VSV chimeric vectors

Abstract

The present invention relates to VSV chimeric vectors, characterized in that the vectors comprise a gene coding for a glycoprotein GP of the Dandenong virus (DANDV) or Mopeia vims (MOPV) and lack a functional gene coding for envelope protein G of the VSV. The invention also provides VSV chimeric vector systems. In addition, the invention relates to uses of the VSV chimeric vectors and systems of the invention, including the use in medicine such as in the treatment of solid tumors.

Claims

1. VSV chimeric vector, comprising a gene coding for a glycoprotein GP of the Dandenong virus and lacking a functional gene coding for envelope protein G of the VSV.

2. The vector of claim 1, wherein the vector further comprises at least one transgene.

3. A pharmaceutical composition comprising the VSV chimeric vector of claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a table (Table 1): Comparison of Old World Arenavirus (A) and New World Arenavirus clade C (B) GPC sequences.

(2) Sequences were obtained from NCBI. The numbers of N-linked glycosylation signals NT or NS were inferred from the translated nucleotide sequence and ranges from 6 (LCMV) to 10 (IPPV, OLIW) signals. Given amino acid positions correspond to the sequence position of the individual sequences starting at the methionine of the translation start. GPC sequences were compared to LCMV-GP WE HPI by pairwise MUSCLE alignments [17]. Relative Sequence Identity (% Seq Id.), absolute identity (n) and sequence similarity is shown. aa: amino acid, nt: nucleotide.

(3) FIG. 2: Multiple sequence alignment of arenavirus glycoproteins.

(4) Amino acid sequences of LCMV-GP WE HPI, DANVD GP, IPPYV GP, MOPV GP, OLIW GP and LATV GP were aligned using MUSCLE [17]. Black arrows indicate the cleavage sites of the signal peptidase and the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P), that cleave the precursor glycoprotein GPC into SSP, GP1 and GP2. Predicted N-linked glycosylation signal sequences NT or NS within the GP1 peptides of the arenavirus GP were boxed by dashed lines.

(5) FIG. 3 is a table (Table 2): Nucleotide and amino acid sequence identity in the GPC region among the selected Old World and New World clade C arenaviruses.

(6) FIG. 4: GFP expression in BHK21CI.13 cells infected with trans-complemented VSV*M.sub.QG virus.

(7) Non-cytopathic VSV*M.sub.QG virus, carrying the mutations M33A, M51R, V221F and S226R within the VSV matrix protein and with replacement of the entire coding sequence for the G protein by the eGFP sequence [18], was trans-complemented in BHK21CI.13 cells. 24 h before infection with VSV*M.sub.QG at a multiplicity of infection (MOI) of 3, BHK2101.13 were transiently transfected with expression plasmids pCAG-DANDV-GP, pCAG-IPPYV-GP, pCAG-LATV-GP, pCAG-MOPV-GP or pCAG-OLIW-GP to express the respective arenavirus GPs in the cells. For control, cells were mock transfected. Supernatants containing trans-complemented viruses were harvested at 24 h post infection and passaged undiluted (neat) or in serial ten-fold dilutions in a range of 1:10 to 1:1000 either on BHK21CI.13 cells (A) or on BHK-556 cells (B). In BHK-566 cells which stably expresses the LCMV-GP, VSV G GFP viruses trans-complemented with DANDV-GP, LATV-GP, MOPV-GP or OLIW-GP were able to spread within the cell culture even at high dilutions, leading to a ubiquitous GFP expression after 48 h post infection. IPPY-GP did not trans-complement VSV*M.sub.QG virus, hence BHK-566 cells did not express GFP at levels higher than the mock control. GFP fluorescence was imaged at 48 h post infection with identical exposure times on a Leica DM2500 fluorescent microscope.

(8) FIG. 5 is a table (Table 3): Cytopathic effects of VSV*M.sub.cpG trans-complemented virus.

(9) VSV*M.sub.cpG virus [18] was trans-complemented in BHK21CI.13 cells. 24 h before infection with VSV*M.sub.cpG at a M01=3, BHK21CI.13 were transiently transfected with expression plasmids pCAG-DANDV-GP, pCAG-IPPYV-GP, pCAG-LATV-GP, pCAG-MOPV-GP or pCAG-OLIW-GP. For control, cells were mock transfected. Supernatants containing trans-complemented viruses were harvested after 24 hpi and passaged undiluted (neat) or in serial ten-fold dilutions in a range of 1:10 to 1:1000 either on BHK21CI.13 cells (A) or on BHK-556 cells (B). In BHK-566 cells which stably expresses the LCMV-GP, VSV*M.sub.cpG viruses trans-complemented with DANDV-GP, LATV-GP, MOPV-GP or OLIVV-GP were able to spread within the cell culture even at high dilutions. Cytopathic effect (CPE) was monitored at 48 h post infection under bright-field microscope. Classification of CPE ranges from strong CPE (++++) to no CPE visible ().

(10) FIG. 6: VSV-G(x) DANDV and MOPV replication kinetics.

(11) Vero cells were seeded for infection in T75 cell culture flasks at a density of 410.sup.4 cells/cm.sup.2, and infected 24 h later with the indicated MOI (Multiplicity of Infection, 0.05 or 0.0005) of VSV-GP as control and the two variants VSV-G(x) DANDV and VSV-G(x) MOPV. Samples were taken after 24, 30 and 42 h and the infectious titer was determined by TCID50 (A) and the genomic titer (B) was determined by qPCR.

(12) FIG. 7: Type I interferon (IFN) limited VSV-G(x) replication in human tumor cell lines.

(13) Human Calu6 (A) and 22Rv1 (B) cells were pre-incubated with indicated amounts of IFN for 16 hours. Subsequently, cells were infected in quadruplicates with VSV-GP, VSV-G(x) DANDV, VSV-G(x) MOPV or VSV-G(x) OLIVV at an MOI of 0.1, 1, or 10, or left uninfected as a negative control. Three days after infection, cells were analyzed for viability using an MTT assay. The graph shows meanSEM, the viability of non-infected cells that were not pre-treated with IFN was normalized to 100%.

(14) FIG. 8: Type I interferon (IFN) limited VSV-G(x) replication in murine tumor cell lines.

(15) Murine SCCVII (A), Ct26CI.25 (B) or LLC1 (C) cells were pre-incubated with indicated amounts of IFN for 16 hours. Subsequently, cells were infected in quadruplicates with VSV-GP, VSV-G(x) DANDV, VSV-G(x) MOPV or VSV-G(x)

(16) OLIVV at an MOI of 0.1, 1, or 10, or left uninfected as a negative control. Three days after infection, cells were analyzed for viability using an MTT assay. The graph shows meanSEM, the viability of non-infected cells that were not pre-treated with IFN was normalized to 100%.

(17) FIG. 9: Schedule of rabbit treatments and monitoring.

(18) The schedule shows the design of one treatment out of three. All cycles of treatment were identical and started at D0, D14 and D28 with the i.v. injection of VSV-GP or VSV-G(x) viruses. Time points at which rabbits were handled are indicated by arrows. nAb, neutralizing antibody; BW, body weight; BT, body temperature; WBC, whole blood count.

(19) FIG. 10: Neutralizing antibody induction in rabbits immunized with LCMV-GP, DANDV-GP and MOPV-GP recombinant VSV vectors.

(20) Sera collected at day 4, at day 10 after each virus treatment (prime, 1.sup.st boost, 2.sup.nd boost) and at the end of the experiment were analysed for neutralizing antibodies against the autologous arenavirus glycoprotein. Sera were tested for their neutralizing capacities using VSV G SEAP pseudo-typed with either LCMV-GP WE HPI, DANDV-GP or MOPV-GP described in detail in the method section. (A) The infection rate [in %], normalized to the no-serum control, was plotted versus the serum dilutions ranging from 1:10 to 1:31.250. After the prime immunization (open squares), none of the rabbits show nAbs directed against the arenavirus GPs of LCMV, DANDV or MOPV. After the first boost sera of rabbits immunized with VSV-G(x) DANDV and MOPV show only partial neutralization of the autologous VSV G SEAP virus when compared to sera of VSV-GP immunized rabbits, hence calculation of the corresponding EC50 values was not applicable (n.a.). EC50 values were calculated after non-linear curve fit (B). Neutralization of VSV-G SEAP GP by the LCMV-GP neutralizing antibody KL25 was used as inter assay control (C).

(21) FIG. 11: Efficacy of intratumoral treatment with different arenavirus glycoprotein GP recombinant VSV vectors in a lung cancer xenograft mouse model.

(22) 510.sup.6 Calu6 lung cancer cells were injected subcutaneously into the right flank of 8 week old NMRI nude mice. Treatment started, when the mean size of tumors reached a volume between 0.05 and 0.07 cm.sup.3. Mice were treated three times 4 days apart either with PBS (n=8) mice intratumorally (A), or with 110.sup.7 TCID50 VSV-GP (B), VSV-G(x) DANDV or VSV-G(x) MOPV (D) Animals were monitored for tumor growth every 2-3 days after start of treatment, and sacrificed when tumor volume reached 0.8 cm.sup.3 or tumors ulcerated. Kaplan-Meier survival curve (E). Dotted lines indicate time points of virus injection.

(23) FIG. 12 is a table (Table 4): Scores for the evaluation of virus induced neurotoxicity.

(24) Neurotoxicity scoring of infected mice is based on different categories such as general appearance, clinical observations, body condition provoked behaviour motility and respiration. The scores in the different categories range from 0-3. The cumulative tox score shown in FIG. 12 is calculated by addition of the scores of each category.

(25) FIG. 13: Neurotoxicity in Swiss CD1 mice.

(26) Swiss CD-1 mice received a single intracranial injection of 3 l containing 110.sup.6 TCID50 via stereotactic injection into the right striatum. PBS was administered i.c. in the control group. Animals were monitored daily for signs of neurotoxicity and general well-being according to FIG. 12 for 42 days. (A) Mouse survival of PBS (diamonds), VSV-G DsRed (squares), VSV-GP (dots), VSV-GP(x) Dandenong (triangles) and VSV-GP(x) Mopeia (reversed triangles) experimental groups were plotted as Kaplan-Meier curves. Kaplan-Meier analysis indicates that none of the tested virus variants show neurotoxicity in mice. (B) Cumulative tox score, calculated according to FIG. 12, indicates that there is no neurotoxicity observed within in the groups. Only the VSV-G DsRed control group, which contains the wildtype VSV glycoprotein at the virion surface developed neurological signs leading to euthanasia within the first week after i.c. infection.

DETAILED DESCRIPTION

(27) As described above, the invention generally relates to the provision of chimeric VSV vectors, wherein the VSV vectors lack a functional gene coding for the envelope protein GP of VSV and, instead, comprise a gene coding for a glycoprotein GP of an arenavirus, in particular the Dandenong virus or Mopeia (MOPV) virus. Alternatively, the vectors may comprise the GP of the Ippy virus, Latino virus or Olivero virus. As the skilled person is aware, the glycoprotein GP is an envelope protein present on the surface of virions, which is responsible for binding between the virion and a cell of a host organism. Thus, the envelope protein determines the tropism of the virion. By altering the tropism of VSV, chimeric VSV vectors may be prepared that are suitable for use in medicine. Such chimeric vectors/virions have been provided in the prior art, e.g. in WO 2010/040526. However, there is a need for further improved chimeric vectors that provide even more efficient treatment options and an even better suitability for medical use. This need is fulfilled by the present invention.

(28) Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. The following definitions are provided for clarity only and are not intended to limit the claimed invention.

(29) The terms a or an refers to one or more, for example, a gene is understood to represent one or more such genes. As such, the terms a (or an), one or more, and at least one are used interchangeably herein. As used herein, the term about means a variability of 10% from the reference given, unless otherwise specified.

(30) With regard to the following description, it is intended that each of the compositions herein described, is useful, in another embodiment, in the methods of the invention. In addition, it is also intended that each of the compositions herein described as useful in the methods, is, in another embodiment, itself an embodiment of the invention. While various embodiments in the specification are presented using comprising language, under other circumstances, a related, embodiment is also intended to be interpreted and described using consisting of or consisting essentially of language.

(31) In this respect, the vectors of the invention are based on the virus of the vesicular stomatitis and have at least the below general advantages over retroviral vectors: (i) VSV vectors are oncolytic and have a particularly high oncolytic activity compared to other oncolytic viral vectors. (ii) VSV vectors replicate preferentially in tumor cells and have a particularly high replication capability compared to other oncolytic viral vectors. (iii) VSV vectors infect actively dividing cells as well as resting cells. (iv) VSV vectors induce a strong innate humoral and cellular immune response. (v) VSV vectors replicate purely cytoplasmatically, i.e., as RNA viruses they cannot integrate into the host cell genome or recombine into replication-competent viruses. (vi) VSV vectors are easy to package. (vii) The VSV glycoprotein is interchangeable with a foreign envelope protein. Examples for glycoproteins which were previously incorporated in the VSV envelope are: HIVgp160 [19], HCVE1/E2 [20], SARS S [21], Lassa GP [22] or the GP of LCMV.

(32) However, the vectors of the invention have additional advantages over the prior art and, in particular, VSV pseudotyped with GP of LCMV.

(33) Specifically, the vectors of the invention surprisingly show a reduced induction of neutralizing antibodies as compared to a VSV vector pseudotyped with GP of LCMV under identical conditions. The skilled person is aware of assays that may be employed in order to determine induction of neutralizing antibodies.

(34) By using the assay as described in Example 5, the inventors surprisingly found that induction of neutralizing antibodies is reduced. Such an effect could not have been reasonably expected by the skilled person. Moreover, the significance of this effect proves that the vectors and further means provided herein provide clear advantages. In this respect, it is preferred that induction of neutralizing antibodies by the vectors/virions provided herein is reduced as compared to a VSV vector pseudotyped with GP of LCMV.

(35) The vectors/virions of the invention have not only proven to reduce neutralizing antibodies, but additionally show increased killing of tumor cells as compared to a VSV vector pseudotyped with GP of LCMV under identical conditions. The skilled person is aware of assays that may be employed to determine/quantify killing of tumor cells. It is preferred that killing of tumor cells by the vectors/virions of the invention is increased as compared to a VSV vector pseudotyped with GP of LCMV.

(36) In order to achieve the above-described surprising effects, the GP of VSV is non-functional while GP of Dandenong virus or Mopeia (MOPV) virus is incorporated or the GP of VSV is replaced by the GP of Dandenong virus or Mopeia (MOPV) virus. Alternatively, the GP of VSV is non-functional while the GP of Ippy virus, Latino virus or Olivero virus is incorporated or the GP of VSV is replaced by the GP of Ippy virus, Latino virus or Olivero virus. There are various possibilities how a VSV vector can be pseudotyped with any of the above GPs. Thus, the precise nucleic acid sequence of the vectors of the invention may vary as long as the GP of VSV is essentially absent from the surface of virions while the GP of any of the viruses above is expressed on the surface of the virions. Within the present invention, exemplary preferred vectors comprise a nucleic acid sequence comprising SEQ ID NOs: 1 or 3, 5, 7 or 9 or a nucleic acid sequence comprising a nucleic acid sequence at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identical to any one of SEQ ID NOs: 1, 3, 5, 7 or 9. In this respect, SEQ ID NO:1 corresponds to a preferred nucleic acid sequence coding for the GP of the Dandenong virus, SEQ ID NO:3 corresponds to a preferred nucleic acid sequence coding for the GP of the Mopeia virus, SEQ ID NO:5 corresponds to a preferred nucleic acid sequence coding for the GP of the Ippy virus, SEQ ID NO:7 corresponds to a preferred nucleic acid sequence coding for the GP of the Latino virus and SEQ ID NO:9 corresponds to a preferred nucleic acid sequence coding for the GP of the Olivero virus.

(37) Percent (%) nucleic acid sequence identity with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the reference sequence. Methods to determine the percentage of identical nucleotides are known to the person skilled in the art. Within the present invention, it is preferred to use computer software such as BLAST. Parameters can readily be determined by the skilled person.

(38) While the nucleic acid sequences coding for any of the GP genes used for pseudotyping VSV may vary, it is important that the functionality conferred by the GP encoded by the preferred gene sequence provided above is maintained. The functionality parameters that are maintained are, preferably, the induction of neutralizing antibodies, the killing of tumor cells and/or the tropism. In this respect, the above provided assays can be employed. Moreover, the skilled person is aware of assays that may be employed in order to determine tropism of a viral particle/virion.

(39) As the skilled person will appreciate, a nucleic acid sequence may be varied with or without changing the primary sequence of the encoded polypeptide. It is preferred within the present invention that the polypeptide sequences encoded by the genes used for pseudotyping remain unchanged with respect to the GP proteins encoded by SEQ ID NOs: 1, 3, 5, 7 or 9, respectively. That is, it is preferred that the GP protein comprised in the virions of the invention corresponds to the GP protein of the Dandenong or Mopeia virus. More particularly, it is preferred that the virions of the invention comprise GP proteins comprising any one of the amino acid sequences shown by SEQ ID NOs: 2, 4, 6, 8 or 10. However, the skilled person is aware that the amino acid sequence of a polypeptide may be altered without affecting its functionality. Thus, polypeptides comprising alternative sequences are also encompassed by the present invention as long as the functionality of any one of the above amino acid sequences is essentially maintained.

(40) Thus, in certain embodiments, virions comprising amino acid sequence variants of the GP proteins provided herein are contemplated. Amino acid sequence variants may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the respective GP, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the GP. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., induction of neutralizing antibodies, killing of tumor cells and/or tropism.

(41) In certain embodiments, variants having one or more amino acid substitutions are provided. Conservative substitutions are shown in Table 5 under the heading of preferred substitutions. More substantial changes are provided in Table 5 under the heading of exemplary substitutions, and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into a GP of interest and the products/virions screened for a desired activity.

(42) Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

(43) TABLE-US-00001 TABLE 5 Amino acid substitutions Original Residue Exemplary Substitutions Preferred Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

(44) Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Within the scope of the present invention, the variant GP proteins comprised in the virions of the invention maintain the functionality conferred on the virions by the non-altered GP proteins of Dandenong virus, Mopeia virus, Ippy virus, Latino virus or Olivero virus, respectively. Such variant GP proteins may have 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO:2, or 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO:4, or 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO:6, or 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:8 or 60, 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO:10, wherein the envelope protein maintains the cellular tropism and functionality of a chimeric VSV virion comprising as envelope protein a GP comprising an amino acid sequence as shown in any one of SEQ ID NOs:2, 4, 6, 8 or 10.

(45) Percent (%) amino acid acid sequence identity with respect to a reference amino acid sequence is defined as the percentage of amino acids in a candidate sequence that are identical with the amino acids in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

(46) In a further embodiment, the invention relates to variant GP sequences having the functionality of any one of the Dandenong or Mopeia GP proteins, or the Ippy, Latino or Olivero GP, wherein the variant GP protein comprises 20, 15, 10, 5 or preferably 4, 3, 2 or 1 alteration(s).

(47) In addition to their inherent oncolytic properties, the VSV chimeric vectors or vector systems based thereon and as provided herein can further be improved by introducing at least one transgene. The skilled person is aware of techniques that can be employed to introduce such transgenes into the vectors of the invention as well as where to introduce them. One exemplary approach comprises the steps of restriction and ligation or PCR and Gibson assembly. Transgenes that may be employed within the present invention are not particularly limited as long as a particular need is addressed by delivery of the transgene. Non-limiting, yet preferred, examples include transgenes coding for a suicide protein, a cytokine/chemokine, an antibody or antibody fragment binding to immune related receptors, a protein that serves for the purpose of vaccination, a viral fusion protein, a marker protein or fusions thereof.

(48) To increase the safety during the use of replicable viruses in therapeutic uses, a vector system is provided which ensures that replication, oncolysis and the production of VSV viruses takes place only in cells which are infected by at least two replication-deficient mutually complementing vectors.

(49) The invention thus, in one embodiment, relates to a VSV chimeric vector system, characterized in that the system comprises at least two complementary replicating VSV vectors, wherein the system comprises genes n, l, p and m coding for proteins N, L, P and M of the VSV, a gene gp coding for Dandenong-GP or Mopeia-GP, or alternatively Ippy-GP, Latino-GP or Olivero-GP, and lacks a functional gene coding for G protein of the VSV, wherein each vector of the system lacks one of the genes n, l, p, m and gp, and wherein the lacking gene is present on any other vector of the system. Such complementary replicating (cr) VSV vectors can spread to a limited extent within the targeted cell, for example a tumor cell, which increases the efficiency of the gene transfer and the oncolysis. Thus, the vector system according to the invention allows for the preparation of oncolytic VSV chimeric vectors with limited reproduction capability for the gene transfer in targeted cells, in particular tumor cells. The gene gp coding for LCMV-GP as well as possible additional genes such as therapy genes and/or marker genes can be present on any vector of the system.

(50) Different variants of the vector system according to the invention are possible. For example, the vector system can consist of two vectors or more than two vectors. When the vector system consist of two vectors, a first vector may comprise GP of the Dandenong virus or Mopeia virus, or alternatively GP of the Ippy virus, Latino virus or Olivero virus, instead of GP of the VSV and a deletion of the gene p coding for the P protein. Also a second vector may comprise no VSV-G but express the P protein of the VSV. Each vector may express nucleoprotein (N) and polymerase (L) of the VSV as well as a less cytopathogenic variant of the M protein (Mncp). The first vector may carry in addition the marker gene rfp, whereas the second vector may carry a transgene.

(51) The invention also provides a vector system comprising two vectors, wherein one vector comprises the GP of the Dandenong virus or Mopeia virus, or alternatively the GP of Ippy virus, Latino virus or Olivero virus, as defined herein and a second vector comprises the GP of LCMV. The second vector comprising the GP of LCMV may be a vector as described in WO 2010/040526.

(52) The invention furthermore relates to cells producing the chimeric virions of the invention. Virus producing cells in the meaning of the invention include classical packaging cells for the production of virions from non-replicable vectors as well as producer cells for the production of virions from vectors capable of reproduction. Packaging cells usually comprise one or more plasmids for the expression of essential genes which lack in the respective vector to be packaged and/or are necessary for the production of virions. Such cells may be mammalian cells, in particular human cells, monkey cells, mouse cells or hamster cells, more particular HEK293 cells, HeLa cells, or Vero cells. Such cells are known to the skilled person who can select appropriate cell lines suitable for the desired purpose.

(53) In previous studies, packaging cells were used for transferring viral vectors; however, this involved mainly fibroblasts which do not migrate within the tumor (Short et al., 1990, Culver et al., 1992). In contrast, adult stem cells, in particular neuronal (NSC), multipotent adult progenitor cells (MAPC) and mesenchymal stem cells (MSC) have a high migratory potential. They remain confined to the tumor tissue, whereby a very efficient but also specific gene transfer into the tumor tissue is achieved. However, these stem cells have limited passage capacity in vitro.

(54) A subpopulation of adult mesenchymal stem cells, so-called BM-TIC (bone marrow derived tumor infiltrating cells) infiltrate, after injection into experimentally induced gliomas, the entire tumor and, in addition, track individual tumor cells remote from the tumor mass [23]. BM-TIC are isolated from adult bone marrow, have a high expansion potential and can be used as migrating producer cells for MLV [24] and VSV vectors.

(55) The subject matter of the invention is thus virus producing cells which produce VSV chimeric vectors of the invention. In particular, these are tumor-infiltrating producer cells which release the said vectors during their migration within the tumor. Preferred cells are adult stem cells, in particular neuronal (NSC) and mesenchymal stem cells (MSC). Particularly preferred cells are BM-TIC cells derived from MSC.

(56) The virus producing cells of the invention and hence also the VSV chimeric vectors produced by said cells may comprise a gene coding for a mutated M protein. This vector variant is selectively oncolytic for tumor cells, whereas it is not toxic for healthy cells. M variants with amino acid exchange in the 37PSAP40 region of the M protein or with single (M51R) or multiple (V221F and S226R; M33A and M51A) mutations outside of the PSAP region of the M protein are preferred. An M protein with mutations M33A, M51R, V22F and S226R is particularly preferred. In order to ensure an efficient virus production in packaging cells, the M variant can be stably transfected with a viral interferon antagonist.

(57) In one embodiment, the virus producing is characterized in that the cell comprises one or more expression cassettes for the expression of at least one of the genes selected from the group consisting of genes n, l, p and m coding for proteins N, L, P and M of the VSV and a gene gp coding for Dandenong-GP or Mopeia-GP glycoprotein, or alternatively Ippy-GP, Latino-GP or Olivero-GP. The cell may furthermore comprise a gene transfer vector for the packaging into a VSV virion pseudotyped with GP of Dandenong or Mopeia virus, or alternatively Ippy, Latino or Olivero virus, wherein the gene transfer vector comprises a transgene.

(58) In addition, subject matter of the invention is an in vitro method for gene transfer, wherein a VSV chimeric vector according to the invention or a VSV chimeric vector system according to the invention comprising a transgene is introduced into a cell either directly or by means of virus producing cells (packaging cells) according to the invention. If a cr vector system with at least two vectors is used, at least two packaging cells are used, wherein each of the cells produces one of the (replication-incompetent) cr vectors. The production of VSV viruses takes place only in cells which are infected with all vectors of the cr vector system and hence comprise all essential viral genes.

(59) In addition, the invention relates to the use of vectors and virus producing cells according to the invention as drugs in therapeutic methods. In particular, the vectors and virus producing cells according to the invention are used for the therapy of solid cancers. The therapeutic effect is caused by the oncolytic properties of the recombinant vectors and viruses as well as by the use of therapeutic genes, without being bound by theory.

(60) Solid cancer can be brain cancer, colorectal cancer, oropharyngeal squamous cell carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal carcinoma, hepatocellular carcinoma, pancreatic adenocarcinoma, cholangiocarcinoma, bladder urothelial carcinoma, metastatic melanoma, prostate carcinoma, breast carcinoma, glioblastoma, non-small cell lung cancer, brain tumor or small cell lung cancer.

(61) The subject matter of the invention is further a pharmaceutical composition which comprises the vector, the virion, or the virus producing cell of the invention and optionally additives such as a pharmaceutically acceptable carrier and auxiliary substances.

(62) In order to increase the viral oncolysis and the transfer efficiency of the therapeutic genes, tumor-infiltrating virus producing cells which continuously release vectors may be formulated for direct implantation into the tumors. The means provided herein may thus be formulated for intratumoral administration or intravenous administration.

(63) The term pharmaceutical formulation or pharmaceutical composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

(64) A pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

(65) As used herein, treatment (and grammatical variations thereof such as treat or treating) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

(66) Pharmaceutical formulations of the vectors, virions or cells as described herein are prepared by mixing such vectors, virions or cells having the desired degree of purity with one or more optional pharmaceutically acceptable carriers [25], in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

(67) The formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.

(68) Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in [25]. Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, which matrices are in the form of shaped articles, e.g. films, or microcapsules. The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

(69) In one aspect, the vector, virion, cell or pharmaceutical composition according to the invention for use as a medicament is provided. In further aspects, the vector, virion, cell or pharmaceutical composition according to the invention for use in a method of treatment are provided. In certain embodiments, the vector, virion, cell or pharmaceutical composition according to the invention for use in treating cancer are provided. In certain embodiments, the invention provides the vector, virion, cell or pharmaceutical composition of the invention for use in a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of the vector, virion, cell or pharmaceutical composition. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

(70) In a further aspect, the invention provides for the use of a vector, virion, cell or pharmaceutical composition of the invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of cancer, in particular a solid tumor. In a further embodiment, the medicament is for use in a method of treating cancer, in particular a solid cancer, the method comprising administering to an individual having cancer, in particular a solid cancer, an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g. a therapeutic agent that is an antagonist of PD-1 or PD-L1.

(71) In a further aspect, the invention provides a method for treating cancer, in particular a solid cancer. In one embodiment, the method comprises administering to an individual having cancer an effective amount of the vector, virion, cell or pharmaceutical composition of the invention. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.

(72) An individual according to any of the above embodiments may be a human. However, included is any mammal in need of these methods of treatment or prophylaxis, including particularly humans. Other mammals in need of such treatment or prophylaxis include dogs, cats, or other domesticated animals, horses, livestock, laboratory animals, including non-human primates, etc. The subject may be male or female. In one embodiment, the subject has, or is at risk of developing, cancer and more particularly, a solid tumor.

(73) As described above, the invention provides pharmaceutical compositions comprising any of the vector, virion or cell provided herein, e.g., for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the vector, virion, or cell provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the vector, virion, or cell provided herein and at least one additional therapeutic agent, e.g., as described below.

(74) The vector, virion, cell or pharmaceutical composition of the invention can be used either alone or in combination with other agents in a therapy. For instance, the vector, virion, cell or pharmaceutical composition of the invention may be co-administered with at least one additional therapeutic agent.

(75) Therapeutic agents within the meaning of the invention are molecules including, without limitation, polypeptides, peptides, glycoproteins, nucleic acids, synthetic and natural drugs, peptides, polyenes, macrocyles, glycosides, terpenes, terpenoids, aliphatic and aromatic compounds, and their derivatives. In a preferred embodiment, the therapeutic agent is an antagonist of PD-1 or PD-L1. In another preferred embodiment, the therapeutic agent is a checkpoint inhibitory antibody or any other immune therapeutic that stimulates the immune response of the individual. In yet another embodiment, the therapeutic agent is a chemotherapeutic agent. The chemotherapeutic agent may be substantially any agent which exhibits an oncolytic effect against tumor cells in an individual and which does not inhibit or diminish the oncolytic effect of the oncolytic virus of the invention. The agent may be any known or subsequently discovered chemotherapeutic agent. By way of example, known types chemotherapeutic agents include, for example, anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethylenimines, methylmelamines, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing agents, interferons, and interleukins.

(76) Suitable therapeutic agents include, without limitation, those presented in Goodman and Oilman's The Pharmacological Basis of Therapeutics (e.g., 9th Ed.) or The Merck Index (e.g., 12th Ed.). Genera of therapeutic agents include, without limitation, drugs that influence inflammatory responses, drugs that affect the composition of body fluids, drugs affecting electrolyte metabolism, chemotherapeutic agents (e.g., for hyperproliferative diseases, particularly cancer, for parasitic infections, and for microbial diseases), antineoplastic agents, drugs affecting the blood and blood-forming organs, hormones and hormone antagonists, vitamins and nutrients, vaccines, oligonucleotides and gene therapies. It will be understood that compositions comprising combinations, e.g. mixtures or blends of two or more active agents, such as two drugs, are also encompassed by the invention.

(77) Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the vector, virion, cell or pharmaceutical composition of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. The vector, virion, cell or pharmaceutical composition of the invention can also be used in combination with radiation therapy.

(78) The vector, virion, cell or pharmaceutical composition of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional, intrauterine or intravesical administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Also envisaged is direct intratumoral administration. Preferred is intravenous or intratumoral administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

(79) Intratumoral injection, or injection directly into the tumor vasculature is specifically contemplated for discrete, solid, accessible tumors. Local, regional or systemic administration also may be appropriate. For example, for tumors of >4 cm, the volume to be administered may be about 4-10 mL (suitably 10 mL), while for tumors of <4 cm, a volume of about 1-3 mL may be used (suitably 3 ml). In some embodiments, the volume of agent administered can be up to 25% or up to 33% of the tumor volume. Multiple injections delivered as single dose comprise about 0.1 to about 0.5 mL volumes. The viral particles may advantageously be contacted by administering multiple injections to the tumor, spaced at approximately 1 cm intervals. In the case of surgical intervention, the present compositions may be used preoperatively, to render an inoperable tumor subject to resection. Continuous administration also may be applied where appropriate, for example, by implanting a catheter into a tumor or into tumor vasculature. Such continuous perfusion may take place for a period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longer following the initiation of treatment. Generally, the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs. It is further contemplated that limb perfusion may be used to administer therapeutic compositions, particularly in the treatment of melanomas and sarcomas.

(80) The vector, virion, cell or pharmaceutical composition of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder 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 agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The vector, virion, cell or pharmaceutical composition need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of the vector, virion, cell or pharmaceutical composition present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

(81) For the prevention or treatment of disease, the appropriate dosage of the vector, virion, cell or pharmaceutical composition of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of the vector, virion, cell or pharmaceutical composition, the severity and course of the disease, whether the vector, virion, cell or pharmaceutical composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the vector, virion, cell or pharmaceutical composition, and the discretion of the attending physician. The vector, virion, cell or pharmaceutical composition is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of the vector, virion, cell or pharmaceutical composition can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 g/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the vector, virion, cell or pharmaceutical composition would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the vector, virion, cell or pharmaceutical composition). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

(82) Alternatively, the vector, virion, cell or pharmaceutical composition of the invention may be delivered in a volume of from about 50 L to about 10 mL including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume is about 50 L. In another embodiment, the volume is about 70 L. In another embodiment, the volume is about 100 l another embodiment, the volume is about 125 L. In another embodiment, the volume is about 150 L. In another embodiment, the volume is about 175 L. In yet another embodiment, the volume is about 200 L. In another embodiment, the volume is about 250 l. In another embodiment, the volume is about 300 L. In another embodiment, the volume is about 450 L. In another embodiment, the volume is about 500 L. In another embodiment, the volume is about 600 L. In another embodiment, the volume is about 750 L. In another embodiment, the volume is about 850 L. In another embodiment, the volume is about 1000 L. In another embodiment, the volume is about 2000 L. In another embodiment, the volume is about 3000 L. In another embodiment, the volume is about 4000 L. In another embodiment, the volume is about 5000 L. In another embodiment, the volume is about 6000 L. In another embodiment, the volume is about 7000 L. In another embodiment, the volume is about 8000 L. In another embodiment, the volume is about 9000 L. In another embodiment, the volume is about 10000 L. An effective concentration of a virion carrying a nucleic acid sequence encoding the desired transgene under the control of the cell-specific promoter sequence desirably ranges between about 10.sup.8 and 10.sup.13 vector genomes per milliliter (vg/mL). The infectious units may be measured as described in S. K. McLaughlin et al [26]. Preferably, the concentration is from about 1.510.sup.9 vg/mL to about 1.510.sup.12 vg/mL, and more preferably from about 1.510.sup.9 vg/mL to about 1.510.sup.11 vg/mL. In one embodiment, the effective concentration is about 1.510.sup.10 vg/mL. In another embodiment, the effective concentration is about 1.510.sup.11 vg/mL. In another embodiment, the effective concentration is about 2.810.sup.11 vg/mL. In yet another embodiment, the effective concentration is about 1.510.sup.12 vg/mL. In another embodiment, the effective concentration is about 1.510.sup.13 vg/mL. It is desirable that the lowest effective concentration be utilized in order to reduce the risk of undesirable effects. Still other dosages in these ranges may be selected by the attending physician, taking into account the physical state of the subject, preferably human, being treated, the age of the subject, the particular type of cancer and the degree to which the cancer, if progressive, has developed.

(83) It is understood that any of the above formulations or therapeutic methods may be carried out using any one of the vector, virion, cell or pharmaceutical composition of the invention.

(84) In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the disorder and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the vector, virion, cell or pharmaceutical composition of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises the vector, virion, cell or pharmaceutical composition of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition, in particular cancer. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

EXAMPLES

Example 1: Arenavirus GP Sequence Alignments

(85) Arena virus RNA sequences of the S segment were retrieved from the NCBI nucleotide library in gene bank flat file format as follows: LCMV GP WE HPI (accession: AJ297484), MOPV GP (accession: JN561684), DANDV GP (accession: EU136038), IPPYV GP (accession: DQ328877), OLIVV GP (accession: U34248) and LATV (accession: AF485259). GPC glycoprotein sequences were obtained by translation of the GPC open reading frame coded by the S segment. Old World and New World clade C arenavirus GPC sequences were compared on nucleotide and protein level using the Geneious software package version 11.0.5 (Biomatters Ltd.).

(86) N-linked glycosylation signals in the GP1 peptide of the glycoprotein GP were identified by the glycosylation signal sequence NS or NT. Numbering of the glycosylation signal sites corresponds to the parental sequence starting at the ATG methionine translation initiation codon (FIG. 1).

(87) Absolute and relative sequence identities (% Seq Id) were calculated after multiple alignment of the nucleotide sequences or the corresponding GPC amino acid sequences (FIGS. 2 and 3). Sequences were aligned using MUSCLE [17] with a maximum number of eight iterations using a distance matrix by k-mer and pctid clustering. Subsequently, sequences were clustered using the UPMGA method. Sequence similarities of every arenavirus GPC protein compared to LCMV WE HPI were calculated from pairwise alignments using the blosum62 matrix with a threshold1.

Example 2: Arenavirus GPs can Complement for VSV-G

(88) The GP glycoproteins of DANDV, MOPV as well as LATV and OLIVV were able to trans-complement VSV*MQG virus. The resulting VSV pseudotypes were able to infect BHK21CI.13 cells in a subsequent passage and showed increased GFP signals at different dilutions when compared to the mock control (FIG. 4A). The GFP signal that could be observed in BHK21CI.13 cells infected with supernatants of IPPYV GP-complemented VSV*MQG virus was comparable to the mock control and results from the input VSV*MQG virus. To confirm these results, BHK-566 cells which stably expresses the LCMV-GP were infected with the pseudotyped VSV*MQG viruses. Viruses trans-complemented with DANDV-GP, LATV-GP, MOPV-GP or OLIVV-GP were able to spread within the cell culture even at high dilutions, leading to a ubiquitous GFP expression after 48 h post infection (FIG. 4B). Cythopathic effects (CPE) of VSV*M.sub.cpG viruses trans-complemented with the GPs of DANDV, MOPW, IPPYV OLIVV and LATV confirmed the results obtained with the corresponding VSV*MQG pseudotypes and are summarized in FIG. 5.

(89) For trans-complementation, 0.8-110.sup.6 BHK21CI.13 cells were seeded in six-well plates one day before transfection. On the next day, BHK21CI.13 were transfected with 2.5 g pCAG-DANDV-GP, pCAG-IPPYV-GP, pCAG-LATV-GP, pCAG-MOPV-GP or pCAG-OLIW-GP using TransIT-LT1 transfection reagent (Mirus Bio LCC, Madison, Wis. USA) according to the manufacturer's recommendations. 24 h post transfection, transiently transfected BHK21CI.13 cells were infected with VSV*M.sub.cpG or VSV*M.sub.QG [9], carrying the mutations M33A, M51R, V221F and S226R within the VSV matrix protein at an MOI of 3. One hour post infection, cells were washed twice with cGMEM (containing 10% FCS, 2% Glutamine and 1% Tryptose Phosphate Broth) and incubated for another 24 hours. Supernatants were harvested and cell debris was removed by centrifugation at 8000 rpm for 5 min in a bench-top centrifuge. Supernatants were then transferred in serial ten-fold dilutions to fresh BHK21CI.13 or BHK-566 cells, which stably expresses LCMV-GP WE HPI after lentiviral transduction, in 24-well plates. GFP expression from the vector or cytopathic effect was monitored after 24 h and 48 h on a Leica DM2500 fluorescent microscope. Pictures were taken using identical exposure times.

Example 3Replication Kinetics

(90) VSV-chimeric vectors that comprises the VSV vector backbone as described in WO 2010/040526 and the GPs of DANDV, MOPV, OLIVV and LATV were cloned. The GPs in the resulting chimeric VSV-G(x)-DANDV, -MOPV, -OLIVV and LATV vectors replace the GP of LCMV without any changes in the remaining VSV vector backbone. Although there were slightly differences in the replication kinetics of VSV-G(x)-DANDV and -MOPV (VSV-G(x)-LATV and -OLIVV were not tested), both viruses replicate to titers higher than 110.sup.7 TCID50/ml (FIG. 6A) or 110.sup.9 genome copies/ml (FIG. 6B) after 30 hpi. With regard to their replication potential, VSV-G(x)-DANDV and -MOPV might be particularly suitable for OV cancer therapy.

(91) For comparison of replication kinetics, Vero cell monolayers in a T75 cell culture flasks were infected with VSV-GP, VSV-G(x) DANDV or VSV-G(x) MOPV at an MOI of 0.05 or 0.0005. 500 l supernatant of infected cells was collected at the indicated time points. Supernatant was centrifuged at approx. 2000 rpm for 5 min in a bench-top centrifuge to remove cell debris and stored at 80 C. until further processing. Analysis of viral titers by TCID50 assay [27] and of viral genomes by qPCR [28] was performed as described elsewhere.

Example 4Tumor Cell Killing of VSV-G(x) Variants (FIG. 7)

(92) Cell lines. Human Calu6 lung carcinoma cells were obtained from Dr. Edith Lorenz, OncoTyrol (Department of Internal Medicine, Hematology and Oncology, G Zwierzina, Innsbruck). Cells were grown in DMEM medium with 10% fetal bovine serum, 2 mM L-Glutamine and 1% Penicillin/Streptomycin (Pen/Strep). The cells were sub-cultured every 2-3 days when they reach 80% confluency using EDTA-Trypsin 0.05%. 22Rv1 cells derived from a human prostate cancer were kindly provided by Prof. Z. Culig (Department of Urology, Medical University of Innsbruck). Cells were sub-cultured twice a week in RPMI1640 containing 10% FCS, 2 mM Glutamine, 10 mM HEPES, 1 mM sodium-pyruvate and 1% Pen/Strep. Adherent murine squamous cell carcinoma cells (SCCVII) were obtained from Dr. Lukas Mach (Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna) Cells were grown in DMEM containing 10% FCS, 2 mM Glutamine, 0.1 mM non-essential amino-acids (NEAA), 1 mM sodium-pyruvate and 1% Pen/Strep. SCCVII cells were sub-cultured at a ratio of 1:10 three times a week using EDTA-Trypsin 0.05%. CT26CI.25 murine colon carcinoma cells derived from Balb/c that were stable transduced with a LacZ cassette disrupting the IFN-I antiviral response were obtained from ATCC (#CRL-2639). Cells were sub-cultured twice a week at a ratio of 1:10 in RPMI1640 containing 10% FCS, 2% Glutamine, 10 mM HEPES, 0.1 mM NEAA, 1 mM sodium-pyruvate 1% P/S and 400 g/ml G418. LLC1 cells were established from a C57BL/6 mouse bearing a lung tumor after implantation of a primary Lewis Lung Carcinoma. The cells were obtained from ATCC (#CRL-1642) and cultured in DMEM containing 10% FCS, 4 mM Glutamine and 1% P/S. Confluent cell cultures were sub-cultured 1:6 to 1:10 every 3-4 days by resuspension of loosely attached cells.

(93) VSV killing assay. Cells were seeded in 96-well plates and pre-incubated overnight with 10, 100, 500 and 1000 units of universal type-1 IFN (PBL, Piscataway, N.J., USA) in a volume of 100 l/well. The following morning, cells were infected with VSV-GP, VSV-G(x) DANDV, VSV-G(x) MOPV or VSV-G(x) OLIW at an MOI of 0.1, 1, or 10 in a final volume of 120 l/well. For each condition, quadruplicate samples were performed. As a positive, killing control cells were incubated with a final concentration of 6.67 mM of H.sub.2O.sub.2. Three days after infection, cells were analyzed for viability using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based in vitro cytotoxicity assay (Sigma-Aldrich, Saint Louis, Mich., USA), according to the manufacturer's recommendations. Plates were measured in a conventional microplate reader at 550 nm, and blank values of wells without cells were subtracted. Values were normalized to mock-infected cells that were not pre-treated with interferon (IFN), and represented as a percentage of viable cells.

Example 5: Neutralizing Antibodies after i.v. Treatment of NZW Rabbits

(94) The study attempted to evaluate whether the VSV G(x) variants induce no neutralizing antibodies (nAb) or lower levels of nAbs compared to VSV-GP following 3 intravenous (i.v.) administrations in healthy tumor-free New Zealand White (NZW) rabbits. NZW rabbits were treated i.v. three times 14 days apart with 110.sup.9 TCID.sub.50 of VSV-GP or different VSV-G(x) candidates. nAb induction as well as body weight and temperature, viremia, whole blood count and blood chemistry were monitored at regular intervals following treatment as depicted in FIG. 9. The primary objective of this study was to monitor nAb induction by measuring nAb levels 10 days after each administration. The nAb assay is based on previously published work by Kaku et al. [29] J. Virol Methods (2012) 179(1):226-32. The VSV G SEAP virus used in the nAb assays expresses the secreted embryonic alkaline phosphatase (SEAP) instead of the envelope glycoprotein G. This virus was complemented by LCMV GP or the respective other arenavirus glycoproteins ectopically expressed on the cell surface of the producer cell. These trans-complemented viruses were than tested for neutralization by the collected rabbit (FIG. 10).

(95) Safety parameters such as the health status of the animals, including body weight (BW) and body temperature (BT), white blood cell (WBC) counts, blood chemistry (optional) and viremia (TCID50 and N-specific qPCR in plasma/blood) were secondary objectives.

(96) Rabbit handling and housing: Crl:KBL(NZW) rabbits were purchased from Charles River Laboratories, (France). At arrival in the animal facility, rabbits were approx. 7 weeks old. The animals were housed in groups of two individuals in rabbit cages (R-Suite Enriched Rabbit Housing, Techniplast). Individuals were identified by ear tattoos. Rabbit housing was controlled daily and the overall health status was monitored. Food supply ad libitum and daily change of drinking water was guaranteed for the time period of the experiment. The rabbits were acclimated a minimum of 4 days before the rabbits were adapted to handling. Virus treatment started after the rabbits were acclimated and handled for at least one week.

(97) Viruses and treatment of NZW rabbits: Virus dilutions were done with PBS. The final concentration of VSV-GP or VSV-G(x) solutions was 110.sup.9 TCID50 per ml. Virus dilutions were kept on ice until shortly before injection. Rabbits were fixed by wrapping the animal in a cotton towel. The injection site of the tattooed left ear was shaved properly and then cleaned using a sterile alcohol swab. 110.sup.9TCID.sub.50 virus in 1 ml PBS were injected slowly into the left ear vein by a 29 gauge needle attached to a 1 ml syringe.

(98) Rabbit monitoring: Body weight and body temperature were measured regularly according to the Schedule presented in FIG. 9. Temperature was measured rectally with a digital thermometer. Body weight was recorded using an Ultra MBSC balance.

(99) Blood sampling and processing: Blood was collected from the ear artery using a 23G micro needle (Sarstedt, Germany). Before puncture of the artery, the ear was shaved properly and disinfected using an alcohol pad. A minimum of 2 ml blood was collected in a 15 ml tube containing 50 l heparin (5000 IE/ml). 100 l heparin blood was stored at 80 C. for analysis of viral titers by qPCR, the remaining blood was processed to heparin plasma (200 l) for TCID50 determination or clinical chemistry (300 l). At day 4 before the first treatment and at day 10 after each treatment cycle additional 2-3 ml blood was collected in a 15 ml Falcon for serum preparation. In addition, approx. 500 l blood was collected in two pre-coated EDTA microvettes (Microvette CB 300 K2E, Sarstedt, Germany). EDTA blood was used for whole blood count and a minimum of 100 l was archived and used for qPCR. EDTA plasma was prepared from the second microvette and stored at 80 C. until use.

(100) Viremia by TCID.sub.50 and VSV-N specific qPCR: Viremia was measured by TCID50 assay in blood plasma after 8 h and consecutive three days after virus application of each treatment cycle (as described by Urbiola et al. [27]). For VSV-N specific qPCR, 100 l of EDTA and Heparin blood frozen and stored at 80 C. for qPCR was used. RNA was isolated using a commercial available RNA isolation kit for blood samples. Determination of viral titers by qPCR was performed as described e.g. by Jenks et al. [28].

(101) White Blood Count: White blood counts using EDTA blood were measured using the ScilVet ABC blood counter. Approximately 10-20 l of fresh EDTA-blood was needed for the assay. Measurements were performed according to the manufacturer.

(102) Sacrifice: Rabbits were anesthetized by Ketamine/Xylazine according to FELASA and GV SOLAS recommendations. After anesthesia the ear right ear vein was catheterized and rabbits were terminal bleed by heart puncture. Blood without anticoagulants and heparin blood was collected. Rabbits were released from anesthesia with a lethal dose of pentobarbital through the vein catheter according to FELASA and GV SOLAS recommendations. After the rabbits had been sacrificed, spleens were removed and splenocytes were isolated by standard procedures for optional downstream analysis (e.g. VSV-N specific T cell responses). Blood was processed to serum or plasma and aliquots were stored at 80 C. until use.

(103) Neutralizing antibody (nAb) assay: Pre-immune sera collected before the first treatment at D-4 and serum samples taken 10d after each treatment cycle were analyzed for the presence of nAb directed against VSV-G SEAP GP virus or pseudo-typed virus with a different glycoprotein variant. SEAP activity in the supernatant of infected BHK21CI.13 cells was measured by the enzymatic conversion of p-nitro-phenylphosphate (pNPP) to p-nitrophenyl which has a yellow color in solution by the virus coded SEAP. The color reaction was read in a conventional micro plate reader at 405 nm and served as indirect measurement of the infection rate by the SEAP expressing virus. Comparing serum and no serum-treated control samples the nAb assay indicated whether a serum sample contained neutralizing antibodies or not. In the absence of nAbs, the virus was not neutralized and able to infected BHK21CI.13 cells resulting in the expression of SEAP. In contrast, if the virus was neutralized by antibodies in the sera, the virus was not able to infect the cells and hence SEAP was not expressed.

(104) One day before virus neutralization, BHK21CI.13 cells were seeded in a 96-well cell culture plate at a density of 110.sup.4 cells in 100 l cGMEM per well using a multi-channel dispenser pipette. To guarantee low inter assay variation cells were counted using the Luna cell counter (Logos Biosystems). Any other cell counting device that is based on the photographic analysis of cell numbers (e.g. Tecan reader) could be used. Counting cells with a CASY counter (OLS OMNI Life Science) or Kovar Glasstic slides is not recommended. Cells were incubate over night at 37 C., 6% CO2, 95% humidity. Cell culture plates were kept in a moisture chamber to prevent evaporation of the wells at the plate borders.

(105) The next day, serial 1:5 serum dilutions starting at a lowest dilution of 1:5 were prepared first. Briefly, 200 l cGMEM per well were added to a 96-well plate using a multi-channel dispenser pipette. 50 l KL25 monoclonal LCMV GP antibody at a concentration of 100 g/ml (inter assay control) or heat inactivated (1 h, 56 C.) serum sample were added to 200 l GMEM into the wells of row C of the 96-well plate and mixed by pipetting up and down several times. Five-fold serial dilutions starting at row C down to row H of the 96 well plate were done using a multichannel pipette: 50 l from the wells in row C were transferred to the wells of the next row and mixed by pipetting several times up and down. 50 l from the wells of row D were then added to the wells of the next row etc. until finished at row H. 175 l of the serum dilutions of each well were transferred into a clean 96-well plate using a multichannel pipette. Serum dilutions were kept on ice. Row A and B of the 96-well plate served as no-infection control (row A) or no-serum control (row B).

(106) Dilutions of pseudo-typed VSV G SEAP virus were prepared in cGMEM at a MOI of 0.1 or 1, corresponding to 110.sup.3 or 110.sup.4 infectious particles per well, respectively, when 110.sup.4 BHK21CI.13 cells were seeded one day before. The amount of virus needed for each triplicate in the nAb assay was calculated according to the following formulas:

(107) $\mathrm{Volume}\mathrm{of}\mathrm{virus}\mathrm{stock}\mathrm{needed}\mathrm{in}\left[\mathrm{in}\mathrm{.Math.l}\right]=\frac{3.5*{10}^4\mathrm{infectious}\mathrm{particles}\left(\mathrm{MOI}=1\right)}{\mathrm{titer}\mathrm{of}\mathrm{virus}\mathrm{stock}\left[\mathrm{TCID}50/\mathrm{ml}\right])}*1000\mathrm{.Math.l}*\mathrm{number}\mathrm{of}\mathrm{wells}\mathrm{and}\mathrm{Volume}\mathrm{of}\mathrm{cGMEM}\left[\mathrm{in}\mathrm{.Math.l}\right]=\left(175\mathrm{.Math.l}\mathrm{number}\mathrm{of}\mathrm{wells}\right)-\mathrm{Volume}\mathrm{of}\mathrm{virus}\mathrm{stock}\mathrm{needed}\mathrm{in}\left[\mathrm{in}\mathrm{.Math.l}\right]$
175 l virus solution were then added to each well of row B-H containing the 175 l serial serum dilutions to make 350 l serum/virus sample. Virus/serum samples were mix by pipetting up and down several times using a multichannel pipette. 175 l cGMEM were added to the wells of row A (non-infection control). Serum/virus mixtures were incubated for 1 h on ice.

(108) After incubation, 100 l of the serum/virus sample was added in triplicate to BHK21CI.13 cells seeded in 96 well plates one day before. Cells should be at 80-90% confluency. BHK21CI.13 were incubated for additional 24 h at 37 C., 6% CO2 and 95% humidity.

(109) On the next day, the nAb activity was measured as a function of SEAP activity. Briefly, the p-nitro-phenylphosphate (pNPP) substrate solution (SIGMA-FAST, Sigma Aldrich) was prepared according to the manufactures recommendations For each 96-well cell culture pNPP substrate solution was prepared by dissolving one buffer tablet and one pNPP substrate tablet in 21 ml sterile H.sub.2O. 40 l cell culture supernatant from the cells infected one day before were transferred into a new 96 well plate using a multichannel pipette. Then 200 l of the pNNP substrate solution was added to the cell culture supernatant using a multichannel dispenser pipette. Plates were incubated at least 1 h at room temperature in the dark. The OD of the wells was read in a conventional micro plate reader at 405 nm. If the OD.sub.405 of the no-serum control was lower than 1.0, incubation time was prolonged for an additional hour or up to 4 h, before OD measurements were repeated. Measured OD values were plotted versus serum dilutions. By non-linear curve fitting EC50 values were calculated using the GraphPad Prism 5 Software version 5.03 (GraphPad, San Diego, Calif. USA).

Example 6: Treatment of Calu6 Lung Tumor Xenograft Model (FIG. 11)

(110) Recipient mice: Eight-week old female NMRI-Nude mice were purchased from Janvier (France). Animals were housed in individually ventilated cages in groups of 8 animals. For identification purpose, the mice were ear clipped. Before tumor engraftment the mice were acclimated for at least one week.

(111) Tumor cells: Calu6 lung carcinoma cell line were obtained from Dr. Edith Lorenz, OncoTyrol (Department of Internal Medicine, Hematology and Oncology, G Zwierzina, Innsbruck). Cells were grown in DMEM medium with 10% fetal bovine serum, 2 mM L-Glutamine and 1% Penicillin/Streptomycin. The cells were sub-cultured every 2-3 days when they reach 80% confluency using EDTA-Trypsin 0.05%. Cells were seeded at low cell numbers. 24 hrs before grafting into NRMI nude mice, cells were harvested, pooled and then passaged at a ratio 1:2 in T175 flasks.

(112) On the day of engraftment, cells were detached using EDTA-Trypsin 0.05%, complete medium was added and cells were counted in a KOVA Glasstic slide (FisherScientific). To determine cell numbers, 10 l cell suspension were mixed with 90 l trypan blue solution by repeated pipetting in a 96-well plate. 20 l of the counting mix was transferred to a KOVA Glasstic slide and cells in 3 squares (a, b, c) including the top and right gridline were counted omitting the blue labeled dead cells. The cell number per ml was determined by the following equation: ((count (a)+count (b)+count (c)))/3)10.sup.410=cells per ml. 510.sup.6 cells per animal were transferred in a 50 ml Falcon tube and washed with PBS. The cells were resuspended in PBS to obtain a final concentration of 110.sup.8 cells per ml. Subsequently, the cell suspension was divided in 1 ml aliquots and stored on ice for maximum 30 minutes before injection.

(113) Engraftment of Calu6 cells into NMRI-Nude recipient mice: Calu6 cells were prepared as described above and resuspended by gently flicking the tube. Mice were anesthetized by isoflurane inhalation, the injection site was cleaned using a sterile alcohol swab. Using a 0.5 ml syringe with a 27G needle, 510.sup.6 cells in 50 l were administered subcutaneously (s.c.) in the right flank. Following injection of tumor cells, animals were checked bi-weekly for palpable lesions. After detecting a lesion, the animal was weighed and tumor size was measured. Tumors were measured using a caliper, values for length (cm) and width (cm) of the tumor were used to calculate the approximate tumor volume (cm.sup.3) using the following formula: Volume=Length(Width).sup.20.4. Length was defined regardless of orientation to the animal axes as being the longer dimension.

(114) Treatment by recombinant VSV vectors: When the mean size of tumors reached a volume between 0.05 and 0.07 cm.sup.3, tumors were treated. 1 cage of 8 mice was allocated per group. Allocation of the cages was done to obtain similar mean and distribution of tumor size between the different groups. For the negative control group, 30 l of PBS was injected i.t. For the other three treatment groups the final concentration of VSV-GP, VSV-GP(x) DANDV and VSV-GP(x) MOPV was 3.310.sup.8 TCID.sub.50 per ml. Dilutions of virus were done with PBS. Before injection, mice were anesthetized by isoflurane inhalation and the injection site was cleaned using a sterile alcohol swab. With a 0.1 ml syringe with a 29G needle, 110.sup.7TCID.sub.50 VSV-GP, VSV-GP(x) DANDV or VSV-GP(x) MOPV in 30 l was injected i.t. per tumor and animal. Treatments were repeated accordingly two times four days apart.

(115) Monitoring Tumor growth: After treatment, the animal were examined and tumor size was measured at least twice a week with a maximal interval of 4 days. Tumors were measured as described above. If tumor size of one animal was measured in between for ethical reasons (e.g. just before sacrifice), at least all animal of this group were measured. Body weight of mice was determined once a week. The following endpoint criteria require euthanasia: (i) tumor volume exceeds 0.8 cm.sup.3 (ii) mice show weight drop >20% or (iii) tumor ulcerates. Dates of sacrifice were used to calculate Kaplan-Mayer survival curves.

Example 7: Neurotoxicity of VSV-G(x)-DANDV and -MOPV

(116) Wild type VSV infections can cause neurological symptoms when the virus gets access to the brain. These neurological complications include a severe encephalitis that can lead to death of the infected subject. The advantage of using a chimeric VSV-GP is that neuronal infection has been shown to be nearly completely absent rendering the VSV-backbone a safe oncolytic agent. The reason for the attenuated phenotype is thought to be due to an altered virus tropism facilitated via the viral envelope glycoprotein. Alterations or modifications of the GP glycoprotein might affect the tropism profile of VSV-GP therefore a neurotoxicity assessment of VSV-G(x) DANV and of VSV-G(x) MOPV was done. Both VSV-G(x) DANDV and VSV-G(x)MOPV did not show any signs of neurotoxicity after direct intracranial injection of Swiss CD1 mice (FIG. 13) as shown for VSV-LCMV GP. All control mice that received VSV-G DsRed died within the first week post infection.

(117) Mice. 8 weeks old female Swiss CD-1 mice were purchased from Janvier (France). The animals were housed in individually ventilated cages in groups of 5 animals. Mice were ear clipped for identification purpose. The animals were acclimated for at least one week before start of the experiment.

(118) Viruses. For each experimental group the designated virus stock was thawed on ice. The viruses were diluted to 110.sup.6TCID.sub.50/3 l in PBS. The overall volume was 100 l. 80 l of the virus suspension was used for stereotactic injection of one group of mice (n=5). The remaining 20 l were used for titration according to standard procedure. Serial log dilutions of the sample covered the range of 110.sup.5-110.sup.10 TCID50/ml. Virus titration was done within one hour after preparation of the virus dilution for stereotactic injection.

(119) Stereotactic Injection. Mice were weighted and an appropriate dose of Ketamine and Xylazine (100 l per 10 g body weight) was injected i.p. After anesthesia had started, the skull between eye line and occiput was shaved properly and the eyes were protected with ointment (bepanthen crme). The mouse was placed on the stereotactic frame by fixing the ear bars into the external auditory canal bilaterally and enclosing the upper jaw with the jaw bar. The skin was antiseptically cleaned with betaiodine in ethanol before a linear skin incision over the midline along the antero-posterior axis was done. The galea-periosteum was scraped with centrifugal motion away from the midline and the bone was dried with a cotton swab with the same motion. After identification of the Bregma, the needle tip of the Hamilton syringe fixed in the stereotactic frame was adjusted directly over the Bregma. Then the needle was moved to the target location 0.4 mm rostral and 2 mm right lateral of the Bregma and the position was marked with a scalpel tip and the needle was removed again. Using an electric drill a burr hole was made at the target location and bone chips and dust was removed from the lesion with a PBS moistened cotton swab.

(120) The syringe was filled up with prepared virus solution using the fast reverse mode of the automated injector. After fast forwarding the syringe plunger until the fluid forms a little droplet at the tip of the needle, it was cleaned with a moistened cotton swab. After the needle tip was positioned to the burr hole 0.4 mm rostral and 2 mm right lateral of the Bregma, the needle was lowered down to the desired coordinate at 3 mm deep. Then automated injection procedure was started. After the injection was finished, the needle was left in place for 1 min to prevent back-flow through the needle trajectory. Then the needle was slowly withdrawed from brain and scull. Before the wound was closed using Vetbond 3M, the lesion was cleaned with PBS. Then the wound was wiped with betaiodine to prevent infection and the animal was transferred to recovery cage equipped with a heating pad to prevent hypothermia.

(121) Post-operative analgesia was provided in accordance with FELASA requirements for postoperative analgesia in rodents. Briefly, 30 mg/kg/day of Ibuprofen was supplied per os in the drinking water for 72 hours. The animals were monitored from the completion of surgery until recumbence and full mobility was achieved. In the first day animals were monitor several times and then daily for opening, discharge, redness or other possible signs of infection at the wound site.

(122) Evaluation of neurotoxicity. After treatment, the animals were examined twice a day for one week or until all VSV-G animals had been sacrificed. After that, animals were monitored daily until day 40 post treatment. Scoring parameters include body weight, motility, appearance and body condition as well as clinical and provoked behavioral signs of neurotoxicity (FIG. 12). A cumulative toxicity score was calculated by addition of the particular scores. Euthanasia was applied according to defined endpoint criteria.

(123) TABLE-US-00002 SEQUENCES SEQIDNO:1 1 atggggcagctcataacaatgtttgaggctttgccccacatcatcgatgaggttatcaac 61 atagttataattgtgcttgtgataataacaagcataaaggctgtgtacaactttgctacc 121 tgtggcattattgcactgatcagcttttgcctcctagctggcagatcttgtggtttatat 181 ggtgtcaccggtcctgacatttacaaaggactctaccaattcaagtccgtggagttcaac 241 atgtcacagctgaatttgacgatgcccaatgcatgttcagccaacaactcccaccattac 301 atcagtatggggaagtctggcttggagctgacttttacaaatgactccatcatcagtcac 361 aacttttgcaacctgactgatgggttcaagaaaaaaacctttgaccacacgctcatgagt 421 atagtggcaagcctacaccttagcatcagaggaaacaccaactataaggctgtttcctgt 481 gattttaacaatggaatcaccatccaatacaacttgtctttctctgatgcacaaagtgcc 541 atcaatcaatgcagaacttttagaggtagagttctagacatgttcaggacagctttcgga 601 gggaagtacatgaggtccggctatggttggaagggctctgatgggaaaactacttggtgc 661 agtcagaccagttatcaatacctaatcatacagaacagaacatgggagaatcactgtgag 721 tacgccggtccttttggcctctcaagggttctctttgctcaggaaaaaacaaaatttctc 781 actaggagattggcagggacttttacttggacactgtcggattcctcaggaactgagaac 841 ccaggtgggtactgtctgacaaagtggatgcttatagctgctgaactcaagtgttttgga 901 aacactgctgttgctaaatgcaatatcaatcatgatgaggaattctgtgacatgttgagg 961 ctaattgattacaacaaggctgctctgaagaaattcaaagaagacgtagagtctgccctt 1021 catctgttcaagacaactgtgaattctctaatatctgaccagctgttaatgagaaatcat 1081 ttgagggacttaatgggtgtgccctattgcaactactcgaaattctggtacttggagcat 1141 gtgaaaacaggtgatacaagtgttccaaaatgctggttggtttctaacggctcatactta 1201 aatgaaacacatttcagtgaccagatagagcaggaagcagacaacatgatcactgagatg 1261 ctcaggaaagattacatcaagaggcagggaagcactcctttggcactgatggacctatta 1321 atgttctctacatcggcatatttaatcagtgttttccttcatctgatgaaaatcccaact 1381 catagacacattaaaggtggcacatgccctaagccacacaggttaactagtaaaggcatc 1441 tgtagctgtggtgcgttcaaagtgccaggagtgaaaacagtctggaagagacgctga >DanedongNC_010248.1 SEQIDNO:2 MGQLITMFEALPHIIDEVINIVIIVLVIITSIKAVYNFATCGIIALISFCLLAGRSCGLYGVTGPDIYKGLYQFKSVEFNMSQLNLTMPNACSA NNSHHYISMGKSGLELTFTNDSIISHNFCNLTDGFKKKTFDHTLMSIVASLHLSIRGNTNYKAVSCDFNNGITIQYNLSFSDAQSAINQCRTFR GRVLDMFRTAFGGKYMRSGYGWKGSDGKTTWCSQTSYQYLIIQNRTWENHCEYAGPFGLSRVLFAQEKTKFLTRRLAGTFTWTLSDSSGTENPG GYCLTKWMLIAAELKCFGNTAVAKCNINHDEEFCDMLRLIDYNKAALKKFKEDVESALHLFKTTVNSLISDQLLMRNHLRDLMGVPYCNYSKFW YLEHVKTGDTSVPKCWLVSNGSYLNETHFSDQIEQEADNMITEMLRKDYIKRQGSTPLALMDLLMFSTSAYLISVFLHLMKIPTHRHIKGGTCP KPHRLTSKGICSCGAFKVPGVKTVWKRR SEQIDNO:3 1 atggggcagatagtcaccttctttcaagaggtgccacacatccttgaagaagtgat 61 gaacattgtgctgatgaccctctcaatcttggccatcctaaagggcatctacaatgtgat 121 gacctgtggaatcatcggtttgataacatttttgttcttgtgtgggagatcatgctcaag 181 catctataaggacaactatgagttcttctctctcgacctcgacatgtcttcactgaatgc 241 aacgatgcctctctcctgctcaaagaacaactcccatcactacatccaagttgggaatga 301 gacaggcctagagctgacattgacaaacactagcataattgatcataagttttgcaacct 361 gtctgatgctcacaggaggaatctttatgacaaagcacttatgtcaatcttgacaacatt 421 ccacctaagcattccagattttaaccaatatgaggcaatgtcatgtgattttaacggagg 481 gaagatctcaattcaatacaacctgtcccactcaaattatgtggatgccggcaaccactg 541 tggcactattgcaaatggcattatggatgtttttagaagaatgtattggagcacctccct 601 ttcggttgcctctgacataagtgggactcaatgcatacagaccgattataagtatttgat 661 tattcagaacacatcatgggaggatcattgcatgttctcaagaccctcacccatgggatt 721 tttaagccttctgtcacagagaaccaggaatttctacatctcaagaagacttttgggtct 781 ttttacatggactttgagtgactcggaagggaacgacatgccaggtggttactgtctcac 841 aaggtctatgctaatagggcttgatctgaaatgctttgggaacactgccattgcgaagtg 901 taatcaggcacatgatgaagaattctgtgacatgctccgcctttttgattttaataaaca 961 agcaattagcaaactgaggtctgaagttcagcagagcataaatttgataaataaagctgt 1021 caacgccctcatcaatgatcaactggtcatgaggaaccatttgagagatctaatgggtat 1081 tccttattgcaactactctaagttttggtacttgaatgatactaggacagggagaacatc 1141 cctcccaaagtgttggcttgtgaccaatgggtcatacctaaatgaaacccagttctcaac 1201 agaaattgagcaagaagccaataacatgttcactgacatgttgaggaaggagtatgagaa 1261 aaggcagagcacaacacctctggggttagtagatctttttgttttctccactagctttta 1321 tttgatctccgtgttcctccacctaatcaaaattccaacacatagacacataaaaggcaa 1381 gccttgcccgaaaccacacagactcaatcacatggcaatctgttcctgtggcttctataa 1441 gcaaccaggtctccccacacaatggaaaaggtga >MopeiaJN_561684.1 SEQIDNO:4 MGQIVTFFQEVPHILEEVMNIVLMTLSILAILKGIYNVMTCGIIGLITFLFLCGRSCSSIYKDNYEFFSLDLDMSSLNATMPLSCSKNNSHHYI QVGNETGLELTLTNTSIIDHKFCNLSDAHRRNLYDKALMSILTTFHLSIPDFNQYEAMSCDFNGGKISIQYNLSHSNYVDAGNHCGTIANGIMD VFRRMYWSTSLSVASDISGTQCIQTDYKYLIIQNTSWEDHCMFSRPSPMGFLSLLSQRTRNFYISRRLLGLFTWTLSDSEGNDMPGGYCLTRSM LIGLDLKCFGNTAIAKCNQAHDEEFCDMLRLFDFNKQAISKLRSEVQQSINLINKAVNALINDQLVMRNHLRDLMGIPYCNYSKFWYLNDTRTG RTSLPKCWLVTNGSYLNETQFSTEIEQEANNMFTDMLRKEYEKRQSTTPLGLVDLFVFSTSFYLISVFLHLIKIPTHRHIKGKPCPKPHRLNHM AICSCGFYKQPGLPTQWKR SEQIDNO:5 1 atgggacagat 61 aataaccttctttcaggaagtgcctcacattatagaggaagtcatgaacattgttctgat 121 caccctatccctcttggctatcttgaagggtgtgtacaatgtcatgacttgtggtctgat 181 tggattgatttcctttctcttactgtgtgggaagtcctgctctctaatctacaaggacac 241 atataacttctcatccattgaacttgacctctcacacctcaacatgacattgcccatgtc 301 ctgcagccgaaacaactcacaccactatgtcttcttcaacggatctggcttagagatgac 361 attcactaacgattcacttcttaaccacaaattctgcaatctttcagacgcccataaaaa 421 gaatctttacgatcatgcgctaatgggcattgtcacaactttccacttgtcaatcccgaa 481 cttcaatcaatacgaagcaatggcctgtgacttcaatggagggaatattagcattcaata 541 caacttgagccacaatgatcgaacagatgcaatgaaccactgtggcactgtagctaatgg 601 tgtactggatgctttttataggttccattggggcagaaatataacctacatcgctcagtt 661 gccaaatggcgacggcactggcagatggacattctgttatgccacaagttataagtacct 721 ggttatccaaaacattagctgggctgatcattgtcaaatgtcaagaccaactcccattgg 781 ctttgctagcattctgagccaacggattaggagcatttacatttcaagacgactgatgag 841 cacattcacttggtctctatccgacagttcaggtactgagaatccaggtggctattgtct 901 caccagatggatgctttttgcggctgacttgaaatgctttggaaacacagccattgccaa 961 atgtaacctgaatcatgatgaagagttctgcgacatgttgaggcttattgacttcaacaa 1021 gcaagccttgaaaacattcaaatcggaggttaaccatggtctacaactaattacaaaggc 1081 gattaatgccttgatcaatgaccaactcattatgaagaatcacttaagggacctaatggg 1141 tatcccttattgcaattattcaaaattctggtatctaaatgacacaagaacaggtagagt 1201 ctctctgcctaagtgctggatgatcagcaacgggacctatctaaatgagactcatttttc 1261 tgatgagattgaacaggaagcagacaatatgatcacagaaatgttgagaaaagagtatca 1321 agaaagacaagggaagaccccattgggtctagttgatctttttatttttagcacaagctt 1381 ttattcaatcacagtgttcctccatctcataaagattccaacgcacagacacatcgtggg 1441 acaagggtgtcccaagccacacagactaaacagtagagcaatctgctcctgtggtgccta 1501 taaacagccgggactacccaccaagtggaagcgctga >IppyNC_007905.1 SEQIDNO:6 MGQIITFFQEVPHIIEEVMNIVLITLSLLAILKGVYNVMTCGLIGLISFLLLCGKSCSLIYKDTYNFSSIELDLSHLNMTLPMSCSRNNSHHYV FFNGSGLEMTFTNDSLLNHKFCNLSDAHKKNLYDHALMGIVTTFHLSIPNFNQYEAMACDFNGGNISIQYNLSHNDRTDAMNHCGTVANGVLDA FYRFHWGRNITYIAQLPNGDGTGRWTFCYATSYKYLVIQNISWADHCQMSRPTPIGFASILSQRIRSIYISRRLMSTFTWSLSDSSGTENPGGY CLTRWMLFAADLKCFGNTAIAKCNLNHDEEFCDMLRLIDFNKQALKTFKSEVNHGLQLITKAINALINDQLIMKNHLRDLMGIPYCNYSKFWYL NDTRTGRVSLPKCWMISNGTYLNETHFSDEIEQEADNMITEMLRKEYQERQGKTPLGLVDLFIFSTSFYSITVFLHLIKIPTHRHIVGQGCPKP HRLNSRAICSCGAYKQPGLPTKWKR SEQIDNO:7 61 atgggtcaagtgattggattcttccaatcat 121 tgcccgagattattaatgaagcgttgaacattgctctgatctgtgtggctctgcttgcca 181 caatcaagggaatggtcaacatctggaaatctggtttgatacagctcctcttcttcctca 241 cattggcaggaagaagttgttctcattcttttacaattggaagatttcatgaatttcaga 301 gtgtgacagttaactttacacagttcatgtcctatgctccaagctcttgctcagtaaaca 361 acacccaccattatttcaaaggtccccaaaacaccacctggggtcttgagttgactttga 421 ctaatgaatcaatgataaacatcaccaattccatgagggtcttcactaacattcatcaca 481 atgtaaccaattgcgtgcagaacatttccgaacatgagggtgtgctcaaatggctgcttg 541 agacaatgcacctaagcatcagcaagccaggaaaacacatcgcacctgtcatgtgtgaga 601 gacaaaaggggttgctcatcgagtacaatctcactatgaccaaagaccaccacccaaatt 661 attggaatcaagtcttgtatggactggctaaattattgggctcaagtaagcgtttgtggt 721 ttggcgcatgtaacaaggctgactgtcagatgcaatcagaccaccagcacataaaatgca 781 attactcgaattgcaaaggttacaccagctttaaatacttaattattcaaaataccactt 841 gggaaaaccattgtgagtataaccatctgaacaccatccaccttctcatgagctctattg 901 gccaatcctttatcacaagaaggcttcaagcctttttaacctggacactttctgatgcac 961 tagggaatgaccttcctgggggatactgtcttgaacagtgggctgtggtgtggtttggca 1021 taaagtgttttgataacacagcaatggccaagtgcaatcagaatcatgattcagagtttt 1081 gtgatatgctaaggctttttgattacaatagaaatgctattcagagcttaaatgatcaat 1141 ctcaagccagattaaaccttttaactaacaccattaattcacttgtctctgacaatttac 1201 taatgaaaaataagctaagagaactaatgaacgtgccatactgtaattacactagattct 1261 ggtttattaatgatacaaagaatggtagacacactcttccacaatgttggttggtatcag 1321 atggttcgtacttgaatgaaaccagattcagaactcaatggctaagtgaaagtaactctt 1381 tatacacagaaatgctgactgaagaatatgaaaaaagacagggtagaacacctctttctt 1441 tagtggacctatgcttttggtcaactctcttttacatatcaacattatttgcgcatttag 1501 tgggtttcccaactcacagacaccttataggtgagggctgcccaaaaccacacagattaa 1561 cagggtcagggatctgctcatgtggacattatggtattccaggtaaaccagtcagatgga 1621 caaagatgtcaaggtga >LatinoNC_010758.1 SEQIDNO:8 MGQVIGFFQSLPEIINEALNIALICVALLATIKGMVNIWKSGLIQLLFFLTLAGRSCSHSFTIGRFHEFQSVTVNFTQFMSYAPSSCSVNNTHH YFKGPQNTTWGLELTLTNESMINITNSMRVFTNIHHNVTNCVQNISEHEGVLKWLLETMHLSISKPGKHIAPVMCERQKGLLIEYNLTMTKDHH PNYWNQVLYGLAKLLGSSKRLWFGACNKADCQMQSDHQHIKCNYSNCKGYTSFKYLIIQNTTWENHCEYNHLNTIHLLMSSIGQSFITRRLQAF LTWTLSDALGNDLPGGYCLEQWAVVWFGIKCFDNTAMAKCNQNHDSEFCDMLRLFDYNRNAIQSLNDQSQARLNLLTNTINSLVSDNLLMKNKL RELMNVPYCNYTRFWFINDTKNGRHTLPQCWLVSDGSYLNETRFRTQWLSESNSLYTEMLTEEYEKRQGRTPLSLVDLCFWSTLFYISTLFAHL VGFPTHRHLIGEGCPKPHRLTGSGICSCGHYGIPGKPVRWTKMSR SEQIDNO:9 61 atgg 121 gtcaagtcattgggttcttccaatccttacccaacatcattaatgaggcactaaacattg 181 ccctcatctgtgtggcattaattgccatattgaaaggtattgttaacatatggaaatcag 241 gtctgattcagctgttcatattcctcatcctagcggggaggagctgttctcacacctttc 301 agattggccgaaaccatgagttccaaagtatcactctaaacttcacacagtttttgggct 361 acgcacccagctcctgttctgttaacaacactcatcactactttagaggtcctggcaatg 421 tctcttggggcatcgagctcacattgaccaacaactccgtgatcaatgcaagcaactcac 481 tgaaggtgttcaccaacattcaccacaacatcaccaactgcgttcagaatatcgatgagc 541 aagaccacctaatgaaatggctcatagaaacaatgcatctacagatcatgaaaccaggaa 601 agagactaccaccaatcctgtgtgagaaagacaaagggttactaattgaatacaacctaa 661 ccaacattgcatctcgcgaagaaaagcactctgagtactggagccaacttctctatggtc 721 tatcaaaactattaggctccagtaagtcattgtggtttgattactgtcagagagcagatt 781 gcatgatgcaagagcacagctcacacttgaagtgcaactactcagagtgttctggccata 841 ccaccttcaaatacctcattctacaaaacaccacttgggagaaccattgtgagttcaacc 901 atctaaacacaattcacctgttgatgagctctactggtcagtctttcataactagaaggt 961 tgcaggcttttctaacttggactttgtcagatgctacaggcaatgacctccctgggggtt 1021 actgcctagagcagtgggccatagtttgggcaggcatcaagtgctttgggaacacagcag 1081 ttgcaaagtgtaatcaaaatcatgactctgaattctgtgacatgttgaggctctttgatt 1141 acaataggaatgctattaagagtctgaatgatcagtctcaaagtcgtctaaatcttctaa 1201 caaatacaataaattcactgatttcggataatctgttgatgaaaaacaagttggctgaga 1261 taatgaacattccgtattgcaattacacaaaattctggtacatcaacgacactaggactg 1321 gaaggcacacccttccccaatgctggttaatctcaaatgggtcttacttgaatgagacta 1381 aattcagaactcagtggctgtcagagagcaatgcactctatacagaaatgctaacagaag 1441 attatgacaaaaggcagggtagcacaccactctccttggttgatctatgtttttggtcaa 1501 ccttattctatgtgaccacattgtttgctcatcttgttggtttcccaacacacagacaca 1561 tcttagatggtccttgccctaaaccccacagattaacaaagaaaggaatttgttcctgtg 1621 gacattttggaataccgggcaaacctgttcgatgggtgaaacgatcacggtga >OliverosNC_010248.1 SEQIDNO:10 MGQVIGFFQSLPNIINEALNIALICVALIAILKGIVNIWKSGLIQLFIFLILAGRSCSHTFQIGRNHEFQSITLNFTQFLGYAPSSCSVNNTHH YFRGPGNVSWGIELTLTNNSVINASNSLKVFTNIHHNITNCVQNIDEQDHLMKWLIETMHLQIMKPGKRLPPILCEKDKGLLIEYNLTNIASRE EKHSEYWSQLLYGLSKLLGSSKSLWFDYCQRADCMMQEHSSHLKCNYSECSGHTTFKYLILQNTTWENHCEFNHLNTIHLLMSSTGQSFITRRL QAFLTWTLSDATGNDLPGGYCLEQWAIVWAGIKCFGNTAVAKCNQNHDSEFCDMLRLFDYNRNAIKSLNDQSQSRLNLLTNTINSLISDNLLMK NKLAEIMNIPYCNYTKFWYINDTRTGRHTLPQCWLISNGSYLNETKFRTQWLSESNALYTEMLTEDYDKRQGSTPLSLVDLCFWSTLFYVTTLF AHLVGFPTHRHILDGPCPKPHRLTKKGICSCGHFGIPGKPVRWVKRSR

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