Transcription and replication particles and their use as antiviral therapies

Abstract

This document relates to methods and materials for treating viral infections. For example, this document provides viral minigenomes designed to express one or more therapeutic polypeptides within virus infected cells to reduce the production of infecting virus, thereby treating the virus infection.

Description

[0001] TRANSCRIPTION AND REPLICATION PARTICLES AND THEIR USE AS ANTIVIRAL THERAPIES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Patent Application Serial No. 63 / 655.548, filed on June 3, 2024. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

[0004] STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under Al 134937 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007] This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2340W01_SL.xml / ’ The XML file, created on May 27, 2025, is 3,806 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0008] TECHNICAL FIELD

[0009] This document relates to methods and materials for treating viral infections. For example, this document provides viral minigenomes designed to express one or more therapeutic molecules (e.g., anti-viral shRNA molecules and / or therapeutic polypeptides) within virus infected cells to reduce the production of infecting virus, thereby treating the virus infection.

[0010] BACKGROUND

[0011] Filoviruses are filamentous enveloped viruses belonging to the family Filoviridae, in the order Mononegavirales. Some filovirus members, such as Ebola virus (EBOV) and Marburg virus, cause severe hemorrhagic fever in humans and non-human primates. The filovirus ribonucleoprotein complex (the "nucleocapsid") forms a double-layered helical structure in which a non-segmented. single-stranded, negative-sense RNA genome is encapsidated by the nucleoprotein (NP), viral protein 35 (VP35), VP24, VP30 and RNA- dependent RNA polymerase. The inner layer consists of the helical NP-RNA complex, acting as a scaffold for the binding of VP35 and VP24 that constitute the outer layer. Life-cycle modeling of EBOV minigenome and transcription and replication virus-like particles (trVLP) have been used to mimic the biology of this Biohazard Level 4 (BSL-4) pathogen under safer Biohazard level 2 (BSL-2) conditions.

[0012] SUMMARY

[0013] This document provides methods and materials for treating viral infections. For example, this document provides viral minigenomes designed to express one or more therapeutic molecules (e.g., anti-viral shRNA molecules and / or therapeutic polypeptides) within virus infected cells to reduce the production of infecting virus, thereby treating the virus infection. For example, a negative strand RNA viral minigenome construct can be designed to include a 3’ leader sequence followed by a nucleic acid sequence encoding one or more therapeutic molecules (e.g.. shRNA molecules capable of binding nucleic acid of an RNA virus, therapeutic polypeptides such as anti-viral polypeptides, and / or antibodies such as neutralizing antibodies) and optionally one or more reporter polypeptides followed by a 5’ trailer sequence. Such a minigenome construct can be delivered to cells within a mammal (e.g., a human). In some cases, the 3’ leader sequence and 5’ trailer sequence can be designed to be from the target virus to be treated and one or more therapeutic molecules can be designed to be effective against the target virus to be treated. For example, when treating an EBOV infection, a 3’ leader sequence and 5’ trailer sequence can be designed to be an EBOV 3’ leader sequence and an EBOV 5' trailer sequence, and a sequence located between the EBOV 3’ leader sequence and the EBOV 5’ trailer sequence can be designed to encode one or more therapeutic molecules (e.g., shRNA molecules capable of binding nucleic acid of an RNA virus, therapeutic polypeptides such as anti-viral polypeptides, and / or antibodies such as neutralizing antibodies) can be effective against EBOV. Optionally, another sequence located between the EBOV 3’ leader sequence and the EBOV 5’ trailer sequence can be designed to encode one or more reporter polypeptides.

[0014] As described herein, life-cycle modeling of EBOV minigenome and trVLP can be modified to yield trVLP therapies against negative-stranded RNA viruses (e.g., EBOV and other filoviruses). For example, a trVLP RNA can be generated in which the leader and trailer sequences from EBOV or another virus are positioned on either side of an RNA encoding a therapeutic transgene (e.g., an shRNA, antiviral protein, neutralizing antibody cDNA, inhibitor or immunostimulatory cDNA, or vaccine antigen). When this RNA is delivered in VLPs or in lipid nanoparticles, it can be delivered into cells in vivo where it will lie largely dormant until that cell is subsequently or concurrently infected with EBOV or any other virus type for which the therapy is designed. When the incoming virus expresses its replication and packaging proteins, they can amplify the therapeutic gene to inhibit the virus in that cell. The therapeutic genome also can be packaged into new particles that can enter other cells to again he dormant until reactivated.

[0015] In general, one aspect of this document features a nucleic acid comprising an RNA sequence or encoding an RNA sequence, wherein the RNA sequence comprises a 3’ leader of a target RNA virus, a nucleic acid encoding a therapeutic molecule effective against the target RNA virus, and a 5’ trailer of the target RNA virus. The target RNA virus can be a negative strand virus. The target RNA virus can be a filovirus. The target RNA virus can be Ebola virus. The therapeutic molecule can be an shRNA, an antiviral polypeptide, a neutralizing antibody, or a vaccine antigen.

[0016] In another aspect, this document features a method for treating a virus infection, or a suspected virus infection, of a target RNA virus. The method comprises (or consists essentially of or consists of) delivering a nucleic acid to cells of a mammal, wherein the nucleic acid comprises an RNA sequence or encoding an RNA sequence, wherein the RNA sequence comprises a 3’ leader of the target RNA virus, a nucleic acid encoding a therapeutic molecule effective against the target RNA virus, and a 5’ trailer of the target RNA virus, wherein replication of the target RNA virus within the cells drives expression of the therapeutic molecule, thereby reducing production of infectious virus particles of the target RNA virus within the cells. The target RNA virus can be a negative strand virus. The target RNA virus can be a filovirus. The target RNA virus can be Ebola virus. The therapeutic molecule can be an shRNA, an antiviral polypeptide, a neutralizing antibody, or a vaccine antigen.

[0017] Unless otherwise defined, all 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 pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0018] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0019] DESCRIPTION OF DRAWINGS

[0020] FIG. 1 is a diagram illustrating a minigenome structure for treating virus infections, according to one embodiment. In this example, antiviral transgene can be specifically amplified in virus-infected cells by ‘'stealing” viral proteins from ongoing infection and the antiviral transgene, which in turn can antagonize the life cycle of the wild infecting virus.

[0021] FIGS. 2A-2D are diagrams illustrating a proposed scheme of a minigenome approach for treating virus infections. The diagram illustrates delivery of minigenome RNA by particles (e.g., lipid nanoparticles (LNPs) or virus-like particles (VLPs) such as trVLPs) to virus-infected cells. The viral polypeptides can serve as helpers to amplify the antiviral transgene (FIG. 2A and FIG. 2B). In the absence of a viral infection (FIG. 2C and FIG. 2D), this therapeutic minigenome RNA can be silent and can be eventually degraded. In contrast, if the same cells are actively infected by a virus, then the GOI: gene of interest (e.g., therapeutic transgene) can be expressed, thereby treating the virus infection.

[0022] FIG. 3 is a graph plotting expression results showing that minigenome nucleic acid entitled MG-aVP24 reduces psiCHECK-2™-VP24 reporter activity. The minigenome entitled MG-aVP24 encodes for shRNA sequence-specific silencing of the EBOV VP24 gene as verified using a modified dual-luciferase psiCHECK™-2 vector (Promega).

[0023] FIG. 4 plots luciferase activity demonstrating that MG-aVP24 inhibits the expression of surrogate virus. Delivery of MG-aVP24 to surrogate Ebola virus-infected cells using trVLP treatment revealed reduced reporter activity and reduced expression of viral poly peptides VP24 and VP40.

[0024] FIG. 5 plots results demonstrating that MG-aVP24 inhibits 4cis-MG replication over time. The inhibitory effect of MG-aVP24 was examined by quantifying VP24 vRNA and mRNA using RT-qPCR analysis. Treatment with MG-aVP24 significantly reduced RNA quantify across all examined groups of RNA samples over time, compared to both untreated or control-MG treatment.

[0025] FIGS. 6A-6F. Proposed scheme of mimgenome therapeutic approach. FIG. 6A) Full-length EBOV genome. FIG. 6B) Replication and translation of minigenome therapeutic transgene cassette. FIG. 6C) Delivery of MG RNA by LNPs to a filovirus- infected cell. FIG. 6D) Delivery of MG RNA by trVLPs to a filovirus-infected target cell. FIG. 6E) Delivery of MG RNA by LNPs to anon-infected cell. FIG. 6F) Delivery' of MG RNA by trVLPs to a non-infected target cell. GOI: gene of interest (therapeutic transgene).

[0026] FIGS. 7A-7B. Expression of lcis-MG-anti-VP24 and silencing of VP24. FIG. 7A) Schematic illustrations of the working mechanisms of lcis-MG-anti-VP24. FIG. 7B) The indicated plasmids and RNAs were transfected into 293 cells and luciferase activity was measured 24 hours later. The cells were also co-transfected with plasmids expressing EBOV helper proteins. Renilla luciferase activity was normalized with firefly luciferase activity. Data are shown as percentage reduction of luciferase activity (7?em7 / a / firefly) relative to each negative control, which was set at 100%. MG-ctrl: Icis-MG vector, scr siRNA: scramble siRNA. Error bars represent SD of triplicate wells, ns > 0.05, **P < 0.01, **** / * < 0.0001; Ordinary One-way ANOVA was used for statistical analysis in all experiments: * p < 0.05, ** p< 0.01. *** p < 0.001, **** p < 0.0001. If there was no statistical significance for a given comparison, it was indicated as “ns”.

[0027] FIGS. 8A-8D. IVT lcis-MG-anti-VP24 RNA. FIG. 8A) In vitro transcription (IVT) of MG. FIG. 8B) Analysis of the integrity and size of IVT therapeutic MG RNA and control MG RNA using agarose gel electrophoresis. FIG. 8C) Sequence of anti-VP24 transgene (SEQ ID NO: 1) and qPCR primer binding sites. FIG. 8D) Scheme and results of IVT lcis-MG-anti-VP24 RNA amplification in the presence of EBOV helper proteins. Quantification of Icis-MG is measured by RT-qPCR targeting the payload region using primer set (qNP UTR R:5’-GCTTGGGGTAAAACATTGG-3’ (SEQ ID NO:2); qtRNA- F: 5 -ACTTGAACCCTGGACCCTCA-3’ (SEQ ID NO: 3)).

[0028] FIGS. 9A-9C. lcis-MG-anti-VP24 inhibits the replication and transcription of 4cis-MG. FIG. 9A) Illustration of 4cis-MG cassette and co-transfection of IVT Icis-MG- anti-VP24 RNA and 4cis-MG into 293 cells. FIG. 9B) Two-step RT quantitative real-time PCR was performed for quantification of VP24 vRNA level RT using vRNA specific primer and VP24 mRNA level RT with Oligo d(T) in P0 cells. Copy numbers were calculated based on a standard curve generated using serially diluted 4cis-MG plasmid DNA and normalized to copies per ng RNA. FIG. 9C) nLuc reporter activity and viral protein expression in helper plasmid pre-transfected Pl target cells. L+: Cells transfected with 4cis-MG and helper plasmids. L-: Cells transfected with 4cis-MG and helper plasmids except for EBOV L polymerase-expressing plasmid. An empty pCAGGS was used to account for total plasmid deficit.**P < 0.01, *** < 0.001, < 0.0001;

[0029] Ordinary One-way ANOVA was used for statistical analysis in all experiments: * p < 0.05, ** p< 0.01 , *** p < 0.001 , **** p < 0.0001. If there was no statistical significance for a given comparison, it was indicated as “ns”.

[0030] FIGS. 10A-10D. Production and analy sis of Icis-MG trVLP. FIG. 10A) Diagram of Icis-MG-mGL and lcis-MG-RBBP6549-57i-mGL plasmids, and fluorescence images showing mGreenlantem expression in cells co-transfected with plasmids expressing Icis- MG, EBOV helpers, and T7 polymerase. FIG. 10B) A schematic illustration of the Icis- MG-RBBP6 trVLP production. 293T cells are transfected with expression plasmids for all seven viral proteins, including RNP proteins NP, VP35, VP30, and L, and proteins VP40, GP and VP24 that involved in particle budding / packaging / entry, as well as a Icis-MG and the accessory' T7 RNA polymerase (T7) for initial transcription of MG plasmid. The vRNA is replicated through a cRNA intermediate by the viral proteins NP, VP35, and L and transcribed by these proteins and VP30 into mRNAs encoding RBBP6 peptide. vRNA minigenomes are packaged and bud as trVLPs. FIG. 10C) Silver staining of purified Icis-MG-mGL and lcis-MG-RBBP6 trVLPs. FIG. 10D) Both trVLPs either treated with TPCK trypsin (O.lug / ml) or not and producer cell whole cell lysate (WCL) were analyzed for presence of GP, VP40 and 0-tubulin with Western Blotting.

[0031] FIGS. 11A-11D. lcis-MG-RBBP6 trVLP inhibits the expression of4cis-MG.

[0032] FIG. 11A) Illustration of lcis-MG-RBBP6 trVLP infection in 293 cells pre-transfected with 4cis-MG, helper plasmids and Timl plasmid. Supernatant containing trVLPS with lcis-MG-RBBP6 or 4cis-MG from Pl cells was passaged 72 h after infection onto P2 cells that pretransfected with expression plasmids encoding the EBOV helper proteins, as well as Timl. Supernatant of each infection was continuously passaged every 72 h for a total of 4 passages. The means and standard deviations from 3 independent experiments are shown. FIG. 11B) Expression levels of viral protein VP40 and 0-tubulin in cells from each passage. FIG. 11C) Reporter activity in target cells pre-transfected with helper and Timl expressing plasmid. The nLuc signals in control Icis-MG-mGL trVLP-infected cells at each passage were set as 100%. The means and standard deviations from 3 independent experiments were plotted. FIG. 11D) Fluorescence imaging of cells from each passage, including Pl, without helper plasmids pre-transfected. **P < 0.01, ****p < 0.0001; Ordinary One-way ANOVA was used for statistical analysis in all experiments: * p < 0.05, ** p< 0.01. *** p < 0.001, **** p < 0.0001. If there was no statistical significance for a given comparison, it was indicated as “ns”. FIGS. 12A-12B. AAV therapeutic delivery. FIG. 12A) Expression of antiviral peptide RBBP6 and negative control mGL. FIG. 12B) Reporter assay and western blot showing antiviral effects of AAV-RBBP6 vs 4cis-MG.

[0033] FIGS. 13A-13B. In vivo LNP-mRNA delivery. FIG. 13A) Firefly luciferase imaging in mice. FIG. 13B) Western blot for VP40 of mouse liver.

[0034] FIGS. 14A-14C. Cre-activated reporter expression in mT / mG:LSL-Luc hybrid mice. FIG. 14A) Schematic of Cre-sensitive, dual reporter mice. FIG. 14B) Luciferase imaging of AAV-Cre-treated reporter mice. FIG. 14C) Confocal microscopy of the livers of reporter mouse tissues following AAV-Cre intravenous (IV) or intramuscular (IM) injection.

[0035] FIG. 15. PCCA mRNA Biodistribution in Mice Treated with AAVrhlO-P CCA. The bottom dotted line is the lower limit of quantitation of 50.

[0036] DETAILED DESCRIPTION

[0037] In some cases, a minigenome can be a truncated, defective viral genome containing the non-coding terminal regions that are recognized by the viral ribonucleoprotein (RNP) complex for genome replication and transcription. An illustration of an exemplar}' minigenome encoding a therapeutic molecule is shown in FIG. 1. Minigenomes encoding one or more therapeutic molecules (and optionally one or more reporter molecules) can be developed for a diverse range of viruses. As described herein, minigenomes (e.g., a negative-strand RN A minigenome or a positive strand RNA minigenome) can be used as antiviral platforms to express therapeutic genes. Such therapeutic minigenomes can outcompete viral replication and also can amplify expression of therapeutic genes specifically in virus-infected cells by “hijacking” viral proteins from an ongoing virus infection.

[0038] This document provides methods and materials for treating viral infections. For example, this document provides viral minigenomes designed to express one or more therapeutic molecules within virus infected cells to reduce the production of infecting virus, thereby treating the virus infection. For example, a negative strand RNA viral minigenome construct can be designed to include a 3 ’ leader sequence follow ed by a nucleic acid sequence encoding one or more therapeutic molecules (and optionally one or more reporter polypeptides) followed by a 5’ trailer sequence. It is understood that the nucleic acid sequence encoding one or more therapeutic molecules and the optional nucleic acid sequence encoding one or more reporter polypeptides is in the proper orientation and sense to result in production of the desired mRNA (e.g.. a desired siRNA) or production of the desired polypeptide upon expression as shown in FIGS. 2A-2D. In some cases, a positive strand RNA viral minigenome construct can be designed and used to treat positive strand RNA virus infections.

[0039] Any appropriate virus infection can be treated as described herein. For example, RNA virus infections can be treated as described herein. In some cases, a Filoviridae virus infection can be treated as described herein. For example, an EBOV and Marburg virus infection can be treated as described herein. In some cases, a negative strand RNA virus infection can be treated as described herein. In some cases, a positive strand RNA virus infection can be treated as described herein using a positive strand RNA viral minigenome construct.

[0040] Any appropriate mammal having, or at risk of having, a virus infection (e g., RNA virus infections such as an EBOV infection) can be treated as described herein. Examples of mammals that can have, or can be at risk of having, a virus infection (e.g., RNA virus infections such as an EBOV infection) and can be treated as described herein (e.g., by administering a mini genome construct described herein to the mammal) include, without limitation, humans, non-human primates such as monkeys, horses, bovine species, porcine species, dogs, cats, mice, and rats. In some cases, a human having a virus infection (e.g., RNA virus infections such as an EBOV infection) can be treated as described herein.

[0041] In some cases, a plant having, or at risk of having, a virus infection (e.g., RNA virus infections) can be treated as described herein. Examples of plants that can have, or can be at risk of having, a virus infection (e.g., RNA virus infections) and can be treated as described herein (e.g.. by delivering a minigenome construct described herein to the plants) include, without limitation, com, rice, soybean, wheat, beans, peas, tomato, and potato plants.

[0042] In some cases, when designing a minigenome construct to treat a virus infection (e.g., RNA virus infections such as an EBOV infection) as described herein, the 3’ leader sequence and 5’ trailer sequence can be designed to be from the target virus to be treated and one or more therapeutic molecules can be designed to be effective against the target virus to be treated. For example, when treating an EBOV infection, a 3‘ leader sequence and 5’ trailer sequence can be designed to be an EBOV 3‘ leader sequence and an EBOV 5 ’ trailer sequence, and a sequence located between the EBOV 3’ leader sequence and the EBOV 5’ trailer sequence can be designed to encode one or more therapeutic molecules (e.g., shRNA molecules capable of binding nucleic acid of an RNA virus, therapeutic polypeptides such as anti-viral polypeptides, and / or antibodies such as neutralizing antibodies) can be effective against EBOV. Optionally, another one or more sequences located between the EBOV 3’ leader sequence and the EBOV 5’ trailer sequence can be designed to encode one or more reporter polypeptides.

[0043] In another example, when treating a Marburg virus infection, a 3’ leader sequence and 5’ trailer sequence can be designed to be a Marburg virus 3' leader sequence and a Marburg virus 5’ trailer sequence, and a sequence located between the Marburg virus 3’ leader sequence and the Marburg virus 5’ trailer sequence can be designed to encode one or more therapeutic molecules (e.g., shRNA molecules capable of binding nucleic acid of an RNA virus, therapeutic polypeptides such as anti-viral polypeptides, and / or antibodies such as neutralizing antibodies) can be effective against Marburg virus. Optionally, another one or more sequences located between the EBOV 3’ leader sequence and the EBOV 5' trailer sequence can be designed to encode one or more reporter polypeptides.

[0044] A minigenome construct provided herein can be designed to encode any one or more therapeutic molecules. Examples of therapeutic molecules that can be encoded by a nucleic acid located between a 3’ leader sequence and a 5’ trailer sequence of a minigenome construct described herein include, without limitation, shRNA molecules, siRNA molecules, miRNA molecules, aptamer molecules targeting genes (e.g., viral polypeptides or host polypeptides) in negative-stranded or complimentary stranded, antiviral polypeptides (e.g., an interferon, protein kinase R (PKR), myxovirus resistance protein (Mx), or tetherin (BST-2)), intracellularly or extracellularly expressed neutralizing antibodies targeting a viral glycoproteins or other polypeptide, intracellularly or extracellularly expressed single-chain antibodies targeting a viral glycoproteins or other polypeptide, polypeptides that inhibit the viral life cycle (e.g., viral polypeptides functioning as dominant-negative inhibitors (e.g., Ebola virus NP600-615), host polypeptides (e.g., retinoblastoma binding protein 6 (RBBP6) or artificially generated peptides), polypeptides that inhibit or alleviate the host pathogenic immune response (e.g.. TNF inhibitors or IL-6 inhibitors).

[0045] Any appropriate method can be used to deliver a minigenome construct described herein to cells within a mammal (e.g., a human) or non-mammalian animal or plant. For example, particles such as LNPs or VLPs such as trVLPs can be used to deliver a viral minigenome provided herein to living cells (e.g., living cells of a mammal such as a human). In some cases, one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be formulated into a composition (e.g., a pharmaceutical composition) for administration to a mammal (e.g., a mammal having, or at risk of having, a virus infection). For example, LNPs containing one or more minigenome constructs provided herein can be formulated into a pharmaceutically acceptable composition for administration to a mammal having, or at risk of having, a virus infection. In some cases, one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and / or diluents. Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)). xylitol, sorbitol, mannitol, gelatin, polymers (e.g.. polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene- polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline such as phosphate-buffered saline (PBS), protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, and lecithin.

[0046] A composition (e.g., a pharmaceutical composition) containing one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be formulated into any appropriate dosage form. Examples of dosage forms include solid or liquid forms including, without limitation, gums, capsules, tablets (e.g., chewable tablets, and enteric coated tablets), suppositories, liquids, enemas, suspensions, solutions (e.g., sterile solutions), sustained- release formulations, delayed-release formulations, pills, powders, and granules. A composition (e.g., a pharmaceutical composition) containing one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be formulated for local or systemic administration.

[0047] A composition (e g., a pharmaceutical composition) containing one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be designed for oral or parenteral (including subcutaneous, intratumoral, intramuscular, intravenous, topical, and intradermal) administration. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0048] This document also provides methods and materials for using one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein. In some cases, one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be used for treating a mammal (e.g., a human) having a virus infection. For example, one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be administered to a mammal having a virus infection to treat the mammal. In some cases, administering one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein to a mammal (e.g., a human) having a virus infection can reduce production of the infecting virus within the mammal.

[0049] In some cases, one or more mini genome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be used for treating a mammal (e.g., a human) suspected to develop a virus infection. For example, one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be administered to a mammal suspected to develop a virus infection to reduce the severity of an infection from that virus within the mammal. In some cases, administering one or more minigenome constructs provided herein and / or particles containing one or more mini genome constructs provided herein to a mammal (e.g., a human) suspected to develop a virus infection can reduce production of the infecting virus within the mammal should that virus infect that mammal.

[0050] An effective amount of a composition (e.g., a pharmaceutical composition) containing one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be any amount that can reduce the severity' of a virus infection without producing significant toxicity' to the mammal. An effective amount of one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be any' appropriate amount. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., a virus infection) may require an increase or decrease in the actual effective amount administered.

[0051] The frequency of administration of a composition (e.g., a pharmaceutical composition) containing one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be any frequency that can reduce the severity of a virus infection without producing significant toxicity' to the mammal. For example, the frequency of administration can be from about three times a day to about once a w eek, from about twice a day to about twice a week, or from about once a day to about twice a week. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can include rest periods. For example, a composition containing one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be administered daily over a two-week period followed by' atwo-week rest period, and such a regimen can be repeated multiple times. As w ith the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., a virus infection) may require an increase or decrease in administration frequency.

[0052] An effective duration for administering a composition (e.g., a pharmaceutical composition) containing one or more minigenome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be any duration that can reduce the severity of a virus infection without producing significant toxicity to the mammal. For example, the effective duration can vary from several days to several weeks, months, or years. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition being treated.

[0053] In some cases, one or more mini genome constructs provided herein and / or particles containing one or more minigenome constructs provided herein can be administered to a mammal (e.g., a human) as the sole active agent to treat a mammal (e.g., a human) having, or suspected to develop, a virus infection.

[0054] The invention will be further described in the following example, which does not limit the scope of the invention described in the claims.

[0055] EXAMPLES

[0056] Example 1

[0057] EBOV was selected as a target given the limited availability of therapeutic options for treating infections by this virus. A monoci stronic-MG (Icis-MG) was constructed to encode an shRNA targeted to VP24, an EBOV polypeptide that is responsible for condensing viral nucleocapsids to facilitate genome packaging into the virion (fcis-MG- aVP24). The silencing effect of VP24 shRNA expressed from lcis-MG-aVP24 was verified using the Promega psiCHECK™-2 vector system, where the VP24 gene was fused to a reporter gene. The antiviral effect of lcis-MG-aVP24 was then evaluated using a tetra-cistronic EBOV MG (4cis-MG) that encoded nanoLuciferase as well as three EBOV proteins, including VP24, as a surrogate viral infection model. Cells were infected with transcription / replication-competent VLPs (trVLPs) that were produced by coexpressing lcis-MG-aVP24 and 4cis-MG along with RNP proteins, and luciferase activity and genome replication were quantified in the infected cells. Notably, a significant decrease in both luciferase activity and synthesis of 4cis-MG viral RNA was observed, indicating the antiviral effect of lcis-MG-aVP24 against the EBOV surrogate model. This therapeutic approach can be extended to target other RNA viruses, offering a versatile strategy against a wide range of infectious RNA viruses. See, FIGS. 1-5.

[0058] Example 2: Infection-sensing Minigenome Antiviral Molecular Therapies against Filoviruses

[0059] The results in this Example re-present and expand on at least some of the results provided in other Examples.

[0060] This Example describes a molecular therapy that uses the virus’ own proteins to combat themselves. In this approach, infection-sensing RNAs encoding therapeutic genes are flanked by viral promoter and packaging signals in negative-sense orientation. In the absence of a viral infection, these therapeutic minigenome RNAs do not express any transgene so remain silent. If the cell is infected, the virus will act as a helper virus providing viral proteins in trans to transcribe, replicate, and package the therapeutic minigenomes.

[0061] Results

[0062] Proposed Scheme of Therapeutic Minigenome Targeting EBOV

[0063] EBOV has a single-stranded, negative-sense RNA genome which contains seven genes for structural proteins, including nucleoprotein (NP), polymerase cofactor VP35. matrix protein VP40. surface glycoprotein (GP). transcriptional activator VP30, genome packaging VP24, and RNA-dependent RNA-polymerase (L) (Fig. 6A). The termini of the genome contain a 3’ leader, 5’ trailer, and untranslated regions (UTRs) that direct replication, transcription and genome packaging signals.

[0064] The proposed therapeutic MG was designed to encode an antiviral transgene flanked by the 3’ leader and 5’ trailer sequences in negative-sense orientation (Fig. 6B). In the presence of EBOV infection, the therapeutic MG RNA w as replicated and transcribed through the functions of EBOV RNP complex proteins, including NP, VP35, VP30, and L. leading to therapeutic gene expression. Viral ribonucleocapsids containing newly synthesized MG RNA, were condensed by VP24 and packaged into transcription / replication-competent virus-like particles (trVLPs) formed by VP40 (Fig. 6C). The released trVLPs displayed EBOV GP on their surface allowing them to deliver therapeutic MG RNA to EBOV target cells (Fig. 6D). These amplification cycles continued as long as the proteins supplied by EBOV were present. On the other hand, in non-infected cells, the therapeutic MG RNA remained silent and were eventually degraded, resulting in no therapeutic gene expression (Fig. 6E). When the MG RNA was delivered to cells viatrVLPs, it was transcribed by the RNP proteins that were packaged with the MG RNA. However, replication did occur unless the cell later became infected with EBOV (Fig. 6F).

[0065] In this study, the therapeutic MG was designed to encode a single transgene, referred to as monocistronic-minigenome (Icis-MG).

[0066] Therapeutic MG encoding Anti -VP 24 shRNA is Transcribed and Facilitates VP24 Silencing with EBOV RNP Proteins supplied in Trans.

[0067] It was evaluated whether targeting VP24 could selectively inhibit the production of infectious EBOV, while minimally affecting the packaging of therapeutic Icis-MG in trVLPs and its subsequent amplification.

[0068] To do this, a T7 polymerase-driven Icis-MG expression plasmid that encodes anti-VP24 shRNA sequence as a therapeutic payload was generated. Specifically, a single copy of anti-VP24 siRNA sequence flanked by precursor transfer RNA (pre-tRNA) sequences was introduced between the conserved EBOV 3’ and 5' ends (Fig. 7A). This design enabled the cleavage of VP24 shRNA from viral transcripts via pre-tRNA cleavage mediated by RNase P and RNase Z. The resulting anti-VP24 shRNA was further processed by Dicer to generate anti-VP24 siRNA (Fig. 7A).

[0069] The silencing effect of VP24 siRNA expressed from lcis-MG-anti-VP24 was evaluated using a dual-luciferase assay with psiCHECK2-VP24 containing the VP24 target sequence fused to Renilla luciferase (Fig. 7A). The functionality of this system was validated by a significant reduction (approximately 60%) in luciferase activity upon expressing 25 nM of synthetic anti-VP24 siRNA when compared to the scrambled siRNA control (Fig. 7B). When the lcis-MG-anti-VP24 plasmid was co-transfected with psiCHECK2-VP24, this resulted in 20% reduction in luciferase activity in the presence of EBOV RNP proteins when compared to co-transfection with the empty Icis-MG plasmid. Reductions in luciferase activity were not observed when the psiCHECK2 plasmid lacking the VP24 sequence was supplied in the system. These results provide the first evidence that the negative-sense MG RNA can be used to express siRNAs, effectively suppressing target gene expression in a sequence-specific manner.

[0070] In vitro Transcribed Therapeutic MG RNA encoding Anti-VP 24 shRNA amplifies in the presence of EBOV RNP Proteins.

[0071] Using the lcis-MG-anti-VP24 plasmid as a template, lcis-MG-anti-VP24 RNA was generated through in vitro transcription (IVT) by T7 RNA polymerase (Fig. SA). The synthesized MG RNA was resolved on a 0.8% agarose gel by electrophoresis, where it appeared as a single, distinct band corresponding to the expected size (Fig. 8B). To test whether 1 cis-MG-anti-VP24 RNA can replicate in cells, 293 cells were co-transfected with IVT MG RNA and helper plasmids expressing RNP proteins. The quantity of MG RNA was measured by RT-qPCR using primers specific to pre-tRNA and leader sequences on the 1 cis-MG-anti-VP24 RNA (Fig. 8C). The RT-qPCR result showed a significant increase in MG RNA copies from 6 to 24 hours after transfection (Fig. 8D). suggesting that lcis-MG-anti-VP24 IVT RNA was replicated in the presence of RNP complex proteins.

[0072] Therapeutic MG Encoding anti-VP24 shRNA Efficiently Inhibits Surrogate Virus Genome Replication, Transcription and Production of Infectious Particles.

[0073] To assess the antiviral effect of 1 cis-MG-anti-VP24 RNA, an EBOV tetracistronic-MG (4cis-MG) system was used as a surrogate virus. The 4cis-MG encodes three EBOV proteins VP40, GP and VP24 as well as a nanoLuciferase (nLuc) reporter. This design enabled modeling of the EBOV life cycle, including genome replication, transcription, packaging, and the production of infectious trVLPs, when RNP proteins were provided in trans (Fig. 9A). While infectious EBOV was required be handled in biosafety level 4 (BSL-4) laboratories due to its extreme virulence, 4cis-MG could be safely handled under BSL-2 conditions.

[0074] Using this system, the VP24 silencing effects of lcis-anti-VP24 by quantifying levels of VP24 mRNA expressed from 4cis-MG were evaluated. The l cis-anti-VP24 or empty MG w as co-transfected in producer cells (passage 0; P0) with 4cis-MG and helper plasmids (Fig. 9A). Under these conditions, a significant reduction in VP24 mRNA levels was observed in the presence of lcis-MG-anti-VP24 RNA compared to the Icis-MG- empty control (Fig. 9B, left). Although to a lesser extent than the therapeutic MG, the Icis-MG-empty control also exhibited an inhibitory effect relative to the L+ condition, where the cells were transfected with 4cis-MG and all helpers but not the Icis-MG (Fig. 9B, left). A similar trend was also observed in 4cis-MG vRNA levels when using the same qPCR primer set targeting the VP24 region showed a significant reduction in the presence of lcis-anti-VP24 compared to the empty MG control (Fig. 9B, right). These results suggest that lcis-anti-VP24 can effectively silence not only VP24 mRNA but also 4cis-MG vRNA by targeting the VP24 sequence on cRNA.

[0075] To assess whether the VP24 silencing effect negatively impacts the packaging of 4cis-MG into infectious trVLPs, supernatants from the previous PO cells were transferred to target cells (passage 1; Pl) (Fig. 9A-9B), and nLuc reporter signals in Pl cells were subsequently analyzed (Fig. 9C). A significant reduction of over 60% in the nLuc reporter signal with lcis-anti-VP24 RNA transfection was observed when compared to the control-MG (Fig. 9C). Similarly, transfection of 25 nM synthesized VP24 siRNA into PO cells, used as a positive control, reduced nLuc activity7by approximately 50% compared to scrambled siRNA. The luciferase activity in L-absent (L-) condition relative to L+ was less than 10%.

[0076] In addition to the nLuc reporter signal, the expression level of VP40 — one of the EBOV proteins encoded by the 4cis-MG — in Pl cells was evaluated. Consistent with nLuc activity, VP40 levels were reduced in both lcis-MG-anti-VP24 and synthetic VP24 siRNA transfections when compared to their respective controls (Fig. 9C). The level of VP40 expression under L- condition was below the detection limit as expected. Collectively, these findings suggest that lcis-MG-anti-VP24 inhibits the production of infectious 4cis-MG trVLPs through its VP24 silencing effect, thereby effectively suppressing replication of this surrogate virus.

[0077] Development of a Therapeutic Minigenome Encoding a Host Antiviral Protein-Derived Peptide.

[0078] Previous studies showed that the host protein retinoblastoma binding protein 6 (RBBP6) inhibits EBOV RNA synthesis by disrupting the interaction between EBOV NP and VP30 proteins (Batra et al.. Cell, 175: 1917 (2018); and Batra et al., EMBO J., 40:el05658 (2021)).These studies also identified a 23 -amino-acid peptide from RBBP6 (RBBP6549-571) as a mediator of this inhibitory effect. Given this, RBBP6549-571 was selected as an example of using host proteins as a payload in therapeutic MG.

[0079] RBBP6549-571 was fused to the green fluorescent protein mGreenLantem (mGL) at the N-terminus and cloned into the Icis-MG (lcis-MG-RBBP6549-57i-mGL). This construct was compared to its negative control, 1 cis-MG-mGL lacking the RBBP6 peptide, in the subsequent assays (Fig. 10A). When these MGs were transfected into PO cells, similar levels of mGL signals were observed in cells co-transfected with EBOV RNP plasmids (Fig. 10A). To assess the potency of trVLPs for therapeutic MG delivery, trVLPs containing lcis-MG-RBBP6549-57i-mGL or Icis-MG-mGL were then generated and thoroughly characterized. trVLPs were produced by transfecting lcis-MG-RBBP6s49-57i-mGL or Icis-MG-mGL plasmid along with plasmids for all seven EBOV proteins (Fig. 10B). trVLP production was quantified for protein content by BCA assay (data not shown). Silver staining and western blot analyses confirmed the presence of GP and VP40 in both trVLPs (Fig. 10C-10D). The protease protection assay showed complete digestion of GP following trypsin treatment, while VP40 remained intact, confirming the integrity of the viral particles w ith the presence of GP displayed on their surface (Fig. 10D).

[0080] Therapeutic MG RBBP6s49-57i trVLPs Inhibit Surrogate Virus Genome Replication. Transcription and Production of Infectious Particles.

[0081] The therapeutic effect of 1 cis-MG-RB B P6549 -571-mGL, delivered via trVLPs, was ultimately evaluated using the EBOV 4cis-MG surrogate virus (Fig. 11A). 293 cells were transfected with plasmids expressing 4cis-MG and helper proteins and subsequently treated with therapeutic trVLPs (Pl). A more than 50% reduction in nLuc activity with lcis-MG-RBBP6549 -571-mGL trVLPs was observed when compared to Icis-MG-mGL trVLPs (Fig. 11B). The supernatants from these Pl cells were then passaged onto fresh cells expressing helper proteins three times (from P2 to P4) (Fig. 11A). Notably, the inhibitory effect of lcis-MG-RBBP6549-57i-mGL on 4cis-MG replication became more pronounced with increased passages, demonstrating reduct ons of nLuc activity' approximately 60% in P2, 80% in P3, and 90% in P4, when compared to negative control Icis-MG-mGL trVLPs (Fig. 11B). Consistent with these observations, a concurrent decrease in the expression of VP40, encoded by 4cis-MG. was also detected in each passage (Fig. 11C), further supporting the effective inhibition of 1 cis-MG-RBBP6549-57i- mGL on 4cis-MG surrogate virus. Therapeutic MG or control MG were only expressed when helper proteins were provided in trans (Fig. 11D). The negative control trVLPs continued to amplify across passages, evidenced by an increase in mGL signals by passage 4 (Fig. HD) This indicated that control Icis-MG was able to utilize viral proteins provided by helper plasmids and 4cis-MG to replicate itself. In contrast, trVLPs containing Icis-MG- RBBP6s49-57i-mGL showed a progressive decline in mGL signals over several passages (Fig. 11D) in parallel to decreased replication of 4cis-MG (Fig. 11B-11C). These results provide strong support for the concept of therapeutic MG as an infection-sensing mechanism and highlight its safety potency. Together these results demonstrate that therapeutic minigenomes can suppress viral RNA replication and viral protein production when cells were challenged with EBOV trVLPs. These results also demonstrate that these infection-sensing minigenome therapies can be used to treat filoviruses and other viral pathogens.

[0082] Materials & Methods.

[0083] Plasmids and RNA:

[0084] The therapeutics expression minigenome plasmids lcis-MG-anti-VP24. Icis-MG- RBBP6549 -571 -mGL and their negative controls Icis-MG-empty, Icis-MG-mGL were constructed using a T7 polymerase-driven EBOV monocistronic minigenome plasmid backbone. Briefly, the synthesized transgenes mGreenLantem (mGL), RBBP6549-571- mGL, and anti-VP24 shRNA (GenScript Biotech, Piscataway, NJ) were individually cloned into BsmBI-v2 (New England Biolabs. Ipswich, MA)-linearized pATX-T7-lcis- EBOVMG-nLuc using In-Fusion cloning kit (Takara Bio, Shiga, Japan). The Icis-MG- empty construct was created by removing the nLuc reporter gene.

[0085] The T7 polymerase-driven 4cis-MG plasmid, encoding EBOV proteins VP40, GP, VP24 and nLuc luciferase, was constructed as described in Zell et al.. Preprints 2024:2024120940 (2024).

[0086] Expression plasmids for EBOV proteins and T7 polymerase in pCAGGs vectors were as described in Hoenen et al.. J. Virol., 80:7260-7264 (2006); and Watt et al., J. Virol., 88:10511-10524 (2014).31’82

[0087] The psiCHECK2-VP24 plasmid was created by inserting the VP24 full length sequence downstream of the Renilla luciferase gene in the psiCHECK2 plasmid (Promega, Madison, WI).

[0088] Icis-MG RNA was synthesized via in vitro transcription (IVT) using T7 RNA polymerase with the HiScribe® T7 High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA). Therapeutic MG plasmids and their respective negative controls, linearized with Mlul and Notl, were used as templates for synthesizing IVT RNAs.

[0089] Cell culture:

[0090] 293 (American Type Culture Collection [ATCC]; CRL-1573) and 293T (ATCC, CRL-3216) were grown in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 mg / mL streptomycin. All cells were incubated at 37°C in a humidified atmosphere equilibrated to 5% CO2.

[0091] Transfection and infection

[0092] Unless otherwise specified, all transfection reagents were purchased from Minis Bio (Madison, WI). TransIT-LTl was used for DNA transfection, TransIT-mRNA was used for RNA transfection, and TransIT-X2 was used for siRNA or siRNA mixed with DNA transfection, following the manufacturer’s instructions. All transfection complexes were incubated in Opti-MEM (Gibco) at room temperature for the recommended duration.

[0093] Before infecting 293 cells grown in 24-well plate with the trVLP-containing supernatant, the culturing medium was removed, and the cells were gently washed with PBS. Then, 100 pL of supernatant was added on top and incubated in a 37°C incubator with 5% CO2 for 1 hour, gently rocking the plate every 15 minutes. After incubation, 500 pL of DMEM culturing media was added to replace the supernatant. psiCHECK-2 assay

[0094] On the day prior to transfection, 100 pL of 293 cells (IxlO4cells) were added to a 96-well plate. The cells were co-transfected with lcis-MG-anti-VP24 and helper plasmids or synthesized siRNAs along with psiCHECK2-VP24 or psiCHECK2. Forty -eight hours post-transfection, the cells were lysed, and a dual-luciferase reporter assay (Promega, Madison, WI) was performed to measure Renilla luciferase and internal control firefly luciferase signals. The ratio of Renilla to firefly luciferase signals was normalized to the respective negative control, which was assigned a value of 100%. and expressed as a percentage of gene expression.

[0095] Minigenome assay

[0096] 293 cells were seeded at a density of 8 x 104cells per well in 24-well plates 24 hours before transfection. Cells (passage 0; P0 cells) were transfected with plasmids: 200 ng of pCAGGS-L, 25 ng each of pCAGGS-NP and pCAGGS-VP35, 15 ng of pCAGGS- VP30, 50 ng of pCAGGS-T7p, 100 ng of EBOV 4cis-MG-nluc, and 10 ng of internal control pGL4. 10[luc2] (Promega, Madison, WI). Therapeutic plasmids or RNAs were added 6 to 8 hours after transfection. At 72 hours post-transfection, supernatants containing trVLPs w ere harvested and centrifuged at 500 x g for 5 minutes to remove cell debris. One hundred microliters of cleared supernatants were used to infect 293 cells (passage 1; Pl cells), which had been pre-transfected for 24 hours with the same amount of helper plasmids as used for PO. Both PO and Pl cells were lysed in 100 pL of lx passive lysis buffer (Promega, Madison, WI) after 72 hours transfection or infection. Fifty microliters of the lysate were used to evaluate nLuc luciferase expression using the Nano- Glo dual-luciferase reporter assay (Promega, Madison, WI) according to the manufacturer’s instructions. The remaining cell lysate were saved for subsequent Western blot analysis.

[0097] Western Blot

[0098] Whole cell lysates were harvested after the removal of media and a single wash with lx PBS. Cells were extracted in lx passive lysis buffer supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich, Burlington, MA). Samples were incubated for 10 minutes at room temperature with rotation, followed by centrifugation at 16,000 x g at 4°C for 10 minutes to remove cell debris. Equal volumes of samples were mixed with 2x Laemmli sample buffer (Bio-Rad, Hercules. CA) containing 10 mM DTT and boiled on a heat block for 10 minutes before loading onto SDS-PAGE gels. Proteins were transferred to a PVDF membrane using a semi-dry transfer method at 15V for 45 minutes (Bio-Rad, Hercules, CA). Nonspecific binding to the membranes was blocked with 5% (w / v) skim milk powder solutions in TBS-Tween. Blots were probed overnight at 4°C with anti- VP40 (clone 5B12, IBT BioServices, Cat. No. 0201-017) and anti-VP24 (Polyclonal, SinoBiological, Cat.No.40454-T46). Afterward, the membranes were washed three times for 10 minutes each with TBS-Tween buffer, followed by incubation with HRP- conjugated secondary' antibodies. Western blot signals were detected using chemiluminescence by adding SuperSignal West Femto substrate (ThermoFisher, Waltham, MA) and imaged using the Bio-Rad ChemiDoc imaging system. After imaging, the membranes were probed with anti- -tubulin (polyclonal, Abeam, Cat. No. ab6046) for 2 hours at room temperature as a loading control.

[0099] RT-qPCR for mRNA and vRNA.

[0100] The therapeutic effects of lcis-MG-anti-VP24 and lcis-MG-RBBP6549-57i-mGL on 4cis-MG were examined by quantifying 4cis-MG RNA in transfected 293 cells. Cells in 24-well plates were lysed in 500 pL of TRIzol reagent for total RNA extraction at 6-, 24-, and 72-hours post-transfection using Direct-zol RNA Mini prep Kits (Zymo Research, Irvine, CA). The extracted RNA was eluted in 100 pL of RNase-free water and quantified using a NanodropOne (ThermoFisher). Two hundred ng of each isolated RNA was reverse transcribed (RT) with SuperScript IV Reverse Transcriptase (Thermo Fisher, Waltham, MA) for mRNA and vRNA using oligo d(T) and strand-specific primers, respectively, resulting in a 20 pL reaction volume. The resulting cDNAs were diluted in 180 pL of nuclease-free water. For the qPCR reaction, 8 pL of diluted cDNA was used as the template, mixed with 10 pL of SYBR green master mix (Applied Biosystems, Waltham, MA) and 2 pL of paired primer (500 nM). The qPCR reaction was loaded into a standard 96-well PCR plate and spun at 150 x g for 1 minute before running on the QuantStudio 3 Real-time PCR system (Applied Biosystems, Waltham, MA). 4cis-MG plasmid DNA was used to standardize genome copy numbers, expressed as “viral genome copies”. Genome copy numbers for each sample were determined by automatic analysis of threshold cycle (Ct) values with the QuantStudio 3 software and calculated based on the measured total extracted RNA amount. Results were expressed as “Viral genome copies per ng RNA”. Three independent experiments were performed, and one representative experiment with three technical repeats was plotted as mean ± SD with GraphPad Prism 10. trVLPs

[0101] To produce therapeutic Icis-MG containing trVLPs. 293T producer cells (P0) were seeded at a density of 1 x 106cells in 2.5 ml of DMEM supplemented with 10% FBS and 1% Pen / strep per well of 6-well plates. A ratio of 1 pg DNA to 3 pL of TransIT LTI (Mirus Bio, Madison, WI, USA) was used for transfecting the cells with 4 pg of pCAGGS-L, 1 pg of pCAGGS-GP, 0.5 pg of pCAGGS-NP, 1 pg of pCAGGS-VP40, 0.5 pg of pCAGGS-VP35. 0.3 pg of pCAGGS-VP30. 0.03 pg of pCAGGS-VP24, 1 pg of pC AGGS-T7, and 1 pg of 1 cis-MG. The media was changed to DMEM with 10% FBS and 1% Pen / strep 24 hours post-transfection.

[0102] Seventy -two hours post-transfection, 7.5 mb of supernatants from three wells were collected and centrifuged for 5 minutes at 150 x g in a benchtop centrifuge to remove cell debris. Seven mL of clarified supernatants were then applied to a 3 mL 20% sucrose layer in NTE buffer (10 mM Tris, ImM EDTA, 100 mM NaCl) in a Beckman centrifuge tube (Cat. No. C 14293) and centrifuged at 32,000 rpm in a SW32-TI rotor for 2 hours at 4°C using a Beckman L7 ultracentrifuge. Pellets were resuspended in 50 pL of NTE buffer, and total protein concentrations were determined using BCA assay (Pierce™ BCA Protein Assay Kits, Thermo Fisher, Cat. No.23225). Silver staining (Pierce™ Silver Stain Kit, Thermo Fisher, Cat. No. 24612) and Western blot were performed to assess individual viral proteins. PO transfected cells used to generate the trVLPs were lysed in IxPassive lysis buffer and analyzed by Western blot.

[0103] Statistical analyses

[0104] All experiments were performed as at least three independent biological replicates. Statistical analyses were performed using one-way ANOVA with GraphPad Prism 10 version 10.0.2.

[0105] Example 3: Adeno-associated virus (AA V)-mediated therapy

[0106] To test AAV for EBOV, AAV-CMVRBBP6 with RBBP6 fused with mGL was made (Fig. 12A). Infection of cells with AAV-CMV-RBBP mediated significant antiviral effects on EBOV 4cis-MG (Fig. 12B). This suggested that AAV can be used against EBOV.

[0107] Lipid nanoparticle (LNP)-mediated RNA delivery: LNPs were packaged with luciferase or EBOV VP40 mRNA using MC3 ionizable lipid on a NanoAssembler Ignite. 10 pg (by RNA) of these LNPs were injected IV into mice via tail vein and they were imaged on an IVIS imager. LNPs produced strong luciferase expression in the liver (Fig. 13 A) and EBOV VP40 protein expression in liver extracts by 6 hours post-injection (Fig. 13B)

[0108] Cre Reporter Mice Models for Sensitive Detection of In Vivo Trans gene Protein Delivery. A Cre "fingerprinting” system was use to track vector tropism in vivo (Fig. 14). This system uses mice that are transgenic for loxP flanked (floxed) reporter cassettes including a floxed membrane-targeted Tomato (mT) red fluorescence protein followed by an inactive membrane-targeted GFP (mG) (mT / mG mice) and a luciferase whose expression is blocked by an upstream floxed poly adenylation sequence (LSL-Luc mice)(Fig. 14). When the two mouse lines were crossed, their offspring had both cassettes (Fig. 14A). In the absence of Cre, all cells in the mouse fluoresced red and did not express luciferase. If Cre was delivered, mT was deleted and mG and luciferase were both activated allowing in vivo luciferase imaging (Fig. 14B) as well as confocal microscopy to identify tissues and single cells that were modified by a vector (Fig. 14C).

[0109] Compare the therapeutic MG using trVLPs, RNA, and AAV platforms against the 4cis-MG surrogate model in BSL-2 cell culture. AAV vectors were generated to express the negative stranded lcis-anti-VP24 and AAV-lcis-RBBP6 minigenomes by transferring these cassettes from the MG plasmids.

[0110] Because precise termini are needed for the leader and trailer sequences, the first version of the constructs encoded hammerhead ribozymes in the RNA to cleave at these ends. These AAV vectors along with their corresponding negative control transgenes were used to generate AAVrhlO particles from 293T cells. AAVrhlO was the most broadly tropic AAV serotype and transduced all tissues tested (Fig. 15).

[0111] OTHER EMBODIMENTS

[0112] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing descnption is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid comprising an RNA sequence or encoding an RNA sequence, wherein said RNA sequence comprises a 3’ leader of a target RNA vims, a nucleic acid encoding a therapeutic molecule effective against said target RNA virus, and a 5 ' trailer of said target RNA virus.

2. The nucleic acid of claim 1, wherein said target RNA virus is a negative strand vims.

3. The nucleic acid of claim 1, wherein said target RNA virus is a filovirus.

4. The nucleic acid of claim 1, wherein said target RNA virus is Ebola vims.

5. The nucleic acid of claim 1, wherein said therapeutic molecule is an shRNA, an antiviral polypeptide, a neutralizing antibody, or a vaccine antigen.

6. A method for treating a vims infection, or a suspected virus infection, of a target RNA virus, wherein said method comprises delivering a nucleic acid to cells of a mammal, wherein said nucleic acid comprises an RNA sequence or encoding an RNA sequence, wherein said RNA sequence comprises a 3’ leader of said target RNA virus, a nucleic acid encoding a therapeutic molecule effective against said target RNA virus, and a 5‘ trailer of said target RNA virus, wherein replication of said target RNA virus within said cells drives expression of said therapeutic molecule, thereby reducing production of infectious vims particles of said target RNA virus within said cells.

7. The method of claim 6. wherein said target RNA virus is a negative strand virus.

8. The method of claim 6, wherein said target RNA virus is a fdovirus.

9. The method of claim 6, w herein said target RNA virus is Ebola virus.

10. The method of claim 6. wherein said therapeutic molecule is an shRNA, an antiviral polypeptide, a neutralizing antibody, or a vaccine antigen.

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