Uses of lipid nanoparticles containing crispr-associated proteins (CAS) and guide RNA in managing viral infections

Abstract

Disclosed herein are compositions and methods for managing viral infections. In certain embodiments, this disclosure relates to compositions that specifically cleave target sequences in viruses, such as dengue virus. Such compositions include lipid nanoparticles containing nucleic acids encoding a Cas guide-RNA associated endonuclease, with a guide sequence of a viral target sequence. In certain embodiments, contemplated methods include treating a viral infection by administering lipid nanoparticles (LNPs) comprising mRNA encoding Cas13a and a guide RNA or multiple guide RNAs that target regions of a dengue genome or other viral genome.

Description

[0001] USES OF LIPID NANOPARTICLES CONTAINING CRISPR-ASSOCIATED

[0002] PROTEINS (CAS) AND GUIDE RNA IN MANAGING VIRAL INFECTIONS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 671,932 filed July 16, 2024. The entirety of this application is hereby incorporated by reference for all purposes.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under HR0011-19-2-0008 awarded by the Defense Advanced Research Projects Agency (DARPA). The government has certain rights in the invention.

[0007] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM

[0008] The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 23122PCT.xml. The XML file is 10,733 bytes, was created on July 15, 2025, and is being submitted electronically via the USPTO Patent Center.

[0009] BACKGROUND

[0010] Dengue virus infections are typically obtained from mosquitos and considered a substantial global health threat. Human infections often cause a high fever and flu-like symptoms. When severe, also referred to as “dengue hemorrhagic fever,” a person is at risk of spontaneous bleeding and other serious complications. There are no clinically approved antiviral agents specific for dengue viruses. Several dengue vaccines exist, but they have drawbacks as multiple dengue serotypes exist for which current vaccines are not universally effective. In addition, there are reports that certain vaccines increase the risk of severe dengue in those who have not previously been infected. Thus, there is a need to identify improved therapies.

[0011] Li et al. report CRISPR-Casl3a cleavage of dengue virus NS3 gene efficiently inhibits viral replication. Mol Ther Nucleic Acids, 2020, 19: 1460-1469. Blanchard et al. report treatment of influenza and SARS-CoV-2 infections via mRNA- encoded Casl3a in rodents. Nat Biotechnol, 2021, 39, 717-726. See also W02019204210.

[0012] Singsuksawat et al. report a programmable antiviral against dengue virus in primary human cells by Casl3b RNP with short spacer and delivery by VLP. Mol Ther Methods Clin Dev, 2021, 21: 729-740.

[0013] Rotolo et al. report species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung. Nature Materials, 2023, 22: 369-379.

[0014] Xue et al. report engineering CRISPR / Casl3 system against RNA viruses. Bioengineering 2022, 9, 291.

[0015] Ramezannia et al. report CRISPR-Cas system to discover host-virus interactions in Flaviviridae. Virol J, 2023, 20(l):247.

[0016] Obi et al. report current trends in dengue antiviral research. Virol J, 2023, 20:247.

[0017] Dahlman et al. report piperazine-derived lipid nanoparticles deliver mRNA to immune cells in vivo. Nature Comm, 2022, 13: 4766 (2022). See also WO2023 / 164615.

[0018] Kou Zhihua et al. report a nucleic acid target spot of anti-dengue virus based on Casl3a. CN108715849 (2018).

[0019] References cited herein are not an admission of prior art.

[0020] SUMMARY

[0021] Disclosed herein are compositions and methods for treating or preventing viral infections. In certain embodiments, this disclosure relates to compositions that specifically cleave target sequences in viruses, for example, genus Flavivirus / family Flaviviridae viruses, such as dengue virus. In certain embodiments, the compositions include lipid nanoparticles comprising nucleic acids encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas guide- RNA associated nuclease, with a guide RNA having a segment that is in a viral genome, targeting the viral genome.

[0022] In certain embodiments, contemplated methods include treating a viral infection by systemically delivering lipid nanoparticles containing mRNA encoding Casl3a and guide RNAs that are in segments of the viral genome. In certain embodiments, the lipid nanoparticle comprises an ionizable lipids, and PEG lipids. In certain embodiments, the lipid nanoparticles further comprise comprises a sterol. In certain embodiments, the lipid nanoparticles further comprise a phospholipid.

[0023] In certain embodiments, the guide RNA comprises a segment of a dengue virus genome sequence comprising or within SEQ ID NOs: 1, 2, 3, or 4 or consists of a 5’ end of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or segment thereof. In certain embodiments, the segment is greater than 8, 9, 10, 12, 11, 12, 13, 14, 15, 20, or 25 nucleotides. In certain embodiments, the segment is less than 20, 25, 30, or 35 nucleotides.

[0024] In certain embodiments, the lipid nanoparticles comprise an amine-based ionizable lipid, or quaternary ammonium lipid, or 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5- dione (cKK-E12) and a polyethylene glycosylated lipid. In certain embodiments, the lipid nanoparticles further comprise a sterol e g., cholesterol.

[0025] In certain embodiments, this disclosure relates to pharmaceutical compositions having lipid nanoparticles comprising a) a nucleic acid encoding a Casl3a nuclease or a Casl3a nuclease and b) anti-dengue Casl3a guide RNA or a nucleic acid encoding an anti-dengue Casl3a guide RNA, wherein the anti-dengue Casl3a guide RNA comprises a double stranded segment that binds the Casl3a nuclease and a single stranded segment that is a segment of a dengue virus genome sequence.

[0026] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0027] Figure 1 A shows a schematic of a formulated lipid nanoparticle drug containing an mRNA encoding LbuCasl3a and anti-DENV guide RNA. Formulation of lipid and cholesterol components with Casl3a mRNA and guide crRNAs into a LNP are illustrated.

[0028] Figure IB shows a schematic of theoretical anti-DENV mechanism of action of Casl3a and the guide crRNA when delivered to infected cells.

[0029] Figure 2A shows crRNA spacer sequences (SEQ ID NOs: 6-11) tiled against 5 DENV2 strains (SEQ ID NOs: 1-5), including serotypes - used for screening of Lbu Casl3a and guide crRNA against DENV2.

[0030] Figure 2B shows a schematic of a cell-free detection assay used to determine crRNA cutting kinetics.

[0031] Figure 2C shows data of ECso analysis of raw fluorescence reads over time from a cell-free detection assay using guide crRNAs. Figure 2D shows screening data using mRNA expressed Cast 3a and guide crRNA against DENV2. NS5 fold change of DENV2 genomic RNA was measured in Vero cells treated with Cast 3a and the indicated guide crRNA.

[0032] Figure 2E shows representative dsRNA staining data of DENV infected Vero cells treated with Cast 3a mRNA and guide crRNA providing quantification of dsRNA+ area per image.

[0033] Figure 2F shows viral titer data on DENV2 after treatment with Casl3a mRNA and the indicated guide crRNA.

[0034] Figure 3 A illustrates experiments for screening and assessing guides against DENV 3. DENV 3 H87 with position of the PCR primer sets and crRNA target region.

[0035] Figure 3B shows data from screening and assessment of guides against DENV3 using mRNA expressed Casl3a and guide crRNA against DENV3. Fold change of DENV3 genomic RNA (NS5 gene) in Vero cells treated with Casl3a and the indicated guide compared to the virus only condition is provided.

[0036] Figure 3C shows data from representative dsRNA staining of DENV3 infected cells treated with Casl3a mRNA and indicated guide crRNA. Quantification of dsRNA+ area per image is provided.

[0037] Figure 3D shows data on viral titer of DENV3 after treatment with Casl3a mRNA and the indicated guide crRNA.

[0038] Figure 3E show data on ionizable lipid selection in vivo using aVHH expression in different LNP formulations. After injection of the formulations, aVHH mRNA expression analysis was done using flow cytometry. Mean fluorescence intensity (MFI) of aVHH staining of hepatocytes is shown.

[0039] Figure 4A shows data on survival percentage of mice indicating mRNA expressed Casl3a and guide 8645G in LPNs prevents death from DENV2 infections in vivo. In vivo experiments were performed for mitigation of DENV2 New Guinea C strain (NGC) by mRNA expressed Cast 3a and guide crRNA formulated into LNPs in female A 129 mice. Treated mice received 2 mg / kg LNP -formulated with Cast 3a and guide 8645. Control mice received 2 mg / kg LNP formulated Casl3a and a non-targeted (control) (NTCR) guide.

[0040] Figure 4B shows data on survival percentage on day 0 recorded over 11 days of treated and control mice. Treated mice (n=5) received 2 mg / kg LNP-formulated Cast 3a and guide 8645. Control mice received 2 mg / kg LNP-formulated Cast 3a and NTCR guide (n=5). Figure 5 shows data on survival percentage indicating mRNA expressed Casl3a and guide 8645G in LPNs treats DENV3 infections in vivo. In vivo experiments were performed for mitigation of DENV3 H87 by mRNA expressed Casl3a and crRNA formulated into LNPs in AG129 mice. Treated mice received 2 mg / kg LNPs formulated with Casl3a and guide 8645G. Control mice received 2 mg / kg LNP-formulated Cast 3a and NTCR guide.

[0041] DETAILED DISCUSSION

[0042] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to embodiments described, and as such may, of course, vary. An “embodiment” is an example of this disclosure and not necessarily limited to such example. It is also to be understood that the terminology used herein is for describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims or as amended during prosecution.

[0043] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0044] Unless defined otherwise, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0045] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0046] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0047] It is noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0048] Use of the term "about" is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / -2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

[0049] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0050] The term “comprising” in reference to an oligonucleotide having a nucleic acid sequence refers to an oligonucleotide that may contain additional 5’ (5’ terminal end) or 3’ (3’ terminal end) nucleotides, i.e., the term is intended to include the oligonucleotide sequence within a larger nucleic acid.

[0051] "Consisting essentially of' or "consists of' or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel character! stic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.

[0052] The term “consisting of’ in reference to an oligonucleotide having a nucleotide sequence refers an oligonucleotide having the exact number of nucleotides in the sequence and not more or having not more than a range of nucleotide expressly specified in the claim. For example, “5’ sequence consisting of’ is limited only to the 5’ end, i.e., the 3’ end may contain additional nucleotides. Similarly, a “3’ sequence consisting of’ is limited only to the 3’ end, and the 5’ end may contain additional nucleotides. In certain embodiments, this disclosure relates to oligonucleotides disclosed herein consisting of sequences disclosed herein having less than an additional 10 or 50 nucleotides on the 5’ end. In certain embodiments, this disclosure relates to oligonucleotides disclosed herein consisting of sequences disclosed herein having less than an additional 10 or 50 nucleotides on the 3’ end.

[0053] As used herein, "subject" refers to any animal, preferably a human patient, livestock, or domestic pet. In certain embodiments, the subject is a human subject 2, 12, 16, or 20 years old or older. In certain embodiments, the subject is a human subject 2, 12, or 15 years old or older or less than 2, 12, or 16 years old. In certain embodiments, the subject is a human subject 55 or 65 years old or older. In certain embodiments, the subject is a human subject greater than 55, 60, 65, or 70 years of age. In certain embodiments, the subject is an infant, e.g., from one month to two years of age. In certain embodiments, the subject is a human subject such as a child, e.g., from two to twelve years of age. In certain embodiments, the subject is a human subject such as an adolescent, e.g., from twelve to sixteen years of age. In certain embodiments, the subject is a human subject sixteen years of age or older.

[0054] As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity is reduced.

[0055] As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also contemplate treatment that merely reduces symptoms, and / or delays disease progression. As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents.

[0056] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence to the mRNA sequence is identical, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0057] "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

[0058] The term "target nucleic acid" sequence refers to a nucleic acid (often derived from a biological sample), to which the oligonucleotide is designed to specifically hybridize. The target nucleic acid has a sequence that is substantially complementary to the nucleic acid sequence of the corresponding oligonucleotide directed to the target. The term target nucleic acid may refer to the specific subsequence of a larger nucleic acid to which the oligonucleotide is directed or to the overall sequence (e.g., gene or mRNA). The difference in usage will be apparent from context.

[0059] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleic acid bases are used, "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine. Ts are generally found in DNA and U’s are generally found in RNA. However, in any of the sequences disclosed herein, a T may be a U and a U may be a T. Unless otherwise specified, a "nucleotide sequence encoding" an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[0060] A "heterologous" nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or a peptide sequence that does not naturally occur, e.g., because the whole sequence contains a segment from other plants, bacteria, viruses, other organisms, or joinder of two sequences that occur in the same organism but are joined together in a manner that does not naturally occur in the same organism or any natural state.

[0061] The term "recombinant" when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques provided that the entire nucleic acid sequence does not occur in nature, i.e., there is at least one mutation in the overall sequence such that the entire sequence is not naturally occurring even though separately segments may occur in nature. The segments may be joined in an altered arrangement such that the entire nucleic acid sequence from start to finish does not naturally occur. The term "recombinant" when made in reference to a protein or a peptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.

[0062] In certain contexts, an “antibody” refers to a protein-based molecule that is naturally produced by animals in response to the presence of a protein or other molecule or that is not recognized by the animal’s immune system to be a “self’ molecule, i.e., recognized by the animal to be a foreign molecule, i.e., an antigen to the antibody. The immune system of the animal will create an antibody to specifically bind the antigen (or any cell or organism attached to the antigen) and thereby targeting the antigen for degradation or elimination. It is well recognized by skilled artisans that the molecular structure of a natural antibody can be synthesized and altered by laboratory techniques. Recombinant engineering can be used to generate fully synthetic antibodies or fragments thereof providing control over variations of the amino acid sequences of the antibody. Thus, the term “antibody” is intended to include natural antibodies, monoclonal antibody, or non- naturally produced synthetic antibodies, such as specific binding single chain antibodies, bispecific antibodies, or fragments thereof. These antibodies may have chemical modifications. The term "monoclonal antibodies" refers to a collection of antibodies encoded by the same nucleic acid molecule that are optionally produced by a single hybridoma (or clone thereof) or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same antigen. The term "monoclonal" is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc.

[0063] In humans, from a structural standpoint, an antibody is a combination of proteins: two heavy chain proteins and two light chain proteins. Alternatively, other animals produce antibodies from nucleic acids that encode a single protein. In humans, the heavy chains are longer than the light chains. The two heavy chains typically have the same amino acid sequence. Similarly, the two light chains typically have the same amino acid sequence. Each of the heavy and light chains contain a variable segment that contains amino acid sequences which participate in binding to the antigen. The variable segments of the heavy chain do not have the same amino acid sequences as the light chains. The variable segments are often referred to as the antigen binding domains. The antigen and the variable regions of the antibody may physically interact with each other at specific smaller segments of an antigen often referred to as the "epitope." Epitopes usually consist of surface groupings of molecules, for example, amino acids or carbohydrates. The terms “variable region,” "antigen binding domain," and "antigen binding region" refer to that portion of the antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. Small binding regions within the antigenbinding domain that typically interact with the epitope are also commonly alternatively referred to as the "complementarity-determining regions, or CDRs."

[0064] "Single chain antibodies" refer to a single peptide containing naturally or non-naturally occurring sequences, including synthetically modified peptide sequences, derived from an antibody variable region that specifically binds an antigen of interest. Single chain antibodies are sometimes fragments or variants of naturally occurring mammalian antibodies. Such antibodies are sometimes referred to as single-domain antibodies (sdAbsor or VHHs), or camelid singledomain antibodies, e.g., when derived from an animal of Camelidae family, e.g., lamas, camels.

[0065] The term "exogenous" indicates that the nucleic acid or polypeptide is part of, or encoded by, a recombinant nucleic acid construct, or is not in its natural environment. For example, an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct. An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism. An exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found.

[0066] "Percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software using default values, e.g., National Institutes of Health, BLAST: Basic Local Alignment Search Tool.

[0067] CRISPR / Cas system

[0068] Nuclease enzymes, also referred to as “endonucleases” and “exonucleases” are capable of hydrolyzing polynucleotide chains such as DNA and RNA. Endonucleases cleave the internal phosphodiester bond present in the polynucleotide chain, whereas exonucleases cleave the phosphodiester bond from the 5’ and / or 3’ ends.

[0069] The CRISPR / Cas system is an immune system currently found in most bacteria. It is used to cleave DNA or RNA of bacteriophages and other pathogens. In the CRISPR / Cas system, CRISPR is the abbreviation of clustered regularly interspaced short palindromic repeats, which refers to a unique DNA region in the bacterial genome that stores fragments used to guide an immune defense against targets. Cas is the abbreviation for CRISPR-associated proteins (Cas). The Cas proteins cut the target DNA or RNA on the genome of the pathogen. The targeting RNA sequence (also referred to as crRNA) produced by CRISPR's transcription recognizes / guides the Cas to target the foreign genetic material for cleavage (endonuclease activity). Cas9, Cpfl, C2cl and C2c2 (also known as Casl3a) have endonuclease activity.

[0070] The CRISPR associated endonuclease, Cas9, belongs to the type II CRISPR / Cas system and has endonuclease activity to cut target DNA. Cas9 is guided by a mature crRNA that contains about 20 base pairs (bp) of unique target sequence (called spacer) and a trans-activated small RNA (tracrRNA) that serves as a guide for ribonuclease-aided processing of pre-crRNA. The crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA. Cas9 recognizes a trinucleotide protospacer adjacent motif (PAM) to specify the cut site (the 3rdnucleotide from PAM). Recombinant crRNA for targeting is typically referred to as “guide RNA” or “gRNA.” The crRNA and tracrRNA may be expressed separately or engineered into an artificial fusion small guide RNA (gRNA) via a synthetic stem loop to mimic the natural crRNA / tracrRNA duplex. Such single stranded guide RNAs (gRNA) typically contain short hairpin (shRNA) motif that can be synthesized or in vitro transcribed for direct RNA transfection or expressed from an RNA expression vector. Typically, the gRNA or crRNA sequence is designed to have a segment with complementarity to a nucleic acid sequence in a target gene. If the target gene is double stranded, complementarity to a sequence in the target gene will necessarily include a sequence segment in the target gene that is the same sequence as in the gRNA or crRNA sequence. Thus, reference to a guide sequence having segment of a target viral genome (with the understanding that U and T can be substituted for each other) is contemplated to inherently include a segment with complementarity to a target nucleic acid sequence in the viral genome.

[0071] In certain embodiments, this disclosure contemplates that the endonuclease is an RNA- guided endonuclease such as Casl3a, Casl3b, Casl3c or Casl3d. In one embodiment, the RNA- guided endonuclease is Casl3a. Casl3 nucleases function similarly to Cas9, using an approximately 64-nt guide RNA to encode target specificity. The Casl3 protein complexes with the guide RNA via recognition of a short hairpin in the crRNA, and target specificity is encoded by an approximately 28 - 30-nt spacer that is complementary to the target region. Guide RNA sequences according to the present guide can be sense or anti-sense sequences. The guide RNA sequence can be configured as a single sequence or as a combination of one or more different sequences, for example, a multiplex configuration. Multiplex configurations can include combinations of two, three, four, five, six, seven, eight, nine, ten, or more different guide RNAs.

[0072] In some embodiments, several guide RNAs are added to create a mixture to target different categories of sequences. For example, two, five, seven or eleven guide RNAs may be present in a CRISPR mixture targeting three different categories of sequences. However, any number of gRNAs may be introduced into a mixture to target categories of sequences. Casl3 nucleases exhibit collateral activity after recognition and cleavage of a target transcript, leading to non-specific degradation of nearby transcripts regardless of complementarity to the spacer. Exemplary bacteria that express Cast 3 nucleases include but are not limited to Leptotrichia wadei, Leptotrichia shahii, Leptotrichia oral taxon, Leptotrichia buccalis, Lachnospiraceae bacterium, Eubacterium rectale, Clostridium aminophilum, Herbinix hemicellulosilytica, Rhodobacter capsulatus, Paludibacter propionicigenes, Carnob acterium gallinarum, Listeria seeligeri, and Listeria newyorkensis. In one embodiment, the Casl3 enzyme is Casl3a from Leptotrichia wadei, Leptotrichia shahii, Leptotrichia oral taxon, or Leptotrichia buccalis.

[0073] In some embodiments, the endonucleases can be a wild-type endonuclease, a modified endonuclease, or a fragment of a wild type or modified endonuclease. The endonuclease can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. For example, nuclease (i.e., DNase, RNase) domains of endonucleases can be modified, deleted, or inactivated. Alternatively, the endonuclease can be truncated to remove domains that are not essential for the function of the fusion protein. The endonuclease can also be truncated or modified to optimize the activity of the effector domain of the fusion protein.

[0074] In certain embodiments, the endonucleases (RNA-guided) can be derived from a wild type Cast 3a protein or fragment thereof. In certain embodiments, the endonucleases can be a modified Casl3a protein. For example, the amino acid sequence of the Cast 3a protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, etc.) of the protein. Alternatively, domains of the Cast 3a protein not involved in cleavage can be eliminated from the protein such that the modified Casl3a protein is smaller than the wild type Casl3a protein.

[0075] In one embodiment, the Casl3a / gRNA complexes are designed to target active viruses within a cell. In another embodiment, the Casl3a / gRNA complexes are designed to target latent viruses within a cell. Once transfected within a cell, the Casl3a / gRNA complexes cause repeated insertions or deletions to render the viral genome incapacitated, or due to number of insertions or deletions, the probability of repair is significantly reduced.

[0076] In certain embodiments, the Cast 3a nuclease is from Leptotrichia buccalis (Lbu), Listeria seeligeri, Leptotrichia shahii, or Leptotrichia wadei. In certain embodiments, the guide RNA contains a segment of a viral genome sequence that is heterologous to the segment of guide RNA that binds / localizes the Cas endonuclease.

[0077] In certain embodiments, this disclosure relates to guide RNAs against dengue serotypes 2 and 3 with in vitro efficacy. In certain embodiments, this disclosure relates to guide RNAs against dengue serotypes 1 and 2 with in vitro efficacy. In certain embodiments, this disclosure relates to guide RNAs against dengue serotypes 3 and 4 with in vitro efficacy.

[0078] In certain embodiments, the single stranded segment of guide RNA that is to a dengue virus genome sequence is a 10-30 nucleotide segment of SEQ ID NO: 1, 2, 3, 4, or 5 or combinations thereof.

[0079] In certain embodiments, the single stranded segment of guide RNA that is a segment of a dengue virus genome sequence comprises or consists of a 5’ end GUG UCU GUC AUU GCC AUC UGU GUC ACC (SEQ ID NO: 11)(8645G). In certain embodiments, the single stranded segment of guide RNA comprises or consists of a 5’ end UG UCU GUC AUU GCC AUC UGU GUC ACC A (SEQ ID NO: 10)(8644G). In certain embodiments, the single stranded segment of guide RNA comprises or consists of a 5’ end G UCU GUC AUU GCC AUC UGU GUC ACC AU (SEQ ID NO: 9)(8643G). In certain embodiments, the single stranded segment of guide RNA comprises or consists of a 5’ end UCU GUC AUU GCC AUC UGU GUC ACC AUG (SEQ ID NO: 8)(8642G). In certain embodiments, the single stranded segment of guide RNA comprises or consists of a 5’ end CU GUC AUU GCC AUC UGU GUC ACC AUG G (SEQ ID NO: 7)(8641G). In certain embodiments, the single stranded segment of guide RNA comprises or consists of a 5’ end U GUC AUU GCC AUC UGU GUC ACC AUG GG (SEQ ID NO: 6)(8640 G).

[0080] In certain embodiments, the single stranded segment of guide RNA that is a dengue virus genome sequence segment comprises of SEQ ID NO: 6, 7, 8, 9, 10, 11, or combinations thereof or combine segments thereof.

[0081] Methods of use

[0082] In certain embodiments, contemplated methods include treating a viral infection by systemically delivering lipid nanoparticles comprising mRNA encoding Cas endonuclease and guide RNAs that contain segments of the viral genome. In certain embodiments, methods entail delivering mRNA encoding Casl3a and guide RNAs by incorporation of both RNA formulated in lipid nanoparticles (LNPs). In certain embodiments, methods include treating or preventing a high fever comprising administering to a subject in need thereof an effective amount of a lipid nanoparticle comprising: a) a nucleic acid encoding a Cast 3 a nuclease and b) anti-viral Cast 3a guide RNA, wherein the anti-viral Casl3a guide RNA comprises a segment in a viral genome sequence.

[0083] In certain embodiments, methods include treating or preventing dengue hemorrhagic fever comprising administering to a subject in need thereof an effective amount of a lipid nanoparticle comprising: a) a nucleic acid encoding a Casl3a nuclease and b) anti-dengue Casl3a guide RNA, wherein the anti-dengue Casl3a guide RNA comprises a segment that is in a dengue virus genome sequence.

[0084] In certain embodiments, methods include treating or preventing spontaneous bleeding, epistaxis, gum bleeding, internal bleeding, hypermenorrhea, hematuria, or gastrointestinal bleeding comprising administering to a subject in need thereof an effective amount of a lipid nanoparticle comprising: a) a nucleic acid encoding a Casl3a nuclease and b) anti-viral Casl3a guide RNA, wherein the anti-viral Casl3a guide RNA comprises a segment that is in a viral genome sequence. In certain embodiments the subject is at risk of spontaneous bleeding, epistaxis, gum bleeding, internal bleeding, hypermenorrhea, hematuria, or gastrointestinal bleeding due to a viral infection diagnosis.

[0085] In certain embodiments, methods include treating or preventing dengue virus induced spontaneous bleeding, epistaxis, gum bleeding, internal bleeding, hypermenorrhea, hematuria, or gastrointestinal bleeding comprising administering to a subject in need thereof an effective amount of a lipid nanoparticle comprising: a) a nucleic acid encoding a Casl3a nuclease and b) antidengue Casl3a guide RNA, wherein the anti-dengue Casl3a guide RNA comprises a segment that is in a dengue virus genome sequence.

[0086] In certain embodiments, methods include treating or preventing a thrombocytopenia comprising administering to a subject in need thereof an effective amount of a lipid nanoparticle comprising: a) a nucleic acid encoding a Casl3a nuclease and b) anti-viral Casl3a guide RNA, wherein the anti-viral Casl3a guide RNA comprises a segment that is in a viral genome sequence. In certain embodiments the subject is at risk of thrombocytopenia due to a viral infection diagnosis.

[0087] In certain embodiments, methods include treating or preventing dengue induced thrombocytopenia comprising administering to a subject in need thereof an effective amount of a lipid nanoparticle comprising: a) a nucleic acid encoding a Casl3a nuclease and b) anti-dengue Casl3a guide RNA, wherein the anti-dengue Casl3a guide RNA comprises a segment that is in a dengue virus genome sequence.

[0088] In certain embodiments, methods include treating or preventing shock, organ failure, or death comprising administering to a subject in need thereof an effective amount of a lipid nanoparticle comprising: a) a nucleic acid encoding a Casl3a nuclease and b) anti-viral Casl3a guide RNA, wherein the anti-viral Casl3a guide RNA comprises a segment that is in a viral genome sequence. In certain embodiments the subject is at risk of shock, organ failure, or death due to a viral infection diagnosis.

[0089] In certain embodiments, methods include treating or preventing dengue induced shock, organ failure, and death shock, organ failure, or death comprising administering to a subject in need thereof an effective amount of a lipid nanoparticle comprising: a) a nucleic acid encoding a Casl3a nuclease and b) anti-dengue Casl3a guide RNA, wherein the anti-dengue Casl3a guide RNA comprises a segment that is in a dengue virus genome sequence.

[0090] In certain embodiments, contemplated methods include treating a dengue viral infection comprising administering to a subject in need thereof an effective amount of a lipid nanoparticle comprising: a) a nucleic acid encoding a Casl3a nuclease and b) anti-dengue Casl3a guide RNA, wherein the anti-dengue Casl3a guide RNA comprises a segment that is in a dengue virus genome sequence.

[0091] In certain embodiments, the segment that is in a dengue virus genome sequence comprises or consists of a 5’ end GUG UCU GUC AUU GCC AUC UGU GUC ACC (SEQ ID NO: 11)(8645G).

[0092] In certain embodiments, the segment that is in a dengue virus genome sequence comprises or consists of a 5’ end UG UCU GUC AUU GCC AUC UGU GUC ACC A (SEQ ID NO: I0)(8644G).

[0093] In certain embodiments, the segment that is in a dengue virus genome sequence comprises or consists of a 5’ end G UCU GUC AUU GCC AUC UGU GUC ACC AU (SEQ ID NO: 9)(8643G).

[0094] In certain embodiments, the segment that is in a dengue virus genome sequence comprises or consists of a 5’ end UCU GUC AUU GCC AUC UGU GUC ACC AUG (SEQ ID NO: 8)(8642G). In certain embodiments, the segment that is in a dengue virus genome sequence comprises or consists of a 5’ end CU GUC AUU GCC AUC UGU GUC ACC AUG G (SEQ ID NO: 7)(8641G).

[0095] In certain embodiments, the segment that is in a dengue virus genome sequence comprises or consists of a 5’ end U GUC AUU GCC AUC UGU GUC ACC AUG GG (SEQ ID NO: 6)(8640G).

[0096] In certain embodiments, the lipid nanoparticle comprises 3,6-bis(4-(bis(2- hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (cKK-E12) and a polyethylene glycosylated lipid. In certain embodiments, the lipid nanoparticle comprises cholesterol.

[0097] In certain embodiments, delivering mRNA encoding Casl3a and guide RNAs is by incorporation of the RNA formulated in lipid nanoparticles (LNPs). In certain embodiments, contemplated methods include treating dengue by administering, e.g., systemically delivering, mRNA encoding Casl3a and guide RNA that have sequences that target / are in conserved regions of a dengue genome. In certain embodiments, the RNAs targets / are in dengue 1, 2, 3, and / or 4 genomes.

[0098] In certain embodiments, this disclosure relates to administering a single dose or multiple doses of LNPs formulated with mRNA encoding Casl3a, and guide RNAs to subjects in need thereof for uses in mitigating lethal infections of dengue. In certain embodiments, this disclosure contemplates a potent pan-DENV drug, and the expansion of this approach to alternative viral infections.

[0099] In certain embodiments, this disclosure relates to methods of treating a viral infection, e.g., dengue viral infection comprising administering to a subject in need thereof an effective amount of a lipid nanoparticle comprising: a) a nucleic acid encoding a Casl3a nuclease or a Casl3a nuclease and b) anti-viral guide RNA, e.g., anti-dengue Casl3a guide RNA or a nucleic acid encoding an anti-viral guide RNA, e.g., anti-dengue Casl3a guide RNA, wherein the anti-viral guide RNA, e.g., anti-dengue Casl3a guide RNA comprises a double stranded segment that binds the Casl3a nuclease and a single stranded segment that target / or is a viral genome sequence, e.g., dengue virus genome sequence.

[0100] In certain embodiments, an additional anti-viral agent or vaccine, can be administered in combination with the agents, lipid nanoparticles, nucleic acids encoding Cas endonucleases, and guide RNAs reported herein to a subject having or at risk of contracting a viral infection, e.g., dengue virus infection.

[0101] In certain embodiments, contemplated methods include treating a viral infection by systemically delivering mRNA encoding Casl3a and guide RNAs that target / are segments in conserved regions a viral genome. In certain embodiments, the mRNA encoding Casl3a and guide RNAs are formulated in lipid nanoparticles (LNPs).

[0102] In certain embodiments, contemplated methods include treating dengue by systemically delivering mRNA encoding Casl3a and guide RNAs that target / are in to conserved regions a dengue genome. In certain embodiments the RNAs target / are in dengue 1, 2, 3, and / or 4 genomes. In certain embodiments the RNAs target / are in dengue 2 and / or 3 genomes. In certain embodiments the delivering mRNA encoding Casl3a and guide RNAs is by incorporation of the RNA formulated in lipid nanoparticles (LNPs).

[0103] In certain embodiments, this disclosure relates to guide RNAs against serotypes 2 and 3 with in vitro efficacy. In certain embodiments, this disclosure relates to single dose or multiple doses of LNPs formulated, with mRNA encoding Cast 3a, and guide RNAs administered to subjects in need thereof for uses in mitigating lethal infections of dengue. In certain embodiments, this disclosure contemplates a potent pan-DENV drug, and the expansion of this approach to alternative viral infections.

[0104] In certain embodiments, this disclosure relates to methods of treating a dengue viral infection comprising administering to a subject in need thereof an effective amount of lipid nanoparticles comprising: a) a nucleic acid encoding a Casl3a nuclease or a Casl3a nuclease and b) anti-dengue Cast 3a guide RNA or a nucleic acid encoding an anti-dengue Casl3a guide RNA, wherein the anti-dengue Casl3a guide RNA comprises a double stranded segment that binds the Casl3a nuclease and a single stranded segment that is a segment of a dengue virus genome sequence.

[0105] In certain embodiments, the single stranded segment that is a segment of a dengue virus genome sequence comprises any of SEQ ID NO: 1, 2, 3, or 4 of segment thereof or the reverse complement thereof or comprises or consists of a 5’ end of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or segment or the reverse complement thereof.

[0106] In certain embodiments, the single stranded segment that is in a dengue virus genome sequence comprises or consists of a 5’ end GUG UCU GUC AUU GCC AUC UGU GUC ACC (SEQ ID NO: 11)(8645G) or the reverse complement thereof. In certain embodiments, the single stranded segment that is in a dengue virus genome sequence comprises or consists of a 5’ end UG UCU GUC AUU GCC AUC UGU GUC ACC A (SEQ ID NO: 10)(8644G) or the reverse complement thereof. In certain embodiments, the single stranded segment that is in a dengue virus genome sequence comprises or consists of a 5’ end G UCU GUC AUU GCC AUC UGU GUC ACC AU (SEQ ID NO: 9)(8643G) or the reverse complement thereof. In certain embodiments, the single stranded segment that is in a dengue virus genome sequence comprises or consists of a 5’ end UCU GUC AUU GCC AUC UGU GUC ACC AUG (SEQ ID NO: 8)(8642G) or the reverse complement thereof. In certain embodiments, the single stranded segment that is a segment of a dengue virus genome sequence comprises or consists of a 5’ end CU GUC AUU GCC AUC UGU GUC ACC AUG G (SEQ ID NO: 7)(8641G) or the reverse complement thereof. In certain embodiments, the single stranded segment that is a segment of a dengue virus genome sequence comprises or consists of a 5’ end U GUC AUU GCC AUC UGU GUC ACC AUG GG (SEQ ID NO: 6)(8640 G) or the reverse complement thereof.

[0107] In certain embodiments, the target nucleic acid sequence comprises one or more nucleic acid sequences, e.g., mRNA, encoding and non-coding nucleic acid sequences of a virus genome or dengue genome. In embodiments, the target nucleic acid sequence comprises one or more sequences within a sequence encoding structural proteins, non-structural proteins, or combinations thereof. The sequences encoding structural proteins comprise nucleic acid sequences encoding a capsid protein (C), precursor viral membrane protein (prM), viral membrane protein (M), envelop protein (E) or combinations thereof. The sequences encoding non-structural proteins comprise nucleic acid sequences encoding: non-structural protein 1 (NS1), non-structural protein 2A (NS2A), non-structural protein 2B (NS2B), non-structural protein 3 (NS3), non-structural protein 4A (NS4A), nonstructural protein 4B (NS4B), non- structural protein 5 (NS5), or combinations thereof.

[0108] In certain embodiments, a gRNA sequence has a 75%, 80%, 85%, 90%, 95%, or more sequence identity to a target nucleic acid sequences or reverse complement thereof encoding a viral capsid protein (C), precursor viral membrane protein (prM), viral membrane protein (M), envelop protein (E), non- structural protein 1 (NS1), non- structural protein 2A (NS2A), non- structural protein 2B (NS2B), non-structural protein 3 (NS3), non-structural protein 4A (NS4A), nonstructural protein 4B (NS4B), non- structural protein 5 (NS5), or any combination thereof. In certain embodiments, a gRNA has greater than 75%, 80% 85% 90%, 90%, or 95% sequence identity to any one or more of SEQ ID NOs: 1-11 or the reverse complement thereof. In other embodiments, a gRNA comprises any one or more of SEQ ID NOs: 1-11 or the reverse complement thereof.

[0109] In certain embodiments, the guide RNA sequences are in single or multiplex configurations. In certain embodiments, the guide RNA sequences are encoded by the same vector encoding the Casl3a or CRISPR / Cas molecule or are encoded by separate vectors, e.g., a first vector and second vector. In certain embodiments, a gRNA comprises one or more modified nucleic acid bases or chimeric sequences. In certain embodiments, the guide RNA is may be RNA or RNA and DNA or modified bases.

[0110] Lipid Nanoparticles

[0111] In certain embodiments, contemplated lipid nanoparticles comprise a) a nucleic acid encoding endonuclease, e.g., a Casl3a nuclease or a Casl3a nuclease and b) anti-viral guide RNA, e.g., Casl3a guide RNA or a nucleic acid encoding an anti-viral guide RNA, e.g., anti-dengue Casl 3a guide RNA, c) an ionizable lipid, a phospholipid, a sterol, and a polyethylene glycol (PEG) phospholipid.

[0112] In certain embodiments, the lipid nanoparticles comprise an ionizable lipid which is an amine-based lipid, or quaternary ammonium lipid, or 3,6-bis(4-(bis(2- hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (cKK-E12) and a polyethylene glycosylated lipid. In certain embodiments, the lipid nanoparticles further comprise cholesterol. In certain embodiments, the lipid nanoparticles further comprise a phospholipid.

[0113] In certain embodiments, lipid nanoparticles disclosed herein can be formulated to target a specific cell type or tissue.

[0114] In certain embodiments, the disclosed lipid nanoparticles include an ionizable lipid or alternative. Ionizable lipids have a positive or partial positive charge at physiological pH. Exemplary alternative ionizable lipids include but are not limited to 3,6-bis({4-[bis(2- hydroxydodecyl)amino]butyl })piperazine-2, 5-dione (cKK-E12), l-Linoleoyl-2-linoleyloxy-3- dmiethylaminopropane (DLin-2-DMAP), l,2-Dilinoleylcarbanioyloxy-3-dimethylaniinopropane (DLin-C-DAP), l,2-Dilmoleoyl-3-dimethylammopropane (DLm-DAP), 1,2-Dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethy laminomethyl- [l,3]-di oxolane (DLin-K-DMA), 2,2-dilmoleyl-4-(2-dimethylaiiimoethyl)-[l ,3]-dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)-heptatriaeonta-6,9,28,3 1-tetraen- 19-yl 4-(dimethylamino)butanoate (DLin- MC3-DMA), l,2-dioieoyl-3 -dimethylammonium propane (DODAP), N,N-dimethyl-(2,3- dioleyloxy)propylamine (DODMA), dioctadecylamidoglycyocarboxyspermine (DOGS), Spermine cholesterylcarbamate (GL-67), bis-guanidinium-spermidine-cholesterol (BGTC), N-t- butyl-N’-tetradecylamino-propionamidine (diC14-amidine), Dimethyldioctadecylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMR1E), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), Dioleyloxypropyl-3-dimethyl hydroxyethyl ammonium bromide (DORIE), N-(l-(2,3- dioleyloxy3)propyl)-N-2-(spenninecarboxamido)ethyl)-N,N-dimethylamrnonium trifluoracetate (DOSPA), 2-dioleoy trimethyl ammonium propane chloride (DOTAP), N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), Aminopropyl-dimethyl- bis(dodecyloxy)-propanaminiumbromide (GAP-DLRIE), l,2-dioleoyl-sn-3-phosphoethanolamine (“DOPE”), or combinations thereof.

[0115] In certain embodiments, the disclosed lipid nanoparticles include a polyethylene glycol phospholipid or alternative. Such polyethylene glycol phospholipid may be alternately referred to as PEGylated lipids. Inclusion of a PEGylating lipid can be used to enhance lipid nanoparticle colloidal stability in vitro and circulation time in vivo. In some embodiments, the PEGylation is reversible in that the PEG moiety is gradually released in blood circulation. Exemplary PEG-lipids include but are not limited to PEG conjugated to saturated or unsaturated alkyl chains having a length of C3-C22. PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides (PEG-CER), PEG-modified dialkylamines, PEG-modified di acylglycerols (PEG-DAG), PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or a PEG- DSPE lipid. In certain embodiments, the molecular weight of the PEG lipid can be about 1 KDa, 2 KDa, or 3KDa.

[0116] In certain embodiments, the disclosed lipid nanoparticles include an alternative phospholipid moiety that may be selected from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Nonnatural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions may be useful in functionalizing a lipid bilayer to provide a nanoparticle composition with a targeting or imaging moiety (e.g., a dye).

[0117] In certain embodiments, the disclosed lipid nanoparticles include alternative phospholipids such as l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-0- octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), 1 -oleoyl-2-cholesterylhemisuccinoy l-sn-glycero-3 -phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (CI 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3- phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3 -phosphocholine, 1 ,2-diphytanoyl-sn- glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-dilinolenoyl-sn- glycero-3-phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac- (1 -glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), l-stearoyl-2-oleoyl- phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, or lysophosphatidylethanolamine (LPE). RNA-targeting, class II, type VI CRISPR-Cas systems in bacteria

[0118] Casl3a (also known as C2c2) belongs to the Type 2 VI-A CRISPR-Cas system which are responsible for the processing and maturation of crRNA and the degradation of target RNA. In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins.

[0119] The Casl3a-crRNA complex activates when the target RNA complements with crRNA, and the Cas protein initiates RNA cleavage. Here, the application of this platform is extended to viruses with systemic cell targets. Individual guides are programmed to target different sequences, activate after binding to them, and then cut multiple parts of the viral genome. Multiple guides can be implemented to overcome viral diversity and resistance. As viruses evolve, the sequence of the guide targeting the viral genome is changed.

[0120] In certain embodiments, the mRNA or guide RNA construct can include modifications, for example, a nuclear localization sequence, a 5’ cap, a 3’ Poly (A) tail, or modified nucleobases such as N'-methylpseudouridine-5’ -triphosphate, 2’-O-methyladenosine-5’-triphosphate, or 2’-O- methyluridine-5’ -triphosphate, or combinations thereof. In some embodiments, the RNA-guided endonuclease is an RNA-guided RNase.

[0121] Naturally occurring mRNAs bear a cap structure at the 5’ end and a long sequence of poly adenylate residue (Poly (A) tail) at the 3’ end that are added after transcription of the DNA. Synthetic mRNAs without modifications do not have a cap on the 5’ end or a Poly (A) tail on the 3’ end. In one embodiment, the nucleic acid constructs encoding an RNA-guided endonuclease are modified with a 5’ cap structure and a 3’ Poly (A) tail to increase stability and reduce immunogenicity. The 5’ cap can be a 7-methylguanosine cap, including variations or LNA modified analogues.

[0122] The poly(A) tail can be added to the 3’UTR to protect the mRNA from nuclease degradation. In one embodiment, the poly(A) tail has 25-1000 nucleotides. In another embodiment, the poly(A) tail has 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 1000 nucleotides.

[0123] Naturally occurring RNAs are synthesized from four basic ribonucleotides: ATP, CTP, UTP, and GTP. In one embodiment, the nucleic acid constructs encoding an RNA-guided endonuclease can include modifications to the ribonucleotides. Suitable modifications include alterations in one or more nucleotides of a codon such that the codon encodes the same amino acid but is more stable than the codon found in the wild-type version of the nucleic acid. For example, an inverse relationship between the stability of RNA and a higher number of cytidines (C’s) and / or uridines (Us) residues has been demonstrated, and RNA devoid of C and U residues have been found to be stable to most RNases. In some embodiments, the number of C and / or U residues in an mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by substitution of one codon encoding a particular amino acid for another codon encoding the same or a related amino acid. Contemplated modifications to the mRNA nucleic acids of the present disclosure also include the incorporation of pseudouridines. The incorporation of pseudouridines into the mRNA nucleic acids may enhance stability and translational capacity, as well as diminishing immunogenicity in vivo.

[0124] In certain embodiments, the disclosed nucleic acid constructs encoding an RNA-guided endonuclease contain post-transcriptionally modified nucleotides. In one embodiment, the incorporation of modified nucleosides can increase stability and reduce immunogenicity of the mRNA construct. In one embodiment, cytidine and / or uridine are replaced by modified nucleosides. Exemplary modified nucleoside bases include but are not limited to 5 -methylcytidine, 2'-O-methylcytidine, pseudouridine, N6-methyladenosine, N6,2'-O-dimethyladenosine, N6,N6,2'- O-trimethyladenosine, 3,2'-0-dimethyluridine, 7-methylguanosine, 2'-O-methylguanosine, N2,7- dimethylguanosine, N2,N2,7-trimethylguanosine. In one embodiment, the synthetic mRNA is incorporated with N^methylpseudouridine-S’-triphosphate, 2’-O-methyladenosine-5’- triphosphate, or 2’ -O-methyluridine-5 ’-triphosphate.

[0125] Pharmaceutical Compositions

[0126] Pharmaceutical compositions including the disclosed lipid nanoparticles having nucleic acids encoding an endonuclease and a guide RNA as provided herein. Pharmaceutical compositions containing the disclosed compositions can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration. In one embodiment, the nucleic acids encoding an endonuclease and the guide RNA are included in the same pharmaceutical composition. In another embodiment, the nucleic acids encoding an endonuclease and the guide RNA are formulated in separate pharmaceutical compositions.

[0127] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient and a lipid nanoparticle comprising a) a nucleic acid encoding a Casl3a nuclease or a Casl3a nuclease and b) anti-viral Casl3a guide RNA or a nucleic acid encoding an anti-viral Casl3a guide RNA, wherein the anti-viral Casl3a guide RNA comprises a sequence that is a segment of a virus genome or mRNA sequence.

[0128] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient and a lipid nanoparticle comprising a) a nucleic acid encoding a Casl3a nuclease or a Casl3a nuclease and b) anti-dengue Cast 3a guide RNA or a nucleic acid encoding an anti-dengue Casl3a guide RNA, wherein the anti-dengue Casl3a guide RNA comprises a segment that is a segment of a dengue virus genome or mRNA sequence.

[0129] The compositions disclosed herein are administered to a subject in a therapeutically effective amount. The precise dosage will vary according to a variety of factors such as subjectdependent variables (e.g., age, immune system health, etc ), the disease, and the treatment being effected. For the disclosed compositions, as further studies are conducted, information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. For the disclosed activatable nuclease compositions, generally dosage levels of 0.001 to 20 mg / kg of body weight daily are administered to mammals. Generally, for intravenous injection or infusion, dosage may be lower.

[0130] In certain embodiments, compositions disclosed herein, including lipid nanoparticles containing nucleic acids encoding an endonuclease and guide RNA, are administered in an aqueous solution, by parenteral injection. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of lipid nanoparticles, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions optionally include one or more of the following: diluents, sterile water, buffered saline of various buffer content (e.g., Tris- HC1, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20™ (polysorbate-20), TWEEN 80™ (polysorbate-80)), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol™, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacterium retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.

[0131] Lipid nanoparticles (LNP) encapsulating Casl3a mRNA along with a guide targeted against dengue virus

[0132] In certain embodiments, this disclosure contemplates a drug that utilizes lipid nanoparticle (LNP) encapsulated LbuCasl3a mRNA along with a crRNA guide targeted against DENV (Fig. 1A). Intravenous injection of the LNP deliver the mRNA and guide to infected cells in the liver and spleen, where the expressed Cast 3a complex with the guide and specifically degrade DENV genomic RNA. To this end, guides for anti-DENV activity were screened and characterized in vitro using DENV2 infections. Using RNA sequencing (RNAseq), it was identified that Casl3a activity specifically reduces the antiviral cell response, without significant off-target effects. The same strategy potently inhibited DENV3 infections in vitro. In addition, an LNP-formulated Casl3a drug protected mice in both DENV2 and DENV3 lethal challenge models, providing the basis for the development of a pan-DENV mRNA-based Casl3a drug.

[0133] Pharmaceutical product that utilizes lipid nanoparticle (LNP) encapsulated LbuCasl3a mRNA along with a crRNA guide targeted against DENV

[0134] Contemplated are intravenous injection to deliver of LNPs containing the mRNA and guide RNA to infected cells in the liver and spleen, where the expressed Casl3a would complex with the guide and specifically degrade DENV genomic RNA. Guides were screened and characterized for anti-DENV activity in vitro using DENV2 infections. Using RNA sequencing (RNAseq), Cast 3 activity was found to specifically reduces the antiviral cell response, without significant off- target effects. The same strategy potently inhibited DENV3 infections in vitro. An LNP- formulated Casl3 drug protected mice in both DENV2 and DENV3 lethal challenge models, providing the basis for the development of a pan-DENV mRNA-based Casl3 drug.

[0135] Screening and assessment of guides against DENV2

[0136] DENV has the unique challenge of 4 serotypes. To address this, 4 strains of DENV were initially compared, each representing one of the 4 main serotypes. One target, NS5, is the largest protein encoded by the flavivirus genome. It is also the most highly conserved, with approximately 67-82% amino-acid sequence identity between the four main serotypes of dengue virus (DENV1- 4). NS5 functions as both an RNA-dependent RNA polymerase (RdRp) that replicates the viral RNA and an RNA methyltransferase enzyme (MTase) that protects the viral genome by RNA capping. In addition, within the human host, NS5 interacts with several proteins such as those in the JAK-STAT pathway, thereby interfering with anti-viral interferon signaling, making an attractive target. TotargetCasl3a toNS5 for DENV, the sequences of 5 strains, including DENV 1- 4 were aligned (Fig. 2A), finding conserved regions of about 27 nt for all 5 was difficult. A conserved region approximately 46 nt long was found for DENV2 and 3 and “tiled” guides 8640G- 8645G were designed.

[0137] To assess guide function, not just sequence homology, the guides, were screened in vitro in a cell-free detection assay. In a single tube, the LbuCasl3a protein, guide RNA, target RNA and reporter RNA (dual-fluorophore-quencher labeled polyU oligonucleotide) were mixed. Appropriate controls including a non-targeted guide RNA were also used. As the ribonucleoprotein (RNP) complex formed, binding to the target activated the Casl3a protein; the cutting kinetics were monitored in a plate reader. From this assay one can detect guide-dependent differences in RNP complex formation and the ability of each ability guide to activate Casl3a once bound to a target. Guides 8640G-8645G were screened against DENV2 / 3. Five activated Cast 3 similarly, with 8640G exhibiting slower kinetics.

[0138] In order to perform a full assessment of guide function against DENV2, reductions in viral RNA, viral titer and double stranded RNA (a hallmark of flavivirus replication) were characterized. To generate accurate qPCR data, 5 primer-probe sets were examined, one straddling the guide RNA binding site, and 2 sets on either side. This assay was performed because Casl3 does not cut in cis exactly where it binds, as per its structure, but near the binding region. As RNA structure, RNA binding proteins, etc., may affect the cutting frequency at any specific location, the cutting region cannot be predicted. DENV2 viral RNA reduction was examined by qPCR with guides 8640G and 8645G by transfecting cells prior to infection. Primer-probe sets 1 and 5 revealed the highest reduction, with guide 8645G providing about 99% knockdown (KD). Primerprobe set 5 was then selected and used for further evaluations. Viral copy number, dsRNA induction, and viral titer were then quantified using all guides in a treatment format with infected Vero cells transfected at 24 hpi. Quantitative PCR (qPCR) results demonstrated viral RNA reductions between 1 and 2 logs. Guides were also examined using viral double-stranded RNA (dsRNA) immunostaining (J2 antibody) and fluorescence imaging in infected Vero cells. By visual inspection, the guides had a profound effect on dsRNA staining, with guide 8645G having the lowest detectable signal. When quantified, guide 8645G yielded an approximately 2 log reduction in dsRNA. Finally, significant reductions in viral titer of approximately 1 log from all tested guides were observed. It should be noted that unlike the use of AAV, plasmids, or RNP delivery, mRNA- based expression of Cast 3a, even with high levels of activation due to the abundance of viral RNA, does not promote cell death.

[0139] RNAseq reveals a reduction of cell antiviral response with minimal off-target effects

[0140] To ensure that the mitigation of DENV infection due to the enzymatic activity of Cast 3, additional controls were examined, including the use of a “dead” (enzymatically inactive) version of Casl3a (dCasl3a) or a GFP encoding mRNA with guide 8645G. The additional controls were first tested in a “cell-free detection assay”, demonstrating that the only condition that results in RNA degradation, is when an enzymatically active Casl3a and guide 8645G are used together. This experiment was repeated in a cell-based assay, which includes viral infections. While viral RNA knockdown was observed in all conditions, the greatest reduction was observed with the enzymatically active Casl3a and guide 8645G. These results indicated that a fraction of the KD effect may be induced by Casl3a RNP binding, slowing viral RNA production, likely due to steric interference. This binding effect, though, did not result in significant reductions in dsRNA immunostaining and moderate reductions in titer, as only the active enzyme affected these metrics significantly. Using the same experimental conditions, RNAseq was performed; raw read coverage showed that specific regions of the genome were read more efficiently than others. When the reads were normalized to the average of the virus only condition, differences were observed. The normalized sequencing data confirmed that the enzymatic action of Casl3a was needed for RNA knockdown and validated the need for active enzyme to decrease viral replication and titer.

[0141] The effect of Casl3 on differential gene expression was also examined. Four comparisons were made: 1) Casl3a with a targeted (guide 8645G) vs a non-targeted guide (control) (NTCR), to compare DENV2 knockdown in conjunction with transfection, 2) DENV2 infection only vs the active Cast 3a + NTCR, to compare the effect of transfection in conjunction with DENV2 infection, 3) Casl3a + guide 8645G vs dCasl3a + guide 8645G, to determine the effect of bound, but inactive, Casl3 complexes on DENV knockdown, and 4) Casl3a + guide 8645G and a GFP- encoding mRNA with guide 8645G, to compare how the targeted guide in the Casl3a complex affects host gene expression compared to an un-complexed guide during DENV infection. Significant knockdown of genes involved in the antiviral response, such as TNFAIP3, CCL5, and CXCL10 were observed when comparing targeted, active Cast 3a to any of the transfected control groups. Among these, CCL5 and CXCL10 are inflammatory chemokines, typically involved in recruiting immune cells to the site of infection. Interestingly, certain genes were dysregulated during transfection, regardless of enzymatic activity or binding by Cast 3a or guides, such as THBS1, AKR1B10, and C0L4A1. The majority of housekeeping genes were unchanged by either infection or transfection, regardless of enzymatic activity or binding by Casl3a or guides. Together, these data indicate that Cast 3a enzymatic cleavage is only present during viral infections with an active enzyme and targeted guide, and that this anti-viral activity is specific, with minimal off-target effects on highly expressed host mRNAs.

[0142] Screening and assessment of guides against DENV3

[0143] The DENV2 guides were tested against DENV3, H87 strain. Like DENV2, 5 primer-probe sets were tested for DENV3 along with the use of a higher MOI (MOI=1), needed to promote a productive infection. In this case, primer-probe set 3 yielded the lowest RNA levels across all guides; and thus, a more accurate assessment of knockdown. Guide 8645G achieved the highest reduction, approximately 90%. Changes in DENV3-induced dsRNA levels and viral titer were assessed. Double-stranded RNA levels decrease across all of the guides, with a 30% decrease using guide 8645G and approximately 90% decrease using 8641G. When titer was assessed, all guides demonstrated significant reductions in titer. Overall, these results support the use of individual guides to inhibit multiple DENV serotypes. Evaluation of systemic LNP-mediated mRNA delivery to cell types susceptible to DENV

[0144] For DENV, clinical and autopsy findings in humans, as well as nonhuman primates, have indicated that cells of the mononuclear phagocyte lineage are the primary cell targets. Human autopsy studies have described macrophages and lymphocytes located in the spleen, lymph nodes, peripheral blood, lungs, liver, kidney, and stomach, as well as endothelial cells, hepatocytes, and non-neuronal cells of the central nervous system as the primary targets. In mice, DENV has been found in lymph nodes, spleen, bone marrow, and circulating white blood cells. Macrophages and dendritic cells were demonstrated to be targets for DENV2 infection in the spleen identified by flow cytometry and PCR.

[0145] In preparation of an in vivo DENV2 challenge, the assessment of efficient mRNA delivery to DENV targeted myeloid cells was performed. A Cre-LoxP-tdTomato system with a mRNA encoded, GPI-anchored, camelid antibody (VHH) was used. When expressed in cells, the VHH antibodies accumulate on cellular membranes, allowing for sensitive flow cytometry detection with an anti-VHH antibody. Using this strategy, the delivery of a number of LNP “liver” chemistries to hepatocytes and liver and splenic macrophages were compared. LNPs with the following ionizable lipids, cKK-E12 (MIT), MC3, LP01 (Intellia™), and Lipid 5 (Moderna™) were compared. All of these are efficient “liver-tropic” LNPs, with MC3, LP01, and Lipid 5 having been used in mice, NHPs, and humans. The LNP, cKK-E12 (3,6-bis(4-(bis(2- hydroxydodecyl)amino)butyl)piperazine-2, 5-dione) had the highest % delivery and mean cellular fluorescence intensity (MFI) in hepatocytes and was also very efficient in targeting liver and splenic macrophages, as evaluated by flow cytometry.

[0146] 3, 6-his(4-(bi$(2-hydroxydodecyi)amino)butyl)piperaziiie-2, 5-dione

[0147] Therefore, the cKK-E12 LNP was used in vivo efficacy experiments. mRNA expressed Casl3a treats DENV2 infections in vivo

[0148] To demonstrate the potency of this approach in vivo, a stringent challenge study was designed to test whether Casl3a could treat lethal DENV infections in mice and prevent mortality. A129 female mice were infected via an intraperitoneal (IP) injection with 105pfu of DENV 2, New Guinea C strain (NGC). At 24 h post infection, animals received either LNP-encapsulated mRNA encoding Casl3a and guide 8645G or Casl3a and an NTCR guide, while the last set received only PBS, via tail vein IV injection. Mice body weights and physical signs / behavior were monitored daily, with animals euthanized if they lost more than 20% body weight or if they exhibited 3 physical signs of disease such as hunched posture, ruffled fur, difficulty walking, or shaking. Visceral infections with DENV in humans and mice typically do not invade the CNS, and IFN-competent strains of mice are not susceptible to DENV-induced disease. However, DENV traffics (potentially inside infected macrophages) to the brain as the infection progresses in mice with knocking out Type 1 IFN (A129) or Type 1 / 2 IFN (AG129) receptors, and thus will exhibit neurological signs such as difficulty walking and shaking.

[0149] In the PBS group, all 5 animals were euthanized by day 6. For the Casl3a + NTCR animals, 5 of the 6 were euthanized by day 7, and one animal recovered. All 8 animals that received mRNA encoded Cast 3a and the 8645G guide RNA recovered with no observed neurological signs. This experiment was then repeated, and animals were bled on day 2 and 3 for titer assessment. Here, 3 of the 5 animals that received Casl3a mRNA and the NTCR guide were euthanized by day 7, while all 5 of the animals that received Casl3a mRNA and guide 8645G survived, confirming the previous results. Statistically significant decreases in viral titer were measured at both day 2 and 3, with no detectable titer at day 2 in the treated group. Overall, these results demonstrate that targeted mRNA expressed Casl3a potently treat DENV 2-induced disease in vivo. mRNA expressed Casl3a treats DENV3 infections in vivo

[0150] To assess the ability to treat multiple DENV serotypes, a DENV 3 treatment challenge experiment was performed. AG129 mice (male and female) were infected via an intraperitoneal (IP) injection with 105pfu of DENV 3, H87 strain. After 24 hours, mice received mRNA encoded Casl3a and either guide 8645G or NTCR, while the control group received only PBS, via tail vein IV injection. In the case of PBS and NTCR conditions, all animals were euthanized by day 4 and day 7, respectively. All 9 animals that received the mRNA encoded Casl3a and 8645G guide RNA recovered with no adverse clinical signs observed for the duration of the study. In addition, statistically significant decreases in viral titer, up to 4 logs of reduction, were measured at day 3 for both male and female animals. These large decreases in titer likely are responsible for the survival of the treated animals both for DENV 2 and 3. Together, these data demonstrate that treatment with mRNA expressed Casl3a, along with guide 8645G, significantly reduce the effects of DENV 2 and 3 infections in the mouse model.

Claims

CLAIMSWe claim:

1. A method of treating a viral infection comprising administering to a subj ect in need thereof an effective amount of a lipid nanoparticle comprising: a) a nucleic acid encoding a Cas nuclease and b) anti-viral Cas guide RNA, wherein the anti-viral Cas guide RNA comprises a segment that is a virus genome sequence.

2. The method of claim 1, wherein the Cas nuclease is a Casl3a endonuclease.

3. The method of claim 2, wherein the anti-viral Cas guide RNA comprises a segment that is a dengue virus genome sequence.

4. The method of claim 3, wherein the segment that is a dengue virus genome sequence is a greater than 15 nucleotide segment within SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

5. The method of claim 3, wherein the segment that is a dengue virus genome sequence consists of a 5’ end GUGUCU GUC AUU GCC AUC UGU GUC ACC (SEQ ID NO: 11)(8645G).

6. The method of claim 3, wherein the segment that is a dengue virus genome sequence consists of a 5’ end UG UCU GUC AUU GCC AUC UGU GUC ACC A (SEQ ID NO: 10)(8644G).

7. The method of claim 3, wherein the segment that is a dengue virus genome sequence consists of a 5’ end G UCU GUC AUU GCC AUC UGU GUC ACC AU (SEQ ID NO: 9)(8643G).

8. The method of claim 3, wherein the segment that is a dengue virus genome sequence consists of a 5’ end UCU GUC AUU GCC AUC UGU GUC ACC AUG (SEQ ID NO: 8)(8642G).

9. The method of claim 3, wherein the segment that binds a dengue virus genome sequence consists of a 5’ end CU GUC AUU GCC AUC UGU GUC ACC AUG G (SEQ ID NO: 7)(8641G).

10. The method of claim 3, wherein the segment that is a dengue virus genome sequence consists of a 5’ end U GUC AUU GCC AUC UGU GUC ACC AUG GG (SEQ ID NO: 6)(8640G).

11. The method of claim 1, wherein the lipid nanoparticle comprises an ionizable lipid, sterol, and a polyethylene glycosylated lipid.

12. The method of claim 1, wherein the ionizable lipid 3,6-bis(4-(bis(2- hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (cKK-E12).

13. A pharmaceutical composition comprising a lipid nanoparticle comprising: a) a nucleic acid encoding a Cas nuclease and b) anti-viral Cas guide RNA or a nucleic acid encoding an anti-viral Cas guide RNA, wherein the anti-viral Cas guide RNA comprises a segment that is a viral genome sequence.

14. The pharmaceutical composition of claim 13, wherein the Cas nuclease is a Casl3a endonuclease.

15. The pharmaceutical composition of claim 13, wherein the anti-viral Cas guide RNA comprises a segment that is a dengue virus genome sequence.

16. The pharmaceutical composition of claim 15, wherein the segment that is a dengue virus genome sequence is a greater than 15 nucleotide segment withing SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

17. The pharmaceutical composition of claim 15, wherein the single stranded segment that is a dengue virus genome sequence consists of a 5’ end GUG UCU GUC AUU GCC AUC UGU GUC ACC (SEQ ID NO: 11)(8645G).

18. The pharmaceutical composition of claim 15, wherein the segment that is a dengue virus genome sequence consists of a 5’ end UG UCU GUC AUU GCC AUC UGU GUC ACC A (SEQ ID NO: 10)(8644G).

19. The pharmaceutical composition of claim 15, wherein the segment that is a dengue virus genome sequence consists of a 5’ end G UCU GUC AUU GCC AUC UGU GUC ACC AU (SEQ ID NO: 9)(8643G).

20. The pharmaceutical composition of claim 15, wherein the segment that is a dengue virus genome sequence consists of a 5’ end UCU GUC AUU GCC AUC UGU GUC ACC AUG (SEQ ID NO: 8)(8642G).

21. The pharmaceutical composition of claim 15, wherein the segment that is a dengue virus genome sequence consists of a 5’ end CU GUC AUU GCC AUC UGU GUC ACC AUG G (SEQ ID NO: 7)(8641G).

22. The pharmaceutical composition of claim 15, wherein the segment that is a dengue virus genome sequence consists of a 5’ end U GUC AUU GCC AUC UGU GUC ACC AUG GG (SEQ ID NO: 6)(8640G).

23. The pharmaceutical composition of claim 13, wherein the lipid nanoparticle comprises an ionizable lipid, sterol, and a polyethylene glycosylated lipid.

24. The pharmaceutical composition of claim 23, wherein the ionizable lipid 3,6-bis(4-(bis(2- hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (cKK-E12).

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