Helper-free rescue system for negative-strand RNA viruses

Abstract

The present disclosure provides a method for producing a recombinant Mononegavirales virus in at least one host cell. The method comprises delivering a rescue composition to the at least one host cell, wherein the rescue composition comprises (i) a ribonucleic acid (RNA) sequence of a genome or antigenome of a non-segmented, negative-sense, single-stranded RNA virus of the Order Mononegavirales and (ii) at least one messenger RNA encoding trans-acting proteins necessary for encapsidation, transcription and replication of the virus. The method further comprises culturing the at least one host cell under conditions sufficient for production of the recombinant virus. The disclosure also provides compositions and systems for producing recombinant Mononegavirales viruses using the described rescue composition.

Description

HELPER-FREE RESCUE SYSTEM FOR NEGATIVE-STRAND RNA VIRUSESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 585,959, titled "Novel Rescue System for Non-segmented Negative-Sense Single-Stranded RNA Virus and Uses Thereof', filed September 28, 2023, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not Applicable.FIELD

[0003] The present disclosure relates to viral rescue systems, and more particularly to a helper-free rescue system for generating non-segmented negative-sense single-stranded RNA viruses.INTRODUCTION

[0004] Non-segmented negative-sense single-stranded RNA viruses (NNS-RVs) are a diverse group of pathogens that cause significant human and animal diseases. These viruses belong to the order Mononegavirales and include important human pathogens such as rabies virus, measles virus, and respiratory syncy tial virus. The genome of NNS-RVs consists of a single molecule of negative-sense RNA that serves as a template for both transcription and replication.

[0005] Reverse genetics systems have been developed for many NNS-RVs, allowing the generation of recombinant viruses from cloned cDNA. These systems have revolutionized the study of viral biology, pathogenesis, and the development of vaccines and therapeutics. However, current reverse genetics approaches for NNS-RVs often suffer from low efficiency and technical complexity.

[0006] Typical reverse genetics methods for NNS-RVs involve transfecting cells with multiple plasmids encoding the full-length viral antigenome and viral proteins necessary for replication and transcription. These systems frequently require the use of helper viruses, engineered cell lines expressing T7 RNA polymerase, or both. The need for multiplecomponents and specialized reagents can make these approaches cumbersome and limit their broader application.

[0007] The efficiency of virus recovery in existing systems is often low, particularly for attenuated strains or when introducing mutations that affect viral fitness. This inefficiency can be attributed to several factors, including the need to express multiple viral proteins from separate plasmids, potential cytotoxicity of viral proteins, and innate immune responses triggered by plasmid DNA transfection.

[0008] Another challenge in current reverse genetics approaches is the difficulty in recovering viruses in vivo or in primary cells, which may better reflect natural infection conditions. Most existing systems are optimized for use in cell lines that may not fully recapitulate the cellular environment encountered by the virus during natural infection.

[0009] There is a need for improved reverse genetics systems for NNS-RVs that offer greater efficiency, simplicity, and versatility. Such advancements could accelerate research into viral biology, facilitate the development of novel vaccines and therapeutics, and enable new approaches to studying virus-host interactions.SUMMARY

[0010] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0011] According to an aspect of the present disclosure, a method for producing a recombinant Mononegavirales Virus in at least one host cell is provided. The method includes conducting delivery of a rescue composition which comprises (i) a Ribonucleic acid (RNA) sequence of a genome or antigenome of a non-segmented, negative sense, singlestranded RNA virus of the Order Mononegavirales and (ii) at least one messenger RNA of the trans-acting proteins necessary for encapsidation, transcription and replication; under conditions sufficient to the production of the recombinant Virus.

[0012] According to other aspects of the present disclosure, the method may include one or more of the following features. The RNA sequence may comprise a genome or antigenome of a non-segmented, negative-sense, Single-Stranded RNA virus of the Order Mononegavirales that is a chimera of more than one genome or anti-genome Source. The RNA sequence may comprise a genome or antigenome of anon-segmented, negative-sense, Single-Stranded RNA virus of the Order Mononegavirales, that is a recombinant virus. TheRNA virus may be a virus of the Paramyxoviridae Family. The RNA virus may be a virus of the Rhabdoviridae Family. The RNA vims may be a vims of the Filoviridae Family. The RNA vims may be a virus selected from the group consisting of VSV, MV, RSV, PIV, and BPV. The RNA virus may be a virus VSV.

[0013] The RNA may comprise the genome or anti -genome of RSV and the mRNA may encode the trans-acting proteins necessary for encapsidation, transcription, and replication N, P, L. and M2. The RNA may comprise the genome or anti-genome of VSV and the mRNA may encode the trans-acting proteins necessary for encapsidation, transcription, and replication N, P, and L. The RNA may comprise the genome or anti-genome of MV and the mRNA may encode the trans-acting proteins necessary' for encapsidation. transcription, and replication N, P, and L.

[0014] The method may comprise RNAs, including viral antigenomic RNA or viral genomic RNA. The viral antigenomic RNA may include the hepatitis delta virus ribozyme sequence (6) used to generate the correct 3' terminus of the antigenomic RNA. The method may include mRNA of helper genes. The helper genes may be genes encoding trans-acting proteins necessary for virus encapsidation. The helper genes may be genes encoding transacting proteins necessary for virus transcription. The helper genes may be genes encoding trans-acting proteins necessary' for virus replication.

[0015] Helper genes, such as N, P, and L, may be delivered into cells in the form of mRNA for initiating virus replication and / or viral gene expression. The mRNA of helper genes may be organized into a polycistronic messenger RNA. The mRNA of N, P, and L genes may be delivered by their monocistronic messenger RNAs. The recombinant viruses may be generated by delivering the mRNA of N, P, and L into cultured cells or tumor tissue. The recombinant viruses may be generated by delivering engineered viral antigenomic RNA or genomic RNA into cultured cells or tumor tissue.

[0016] The method may include delivering siRNA and / or shRNA that inhibit genes of host antiviral pathways (i.e., PKR, RIG, interferon, etc.) into cells to enhance the virus rescue efficiency. For example, Co-transfect mRNA of the extracellular domain of Type I Interferon receptor (IFNAR) 1 and 2 can boost the VSV rescue efficiency. The extracellular domains neutralize IFN, allowing VSV to spread by preventing the stimulation of neighboring cells.

[0017] The method may include delivering mRNA of factors capable of counteracting host antiviral response to enhance the virus recovery efficiency. The rescue composition may further comprise RNA with unmodified nucleotides and / or modified nucleotides. RNA containing modified nucleotides may help improve the virus recoveryefficiency.

[0018] The disclosed methods may be applicable to produce all non-segmented, negative-sense, single-stranded RNA viruses of the order of designated Mononegavirales. The helper genes delivered by encoding mRNA may perform better than helper genes delivered by encoding DNA vector. The same concept may be applied to produce other viruses, such as lentiviral vectors and Adeno-associated viruses.

[0019] According to another aspect of the present disclosure, a recombinant virus prepared from any of the methods described above is provided.

[0020] According to another aspect of the present disclosure, an RNA composition for producing a recombinant virus as described above is provided.

[0021] These and other features, aspects and advantages of the present teachings will become better understood with reference to the following description, examples and appended claims.DRAWINGS

[0022] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0023] FIG. 1 illustrates a schematic representation of viral genomic and messenger RNA components, according to aspects of the present disclosure.

[0024] FIG. 2 depicts a process for virus recovery using RNA transfection, in accordance with example embodiments.

[0025] FIG. 3 shows a system diagram of the Vesicular Stomatitis Virus rescue and replication cycle, according to an embodiment.DETAILED DESCRIPTION

[0026] Abbreviations and Definitions

[0027] To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below as follows:

[0028] As used herein, the term "Mononegavirales" may refer to an order of viruses characterized by a non-segmented, negative-sense, single-stranded RNA genome. This order may include several families of viruses, such as Paramyxoviridae, Rhabdoviridae, and Filoviridae. Viruses in this order may share common features in their genomic organization and replication strategies, including a linear genome that is complementary to messengerRNA and encodes multiple genes in a single RNA molecule. The genomes of Mononegavirales viruses may be encapsidated by viral proteins to form a ribonucleoprotein complex, which serves as the template for both transcription and replication.

[0029] As used herein, the term "non-segmented negative-sense single-stranded RNA virus" or "NNS-RV" may refer to a virus with a genome consisting of a single molecule of negative-sense RNA. The genome of an NNS-RV may be complementary to messenger RNA and may serve as a template for both transcription and replication. NNS-RVs may belong to the order Mononegavirales and may include viruses from families such as Paramyxoviridae, Rhabdoviridae, and Filoviridae. The genome of an NNS-RV may encode multiple genes in a single RNA molecule and may be encapsidated by viral proteins to form a ribonucleoprotein complex. This genomic structure and organization may distinguish NNS- RVs from other types of RNA viruses, such as those with segmented genomes or positivesense RNA genomes. In some aspects, non-segmented negative-sense single-stranded RNA viruses (NNS-RVs) may include, but are not limited to: 1. Rhabdoviridae family: Vesicular stomatitis virus (VSV), Rabies virus, Chandipura virus, Bovine ephemeral fever virus; 2. Paramyxoviridae family: Measles virus. Mumps virus, Respiratory syncytial virus (RSV), Human parainfluenza viruses (HPIV), Nipah virus, Hendra virus, Canine distemper vims; 3. Filoviridae family: Ebola virus, Marburg virus, Cuevavirus; 4. Bomaviridae family: Boma disease virus; 5. Pneumoviridae family: Human metapneumo virus, Avian metapneumovirus: 6. Nyamiviridae family: Nyavirus; 7. Sunviridae family: Sunshine Coast vims; and 8. Mymonaviridae family: Sclerotinia sclerotiorum negative-stranded RNA virus I . Tn some cases, these viruses may exhibit different host ranges, tissue tropisms, and pathogenicities. The genomic organization and replication strategies of these NNS-RVs may share common features, such as a linear genome encoding multiple genes in a single RNA molecule and the formation of a ribonucleoprotein complex. The helper-free rescue system described in this disclosure may be applicable to various NNS-RVs, potentially facilitating research, vaccine development, and therapeutic applications across this diverse group of viruses.

[0030] As used herein, the term "helper-free rescue system" may refer to a method for generating recombinant non-segmented negative-sense single-stranded RNA viruses (NNS-RVs) without the need for helper vimses or engineered cell lines expressing viral proteins. This system may involve delivering a rescue composition comprising the viral genome or antigenome RNA and messenger RNAs encoding essential viral proteins directly into host cells. The helper-free rescue system may eliminate the requirement for additional viral components or specialized cell lines, potentially simplifying the virus recovery processand enhancing its efficiency. This approach may be applicable to various viruses of the order Mononegavirales and may facilitate the production of recombinant viruses for research, vaccine development, or therapeutic applications.

[0031] As used herein, the term "rescue composition" may refer to a mixture of nucleic acids designed for the production of recombinant viruses. This composition may include a ribonucleic acid (RNA) sequence representing the genome or antigenome of a nonsegmented. negative-sense, single-stranded RNA virus, as well as one or more messenger RNAs encoding trans-acting proteins necessary for viral encapsidation, transcription, and replication. In some aspects, the rescue composition may also contain additional components such as siRNA, shRNA, or mRNA encoding factors capable of modulating host cell responses. The rescue composition may be formulated for delivery into host cells to initiate the production of recombinant viruses without the need for helper viruses or specialized cell lines.

[0032] As used herein, the term "antigenome" may refer to a positive-sense RNA complement of the viral genome in non-segmented negative-sense single-stranded RNA viruses. The antigenome may serve as an intermediate in viral replication, acting as a template for the synthesis of new negative-sense genomic RNA. In some aspects, the antigenome may be encapsidated by viral proteins and may form part of the ribonucleoprotein complex involved in viral replication. The antigenome may contain the full-length sequence of the viral genome in the opposite orientation and may play a crucial role in the life cycle of non-segmented negative-sense RNA viruses.

[0033] As used herein, the term "trans-acting proteins" may refer to viral proteins that are encoded by the viral genome but act on other parts of the viral genome or on other viral components to facilitate viral replication, transcription, or encapsidation. These proteins may be produced separately from the viral genomic RNA and may interact with the viral genome or its products in trans, meaning they act on molecules other than the one that encoded them. In the context of non-segmented negative-sense single-stranded RNA viruses, trans-acting proteins may include the nucleoprotein (N), phosphoprotein (P), and large protein (L), which may form complexes essential for viral RNA synthesis and genome encapsidation. The specific functions and interactions of trans-acting proteins may vary among different virus families within the order Mononegavirales.

[0034] As used herein, the term "encapsidation" may refer to the process by which viral genomic or antigenomic RNA is packaged into a protein shell or capsid. In the context of non-segmented negative-sense single-stranded RNA viruses, encapsidation may involvethe assembly of viral nucleoproteins around the RNA molecule to form a ribonucleoprotein complex. This process may be essential for protecting the viral genetic material, facilitating viral replication and transcription, and enabling the formation of infectious virus particles. Encapsidation may occur in a specific sequence and may be mediated by interactions between viral proteins and RNA sequences or structures. The encapsidated RNA-protein complex may serve as the functional template for viral RNA synthesis and may be incorporated into progeny virions during virus assembly.

[0035] As used herein, the term "polycistronic messenger RNA" may refer to a single messenger RNA molecule that contains coding sequences for two or more distinct proteins. In the context of viral genetics, a polycistronic mRNA may encode multiple viral proteins, such as the N, P, and L proteins of non-segmented negative-sense single-stranded RNA viruses. This type of mRNA may allow for the coordinated expression of multiple genes from a single transcript. The translation of polycistronic mRNAs may involve mechanisms such as internal ribosome entry sites (IRES) or ribosome reinitiation to enable the synthesis of multiple protein products. In some aspects, the use of polycistronic mRNAs in viral rescue systems may enhance the efficiency of recombinant virus production by ensuring the simultaneous expression of essential viral proteins in appropriate ratios.

[0036] As used herein, the term "modified nucleotides" may refer to nucleotides that have been chemically altered from their standard structure. These modifications may include changes to the base, sugar, or phosphate components of the nucleotide. Modified nucleotides may be incorporated into RNA molecules to confer specific properties, such as increased stabi 1 i ty, resistance to nuclease degradation, or altered recognition by cellular machinery7. In some aspects, modified nucleotides may be used in the production of recombinant viruses to enhance the efficiency of virus recovery or to modulate the host immune response. The specific modifications and their effects may vary depending on the type of modification and the context in which the modified nucleotides are used. Nucleotide modifications may include: 1. Base modifications: Methylation (e.g. 5 -methylcytosine, N6-methyladenosine), Pseudouridylation, Inosine substitution, Thiolation (e.g. 2-thiouridine), Hydroxylation (e.g. 5- hydroxymethylcytosine); 2. Sugar modifications: 2'-O-methylation. 2'-fluoro substitution, 2'- O-methoxyethyl modification, Locked nucleic acids (LNA), Unlocked nucleic acids (UNA); 3. Phosphate backbone modifications: Phosphorothioate linkages, Phosphorodithioate linkages, Boranophosphate linkages, Methylphosphonate linkages; 4. Nucleobase analogs: 5- bromouridine, 5-iodouridine, 2-aminopurine, 7-deazaguanosine; 5. Non-standard nucleotides: Xanthosine, Diaminopurine, Zebularine; 6. Fluorescent nucleotide analogs: Cy3 / Cy5-labelednucleotides, Fluorescein-labeled nucleotides; 7. Photoreactive nucleotide analogs: 4- thiouridine, 5 -azidouridine; and 8. Nucleotide mimetics: Peptide nucleic acids (PNA), Morpholino oligonucleotides. In some cases, combinations of these modifications may be used to achieve desired properties such as increased stability, altered binding affinity, or modulation of immune recognition. The specific modifications selected may depend on the intended application and the properties required for the modified RNA molecule.

[0037] As used herein, the term "siRNA" may refer to small interfering RNA, which are short double-stranded RNA molecules typically 20-25 nucleotides in length. siRNA may function in the RNA interference (RNAi) pathway, where it interferes with the expression of specific genes with complementary nucleotide sequences. In some aspects, siRNA may be used to suppress host antiviral responses or modulate gene expression in host cells during virus production. The use of siRNA in viral rescue systems may enhance the efficiency of recombinant virus generation by temporarily inhibiting cellular pathways that could interfere with viral replication or assembly.

[0038] As used herein, the term "shRNA" may refer to short hairpin RNA, which are artificial RNA molecules designed to silence target gene expression through RNA interference. shRNA may consist of a sequence of RNA that forms a tight hairpin turn and can be expressed from within cells using a vector system. When processed by cellular machinery, shRNA may be cleaved to produce siRNA-like molecules. In some aspects, shRNA may be used in viral rescue systems to modulate host cell gene expression or suppress antiviral responses, potentially enhancing the efficiency of recombinant virus production. The use of shRNA may offer advantages over siRNA in certain applications, such as more stable and long-term gene silencing effects.

[0039] As used herein, the term "oncolytic virus" may refer to a virus that preferentially infects and kills cancer cells while sparing normal, healthy cells. These viruses may be naturally occurring or genetically modified to enhance their tumor-specific properties. Oncolytic viruses may replicate within cancer cells, leading to cell lysis and the release of new viral particles that can infect neighboring cancer cells. In some aspects, oncolytic viruses may also stimulate an anti-tumor immune response, potentially enhancing the overall therapeutic effect. The use of oncolytic viruses may represent a novel approach in cancer therapy, potentially offering a targeted treatment strategy with reduced side effects compared to traditional chemotherapy or radiation. In some cases, oncolytic viruses may be engineered to express therapeutic genes or immune-stimulating factors to further enhance their anticancer effects. Examples of oncolytic viruses may include: 1. Talimogene laherparepvec (T-VEC): A modified herpes simplex virus type 1 (HSV-1) approved for the treatment of melanoma; 2. Vesicular stomatitis virus (VSV): An RNA virus that may be engineered to selectively target cancer cells; 3. Adenoviruses: Various modified adenoviruses, such as ONYX-015 and Hl 01, which may preferentially replicate in p53-deficient cancer cells; 4. Vaccinia virus: Strains like JX-594 (Pexa-Vec) that may be modified to enhance tumor selectivity and anti-cancer effects; 5. Reovirus: A naturally occurring virus that may selectively replicate in cells with an activated Ras pathway, common in many cancers; 6. Newcastle disease virus (NDV): An avian paramyxovirus that may exhibit selective replication in cancer cells; 7. Measles virus: Attenuated strains that may be engineered to target specific receptors overexpressed on cancer cells; 8. Coxsackievirus: Particularly the A21 strain (CAVATAK), which may target ICAM-1 overexpressed in some cancers; 9. Poliovirus: Recombinant strains like PVS-RIPO that may be modified to selectively infect and kill cancer cells; 10. Seneca Valley Virus: A naturally occurring picomavirus that may show selective tropism for neuroendocrine cancers; 11. Maraba virus: A rhabdovirus that may be engineered to enhance its oncolytic properties; 12. Myxoma virus: A poxvirus that may exhibit natural oncolytic activity against various human cancers; 13. Parvovirus: Particularly the H-1PV strain, which may show selective replication in transformed cells; 14. Sindbis virus: An alphavirus that may be modified to target tumors through specific receptors; and 15. Bovine herpesvirus 4 (BoHV-4): A virus that may show natural tropism for certain cancer cells.

[0040] As used herein, the term "Pseudo-VSV" may refer to a modified form of Vesicular Stomatitis Virus (VSV) that is designed to mimic certain properties of the wildtype virus while lacking the ability to replicate independently. Pseudo-VSV may be engineered by replacing one or more of the VSV genes, typically the glycoprotein (G) gene, with a gene encoding a different viral envelope protein or a reporter gene. In some aspects, Pseudo-VSV may retain the ability to infect cells and express viral proteins, but may be unable to produce infectious progeny virions without complementation of the missing gene products. This type of virus may be used in various research applications, including studies of viral entry mechanisms, gene delivery, and vaccine development. In some cases. Pseudo- VSV may be produced using helper cell lines that express the missing viral proteins, allowing for the generation of single-cycle infectious particles.

[0041] As used herein, the term "in-situ generation" may refer to the process of producing or creating something directly at its intended site or location of use. In the context of viral production or gene therapy, in-situ generation may involve the synthesis or assemblyof viral particles or genetic elements within the target cells or tissues, rather than producing them externally and then introducing them. This approach may allow for the direct production of desired components in the specific cellular environment where they are needed. In some aspects, in-situ generation may offer advantages such as reduced handling steps, potentially improved efficiency, and the ability to overcome certain delivery7challenges associated with pre-formed particles or complexes.

[0042] As used herein, the term "Lentiviral vectors" may refer to gene delivery tools derived from lentiviruses, a subclass of retroviruses. These vectors may be engineered to efficiently deliver genetic material into both dividing and non-dividing cells, integrating the transferred genes into the host cell genome for long-term expression. Lentiviral vectors may be modified to remove viral genes associated with pathogenicity while retaining the ability to package and deliver genetic cargo. In some aspects, these vectors may be used for various applications in gene therapy, research, and biotechnology7, including the introduction of therapeutic genes, creation of stable cell lines, and generation of transgenic animals. Lentiviral vectors may offer advantages such as large packaging capacity, reduced immunogenicity, and the ability to transduce a wide range of cell types. Lentiviral vectors may include: 1. HIV-1 -based vectors: Derived from Human Immunodeficiency Virus type 1, these may be the most commonly used lentiviral vectors due to their well-characterized biology; 2. HIV-2-based vectors: Derived from Human Immunodeficiency Virus type 2, these may offer some advantages in terms of reduced pathogenicity; 3. SIV-based vectors: Derived from Simian Immunodeficiency Virus, these may be used in certain research applications, particularly in non-human primate studies; 4. FIV -based vectors: Derived from Feline Immunodeficiency Virus, these may be used in veterinary applications and some research settings; 5. EIAV-based vectors: Derived from Equine Infectious Anemia Virus, these may offer unique properties for certain gene therapy applications; 6. BlV-based vectors: Derived from Bovine Immunodeficiency Virus, these may be used in some research and agricultural applications; 7. Self-inactivating (SIN) vectors: These may contain deletions in the U3 region of the 3' LTR, reducing the risk of insertional mutagenesis; 8. Non-integrating lentiviral vectors: These may be engineered to deliver genetic material without integrating into the host genome, potentially reducing long-term risks; 9. Conditional lentiviral vectors: These may incorporate inducible promoters or other regulatory elements to control transgene expression: 10. Bicistronic lentiviral vectors: These may express two genes simultaneously, often using an internal ribosome entry site (IRES) or 2A peptide sequence; 11. Polycistronic lentiviral vectors: These may express multiple genes from a single vector, often using 2Apeptide sequences or IRES elements; 12. Tissue-specific lentiviral vectors: These may incorporate tissue-specific promoters or enhancers to restrict transgene expression to certain cell types; 13. Pseudotyped lentiviral vectors: These may incorporate envelope proteins from other viruses to alter tropism and enhance transduction efficiency in specific cell types; 14. Lentiviral vectors with microRNA target sequences: These may incorporate microRNA target sequences to regulate transgene expression in specific cell types or developmental stages; and 15. Lentiviral vectors with scaffold / matrix attachment regions (S / MARs): These may include S / MAR elements to potentially enhance transgene expression and reduce silencing.

[0043] As used herein, the term "Adeno-associated viruses (AAVs)" may refer to small, non-enveloped viruses belonging to the Parvoviridae family. AAVs may have a singlestranded DNA genome of approximately 4.7 kilobases and may require a helper virus, such as adenovirus or herpesvirus, for efficient replication. In some aspects, AAVs may infect both dividing and non-dividing cells and may integrate into the host genome at a specific site on chromosome 19 or persist as episomes. AAVs may be used as gene delivery vectors in various research and therapeutic applications due to their low immunogenicity, ability to transduce a wide range of cell types, and potential for long-term gene expression. In some cases, recombinant AAV vectors may be engineered to carry therapeutic genes or regulatory elements for gene therapy and vaccine development. Adeno-associated viruses (AAVs) may include: 1. AAV 1 : This serotype may show tropism for skeletal muscle, central nervous system (CNS), and pancreas; 2. AAV2: Often considered the prototype AAV, this serotype may transduce a wide range of tissues, including muscle, liver, and CNS; 3. AAV3: This serotype may demonstrate tropism for liver cells and may be used in hepatic gene therapy applications; 4. AAV4: This serotype may show tropism for retinal pigment epithelium and may be used in ocular gene therapy; 5. AAV5: This serotype may efficiently transduce lung epithelial cells and CNS, and may be used in gene therapies targeting these tissues; 6. AAV6: This serotype may show high transduction efficiency in skeletal muscle and may be used in muscular dystrophy gene therapies; 7. AAV7: This serotype may efficiently transduce skeletal muscle and retina, and may be used in gene therapies targeting these tissues; 8. AAV8: This serotype may show high liver tropism and may be used in liver-directed gene therapies; 9. AAV9: This serotype may cross the blood-brain barrier and may be used in CNS-directed gene therapies; 10. AAVrhlO: This rhesus monkey-derived serotype may show widespread CNS transduction and may be used in neurological disorder treatments; 11. AAV-DJ: This engineered serotype may be a chimera of multiple AAV serotypes and may show high transduction efficiency in multiple cell types; 12. AAV-PHP.B: This engineeredserotype may show enhanced CNS tropism and may be used in neurological gene therapies; 13. AAV -PHP. eB: This further evolved version of AAV-PHP.B may show even higher CNS transduction efficiency; 14. AAV-TT: This engineered serotype may show enhanced transduction in the retina and may be used in ocular gene therapies; 15. AAV-Anc80: This ancestral AAV seroty pe may show broad tissue tropism and may be used in various gene therapy applications; 16. AAV-LK03: This engineered serotype may show high liver tropism and may be used in liver-directed gene therapies; 17. AAV-NP59: This novel primate serotype may show enhanced transduction in neural progenitor cells and may be used in CNS-directed therapies; 18. AAV-NP66: Another novel primate serotype that may show enhanced transduction in the CNS and may be used in neurological disorder treatments; 19. AAV-F: This serotype may be isolated from human heart tissue and may show cardiac tropism, potentially useful in cardiovascular gene therapies; 20. AAV-pol : This porcine- derived serotype may show unique tissue tropism and may be used in xenotransplantation studies. In some cases, these AAV serotypes may be further modified or engineered to enhance their transduction efficiency, tissue specificity, or immune evasion properties. The choice of AAV serotype may depend on the specific target tissue, the therapeutic gene to be delivered, and the desired outcome of the gene therapy or research application.

[0044] As used herein, the term "Ribonucleoprotein complex" may refer to a macromolecular assembly composed of both RNA and proteins. In the context of viruses, particularly non-segmented negative-sense single-stranded RNA viruses, a ribonucleoprotein complex may consist of the viral genomic or antigenomic RNA encapsidated by viral nucleoproteins and associated with other viral proteins such as the RNA-dependent RNA polymerase. This complex may serve as the functional unit for viral transcription and replication. In some aspects, the formation of a ribonucleoprotein complex may protect the viral RNA from degradation and may be essential for the initiation of viral gene expression and genome replication. The specific composition and structure of ribonucleoprotein complexes may vary among different virus families and may play crucial roles in various stages of the viral life cycle.

[0045] Helper-Free Rescue System for Negative-Strand RNA Viruses

[0046] The following description sets forth exemplary aspects of the present disclosure. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure. Rather, the description also encompasses combinations and modifications to those exemplary aspects described herein.

[0047] The present disclosure provides a novel method for generating nonsegmented negative-sense single-stranded RNA viruses (NNS-RVs). This method, referred to as a helper-free rescue system, offers a significant improvement over existing methods in terms of efficiency, simplicity, and versatility. The disclosed system eliminates the need for helper viruses or engineered cell lines, which are typically required in conventional reverse genetics approaches for NNS-RVs. This simplifies the rescue procedure and dramatically enhances the rescue efficiency.

[0048] The disclosed method is particularly advantageous for generating recombinant NNS-RVs, which have significant applications in virotherapy and vaccine development. The method can be adapted for in vitro and in vivo virus recovery, offering flexibility in its application. Furthermore, the method can potentially enable the in-situ generation of oncolytic viruses directly from tumor tissues, which could revolutionize the production process of oncolytic viruses and significantly reduce the time and cost associated with virus production and purification.

[0049] In some embodiments, the method involves the delivery of a rescue composition comprising a Ribonucleic acid (RNA) sequence of a genome or antigenome of a NNS-RV and at least one messenger RNA of the trans-acting proteins necessary for encapsidation, transcription, and replication. The RNA sequence and the messenger RNA are delivered into a host cell under conditions sufficient for the production of the recombinant virus.

[0050] The present disclosure also encompasses various modifications to the described method, including the use of different RNA sequences and messenger RNAs, the use of additional components to enhance virus recovery efficiency, and the application of the method to different types of NNS-RVs.

[0051] Referring to FIG. 1, the figure illustrates the components and organization of the viral genomic RNA and messenger RNA (mRNA). The genomic RNA, as shown in part A of FIG. 1 , is a linear arrangement of viral genes, represented by boxes labeled N, P, M, G, and L. These genes are connected by short lines, indicating their sequential order in the genome. At the end of the sequence, there is a small box labeled with the Greek letter delta (5). This delta symbol represents the hepatitis delta virus ribozy me sequence, which is used to generate the correct 3' terminus of the antigenomic RNA. This is a critical step in the process of viral replication, as it ensures the correct formation of the viral genome.

[0052] In contrast, the structure of the viral mRNA, as shown in part B of FIG. 1. includes additional elements not present in the genomic RNA. The mRNA structure beginswith a "Cap" at the 5' end, followed by an untranslated region (UTR). The coding sequence includes the N gene, followed by an intergenic region (I), then the P gene, another intergenic region, and finally the L gene. At the 3' end of the mRNA, there is another UTR followed by a poly(A) tail. These additional elements in the mRNA, such as the cap, UTRs, and poly(A) tail, play crucial roles in the process of protein synthesis in the host cell.

[0053] In some aspects, the helper genes N, P, and L are organized into a polycistronic messenger RNA. as shown in part B of FIG. 1. This arrangement allows for the simultaneous expression of multiple genes from a single mRNA molecule, which can enhance the efficiency of viral protein production. The use of a polycistronic mRNA for the delivery' of helper genes is a novel aspect of the disclosed method, which distinguishes it from traditional methods that typically involve the delivery of helper genes via separate mRNA molecules or DNA plasmids. This innovative approach can significantly improve the efficiency of the virus rescue process and facilitate the generation of recombinant NNS-RVs.

[0054] Referring to FIG. 2. the figure illustrates a process for virus recovery' using RNA transfection. The process begins with the initial RNA components, including the viral antigenomic RNA and the messenger RNA (mRNA) of the helper genes N, P, and L. These RNA components are combined to form a transfection mixture, which is then introduced into a host environment, such as cultured cells or tumor tissue.

[0055] In some aspects, the host environment may be a cultured cell line that is compatible with the virus. Suitable cell lines may include, but are not limited to, BHK cells, Vero cells, HEK293 cells, or other cell lines that are permissive for the virus. The transfection mixture is introduced into the cells, where the mRNA of the helper genes is translated to produce the necessary' viral proteins, and the antigenomic RNA is encapsidated to form a functional nucleocapsid template.

[0056] The viral proteins N, P, and L, produced from the mRNA, play crucial roles in the virus rescue process. The N protein encapsidates the antigenomic RNA to form a nucleocapsid, which serves as a template for the synthesis of genomic RNA. The P and L proteins form a complex that acts as the viral RNA-dependent RNA polymerase, which engages the nucleocapsid template to achieve transcription and replication.

[0057] Following the transcription and replication processes, the newly synthesized genomic RNA serves as a template for the synthesis of more mRNAs and for the replication to produce more antigenomic RNA. The genomic RNA and viral proteins are then packaged into new virus particles, which are released from the host cell to complete the virus recovery process.

[0058] In some cases, the disclosed method may also be applied for in-situ generation of oncolytic VS V directly from tumor tissue. This involves delivering the viral antigenomic RNA and the mRNA of the helper genes N, P, and L directly into the tumor tissue. The in-situ generation of oncolytic VSV can potentially revolutionize the production process of oncolytic viruses, significantly reducing the time and cost associated w ith virus production and purification. This approach also alleviates the regulatory burden associated with removing confounding cellular components from the virus preparation.

[0059] Referring to FIG. 3, the figure illustrates a system diagram of the Vesicular Stomatitis Virus (VSV) rescue and replication cycle. The diagram is divided into two main sections: VSV rescue on the left and VSV replication cycle on the right.

[0060] In the VSV rescue section, the initial steps of virus generation are depicted. The process begins with the delivery of N, P, and L mRNA and antigenomic RNA into a host cell, such as a cultured cell or tumor tissue. Upon entry into the host cell, the mRNA undergoes translation to produce N, P, and L proteins. These proteins play crucial roles in the virus rescue process. The N protein encapsidates the antigenomic RNA to form a nucleocapsid, which serves as a functional template for the synthesis of genomic RNA. The P and L proteins associate with the nucleocapsid to form a ribonucleoprotein complex, which then generates genomic RNA.

[0061] In the VSV replication cycle section, the subsequent stages of virus production are illustrated. The genomic RNA serves as a template for the synthesis of messenger RNAs (mRNAs) and for the replication to produce more antigenomic RNA. The mRNAs are translated to produce N, P, M, G, and L proteins. The newly synthesized antigenomic RNA serves as a template for generating further copies of the genomic RNA. The genomic RNA and viral proteins are then packaged into new virus particles, which are shown budding from the cell membrane. Released VSV particles are illustrated at the top of the diagram.

[0062] This system provides a visual representation of how the initial rescue components lead to the establishment of a full replication cycle, resulting in the production and release of new virus particles. In some aspects, the disclosed method may be used to generate recombinant NNS-RVs by delivering the viral antigenomic RNA and the mRNA of the helper genes into a host cell or tumor tissue. The immediate expression of the helper genes from their mRNA can significantly improve the efficiency of the virus rescue process and facilitate the generation of recombinant NNS-RVs. In some cases, the method may also be applied for in-situ generation of oncolytic VSV directly from tumor tissue, which couldrevolutionize the production process of oncolytic viruses and significantly reduce the time and cost associated with virus production and purification.

[0063] Other procedures were performed to illustrate a comparison of Vesicular Stomatitis Virus (VSV) recover}' efficiency between two different methods: plasmid-based and RNA-based. Plaque Forming Units / mL were measured, and the RNA method performed significantly better than the plasmid-based method generating approximately 350 plaqueforming units per mL. This indicates a higher efficiency of VSV recover}' using the RNA- based method compared to the plasmid-based method.

[0064] In some aspects, the significant difference in plaque-forming units between the two methods suggests that the RNA-based method may provide a more efficient approach for VSV recovery. This could be due to the immediate expression of helper genes from their mRNA in the RNA-based method, which can significantly improve the efficiency of the virus rescue process. In contrast, the plasmid-based method may involve a more complex and timeconsuming process, including the transcription and translation of helper genes from DNA plasmids, which could potentially limit the efficiency of VSV recovery.

[0065] In some cases, the higher efficiency of the RNA-based method may also facilitate the generation of recombinant NNS-RVs, which have significant applications in virotherapy and vaccine development. The RNA-based method may also be advantageous for in-situ generation of oncolytic VSV directly from tumor tissue, which could revolutionize the production process of oncolytic viruses and significantly reduce the time and cost associated with virus production and purification.

[0066] In other embodiments, the RNA-based method may be combined with other techniques or modifications to further enhance VSV recovery efficiency. For example, additional components may be included in the transfection mixture to enhance virus recovery, such as siRNA and / or shRNA that inhibit genes of host antiviral pathways, or mRNA of factors that can counteract host antiviral response. In some cases, incorporating modified nucleotides into RNA can further improve virus recovery efficiency by sneaking from immune detection.

[0067] In some aspects, the helper genes N, P. and L may be delivered as separate monocistronic messenger RNAs (mRNAs). This approach allows for the independent expression of each helper gene, which may provide additional flexibility in controlling the timing and level of expression of each gene. For example, the expression of the N gene may be initiated earlier than the P and L genes to ensure the formation of nucleocapsids prior to the initiation of transcription and replication. Alternatively, the expression of the P and Lgenes may be delayed to allow for the accumulation of sufficient N protein for encapsidation. This flexibility in controlling the expression of helper genes may further enhance the efficiency of the virus rescue process.

[0068] In some cases, the RNA used in the rescue composition may contain modified nucleotides. The incorporation of modified nucleotides into RNA can provide several advantages in the virus rescue process. For instance, modified nucleotides may enhance the stability of the RNA, thereby increasing the efficiency of virus recovery. Modified nucleotides may also help to evade immune detection, which can be particularly beneficial in in vivo applications where the host immune response may limit the efficiency of virus recovery.

[0069] In other embodiments, the rescue composition may also include siRNA and / or shRNA that inhibit host antiviral pathways. The host antiviral response is a major obstacle in the recovery of NNS-RVs, as it can inhibit the initiation of viral replication and the expression of viral genes. By co-transfecting siRNA and / or shRNA that target key components of the host antiviral pathways, the antiviral response can be suppressed, thereby enhancing the efficiency of virus recovery.

[0070] In yet other aspects, the rescue composition may include mRNA of factors capable of counteracting the host antiviral response. These factors may include viral proteins or host proteins that are known to inhibit the antiviral response. The expression of these factors from their mRNA can help to establish a favorable environment for the initiation of viral replication and the expression of viral genes, thereby enhancing the efficiency of virus recovery.

[0071] These strategies and modifications provide additional tools for enhancing the efficiency and versatility of the helper-free rescue system for non-segmented negative-sense RNA viruses. By combining these strategies with the basic components of the rescue composition, it is possible to tailor the rescue process to specific applications and to overcome specific challenges associated with the recovery' of NNS-RVs.

[0072] In some aspects, the helper-free rescue system may be applied to produce other types of viral vectors. For instance, lentiviral vectors and Adeno-associated viruses (AAVs) may be produced using the disclosed method. These viral vectors are widely used in gene therapy and vaccine development due to their ability to deliver genetic material into host cells. By delivering the viral genome or antigenome and the mRNA of the helper genes into a host cell, the lentiviral vectors or AAVs can be generated efficiently. This approach may simplify the production process of these viral vectors and potentially enhance their yield.

[0073] In other cases, the helper-free rescue system may be used to generate non- replicative pseudo-VSV without supplementing VSV-G in trans for VSV-G / AG-VSV virus. Traditional pseudo-VSV using VSV-G / AG-VSV virus infecting cells transfected by the desired glycoprotein usually results in a substantial amount of VSV-G co-existing with the desired glycoprotein on the surface. However, with the disclosed method, only the desired glycoprotein on the surface of pseudo-VSV is developed. This is due to the superior recoveryefficiency of the disclosed method, which enables the in-situ generation of the oncolytic virus by delivering the (anti-)genomic RNA and mRNA of helper genes into tumor tissue, significantly saving the time and cost for virus production and purification. Moreover, there are fewer concerns about the contamination with host cell components and unknown or undetectable viruses potentially carried by animal-origin material. Therefore, the present invention also alleviates the regulatory burden.

[0074] EXAMPLES

[0075] Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.

[0076] Example 1 - NNS-RV Generation

[0077] The present invention relates methods for generating non-segmented negative-sense single-stranded RNA viruses (NNS-RVs). The disclosed methods simplify rescue procedures and dramatically improve rescue efficiency. The disclosed methods can be modified and applied to rescuing all engineered NNS-RVs.

[0078] The antigenomic or genomic RNA of NNS-RV is initially synthesized outside the host cell. Hepatitis delta virus ribozyme sequence is employed to generate the correct 3' terminus of the antigenomic RNA, which is placed directly after the 3’ terminus of the antigenomic RNA. The mRNA of the nucleocapsid protein (N) gene, polymerase cofactor phosphoprotein (P) gene, and RNA polymerase large protein (L) gene were co-delivered with antigenomic or genomic RNA into cells.

[0079] Upon entry into eukaryotic cells, the viral genes of N, P. and L will be expressed immediately from their mRNA. The newly synthesized N protein will encapsulate the naked, single-stranded viral antigenomic or genomic RNA into functional nucleocapsid templates. The viral polymerase (L) protein and its associated phosphoprotein (P) will comprise the active viral RNA-dependent RNA polymerase, which must engage this nucleocapsid template to achieve transcription and replication. The antigenomic strand is thetemplate for generating further copies of the genomic RNA. The genomic RNA serves as a template for synthesizing messenger RNAs (mRNAs) and the antigenomic strand (Figure 3).

[0080] 1. Generating non-replicative pseudo-VSV from cultured cells.

[0081] The disclosed method is suitable for generating non-replicative pseudo-VSV without supplementing VS V-G in trans for VSV-G / AG-VSV virus. Traditional pseudo-VSV using VSV-G / AG-VSV virus infecting cells transfected by the desired glycoprotein, the resulting pseudo-VSV usually has a substantial amount of VS V-G co-existed with the desired glycoprotein on the surface7. VSV-G is indispensable for amplifying recovered viruses when the virus rescue efficiency is low by the traditional recovery method. This disclosed recovery method is super-efficient, and VSV-G is unnecessary for generating pseudo-VSV with another glycoprotein. Only the desired glycoprotein on the surface of pseudo-VSV is developed with the disclosed method. To produce Pseudo-VSV in-vitro, seed cells (i.e., BHK cell, Vero cell, HEK293 cell, or other compatible cells for VS V) 20 hours before transfection. Ideally, the cells reach 70% to 80% confluency by the time of transfecting RNA complexes. Then, co-transfect cells with antigenomic RNA of AG-VSV, N, P, and L, mRNA of the desired glycoprotein for pseudotyping AG-VSV. Forty-eight hours later, the generated virus can be collected from the culture supernatant. The N, P, and L mRNA can be organized as shown in Figure IB, or their separate mRNA contains a 5’ cap and 3’ polyA tail for each gene. Figure 2 & 3 is the schematic process of this disclosed method.

[0082] 2. Generating replicative VSV from cultured cells.

[0083] To produce infectious VSV from cultured cells, seed cells (i.e., BHK cell, Vero cell, HEK293 cell, or other compatible cell lines for VSV) 20 hours before transfection. Ideally, the cells reach 70% to 80% confluency by the time of transfecting RNA complexes. Then, transfected cells with antigenomic RNA of recombinant VSV, mRNA of N, P. and L. Infectious VSV can be collected 24 hours after transfecting RNAs into cells. The N, P, and L mRNA can be organized as shown in Figure IB or the form of separate mRNA with a 5’ cap and 3’ polyA tail for each gene. Figure 2 & 3 is the schematic process of this disclosed method.

[0084] 3. In-situ generation of oncolytic VSV in tumor tissue.

[0085] When the engineered VSV is served as an oncolytic virus, the oncolyticVSV can be generated by intratumorally delivering its antigenomic RNA, mRNA of N, P, and L, as shown in Figure 2. The N, P, and L mRNA can be organized as shown in Figure IB or the form of separate mRNA with a 5’ cap and 3’ poly A tail for each gene.

[0086] To enhance the efficiency of recovering oncolytic VSV directly from cancer tissue, siRNA and / or shRNA that inhibiting host antiviral pathways (i.e., PKR, RIG, etc.), mRNA of factors can counteract host antiviral response can also be co-transfected. Incorporating modified nucleotides into RNA can further improve virus recovery efficiency by sneaking from immune detection.

[0087] The present invention is not to be limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the invention, and any constructs, viruses, or proteins that are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention, in addition to those shown and described herein, come apparent to those skilled in the art from the preceding description and accompanying drawings. The concept of delivering virus helper genes in the form of mRNA can significantly improve virus recovery and / or replication. It can be applied to producing other viruses, such as lentiviral vectors and Adeno-associated viruses.

[0088] This is further detailed in the Figures. Figure 1 provides a diagram of antigenomic RNA of recombinant VS V and the organization of polycistronic mRNA of helper genes. A. The antigenomic RNA of the VSV. The hepatitis delta virus ribozyme sequence (5) is used to generate the correct 3’ terminus of the antigenomic RNA. B. Their mRNA delivers the helper genes N, P, and L. In this exemplary figure, the translation of N is initiated by the 5’ cap, and the translation of P and L is undertaken by the internal ribosome entry site (I).

[0089] Figure 2 provides a diagram of VSV recovery process. The stages of virus rescue are shown schematically. mRNA of N, P, and L genes are co-transfected with antigenomic RNA of VSV; upon translation of the transfected mRNA, the replicative RNP is reconstituted with nucleocapsids, L and P protein. RNP will then initiate the expression of all viral genes and the replication of genomic RNA. Recombinant virus can be recovered within 24 hours post RNA transfection.

[0090] Figure 3 provides an overview diagram of exemplary recombinant VS V generation without helper plasmids, helper viruses, and engineered cell lines. Events that occur in the cell include (1) expression of the N, P and L genes from mRNA; (2) encapsidation of antigenomic RNA with N protein to form nucleocapsids; (3) Nucleocapsidsserved as the template for the synthesis of genomic strand (+), P protein and L protein form a complex of RNA dependent RNA polymerase; (4) Nucleocapsids of genomic RNA served as the template for the synthesis of messenger RNAs (mRNAs) as well as for that of the antigenomic strand (+), (6) encapsidation and packaging of nascent nucleocapsids, and (7) releasing packaged V SV.

[0091] Example 2 - Virus Plaque Assay Protocol

[0092] DEFINITIONS:

[0093] Plaque: an area of cells in a monolayer that display a cytopathic effect, e.g. appearing round and darker than other cells under the microscope or as white spots when visualized by the eye; the plaque centers may lack cells due to virus-induced lysis.

[0094] Plaque-forming unit (PFU): a virus or group of viruses that cause a plaque.

[0095] MATERIALS AND EQUIPMENT

[0096] Cell Growth Media (Advanced DMEM with 4 mM glutamine or glutamax, 5% fetal bovine serum)

[0097] Plaquing Medium (Advanced DMEM with 4 mM glutamine or glutamax, 2% fetal bovine serum)

[0098] PBS, without Ca2+and Mg2+

[0099] Agarose, UltraPure (Gibco Cat. No. 15510-019 or equivalent)

[0100] Tissue culture grade water (WFI)

[0101] Pipettors

[0102] Tubes to make your dilutions

[0103] 65°C water bath

[0104] Microwave oven

[0105] Vortex

[0106] Various tissue culture-grade sterile bottles of appropriate size (100-250 ml)

[0107] MTT (Sigma, M2128)

[0108] 6 well tissue culture dishes

[0109] Certain cell lines, such as Vero.

[0110] HARVEST AND PLATING OF CELLS IN 6-WELL DISHES

[0111] Make a solution of cells at 1 x 10A6 cells per ml in Growth Media.

[0112] Add 2.0 ml of grow th media with cells to each well. Jiggle the plate todistribute cells. 12 evenly to 6 o'clock and 9 to 3 o’clock (left-right, top-bottom). Incubate plates in a 37 °C. CO2 incubator.

[0113] After -12-24 hours, the cells should be -90-100% confluent. Go on to the next step immediately. Waiting several more days for cells to become completely confluent is not optimal.

[0114] VIRUS DILUTION AND INFECTION

[0115] Prepare dilutions of the virus in the DMEM medium. Never make more than a 100-fold dilution; always pipette at least 10 pl of virus or solution to reduce pipetting error.

[0116] Prepare 4 tubes; each tube will contain 1.5 ml of DMEM medium.

[0117] Transfer 15 pl of the virus sample into the first tube. Thoroughly mix by flipping the tube up and down.

[0118] Repeat the dilution process through all four tubes.

[0119] The tubes will now have the following effective virus dilutions: 10'2(l / l 00), IO’4(1 / 10,000), IO'6(1 / 1,000,000), and 10'8(1 / 100,000.000).

[0120] INFECTION OF THE MONOLAYERS

[0121] Pipette off and discard 1 mL of media from each well. One ml of media should now remain on each monolayer.

[0122] Add 100 pL or 10 pl of each dilution in duplicate to each well, letting the virus flow gently into the media.

[0123] [Consider this: If lOOpl of a 1 O'6dilution yields 25 plaques, the titer of the virus is 2.5x108PFU / ml. If lOpl of a 10'6dilution yields 30 plaques, the titer of the virus is 3.0 xlO9PFU / ml.]

[0124] Incubate the infected monolayers at 37 °C for 1 hour; shake the plates gently several times during this adsorption period.

[0125] AGAR OVERLAY

[0126] Prepare a sterile solution of 4% agarose in ddH2O by autoclaving at 121 °C for 20 minutes. Agarose may be stored on the shelf at room temp or used immediately after equilibrating in a 60 °C w ater bath. Alternatively, 100 ml aliquots of solidified agarose can be melted in a microw ave for about 1 minute and cooled to 60 °C in a water bath. Agarose can bum in the autoclave, so don't leave the agarose solution in a hot autoclave overnight.

[0127] Warm the plaquing media (see above, 2 ml per well to be overlaid) in a 37°C water bath until equilibrated. Make sure your media is at 37 °C.

[0128] Gently draw media from each virus-infected monolayer well and discard. An aspirating pipette attached to a vacuum is suggested.

[0129] Mix the volume of media you need (i.e., 5 plates, 30 wells, 60 ml) in a 37 °C pre-warmed container and add 10% volumes of liquid agarose to the bottle with swirling (1: 10 dilution). Immediately mix well and avoid bubbles.

[0130] With a new pipette, immediately but gently add 2 ml of the agarose / growth media mixture to each well, pipetting it down the side of the well.

[0131] Let the plate(s) sit for 15 minutes in the level hood at RT as the agar overlay turns solid.

[0132] Move the plate(s) to a humidified incubator at 37 °C and 5% CO2.

[0133] PLAQUE VISUALIZATION

[0134] Plaques will be visible by days 2 to 5 after infection, and if you desire, you can stain the monolayer to visualize and count the plaques on the final day of plaque development. With the naked eye, look for white dots on the monolayer. Viewing the plate with oblique light may make these dots more easily visualized. Note: It is critical to confirm that the dots are plaques by inspection under a microscope.

[0135] If desired, stain the plate(s) 2-5 days post-infection or when plaques fully develop. Stain for 3 hr at 37°C by adding 0. 1 volume of MTT solution (5 mg / ml in PBS) (Sigma, M2128). You can pick viable virus plaques through the MTT staining. Sterilize the MTT solution by filtration if plaques will be picked. The monolayer will appear blue / black, and the plaques will be clear areas. This method of staining gives much more evident plaques than neutral red staining.

[0136] PFU / ml CALCULATION

[0137] After counting plaques, you can calculate the concentration of the initial viral suspension in PFU / ml.

[0138] Take the dilution of the virus you plated and the volume of the virus solution you placed on the single monolayer to determine the PFU / ml.

[0139] Example: If you placed 10 pl of a 10'6dilution on a well and see 44 plaques, the titer from that well is 4.4 x 109PFU / ml; your initial virus suspension is 4.4 x 109PFU / ml.

[0140] Example: If you placed 100 pl of a 10 8 dilution on a well and see 88 plaques, the titer from that well is 8.8 x 1010PFU / ml.

[0141] Example 3 - Transfecting mRNA with Lipofectamine 2000

[0142] MATERIALS AND EQUIPMENT:

[0143] Advanced DMEM medium (Thermo Fisher, Cat#: 12491015)

[0144] Glutamax Supplement (Thermo Fisher, Cat#: 35050061)

[0145] Fetal Bovine Serum (Thermo Fisher. Cat#: 16140071)

[0146] PBS. without Ca2+and Mg2+

[0147] Cell culture flask or plate

[0148] Fluorescent Microscope

[0149] Opti-MEM MEM (Thermo Fisher, Cat#: 31985062)

[0150] Cells to be transfected.

[0151] TrypLE (Thermo Fisher, Cat#: 12605010)

[0152] Pipettors

[0153] Microcentrifuge tubes

[0154] Lipofectamine 2000 Transfection Reagent (Thermo Fisher, Cat#: 11668027)

[0155] PROCEDURES:

[0156] Prepare cell growth complete media: Advanced DMEM with 4 mM glutamax. 5% fetal bovine serum.

[0157] One day (typically 20 hrs) before transfection, seed cells to be 70-90% confluent at transfection.

[0158] Calculate the amount of RNA. lipofectamine. and Opti-MEM needed based on the following formulation: I pg RNA per ml cell culture. 1 pl lipofectamine per pg RNA, 100 pl Opti- MEM medium per pl lipofectamine.

[0159] Dilute the amounts of Lipofectamine reagent in Opti-MEM and mix well with a pipette.

[0160] Dilute RNA in Opti-MEM medium and mix well with a pipette.

[0161] Add diluted DNA to diluted Lipofectamine 2000 reagent (1:1 ratio) and mix well with a pipette.

[0162] Incubate the mixture for 5 to 10 minutes at room temperature.

[0163] Add RNA-Lipid complex to cells, and then incubate cells in a humidified, 37 °C. CO2 incubator for four hours.

[0164] Replace the cell culture media with pre-warm fresh cell culture media.

[0165] Visualize the transfected cells under the fluorescent microscope. Reporter gene expression can be detected 3 hrs post-transfection.

[0166] Analyze transfected cells. For the VSV rescuing experiment, collect cell culturesupernatant and perform a plaque assay to determine the virus titer.

[0167] Other Embodiments

[0168] The detailed description set-forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the present inventive discovery'. Such modifications are also intended to fall within the scope of the appended claims.

[0169] References Cited

[0170] All publications, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.

[0171] Specifically intended to be within the scope of the present invention, and incorporated herein by reference in its entirety, is the following publications:

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Claims

CLAIMSWhat is claimed is:

1. A method for producing a recombinant Mononegavirales virus in at least one host cell, the method comprising: delivering a rescue composition to the at least one host cell, wherein the rescue composition comprises (i) a ribonucleic acid (RNA) sequence of a genome or antigenome of a non-segmented. negative-sense, single-stranded RNA virus of the Order Mononegavirales and (ii) at least one messenger RNA encoding trans-acting proteins necessary for encapsidation, transcription and replication of the virus; and culturing the at least one host cell under conditions sufficient for production of the recombinant virus.

2. The method of claim 1, wherein the RNA sequence comprises a genome or antigenome of a non-segmented, negative-sense, single-stranded RNA virus of the Order Mononegavirales that is a chimera of more than one genome or anti-genome source.

3. The method of claim 1. wherein the RNA virus is selected from the group consisting of viruses of the Paramyxoviridae Family, viruses of the Rhabdoviridae Family, and viruses of the Filoviridae Family.

4. The method of claim 3, wherein the RNA virus is vesicular stomatitis virus (VSV).

5. The method of claim 1. wherein the at least one messenger RNA encodes the trans-acting proteins N, P, and L.

6. The method of claim 5, wherein the at least one messenger RNA is a polycistronic messenger RNA encoding the N, P, and L proteins.

7. The method of claim 1. wherein the rescue composition further comprises at least one of: siRNA, shRNA, or mRNA encoding factors capable of counteracting a host antiviral response.

8. The method of claim 1, wherein the RNA in the rescue composition comprises modified nucleotides.

9. The method of claim 1. wherein the at least one host cell is a tumor cell and the method produces an oncolytic virus.

10. The method of claim 1, wherein the extracellular domain of Type I Interferon receptor (IFNAR) 1 and 2 is also delivered.

11. A composition for producing a recombinant Mononegavirales virus, comprising:(i) a ribonucleic acid (RNA) sequence of a genome or antigenome of a nonsegmented. negative-sense, single-stranded RNA virus of the Order Mononegavirales; and(ii) at least one messenger RNA encoding trans-acting proteins necessary for encapsidation, transcription and replication of the virus.

12. The composition of claim 11, wherein the RNA sequence comprises a genome or antigenome of a non-segmented, negative-sense, single-stranded RNA virus of the Order Mononegavirales that is a chimera of more than one genome or anti-genome source.

13. The composition of claim 11, wherein the RNA virus is selected from the group consisting of viruses of the Paramyxoviridae Family, viruses of the Rhabdoviridae Family, and viruses of the Filoviridae Family.

14. The composition of claim 13, wherein the RNA virus is vesicular stomatitis virus (V SV).

15. The composition of claim 11, wherein the at least one messenger RNA encodes the trans-acting proteins N, P. and L.

16. The composition of claim 15, wherein the at least one messenger RNA is a polycistronic messenger RNA encoding the N, P, and L proteins.

17. The composition of claim 11, further comprising at least one of: siRNA, shRNA, or mRNA encoding factors capable of counteracting a host antiviral response.

18. A system for producing a recombinant Mononegavirales virus, comprising: a host cell; and a rescue composition comprising (i) a ribonucleic acid (RNA) sequence of a genome or antigenome of a non-segmented, negative-sense, single-stranded RNA virus of the Order Mononegavirales and (ii) at least one messenger RNA encoding trans-acting proteins necessary for encapsidation, transcription and replication of the virus.

19. The system of claim 18, wherein the RNA sequence comprises a genome or antigenome of a non-segmented, negative-sense, single-stranded RNA virus of the Order Mononegavirales that is a chimera of more than one genome or anti-genome source.

20. The system of claim 18, wherein the RNA virus is vesicular stomatitis virus (VSV) and the at least one messenger RNA encodes the trans-acting proteins N, P, and L.

21. The system of claim 20, wherein the at least one messenger RNA is a polycistronic messenger RNA encoding the N, P, and L proteins, and wherein the rescue composition further comprises at least one of: siRNA. shRNA, or mRNA encoding factors capable of counteracting a host antiviral response.

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