rev. After transfection with the transfer plasmid and the helper plasmid, the packaging cell line produces infectious lentiviral particles that contain a nucleic acid encoding RTM. In some embodiments, VSV-G-pseudotyped lentiviral vectors may be produced in combination with the viral envelope glycoprotein G of vesicular stomatitis virus (VSV) to produce pseudotyped lentiviral particles.

[0086] Recombinant cells, e.g., recombinant mammalian cells, are provided that contain a nucleic acid encoding an RTM as described herein, or a viral expression vector that includes a nucleic acid encoding an RTM as described herein, which may be useful, e.g., in producing viral particles as described herein.

[0087] Viral particles can be harvested from cell supernatant and stored and / or concentrated for use as described herein. Many known techniques and protocols for nucleic acid manipulation and transformation, for example, in preparing nucleic acid constructs, introducing DNA into cells, and gene expression, are described in detail in Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992. Reagents for generating viral vectors are available from commercial sources (e.g., Dharmacon). Suitable techniques for preparing viral vectors are known in the art (see, for example, Dunol, T., et al (1998). J. Virol. 72, 8463-8471; Merten et al (2016) Mol Ther Methods Clin Dev. 2016; 3: 16017).

[0088] While it is possible for the RTM, nucleic acid, expression vector or viral particle described herein to be used (e.g., administered) alone, it is often preferable to provide it in the form of a pharmaceutical composition, which may contain at least one component in addition to the RTM, nucleic acid, expression vector or viral particle. Thus, a pharmaceutical composition may contain, in addition to the RTM, nucleic acid, expression vector or viral particle, a pharma- ceutical acceptable excipient, carrier, buffer, stabilizer or other material known to those skilled in the art.

[0089] The term "pharmacologically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are within the scope of sound medical judgment, suitable for use in contact with the tissues of a target subject (e.g., human) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, e.g., Remington: The Science and Practice of Pharmacy, 23rd edition, Academic Press.

[0090] Pharmaceutical compositions and formulations can be conveniently provided in unit dosage form and can be prepared by any method known in the art of pharmacy. Such methods include the step of combining the RTM, nucleic acid, expression vector, or viral particle with a carrier which constitutes one or more accessory ingredients. In general, compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier.

[0091] Pharmaceutical compositions containing RTM, nucleic acids, expression vectors or viral particles can be administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition being treated. For example, pharmaceutical compositions containing RTM, nucleic acids, expression vectors or viral particles can be administered in combination with a cytotoxic agent that is activated by a suicide protein encoded by the coding sequence of the RTM.

[0092] In some embodiments, the pharmaceutical composition may further comprise a cytotoxic agent that is activated by the suicide protein. For example, the composition may comprise a pro-form of a cytotoxic agent that is converted to an active cytotoxic agent by the suicide protein.

[0093] The RTM described herein is useful in therapy, for example, in the treatment of cancer, by selectively killing cells expressing HERV genes. The RTM, nucleic acid, expression vector, viral particle or pharmaceutical composition described herein can be used in a method of treatment of the human or animal body. The method of treatment may include administering the RTM, nucleic acid, expression vector, viral particle or pharmaceutical composition to an individual in need thereof. Therapeutic applications of RTM are established in the art (see Hong et al (2020) Br Med Bull 136 4-20). Preferably, the method is a method of treatment of cancer. The method of treatment of cancer described herein may include administering the RTM, nucleic acid, vector, viral particle or pharmaceutical composition described herein to an individual in need thereof.

[0094] The method may further comprise administering to the individual a cytotoxic agent that is activated by the suicide protein encoded by the coding sequence of the RTM. For example, the individual may be administered a pro-form of the cytotoxic agent that is converted to an active cytotoxic agent by the suicide protein.

[0095] Another aspect provides an RTM, a nucleic acid, a vector, a viral particle or a pharmaceutical composition described herein for use in a method for treating cancer, and the use of an RTM, a nucleic acid, a vector, a viral particle or a pharmaceutical composition described herein in the manufacture of a medicament for use in a method for treating cancer.

[0096] Cancers suitable for treatment as described herein may be characterized by the presence of one or more cancer cells expressing a HERV gene. In some preferred embodiments, one or more cancer cells may express a HERV-K gene, such as a HERV-K HML-2 env gene. For example, the cancer cells may express a HERV-K HML-2 group 1 Np9 gene and / or a HERV-K HML-2 group 1 Rec gene.

[0097] Suitable cancers may be familial or sporadic. Suitable cancers may be metastatic or non-metastatic.

[0098] For example, the cancer may be any type of solid or non-solid cancer, or malignant lymphoma. melanoma), head and neck cancer, kidney cancer, sarcoma, germ cell cancer (e.g. teratocarcinoma), liver cancer (e.g. hepatocellular carcinoma), lymphoma (e.g. Hodgkin's or non-Hodgkin's lymphoma), acute myeloid or myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CML), chronic myeloid or myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (P-TLL), large granular lymphocytic leukemia, adult T-cell leukemia, leukemia, skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, esophageal cancer, pancreatic cancer (such as pancreatic ductal adenocarcinoma), stomach cancer, brain cancer, preferably ovarian cancer, colon cancer, breast cancer, melanoma, leukemia, testicular cancer, and prostate cancer.

[0099] In some preferred embodiments, the cancer may be a liver cancer, such as hepatocellular carcinoma, or a pancreatic cancer, such as pancreatic ductal adenocarcinoma.

[0100] In some embodiments, the cancer for treatment as described herein may be previously identified as expressing a HERV gene or expressing a HERV gene above a threshold. In another embodiment, the method may include identifying the cancer as expressing a HERV gene or expressing a HERV gene above a threshold and treating the cancer as described herein. The method for selecting an individual with a cancer likely to respond to treatment with RTM as described herein includes: determining expression of HERV genes in a sample obtained from the individual, Expression of the above HERV genes indicates that an individual is likely to respond to treatment.

[0101] The sample may be a sample of cancer cells, or a sample of blood or other biological fluid in the individual that contains cell-free nucleic acid derived from cancer cells.

[0102] Suitable techniques for determining the expression of HERV genes are well known in the art.

[0103] An individual suitable for such treatment may be a mammal, such as a rodent (e.g., guinea pig, hamster, rat, mouse, etc.), murine (e.g., mouse), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), primate, ape (e.g., monkey or ape), monkey (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon, etc.), or human.

[0104] In some preferred embodiments, the individual is a human. In other preferred embodiments, a non-human mammal can be used, particularly a mammal that is traditionally used as a model for demonstrating therapeutic efficacy in humans (e.g., a mouse, a primate, a pig, a dog, or a rabbit animal).

[0105] Administration is typically in a "therapeutically effective amount" or a "prophylactically effective amount" that is sufficient to show benefit to the patient. Such benefit may be at least an amelioration of at least one symptom. The actual amount administered, rate of administration, and time-course of administration will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration, and other factors known to the physician.

[0106] The RTMs, nucleic acids, expression vectors, viral particles or pharmaceutical compositions described herein may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the circumstances of the individual being treated.

[0107] Prescribing treatment, e.g., determining dosage, is within the responsibility of the attending physician and other physicians and may depend on the severity of symptoms and / or progression of the disease being treated. Appropriate dosages of therapeutic polypeptides are well known in the art (Ledermann JA et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe KD et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages are described herein or in the Physician's Desk Reference (2003), and appropriate amounts can be used depending on the type of pharmaceutical agent being administered. The therapeutically effective amount or appropriate dosage of the RTM, nucleic acid, expression vector, viral particle or pharmaceutical composition described herein can be determined by comparing its in vitro activity and in vivo activity in animal models. Methods are known for extrapolating effective dosages in mice and other test animals to humans. The exact dose will depend on many factors, including whether the RTM, nucleic acid, expression vector, viral particle or pharmaceutical composition described herein is for prophylaxis or treatment, the size and location of the area to be treated, and the exact nature of the RTM.

[0108] Treatments may be repeated at daily, twice-weekly, weekly, or monthly intervals, at the discretion of the physician. The treatment schedule for an individual may depend on the immunological, pharmacokinetic, and pharmacodynamic properties of the RTM, nucleic acid, expression vector, viral particle, or pharmaceutical composition, the route of administration, and the nature of the condition being treated.

[0109] Following treatment with the RTM, nucleic acid, expression vector, viral particle or pharmaceutical composition, treatment with the activatable cytotoxic agent may be periodic, with the administration interval being about one week or more, e.g., about two weeks or more, about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment may be administered every 2-4 weeks or every 4-8 weeks. This may be useful, for example, to selectively kill cells that have become cancerous after initial treatment, preventing or reducing the risk of recurrence.

[0110] Treatment with the RTM, nucleic acid, expression vector, viral particle or pharmaceutical composition and / or pro-form of a cytotoxic agent can occur before and / or after surgery and / or can be administered or applied directly to the anatomical site of trauma, surgical treatment or invasive procedure. Suitable formulations and routes of administration are described above.

[0111] The RTM, nucleic acid, expression vector, viral particle or pharmaceutical composition may be administered in combination with a cytotoxic agent that is activated by a suicide protein encoded by the coding sequence of the RTM. The pro-form of the cytotoxic agent that can be converted to an active agent by the suicide protein can be readily administered to the patient using standard medical approaches. Methods known in the art can also be used to determine the most appropriate dose and route for administration of the pro-form. For example, ganciclovir can be administered systemically (e.g., orally or parenterally) at a dose of about 1-20 mg / kg body weight / day, acyclovir can be administered at a dose of about 1-100 mg / kg body weight / day, and fiau can be administered at a dose of about 1-50 mg / kg body weight / day.

[0112] As used herein, the terms "cancer," "neoplasm," and "tumor" are used interchangeably and refer, in either singular or plural, to cells that have undergone a malignant transformation that becomes pathological to the host organism.

[0113] Primary cancer cells can be easily distinguished from non-cancerous cells by well-established techniques, especially histological examination. The definition of cancer cells used herein includes not only primary cancer cells, but also any cells derived from cancer cell ancestors. This includes metastasized cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that usually appear as solid tumors, a "clinically detectable" tumor is one that can be detected based on tumor mass, for example, by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound, or palpation during physical examination.

[0114] Cancer cells that express HERV genes and are selectively targeted by the RTMs described herein are more susceptible and sensitive than other cells to treatment with cytotoxic agents because they are exposed to the active cytotoxic agent resulting from the conversion of the pro-form by the suicide protein. Furthermore, the activated cytotoxic agent can passively diffuse to neighboring cancer cells, further enhancing cancer cell death. This "bystander effect" enhances the efficacy of the treatment.

[0115] An individual with cancer may show at least one distinguishable sign, symptom, or examination finding that is sufficient to diagnose cancer according to clinical criteria known in the art.Examples of such clinical criteria can be found in medical textbooks such as Harrison's Principles of Internal Medicine, 15th Ed., Fauci AS et al., eds., McGraw-Hill, New York, 2001.In some cases, diagnosing cancer in an individual may include identifying a specific cell type (e.g., cancer cell) in a sample of body fluid or tissue obtained from the individual.

[0116] Treatment can be any treatment or therapy, whether in humans or animals (e.g., veterinary uses), that achieves some desired therapeutic effect, e.g., inhibiting or slowing the progression of a condition, including slowing the rate of progression, halting the rate of progression, ameliorating the condition, curing or remission (whether partial or complete) of the condition, preventing, slowing, alleviating or arresting one or more symptoms and / or signs of the condition, or prolonging the survival of a subject or patient beyond that expected in the absence of treatment.

[0117] In particular, treatment may include inhibition of cancer growth, including complete remission of cancer, and / or inhibition of cancer metastasis. Cancer growth generally refers to any one of a number of indicators that indicate a change in cancer to a more developed form. Thus, indicators for measuring inhibition of cancer growth include a reduction in cancer cell viability, a reduction in tumor volume or morphology (e.g., determined using computed tomography (CT), ultrasound, or other imaging methods), slowing of tumor growth, or destruction of tumor vasculature. Administration of the RTM, nucleic acid, vector, viral particle, or pharmaceutical composition described herein may improve an individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and / or reduce the tendency of cancer growth in the individual.

[0118] The RTMs described herein may also be useful for selectively killing cells expressing HERV genes and preventing the onset or recurrence of cancer in individuals undergoing cell therapy, i.e., individuals receiving autologous or allogeneic cells for the treatment of a condition such as cancer. The cells of an individual may express HERV genes if they are cancerous or in the process of becoming cancerous. These cells can be killed as described herein before a cancerous condition occurs in the individual. For example, a method of preventing the onset or recurrence of cancer in an individual to whom a population of cells has been administered includes administering the population of cells to an individual in need thereof, wherein the cells comprise an RTM, a nucleic acid, an expression vector, or a viral particle described herein. A cytotoxic compound activated by the suicide protein is then administered to the individual, resulting in activation of the cytotoxic compound in the cells of the population that have become cancerous or are becoming cancerous in the individual.

[0119] Cells suitable for use in cell therapy are well known in the art and can include, for example, hematopoietic cells such as T cells, hematopoietic stem cells and bone marrow cells.

[0120] The RTMs, nucleic acids, vectors, viral particles or pharmaceutical compositions described herein may be useful in delivering a nucleic acid encoding a suicide protein in vitro or ex vivo, for example for use in killing HERV gene expressing cells. Methods for delivering a nucleic acid encoding a suicide protein to a cell in vitro include: This may involve introducing an RTM, nucleic acid, vector or viral particle as described herein into a cell, such that expression of the HERV gene in the cell causes expression of the suicide protein.

[0121] The cell may express the HERV gene or may undergo a transformation event that results in the expression of the HERV gene. The RTM in the HERV gene-expressing cell may be trans-spliced ​​into the HERV pre-mRNA to form a chimeric mRNA molecule that includes a nucleotide sequence encoding a suicide protein. The chimeric mRNA may be further processed and translated such that the suicide protein is expressed in the cell. The cell may be contacted with a cytotoxic agent that is activated by the suicide protein, such that the cytotoxic agent is activated by the suicide protein and kills the cell.

[0122] The methods described herein may be useful for killing cells in vitro or ex vivo. Methods for killing cells in vitro or ex vivo include: Introducing a cell with the RTM, nucleic acid, vector or viral particle described herein, Expression of HERV genes in cells leads to the expression of suicide proteins, contacting the cells with a cytotoxic agent that is activated by the suicide protein; and activating the cytotoxic agent via the suicide protein following expression of the HERV gene in the cell.

[0123] The methods described herein can be useful for depleting HERV gene-expressing cells in a cell population. For example, a method for depleting HERV gene-expressing cells in a population includes: introducing an RTM, nucleic acid, vector or viral particle as described herein into a cell population, such that the suicide protein is expressed in HERV gene-expressing cells in the cell population; contacting the population with a cytotoxic compound that is activated by the suicide protein, thereby activating the cytotoxic compound in HERV gene-expressing cells in the population, thereby depleting HERV gene-expressing cells in the population.

[0124] The HERV gene-expressing cells can include cancer cells or precancerous cells. The methods described herein can be useful for depleting or removing cancer cells or precancerous cells from a cell population.

[0125] The population of cells may be a sample of cells derived from or obtained from an individual, or cultured cells derived therefrom. Suitable cells include mammalian, preferably human, cells. For example, suitable cells may include cells for use in cell therapy, such as T cells, hematopoietic cells, such as hematopoietic stem cells and bone marrow cells.

[0126] The RTM, nucleic acid, vector or viral particle may be introduced into cells in vitro or ex vivo by any convenient technique such as transfection, lipofection, transduction, electroporation, nucleofection or transformation.

[0127] Other aspects and embodiments of the present invention provide for replacing the term "comprising" in the above-mentioned aspects and embodiments with the term "consisting of," as well as for replacing the term "comprising" in the above-mentioned aspects and embodiments with the term "consisting essentially of."

[0128] As used herein, the term "downstream" refers to the 5' to 3' direction of the nucleic acids described herein, and the term "upstream" as used herein refers to the 3' to 5' direction of the nucleic acids described herein.

[0129] Reference to a nucleotide sequence set forth herein includes DNA molecules having the particular sequence, and includes RNA molecules having the particular sequence in which U is substituted for T, unless the context requires otherwise.

[0130] It is to be understood that the present application discloses any and all combinations of the above aspects and any of the above-described embodiments with each other, unless the context requires otherwise.Similarly, the present application discloses any and all combinations of preferred and / or optional features, either alone or in combination with any of the other aspects, unless the context requires otherwise.

[0131] Modifications of the above embodiments, further embodiments, and modifications thereof will be apparent to those of skill in the art upon reading this disclosure, and therefore are within the scope of the invention.

[0132] All documents and sequence database entries referred to herein are incorporated by reference in their entirety for all purposes.

[0133] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, without regard to the presence or absence of the other. For example, "A and / or B" should be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein. EXAMPLES

[0134] The described RNA trans-splicing invention uses a universal binding domain sequence to target the env intron and thereby target multiple Np9 encoding class I proviruses, or Rec from class II proviruses. As a result of the RNA trans-splicing reaction, an optimized HSV-tk gene is expressed to eliminate aberrant HSV-tk isoform production and the resulting undesirable off-target effects.

[0135] Materials and Methods Generation of RNA trans-splicing binding domains The Np9 target RNA trans-splicing binding domain was generated from the following endogenous retroviral sequence, Homo sapiens isolate HML-2_22q11.21 endogenous virus HERV-K, complete sequence (GenBank: JN675087.1), based on a previously described method (Ingemarsdotter, C K et al (2017) Mol. Ther. Nucleic acids, 7, 140-154). Briefly, minimum free energy (MFE) calculations of the reverse complement of the target sequence were performed with a window size of 50 nucleotides and a step size of 1 using the Foldanalyze software within the Bioinformics HUSAR server (German Cancer Research Center). The HML-2_22q11.21 sequence (GenBank: JN675087.1) was subjected to splice site prediction using the Neural Network Server in the Berkeley Drosophila Genome Project and the CrypSkip software in the Bioinformatics HUSAR server. Based on high minimum free energy and unpaired nucleotides near and downstream of the Np9 splice donor site, potential binding domain sequences were selected with Foldanalyze for further RNA secondary structure prediction (Armbruester, V. et al (2002). Clin. Cancer. Res. 8, 1800-1807).

[0136] The RNA secondary structure of the selected potential binding domain regions was predicted using the web servers Mfold (Zuker, M., (2003) Nucleic acids research, 31, 3406-3415 and RNAfold). The selected binding domain regions were refolded in an RNA trans-splicing cassette backbone to exclude long-range interactions of the binding domains. The binding domain structure was optimized by introducing wobble base pairs (C-U or A-G modifications) to reduce duplex formation. To prevent effects caused by long double-stranded RNA, two mismatched nucleotides were introduced as previously described (Chalupnikova, K. et al (2013). Methods Mol. Biol. 942, 291-314).

[0137] In addition, the Bbv CI restriction enzyme site and adjacent nucleotides flanking the binding domain in the RNA trans-splicing cassette are [ka] from [ka] to destroy potential splice acceptor sites identified by splice site prediction using the Neural Network Server in the Berkeley Drosophila Genome Project. After selecting promising binding domain structures and optimizing the binding domain structures, the RNA trans-splicing binding domain and the flanking regions (between NheI and MluI restriction enzyme sites) in the RNA trans-splicing binding cassette sequence were analyzed for potential splice donor and splice acceptor sites by splice site prediction using the Neural Network Server in the Berkeley Drosophila Genome Project. After any mutations were introduced, to avoid potential splice donor and splice acceptor sites, the binding domain and the flanking regions (NheI and MluI) were reanalyzed by splice site prediction using the Neural Network Server in the Berkeley Drosophila Genome Project to confirm that the introduced mutations did not introduce novel splice donor and splice acceptor sites.

[0138] Generation of HSV-tk translation initiation mutants The Np9 target binding domain and flanking sequences were synthesized by GeneArt Synthesis with flanking NheI and MluI restriction enzyme sites (Life Technologies). The insert was cloned into the CMV promoter-driven BD100-HSVtk and BD100-HSVtk opt2 pVAX-1 backbones using NheI and MluI restriction enzyme (Thermo Scientific) digestion and ligation, replacing the HIV target binding domain BD100 region (Ingemarsdotter et al (2017) Mol. Ther. Nucleic Acids, 7, 140-154). The BD100 and BD72 binding domain regions correspond to the binding domain regions BD1-D4 and BD2- D4, respectively, as described by Ingemarsdetter et al. For the HIV-targeting RNA trans-splicing construct CkRhsp-BD72-pVAX-1, 10 ng of the plasmid CkRhsp-BD72-pVAX-1 as a template and the site-directed mutagenesis primers were used to generate the HSV-tk ATG by site-directed mutagenesis using a QuickChange II XL site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer's instructions. 46 and A.T.G. 60 We generated HSV-tk translation initiation mutants in Forward: 5'-GAAACTGCCCACGCTACTGCGGGTTTATATAGACGGTCCCCACGGGATCGGG-3' Reverse: 5'-CCCGATCCCGTGGGGACCGTCTATATAAACCCGCAGTAGCGTGGGCAGTTTC-3'.

[0139] Second ATG, ATG 46 Mutagenesis of the HSV-tk ATG was confirmed by sequencing, and 10 ng of the resulting plasmid DNA was used as template in a second round of mutagenic PCR to amplify the third HSV-tk ATG, ATG 60 was mutated. Forward; 5'-ACGGTCCCCACGGGATCGGGAAAACCAC-3', and tktr 3 mut Reverse: 5'-GTGGTGGTTTTCCCGATCCCGTGGGGACCGT-3'

[0140] Phusion HF DNA polymerase and 5x Phusion HF reaction buffer (New England Biolabs), 125 ng of forward and reverse mutagenesis primers, 1 μl of dNTPmix (Agilent Technologies), and 3 μl of Quicksolution (Agilent Technologies) were used. Mutagenesis PCR conditions: -95°C for 1 min, followed by 18 cycles of 95°C for 50 s, 60°C for 50 s, 68°C for 4 min, followed by 68°C for 7 min. PCR products were digested with Dpn1 for 1 h at 37°C and transformed into XL10-Gold ultracompetent cells (Agilent Technologies). The resulting HSV-tk opt domain was subcloned into the ChkRhsp-BD100 opt1-pVAX-1 backbone using PstI and MluI restriction digestion, ligation, and subcloning to generate ChkRhsp-BD100 opt2-pVAX-1. The ChkRhsp promoter was replaced with the CMV promoter in the ChkRhsp-BD100 HSV-tk opt2-pVAX-1 and BD100-HSV-tk-pVAX-1 backbones using SpeI and NheI restriction enzyme digestion, ligation and subcloning.

[0141] The Np9 target binding domain and flanking sequences were synthesized by GeneArt Synthesis with flanking NheI and MluI restriction enzyme sites (Life Technologies). The insert was cloned into the CMV-BD100-HSVtk and CMV-BD100-HSVtk opt2 pVAX-1 backbones using NheI and MluI restriction enzyme (Thermo Scientific) digestion and ligation, replacing the HIV target binding domain BD100 region described in Ingemarsdotter et al (2017) with the Np9 target binding domain.

[0142] Generation of RNA trans-splicing pVAX-1 shuttle plasmid To facilitate subcloning of the RNA trans-splicing cassette from pVAX-1 into the third-generation lentiviral gene transfer plasmid pSico (Addgene, ref: 11578), restriction enzyme sites XbaI and XhoI were introduced adjacent to the HIV-targeting RNA trans-splicing cassette, XbaI at the 5' end and XhoI at the 3' end upstream of the polyA tail within the RNA trans-splicing cassette within the CkRhsp-BD100-opt2 pVAX-1 backbone (Ingemarsdotter et al. (2017)), using the QuickChange XL site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer's instructions and with 10 ng of plasmid CkRhsp-BD100-opt2-pVAX-1 as template.

[0143] In the first mutagenic PCR, the following mutagenic primers were used to introduce an XbaI restriction enzyme site at the 5′ end of the RNA trans-splicing cassette; 5'XbaI forward;5'-GACATTGATTATTGTCTAGAACTAGTTGAGCCCCACG-3'. 5'XbaI Reverse;5'-CGTGGGGCTCAACTAGTTCTAGACAATAATCAATGTC-3'.

[0144] The PCR products were digested with DpnI for 1 h at 37°C and transformed into XL-10Gold ultracompetent cells (Agilent Technologies). Positive clones were confirmed by sequencing and used as templates for a second round of mutagenic PCR to introduce a XhoI site at the 3' end within the RNA trans-splicing cassette upstream of the polyA tail. The primers used for the second round of mutagenic PCR were as follows; 3'XhoI forward; 5'-GGGAGGCGAACTGACTCGAGAACTTGTTTATTGC-3' and 3'XhoI reverse; 5'-GCAATAAACAAGTTCTCGAGTCAGTTCGCCTCCC-3'.

[0145] The Np9 target trans-splicing domain was cloned into pVAX-1 shuttle 5'XbaI-BD100-opt1 and 5'XbaI-BD100-opt2 plasmids using digestion and ligation with SpeI and PvuI restriction enzymes. The resulting plasmids, HSVtk-ts-pVAX shuttle and HSVtk-ts-opt-pVAX shuttle, were digested with XbaI, XhoI and NsbI and ligated into the pSico lentiviral backbone at the XbaI and XhoI restriction enzyme sites to isolate the Np9 target RNA trans-splicing cassette between XbaI and XhoI.

[0146] Amplification of the thyroxine-binding globulin promoter The thyroxine-binding globulin promoter (TBG) was amplified by PCR from the plasmid pAAV.TBG.PI.Null.bGH (Addgene plasmid: 105536) using the following forward primers with flanking BcuI restriction enzyme sites: TG BcuI forward, [ka] and a reverse primer with an adjacent NheI restriction enzyme site TG NheI reverse, [ka]

[0147] 20 ng of plasmid pAAV.TBG.PI.Null.bGH (Addgene plasmid: 105536) was used as template in a PCR reaction containing 1x GoTaq reaction buffer (Promega), 500 nM forward primer and 500 nM reverse primer, 200 μM PCR nucleotide mix (Promega), 1.25 U GoTaq G2 DNA polymerase in a final reaction volume of 50 μl. PCR cycle conditions were 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, followed by a 72°C for 5 min and a 4°C hold. The TBG promoter was cloned into -HSVtk-ts-pVAX shuttle and HSVtk-ts-opt-pVAX shuttle plasmids to replace the CMV promoter using BcuI and NheI double digestion and cloning. The resulting TBG-HSVtk-ts-pVAX shuttle and TBG-HSVtk-ts-opt-pVAX shuttle were digested with XbaI, XhoI and NsbI and ligated into the pSico lentiviral backbone to isolate the Np9 target RNA trans-splicing cassette between XbaI and XhoI.

[0148] cell culture Hep3B (ATCC), HuH-7 (JCRB Cell Bank) engineered to express mRFP (HuH-7-mRFP), HEK 293T, MIA PaCa-2 (PHE(ECACC) and Panc-1 cells (PHE(ECACC) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco). Aspc-1 cells (ATCC) were cultured in RPMI medium (Sigma) supplemented with 10% FBS and 1% penicillin-streptomycin. All cells were maintained at 37 °C in a humidified 5% CO2 atmosphere.

[0149] Western blot HEK 293T cells were cultured at 1 x 10 in 1 mL / well of supplemented DMEM. 5Cells were seeded in 24-well plates at a density of 1000 cells / well. After 24 hours, cells were transfected with 0.5 μg of plasmids (pSico[CMV-GFP], pS-HSV-tk-ts, pS-HSV-tk-ts-opt, pS-CMV-HSV-tk). Three days after transfection, cells were washed with phosphate-buffered saline (PBS; Sigma) and harvested using 1x cell culture lysis reagent (Promega) and incubated at room temperature for 15 min. Lysates were then spun at 12000 rpm for 2 min. Supernatants were collected and treated with protease inhibitor cocktail (Halt). Protein concentrations were quantified using bovine serum albumin (BSA; Sigma Aldrich) standards and Bradford dye reagent (Bio-Rad). Then, β-mercaptoethanol-containing loading dye was added to 5 μg of protein and boiled at 95 °C for 5 min. The prepared samples were electrophoresed on a 10% SDS-PAGE gel and transferred to nitrocellulose paper using transfer buffer (25 mM Tris base, 150 mM glycine, 10% ethanol) at 100 V for 1 h. The membrane was blocked with blocking buffer (BSA 4%, Tween 0.05% in PBS) for 1 h at room temperature and incubated with goat anti-HSV-1 thymidine kinase antibody vN-20 (1:5000; Santa Cruz Biotechnology sc28037) in staining solution (BSA 1.5%, Tween 0.05% in PBS) overnight at 4°C. The membrane was then washed three times with PBS-0.05% Tween, incubated with rabbit anti-goat HRP-conjugated antibody (1:2000 Dako P0160) in staining solution for 1 h at room temperature, and finally washed three more times with PBS-0.05% Tween. Stained membranes were developed with ECL Western blot substrate (Promega) and visualized on high-contrast blue-sensitive X-ray membranes. Blots were then washed twice with PBS, incubated in stripping buffer (Thermo Scientific) for 15 min, and washed an additional three times with PBS.Gels were restained with rabbit anti-vinculin antibody (1:5000; Invitrogen 700062) and goat anti-rabbit HRP-conjugated secondary antibody (1:5000; Invitrogen 656120) and visualized with ECL Western blot substrate. Western blot quantification was completed using Fiji software (https: / / imagej.net / Fiji).

[0150] MTT cell viability assay Cell viability was assessed based on a previously established method (Pannecoque et al. (2008). Nature protocols, 3, 427-434). Briefly, on day 1, Hep3B, HuH7-mRFP, HEK 293T, MIA PaCa-2, Panc-1, and Aspc-1 cells were seeded at 2 × 10 4Cells were seeded at a density of 100 cells / well in 100 μl medium / well. On day 2, HEK 293T, MIA PaCa-2, Panc-1, and Aspc-1 cells were transduced with lentivirus (Lv-CMV-GFP, Lv-HSV-tk-ts, Lv-HSV-tk-ts-opt, Lv-CMV-HSV-tk) at an MOI of 1 or 2. On day 4, cells were treated with 1000 μM GCV (Sigma). Hep3B or HuH7-mRFP cells were transduced with lentiviral vectors; pSico (negative control), trans-splicing vectors, 3'ER-HSVtk, 3'ER-HSVtk opt, or HSVtk (positive control) at an MOI of 1. Hep3B and HuH7-mRFP cells were treated with GCV at various concentrations on days 3 and 4. Finally, on day 8, the tetrazolium dye MTT (3-[4,5-dimethyl-2-thiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (Sigma) was added to the medium in the wells to a final concentration of 10% (0.5 mg / ml) and incubated at 37°C for 2–2.5 h. The medium was then removed and replaced with an acidified isopropanol solution of Triton X-100 (0.04 N HCl, 6% Triton X-100) and incubated at room temperature for 15 min. Absorbance was measured at both 595 nm and 655 nm in an iMark Microplate Absorption Reader (Bio-Rad). Background fluorescence readings at 655 nm were subtracted from the readings at 595 nm. Cell viability was calculated by setting the mean fluorescence of all untreated wells on each plate to 100%.

[0151] Production of lentiviral vectors Lentiviral vectors were produced as previously described (Dull et al (1998) Journal of Virology, 72, 8463-8471) with the addition of benzonase treatment to eliminate plasmid carryover. Briefly, HEK 293T cells were cultured at 5 × 10 6Cells were seeded in 10 cm dishes at a density of 10 cells / plate. After 24 hours, cells were transfected with 10 μg of transfer plasmids (pSico, pS-HSV-tk-ts, pS-HSV-tk-ts-opt, pS-CMV-HSV-tk) or (3'ER-HSVtk, 3'ER-HSVtk opt, or TBG-HSVtk driven by the TBG promoter in the pSico plasmid backbone (positive control)), 6.5 μg of pMDLg / pRRE (Gag and Pol expressing packaging plasmid), 2.5 μg of pRSV-REV (Rev expressing packaging plasmid), and 3.5 μg of VSV-G envelope expressing plasmid) using TransIT-LT1 transfection reagent (Mirus) as described previously (Dull et al. (1998) supra). 24 hours after transfection, the medium was removed and replaced with fresh DMEM. 48 hours after transfection, the medium was collected and centrifuged at 3000 rpm for 10 minutes to remove cell debris. The supernatant was collected, treated with 50 U / mL benzonase (Sigma) and incubated at 37°C for 30 minutes. To remove remaining debris, the supernatant was then passed through a 0.45 μm detergent-free cellulose acetate syringe filter (Sartorius Minisart) into an appropriate ultracentrifuge tube, weighed, and centrifuged at 20,000×g for 90 minutes at 4°C. The supernatant was removed from the tube and the lentiviral pellet was resuspended in 500 μl of PBS.

[0152] Calculation of infectious titers using qPCR Briefly, HEK 293T cells were cultured at 2 × 10 in 2 mL of DMEM. 5Cells were seeded in 12-well plates at 1000 ng / well. After 24 h, cells were transduced in duplicate wells with 1 μL, 5 μL, or 10 μL of each lentiviral vector (Lv-CMV-GFP, Lv-HSV-tk-ts, Lv-HSV-tk-ts-opt, Lv-CMV-HSV-tk). Three days after transduction, DNA from one well was harvested from each transduction using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's instructions, extending the incubation time to 30 min at 56 °C. The remaining cells were harvested 6 days after transformation as described above. psi copy numbers were determined by amplifying 100 ng of genomic DNA during psi qPCR quantification using 20 nM fwd / rev primers and SYBR Green PCR master mix (Applied Biosystems) in parallel with a standard curve based on serial dilutions of pSico plasmid DNA. For albumin qPCR quantification, 100 ng of genomic DNA was amplified using 100 nM fwd / rev primers and SYBR Green PCR master mix (Applied Biosystems), and albumin copy number was measured in parallel with a standard curve based on HEK 293T genomic DNA and used to estimate cell number.

[0153] Both used the following qPCR cycling protocol: an initial temperature ramp of 50°C for 2 min and 95°C for 20 s, 40 cycles of 95°C for 3 s and 60°C for 30 s, and a final melting curve with temperature cycling between 95°C and 60°C (95°C for 15 s, 60°C for 1 min, 95°C for 15 s and 60°C for 15 s). Infectivity titers were determined based on previously described methods (Kutner, RH et al (2009) Nature Protocols 4 495-505). Psi and albumin primers previously designed by Charrier et al. (2007) Gene Therapy 14 415-428. Psi-Fwd5'-CAGGACTCGGCTTGCTGAAG-3', Psi-Rev5'-TCCCCCGCTTAATACTGACG-3' Albumin-Fwd5'-GCTGTCATCTCTTGGGCTGT-3, Albumin-Rev 5'-ACTCATGGGAGCTGCTGGTTC-3'.

[0154] Lentiviral vectors pSico (negative control), 3'ER-HSVtk, 3'ER-HSVtk opt, or TBG promoter-driven HSVtk (positive control) were titrated in HEK293T cells to measure infectious titers. Genomic DNA was isolated as described above 3 days after transduction with 5 μl or 10 ul of lentiviral vector / well in triplicate wells, and qPCR reactions were performed using 20 ng genomic DNA as template, 20 nM WPRE-Fwd and 20 nM WPRE-Rev primers, and 2x TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific).

[0155] (WPRE-Fwd 5'-GGCACTGACAATTCCGTGT-3', WPRE-Rev 5'-AGGGACGTAGCAGAAGGACG-3') and 100 nM WPRE probe 5'6FAM-ACGTCCTTTCCATGCTGCTCGC-TAM3'. WPRE primer and probe sequences previously designed by Charrier et al.(2007)Gene Therapy 14 415-428. WPRE copy number was determined using a standard curve of serially diluted pSico plasmid DNA. qPCR cycling conditions were 50°C for 2 min, 95°C for 20 s, 95°C for 3 s, and 60°C for 30 s repeated for 40 cycles. Albumin primers were used in qPCR reactions using 100 ng of genomic DNA as template to estimate cell number by comparison with a standard curve of serially diluted genomic DNA isolated from HEK 293T cells.

[0156] statistical analysis Statistical analysis for Figures 5 and 7 was performed using Prism 7 (Graphpad software). Expression levels of HSV-tk P1, P3 proteins were compared using a two-tailed unpaired t-test, assuming parametric distribution. In Figure 7, all MTT cell viability data were compared using a two-way analysis of variance (ANOVA) with Tukey's post hoc multiple comparison test. In Figure 6, statistical analysis was performed using Student's t-test (Excel), assuming unequal variances. All p-values ​​less than 0.05 were considered significant. All replicates used in statistical analysis are biological replicate wells.

[0157] result To target HCC or PDAC, we generated RNA trans-splicing constructs with a novel binding domain directed to the HERV-K env pre-mRNA containing an incomplete herpes simplex virus thymidine kinase gene (Figure 1). To generate a preferred structure and avoid potential RNA editing, the binding domain sequence contained four wobble bases (C~U and A~G), or two mismatches (2xA~U) (Chalupnikova et al (2013) Methods in Molecular Biology, 942, 291-314). Sequence alignment showed 89% complementarity to the HERV-K target sequence (Genbank sequence number JN675087.1) (Figure 2D).

[0158] RNA secondary structure predictions of isolated binding domains, when analyzed by RNA fold, showed similar structures with many unbound nucleotides in either minimum free energy (MFE) or centroid RNA secondary structure predictions (Figure 3A). To confirm that the binding domain contains unstructured regions within the RNA trans-splicing cassette backbone, the RNA binding domain was folded in the context of the RNA trans-splicing cassette. MFE and centroid folding confirmed stretches of up to 18 unbound nucleotides when folded within the RNA trans-splicing cassette (Figure 3B).

[0159] To further improve the RNA trans-splicing approach, Np9 targeting was combined with mutations within the RTM to reduce potential off-target splicing in cis (Figure 4B, top) and aberrant HSV-tk peptide production from the second (ATG46) and third (ATG60) canonical HSV-tk translation initiation codons (Figure 4B, bottom).

[0160] To examine the effect of HSV-tk translation initiation mutants on HSV-tk isoform production, the Np9-targeted RNA trans-splicing construct HSV-tk-ts opt was tested in transfection studies in HEK293T cells against HSV-tk-ts, which lacks the first ATG start codon of HSV-tk and carries wild-type ATG46 and ATG60 (Figure 5A), along with HSV-tk containing all the canonical translation initiation sites of wild-type HSV-tk as a positive control, alongside a negative control construct expressing GFP from the CMV promoter. Translation products generated from the different HSV-tk translation initiation sites are shown in Figure 5B, and predicted HSV-tk isoforms generated from the different constructs are shown in Figure 5C. Western blot analysis confirmed similar levels of P1 production when comparing HSV-tk-ts and HSV-tk-ts-opt (Figure 5D), suggesting successful trans-splicing followed by production of HSV-tk P1 from the Np9 translation initiation codon. Quantification of the bands normalized to the level of vinculin (loading control) confirmed this observation (P<0.2877) (Figure 5E). In contrast, the expression level of P3 was significantly reduced after transfection with HSV-tk-ts-opt compared to HSV-tk-ts, where P3 isoform expression is overexpressed over wild-type HSV-tk (Figure 5D). Quantification showed an 18-fold reduction in P3 expression in cells transfected with HSV-tk-ts-opt compared with HSV-tk-ts (P = 0.0047), confirming that the use of the optimized RNA trans-splicing construct HSV-tk-ts-opt reduced the aberrant production of HSV-tk peptides.

[0161] Trans-splicing lentiviral vectors induce cell death in liver and pancreatic cancer cell lines To investigate the effect of Np9-targeting RNA trans-splicing lentiviral vectors on cell viability, we evaluated the effect by MTT assay and compared vector-transduced cells before and after GCV treatment for each vector type. In hepatocellular carcinoma cell line Hep3B, a significant decrease in cell viability was observed after transduction with the trans-splicing vector 3'ER-HSV-tk, with 25.54 ± 0.48% (P < 0.001) surviving cells after two doses of GCV at a concentration of 300 μM, comparable to the HSV-tk positive control vector, where 21.67 ± 2.21% (P < 0.01) of cells survived after GCV administration. With the tk-optimized trans-splicing vector, 3'ER-HSV-tk-opt, the viability was significantly decreased to 46.01 ± 4.39% (P < 0.05) (Figure 6A).

[0162] A similar trend was observed in HuH7-mRFP cells, where in cells transduced with the trans-splicing vectors 3'ER-HSV-tk or 3'ER-HSV-tk-opt, cell viability after GCV treatment was reduced to 41.33 ± 6.11% (P < 0.01) or 64.96 ± 1.77% (P < 0.001), respectively. In HuH7-mRFP cells transduced with the HSV-tk positive control vector, 24.67 ± 4.73% (P < 0.001) of cells remained viable after GCV addition (Figure 6B).

[0163] In Hep3B cells, a significant decrease in cell viability was observed when GCV concentration was reduced to two doses, 10uM or 100uM, with viable cell remaining at 20.50±5.5% (P<0.05) for 3'ER-HSV-tk and 39.58±2.07% (P<0.01) for 3'ER-HSV-tk-opt in combination with 10uM GCV treatment. Furthermore, two doses of 100uM GCV reduced cell viability to 7.15±3.3% (P<0.01) for 3'ER-HSV-tk and 14.62±3.38% (P<0.05) for 3'ER-HSV-tk-opt. At these GCV concentrations, 19.55 ± 2.90% (P < 0.05) and 5.24 ± 1.71% (P < 0.05) of cells survived transduction with the HSV-tk positive control vector in combination with 10 μM or 100 μM, respectively (Figure 6C).

[0164] Next, the Np9-targeting RNA trans-splicing lentiviral vector was tested in HEK 293T cells, which were transduced at MOIs of 1 and 2 and treated with 1000 μM GCV, and then analyzed for cell viability by MTT assay. After treatment with GCV, administration of the trans-splicing lentiviral vectors Lv HSV-tk-ts and Lv HSV-tk-ts-opt at an MOI of 1 significantly reduced cell viability to 81.31 ± 3.25% (P < 0.0001) and 68.80 ± 5.54% (P < 0.0001), respectively, corresponding to a loss of cell viability of 89.73 ± 3.83% (P < 0.0001) in samples treated with GCV and the positive control CMV-HSV-tk (Figure 7A). At an MOI of 2, the trans-splicing construct resulted in an almost complete loss of cell viability, with only 6.19 ± 1.27% and 2.96 ± 2.08% of cells remaining in the Lv HSV-tk-ts and Lv HSV-tk-ts-opt treated samples (Fig. 7B ).

[0165] Furthermore, the efficacy of the trans-splicing lentiviral vectors was evaluated in pancreatic cancer cell lines. MIA Paca-2, Panc-1, and Aspc-1 cells were transduced with Lv CMV-GFP, Lv HSV-tk-ts, Lv HSV-tk-ts-opt, or Lv CMV-HSV-tk at an MOI of 1 or 2 and treated with 1000 μM GCV 2 days later. Both trans-splicing vectors significantly reduced cell viability in all cell types tested at both MOIs in the presence of GCV, whereas Lv HSV-tk-ts-opt had no significant effect at an MOI of 1 in Panc-1 cells (Figures 7C, 7D, 7E, 7F, 7G, 7H).

[0166] In Panc-1 cells, Lv HSV-tk-ts and Lv HSV-tk-ts-opt exhibited HSV-tk / GCV-induced cytotoxicity at an MOI of 1, with cell viabilities of 44.37 ± 5.80% (P < 0.0001) and 80.20 ± 5.16% (P = 0.8408), respectively, whereas at an MOI of 2, they exhibited only 16.94 ± 2.13% (P < 0.0001) and 52.38 ± 4.95% (P = 0.0011), respectively, indicating highly significant cytotoxicity (Figures 7C, 7D). The trans-splicing lentiviral vectors (Lv HSV-tk-ts and Lv HSV-tk-ts-opt) were also effective in Aspc-1 cells to an even greater extent than in Panc-1 cells, with viability of only 45.16 ± 11.99% (P = 0.0005) and 59.69 ± 5.21% (P = 0.0244) when treated with an MOI of 1, but only 35.67 ± 1.21% (P = 0.0002) and 43.95 ± 8.47% (P < 0.0001) when treated with an MOI of 2 (Figures 7E, 7F). Finally, in MIA PaCa-2 cells, as in HEK 293T cells, there was an almost complete loss of cell viability after treatment with the trans-splicing lentiviral vectors and 1000 μM GCV. At an MOI of 1, only 14.07 ± 2.82% (P < 0.0001) and 8.03 ± 2.19% (P < 0.0001) of cells remained after treatment with Lv HSV-tk-ts and Lv HSV-tk-ts-opt, respectively (Figure 7G). At an MOI of 2, this effect was even more pronounced, as only 4.11 ± 4.54% (P < 0.0001) and 1.22 ± 0.31% (P = 0.0002) of cells remained viable after treatment with Lv HSV-tk-ts and Lv HSV-tk-ts-opt, respectively, comparable to that observed in samples treated with the positive control, Lv CMV-HSV-tk, where only 2.09 ± 0.53% (P < 0.0001) cell viability after treatment was observed (Figure 7H).

[0167] Taken together, this confirms the efficacy of targeting HERV-K Env by RNA trans-splicing to kill liver and pancreatic cancer cells.

[0168] Reference sequence TGGATAATCCTATAGAAGTATATGTTAATGATAGCGAATGGGTA SEQ ID NO:1 - Binding region of HERV-K target sequence (DNA) UGGAUAAUCCUAUAGAAGUAUAUGUUAAUGAUAGCGAAUGGGUA SEQ ID NO:2 - Binding region of HERV-K target sequence (RNA) TACCCATTCGCTATCATTAACATATACTTCTATAGGATTATCCA SEQ ID NO:3 - Reverse complement of HERV-K target sequence (DNA) UACCCAUUCGCUAUCAUUAACAUAUACUUCUAUAGGAUUAUCCA SEQ ID NO:4 - Reverse complement of HERV-K target sequence (RNA) UACUCAUUCGCUAUCAUUUUCAUAUACUUCUGUGGGAUUAUCCA SEQ ID NO:5 - Modified reverse complement of HERV-K target sequence (RNA) 001 masypchqha safdqaarsr ghsnrrtalr prrqqeatev rpeqkmptll rvyidgphgm 061 gkttttqllv algsrddivy vpepmtywrv lgasetiani yttqhrldqg eisagdaavv 121 mtsaqitmgm pyavtdavla phiggeagss happpaltli fdrhpiaall cypaarylmg 181 smtpqavlaf valipptlpg tnivlgalpe drhidrlakr qrpgerldla mlaairrvyg 241 llantvrylq gggswredwg qlsgtavppq gaepqsnagp rphigdtlft lfrapellap 301 ngdlynvfaw aldvlakrlr pmhvfildyd qspagcrdal lqltsgmvqt hvttpgsipt 361 icdlartfar emgean Array number 6 - Herpes simplex virus thymidine kinase (HSV - tk) amino acid sequence (AAC16235.1) 306 atggc ttcgtacccc tgccatcaac acgcgtctgc gttcgaccag gctgcgcgtt 361 ctcgcggcca tagcaaccga cgtacggcgt tgcgccctcg ccggcagcaa gaagccacgg 421 aagtccgccc ggagcagaaa atgcccacgc tactgcgggt ttatatagac ggtccccacg 481 ggatggggaa aaccaccacc acgcaactgc tggtggccct gggttcgcgc gacgatatcg 541 tctacgtacc cgagccgatg acttactggc gggtgctggg ggcttccgag acaatcgcga 601 acatctacac cacacaacac cgcctcgacc agggtgagat atcggccggg gacgcggcgg 661 tggtaatgac aagcgcccag ataacaatgg gcatgcctta tgccgtgacc gacgccgttc 721 tggctcctca tatcgggggg gaggctggga gctcacatgc cccgcccccg gccctcaccc 781 tcatcttcga ccgccatccc atcgccgccc tcctgtgcta cccggccgcg cgatacctta 841 tgggcagcat gaccccccag gccgtgctgg cgttcgtggc cctcatcccg ccgaccttgc 901 ccggcacaaa catcgtgttg ggggcccttc cggaggacag acacatcgac cgcctggcca 961 aacgccagcg ccccggcgag cggcttgacc tggctatgct ggccgcgatt cgccgcgttt 1021 acgggctgct tgccaatacg gtgcggtatc tgcagggcgg cgggtcgtgg cgggaggatt 1081 ggggacagct ttcggggacg gccgtgccgc cccagggtgc cgagccccag agcaacgcgg 1141 gcccacgacc ccatatcggg gacacgttat ttaccctgtt tcgggcccccc gagttgctgg 1201 cccccaacgg cgacctgtat aacgtgtttg cctgggcctt ggacgtcttg gccaaacgcc 1261 tccgtcccat gcacgtcttt atcctggatt acgaccaatc gcccgccggc tgccgggacg 1321 ccctgctgca acttacctcc gggatggtcc agacccacgt caccacccca ggctccatac 1381 cgacgatctg cgacctggcg cgcacgtttg cccgggagat gggggaggct aactga SEQ ID NO:7 - Herpes simplex virus thymidine kinase (HSV - tk) coding sequence (AF057310.1) ASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRPEQKLPTLLRVYIDGPHGI GKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQHRLDQGEISAGDAAVV MTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAAPPALTLIFDRHPIAALLCYPAARYLMG SMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRVYG LLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAP NGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPT ICDLARTFAREMGEAN SEQ ID NO:8 - Modified HSV thymidine kinase (HSV-tk) amino acid sequence [ka] SEQ ID NO:9 - Modified HSV thymidine kinase (HSV-tk) coding sequence GGTGACGAAAA CGTGCTATCA GTTCGCTCCC CCACTCCC SEQ ID NO:10 - Spacer sequence GSGATNFSLL KQAGDVEENP GP SEQ ID NO:11 - P2A cleavage sequence GGGCCTGGGC CTGGGTACTA ACACGATCGT TTTTTTCCCT TTTTTTCCAG G SEQ ID NO:12 - Trans-splicing domain sequence GCUAGCUACU CAUUCGCUAU CAUUUUCAUA UACUUCUGUG GGAUUAUCCA CCUCGCGGUG 60 ACGAAAACGU GCUAUCAGUU CGCUCCCCCA CUCCCGCUUU CAUUUUUGUC UUGUCUUUU 120 UUAACCUGGGG CCUGGGCCUG GGUACUAACA CGAUCGUUUU UUUCCCUUUU UUUCCAGGGG 180 AAGCGGAGCU ACUAACUUCA GCCUGCUGAA GCAGGCUGGA GACGUGGAGG AGAACCCUGG 240 GCCUGCUUCG UACCCCUGCC AUCAAACACGC GUCUGCGUUC GACCAGGCGG CGCGAUCACG 300 GGGACACAGC AACCGACGGA CGGCGUUGCG CCCUCGCCGG CAGCAAGAAG CCACGGAAGU 360 CGCCCGGAG CAGAAAAUGC CCACGCUACU GCGGGUUUAU AUAGACGGUC CCCACGGGAU 420 GGGGAAAACC ACCACCACGC AACUGCUGGU GGCCCUGGGU UCGCGCGACG AAUUCGUCUA 480 CGUACCCGAG CCGAUGACUU ACUGGCGGGU GCUGGGGGCU UCCGAGACAA UCGCGAACAU 540 CUACACCACA CAACACCGCC UCGACCAGGU AAGUAUCAAG GUUACAAGAC AGGUUAAGG 600 AGACCAUAG AAACUGGGCU UGUCGAGACA GAGACGACUC UUGCGUUUCU GAUAGGCACC 660 UAUUGGUCUU ACUGACAUCC ACUUGCUU UCUCUCCACA GGGUGAGAUA UCGGCCGGGG 720 ACGCGGCGGU GGUAAUGACA AGCGCCCAGA UACAAUGGG CAUGCCUUAU GCCGUGACCG 780 ACGCCGUUCU GGCUCCUCAU AUCGGGGGGG AGGCUGGGAG CUCACAUGCC CCUCCUCCGG 840 CCCUCACCCU CAUCUUCGAC CGCCAUCCCA UCGCCGCCCU CCUGUGCUAC CCGGCCGCGC 900 GAUACCUUAU GGGCAGCAUG ACCCCCCAGG CCGUGCUGGC GUUCGUGGCC CUCAUCCCGC 960 CGACCUUGCC CGGCACAAAC AUCGUGUUGG GGGCCCUUCC GGAGGACAGA CACAUCGACC 1020 GCCUGGCCAA ACGCCAGCGC CCCGGCGAGC GGCUUGACCU GGCUAUGCUG GCCGCGAUUC 1080 GCCGCGUUUA CGGGCUGCUU GCCAAUACGG UGCGGUAUCU GCAGGGCGGC GGGUCGUGGC 1140 GGGAGGAUUG GGGACAGCUU UCGGGGACGG CCGUGCCGCC UCAGGGUGCC GAGCCUCAGA 1200 GCAACGCGGG CCCACGACCC CAUAUCGGGG ACACGUUAUU UACCCUGUUU CGGGCCCCCG 1260 AGUUGCUGGC CCCCAACGGC GACCUGUAUA ACGUGUUUGC CUGGGCCUUG GACGUCUUGG 1320 CCAAACGCCU CCGUCCCAUG CACGUCUUUA UCCUGGAUUA CGACCAAUCG CCCGCCGGCU 1380 GCCGGGACGC CCUGCUGCAA CUUACCUCCG GGAUGGUCCA GACCCACGUC ACCACCCCAG 1440 GCUCCAUACC GACGAUCUGC GACCUGGCGC GCACGUUUGC GCGGGAGAUG GGGGAGGCGA 1500 ACUGACUCGA G 1511 SEQ ID NO:13 - RTM sequence [ka] Bases 1-6 Nhe1 site Bases 7-50 Binding domain Solid underline Bases 51-56: Modified BbvC1 site Bases 57-95 spacer dotted underline Bases 128-142 ISE double underline Bases 143-150 Branch point (dashed underline) Bases 150-156 Pvu1 site Bases 157-175 Polypyrimidine tract Dotted line + dashed underline Bases 176-178 Splice site Solid underline Bases 179-244 P2A sequence Dotted underline Bases 245 to 1505 HSVtk sequence Wavy underline Bases 569-711 intron italics Bases 1506-1511 Xho1 site SEQ ID NO:14 - Coding DNA sequence of RTM (SEQ ID NO:13) GCUAGCUACU CAUUCGCUAU CAUUUUCAUA UACUUCUGUG GGAUUAUCCA CCUCGCGGUG 60 ACGAAAACGU GCUAUCAGUU CGCUCCCCCA CUCCCGCUUU CAUUUUUGUC UUGUCUUUUU 120 UUAACCUGGG CCUGGGCCUG GGUACUAACA CGAUCGUUUU UUUCCCUUUU UUUCCAGGGG 180 AAGCGGAGCU ACUAACUUCA GCCUGCUGAA GCAGGCUGGA GACGUGGAGG AGAACCCUGG 240 GCCUGCUUCG UACCCCUGCC AUCAAACACGC GUCUGCGUUC GACCAGGCGG CGCGAUCACG 300 GGGACACAGC AACCGACGGA CGGCGUUGCG CCCUCGCCGG CAGCAAGAAG CCACGGAAGU 360 CCGCCCGGAG CAGAAACUGC CCACGCUACU GCGGGUUUAU AUAGACGGUC CCCACGGGAU 420 CGGGAAAACC ACCACCACGC AACUGCUGGU GGCCCUGGGU UCGCGCGACG AUAUCGUCUA 480 CGUACCCGAG CCGAUGACUU ACUGGCGGGU GCUGGGGGCU UCCGAGACAA UCGCGAACAU 540 CUACACCACA CAACACCGCC UCGACCAGGU AAGUAUCAAG GUUACAAGAC AGGUUAAGG 600 AGACCAUAG AAACUGGGCU UGUCGAGACA GAGACGACUC UUGCGUUUCU GAUAGGCACC 660 UAUUGGUCUU ACUGACAUCC ACUUGCUU UCUCUCCACA GGGUGAGAUA UCGGCCGGGG 720 ACGCGGCGGU GGUAAUGACA AGCGCCCAGA UACAAUGGG CAUGCCUUAU GCCGUGACCG 780 ACGCCGUUCU GGCUCCUCAU AUCGGGGGGG AGGCUGGGAG CUCACAUGCC CCUCCUCCGG 840 CCCUCACCCU CAUCUUCGAC CGCCAUCCCA UCGCCGCCCU CCUGUGCUAC CCGGCCGCGC 900 GAUACCUUAU GGGCAGCAUG ACCCCCCAGG CCGUGCUGGC GUUCGUGGCC CUCAUCCCGC 960 CGACCUUGCC CGGCACAAAC AUCGUGUUGG GGGCCCUUCC GGAGGACAGA CACAUCGACC 1020 GCCUGGCCAA ACGCCAGCGC CCCGGCGAGC GGCUUGACCU GGCUAUGCUG GCCGCGAUUC 1080 GCCGCGUUUA CGGGCUGCUU GCCAAUACGG UGCGGUAUCU GCAGGGCGGC GGGUCGUGGC 1140 GGGAGGAUUG GGGACAGCUU UCGGGGACGG CCGUGCCGCC UCAGGGUGCC GAGCCUCAGA 1200 GCAACGCGGG CCCACGACCC CAUAUCGGGG ACACGUUAUU UACCCUGUUU CGGGCCCCCG 1260 AGUUGCUGGC CCCCAACGGC GACCUGUAUA ACGUGUUUGC CUGGGCCUUG GACGUCUUGG 1320 CCAAACGCCU CCGUCCCAUG CACGUCUUUA UCCUGGAUUA CGACCAAUCG CCCGCCGGCU 1380 GCCGGGACGC CCUGCUGCAA CUUACCUCCG GGAUGGUCCA GACCCACGUC ACCACCCCAG 1440 GCUCCAUACC GACGAUCUGC GACCUGGCGC GCACGUUUGC GCGGGAGAUG GGGGAGGCGA 1500 ACUGACUCGA G 1511 SEQ ID NO:15 - RTM sequence with modified HSV-tk coding sequence [ka] Bases 1-6 Nhe1 site Bases 7-50 Binding domain Solid underline Bases 51-56: Modified BbvC1 site Bases 57-95 spacer dotted underline Bases 128-142 ISE double underline Bases 143-150 Branch point (dashed underline) Bases 150-156 Pvu1 site Bases 157-175 Polypyrimidine tract Dotted line + dashed underline Bases 176-178 Splice site Solid underline Bases 179-244 P2A sequence Dotted underline Bases 245 to 1505 HSVtk sequence Wavy underline Bases 377-380 Mutated ATG (bold) Bases 419-421 Mutated ATG (bold) Bases 569-711 intron italics Bases 1506-1511 Xho1 site SEQ ID NO:16 - Coding DNA sequence of RTM with modified HSV-tk coding sequence (SEQ ID NO:15) GGAAGCGGAGCT ACTAACTTCA GCCTGCTGAA GCAGGCTGGA GACGTGGAGG AGAACCCTGG GCCT SEQ ID NO:17 - Nucleotide sequence encoding the P2A peptide.

Claims

1. (i) a binding region specific to HERV pre-mRNA; (ii) a trans-splicing domain; and (iii) a coding sequence for a suicide protein; and An RNA trans-splicing molecule (RTM).

2. The RTM of claim 1, wherein the binding region specifically binds to HERV pre-mRNA in a cell, thereby causing the coding sequence to be trans-spliced ​​with the HERV mRNA and expressing the suicide protein in the cell.

3. The RTM of claim 1 or claim 2, wherein the HERV pre-mRNA is a HERV-K pre-mRNA.

4. The RTM of claim 3, wherein the HERV-K pre-mRNA is HERV-K Np9 or HERV-K Rec pre-mRNA.

5. The RTM of claim 3, wherein the binding region specifically binds to the nucleotide sequence of SEQ ID NO: 2 in the HERV-K Np9 or HERV-K Rec pre-mRNA.

6. The RTM according to any one of claims 1 to 5, wherein the binding region comprises the nucleotide sequence of SEQ ID NO: 4 or a variant thereof.

7. The RTM of claim 6, wherein the binding region comprises a nucleotide sequence that is a variant of SEQ ID NO: 4 modified relative to SEQ ID NO: 4 at one or more positions corresponding to positions 4, 19, 20, 32 and 34 of SEQ ID NO:

4.

8. The RTM of claim 7, wherein the binding region comprises a nucleotide sequence that is a variant of SEQ ID NO: 4 having a C to U substitution at position corresponding to position 4 of SEQ ID NO: 4, an A to U substitution at position corresponding to position 19, an A to U substitution at position corresponding to position 20, an A to G substitution at position corresponding to position 32, and an A to G substitution at position corresponding to position 34.

9. The RTM according to any one of claims 1 to 8, wherein the binding region comprises the nucleotide sequence of SEQ ID NO: 5 or a variant thereof.

10. The RTM of any one of claims 1 to 9, wherein the suicide protein is herpes simplex virus (HSV) thymidine kinase.

11. The RTM of claim 10, wherein the HSV thymidine kinase comprises the amino acid sequence of SEQ ID NO:6 or a variant thereof.

12. The RTM of claim 10 or 11, wherein the HSV thymidine kinase comprises an amino acid sequence that is a variant of SEQ ID NO: 6, modified relative to SEQ ID NO: 6 at positions corresponding to positions 46 and 60 of SEQ ID NO: 6, and optionally at positions corresponding to positions 1, 46 and 60 of SEQ ID NO:

6.

13. The RTM of claim 12, wherein the HSV thymidine kinase comprises an amino acid sequence that is a variant of SEQ ID NO: 6 having an M to L substitution at position corresponding to position 46 of SEQ ID NO: 6, an M to I substitution at position corresponding to position 60, and optionally a deletion of M at position corresponding to position 1 of SEQ ID NO:

6.

14. The RTM according to any one of claims 10 to 13, wherein the HSV thymidine kinase comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof.

15. The RTM of any one of claims 1 to 14, further comprising a spacer disposed between the binding region and the trans-splicing domain in the RTM.

16. The RTM of claim 15, wherein the spacer comprises the sequence of SEQ ID NO: 10 or a variant thereof.

17. The RTM according to any one of claims 1 to 16, comprising the binding region, the trans-splicing domain and the coding sequence in the 5' to 3' direction.

18. The RTM of claim 17 , wherein the trans-splicing domain comprises an acceptor site.

19. The RTM of claim 18, wherein the trans-splicing domain comprises the nucleotide sequence of SEQ ID NO: 12 or a variant thereof.

20. 20. The RTM of any one of claims 17 to 19, further comprising a sequence encoding a 2A self-cleaving peptide, said sequence being located between said trans-splicing domain and said coding sequence in said RTM.

21. The RTM described in claim 20, wherein the 2A cleavage site comprises the sequence of SEQ ID NO: 11 or a variant thereof.

22. The RTM of any one of claims 17 to 21, wherein the coding sequence of the suicide protein lacks a start codon.

23. An RTM described in any one of claims 17 to 22, comprising a nucleic acid sequence of SEQ ID NO: 13, a variant of SEQ ID NO: 13, a nucleic acid sequence of SEQ ID NO: 15, or a variant of SEQ ID NO:

15.

24. The RTM according to any one of claims 1 to 16, comprising the coding sequence, the trans-splicing domain and the binding region in the 5' to 3' direction.

25. The RTM of claim 23 , wherein the trans-splicing domain comprises a splice donor site.

26. 26. The RTM of claim 24 or 25, wherein the coding sequence of the suicide protein comprises a start codon.

27. The RTM of any one of claims 24 to 26, further comprising a ribozyme sequence located downstream (3') of the binding region, said ribozyme sequence cleaving the RTM 3' of the binding region to remove a downstream nucleotide sequence.

28. A nucleic acid encoding an RTM described in any one of claims 1 to 27.

29. A nucleic acid encoding an HSV thymidine kinase comprising an amino acid sequence which is a variant of SEQ ID NO:6, modified relative to SEQ ID NO:6 at positions corresponding to positions 46 and 60 of SEQ ID NO:6, and optionally at positions corresponding to positions 1, 46 and 60 of SEQ ID NO:

6.

30. 30. The nucleic acid of claim 29, wherein the HSV thymidine kinase comprises an amino acid sequence that is a variant of SEQ ID NO: 6 having an M to L substitution at position corresponding to position 46, an M to I substitution at position corresponding to position 60, and optionally a deletion of M at position corresponding to position 1 of SEQ ID NO:

6.

31. 31. The nucleic acid of claim 29 or 30, wherein the HSV thymidine kinase comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof.

32. 32. The nucleic acid according to any one of claims 29 to 31, comprising the nucleotide sequence of SEQ ID NO: 9 or a variant thereof.

33. The nucleic acid according to any one of claims 29 to 32, wherein the nucleic acid is an RTM further comprising a binding region specific for a target pre-mRNA and a trans-splicing domain.

34. 29. An expression vector comprising the nucleic acid of claim 28.

35. The expression vector of claim 34 , wherein the vector is a viral vector.

36. 37. A viral particle comprising a nucleic acid according to claim 28 or an expression vector according to claim 34 or claim 35.

37. An isolated host cell comprising the RTM of any one of claims 1 to 27, the nucleic acid of claim 28, or the expression vector of claim 34 or claim 35.

38. A pharmaceutical composition comprising an RTM according to any one of claims 1 to 27, a nucleic acid according to claim 28, an expression vector according to claim 34 or claim 35, a viral particle according to claim 36, or an isolated host cell according to claim 37.

39. 39. The pharmaceutical composition of claim 38 for the treatment of cancer.

40. Use of the RTM of any one of claims 1 to 27, the nucleic acid of claim 28, the expression vector of claim 34 or claim 35, the viral particle of claim 36, the isolated host cell of claim 37, or the pharmaceutical composition of claim 38 in the manufacture of a medicament for use in the treatment of cancer.

41. After administering the pharmaceutical composition of claim 39 to an individual, further comprising:

40. The pharmaceutical composition of claim 39, which is used to administer to an individual as a first treatment an inactive pro-form of the cytotoxic compound that is activated by the suicide protein so that the cytotoxic compound is activated in the individual's cancer cells.

42. 42. The pharmaceutical composition of claim 41, wherein the cytotoxic compound is used to administer to the individual one or more further treatments with the inactive pro-form such that the cytotoxic compound is activated in cells that have become or are becoming cancer cells in the individual after the initial treatment.

43. 43. The pharmaceutical composition of claim 42, wherein the one or more further treatments are administered at least one week after the first treatment.

44. The pharmaceutical composition of any one of claims 39 and 41 to 43, wherein the cancer is characterized by expression of a HERV gene.

45. The pharmaceutical composition according to any one of claims 39 and 41 to 44, wherein the cancer is pancreatic cancer or liver cancer.

46. 1. A method for killing cells in vitro, comprising: Contacting a cell with an RTM according to any one of claims 1 to 27, a nucleic acid according to claim 28, an expression vector according to claim 34 or claim 35, or a viral particle according to claim 36, such that the cell expresses the suicide protein; contacting the cell with an inactive pro-form of a cytotoxic compound that is activated by the suicide protein, such that the cytotoxic compound is activated by the suicide protein, thereby killing the cell.

47. The method of claim 46, wherein the cell expresses a HERV gene.

48. 1. A method for depleting HERV gene-expressing cells in a population, comprising: contacting the cell population with the RTM of any one of claims 1 to 27, the nucleic acid of claim 28, the expression vector of claim 34 or claim 35, the viral particle of claim 36, and / or the pharmaceutical composition of claim 38, such that HERV gene-expressing cells in the cell population express the suicide protein; contacting the population with an inactive pro-form of a cytotoxic compound that is activated by the suicide protein, such that the suicide protein activates the cytotoxic compound in HERV gene-expressing cells in the population, thereby depleting HERV gene-expressing cells in the population.

49. The method of claim 48, wherein the HERV gene-expressing cell is a cancer cell.

50. 50. The method of claim 48 or 49, wherein the cell population is a sample of cells obtained from an individual or their descendants.

51. The method of any one of claims 48 to 50, wherein the cell is a hematopoietic cell.

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