US 2017.0196966A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0196966 A1 HENDERSON (43) Pub. Date: Jul. 13, 2017

(54) MICRONEEDLE COMPOSITIONS AND A6IR 9/00 (2006.01) METHODS OF USING SAME A6IR 48/00 (2006.01) (52) U.S. Cl. (71) Applicant: Verndari, Inc., Sacramento, CA (US) CPC ...... A61K 39/145 (2013.01); A61K 48/005 (2013.01); C12N 7/00 (2013.01); A61K 9/002I (72) Inventor: Daniel R. HENDERSON, Sacramento, (2013.01); CI2N 27.60/16(34 (2013.01); CI2N CA (US) 2760/16234 (2013.01); C12N 2770/36143 (2013.01); A61 K 2039/53 (2013.01) (21) Appl. No.: 15/403,989 (57) ABSTRACT (22) Filed: Jan. 11, 2017 Described herein, are microneedle devices comprising a recombinant alphavirus replicon encoding an exogenous Related U.S. Application Data polypeptide, wherein the recombinant alphavirus replicon is (60) Provisional application No. 62/277.312, filed on Jan. coated onto or embedded into a plurality of microneedles. 11, 2016. Also described herein are methods of preparing a microneedle device comprising a recombinant alphavirus replicon encoding an exogenous polypeptide. Also disclosed Publication Classification herein are methods of inducing an immune response in an (51) Int. Cl. individual comprising contacting the individual with a A6 IK 39/45 (2006.01) microneedle device comprising a recombinant alphavirus CI2N 7/00 (2006.01) replicon encoding an exogenous polypeptide.

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MCRONEEDLE COMPOSITIONS AND antigen associated with a cancer. In some embodiments, the METHODS OF USING SAME foreign antigen is an antigen associated with an infectious agent. In some embodiments, the recombinant alphavirus CROSS REFERENCE TO RELATED replicon is present in an amount effective to induce an APPLICATIONS immune response to the foreign or self-antigen. In some 0001. This application claims the benefit of U.S. Provi embodiments, the exogenous polypeptide is an influenza sional Application No. 62/277.312, filed on Jan. 11, 2016, virus HA or NA polypeptide. In some embodiments, the which is incorporated by reference herein in its entirety. influenza virus HA polypeptide is an influenza A virus HA polypeptide or an influenza B virus HA polypeptide. In some SEQUENCE LISTING embodiments, the influenza virus HA polypeptide is from a 0002 The instant application contains a Sequence Listing viral strain of a group 1 influenza A virus Subtype selected which has been submitted electronically in ASCII format from H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and is hereby incorporated by reference in its entirety. Said or H18. In some embodiments, the influenza virus HA ASCII copy, created on Jan. 31, 2017, is named polypeptide is from a viral strain of a group 2 influenza A 47750701201 SL.txt and is 514 bytes in size. virus subtype selected from H3, H4, H7, H10, H14, or H15. In some embodiments, the influenza virus HA polypeptide is BACKGROUND OF THE INVENTION from a viral strain of an influenza B virus. In some embodi ments, the influenza virus HA polypeptide is from a viral 0003 Delivery of compositions to a target cell or tissue strain of an influenza A virus H1 subtype. In some embodi faces various transport barriers. Nucleic acids that encode ments, the influenza virus HA polypeptide is from a viral gene products, such as proteins, and non-coding RNA (e.g., strain of an influenza A virus H3 subtype. In some embodi siRNAs) can be delivered directly to a desired vertebrate ments, the influenza virus HA polypeptide is from a viral subject, or can be delivered ex vivo to cells obtained or strain of an influenza B virus Yamagata or Victoria lineage. derived from the subject, and the cells can be re-implanted In some embodiments, the recombinant alphavirus replicon into the subject. Delivery of such nucleic acids to a verte encodes an exogenous polypeptide comprising: (a) an HA brate subject is desirable for many purposes, such as, for polypeptide from a viral strain of an influenza A virus H1 gene therapy, to induce an immune response against an subtype; (b) an HA polypeptide from a viral strain of an encoded polypeptide, or to regulate the expression of endog influenza A virus H3 subtype; (c) an HA polypeptide from a enous genes. The use of this approach has been hindered viral strain of an influenza B virus Yamagata lineage; (d) an because free DNA is not readily taken up by cells and free HA polypeptide from a viral strain of an influenza B virus RNA is rapidly degraded in vivo. Moreover, delivery can Victoria lineage; or (e) any combinations thereof. In some also be problematic. For instance, Subcutaneous or intra embodiments, the recombinant alphavirus replicon encodes muscular injections using hypodermic needles can cause at least two exogenous polypeptides comprising: (a) an HA pain, trauma, and anxiety in a Subject. polypeptide from a viral strain of an influenza A virus H1 0004 Delivery of one or more polypeptides, whether subtype; (b) an HA polypeptide from a viral strain of an directly as protein or indirectly by an encoding polynucle influenza A virus H3 subtype; (c) an HA polypeptide from a otide, has many useful applications, including vaccination. viral strain of an influenza B virus Yamagata lineage; or (d) Vaccination has proven an effective means to fight and even an HA polypeptide from a viral strain of an influenza B virus eradicate infectious diseases. The influenza vaccine, for Victoria lineage. In some embodiments, each of the exog example, is currently recommended by the CDC as the enous polypeptides are encoded on a single recombinant primary method for preventing influenza. However, influ alphavirus replicon. In some embodiments, the exogenous enza virus has a high rate of mutation and antigenic variation polypeptides are encoded on different recombinant alphavi and a new vaccine is typically produced each year based rus replicons. In some embodiments, the exogenous poly upon the predicted circulating pathogenic strains. This poses peptide is a hepatitis B virus Surface antigen (HBSAg). In a number of challenges. For instance, the effectiveness of the Some embodiments, the recombinant alphavirus replicon vaccine is only as good as the prediction. If the prediction of encodes an exogenous polypeptide comprising: (a) an anti the dominant strain is incorrect, the vaccine will have gen from a polio virus; (b) an antigen from Clostridium limited effectiveness for most people. Further, it can take tetani; (c) an antigen from a rabies virus; or (d) any months to produce enough influenza vaccine to vaccinate a combinations thereof. In some embodiments, the recombi population. nant alphavirus replicon encodes an exogenous polypeptide comprising: (a) an antigen from a polio virus; (b) an antigen SUMMARY OF THE INVENTION from Clostridium tetan; and (c) an antigen from a rabies 0005 Disclosed herein, in some embodiments, are virus. In some embodiments, each of the exogenous poly microneedle devices for administering an RNA molecule, peptides are encoded on a single recombinant alphavirus comprising: (a) a Substrate comprising a plurality of replicon. In some embodiments, the exogenous polypeptides microneedles; and (b) a composition comprising an RNA are encoded on different recombinant alphavirus replicons. encoding an exogenous polypeptide coated onto or embed In some embodiments, the recombinant alphavirus replicon ded into the plurality of microneedles. In some embodi encodes an exogenous polypeptide comprising: (a) an anti ments, the RNA molecule is a recombinant alphavirus gen from a Marburg virus; (b) an antigen from an Ebola replicon. In some embodiments, the RNA molecule is dehy Sudan virus; (c) an antigen from an Ebola Zaire virus; or (d) drated. In some embodiments, the plurality of microneedles any combinations thereof. In some embodiments, the recom are dissolvable, biosoluble, or biodegradable. In some binant alphavirus replicon encodes an exogenous polypep embodiments, the exogenous polypeptide is a foreign or a tide comprising: (a) an antigen from a Marburg virus; (b) an self-antigen. In some embodiments, the self-antigen is an antigen from an Ebola Sudan virus; and (c) an antigen from US 2017/O 196966 A1 Jul. 13, 2017

an Ebola Zaire virus. In some embodiments, each of the embodiments, the dendrimer is a PAMAM dendrimer. In exogenous polypeptides are encoded on a single recombi some embodiments, the PAMAM dendrimer comprises nant alphavirus replicon. In some embodiments, the exog amino Surface reactive groups. In some embodiments, the enous polypeptides are encoded on different recombinant PAMAM dendrimer is a G5 or G9 PAMAM dendrimer alphavirus replicons. In some embodiments, the comprising amino Surface reactive groups. In some embodi microneedle device is effective in inducing an immune ments, the dendrimer-replicon nanoparticle is produced by a response to the exogenous polypeptide after storage for at microfluidic mixing device. In some embodiments, the least one month at room temperature. In some embodiments, recombinant alphavirus replicon is coated onto the plurality a second bioactive agent coated onto or embedded into the of microneedles using a microfluidic dispensing device. plurality of microneedles. In some embodiments, the second bioactive agent is a polypeptide. In some embodiments, the 0008 Also disclosed herein, in some embodiments, are second bioactive agent enhances an immune response in an methods of inducing an immune response in an individual in individual. In some embodiments, the second bioactive need thereof, comprising: (a) contacting the dermal Surface agent is an adjuvant. In some embodiments, the recombinant of the individual with a microneedle device comprising (i) a alphavirus replicon is formulated as a dendrimer-replicon plurality of microneedles comprising a recombinant nanoparticle. In some embodiments, the dendrimer is a alphavirus replicon encoding an exogenous polypeptide PAMAM dendrimer. In some embodiments, the PAMAM coated onto or embedded into the plurality of microneedles, dendrimer comprises amino Surface reactive groups. In and (b) delivering the recombinant alphavirus replicon to some embodiments, the PAMAM dendrimer is a G5 or G9 the individual, thereby inducing an immune response in the PAMAM dendrimer comprising amino surface reactive individual. In some embodiments, the recombinant alphavi groups. In some embodiments, the PAMAM dendrimer rus replicon is dehydrated. In some embodiments, the plu comprises modified amino Surface reactive groups. In some rality of individual microneedles are dissolvable, biosoluble, embodiments, the modified amino Surface reactive groups or biodegradable. In some embodiments, the exogenous are modified with a fluorinating agent, an N-hydroxysuc polypeptide is a foreign or a self-antigen. In some embodi cinimide ester, or an amino acid. In some embodiments, the ments, the self-antigen is an antigen associated with a N-hydroxysuccinimide ester is an N-hydroxysuccinimide cancer. In some embodiments, the foreign antigen is an ester of PEG or an N-hydroxysuccinimide ester of a cell antigen associated with an infectious agent. In some penetrating peptide. In some embodiments, the fluorinating embodiments, the recombinant alphavirus replicon is pres agent is heptafluoro butyric acid anhydride. In some ent in an amount effective to alone induce an immune embodiments, the amino acid is arginine or histidine. In response to the foreign or self-antigen. In some embodi Some embodiments, the dendrimer-replicon nanoparticle is ments, the exogenous polypeptide is an influenza virus HA formulated by a microfluidic mixing device. In some or NA polypeptide. In some embodiments, the influenza embodiments, the recombinant alphavirus replicon is coated virus HA polypeptide is an influenza A virus HA polypeptide onto the plurality of microneedles using a microfluidic or an influenza B virus HA polypeptide. In some embodi dispensing device. ments, the influenza virus HA polypeptide is from a viral strain of a group 1 influenza A virus Subtype selected from 0006 Disclosed herein, in some embodiments, are H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, or H18. microneedle devices for administering an RNA molecule, In some embodiments, the influenza virus HA polypeptide is comprising: (a) a Substrate comprising a plurality of from a viral strain of a group 2 influenza A virus Subtype microneedles; and (b) a pharmaceutical composition com selected from H3, H4, H7, H10, H14, or H15. In some prising a recombinant alphavirus replicon encoding an embodiments, the influenza virus HA polypeptide is from a exogenous polypeptide and a pharmaceutically acceptable viral strain of an influenza B virus. In some embodiments, carrier or excipient coated onto or embedded into the the influenza virus HA polypeptide is from a viral strain of plurality of microneedles. an influenza A virus H1 subtype. In some embodiments, the 0007 Disclosed herein, in some embodiments, are meth influenza virus HA polypeptide is from a viral strain of an ods of delivering a polypeptide to an individual in need influenza A virus H3 subtype. In some embodiments, the thereof, comprising administering to the individual any one influenza virus HA polypeptide is from a viral strain of an of the microneedle devices described herein. Also disclose influenza B virus Yamagata or Victoria lineage. In some herein, in some embodiments, are methods of preparing a embodiments, the recombinant alphavirus replicon encodes microneedle device, comprising: (a) obtaining a substrate an exogenous polypeptide selected from: (a) an HA poly comprising a plurality of microneedles; and (b) coating or peptide from a viral strain of an influenza A virus H1 embedding a composition comprising an RNA molecule subtype; (b) an HA polypeptide from a viral strain of an encoding an exogenous polypeptide onto or into the plurality influenza A virus H3 subtype; (c) an HA polypeptide from a of microneedles. In some embodiments, the RNA molecule viral strain of an influenza B virus Yamagata lineage; (d) an is a recombinant alphavirus replicon. In some embodiments, HA polypeptide from a viral strain of an influenza B virus the recombinant alphavirus replicon is dehydrated. In some Victoria lineage; or (e) any combinations thereof. In some embodiments, the recombinant alphavirus replicon is dehy embodiments, the recombinant alphavirus replicon encodes drated prior to being coated onto or embedded into the at least two exogenous polypeptides selected from: (a) an plurality of microneedles. In some embodiments, the recom HA polypeptide from a viral strain of an influenza A virus binant alphavirus replicon is dehydrated after being coated H1 subtype; (b) an HA polypeptide from a viral strain of an onto or embedded into the plurality of microneedles. In influenza A virus H3 subtype; (c) an HA polypeptide from a some embodiments, the plurality of individual microneedles viral strain of an influenza B virus Yamagata lineage; or (d) are dissolvable, biosoluble, or biodegradable. In some an HA polypeptide from a viral strain of an influenza B virus embodiments, the recombinant alphavirus replicon is for Victoria lineage. In some embodiments, each of the exog mulated as a dendrimer-replicon nanoparticle. In some enous polypeptides are encoded on a single recombinant US 2017/O 196966 A1 Jul. 13, 2017

alphavirus replicon. In some embodiments, the exogenous nsP4) and five structural genes (capsid, E3, E2, 6K, E1) is polypeptides are encoded on different recombinant alphavi also illustrated. Typically, one or more, but preferably all, of rus replicons. In some embodiments, the exogenous poly the structural genes are excluded from the recombinant peptide is a hepatitis B virus Surface antigen (HBSAg). In alphavirus replicon encoding an exogenous polypeptide. Some embodiments, the recombinant alphavirus replicon is After generation of the recombinant alphavirus replicon, it formulated as a dendrimer-replicon nanoparticle. In some is packaged into or onto a microneedle or microneedle array. embodiments, the dendrimer is a PAMAM dendrimer. In The microneedle is applied to the skin of a subject for some embodiments, the PAMAM dendrimer comprises intradermal administration of the replicon. amino Surface reactive groups. In some embodiments, the 0012 FIG. 2 shows an illustration of a microneedle dendrimer is a G5 or G9 PAMAM dendrimer comprising useful for delivery of a bioactive agent. In some embodi amino Surface reactive groups. In some embodiments, the ments, needles have a height of about 1500 microns and a dendrimer-replicon nanoparticle is formulated by a micro base with a diameter of about 670 microns. In some embodi fluidic mixing device. In some embodiments, the recombi ments, microneedles are provided in an array, such as an nant alphavirus replicon is coated onto the plurality of array on a patch having an area of about 1 cm. microneedles using a microfluidic dispensing device. In 0013 FIG. 3 exemplifies an EGFP-replicon containing Some embodiments, the recombinant alphavirus replicon is DNA plasmid puC57-Kan-T7-VEEV-EGFP. present in an amount effective to induce an immune response 0014 FIG. 4 exemplifies an influenza HA-replicon con in the individual to the exogenous polypeptide. In some taining DNA plasmid puC57-Kan-T7-VEEV-HA. embodiments, a second bioactive agent packaged in or on the microneedles. In some embodiments, the second bioac 0015 FIG. 5 exemplifies a hepatitis B surface antigen tive agent is a polypeptide. In some embodiments, the replicon DNA plasmid puC57-Kan-T7-VEEV-HBSAg2. second bioactive agent enhances an immune response. In 0016 FIG. 6 exemplifies a replicon with no polypeptide Some embodiments, the second bioactive agent is an adju (“empty replicon'); DNA plasmid puC57-Kan-T7-VEEV. vant. In some embodiments, the dermal Surface is pre (0017 FIG. 7 exemplifies linear, self-replicating RNA treated prior to contacting the individual with the replicons. microneedle device. In some embodiments, the individual is 0018 FIG. 8 is an exemplary agarose gel showing suc contacted with a second microneedle device comprising a cessful in vitro transcription of replicon RNA. plurality of microneedles, wherein each microneedle com prises the recombinant alphavirus replicon encoding the (0019 FIG. 9 is an exemplary EGFP western blot of 24 exogenous polypeptide coated onto or embedded into the hour and 48 hour cell lysates obtained from EGFP-replicon microneedle. In some embodiments, the individual is admin transfected HEK-293T cells. Native replicon EGFP is a 26.9 istered a second composition comprising the alphavirus kDa protein while the commercially-obtained positive con replicon encoding the exogenous polypeptide by a second trol EGFP protein is a 32.7 kDa protein. route of administration. In some embodiments, the second 0020 FIG. 10 is an exemplary influenza HA protein route of administration is oral. In some embodiments, oral western blot of cell lysates obtained from HA-replicon administration of the second composition is administered transfected HEK-293T cells. Molecular weight ladders are prior to contacting the individual with the microneedle shown in lanes 1 and 7, while HA protein positive control device. In some embodiments, oral administration of the (72 kDa) is in lane 2. HA-replicon transfected samples are second composition is administered after contacting the present in lanes 4, 5, and 6. Although the anti-HA antibody individual with the microneedle device. demonstrates some cross-reactivity with HEK-293 cell 0009. Also disclosed herein, in some embodiments, are lysates, there is no 72 kDa band present in the lysate methods of monitoring an immune response in an individual buffer-only negative control in lane 3. comprising: (a) administering to the individual the (0021 FIG. 11 is an exemplary analysis of EGFP fluores microneedle device of any one of claims 1-46; and (b) cence from HEK-293T cell lysates obtained 24–48 hours assaying a sample from the individual to determine a level post EGFP-replicon transfection. of an immune response in the individual against the exog 0022 FIG. 12 is an exemplary comparison of the enous polypeptide. amounts of EGFP fluorescence of an EGFP-replicon RNA versus an EGFP mRNA. BRIEF DESCRIPTION OF THE DRAWINGS 0023 FIG. 13 is an exemplary Malvern Zetasizer char 0010. The novel features of the invention are set forth acterization of the size of replicon RNA, G9 dendrimer, and with particularity in the appended claims. A better under the dendrimer-replicon complex. standing of the features and advantages of the present 0024 FIG. 14 is an exemplary Malvern Zetasizer char invention will be obtained by reference to the following acterization of the size of replicon particles made with lipids detailed description that sets forth illustrative embodiments, or dendrimers following nanoparticle assembly in either the in which the principles of the invention are utilized, and the Precision NanoSystems microfluidic mixing Nano Assemblr accompanying drawings of which: (Rep. Lipid Nano particle, Rep. Lipid Nano particle #2) or 0011 FIG. 1 illustrates an exemplary method of produc mixed by hand (remaining samples). ing recombinant alphavirus replicons for transdermal (0025 FIG. 15 is an exemplary Malvern Zetasizer char administration to a subject. A linear DNA template com acterization of the polydispersity index (PDI) of replicon prises a sequence (SEQ ID NO: 1) that encodes a recombi particles made with lipids or dendrimers following nano nant alphavirus replicon encoding an exogenous polypep particle assembly with either the Precision NanoSystems tide. The T7 RNA polymerase transcribes the DNA sequence microfluidic mixing Nano Assemblr (Rep. Lipid Nano par into an RNA transcript. A wild-type alpha virus replicon ticle, Rep Lipid Nano particle #2) or mixed by hand (remain cassette containing four non-structural (nsP1, nsP2, nsP3, ing samples). US 2017/0196966 A1 Jul. 13, 2017

0026 FIG. 16 exemplifies the recovery of EGFP mRNA Certain Terminology from a BioDot printed microneedle array as measured by a 0035). The terms “polynucleotide,” “nucleotide, cubit fluorimeter. “nucleotide sequence.” “nucleic acid.” and "oligonucle 0027 FIG. 17 is a CAD drawing of an exemplary indi otide' are used interchangeably. They refer to a polymeric vidual microneedle. form of nucleotides of any length, either deoxyribonucle 0028 FIG. 18 is a photograph of an exemplary 1 cm otides or ribonucleotides, or analogs thereof. The following microneedle array loaded with 5nL 1% Congo Red/well (see are non-limiting examples of polynucleotides: coding or arrowhead). non-coding regions of a gene or gene fragment, loci (locus) 0029 FIG. 19 exemplifies loaded microneedles in the Z defined from linkage analysis, exons, introns, messenger plane. RNA (mRNA), transfer RNA (tRNA), ribosomal RNA 0030 FIG. 20 exemplifies a microneedle array patch (rRNA), short interfering RNA (siRNA), short-hairpin RNA applied to the dorsal skin of a mouse. (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recom 0031 FIG. 21 exemplifies EGFP fluorescence in the binant polynucleotides, branched polynucleotides, plasmids, dorsal skin of a mouse treated with a microneedle array vectors, isolated DNA of any sequence, isolated RNA of any patch containing EGFP protein. sequence, nucleic acid probes, and primers. In some 0032 FIG. 22 exemplifies the production and titer of embodiments, a polynucleotide comprises one or more EGFP antibodies obtained from the 28-day sera of mice modified nucleotides, such as methylated nucleotides and treated with an EGFP-replicon RNA coated microneedle nucleotide analogs. In some embodiments, modifications to array patch. the nucleotide structure are imparted before or after assem bly of the polymer. In some embodiments, the sequence of DETAILED DESCRIPTION OF THE nucleotides is interrupted by non-nucleotide components. In INVENTION some embodiments, the polynucleotide is further modified after polymerization, such as by conjugation with a labeling 0033 Disclosed herein, in some embodiments, are com component. positions comprising self-replicating RNAs or replicon 10036). The terms “polypeptide,” “peptide,” and “protein' RNAs that are formulated for delivery to a subject in need are used interchangeably herein to refer to polymers of thereof, methods of preparing the same, and methods of amino acids of any length. In some embodiments, a poly administering the compositions to a subject in need thereof, peptide is any protein, peptide, protein fragment, or com Such as for vaccination or gene therapy. ponent thereof. In some embodiments, a polypeptide is a 0034) In some embodiments, the compositions comprise protein naturally occurring in nature or a protein that is a bioactive agent (e.g., a polypeptide, or a recombinant ordinarily not found in nature. In some embodiments, a alphavirus replicon or RNA molecule that encodes an exog polypeptide consists largely of the standard twenty protein enous polypeptide), coated onto or embedded into or a building amino acids or it is modified to incorporate non microneedle or microneedle array. In some embodiments, Standard amino acids. In some embodiments, a polypeptide the bioactive agent is coated onto or embedded into the is modified, typically by the host cell, for example, by microneedle and the bioactive agent is in a dehydrated form. adding any number of biochemical functional groups, In Some embodiments, the bioactive agent comprises a including phosphorylation, acetylation, acylation, formy recombinant alpha virus replicon. In some embodiments, the lation, alkylation, methylation, lipid addition (e.g., palmi recombinant alpha virus replicons are embedded into a toylation, myristoylation, prenylation, etc.) and carbohy microneedle. In some embodiments, the recombinant drate addition (e.g., N-linked and 0-linked glycosylation, alphavirus replicons are coated onto a microneedle. In some etc.). In some embodiments, polypeptides undergo structural embodiments, the recombinant alpha virus replicons are changes in the host cell such as the formation of disulfide coated onto a microneedle by dipping, followed by dehy bridges or proteolytic cleavage. dration of the coated alphavirus replicon. In some embodi 0037. In general, "sequence identity” refers to an exact ments, the recombinant alphavirus replicons are coated onto nucleotide-to-nucleotide or amino acid-to-amino acid cor a microneedle by a microfluidic dispensing device, followed respondence of two polynucleotides or polypeptide by dehydration of the coated alphavirus replicon. In some sequences, respectively. Typically, techniques for determin embodiments, the microfluidic dispensing device is a Bio ing sequence identity include determining the nucleotide Dot printer, or similar device. In some embodiments, the sequence of a polynucleotide and/or determining the amino alphavirus replicons are formulated into an alphavirus rep acid sequence encoded thereby, and comparing these licon-dendrimer nanoparticle. In some embodiment, the sequences to a second nucleotide or amino acid sequence. dendrimer is a PAMAM dendrimer. In some embodiments, Any suitable technique is contemplated by the disclosure the dendrimer is a PAMAM dendrimer with amino surface herein. In some embodiments, two or more sequences (poly reactive groups. In some embodiments, the PAMAM den nucleotide or amino acid) are compared by determining their drimer with amino surface reactive groups is a G5 or G9 percent identity.” In some embodiments, ranges of desired PAMAM dendrimer. In some embodiments, the dendrimer is degrees of sequence identity are approximately 80% to a PAMAM dendrimer that comprises modified (e.g., fluori 100% and integer values there between. In some embodi nated) amino surface reactive groups. In some embodiments, ments, the percent identities between a disclosed sequence the alphavirus replicon-dendrimer nanoparticle is formu and a claimed sequence are at least 80%, at least 85%, at lated by hand mixing. In some embodiments, the alphavirus least 90%, at least 95%, or at least 98%. replicon-dendrimer nanoparticle is formulated by a micro 0038. The terms “subject,” “individual,” and “patient” fluidic mixing device. In some embodiments, the microflu are used interchangeably herein to refer to a vertebrate, idic mixing device is a Precision NanoSystems NanoAs preferably a mammal, more preferably a human. In some semblr, or similar device. embodiments, mammals include, but are not limited to: US 2017/O 196966 A1 Jul. 13, 2017

murines, simians, humans, farm animals, sport animals, and which greatly increases the stability of the composition at pets. In some embodiments, tissues, cells, and their progeny room temperature. Microneedle administration is painless, of a biological entity obtained in vivo or cultured in vitro are making it a more tolerated form of administration. encompassed. None of these terms, as used herein, entail 0043. In some embodiments, microneedles are solid Supervision of a medical professional. structures. In some embodiments, microneedles are hollow 0039. As used herein, the term “about’ is used to indicate structures. In some embodiments, bioactive agents for deliv that a value includes the standard deviation of error for the ery (e.g., polypeptides or recombinant alphavirus replicons device or method being employed to determine the value. or RNA molecules) are released through hollow structures 0040. Unless defined otherwise, all technical and scien (e.g., a liquid composition is injected or infused into the tific terms used herein have the same meaning as is com skin). In some embodiments, bioactive agents (e.g., poly monly understood by one of skill in the art to which the peptides or recombinant alphavirus replicons or RNA mol claimed subject matter belongs. It is to be understood that ecules) are packaged onto a microneedle (for example, the foregoing general description and the following detailed coated onto a surface of the microneedle after formation). In description are exemplary and explanatory only and are not Some embodiments, the bioactive agents are packaged onto restrictive of any Subject matter claimed. In this application, a microneedle as a dried form. In some embodiments, the the use of the singular includes the plural unless specifically bioactive agents are dehydrated after being packaged onto a stated otherwise. It must be noted that, as used in the microneedle. In some embodiments, compositions are pack specification and the appended claims, the singular forms aged into a microneedle (for example, forming part of the “a,” “an,” and “the' include plural referents unless the microneedle itself, such as by deposition into the interior of context clearly dictates otherwise. In this application, the use the microneedle, or by inclusion in a mixture used to form of “or” means “and/or unless stated otherwise. Further the microneedle). In some embodiments, the replicon is more, use of the term “including as well as other forms, dissolved in the skin compartment. In some embodiments, such as “include,” “includes, and “included,” is not the replicon is injected into the skin. In some embodiments, intended to be limited solely to the recited items. The section microneedles are formed in an array comprising a plurality headings used herein are for organizational purposes only of microneedles. In some embodiments, the microneedle and are not to be construed as limiting the Subject matter array is a 5x5 array of microneedles. In some embodiments, described. the microneedle array is physically or operably coupled to a Solid Support or Substrate. In some embodiments, the solid Microneedles and Microneedle Arrays Support is a patch. In some embodiments, the microneedle 0041 Disclosed herein, in some embodiments, are array is applied directly to the skin for intradermal admin microneedle devices for administering a recombinant istration of a composition. alphavirus replicon or RNA molecule encoding an exog 0044. A microneedle array patch can be any suitable enous polypeptide comprising: a Substrate comprising a shape or size. In some embodiments, a microneedle array plurality of microneedles; and a composition comprising a patch is shaped to mimic facial features, e.g., an eyebrow. In recombinant alphavirus replicon or RNA molecule encoding Some embodiments, a microneedle array patch is the Small an exogenous polypeptide coated onto or embedded into the est size allowable to deliver a selected amount of bioactive plurality of microneedles. Also disclosed herein, in some agent. embodiments, are methods of preparing a microneedle 0045. The size and shape of the microneedles varies as device, comprising: obtaining a substrate comprising a plu desired. In some embodiments, microneedles include a rality of microneedles; and coating or embedding a recom cylindrical portion physically or operably coupled to a binant alphavirus replicon encoding an exogenous polypep conical portion having a tip. In some embodiments, tide onto or into the plurality of microneedles. Also microneedles have an overall pyramidal shape or an overall disclosed herein, in some embodiments, are methods of conical shape. In some embodiments, the microneedle inducing an immune response in an individual in need includes a base and a tip. In some embodiments, the tip has thereof, comprising: (a) contacting the dermal Surface of an a radius that is less than or equal to about 1 micrometer. In individual with a microneedle device comprising (i) a plu Some embodiments, the microneedles are of a length Sufi rality of microneedles comprising a recombinant alphavirus cient to penetrate the stratum corneum and pass into the replicon encoding an exogenous polypeptide coated onto or epidermis or dermis. In certain embodiments, the embedded into the plurality of microneedles, and (b) deliv microneedles have a length (from their tip to their base) ering the recombinant alphavirus replicon to the individual, between about 0.1 micrometer and about 5 millimeters in thereby inducing an immune response in the individual. length, for instance about 5 millimeters or less, 4 millimeters 0042 Microneedles are structures of typically microm or less, between about 1 millimeter and about 4 millimeters, eter to millimeter size, and preferably designed to pierce the between about 500 micrometers and about 1 millimeter, skin and deliver a composition to the epidermis or dermis of between about 10 micrometers and about 500 micrometers, a Subject. Microneedles offer Some advantages over tradi between about 30 micrometers and about 200 micrometers, tional Sub-cutaneous or intramuscular injections. In some or between about 250 micrometers to about 1,500 microm embodiments, microneedles are used to deliver the replicon eters. In some embodiments, the microneedles have a length directly to the immune cells in the skin, which is advanta (from their tip to their base) between about 400 micrometers geous for immunization purposes. The amount of replicon to about 600 micrometers. needed for microneedle administration, compared to tradi 0046. In some embodiments, the size of individual tional Sub-cutaneous or intramuscular injections, is Smaller microneedles is optimized depending upon the desired tar and can reduce production cost and time. In some embodi geting depth or the strength requirements of the needle to ments, the microneedle is self-administered. In some avoid breakage in a particular tissue type. In some embodi embodiments, the replicon is dried onto the microneedle, ments, the cross-sectional dimension of a transdermal US 2017/O 196966 A1 Jul. 13, 2017

microneedle is between about 10 nm and 1 mm, or between dissolvable portion (such as the base of a microneedle). Such about 1 micrometers and about 200 micrometers, or between that a portion of the microneedle structure dissolves in the about 10 micrometers and about 100 micrometers. In some skin. In some embodiments, the dissolvable microneedle embodiments, the outer diameter of a hollow needle is encompasses the entire microneedle, such that the entire between about 10 micrometers and about 100 micrometers microneedle structure dissolves in the skin. In some embodi and the inner diameter of a hollow needle is between about ments, a dissolvable coating is formed on a non-dissolvable 3 micrometers and about 80 micrometers. Support structure Such that only the coating dissolves in the 0047 Microneedles can be arranged in a variety of dif skin. In some embodiments, the microneedle is coated with ferent patterns. In some embodiments, the microneedles are a polymer that is dissolvable, biodegradable, biosoluble, or spaced apart in a uniform manner, Such as in a rectangular any combinations thereof. or square grid or in concentric circles. In some embodi 0050. In some embodiments, replicon compositions are ments, the microneedles are spaced on the periphery of the directly coated onto the dissolvable, biodegradable, or bio Substrate. Such as on the periphery of a rectangular grid. In soluble microneedle. In some embodiments, replicon com Some embodiments, the spacing depends on numerous fac positions are contained within the dissolvable, biodegrad tors, including height and width of the microneedles, the able, or biosoluble microneedle itself (e.g., by forming part characteristics of a film to be applied to the surface of the of the dissolvable polymer matrix). In some embodiments, microneedles, as well as the amount and type of a Substance replicon compositions are mixed with a polymer matrix that is intended to be moved through the microneedles. In prior to molding and polymerization of microneedle struc Some embodiments, the arrangement of microneedles is a tures. “tip-to-tip” spacing between microneedles of about 50 0051 A variety of methods for manufacturing micrometers or more, about 100 micrometers to about 800 microneedles are available and any suitable method for micrometers, or about 200 micrometers to about 600 manufacturing microneedles or microneedle arrays are con micrometers. templated for use with the compositions and methods dis 0048. In some embodiments, the microneedle composi closed herein. In some embodiments, microneedles are tion is of any suitable material. Example materials include manufactured using any suitable method, including, but not metals, ceramics, semiconductors, organics, polymers, and limited to: molding (e.g., self-molding, micromolding, composites. In some embodiments, materials of construction microembossing, microinjection, and the like), casting (e.g., include, but are not limited to: pharmaceutical grade stain die-casting), or etching (e.g., Soft microlithography tech less steel, gold, titanium, nickel, iron, gold, tin, chromium, niques). The method of manufacture used depends on the copper, alloys of these or other metals, silicon, silicon materials employed. dioxide, and polymers. In some embodiments, the polymer 0052. In some embodiments, a microneedle composition is a biodegradable polymer or a non-biodegradable polymer. comprises a bioactive agent. In some embodiments, the Representative biodegradable polymers include, but are not terms “biologically active' and “bioactive' refers to a com limited to: polymers of hydroxy acids such as lactic acid and position or compound itself that has a biological effect, or a glycolic acid polylactide, polyglycolide, polylactide-co-gly composition or compound that binds, reacts, modifies, collide, and copolymers with PEG, polyanhydrides, poly causes, promotes, enhances, blocks, or reduces the activity (ortho)esters, polyurethanes, poly(butyric acid), poly(Valeric of a second molecule that has a biological effect. In some acid), and poly(lactide-co-caprolactone). Representative embodiments, the second molecule is an endogenous mol non-biodegradable polymers include polycarbonate, ecule. In some embodiments, the second molecule is an polymethacrylic acid, ethylenevinyl acetate, polytetrafluor exogenous molecule. In some embodiments, a “biological ethylene and polyesters. effect” includes, but is not limited to an effect that: stimu 0049. In some embodiments, the microneedle is dissolv lates or causes an immunoreactive response; impacts a able, biosoluble, biodegradable, or any combinations biological process in a cell, tissue, or organism (e.g., in an thereof. “Biodegradable' is used to refer to any substance or animal); impacts a biological process in a pathogen or object that is decomposed by bacteria or another living parasite; or generates or causes to be generated a detectable organism. Any suitable dissolvable, biosoluble, and/or bio signal. In some embodiments, biologically active composi degradable microneedles are contemplated for use with the tions, complexes, or compounds are used in investigative, compositions and methods disclosed herein. In some therapeutic, prophylactic, and/or diagnostic methods and embodiments, the dissolvable, biosoluble, or biodegradable compositions. In some embodiments, biologically active microneedles are composed of water Soluble materials. In compositions, complexes, or compounds act to cause or Some embodiments, these materials include chitosan, colla stimulate a desired effect upon a cell, tissue, organ, or gen, gelatin, maltose, dextrose, galactose, alginate, agarose, organism (e.g., an animal) Non-limiting examples of desired cellulose (such as carboxymethylcellulose or hydroxypro effects include, but are not limited to: modulating, inhibiting, pylcellulose), starch, hyaluronic acid, or any combinations or enhancing gene expression in a cell, tissue, organ, or thereof. In some embodiments, a selected material is resil organism; preventing, treating, or curing a disease or con ient enough to allow for penetration of the skin. In some dition in an animal Suffering therefrom; limiting the growth embodiments, the dissolvable microneedle dissolves in the of or killing a pathogen in an animal infected thereby; skin within seconds, such as within about 5, 10, 15, 20, 25, augmenting the phenotype or genotype of an animal; stimu 30, 45, 50, 60, 120, 180, or more seconds. In some embodi lating a prophylactic immunoreactive response in an animal; ments, the dissolvable microneedle dissolves in the skin and diagnosing a disease or disorder in an animal. within minutes, such as within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 0053. In some embodiments, a microneedle composition 10, 15, 20, 30, 60, 120 or more minutes. In some embodi comprises a bioactive agent. In some embodiments, the ments, the dissolvable microneedle comprises a dissolvable bioactive agent is the recombinant alphavirus replicons portion (Such as the tip of the microneedle) and a non described herein. In some embodiments, the bioactive agent US 2017/O 196966 A1 Jul. 13, 2017 is an RNA molecule encoding an exogenous polypeptide. In viral strain are encoded on a separate alphavirus replicon. In Some embodiments, a bioactive agent comprises a polypep Some embodiments, the microneedle composition comprises tide. Such as any of the polypeptides described herein. In two bioactive agents, such as a polypeptide and a recombi Some embodiments, the bioactive agent comprises an anti nant alphavirus replicon encoding the same polypeptide gen. In some embodiments, the bioactive agent comprises an (e.g., an influenza H1 polypeptide and a replicon encoding epitope of an antigen. In some embodiments, a microneedle the influenza H1 polypeptide). In some embodiments, the composition comprises a recombinant alphavirus replicon microneedle composition comprises two different bioactive encoding an exogenous polypeptide and further comprises a agents, such a polypeptide and a recombinant alphavirus polypeptide. For example, a polypeptide useful as a bioac replicon encoding a different polypeptide (e.g., an influenza tive agent is an influenza virus antigen, e.g., hemagglutinin H1 polypeptide and a replicon encoding an influenza H3 (HA). In some embodiments, a replicon composition com polypeptide). prises a replicon RNA encoding an HA protein and a 0056. In some embodiments, the microneedle composi bioactive molecule comprising an epitope of one or more tion comprises three bioactive agents. In some embodi different HA proteins. In some embodiments, a replicon ments, the microneedle composition comprises three differ composition comprises a replicon RNA encoding a hepatitis ent recombinant alphavirus replicons, each encoding for a B surface antigen (HBSAg). In some embodiments, a bio different polypeptide. In some embodiments, the active agent is a polypeptide that enhances an immune microneedle composition comprises a single recombinant response (e.g., an antigen or an adjuvant). alphavirus replicon that encodes for three different polypep 0054. In some embodiments, the microneedle composi tides. In some embodiments, the microneedle composition tion comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different comprises a replicon encoding an HA polypeptide from an bioactive agents. In some embodiments, the microneedle influenza H1 Viral strain, a replicon encoding an HA poly composition comprises 2 different bioactive agents. In some peptide from an influenza H3 viral strain, and a replicon embodiments, the microneedle composition comprises 3 encoding an HA polypeptide from an influenza B HA viral different bioactive agents. In some embodiments, the strain (trivalent vaccine). In some embodiments, a single microneedle composition comprises 4 different bioactive alphavirus replicon encodes the HA polypeptide from an agents. In some embodiments, the microneedle composition influenza H1 viral strain, the HA polypeptide from an comprises 5 different bioactive agents. In some embodi influenza H3 viral strain, and the HA polypeptide from an ments, the microneedle composition comprises 6 different influenza B HA viral strain. In some embodiments, the HA bioactive agents. In some embodiments, the microneedle polypeptide from an influenza H1 viral strain, the HA composition comprises 7 different bioactive agents. In some polypeptide from an influenza H3 viral strain, and the HA embodiments, the microneedle composition comprises 8 polypeptide from an influenza BHA viral strain are encoded different bioactive agents. In some embodiments, the on three separate alphavirus replicons. In some embodi microneedle composition comprises 9 different bioactive ments, the microneedle composition comprises three differ agents. In some embodiments, the microneedle composition ent bioactive agents, wherein at least one of the bioactive comprises 10 different bioactive agents. In some embodi agents is a polypeptide. ments, the bioactive agents are of the same type (e.g., 0057. In some embodiments, the microneedle composi multiple alphavirus replicons, each encoding a different tion comprises four bioactive agents. In some embodiments, polypeptide). In some embodiments, a single alphavirus the microneedle composition comprises four different replicon encodes multiple bioactive agents (e.g., multiple recombinant alphavirus replicons, each encoding for a dif exogenous polypeptides). In some embodiments, each exog ferent polypeptide. In some embodiments, the microneedle enous polypeptide is encoded by a separate alphavirus composition comprises a single recombinant alphavirus replicon. In some embodiments, the bioactive agents are of replicon that encodes for four different polypeptides. In different type (e.g., comprises an alphavirus replicons and a Some embodiments, the microneedle composition comprises polypeptide). a replicon encoding an HA polypeptide from an influenza 0055. In some embodiments, the microneedle composi H1 viral strain, a replicon encoding an HA polypeptide from tion comprises two bioactive agents. In some embodiments, an influenza H3 viral strain, a replicon encoding an HA the microneedle composition comprises two different bio polypeptide from an influenza B Yamagata lineage viral active agents, such as two different recombinant alphavirus strain, and a replicon encoding an HA polypeptide from an replicons, two different polypeptides, or a recombinant influenza B Victoria lineage viral strain (quadrivalent vac alphavirus replicon and a polypeptide. In some embodi cine). In some embodiments, a single alphavirus replicon ments, the microneedle composition comprises two different encodes the HA polypeptide from an influenza H1 viral recombinant alphavirus replicons, each encoding for a dif strain, the HA polypeptide from an influenza H3 viral strain, ferent polypeptide. In some embodiments, the microneedle the HA polypeptide from an influenza B Yamagata lineage composition comprises a single recombinant alphavirus viral strain, and the HA polypeptide from an influenza B replicon that encodes for two different polypeptides. In some Victoria lineage viral strain. In some embodiments, the HA embodiments, the microneedle composition comprises a polypeptide from an influenza H1 viral strain, the HA replicon encoding an HA polypeptide from an influenza H1 polypeptide from an influenza H3 viral strain, the HA viral strain and a replicon encoding an HA polypeptide from polypeptide from an influenza B Yamagata lineage viral an influenza H3 viral strain (bivalent vaccine). In some strain, and the HA polypeptide from an influenza B Victoria embodiments, a single alphavirus replicon encodes an HA lineage viral Strain are encoded on four separate alphavirus polypeptide from an influenza H1 viral strain and an HA replicons. In some embodiments, the microneedle compo polypeptide from an influenza H3 viral strain. In some sition comprises four different bioactive agents, wherein at embodiments, the HA polypeptide from an influenza H1 least one of the bioactive agents is a polypeptide. In some viral strain and the HA polypeptide from an influenza H3 embodiments, a single alphavirus replicon encodes five US 2017/O 196966 A1 Jul. 13, 2017 exogenous polypeptides, such as any of the exogenous Self-Replicating RNA Molecules polypeptides disclosed herein. 0060. In some embodiments, the bioactive agents pack 0058. In some embodiments, a bioactive agent is an aged for delivery into or onto microneedles are replicons. A adjuvant. Exemplary adjuvants include, without limitation: “replicon refers to a DNA or RNA molecule that is capable aluminum salts (e.g., aluminum phosphate, aluminum of undergoing self-replication, in whole or in part, Such as in hydroxide); squalene; saponins (QS21, ISCOMS), saponins a self-replicating nucleic acid molecule. In preferred complexed to membrane protein antigens (immune stimu embodiments, the replicon is an RNA molecule. Replicon lating complexes); pluronic polymers with mineral oil, RNA can substantially amplify the production of an encoded protein, leading to Sustained translation and protein produc killed Mycobacteria in mineral oil, a water-in-mineral-oil tion in a target cell. In some embodiments, RNA replicons emulsion which contains killed/dried mycobacteria in the oil are based on or derived from viruses. A variety of suitable phase, a weaker formulation without the mycobacteria; viruses (e.g., RNA viruses) are available, including, but not Freund's complete adjuvant; Freund's incomplete adjuvant; limited to, picornavirus, flavivirus, coronavirus, pestivirus, bacterial products, such as muramyl dipeptide (MDP) and rubivirus, calcivirus, and hepacivirus. In preferred embodi lipopolysaccharide (LPS), MPL as well as lipid A; lipo ments, the RNA replicon is derived from an alpha virus. In Somes, a membrane active glucoside extracted from the tree Some embodiments, replicons are positive-stranded so that Quillia saponaria, nonionic block copolymer Surfactants; they are directly translated by the host cell without requiring non-metabolised synthetic molecules which tend to bind intermediate replication steps, such as reverse transcription. proteins to cell surfaces; Infectious particles; Oil-in-water In some embodiments, replicons are derived from negative emulsions (e.g., MF59); CpG (Oligonucleotides)-TLR ago Stranded viruses. In some embodiments, the negative nists; and other TLR agonists like imiquimod and immuno stranded virus derived replicon is from Sendai virus or peptides. vesicular stomatitis virus. In some embodiments, when a 0059. In some embodiments, bioactive agents are pack positive-stranded replicon is delivered to a host cell, it is aged onto microneedles. In some embodiments, bioactive directly translated, generating an RNA-dependent RNA agents are packaged or embedded into microneedles. In polymerase which then produces both antisense and sense some embodiments, the bioactive agent is an RNA molecule transcripts from the delivered RNA. In some embodiments, encoding an exogenous polypeptide. In some embodiments, these RNA transcripts are translated directly such that the the bioactive agent is a recombinant alphavirus replicon. In host cell produces an encoded polypeptide, or they are some embodiments, the alphavirus replicon is dehydrated further transcribed to produce more transcripts that are before packaging into or onto the microneedle. In some translated by the host cell to produce more encoded poly embodiments, the alphavirus replicon is dehydrated after peptide. packaging into or onto the microneedle. In some embodi 0061. In preferred embodiments, the replicon is derived ments, the microneedle is packaged individually at a unit from an alphavirus. The alphavirus genus belongs to the dose of replicon. In some embodiments, the unit dose is Togaviridae family and contains 28 known virus species. effective in inducing an immune response in a Subject to the The positive-sense alphavirus genome typically contains exogenous polypeptide. In some embodiments, the unit dose two open-reading frames and encodes four non-structural is effective in inducing an immune response in a Subject to proteins (nsP1-4) and five structural proteins (capsid, E3, the exogenous polypeptide after storage for at least about E2, E1, and 6K). In some embodiments, the alphavirus one week (e.g., about or more than about 1, 2, 3, 4, 6, 8, 12. replicons lack the genes encoding the structural proteins. or more weeks) at room temperature. In some embodiments, Excluding the genes encoding the structural proteins will the unit dose is effective in inducing an immune response in prevent viral replication. In some embodiments, the genes a Subject to the exogenous polypeptide after storage for at encoding the structural proteins are replaced with mRNA least about one month (e.g., about or more than about 1, 2, encoding an exogenous polypeptide Such that the host cell 3, 4, 5, 6, 8, 10, 12, or more months) at room temperature. will produce a large amount of exogenous polypeptide, but In some embodiments, the recombinant alphavirus replicon will not produce infectious viral particles. is present in an amount effective to induce an immune 0062) A variety of alphaviruses suitable for the provision response in the Subject to the exogenous polypeptide. In of elements of a replicon in accordance with the disclosure Some embodiments, the recombinant alphavirus replicon is are available. In some embodiments, replicon sequences present in an amount effective to alone induce an immune comprise a wild-type alphavirus sequence. In some embodi response to the foreign or self-antigen. In some embodi ments, replicon sequences comprise a mutated alphavirus ments, the amount of recombinant alphavirus replicon Var sequence. In some embodiments, a replicon derived from an ies depending upon the health and physical condition of the alphavirus comprises sequence elements (e.g., elements that individual to be treated, age, the taxonomic group of indi promote replication of a sequence encoding an exogenous vidual to be treated (e.g., non-human primate, primate, polypeptide) that are at least about 80%, 85%, 90%, 95% or human, etc.), the capacity of the individual’s immune sys more identical to a reference wild-type alphavirus replicon, tem to synthesize antibodies, the degree of protection or a portion thereof (e.g., non-structural genes or portions desired, the formulation of the vaccine, and other relevant thereof). In some embodiments, sequence elements derived factors. In some embodiments, the polypeptide and RNA from an alphavirus are 500, 1000, 1500, 2000, 3000, 4000, content of certain compositions is expressed in terms of an 5000, or more nucleotides in length. Example alpha virus es amount of RNA per dose. In some embodiments, a dose has are available from depositories such as the American Type s100 ug RNA (e.g., from 10-100 ug, such as about 10 ug, 25 Culture Collection (ATCC), and include Aura (ATCC ug, 50 ug, 75 ug or 100 ug). In some embodiments, expres VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), sion is seen at much lower levels (e.g., s1 Lig/dose, s100 Cabassou (ATCC VR-922), Chikungunya virus (ATCC ng/dose, s10 ng/dose, s1 ng/dose). VR-64, ATCC VR-1241), Eastern equine encephalomyelitis US 2017/O 196966 A1 Jul. 13, 2017

virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC Some embodiments, the template DNA comprises an VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), alphavirus replicon comprising four non-structural genes Kyzylagach (ATCC VR-927), Mayaro (ATCC VR-66), (e.g., nsP1-4) and a sequence encoding the exogenous Mayaro virus (ATCC VR-1277), Middleburg (ATCC polypeptide such that the mRNA transcript comprises the VR-370), Mucambo virus (ATCC VR-580, ATCC recombinant alphavirus replicon encoding the alphavirus VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC non-structural genes (e.g., nsP1-4) and the exogenous poly VR-372, ATCC VR-1245), Ross River virus (ATCC peptide. Methods for producing alphavirus replicons are VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, available and any Suitable method for producing an alphavi ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC rus replicon is contemplated for use with the compositions VR-1248), Tonate (ATCC VR-925), Triniti (ATCC and methods disclosed herein. VR-469), Una (ATCC VR-374), Venezuelan equine 0065. The disclosed recombinant alpha virus replicons in encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCC accordance with the present disclosure have various lengths. VR-1250 ATCC VR-1249, ATCC VR-532), Western equine In some embodiments, the recombinant alphavirus replicons encephalomyelitis (ATCC VR-70, ATCC VR-1251, ATCC are about 4000, 5000, 6000, 7000, 8000, 9000, 10000, VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and 15000, 20000, 30000, or more nucleotides in length. In some Y-62-33 (ATCC VR-375). In some embodiments, chimeric embodiments, the recombinant alpha virus replicon is 5000 alphavirus replicons, which include sequences from mul 25000 nucleotides in length (e.g., 8000-15000 nucleotides, tiple different alphaviruses, are used. In some embodiments, or 9000-12000 nucleotides). In some embodiments, a rep the alphavirus replicon is derived from Semliki Forest virus, licon comprises a 5' cap. In some embodiments, the 5' cap Sindbis virus, Venezuelan equine encephalomyelitis virus, is a 7-methylguanosine. In some embodiments, the 5' cap or any combinations thereof. In preferred embodiments, the enhances in vivo translation of the RNA. alphavirus replicon is derived from a Venezuelan equine 0066. In some embodiments, the 5' nucleotide of a rep encephalomyelitis virus. licon has a 5' triphosphate group. In some embodiments, in 0063. In some embodiments, the RNA replicon is used as a capped RNA, the 5' triphosphate group is linked to a a vector to deliver a nucleic acid encoding an exogenous 7-methylguanosine via a 5'-to-5' bridge. In some embodi transcript or polypeptide to a host cell. In some embodi ments, a 5' triphosphate enhances RIG-I binding and thus ments, the RNA replicon contains an RNA sequence that, promotes adjuvant effects. In some embodiments, a replicon when delivered to a host cell, results in the production of a comprises a 3' poly-A tail. In some embodiments, the polypeptide or active transcript. In some embodiments, the replicon includes a poly-Apolymerase recognition sequence RNA sequence contains the genetic code for a selected (e.g., AAUAAA) near its 3' end. In some embodiments, a polypeptide and the RNA replicon is said to “encode' that replicon for delivery to a Subject is single-stranded. In some polypeptide or active RNA. The term “exogenous” is used to embodiments, single-stranded RNAS initiate an adjuvant refer to any molecule (e.g., polypeptide, nucleic acid, and effect by binding to TLR7, TLR8, RNA helicases, and/or the like) that is not ordinarily encoded by the viral replicon. PKR. In some embodiments, RNA is delivered in double In some embodiments, an exogenous sequence is inserted stranded form (dsRNA) and binds to TLR3. In some into an alphavirus replicon by way of recombinant tech embodiments, TLR3 is triggered by dsRNA which is formed niques or artificial synthesis to produce a recombinant either during replication of a single-stranded RNA or within polynucleotide comprising the exogenous sequence. In some the secondary structure of a single-stranded RNA. In some embodiments, an exogenous polypeptide is derived from an embodiments, a replicon comprises (in addition to any 5' cap organism other than the alphavirus from which the alphvirus structure) one or more nucleotides having a modified nucle sequence portions of the recombinant replicon are derived. obase. In some embodiments, a self-replicating RNA com In some embodiments, the replicon is engineered to encode prises one or more modified pyrimidine nucleobases, such as an exogenous polypeptide such that delivery of the engi pseudouridine and/or 5-methylcytosine residues. In some neered RNA replicon into a host cell results in the produc embodiments, the RNA includes no modified nucleobases. tion of a large amount of exogenous polypeptide by the host In some embodiments, the RNA includes no modified cell. nucleotides (e.g., all of the nucleotides in the RNA are 0064. A variety of methods are available for producing a standard A, C, G and U ribonucleotides, except for any 5' recombinant replicon. In some embodiments, a recombinant cap structure, which, in Some embodiments, comprise a replicon is generated in the laboratory by in vitro transcrip 7-methylguanosine). In some embodiments, the replicon is tion (IVT) techniques. In some embodiments, IVT uses a an RNA comprising a 5' cap comprising a 7'-methylguanos linear DNA template, such as a cDNA, linearized bacterial ine, and the first 1, 2, 3, or more 5' ribonucleotides are plasmid, or PCR product. In some embodiments, the tem modified at the 2' OH position of the ribose. A variety of 2 plate DNA has a promoter sequence specific for a DNA OH ribose modifications are available and contemplated for dependent RNA polymerase enzyme to initiate RNA syn use with the compositions and methods disclosed herein. thesis. Although any suitable technique is contemplated, Exemplary of 2 OH modifications include, but are not DNA templates are typically generated and propagated in a limited to: 2'-O-Me, 2'-MOE, 2'-amino, and 2'-F. In some bacterial plasmid or are created synthetically (e.g., PCR or embodiments, an RNA replicon comprises only phosphodi other synthetic DNA methods known in the art). In some ester linkages between nucleosides. In some embodiments, embodiments, the linear DNA molecule acts as a template the RNA replicon comprises phosphoramidate, phosphoro for an in vitro enzymatic reaction using a polymerase (e.g., thioate, methylphosphonate, or other linkages (such as 2'-4'- a bacteriophage RNA polymerase) that results in an RNA locked/bridged sugars (e.g., LNA, ENA, or UNA)). transcript ("copy') of the template DNA molecule. 0067. In some embodiments, RNA replicons encoding Examples of bacteriophage RNA polymerases useful in such exogenous polypeptides are prepared for delivery to a Sub processes include T7, T3, and SP6 RNA polymerases. In ject in need thereof. In some embodiments, a recombinant US 2017/O 196966 A1 Jul. 13, 2017 alphavirus replicon in accordance with the present disclo are used, such that an immunogenic response to more than Sure is used for vaccination. In some embodiments, a one HA is elicited. In some embodiments, the replicon recombinant alphavirus replicon in accordance with the composition is monovalent (the vaccine protects against one present disclosure is used for vaccination or gene therapy. In influenza strain, Such as one HA subtype antigen, e.g., H1). Some embodiments, replicons used for vaccination encode bivalent (the vaccine protects against two influenza Strains, one or more antigenic polypeptides (also referred to as an Such as two HA Subtype antigens, e.g., H1 and H3), trivalent 'antigen' or “immunogen') and are capable of generating an (the vaccine protects against three influenza Strains, such immunogenic response in the Subject, such as by activating three HA Subtype antigens, e.g., H1, H3, and a circulating the Subject's adaptive immune system mediated by a influenza B strain); quadrivalent (the vaccine protects humoral immune response, cell-mediated immune response, against four influenza strains, such as four HA Subtype or both. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, antigens, e.g., H1, H3, influenza B Yamagata, and influenza or more antigens are encoded in one or more replicons (e.g., B Victoria); or still higher valencies. In some embodiments, one replicon encoding multiple antigens, or multiple repli the HA antigens are determined based on the dominant cons encoding different antigens). In some embodiments, influenza strains (or the predicted most dominant strains) antigens are foreign antigens or self-antigens. In general, the causing pathogenesis in any particular season. In some term “self-antigen” refers to an immunogenic antigen or embodiments, the microneedle composition is bivalent and antigenic determinant which is native to the Subject. In some comprises alphavirus replicons that encode an HA polypep embodiments, the self-antigen is an antigen associated with tide from a viral strain of an influenza AH1 virus; and an HA cancer. A “foreign antigen” refers to those antigens that are polypeptide from a viral strain of an influenza A H3 virus. not self-antigens, such as antigens derived from an infec In some embodiments, the bivalent HA polypeptides are tious agent, a chemical, pollen, and the like. In some encoded in the same alphavirus replicon. In some embodi embodiments, a foreign antigen is derived from an infec ments, the bivalent HA polypeptides are encoded on sepa tious agent. Exemplary infectious agents include, but are not rate alphavirus replicons. In some embodiments, the limited to, bacteria, virus, protozoa, fungi, prions, helminths microneedle composition is trivalent and comprises alphavi and other parasites, and any combinations thereof. In some rus replicons that encode an HA polypeptide from a viral embodiments, foreign antigens derived from infectious strain of an influenza A H1 virus; an HA polypeptide from agents in the present disclosure are derived from infectious a viral strain of an influenza A H3 virus; and an HA agents that are pathogenic. In some embodiments, the rep polypeptide from a viral Strain of a circulating influenza B licon or polypeptide based vaccine disclosed herein provides virus. In some embodiments, the trivalent HA polypeptides a protective effect, Such as by reducing the incidence of are encoded in the same alphavirus replicon. In some infection (such as by at least about 10%, 20%, 30%, 40%, embodiments, the trivalent HA polypeptides are encoded on 50%. 60%, 70%, 80%, 90%, or more), increasing the separate alphavirus replicons. In some embodiments, the average number of infectious particles necessary to establish microneedle composition is quadrivalent and comprises an infection (such as by at least about 25%, 50%, 100%, alphavirus replicons that encode an HA polypeptide from a 250%, 500%, or more), or decreasing the average duration viral strain of an influenza A H1 virus; an HA polypeptide of an associated disease (such as by at least about 10%, 20%, from a viral strain of an influenza A H3 virus; an HA 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more), among a polypeptide from a viral strain of an influenza B Yamagata treated population relative to an untreated population. In lineage virus; and an HA polypeptide from a viral strain of Some embodiments, protective effects are measured in an an influenza B Victoria lineage virus. In some embodiments, appropriate animal model, or through an epidemiological the quadrivalent HA polypeptides are encoded in the same study of treated and untreated populations. In some embodi alphavirus replicon. In some embodiments, the quadrivalent ments, an antigen comprises or consists of about 5, 10, 15. HA polypeptides are encoded on separate alphavirus repli 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 300, 400, 500, cons. In some embodiments, the HA subtype chosen for the 1000, 2000, 3000, 5000, or more amino acids. In some immunogenic composition is dictated by the most dominant embodiments, an antigen derived from a particular source influenza strain(s) (or influenza strain(s) predicted to be the typically has about 80%, 90%. 95%, 99%, or more sequence most dominant) during any given season. In some embodi identity at the nucleotide or amino acid level when optimally ments, the replicon encoding the HA antigen generates an aligned to the sequence from which it was derived over the immune response (especially an antibody response), to the length of the antigen. influenza virus antigen in a subject when delivered to the Subject. 0068. In some embodiments, the antigen encoded by a recombinant alphavirus replicon of the disclosure is an 0069. In some embodiments, the polypeptide encoded by influenza virus hemagglutinin (HA) protein. In some the recombinant alphavirus replicon is an antigen derived embodiments, the antigen encoded by a recombinant from hepatitis B virus. In some embodiments, the polypep alphavirus replicon of the disclosure is an influenza virus tide encoded by the recombinant alphavirus replicon is neuraminidase (NA) protein. In some embodiments, HA is hepatitis B Surface antigen (HBSAg). derived from a viral strain of an influenza A, B, or C virus. 0070. In some embodiments, the polypeptide encoded by In some embodiments, the antigen is derived from a viral the recombinant alphavirus replicon is an antigen from a strain of influenza A virus and comprises one or more HA polio virus. In some embodiments, the polypeptide encoded subtypes including H1, H2, H3, H4, H5, H6, H7, H8, H9, by the recombinant alphavirus replicon is an antigen from H10, H11, H12, H13, H14, H15, H16, H17, or HA18. In Clostridium tetani. In some embodiments, the polypeptide Some embodiments, multiple replicons each encoding a encoded by the recombinant alphavirus replicon is an anti different HA are used. Such that an immunogenic response gen from a rabies virus. In some embodiments, the to more than one HA is elicited. In some embodiments, a microneedle devices disclosed herein comprise a recombi single replicon encoding multiple different HA polypeptides nant alphavirus replicon that encodes an exogenous poly US 2017/O 196966 A1 Jul. 13, 2017 peptide from: (a) an antigen from a polio virus; (b) an (ExPEC) and/or enterohemorrhagic E. coli (EHEC). ExPEC antigen from Clostridium tetani; and (c) an antigen from a strains include uropathogenic E.coli (UPEC) and meningitis/ rabies virus. In some embodiments, each of the exogenous sepsis-associated E.coli (MNEC). A useful immunogen for polypeptides are encoded on a single recombinant alphavi several E.coli types is Acfl); Bacillus anthracis. In some rus replicon. In some embodiments, the exogenous poly embodiments, antigens are derived from Yersinia pestis, peptides are encoded on different recombinant alphavirus Staphylococcus epidermis, Clostridium perfingens or replicons. Any suitable polio virus, Clostridium tetani; or Clostridium botulinums, Legionella pneumophila, Coxiella rabies virus antigens are contemplated by the disclosure burnetii, Brucella, such as B. abortus, B. canis, B. meliten herein. sis, B. neotomae, B. ovis, B. suis, B. pinnipediae, Franci 0071. In some embodiments, the polypeptide encoded by sella, Such as F. novicida, F. philomiragia, F. tularensis, the recombinant alphavirus replicon is an antigen from a Neisseria gonorrhoeae, Treponema pallidum, Haemophilus Marburg virus. In some embodiments, the polypeptide ducreyi, Enterococcus faecalis, Enterococcus faecium, encoded by the recombinant alphavirus replicon is an anti Staphylococcus saprophyticus, Yersinia enterocolitica, gen from an Ebola Sudan virus. In some embodiments, the Mycobacterium tuberculosis, Rickettsia, Listeria monocy polypeptide encoded by the recombinant alphavirus repli togenes, Vibrio cholerae, Salmonella typhi; Borrelia burg con is an antigen from an Ebola Zaire virus. In some dorferi; Porphyromonas gingivalis; and Klebsiella. embodiments, the microneedle devices disclosed herein 0074. In some embodiments, the polypeptide encoded by comprise a recombinant alphavirus replicon that encodes an the recombinant alphavirus replicon is an antigen derived exogenous polypeptide from: (a) an antigen from a Marburg from a virus. In some embodiments, the viral antigen is virus; (b) an antigen from an Ebola Sudan virus; and (c) an derived from any one or more of Orthomyxovirus; influenza antigen from an Ebola Zaire virus. In some embodiments, A, B or C virus, such as the hemagglutinin, neuraminidase each of the exogenous polypeptides are encoded on a single or matrix M2 proteins: Paramyxoviridae viruses such as recombinant alphavirus replicon. In some embodiments, the those derived from Pneumoviruses (e.g. respiratory syncy exogenous polypeptides are encoded on different recombi tial virus, RSV), Rubula viruses (e.g. mumps virus), nant alphavirus replicons. Any Suitable Marburg virus, Paramyxoviruses (e.g. parainfluenza virus), Metapneumovi Ebola Sudan virus, or Ebola Zaire virus antigens are con ruses and Morbilliviruses (e.g. measles); Poxviridae includ templated by the disclosure herein. ing those derived from Orthopoxvirus such as Variola vera, 0072. In some embodiments, the polypeptide encoded by including but not limited to, Variola major and Variola the recombinant alpha virus replicon is an antigen derived minor; Picornavirus such as those derived from Enterovi from human immunodeficiency virus (HIV). In some ruses, Rhinoviruses, Heparnaviruses, Cardioviruses and embodiments, the HIV antigen is Gp120, Gp160, Gag, Pol, Aphthoviruses; Bunyavirus including those derived from an and Nef proteins or portions thereof. Examples of other HIV Orthobunyavirus, such as California encephalitis virus, a antigens include Tat, Rev, Vif Vpr and Vpu proteins. The Phlebovirus, such as Rift Valley Fever virus, or a Nairovirus, replicon encoding an HIV antigen will preferably generate Such as Crimean-Congo hemorrhagic fever virus; Heparna an immune response (such as an antibody response), to the virus such as hepatitis A virus (HAV); Filovirus such as an HIV virus epitope in a subject when delivered to the subject. Ebola virus (including a Zaire, Ivory Coast, Reston or Sudan 0073. In some embodiments, the polypeptide encoded by ebolavirus) or a Marburg virus, Togavirus including a the recombinant alphavirus replicon is an antigen derived Rubivirus, an Alphavirus, or an Arterivirus including from a bacterium. In some embodiments, antigens derived Rubella virus, Flavivirus such as Tick-borne encephalitis from a bacterium include those derived from any one or (TBE) virus, Dengue (types 1, 2, 3 or 4) virus, Yellow Fever more of Neisseria meningitidis, including membrane pro virus, Japanese encephalitis virus, Kyasanur Forest Virus, teins such as adhesins, autotransporters, toxins, iron acqui West Nile encephalitis virus, St. Louis encephalitis virus, sition proteins, and factor H binding protein; Streptococcus Russian spring-Summer encephalitis virus, Powassan pneumoniae Such as the RrgB pilus subunit, the beta-N- encephalitis virus, Pestivirus such as Bovine viral diarrhea acetyl-hexosaminidase precursor (spr0057), sprO096, Gen virus (BVDV), Classical swine fever (CSFV) or Border eral stress protein GSP-781 (spr2021, SP2216), serine/threo disease (BDV); Hepadnavirus such as Hepatitis B virus (e.g. nine kinase StkP (SP1732), and pneumococcal surface hepatitis B virus surface antigen (HBSAg)); other hepatitis adhesin PsaA; Streptococcus pyogenes Such as streptococcal viruses Such as hepatitis C virus, delta hepatitis virus, group A antigen; Moraxella catarrhalis, Bordetella pertus hepatitis E virus, or hepatitis G. virus; Rhabdovirus including sis such as pertussis toxin or toxoid (PT), filamentous a Lyssavirus (e.g. a Rabies virus) and Vesiculovirus (VSV): haemagglutinin (FHA), pertactin, and agglutinogens 2 and Caliciviridae including Norwalk virus (Norovirus), and Nor 3: Staphylococcus aureus such as hemolysin, esXA, esXB, walk-like Viruses, such as Hawaii Virus and Snow Mountain ferri chrome-binding protein (sta(006) and/or the staOl 1 Virus; Coronavirus including immunogens derived from a lipoprotein; Clostridium tetani Such as tetanus toxoid; SARS coronavirus, avian infectious bronchitis (IBV), Cornynebacterium diphtheriae such as diphtheria toxoid; Mouse hepatitis virus (MHV), and Porcine transmissible Haemophilus influenzae, Pseudomonas aeruginosa, Strep gastroenteritis virus (TGEV). In some embodiments, the tococcus agalactiae, Chlamydia trachomatis Such as Pep.A, coronavirus immunogen is a spike polypeptide; Retrovirus LcrE, ArtJ, DnaK, CT398, Omph-like, L7/L12, OmcA, such as an Oncovirus, a Lentivirus (e.g. HIV-1 or HIV-2) or AtoS, CT547, Eno, Htra and MurG; Chlamydia pneumo a Spumavirus; Reovirus including an Orthoreovirus, a Rota niae, Helicobacter pylori such as CagA. VacA, NAP and/or virus, an Orbivirus, or a Coltivirus, Parvovirus including urease; Escherichia coli Such as immunogens derived from Parvovirus B19. Herpesvirus including a human herpesvi enterotoxigenic E. coli (ETEC), enteroaggregative E. coli rus, Such as, by way of example only, Herpes Simplex (EAggEC), diffusely adhering E. coli (DAEC), enteropatho Viruses (HSV) (e.g. HSV types 1 and 2), Varicella-zoster genic E. coli (EPEC), extraintestinal pathogenic E. coli virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus US 2017/O 196966 A1 Jul. 13, 2017

(CMV), Human Herpesvirus 6 (HHV6), Human Herpesvi Sacharomyces cerevisae, Saccharomyces boulardii, Saccha rus 7 (HHV7), and Human Herpesvirus 8 (HHV8); Papova romyces pombe, Scedosporium apiosperum, Sporothrix viruses including those derived from Papillomaviruses and schenckii, TrichospOron beigelii, Toxoplasma gondii, Peni Polyomaviruses. In some embodiments, the (human) papil cillium marineffei, Malassezia spp., Fonsecaea spp., Wangi lomavirus is of serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, ella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65, e.g., from one spp., Rhizopus spp., Mucor spp., Absidia spp., Mortierella or more of serotypes 6, 11, 16 and/or 18; Adenovirus spp., Cunninghamella spp., Saksenaea spp., Alternaria spp., including adenovirus serotype 36 (Ad-36). Examples of Curvularia spp., Helminthosporium spp., Fusarium spp., enterovirus include poliovirus, e.g., a type 1, type 2 and/or Aspergillus spp., Penicillium spp., Monolinia spp., Rhizoc type 3 poliovirus; an EV71 enterovirus; a coxsackie A or B tonia spp., Paecilomyces spp., Pithomyces spp., and Cla virus dosporium spp. 0075. In some embodiments, the polypeptide encoded by 0077. In some embodiments, the polypeptide encoded by the recombinant alphavirus replicon is an antigen derived the recombinant alphavirus replicon is an antigen derived from a virus which infects fish or other animals, such as: from a parasite. Exemplary parasites include, but are not infectious salmon anemia virus (ISAV), salmon pancreatic limited to, those from the Plasmodium genus, such as P disease virus (SPDV), infectious pancreatic necrosis virus falciparum, P. vivax, P malariae or P ovale. In some (IPNV), channel catfish virus (CCV), fish lymphocystis embodiments, the antigen elicits an immune response disease virus (FLDV), infectious hematopoietic necrosis against a parasite from the Caligidae family, particularly virus (IHNV), koi herpesvirus, salmon picorna-like virus those from the Lepeophtheirus and Caligus genera e.g., sea (also known as picorna-like virus of atlantic salmon), land lice Such as Lepeophtheirus salmonis or Caligus roger locked salmon virus (LSV), atlantic salmon rotavirus (ASR), cresseyi. trout strawberry disease virus (TSD), coho salmon tumor 0078. In some embodiments, the polypeptide encoded by virus (CSTV), or viral hemorrhagic septicemia virus the recombinant alphavirus replicon is an antigen derived (VHSV). from an allergen. Exemplary allergens include, but are not 0076. In some embodiments, the polypeptide encoded by limited to: pollen allergens (tree, herb, weed, and grass the recombinant alphavirus replicon is an antigen derived pollen allergens); insect or arachnid allergens (inhalant, from a fungus. Exemplary fungal antigens include, but are saliva, and Venom allergens, mite allergens, cockroach and not limited to, antigens from any of Absidia, Acremonium, midges allergens, hymenopthera Venom allergens); animal Alternaria, Aspergillus, Basidiobolus, Bipolaris, Blastomy hair and dandruff allergens (from e.g., dog, cat, horse, rat, ces, Candida, Chlamydia, Coccidioides, Conidiobolus, mouse, etc.); and food allergens (e.g., a gliadin). Exemplary Cryptococcus, Curvalaria, Epidermophyton, Exophiala, pollen allergens from trees, grasses and herbs are Such Geotrichum, Histoplasma, Madurella, Malassezia, originating from the taxonomic orders of Fagales, Oleales, Microsporum, Moniliella, Mortierella, Mucor, Paecilomy Pinales and platanaceae including, but not limited to, birch ces, Penicillium, Phialemonium, Phialophora, Prototheca, (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpi Pseudallescheria, Pseudomicrodochium, Pythium, Rhino nus) and Olive (Olea), cedar (Cryptomeria and Juniperus), sporidium, Rhizopus, Scolecobasidium, Sporothrix, Stem plane tree (Platanus), the order of Poales including grasses phyllium, Trichophyton, TrichospOron, and Xvlohypha. In of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Some embodiments, fungal antigens are be derived from any Holcus, Phalaris, Secale, and Sorghum, the orders of Aster of Epidermophyton floccusum, Microsporum audouini, ales and Urticales including herbs of the genera Ambrosia, Microsporum canis, Microsporum distortum, Microsporum Artemisia, and Parietaria. Other exemplary inhalation aller equinum, Microsporum gypsum, Microsporum nanum, gens are those from house dust mites of the genus Der Trichophyton concentricum, Trichophyton equinum, matophagoides and Euroglyphus, storage mite e.g., Lepido Trichophyton gallinae, Trichophyton gypseum, Trichophy glyphys, Glycyphagus and Tyrophagus, those from ton megnini, Trichophyton mentagrophytes, Trichophyton cockroaches, midges and fleas e.g., Blatella, Periplaneta, quinckeanum, Trichophyton rubrum, Trichophyton Schoen– Chironomus and Ctenocepphalides, and those from mam leini, Trichophyton tonsurans, Trichophyton verrucosum, T. mals such as cat, dog and horse, venom allergens including verrucosum var. album, var. discoides, var. ochraceum, Such originating from stinging orbiting insects such as those Trichophyton violaceum, and/or Trichophyton faviforme; or from the taxonomic order of Hymenoptera including bees from Aspergillus fumigatus, Aspergillus flavus, Aspergillus (Apidae), wasps (Vespidea), and ants (Formicoidae). niger; Aspergillus nidulans, Aspergillus terreus, Aspergillus 0079. In some embodiments, the polypeptide encoded by Sydowi, Aspergillus flavatus, Aspergillus glaucus, Blasto the RNA or recombinant alphavirus replicon is an antigen schizomyces capitatus, Candida albicans, Candida enolase, derived from a tumor antigen. In some embodiments, the Candida tropicalis, Candida glabrata, Candida krusei, Can tumor antigen is selected from any one or more of the dida parapsilosis, Candida Stellatoidea, Candida kusei, following, or portions thereof: (a) cancer-testis antigens Candida parakwisei, Candida lusitaniae, Candida pseudo such as NY-ESO-1, SSX2, SCP1 as well as RAGE, BAGE, tropicalis, Candida guilliermondi, Cladosporium carrionii, GAGE and MAGE family polypeptides, for example, Coccidioides immitis, Blastomyces dermatidis, Cryptococ GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, cus neoformans, Geotrichum clavatum, Histoplasma capsu MAGE-4, MAGE-5, MAGE-6, and MAGE- 12; (b) mutated latum, Klebsiella pneumoniae, Microsporidia, Encephalito antigens, for example, p53, p21/Ras, CDK4, MUM1, cas zoom spp., Septata intestinalis and Enterocytozoon bieneusi: pase-8, CIA 0205, HLA-A2-R1701, beta catenin, TCR, the less common are Brachiola spp., Microsporidium spp., BCR-abl, triosephosphate isomerase, KIA 0205, CDC-27, Nosema spp., Pleistophora spp., Trachipleistophora spp., and LDLR-FUT; (c) over-expressed antigens, for example, Vittaforma spp., Paracoccidioides brasiliensis, Pneumocys mesothelin, livin, Survivin, ICAM-1, galectin 4, galectin 9. tis carinii, Pythiumn insidiosum, Pityrosporum ovale, proteinase 3, WT 1, carbonic anhydrase, aldolase A, US 2017/O 196966 A1 Jul. 13, 2017

PRAME. HER-2/neu, mammaglobin, alpha- fetoprotein, chemokines. In some embodiments, polypeptides are KSA, gastrin, telomerase catalytic protein, MUC-1, G-250, derived from mammals (e.g., without limitation, human, p53, granulocyte-macrophage colony-stimulating factor dog, cat, monkey, sheep, goat, horse, cow, and the like), (GM-CSF), and carcinoembryonic antigen; (d) shared anti bacteria, viruses, fungi, or parasites. In some embodiments, gens, for example, melanoma-melanocyte differentiation the encoded polypeptide is derived from a human. In some antigens such as MART-1/Melan A, Gp 100, MC1R, mel embodiments, polypeptides are naturally occurring, modi anocyte-stimulating hormone receptor, tyrosinase, tyrosi fied from the natural state, chimeric proteins containing nase related protein-1/TRP1 and tyrosinase related protein fragments of proteins from at least two different proteins, 2/TRP2; (e) prostate associated antigens such as PAP, PSA, engineered proteins with enhanced function or activity, PSMA, PSH-P1, PSM-P1, PSM-P2: (f) immunoglobulin engineered proteins with decreased function or activity, or idiotypes; (g) or any combinations thereof. In certain targeted to a specific compartment of a cell, e.g., cytoplas embodiments, tumor immunogens include, but are not lim mic, membrane, or nucleus, or any combinations thereof. In ited to, pi 5, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, Some embodiments, the encoded polypeptide is human IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, insulin useful for the treatment of diabetes, e.g., type I or human papillomavirus (HPV) antigens, including E6 and type II diabetes. E7, hepatitis B and C virus antigens, human T-cell lympho tropic virus antigens, TSP-180, p.185erbB2, p 180erbB-3, I0082 Examples of polypeptides that are delivered, either c-met, mn-23H1, TAG-72-4, CA19-9, CA 72-4, CAM 17.1, directly as polypeptides or indirectly by delivering a recom NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 binant alphavirus replicon or other polynucleotide encoding Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA the polypeptide, include, but are not limited to: VEGF, 27.29YBCAA), CA 195, CA 242, CA-50, CAM43, VEGF-R1, VEGF-R2, VEGF-R3, Her-1, Her-2, Her-3, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, EGF-1, EGF-2, EGF-3, EGF-R, c-Met, ICOS, CD4OL, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, LFA-1, OX40, TACT-5, IgE, BAFF/BLys, TPO-R, CD2F RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein cyclo 10/SLAMF9, CD2, CD3, CD4, CDS, CDSL, CD6, CD8, philin C-associated protein), TAAL6, TAG72, TLP, TPS, or CD9, CD14, CD19, CD20, CD22, CD23/Fc epsilon R2, DPPIV/CD26, CD27/TNFRSF7, CD28, CD30/TNFRSF8, any combinations thereof. CD31/PECAM-1, CD33, CD34, CD36/SR-B3, CD38, 0080. In some embodiments, replicons are used to deliver CD40, CD43, CD44, CD45, CD46, CD47, CD48/SLAMF2, an exogenous polypeptide to a subject. In some embodi CD52, CD55/DAF, CD58/LFA-3, CD59, CEACAM-1/ ments, the replicons encode, for example, and without CD66a, CD68, CD69, CD72, CD74, CD83, CD84, CD90/ limitation, a functional, therapeutic polypeptide to replace a Thyl, C1q R1/CD93, CD94, CD95, CD97, EMMPRIN/ mutated or missing polypeptide in the Subject. In some CD147, DEP-1/CD148, CD151, CD155/PVR, CD160, embodiments, the replicons encode a polypeptide that inhib CD163, CD164, CD200, CD200-R1, CD229/SLAMF3, its or interferes with the function of an endogenous poly TNFa. TRAIL, TRAIL-R1, TRAIL-R2, TRAIL-R3, peptide in the Subject. In some embodiments, the polypep TRAIL-R4, Complement Receptor 1. FGFa, Osteopontin, tide encoded by the replicon is an exogenous polypeptide Vitronectin, alpha-2-macroglobulin, CCL1, CCL2. CCL3, that provides therapeutic benefit to the subject. In some CCL4, CCL5, CCL6/C10, CCL7, CXCL8, CXCL9, embodiments, the therapeutic benefit means the treatment CXCL10, CCL11, CXCL11/Eotaxin, CXCL12, CCL13, of reduced incidence of alleviation of the symptoms of, or CXCL13, CXCL14. CCL14. CCL15, CXCL16, CCL16, cure of a disease, condition, or disorder. In some embodi CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL24/ ments, the polypeptide is an antigen. In some embodiments, Eotaxin-2, CCL26/Eotaxin-3, CCL27, CCL28, PDGF, replicons encode a polypeptide delivered for cosmetic pur TGFb, GM-CSF, SCF. p40, IL1a, IL1-R1, IL-1b, IL2, IL2 poses. In some embodiments, the polypeptide is delivered R, IL3, IL4, IL5, IL6, IL6-R, IL8, IL 10, IL12, IL15, IL23, according to the compositions and methods of the disclosure Fas, FasL, F10, 41 BB, ACE, ACE-2, KGF, FGF-7, SCF, (e.g., by a microneedle device). In some embodiments, the Netrin1, Netrin2, IFNa, IFNb, IFNg, ADAMS1, ADAMS5, polypeptide includes any polypeptide found in nature, engi ADAMS, ADAMS, ADAM10, ADAM12, ADAM15, neered polypeptides, chimeric polypeptides (e.g., containing TACE/ADAM17, ADAM19, ADAM33, ADAMTS1, fragments or portions from more than one source of poly ADAMTS4, ADAMTS5, ADAMTSL-1/Punctin, ALCAM, peptide), and the like. The polypeptides and polypeptide ALK-1, APRIL. Angiogenin, Amphiregulin, Angiopoietin 1, encoded replicons disclosed herein can be of any Suitable Angiopoietin 2, Angiopoietin 3, Angiopoietin 4, B7-1/ length. In some embodiments, the polypeptide contains less CD80, B7-2/CD86, B7-H1, B7-H2, B7-H3, B7-H4, BACE than about twenty amino acids (e.g., less than 20, 19, 18, 17. 1, BACE-2, BAK, BCAM, BDNF, bNGF, bECGF, BMP1, 16, 15, 14, 13, 12, 11, 10, or fewer amino acids); more than BMP 2, BMP3, BMP-3b/GDF-10, BMP4, BMP5, BMP 6, about 20 amino acids but less than about 50 amino acids; or BMP 7, BMP8, BMP9, BMP10, BMP-15/GDF-9B, BMPR a protein, containing more than about 50 amino acids. In 1A/ALK-3, BMPR-1B/ALK-6, BMPR-2, CRP, E-Cadherin/ Some embodiments, the polypeptide contains about or more Cadherin 1, N-Cadherin/Cadherin 2, P-Cadherin/Cadherin than about 100, 1000, 2000, 3000, 4000, 5000, 7500, 10000, 3, Cadherin-4/R-Cadherin, VE-Cadherin/Cadherin 5, Cad 20000, or more amino acids. herin 6, Cadherin 8, Cadherin 11, Cadherin 12, Cadherin 13, 0081. In some embodiments, replicons encode a poly Cadherin 17, Cathepsin 1, Cathepsin 3, Cathepsin 6, Cathe peptide for use in providing a therapeutic and/or cosmetic psin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D, benefit to a subject. In some embodiments, the polypeptide Cathepsin E. Cathepsin F. Cathepsin H. Cathepsin L. Cathe for use in providing a therapeutic and/or cosmetic benefit psin O. Cathepsin S, Cathepsin V. Cathepsin X/Z/P. LFA-3, includes enzymes, enzyme inhibitors, hormones, immuno GP2b3a, GH receptor, RSV F protein, CTLA-4, Integrin globulins such as natural, modified, or chimeric immuno C.4B1, Integrin C4 B7, Lymphotoxin, Digoxin, Rhod, TNF-R globulins or fragments thereof, lymphokines, cytokines, and family, Lymphotoxin a/b receptor, TL1A/TNFSF15, BAFF US 2017/O 196966 A1 Jul. 13, 2017

R/TNFRSF13C, Fas/TNFRSF6, DR3/TNFRSF25, HVEM/ A4, Clusterin, Carboxypeptidase B1, Clusterin-like 1, Car TNFRSF14, TROY/TNFRSF19, CD40 Ligand/TNFSFS, boxypeptidase E/CPE, CMG-2, Carboxypeptidase X1, BCMA/TNFRSF17, LIGHT/TNFSF14, 4-1BB/TNFRSF9, CMV UL146, Cardiotrophin-1, CMV UL147, Camosine GITR/TNFRSF18, Osteoprotegerin/TNFRSF11B, RANK/ Dipeptidase 1, CNP. Caronte, CNTF, CART, CNTF Ralpha, TNFRSF11 A TRANCE/RANKL/TNFSF11, 4-1BB Coagulation Factor II/Thrombin, Caspase-1, Coagulation Ligand/TNFSF9, TWEAK/TNFSF12, CD40 Ligand/ Factor III/Tissue Factor, Caspase-2, Coagulation Factor VII, TNFSFS, Fas Ligand/TNFSF6, RELT/TNFRSF19L, Caspase-3, Coagulation Factor X. Caspase-4, Coagulation APRILFTNFSF13 s DcR3/TNFRSF6B, TNFR-1A Factor XI, Caspase-6, Coagulation Factor XIV/Protein C, TNFRSF1A, CD30 Ligand/TNFSF8, GITR Ligand/ Caspase-7, COCO, Caspase-8, Cohesin, Caspase-9, Colla TNFSF18, TNFSF18, TAC1/TNFRSF13B, NGFR/ gen I, Caspase-10, Collagen II, Caspase- 12, Collagen IV. TNFRSF16, OX40 Ligand/TNFSF4, TWEAK-R/ Caspase-13, Common gamma Chain/IL-2R gamma, Cas TNFRSF12, DR6/TNFRSF21, Pro-TNF-alpha/TNFSF1-A, pase Peptide Inhibitors, COMP/Thrombospondin-5, Cata TNF-beta/TNFSF 1B, PGRP-S, TNFR-2/TNFRSF1B, lase, Complement Component C1rLP. beta-Catenin, EDA-A2, EDAR, XEDAR, 4EBP1, 14-3-3 Zeta, 53BP1, Complement Component C1q.A. Complement Component 2B4/SLAMF4, 8D6A, A2B5, Aminopeptidase LRAP/ C1qC. Complement Factor D. Complement Factor I, ERAP2, A33, Aminopeptidase N/ANPEP, Aag. Aminopep Complement MASP3, Connexin 43, Contactin-1, Contactin tidase P2/XPNPEP2, ABCG2, Aminopeptidase P1/XPN 2/TAG1, Contactin-4, Contactin-5, Corin, Cornulin, PEP1, ACE-1, Aminopeptidase PILS/ARTS1, ACE-2, CORS26/C1qTNF3, COUP-TF I/NR2F1, CBP, COUP-TF Amnionless, Actin, Amphiregulin, beta-Actin, Activin A, II/NR2F2, CCI, COX-1, CCKAR, COX-2, CRACC/ AMPK alpha 1, Activin AB, AMPK alpha 2, Activin B, SLAMF7, CCR1, C-Reactive Protein, CCR2, Creatine AMPK beta 1, Activin C, AMPK beta 2, Activin R12A/ Kinase, Muscle/CKMM, CCR3, CCR4, CREB, CCR5, ALK-2, Androgen R/NR3C4, Activin R1B/ALK-4, Angio CREG, CCR6, CRELD1, CCR7, CRELD2, CCR8, CRHBP, genic Activin R2A, Activin R2B, ADAM 10, Angiopoietin CCR9, CRHR-1, CCR10, CRIM1, Cripto, CRISP-2, like 1 Angiopoietin-like 2, Angiopoietin-like 3. CRISP-3, Crossveinless-2, CRTAM, CRTH-2, CRY1, Cryp Angiopoietin-like 4, Angiopoietin-like 7/CDT6. Angiosta tic, CSB/ERCC6, CTGF/CCN2, CTLA-4, Cubilin, tin, Annexin A1/Annexin 1, Annexin A7, Annexin A10. CX3CR1, CXADR, CD27 LigandVTNFSF7, CXCR3, Annexin V, Adiponectin/Acrp30, ANP, AEBSF, Aggrecan, CXCR4, CXCR5, CD30 Ligand/TNFSF8, CXCR6, Cyclo APAF-1, Agrin, APC, AgRP APE, AGTR-2, APJ, AIF, philin A, Cyró1/CCN1, Cystatin A, Cystatin B. Cystatin C, APLP-1, APLP-2, Akt1, Apollipoprotein A1, Akt2, Apolipo Cystatin D, Cystatin E/M, Cystatin F. Cystatin H, Cystatin protein B, Akt3, APP Serum Albumin, ALCAM, ARC, H2, Cystatin S. Cystatin SA, Cystatin SN, Cytochrome c, ALK-1, Artemin, ALK-7, Arylsulfatase A/ARSA, Alkaline Apocytochrome c, Holocytochrome c, Cytokeratin 8, Cytok Phosphatase, ASAH2/N-acylsphingosine Amidohydrolase eratin 14, Cytokeratin 19, Cytonin, D6, DISP1, DAN, Dkk 2, alpha-2u Globulin, ASC, alpha-1-Acid Glycoprotein, 1, DANCE, Dkk-2, DARPP-32, Dkk-3, DAX1/NROB1, ASGR1, alpha-Fetoprotein, ASK1, ALS, ATM, Ameloblas Dkk-4, DCC, DLEC, CLEC4A, DLL1, DCAR, DLJL4, tic ATRIP, AMICA/JAML, Aurora A, AMIGO, Aurora B, DcR3/TNFRSF6B, DC-SIGN, DNA Ligase IV, DC-SIGNR/ AMIGO2, Axin-1, AMIGO3, Ax1, Aminoacylase/ACY1, CD299, DNA Polymerase beta, DcTRAIL-R1/TNFRSF23, AZurocidin/CAP37/HBP. Aminopeptidase A/ENPEP. DNAM-1, DcTRAIL-R2/TNFRSF22, DNA-PKcs, DDR1, B4GALT1, BIM, 6-Biotin-17-NAD, BLAME/SLAMF8, DNER, DDR2, Dopa Decarboxylase/DDC, DEC-205, BLIMP1, Bik, BMI-1, Bad, Bag-1, BAK, BAMBI/NMA, DPCR-1, Decapentaplegic, DPP6, Decorin, DPPA4, Dectin BARD1, Bax, Bcl-10, Bcl-2, Bcl-2 related protein A1, 1/CLECyA, DPPA5/ESG1, Dectin-2/CLEC6A, DPPII/QPP/ Bcl-w, Bcl-X, BNIP3L, Bcl- XL, BOC, BOK, BPDE, DPP7. Desert Hedgehog, Desmin, Desmoglein-1, DSCAM, Brachyury, B-Raf, beta IG-H3, Betacellulin, BRCA1, beta Desmoglein-2, DSCAM-L1, Desmoglein-3, DSPG3, Defensin2. BRCA2, BID, BTLA, Biglycan, Bub-1, Bik-like Dishevelled-1, Dtk, Dishevelled-3, Dynamin, EAR2/ Killer Protein, c-jun, c-Rel, C1qTNF1, C1qTNF4, NR2F6, EphA5, ECE-1, EphA6, ECE-2, EphA7, ECF-L/ C1qTNF5, Complement Component C1r, Complement CHI3L3, Eph A8, ECM-I, EphBl, Ecotin, EphB2, EDA, Component C1s, Complement Component C2, Complement EphB3, EDA-A2, EphB4, EDAR, EphB6, EDG-1, EDG-5, Component C3a, Complement Component C3d, Comple Ephrin-Al, EDG-8, Ephrin-A2, eEF-2, Ephrin-A3, Ephrin ment Component C5a, CDC2, CDC25A, CDC25B, CDCP1, A4, Ephrin-A5, EGR1, Ephrin-B, EG-VEGF/PK1, Ephrin CDO, CDX4, CEACAM-6, Cerberus 1, CFTR, Calbindin B1, eIF2 alpha, Ephrin-B2, eIF4E, Ephrin-B3, EIk-1, Epi D., Calcineurin A, Chem R23, Calcineurin B, Chemerin, gen, EMAP-II, Epimorphin/Syntaxin 2Epiregulin, CXCL5/ CaM Kinase 2, Chitinase 3-like 1, Chitotriosidase/CHIT1, ENA, EPR-1/Xa Receptor, Endocan, ErbB2, Endoglin/ Cannabinoid R1, Chk1, Cannabinoid R2/CB2/CNR2, Chk2, CD105, ErbB3, Endoglycan, ErbB4, Endonuclease III, CAR/NR1 I3, CHL-1/L1CAM-2, Carbonic Anhydrase I, ERCC1, Endonuclease IV, ERCC3, Endonuclease V, Endo Choline Acetyltransferase/ChaT, Carbonic Anhydrase II, nuclease VIII, ERK1, Endorepellin/Perlecan, ERK2, Chondrollectin, Carbonic Anhydrase III, Chordin, Carbonic Endostatin, ERK3, Endothelin-1, ERK5/BMK1, Engrailed Anhydrase IV. Chordin-Like 1, Carbonic Anhydrase VA, 2, ERR alpha/NR3B1, EN-RAGE, ERR beta/NR3B2, Chordin-Like 2. Carbonic Anhydrase VB, CINC-1, Car Enteropeptidase/Enterokinase, ERR gamma/NR3B3, Eryth bonic Anhydrase VI, CINC-2, Carbonic Anhydrase VII, ropoietin, Erythropoietin R. CCL26/Eotaxin-3, ESAM, CINC-3, Carbonic Anhydrase VIII, Claspin, Carbonic EpCAM/TROP-1, ER alpha/NR3A1. EPCR, ER beta/ Anhydrase IX, Claudin-6, Carbonic Anhydrase X, CLCN5. NR3A2, Eph, Exonuclease III, Eph Al, Exostosin-like Carbonic Anhydrase XII, CLEC-1, Carbonic Anhydrase 2/EXTL2, EphA2, Exostosin-like 3/EXTL3, EphA3, XIII, CLEC-2, Carbonic Anhydrase XIV, CLECSF-13/ FABP1, FGF-BP, FABP2, FGFR1, FGF R2, FGFR3, FGF CLEC-4F, CLECSF8, Carboxypeptidase A1/CPA1, CLF-1, R4, FABP3, FABP4, FABPS, FABP7, FABP9, FGF R5, Carboxypeptidase A2, CL-P1/COLEC12, Carboxypeptidase Complement Factor B, FHRS, FAM3A, Fibronectin,

US 2017/O 196966 A1 Jul. 13, 2017

Tryptase beta-1/MCPT-7, TIM-2, Tryptase beta-2/TPSB2, intracellular channel protein 5: CHRNA3 protein; Clcn3e TIM-3, Tryptase epsilon/BSSP-4, TIM-4, Tryptase protein; CLCNKB protein; CNGA4 protein: Cullin-5: gamma-1 /TPSG1, TIM-5, Tryptophan Hydroxylase, TIM Cyclic-GMP gated potassium channel: Cyclic-nucleotide 6, TSC22, TIMP-1, TSG, TIMP-2, TSG-6, TIMP-3, TSK, gated cation channel 4; Cyclic-nucleotide-gated cation chan TIMP-4, TSLP, TL1A/TNFSF15, TSLP R, TLR1, TSP50, nel alpha 3: Cyclic-nucleotide-gated cation channel beta 3; TLR2, beta-III Tubulin, TLR3, TWEAK/TNFSF12, TLR4, Cyclic-nucleotide-gated olfactory channel: Cystic fibrosis TWEAK R/TNFRSF 12, TLRS, Tyk2, TLR6, TLR9, Tyro transmembrane conductance regulator; Cytochrome B-245 sine Hydroxylase, TLX/NR2E1, Ubiquitin, UNC5H3, Ugi, heavy chain; Dihydropyridine-sensitive L-type, calcium UNC5H4, UGRP1, UNG, ULBP-1, uPA, ULBP-2, uPAR, channel alpha-2/delta subunits precursor; FXYD domain ULBP-3, URB, UNC5H1 UVDE, UNC5H2, Vanilloid R1, containing ion transport regulator 3 precursor: FXYD VEGF R, VASA, VEGF R1/Flt-1, Vasohibin, VEGF domain-containing ion transport regulator 5 precursor; R2/KDR/Flk-1, Vasorin, VEGFR3/Flt-4, Vasostatin, Versi FXYD domain-containing ion transport regulator 6 precur can, Vav-1, VGSQ, VCAM-1, VHR, VDR/NR1I1, Vimen Sor, FXYD domain-containing ion transport regulator 7: tin, VEGF, Vitronectin, VEGF-B, VLDLR, VEGF-C. v WF FXYD domain-containing ion transport regulator 8 precur A2, VEGF-D, Synuclein-alpha , Ku70, WASP Wnt-7b, sor; G protein-activated inward rectifier potassium channel WIF-1, Wnt-8a WISP-1/CCN4, Wnt-8b, WNK1, Wnt-9a, 1; G protein-activated inward rectifier potassium channel 2: Wnt-1, Wnt-9b, Wnt-3a, Wnt-10a, Wnt-4, Wnt-5a, Wnt-11, G protein-activated inward rectifier potassium channel 3: G Wnt-5b, winvNS3, Wnt7a, XCR1, XPE/DDB1, XEDAR, protein-activated inward rectifier potassium channel 4; XPE/DDB2, Xg, XPF, XIAP, XPG, XPA, XPW, XPD, Gamma-aminobutyric-acid receptor alpha-1 subunit precur XRCC1, Yes, YY1, EphA4. sor; Gamma-aminobutyric-acid receptor alpha-2 subunit 0083. In some embodiments, selected polypeptides precursor; Gamma-aminobutyric-acid receptor alpha-3 Sub include any number of ion-channels, including: 5-hy unit precursor; Gamma-aminobutyric-acid receptor alpha-4 droxytryptamine 3 receptor B subunit; 5-hydroxytryptamine Subunit precursor; Gamma-aminobutyric-acid receptor 3 receptor precursor; 5-hydroxytryptamine receptor 3 sub alpha-5 Subunit precursor; Gamma-aminobutyric-acid unit C: AAD 14 protein: Acetylcholine receptor protein, receptor alpha-6 Subunit precursor; Gamma-aminobutyric alpha subunit precursor, Acetylcholine receptor protein, beta acid receptor beta-1 subunit precursor; Gamma-aminobu Subunit precursor, Acetylcholine receptor protein, delta Sub tyric-acid receptor beta-2 Subunit precursor; Gamma-amin unit precursor, Acetylcholine receptor protein, epsilon Sub obutyric-acid receptor beta-3 subunit precursor; Gamma unit precursor; Acetylcholine receptor protein, gamma sub aminobutyric-acid receptor delta subunit precursor: unit precursor; Acid sensing ion channel 3 splice variant b: Gamma-aminobutyric-acid receptor epsilon Subunit precur Acid sensing ion channel 3 splice variant c, Acid sensing ion sor; Gamma-aminobutyric-acid receptor gamma-1 subunit channel 4; ADP-ribose pyrophosphatase, mitochondrial pre precursor; Gamma-aminobutyric-acid receptor gamma-3 cursor; AlphalA-Voltage-dependent calcium channel; Subunit precursor; Gamma-aminobutyric-acid receptor pi Amiloride-sensitive cation channel 1, neuronal; Amiloride Subunit precursor; Gamma-aminobutyric-acid receptor sensitive cation channel 2, neuronal; Amiloride-sensitive rho-1 subunit precursor; Gamma-aminobutyric-acid recep cation channel 4, isoform 2: Amiloride-sensitive sodium tor rho-2 Subunit precursor; Gamma-aminobutyric-acid channel; Amiloride-sensitive Sodium channel alpha-Subunit; receptor theta subunit precursor; GluR6 kainate receptor; Amiloride-sensitive sodium channel beta-subunit; Glutamate receptor 1 precursor; Glutamate receptor 2 pre Amiloride-sensitive Sodium channel delta-Subunit; cursor, Glutamate receptor 3 precursor, Glutamate receptor Amiloride-sensitive sodium channel gamma-Subunit; 4 precursor; Glutamate receptor 7: Glutamate receptor B; Annexin A7: Apical-like protein; ATP-sensitive inward rec Glutamate receptor delta-1 subunit precursor; Glutamate tifier potassium channel 1; ATP-sensitive inward rectifier receptor, ionotropic kainate 1 precursor, Glutamate receptor, potassium channel 10; ATP-sensitive inward rectifier potas ionotropic kainate 2 precursor, Glutamate receptor, iono sium channel 11; ATP-sensitive inward rectifier potassium tropic kainate 3 precursor, Glutamate receptor, ionotropic channel 14; ATP-sensitive inward rectifier potassium chan kainate 4 precursor; Glutamate receptor, ionotropic kainate nel 15: ATP-sensitive inward rectifier potassium channel 8: 5 precursor; Glutamate NMDA receptor subunit 3A pre Calcium channel alpha12.2 Subunit; Calcium channel alpha cursor; Glutamate NMDA receptor subunit 3B precursor: IE subunit, delta 19 delta40 delta46 splice variant; Calcium Glutamate NMDA receptor subunit epsilon 1 precursor: activated potassium channel alpha Subunit 1; Calcium-acti Glutamate NMDA receptor subunit epsilon 2 precursor: vated potassium channel beta Subunit 1; Calcium-activated Glutamate NMDA receptor subunit epsilon 4 precursor: potassium channel beta Subunit 2: Calcium-activated potas Glutamate NMDA receptor subunit Zeta 1 precursor; Gly sium channel beta subunit 3: Calcium-dependent chloride cine receptor alpha- 1 chain precursor, Glycine receptor channel-1; Cation channel TRPM4B, CDNA FLJ90453 fis, alpha-2 chain precursor, Glycine receptor alpha-3 chain clone NT2RP300 1542, highly similar to Potassium channel precursor; Glycine receptor beta chain precursor; H/ACA tetramerisation domain containing 6: CDNA FLJ90663 fis, ribonucleoprotein complex subunit 1; High affinity immu clone PLACE 1005031, highly similar to Chloride intracel noglobulin epsilon receptor beta-subunit; Hypothetical pro lular channel protein 5: CGMP-gated cation channel beta tein DKFZp31310334; Hypothetical protein subunit; Chloride channel protein; Chloride channel protein DKFZp761M1724; Hypothetical protein FLJ12242: Hypo 2: Chloride channel protein 3: Chloride channel protein 4; thetical protein FLJ14389; Hypothetical protein FLJ14798: Chloride channel protein 5: Chloride channel protein 6: Hypothetical protein FLJ14995: Hypothetical protein FLJ1 Chloride channel protein CIC-Ka; Chloride channel protein 6180: Hypothetical protein FLJ1 6802; Hypothetical protein CIC-Kb; Chloride channel protein, skeletal muscle; Chlo FLJ32069: Hypothetical protein FLJ37401; Hypothetical ride intracellular channel 6: Chloride intracellular channel protein FLJ38750; Hypothetical protein FLJ40162: Hypo protein 3: Chloride intracellular channel protein 4; Chloride thetical protein FLJ41415: Hypothetical protein FLJ90576: US 2017/O 196966 A1 Jul. 13, 2017

Hypothetical protein FLJ90590: Hypothetical protein Potassium Voltage-gated channel Subfamily A member 6: FLJ90622; Hypothetical protein KCTD15: Hypothetical Potassium voltage-gated channel subfamily B member 1: protein MGC 15619; Inositol 1,4,5-trisphosphate receptor Potassium Voltage-gated channel Subfamily B member 2; type 1; Inositol 1,4,5-trisphosphate receptor type 2: Inositol Potassium Voltage-gated channel Subfamily C member 1, 1,4,5-trisphosphate receptor type 3: Intermediate conduc Potassium voltage-gated channel subfamily C member 3: tance calcium-activated potassium channel protein 4; Potassium Voltage-gated channel Subfamily C member 4, Inward rectifier potassium channel 13: Inward rectifier Potassium Voltage-gated channel Subfamily D member 1, potassium channel 16: Inward rectifier potassium channel 4; Potassium Voltage-gated channel Subfamily D member 2; Inward rectifying K(+) channel negative regulator Kir2.2V. Potassium voltage-gated channel subfamily D member 3: Kainate receptor subunit KA2a: KCNHS protein; KCTD 17 Potassium Voltage-gated channel Subfamily E member 1, protein; KCTD2 protein; Keratinocyte-associated trans Potassium Voltage-gated channel Subfamily E member 2; membrane protein 1; KV channel-interacting protein 4; Potassium voltage-gated channel subfamily E member 3: Melastatin 1: Membrane protein MLC1; MGC 15619 pro Potassium Voltage-gated channel Subfamily E member 4, tein; Mucolipin-1; Mucolipin-2: Mucolipin-3; Multidrug Potassium Voltage-gated channel Subfamily F member 1, resistance-associated protein 4: N-methyl-D-aspartate Potassium Voltage-gated channel Subfamily G member 1, receptor 2C subunit precursor, NADPH oxidase homolog 1: Potassium Voltage-gated channel Subfamily G member 2; Nav1.5; Neuronal acetylcholine receptor protein, alpha-10 Potassium voltage-gated channel subfamily G member 3: Subunit precursor; Neuronal acetylcholine receptor protein, Potassium Voltage-gated channel Subfamily G member 4, alpha-2 Subunit precursor; Neuronal acetylcholine receptor Potassium Voltage-gated channel Subfamily H member 1, protein, alpha-3 Subunit precursor, Neuronal acetylcholine Potassium Voltage-gated channel Subfamily H member 2; receptor protein, alpha-4 Subunit precursor, Neuronal ace Potassium voltage-gated channel subfamily H member 3: tylcholine receptor protein, alpha-5 subunit precursor: Neu Potassium Voltage-gated channel Subfamily H member 4, ronal acetylcholine receptor protein, alpha-6 Subunit precur Potassium voltage-gated channel subfamily H member 5: sor; Neuronal acetylcholine receptor protein, alpha-7 Potassium voltage-gated channel subfamily H member 6: Subunit precursor; Neuronal acetylcholine receptor protein, Potassium voltage-gated channel subfamily H member 7; alpha-9 subunit precursor: Neuronal acetylcholine receptor Potassium voltage-gated channel subfamily H member 8: protein, beta-2 subunit precursor; Neuronal acetylcholine Potassium voltage-gated channel subfamily KQT member 1: receptor protein, beta-3 subunit precursor; Neuronal acetyl Potassium voltage-gated channel subfamily KQT member 2: choline receptor protein, beta-4 subunit precursor; Neuronal Potassium voltage-gated channel subfamily KQT member 3: voltage-dependent calcium channel alpha 2D subunit; P2X Potassium voltage-gated channel subfamily KQT member 4: purinoceptor 1; P2X purinoceptor 2; P2X purinoceptor 3: Potassium voltage-gated channel subfamily KQT member 5: P2X purinoceptor 4: P2X purinoceptor 5: P2X purinoceptor Potassium Voltage-gated channel Subfamily S member 1, 6; P2X purinoceptor 7: Pancreatic potassium channel Potassium Voltage-gated channel Subfamily S member 2; TALK-Ib; Pancreatic potassium channel TALK-Ic; Pancre Potassium voltage-gated channel subfamily S member 3: atic potassium channel TALK-Id; Phospholemman precur Potassium Voltage-gated channel Subfamily V member 2; sor; Plasmolipin; Polycystic kidney disease 2-related pro Potassium Voltage-gated channel, Subfamily H. member 7, tein; Polycystic kidney disease 2-like 1 protein; Polycystic isoform 2; Potassium/sodium hyperpolarization-activated kidney disease 2-like 2 protein; Polycystic kidney disease cyclic nucleotide-gated channel 1; Potassium/sodium hyper and receptor for egg jelly-related protein precursor; Poly polarization-activated cyclic nucleotide-gated channel 2: cystin-2, Potassium channel regulator, Potassium channel Potassium/sodium hyperpolarization-activated cyclic subfamily K member 1: Potassium channel subfamily K nucleotide-gated channel 3; Potassium/sodium hyperpolar member 10; Potassium channel subfamily K member 12; ization-activated cyclic nucleotide-gated channel 4; Prob Potassium channel subfamily K member 13; Potassium able mitochondrial import receptor subunit TOM40 channel subfamily K member 15; Potassium channel sub homolog: Purinergic receptor P2X5, isoform A: Putative 4 family K member 16; Potassium channel subfamily K repeat Voltage-gated ion channel; Putative chloride channel member 17; Potassium channel subfamily K member 2: protein 7; Putative GluR6 kainate receptor; Putative ion Potassium channel subfamily K member 3: Potassium chan channel protein CATSPER2 variant i: Putative ion channel nel subfamily K member 4: Potassium channel subfamily K protein CATSPER2 variant 2: Putative ion channel protein member 5: Potassium channel subfamily K member 6: CATSPER2 variant 3: Putative regulator of potassium chan Potassium channel subfamily K member 7: Potassium chan nels protein variant 1; Putative tyrosine-protein phosphatase nel subfamily K member 9; Potassium channel tetramerisa TPTE: Ryanodine receptor 1: Ryanodine receptor 2: Ryano tion domain containing 3; Potassium channel tetramerisation dine receptor 3; SH3KBP1 binding protein 1: Short transient domain containing protein 12; Potassium channel tetrameri receptor potential channel 1; Short transient receptor poten sation domain containing protein 14; Potassium channel tial channel 4; Short transient receptor potential channel 5; tetramerisation domain containing protein 2; Potassium Short transient receptor potential channel 6: Short transient channel tetramerisation domain containing protein 4; Potas receptor potential channel 7; Small conductance calcium sium channel tetramerisation domain containing protein 5: activated potassium channel protein 1; Small conductance Potassium channel tetramerization domain containing 10; calcium-activated potassium channel protein 2, isoform b: Potassium channel tetramerization domain containing pro Small conductance calcium-activated potassium channel tein 13; Potassium channel tetramerization domain-contain protein 3, isoform b; Small-conductance calcium-activated ing 1; Potassium Voltage-gated channel Subfamily A member potassium channel SK2. Small-conductance calcium-acti 1; Potassium Voltage-gated channel Subfamily A member 2; vated potassium channel SK3; Sodium channel; Sodium Potassium Voltage-gated channel Subfamily A member 4, channel beta-1 subunit precursor, Sodium channel protein Potassium Voltage-gated channel Subfamily A member 5: type II alpha subunit; Sodium channel protein type III alpha US 2017/O 196966 A1 Jul. 13, 2017 subunit; Sodium channel protein type IV alpha subunit; line Vertebrate type 4: Adrenoceptors such as Alpha Adre Sodium channel protein type IX alpha subunit; Sodium noceptors type 1: Alpha Adrenoceptors type 2: Beta Adre channel protein type V alpha Subunit, Sodium channel noceptors type 1; Beta Adrenoceptors type 2: Beta protein type VII alpha subunit; Sodium channel protein type Adrenoceptors type 3: Dopamine Vertebrate type 1; Dop VIII alpha subunit; Sodium channel protein type X alpha amine Vertebrate type 2: Dopamine Vertebrate type 3: Dop subunit; Sodium channel protein type XI alpha subunit; amine Vertebrate type 4: Histamine type 1; Histamine type Sodium-and chloride-activated ATP-sensitive potassium 2: Histamine type 3: Histamine type 4: Serotonin type 1: channel; Sodium/potassium-transporting ATPase gamma Serotonin type 2: Serotonin type 3: Serotonin type 4: Sero chain; Sperm-associated cation channel 1; Sperm-associated tonin type 5: Serotonin type 6: Serotonin type 7: Serotonin cation channel 2, isoform 4, Syntaxin-1B1; Transient recep type 8; other Serotonin types; Trace amine, Angiotensin type tor potential cation channel subfamily A member 1: Tran 1; Angiotensin type 2: Bombesin; Bradykinin; C5a anaphy sient receptor potential cation channel subfamily M member latoxin; Fmet-leu-phe, APJ like, Interleukin-8 type A: Inter 2: Transient receptor potential cation channel subfamily M leukin-8 type B; Interleukin-8 type others, C-C Chemokine member 3; Transient receptor potential cation channel Sub type 1 through type 11 and other types: C-X-C Chemokine family M member 6; Transient receptor potential cation (types 2 through 6 and others); C-X3-C Chemokine; Cho channel subfamily M member 7: Transient receptor potential lecystokinin CCK; CCK type A, CCK type B; and others: cation channel subfamily V member 1: Transient receptor Endothelin; Melanocortin (Melanocyte stimulating hor potential cation channel subfamily V member 2: Transient mone, Adrenocorticotropic hormone, Melanocortin hor receptor potential cation channel subfamily V member 3: mone); Duffy antigen; Prolactin-releasing peptide (GPR10); Transient receptor potential cation channel subfamily V Neuropeptide Y (type 1 through 7); Neuropeptide Y. Neu member 4, Transient receptor potential cation channel Sub ropeptide Y other, Neurotensin; Opioid (type D, K, M, X): family V member 5: Transient receptor potential cation Somatostatin (type 1 through 5); Tachykinin (Substance P channel subfamily V member 6; Transient receptor potential (NK1), Substance K (NK2); Neuromedin K (NK3); Tachy channel 4 epsilon splice variant; Transient receptor potential kinin-like 1; Tachykinin-like 2; Vasopressin/vasotocin (type channel 4 Zeta splice variant; Transient receptor potential 1 through 2); Vasotocin, Oxytocin/mesotocin, Conopressin; channel 7 gamma splice variant; Tumor necrosis factor, Galanin like: Proteinase-activated like (PARI, PAR2, PAR3, alpha-induced protein 1, endothelial; Two-pore calcium PAR4); Orexin & neuropeptides; FFJQRFP; Chemokine channel protein 2; VDAC4 protein; Voltage gated potassium receptor-like; Neuromedin U-like (Neuromedin U, PRXam channel Kv3.2b; Voltage gated sodium channel betalB sub ide); hormone protein (Follicle stimulating hormone, Lutro unit; Voltage-dependent anion channel; Voltage-dependent pin-choriogonadotropic hormone, Thyrotropin, Gonadotro anion channel 2; Voltage-dependent anion-selective channel pin type I, Gonadotropin type II); (Rhod).opsin, Rhodopsin protein 1; Voltage-dependent anion-selective channel pro Vertebrate (types 1-5); Rhodopsin Vertebrate type 5; Rho tein 2, Voltage-dependent anion-selective channel protein 3. dopsin Arthropod; Rhodopsin Arthropod type 1; Rhodopsin Voltage-dependent calcium channel gamma-1 subunit; Volt Arthropod type 2: Rhodopsin Arthropod type 3: Rhodopsin age-dependent calcium channel gamma-2 Subunit; Voltage Mollusc.; Rhodopsin; Olfactory (Olfactory II family 1 dependent calcium channel gamma-3 subunit; Voltage-de through 13); Prostaglandin (Prostaglandin E2 subtype EP1, pendent calcium channel gamma-4 Subunit; Voltage Prostaglandin E2/D2 subtype EP2, Prostaglandin E2 sub dependent calcium channel gamma-5 Subunit; Voltage type EP3, Prostaglandin E2 subtype EP4, Prostaglandin dependent calcium channel gamma-6 Subunit; Voltage F2-alpha); Prostacyclin; Thromboxane: Adenosine type 1 dependent calcium channel gamma-7 Subunit; Voltage through 3: Purinoceptors; Purinoceptor P2RY 1-4, 6,11; dependent calcium channel gamma-8 subunit; Voltage GPR 91; Purinoceptor P2RY5, 8, 9, 10; GPR35; GPR 92: dependent L-type calcium channel alpha-1C Subunit; GPR 174; Purinoceptor P2RY 12-14; GPR87 (UDP-Glu Voltage-dependent L-type calcium channel alpha-ID Sub cose); Cannabinoid; Platelet activating factor; Gonadotro unit; Voltage-dependent L-type calcium channel alpha-1S pin- releasing hormone; Gonadotropin-releasing hormone Subunit; Voltage-dependent L-type calcium channel beta-1 type I: Gonadotropin-releasing hormone type II: Adipoki Subunit; Voltage-dependent L-type calcium channel beta-2 netic hormone like; Corazonin; Thyrotropin-releasing hor Subunit; Voltage-dependent L-type calcium channel beta-3 mone & Secretogogue; Thyrotropin- releasing hormone; Subunit; Voltage-dependent L-type calcium channel beta-4 Growth hormone secretagogue; Growth hormone secret Subunit; Voltage-dependent N-type calcium channel alpha agogue-like: Ecdysis-triggering hormone (ETHR); Mela IB subunit; Voltage-dependent P/O-type calcium channel tonin; Lysosphingolipid & LPA (EDG); Sphingosine 1-phos alpha-1 A Subunit; Voltage-dependent R-type calcium chan phat; Edg-1; Edg-2, Edg-3. Edg-4, Edg-5; Edg-6: Edg-7; nel alpha-1 E. Subunit; Voltage-dependent T-type calcium Edg-8; Leukotriene B4 receptor BLT 1: Leukotriene B4 channel alpha-1 G subunit; Voltage-dependent T-type cal receptor BLT2: Class A Orphan/other; Putative neurotrans cium channel alpha-1H subunit; Voltage-dependent T-type mitters; SREB; Mas proto-oncogene & Mas-related calcium channel alpha-11 subunit; Voltage-gated L-type (MRGs); GPR45-like: Cysteinyl leukotriene; G-protein calcium channel alpha-1 subunit; Voltage-gated potassium coupled bile acid receptor; Free fatty acid receptor (GP40, channel beta-1 subunit; Voltage-gated potassium channel GP41, GP43); Class B Secretin-like: Calcitonin; Corticotro beta-2 subunit; Voltage-gated potassium channel beta-3 Sub pin-releasing factor; Gastric inhibitory peptide; Glucagon; unit; Voltage-gated potassium channel KCNA7. Growth hormone-releasing hormone; Parathyroid hormone: 0084. In some embodiments, exemplary G-protein PACAP; Secretin; Vasoactive intestinal polypeptide; Latro coupled receptors (GPCRs) include, but are not limited to: philin: Larrophilin type 1; Latrophilin type 2: Latrophilin Class A Rhodopsin like receptors such as Muse, acetylcho type 3: ETL receptors; Brain-specific angiogenesis inhibitor line Vertebrate type 1; Muse, acetylcholine Vertebrate type (BAI); Methuselah-like proteins (MTH): Cadherin EGF 2: Muse, acetylcholine Vertebrate type 3: Muse, acetylcho LAG (CELSR); Very large G-protein coupled receptor; US 2017/O 196966 A1 Jul. 13, 2017 20

Class C Metabotropic glutamate/pheroraone; Metabotropic a three-dimensional globular shape, monodispersity, and glutamate group I through III; Calcium-sensing like: Extra nanometric size range. The globular shape of a dendrimer cellular calcium-sensing; Pheromone, calcium-sensing like allows for a large number of terminal functional groups other; Putative pheromone receptors; GABA-B; GABA-B (“surface groups' or “surface reactive groups') whose subtype 1; GABA-B subtype 2: GABA-B like; Orphan chemical composition can be can be tailored to the desired GPRCS: Orphan GPCR6: Bride of sevenless proteins application (e.g., drug immobilization). Because dendrimers (BOSS): Taste receptors (T1R), Class D Fungal pheromone: are synthesized by the attachment of monomeric units that Fungal pheromone A-Factor like (STE2, STE3); Fungal branch out in a stepwise manner, precise control of the pheromone B like (BAR, BBR, RCB, PRA); Class E. cAMP molecular size, shape, density, polarity, flexibility, and solu receptors; Ocular albinism proteins: Frizzled/Smoothened bility is feasible. The choice of different branching units and family; frizzled Group A (FZ 1, 2, 4, 5, 7-9); frizzled Group functional Surface groups determine the final physical and B (FZ 3 & 6); frizzled Group C; Vomeronasal receptors: chemical characteristics of the dendrimer-based nanostruc Nematode chemoreceptors; Insect odorant receptors; and ture. Various types of dendrimers are available and any Class Z Archaeal/bacterial/fungal opsins. Suitable dendrimer is contemplated for use in the composi 0085. In some embodiments, the replicon encodes a tions and methods disclosed herein. Exemplary dendrimers polypeptide that provides a cosmetic benefit to the subject. include, but are not limited to: polyamidoamine (PAMAM) In some embodiments, the replicon encodes the polypeptide dendrimers, polyglutamic acid dendrimers, poly-L-lysine Botulinum toxin derived from the bacterium Clostridium dendrimers, polypropyleneimine (PPI) dendrimers, botulinum, or a homolog or ortholog thereof. Botulinum polymelamine dendrimers, triazine dendrimers, carbosilane toxin at high doses can cause botulism, often a fatal condi dendrimers, DETM dendrimers, phosphorus dendrimers, tion, but localized delivery of a small concentration of polyester dendrimers, polyether dendrimers, PAMAMOS botulinum toxin delivered into the skin can provide a dendrimers, poly(N,N-dimethylaminoethyl methacrylate) cosmetic benefit. Currently, two types of Botulinum toxin (PDMAEMA) dendrimers, diethylaminoethyldextran (BTX) are used therapeutically; type A (BTX-A) (Trade (DEAE-dextran) dendrimers, tecto dendrimers, multilingial names: Botox, Dysport, and Xeomin) and type B (BTX-B) dendrimers, amphiphilic dendrimers, chiral dendrimers, and (Trade name: MyoBloc). BTX is used therapeutically to micellar dendrimers. treat Strabismus (crossed eyes), blepharospasm (uncon 0089. In some embodiments, the dendrimer or surface trolled twitching or blinking of the eye), upper motor neuron reactive groups of the dendrimer are modified. Any suitable syndrome, severe primary axillary hyperhidrosis (excessive dendrimer modification is contemplated by the disclosure Sweating), cervical dystonia, chronic migraine, achalasia, herein. Exemplary dendrimer modifications include, but are chronic focal neuropathies, bruxism, idiopathic and neuro not limited to: lipid-modified dendrimers (e.g., lipid-modi genic detrusor overactivity, incontinence, anal fissure, vag fied PAMAM or PPI dendrimers), fluorinated dendrimers inismus, movement disorders associated with injury or dis (e.g., fluorinated PAMAM dendrimers), N-hydroxysuccin ease of the central nervous system, focal dystonias, gastric imide ester modified dendrimers (e.g., N-hydroxysuccinim cancer, temporomandibular joint pain disorders, diabetic ide esters of PEG or an hydroxysuccinimide ester of a cell neuropathy, wound healing, excessive salivation, vocal cord penetrating peptide), amino acid modified dendrimers (e.g., dysfunction, allergic rhinitis, and is used for cosmetic pur arginine and lysine modified PAMAM dendrimers), protein poses (e.g. to treat Wrinkles). In some embodiments, com and peptide modified dendrimers, Saccharide modified den positions described herein are used for localized delivery of drimers (e.g., cyclodextrin modified PAMAM dendrimers), BTX directly, e.g., to the skin. polymer modified dendrimers (e.g., PEGylation, hyaluronic I0086. In some embodiments, the replicon encodes one or acid modified dendrimers, or chitosan modified dendrimers), more BTX, and is used cosmetically (e.g., to reduce the nanoparticle modified dendrimers (e.g., carbon nanotubes, appearance of wrinkles). In other embodiments, the BTX in graphene, quantum dots, gold nanoparticles, magnetic nano the composition is in the form of a BTX polypeptide, instead particles, upconversion nanoparticles, or silicon nanomate of being encoded in a recombinant alphavirus replicon, Such rials), and cationic moiety modified dendrimers (e.g., oli that a defined dose of protein is provided (e.g., in a unit goamine, tertiary amine, quaternary ammonium, dose). imidazolium, guanidium, or phosphonium modified den 0087. Other polypeptides that can be used for cosmetic drimers). In a preferred embodiment, the recombinant purposes include, but are not limited to, calcitonin, adreno alphavirus replicon is formulated in a PAMAM dendrimer corticotropic hormone, parathyroid hormone (PTH), hPTH nanoparticle. In some embodiments, the PAMAM dendrimer (1->34), EGF, insulin, secretin, oxytocin, angiotensin, B-en is a modified PAMAM dendrimer. In some embodiments, dorphin, glucagon, Vasopressin, Somatostatin, gastrin, the amino surface reactive groups of a PAMAM dendrimer luteinizing hormone-releasing hormone, enkephalin, neuro is modified by fluorination (e.g., by heptafluorobutyric acid tensin, atrial natriuretic peptide. Somatotropin, Somatotro anhydride). When the surface reactive groups of a dendrimer pin-releasing hormone, bradykinin, Substance P. dynorphin, are modified, in some embodiments, only a portion of the thyroid stimulating hormone, mammotrophic hormone, Surface reactive groups are modified. For instance, in some interferon, interleukin, G-C SF, glutathione peroxidase, embodiments, a fluoridated PAMAM dendrimer comprises Superoxide dismutase, desmopressin, Somatomedin, and both unmodified amino Surface reactive groups and fluori endothelin. nated amino Surface reactive groups. (0090 Polyamidoamine (PAMAM) dendrimers are hyper Dendrimers and Dendrimer Nanoparticles branched polymers that exhibit molecular uniformity, nar 0088. The term “dendrimer' is derived from its tree-like row molecular weight distribution, defined size and shape branching structure and are defined as synthetic macromol characteristics, and a multifunctional terminal Surface. ecules characterized by a large number of branching points, These nanoscale polymers are comprised of an ethylenedi US 2017/O 196966 A1 Jul. 13, 2017 amine core, a repetitive branching amidoamine internal dimethyl-3-aminopropane (DLenDMA). Zwitterionic lipids structure, and a primary amine Surface reactive group. include, but are not limited to, acyl Zwitterionic lipids and Dendrimers are “grown' off a central core in an iterative ether Zwitterionic lipids. Examples of useful Zwitterionic manufacturing process, with each Subsequent step represent lipids are DPPC, DOPC and dodecylphosphocholine. In ing a new 'generation of dendrimer. Increasing generations Some embodiments, the lipids are saturated. In some produce macromolecules with larger molecular diameters, embodiments, the lipids are unsaturated. twice the number of reactive surface sites, and approxi 0095. In some embodiments, liposomes are formed from mately double the molecular weight of the preceding gen a single lipid or from a mixture of lipids. In some embodi eration. ments, a mixture comprises: (i) a mixture of anionic lipids; 0091 PAMAM dendrimers are highly effective agents for (ii) a mixture of cationic lipids; (iii) a mixture of Zwitterionic the delivery of a wide variety of genetic materials into many lipids; (iv) a mixture of anionic lipids and cationic lipids; (v) cells. As synthetic non-viral vectors, PAMAM dendrimers a mixture of anionic lipids and Zwitterionic lipids; (vi) a can be specifically designed to minimize immune responses, mixture of Zwitterionic lipids and cationic lipids; or (vii) a cytotoxicity, and effectively stabilize polynucleic acids mixture of anionic lipids, cationic lipids and Zwitterionic against nucleases for efficient delivery. PAMAM dendrimers lipids. In some embodiments, a mixture comprises both contain a diverse range of available Surface modifications, saturated and unsaturated lipids. In some embodiments, a including, but not limited to: primary amino, carboxylate, mixture comprises DSPC (Zwitterionic, saturated), hydroxy, mixed amine/hydroxyl, C12 hydrophobe, and Suc DlinDMA (cationic, unsaturated), and/or DMPG (anionic, cinamic acid Surface reactive groups. saturated). In some embodiments, where a mixture of lipids 0092. In some embodiments, the recombinant alphavirus is used, not all of the component lipids in the mixture need replicon is formulated in a PAMAM dendrimer nanoparticle, to be amphiphilic, e.g., one or more amphiphilic lipids are wherein the PAMAM dendrimer comprises a primary amine mixed with cholesterol. Surface reactive group. In some embodiments, the dendrimer is a generation 0 (GO), generation 1 (G1), generation 2 (G2), 0096. In some embodiments, the hydrophilic portion of a generation 3 (G3), generation 4 (G4), generation 5 (G5), lipid is modified by attachment (e.g., covalent attachment) generation 6 (G6), generation 7 (G7), generation 8 (G8), of a polyethylene glycol (also referred to as PEGylation). In generation 9 (G9), or generation 10 (G10) PAMAM den some embodiments, PEGylation increases stability and pre drimer that comprises amino Surface reactive groups. In a vent non-specific adsorption of the liposomes. In some preferred embodiment, the alphavirus replicon is formulated embodiments, lipids are conjugated to PEG using any Suit in a PAMAM dendrimer nanoparticle, wherein the PAMAM able technique. dendrimer is a G5 or G9 PAMAM dendrimer comprising 0097 Liposomes are usually divided into three groups: amino Surface reactive groups. multilamellar vesicles (MLV); small unilamellar vesicles 0093. In some embodiments, the recombinant alphavirus (SUV); and large unilamellar vesicles (LUV). MLVs have replicon is formulated using a microfluidic mixing device. multiple bilayers in each vesicle, forming several separate Microfluidic mixing devices facilitate controlled, bottom aqueous compartments. SUVs and LUVs have a single up, molecular self-assembly of nanoparticles via custom bilayer encapsulating an aqueous core: SUVs typically have engineered microfluidic mixing cartridges that allow milli a diameter >50 nm, and LUVs have a diameter >50 nm. In second mixing of nanoparticle components at the nanoliter some embodiments, liposomes are LUVs with a diameter in scale. Rapid mixing on a small scale allows reproducible the range of 50-220 nm. In some embodiments, for a control over particle formation compared to traditional composition comprising a population of LUVs with different methods of nanoparticle formation (such as hand mixing). In diameters: (i) at least 80% by number have diameters in the Some embodiments, the recombinant alphavirus replicon is range of 20-220 nmi; (ii) the average diameter (Zav, by formulated as a PAMAM dendrimer nanoparticle using a intensity) of the population are in the range of 40-200 nm: microfluidic device. In some embodiments, the microfluidic and/or (iii) the diameters have a polydispersity index <0.2. device is a NanoAssemblr (Precision NanoSystems). A variety of techniques for preparing Suitable liposomes are available and any Suitable liposomal preparation technique Encapsulation in Liposomes is contemplated. 0094. In some embodiments, the one or more bioactive 0098. In some embodiments, encapsulating a bioactive agents (e.g., polypeptides or recombinant alphavirus repli agent increases the stability of the bioactive agent. In some cons) are encapsulated in a liposome. In some embodiments, embodiments, the time that a unit dose of a microneedle various amphiphilic lipids form bilayers in an aqueous composition comprising a recombinant alphavirus particle environment to encapsulate a bioactive agent-containing encoding an antigen remains effective in inducing a detect aqueous core as a liposome. In some embodiments, these able immune response to the antigen is increased by encap lipids have an anionic, cationic or Zwitterionic hydrophilic Sulating the replicon in liposomes, relative to the replicon head group. In some embodiments, some phospholipids are without encapsulation. In some embodiments, the increase is anionic whereas others are Zwitterionic. Suitable classes of about or more than about 10%, 25%, 50%, 75%, 100%, phospholipid include, but are not limited to, phosphatidyle 200%, 500%, or more. In some embodiments, the stability of thanolamines, phosphatidylcholines, phosphatidylserines, the bioactive agent is more than doubled by encapsulating in and phosphatidylglycerols. Exemplary cationic lipids liposomes. In some embodiments, encapsulating a bioactive include, but are not limited to, dioleoyl trimethylammonium agent in a liposome is effective in increasing efficiency of propane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3- delivering the bioactive agent to a cell. In some embodi aminopropane (DSDMA), 1,2-dioleyloxy-N,Ndimethyl-3- ments, encapsulating in liposomes decrease the amount of aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl bioactive agent necessary to achieve a desired result (e.g., a 3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N- protective immune response) by 10%, 20%, 30%, 40%, US 2017/O 196966 A1 Jul. 13, 2017 22

50%. 60%, 70%, 80%, 90%, 95%, or more, as compared to composition that has been reduced to a dried composition. In the amount required if the bioactive agent were delivered Some embodiments, a dehydrated replicon composition has without encapsulation. increased stability at room temperatures (e.g., temperatures between 21° C. to 28°C.). In some embodiments, a dehy Dehydrated Compositions drated replicon composition has increased shelf-life at room 0099 Disclosed herein, in some embodiments, are temperatures. In some embodiments, the increase in stability microneedle devices for administering a recombinant at room temperature is about or more than about 10%, 25%, alphavirus replicon or RNA molecule encoding an exog 50%, 75%, 100%, 200%, 500%, or more, relative to the enous polypeptide comprising: a Substrate comprising a composition before dehydration, or an equivalent composi plurality of microneedles; and a composition comprising a tion provided in liquid form. recombinant alphavirus replicon or RNA molecule encoding 0102. In some embodiments, dehydration is accom an exogenous polypeptide coated onto or embedded into the plished by any number of methods. In one embodiments, plurality of microneedles. Also disclosed herein, in some dehydration of the replicon composition is accomplished by embodiments, are methods of preparing a microneedle freeze-drying, also referred to as “lyophilization.” In some device, comprising: obtaining a substrate comprising a plu embodiments, lyophilization involves the steps of freezing rality of microneedles; and coating or embedding a recom the water in a composition to the solid state, followed by binant alphavirus replicon encoding an exogenous polypep Sublimation. In some embodiments, Sublimation is per tide onto or into the plurality of microneedles. Also formed in a vacuum. In some embodiments, heat is applied disclosed herein, in some embodiments, are methods of to the composition to accelerate the Sublimation process. In inducing an immune response in an individual in need Some embodiments, lyophilization removes Substantially all thereof, comprising: (a) contacting the dermal Surface of an of the water content of the composition. In some embodi individual with a microneedle device comprising (i) a plu ments, lyophilization is used when low-temperature drying rality of microneedles comprising a recombinant alphavirus methods are desired. In some embodiments, freeze-drying replicon encoding an exogenous polypeptide coated onto or machines are readily available from a number of different embedded into the plurality of microneedles, and (b) deliv manufacturers. ering the recombinant alphavirus replicon to the individual, 0103) In some embodiments, dehydration methods are thereby inducing an immune response in the individual. not limited to lyophilization, and other methods of dehydra 0100. In some embodiments, the RNA compositions tion are contemplated. In some embodiments, a replicon described herein are provided in a dehydrated form. In some composition is air-dried. In some embodiments, the repli embodiments, a recombinant alphavirus replicon is in a cons are washed with a volatile alcohol (e.g., isopropanol or dehydrated form, Such as before or after applying to or ethanol). In some embodiments, this step replaces the water embedding within microneedles. In some embodiments, the content in the composition with the volatile alcohol and replicon encapsulated in a liposome is in a dehydrated form. precipitates the nucleic acid molecule. In some embodi In some embodiments, dehydration offers several advan ments, after a step of centrifugation to pellet the precipitated tages including increased Stability of the composition, nucleic acid molecule, the Volatile alcohol is removed. Such increased shelf-life of the composition, and reduced weight as by pipetting or pouring off the volatile alcohol, and the of the composition. pelleted nucleic acid molecule is allowed to air-dry. In some 0101. In general, the term “dehydration” refers to the embodiments, dehydration comprises the use of a desiccant. removal of an amount of water from a composition. In some Examples of desiccants include alumina, aluminum amal embodiments, a composition is dehydrated so as to remove gam, barium oxide, barium perchlorate, boric anhydride, about or at least about 10%, 20%, 30%, 40%, 50%, 60%, calcium chloride (anhydrous), calcium oxide, calcium Sul 70%, 80%, 90%, 95% or more of a starting amount of water. phate (anhydrous), copper (II) sulfate (anhydrous), magne In some embodiments, at least 50% of a starting amount of sium amalgam, magnesium perchlorate (anhydrous), mag water is removed. In some embodiments, the amount of nesium Sulphate (anhydrous), phosphorus pentoxide, water desired to be removed depends on the starting amount potassium, potassium carbonate (anhydrous), potassium of water, so as to arrive at an amount of water at or below hydroxide, silica gel, Sodium, Sodium hydroxide, sodium a target amount. In some embodiments, a composition is potassium alloy, Sodium Sulphate (anhydrous), Sulfuric acid, dehydrated so as to contain about or less than about 10%, and the like. Other available methods of dehydration include 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, or heat drying, freeze-drying with liquid nitrogen, and spray less water. In some embodiments, the dehydrated form drying. contains less than 1% water. In some embodiments, a 0104. In some embodiments, dehydrated bioactive agents composition, or component thereof, is dehydrated to remove are coated directly onto microneedle structures for admin substantially all, e.g., 90%, 91%, 92%, 93%, 94%, 95%, istration to a subject. In some embodiments, a liquid repli 96%, 97%, 98%, 98.5%, 99%, or more water content. In con composition is spray-dried directly onto the microneedle Some embodiments, a dehydrated replicon composition is to coat the structure. In another example, the microneedle is substantially free of water content. In some embodiments, dipped into the liquid replicon composition and then the 98% of the water content of a composition is removed. In composition is air-dried onto the structure. In some embodi Some embodiments, the starting form of a composition to be ments, the liquid replicon or polypeptide composition is dehydrated is liquid, semi-liquid, semi-solid, Solid, or a gel. coated onto the microneedle using a microfluidic device In general, a composition that has been dehydrated is (e.g., the BioDot printer described herein). In some embodi referred to as a “dried composition. In some embodiments, ments, a dehydrated replicon composition is coated directly a dried composition is in a powdered form. In some embodi onto a metal microneedle structure. In other embodiments, ments, a dried composition is gel-like. In some embodi the dehydrated replicon composition is coated directly onto ments, a dehydrated composition includes a liquid replicon a polymer microneedle structure. In yet other embodiments, US 2017/0196966 A1 Jul. 13, 2017 the dehydrated replicon composition is coated directly onto between 200 mOsm/kg and 400 mOsm/kg, e.g., between a polymer-coated microneedle structure. In some embodi 240-360 mosm/kg, or between 290-310 mOsm/kg. In some ments, the composition including the microneedles them embodiments, pharmaceutical compositions include one or selves is dehydrated. more preservatives, such as thiomersal or 2-phenoxyethanol. 0105. In some embodiments, the recombinant alphavirus In some embodiments, pharmaceutical compositions are replicon is packaged onto a microneedle using a microfluidic mercury-free. In some embodiments, the pharmaceutical dispensing device. Microfluidic dispensing devices are non composition is preservative-free. In some embodiments, contact liquid handling systems with high speed aspirating pharmaceutical compositions are sterile or sterilized. In and dispensing capabilities that reproducibly dispense an Some embodiments, pharmaceutical compositions are non accurate printing Volume onto a microneedle, plurality of pyrogenic, e.g., containing <1 EU (endotoxin unit, a stan microneedles, or microneedle array. A microfluidic dispens dard measure) per dose, and in some cases <0.1 EU per dose. ing device typically utilizes a moveable stage holding a In some embodiments, pharmaceutical compositions contain ceramic needle which accurately picks up a pre-determined an RNAse inhibitor. Any suitable RNAse inhibitor is con Volume of reagent (e.g., replicon RNA) and then ejects templated for use herein, such as those sold commercially (prints) nanoliter Volumes onto a substrate (e.g., a by, e.g., Life Technologies. Sigma-Aldrich, & Roche. In microneedle array) positioned on a printing table. In some Some embodiments, pharmaceutical compositions are pre embodiments, the microfluidic dispensing device is a Bio pared in unit dose form. Dot AD1520 tabletop workstation. 0108. In some embodiments, the pharmaceutical compo 0106. In some embodiments, a dehydrated bioactive sitions disclosed herein further comprise a small molecule agent (e.g., a polypeptide or replicon) is incorporated into immunopotentiators. In some embodiments, the pharmaceu the microneedle itself (e.g., embedded into the microneedle). tical composition includes a TLR2 agonist (e.g., In some embodiments, a dehydrated replicon is mixed with Pam3CSK4), a TLR4 agonist (e.g., an aminoalkyl glu a polymer before molding and polymerization. In some cosaminide phosphate, such as E6020), a TLR7 agonist embodiments, the replicon-polymer composition is then (e.g., imiquimod), a TLR8 agonist (e.g., residuimod) and/or molded and polymerized to form a solid microneedle struc a TLR9 agonist (e.g., IC31). In some embodiments, any such ture wherein the replicon is contained within the agonist is selected to have a molecular weight of <2000 Da. microneedle structure. In some embodiments, the polymer 0109. In some embodiments, replicon compositions are Substance is dissolvable, biodegradable, biosoluble, or a stored at low temperatures to maintain integrity of the combination thereof such that upon application of the replicon molecule. In some embodiments, suitable storage microneedle to the skin of a subject, the polymer is dis temperatures are -80° C. to +4° C. In some embodiments, solved, biodegraded, and/or solubilized and the replicon is the replicon composition is in a liquid form and is stored at released. -80° C. to +4°C. In other examples, the replicon compo sition is in a dehydrated form and is stored at room tem Pharmaceutical Compositions perature. 0107. In some embodiments, the microneedle devices of the disclosure include the active components (e.g., a recom Methods of Use binant alphavirus replicon and/or a polypeptide) formulated 0110 Disclosed herein, in some embodiments, are as a pharmaceutical composition. In some embodiments, the microneedle devices for administering a recombinant pharmaceutical composition comprises a recombinant alphavirus replicon or RNA molecule encoding an exog alphavirus replicon and a pharmaceutically acceptable car enous polypeptide comprising: a substrate comprising a rier or excipient. In some embodiments, the microneedle plurality of microneedles; and a composition comprising a devices of the present disclosure include a recombinant recombinant alpha virus replicon or RNA molecule encoding alphavirus replicon in water or in a buffer (e.g., a phosphate an exogenous polypeptide coated onto or embedded into the buffer, a Tris buffer, a borate buffer, a succinate buffer, a plurality of microneedles. Also disclosed herein, in some histidine buffer, or a citrate buffer), or any other pharma embodiments, are methods of preparing a microneedle ceutically acceptable carrier or excipient. Any suitable phar device, comprising: obtaining a substrate comprising a plu maceutically acceptable carriers or excipients are contem rality of microneedles; and coating or embedding a recom plated by the disclosure herein. In some embodiments, binant alpha virus replicon encoding an exogenous polypep buffer salts, when present, are included in the 5-20 mM tide onto or into the plurality of microneedles. Also range. In some embodiments, pharmaceutical compositions disclosed herein, in some embodiments, are methods of have a pH between 5.0 and 9.5, e.g., between 6.0 and 8.0. In inducing an immune response in an individual in need Some embodiments, compositions include sodium salts (e.g., thereof, comprising: (a) contacting the dermal surface of an Sodium chloride) to give tonicity. In some embodiments, a individual with a microneedle device comprising (i) a plu concentration of 10+2 mg/ml NaCl is typical, e.g., about 9 rality of microneedles comprising a recombinant alphavirus mg/ml. In some embodiments, pharmaceutical compositions replicon encoding an exogenous polypeptide coated onto or include metalion chelators. In some embodiments, chelators embedded into the plurality of microneedles, and (b) deliv prolong RNA stability by removing ions which accelerate ering the recombinant alphavirus replicon to the individual, phosphodiester hydrolysis. Examples of chelators include, thereby inducing an immune response in the individual. but are not limited to, EDTA, EGTA, BAPTA, pentetic acid, 10111 Conventional injection methods for delivering etc., which, in some embodiments, are present at between compounds (such as in vaccination with an antigen) bypass 10-500 LM, e.g., 0.1 mM. In some embodiments, a citrate the skin's immune system and the compound is injected salt, such as sodium citrate, act as a chelator, while advan directly into sub-cutaneous tissue or muscle. The skin con tageously also providing buffering activity. In some embodi tains a large number of immune cells, including epidermal ments, pharmaceutical compositions have an osmolality of Langerhans cells and dermal dendritic cells. Without wish US 2017/O 196966 A1 Jul. 13, 2017 24 ing to be bound by theory, these cells are thought to induce megakaryoblastic leukemia, Acute monocytic leukemia, cell-mediated immune responses as well as enhance the Acute myeloblastic leukemia with maturation, Acute production of antibodies by the antibody-releasing lympho myeloid dendritic cell leukemia, Acute myeloid leukemia, cytes, B cells. In some embodiments, intradermal adminis Acute promyelocytic leukemia, Adamantinoma, Adenocar tration of vaccine is employed to trigger an immunogenic cinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid effect. odontogenic tumor, Adrenocortical carcinoma, Adult T-cell 0112 Disclosed herein, in some embodiments, are meth leukemia, Aggressive NK-cell leukemia, AIDS-Related ods of preparing a microneedle device, comprising: obtain Cancers, AIDS-related lymphoma, Alveolar soft part sar ing a Substrate comprising a plurality of microneedles; and coma, Ameloblastic fibroma, Anal cancer, Anaplastic large coating or embedding a recombinant alphavirus replicon cell lymphoma, Anaplastic thyroid cancer, Angioimmuno encoding an exogenous polypeptide onto or into the plurality blastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, of microneedles. In some embodiments, the method com Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid prises packaging the recombinant alphavirus in or on (e.g., tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell by coating onto or embedding into) microneedles and dehy leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary drating the recombinant alphavirus before packaging. In tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone Some embodiments, the method comprises packaging the tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, recombinant alphavirus in or on (e.g., by coating onto or Brenner tumor, Bronchial Tumor, Bronchioloalveolar carci embedding into) microneedles and dehydrating the recom noma, Brown tumor, Burkitt's lymphoma, Cancer of binant alphavirus after packaging. Materials and methods Unknown Primary Site, Carcinoid Tumor, Carcinoma, Car for preparing recombinant alphavirus replicons and further cinoma in situ, Carcinoma of the penis, Carcinoma of packaging, dehydrating, and/or encapsulating can include Unknown Primary Site, Carcinosarcoma, Castleman's Dis any such materials and methods described herein, including ease, Central Nervous System Embryonal Tumor, Cerebellar with regard to any other aspect of the disclosure. Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cho 0113 Disclosed herein, in some embodiments, are meth langiocarcinoma, Chondroma, Chondrosarcoma, Chor ods of inducing an immune response in an individual in need doma, Choriocarcinoma, Choroid plexus papilloma, thereof, comprising: (a) contacting the dermal Surface of an Chronic Lymphocytic Leukemia, Chronic monocytic leuke individual with a microneedle device comprising (i) a plu mia, Chronic myelogenous leukemia, Chronic Myeloprolif rality of microneedles comprising a recombinant alphavirus erative Disorder, Chronic neutrophilic leukemia, Clear-cell replicon encoding an exogenous polypeptide coated onto or tumor, Colon Cancer, Colorectal cancer, Craniopharyn embedded into the plurality of microneedles, and (b) deliv gioma, Cutaneous T-cell lymphoma, Degos disease, Der ering the recombinant alphavirus replicon to the individual, matofibrosarcoma protuberans, Dermoid cyst, Desmoplastic thereby inducing an immune response in the individual. The small round cell tumor. Diffuse large B cell lymphoma, "dermal surface’ generally refers to the outer layer or Dysembryoplastic neuroepithelial tumor, Embryonal carci substantially the outer layer of the epidermis (the layer of noma, Endodermal sinus tumor, Endometrial cancer, Endo epidermal cells exposed to the outside environment). In metrial Uterine Cancer, Endometrioid tumor, Enteropathy Some embodiments, replicons are packaged into or on associated T-cell lymphoma, Ependymoblastoma, microneedles for intradermal administration to a subject. In Ependymoma, Epithelioid sarcoma, Erythroleukemia, Some embodiments, other routes of administration are pos Esophageal cancer, Esthesioneuroblastoma, Ewing Family sible depending on the target tissue to which a microneedle of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, composition is to be applied. In some embodiments, other Extracranial Germ Cell Tumor, Extragonadal Germ Cell routes of administration include, but are not limited to, Tumor, Extrahepatic Bile Duct Cancer, Extramammary Pag application to muscular tissue, intraperitoneal tissue, intra et's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, dermal tissue, Subcutaneous tissue, and buccal tissue (e.g., Fibrosarcoma, Follicular lymphoma, Follicular thyroid can cheek or tongue). In some embodiments, administration of a cer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, dehydrated replicon occurs in a number of different ways. In Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gas one example, the dehydrated replicon is coated onto the trointestinal cancer, Gastrointestinal Carcinoid Tumor, Gas surface of the microneedle and is dissolved upon direct trointestinal Stromal Tumor, Gastrointestinal stromal tumor, application of the microneedle to the skin of a Subject, or Germ cell tumor, Germinoma, Gestational choriocarcinoma, upon penetration of a dermal Surface. In some embodiments, Gestational Trophoblastic Tumor, Giant cell tumor of bone, a dehydrated replicon dissolves in seconds or minutes. In Glioblastoma multiforme, Glioma, Gliomatosis cerebri, some embodiments, the dehydrated replicon dissolves Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa within about 5, 10, 15, 20, 25, 30, 45, 50, 60, 120, 180, or cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head more seconds; or within minutes, such as within about 1, 2, and Neck Cancer, Head and neck cancer, Heart cancer, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 60, 120 or more minutes. Hemangioblastoma, Hemangiopericytoma, Hemangiosar 0114. In some embodiments, replicon compositions are coma, Hematological malignancy, Hepatocellular carci used to deliver a polypeptide to a subject in need thereof. In noma, Hepatosplenic T-cell lymphoma, Hereditary breast Some embodiments, the replicon encoding a tumor antigen ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin’s is used to treat a cancer. Examples of cancer antigens for use lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, in accordance with these methods have been described. Inflammatory breast cancer, Intraocular Melanoma, Islet cell Examples of cancers that are treated using microneedle carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leu compositions of the disclosure include, but are not limited kemia, Sarcoma, Kaposi's sarcoma, Kidney Cancer, to: Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Acral lentiginous melanoma, Acrospiroma, Acute eosino Laryngeal cancer, Lentigo maligna melanoma, Leukemia, philic leukemia, Acute lymphoblastic leukemia, Acute Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung US 2017/O 196966 A1 Jul. 13, 2017

cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, noma, Urachal cancer, Urethral cancer, Urogenital neo Lymphoepithelioma, Lymphoid leukemia, Lymphoma, plasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Macroglobulinemia, Malignant Fibrous Histiocytoma, Verner Morrison syndrome, Verrucous carcinoma, Visual Malignant fibrous histiocytoma, Malignant Fibrous Histio Pathway Glioma, Vulvar Cancer, Waldenstrom's macro cytoma of Bone, Malignant Glioma, Malignant Mesothe globulinemia, Warthin's tumor, Wilms tumor. lioma, Malignant peripheral nerve sheath tumor, Malignant 0.115. In some embodiments, therapeutic efficacy of the rhabdoid tumor, Malignant triton tumor, MALT lymphoma, methods and compositions disclosed herein, with regard to Mantle cell lymphoma, Mast cell leukemia, Mediastinal the treatment of a cancer, whether benign or malignant, is germ cell tumor, Mediastinal tumor, Medullary thyroid measured by the degree to which the methods and compo cancer, Medulloblastoma, Medulloblastoma, Medulloepi sitions promote inhibition of tumor cell proliferation, the thelioma, Melanoma, Melanoma, Meningioma, Merkel Cell inhibition of tumor vascularization, the eradication of tumor Carcinoma, Mesothelioma, Mesothelioma, Metastatic cells, and/or a reduction in the size of at least one tumor Such Squamous Neck Cancer with Occult Primary, Metastatic that a subject is treated for the proliferative disorder. In some urothelial carcinoma, Mixed Mullerian tumor, Monocytic embodiments, several parameters are considered in the leukemia, Mouth Cancer. Mucinous tumor, Multiple Endo determination of therapeutic efficacy are discussed herein. In crine Neoplasia Syndrome, Multiple Myeloma, Multiple Some embodiments, the proper combination of parameters myeloma, Mycosis Fungoides, Mycosis fungoides, Myelo for a particular situation are established by the clinician. dysplastic Disease, Myelodysplastic Syndromes, Myeloid Progress in treating cancer (e.g., reducing tumor size or leukemia, Myeloid sarcoma, Myeloproliferative Disease, eradicating cancerous cells) are ascertained using any Suit Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, able method, such as those methods currently used in the Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuro clinic to track tumor size and cancer progress. In some blastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodu embodiments, one efficacy parameter used to evaluate the lar melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin treatment of cancer is a reduction in the size of a tumor. lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Tumor size can be figured using any suitable technique. Such Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligo as measurement of dimensions, or estimation of tumor dendroglioma, Oncocytoma, Optic nerve sheath menin Volume using available computer Software. Such as gioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, FreeFlight software developed at Wake Forest University Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian that enables accurate estimation of tumor volume. In some cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell embodiments, tumor size is determined by tumor visualiza Tumor, Ovarian Low Malignant Potential Tumor, Paget’s tion using, for example, CT, ultrasound, SPECT, spiral CT, disease of the breast, Pancoast tumor, Pancreatic Cancer, MRI, photographs, and the like. In some embodiments, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, where a tumor is Surgically resected after completion of the Paraganglioma, Paranasal Sinus Cancer, Parathyroid Can therapeutic period, the presence of tumor tissue and tumor cer, Penile Cancer, Perivascular epithelioid cell tumor, Pha size is determined by gross analysis of the tissue to be ryngeal Cancer, Pheochromocytoma, Pineal Parenchymal resected, and/or by pathological analysis of the resected Tumor of Intermediate Differentiation, Pineoblastoma, tissue. In some embodiments, the growth of a tumor is Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma stabilized (e.g., one or more tumors do not increase more Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, than 1%. 5%, 10%, 15%, or 20% in size, and/or do not Precursor T-lymphoblastic lymphoma, Primary central ner metastasize) as a result of treatment. In some embodiments, Vous system lymphoma, Primary effusion lymphoma, Pri a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, mary Hepatocellular Cancer, Primary Liver Cancer, Primary 9, 10, 11, 12, or more weeks; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, peritoneal cancer, Primitive neuroectodermal tumor, Pros 11, 12, or more months. In some embodiments, the size of tate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal a tumor is reduced by at least about 5% (e.g., at least about cell carcinoma, Respiratory Tract Carcinoma Involving the 10%, 15%, 20%, or 25%). In some embodiments, tumor size NUT Gene on Chromosome 15, Retinoblastoma, Rhab is reduced at least about 30% (e.g., at least about 35%, 40%, domyoma, Rhabdomyosarcoma, Richter's transformation, 45%, 50%, 55%, 60%, or 65%). In some embodiments, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, tumor size is reduced at least about 70% (e.g., at least about Schwannomatosis, Sebaceous gland carcinoma, Secondary 75%, 80%, 85%, 90%, or 95%). Most preferably, the tumor neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell is completely eliminated, or reduced below a level of tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet detection. In some embodiments, a Subject remains tumor ring cell carcinoma, Skin Cancer, Small blue round cell free (e.g., in remission) for at least about 1, 2, 3, 4, 5, 6, 7, tumor, Small cell carcinoma, Small Cell Lung Cancer, Small 8, 9, 10, 11, 12, or more weeks following treatment. In some cell lymphoma, Small intestine cancer, Soft tissue sarcoma, embodiments, a Subject remains tumor free for at least about Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months following tumor, Splenic marginal Zone lymphoma, Squamous cell treatment. In some embodiments, a Subject remains tumor carcinoma, Stomach cancer, Superficial spreading mela free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years noma, Supratentorial Primitive Neuroectodermal Tumor, after treatment. Surface epithelial-stromal tumor, Synovial sarcoma, T-cell 0116. In some embodiments, the present disclosure fur acute lymphoblastic leukemia, T-cell large granular lympho ther provides for a method of monitoring an immune cyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell response in an individual. In some embodiments, the method prolymphocytic leukemia, Teratoma, Terminal lymphatic comprises administering to the Subject an alphavirus repli cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic con composition; and assaying a sample from the Subject to Carcinoma, Thymoma, Thyroid cancer, Transitional Cell determine a level of an immune response in the individual Cancer of Renal Pelvis and Ureter, Transitional cell carci against the exogenous polypeptide. US 2017/O 196966 A1 Jul. 13, 2017 26

0117. In some embodiments, the replicons are screened treatment. In some embodiments, a microneedle containing or analyzed to confirm their therapeutic properties using any the BTX polypeptide is applied to an area of the face that suitable in vitro or in vivo testing methods available. In received the facial, and preferably is a site of desired Some embodiments, vaccines composed of replicons are treatment. In some embodiments, BTX is delivered via tested for their effect on induction of proliferation or effector intradermal administration to the Subject. function of the particular lymphocyte type of interest, e.g., B cells, T cells, T cell lines, and T cell clones. In some Oral Compositions embodiments, spleen cells from immunized mice are iso 0121 Disclosed herein, in some embodiments, are meth lated and the capacity of cytotoxic T lymphocytes to lyse ods for producing an oral composition and administering the autologous target cells that contain a replicon that encodes oral composition to a subject. In some embodiments, the oral the immunogen. In some embodiments, T helper cell differ composition is used to deliver an RNA replicon or polypep entiation is analyzed by measuring proliferation or produc tide to a subject. In some embodiments, the oral composition tion of TH1 (IL-2 and IFN-gamma) and/or TH2 (IL-4 and comprises a polynucleotide. In some embodiments, a poly IL-5) cytokines by ELISA or directly in CD4+ T cells by nucleotide is any nucleic acid molecule as described herein cytoplasmic cytokine staining and flow cytometry. (i.e., DNA, RNA, or combinations thereof). 0118. In some embodiments, replicons that encode an I0122. In some cases, the polynucleotide is an mRNA. In antigen are tested for their ability to induce humoral immune some embodiments, the mRNA is any RNA molecule com responses, as evidenced, for example, by induction of B cell prising a coding sequence. In some embodiments, RNAS are production of antibodies specific for an antigen of interest. generated by in vitro transcription, the methods of which In some embodiments, these assays are conducted using have been described herein. In some embodiments, RNAs peripheral B lymphocytes from immunized individuals, but comprise a coding sequence or coding region that encodes a any suitable assay method is contemplated. In some embodi polypeptide. The polypeptide can be any of the polypeptide ments, assays used to characterize replicons involve detect as disclosed herein. In some embodiments, the polypeptide ing expression of the encoded antigen by the target cells. In is an antigen Suitable for use as a vaccine. Examples of Some embodiments, FACS is used to detect antigen expres antigens Suitable for use as vaccines are provided herein. In sion on the cell surface or intracellularly. In some embodi Some cases, the antigen is a foreign antigen. In some ments, FACS selection sorts for different levels of expres embodiments, the foreign antigen is any foreign antigen Sion. In some embodiments, lower expression is desired. In disclosed herein. In some embodiments, the foreign antigen some embodiments, other suitable method for identifying is an antigen associated with influenza virus (e.g., hemag cells which express a particular antigen involve panning glutinin (HA) or neuraminidase (NA)). In some embodi using monoclonal antibodies on a plate or capture using ments, the antigen is a self-antigen (i.e., an antigen associ magnetic beads coated with monoclonal antibodies. ated with cancer). In some embodiments, an antigen that is 0119. In some embodiments, a subject is pre-treated with encoded by a replicon is delivered to a subject. In some an agent that facilitates delivery of the microneedle compo embodiments, the antigen elicits an immune response in the sition through the skin. In some embodiments, the pre subject. In some embodiments, mRNAs comprise a 7-meth treatment is a physical disruption of the skin barrier. In some ylguanosine cap (i.e., a 5' cap). In some embodiments, embodiments, the pre-treatment includes microdermabra mRNAS comprise a polyadenylation tail. In some embodi Sion, exfoliation, thermal ablation, chemical ablation, laser ments, mRNAs comprise a 5' untranslated region (5' UTR), treatment, electric currents, electroporation, Sonophoresis, a 3' untranslated region (3' UTR), or both. and the like. In some embodiments, the pre-treatment is an I0123. In some embodiments, the oral composition com application of a chemical agent. In some embodiments, the prises an RNA replicon. In some embodiments, RNA rep pre-treatment is a combination of physical disruption and a licons are any RNA replicon as disclosed herein. In some chemical agent. In some embodiments, the pre-treatment is embodiments, RNA replicons are derived from picornavirus, a common cosmetic procedure performed before delivery of flavivirus, coronavirus, pestivirus, rubivirus, calcivirus, the bioactive agent. In some embodiments, the pre-treatment hepacivirus, or alphavirus . In some embodiments, RNA is a facial. In some embodiments, the pre-treatment is replicons comprise a coding sequence or coding region that cleaning and/or disinfecting the Surface to be treated. In encodes a polypeptide. In some embodiments, the polypep Some embodiments, a Subject undergoing cosmetic treat tide is any polypeptide as disclosed herein. In some embodi ment with a bioactive agent receives treatment at a derma ments, the polypeptide is an antigen Suitable for use as a tologists office. In some embodiments, treatment comprises vaccine. Examples of antigens Suitable for use as vaccines administering a microneedle composition comprising a bio are provided herein. In some cases, the antigen is a foreign active agent (e.g., a recombinant alphavirus replicon) after antigen. In some embodiments, the foreign antigen is any pretreatment of the surface to which the microneedle com foreign antigen disclosed herein. In some embodiments, the position is applied. In some embodiments, the pretreatment foreign antigen is an antigen associated with influenza virus comprises wiping the Surface to be treated with a wipe, (e.g., hemagglutinin (HA) or neuraminidase (NA)). In some Swab, or other material. In some embodiments, the wipe, embodiments, the antigen is a self-antigen (i.e., an antigen Swab, or other material is Supplied in a prepackaged form. associated with cancer). In some embodiments, any antigen 0120 In some embodiments, the treatment comprises that is encoded by a replicon is delivered to a subject. In administering a microneedle composition comprising a BTX Some embodiments, the antigen elicits an immune response polypeptide for the treatment of wrinkles and frown lines. In in the subject. Some embodiments, treatment is coupled with another cos 0.124. In some embodiments, the oral composition com metic procedure, for example, a facial. In some embodi prises one or more replicon molecules encoding one or more ments, the facial facilitates delivery of the replicon. In some polypeptides. In some embodiments, the one or more rep embodiments, the Subject receives the facial as a primary licon molecules encode one or more, two or more, three or US 2017/O 196966 A1 Jul. 13, 2017 27 more, four or more, five or more, six or more, seven or more, RNA replicons encapsulated in liposomes suitable for oral eight or more, nine or more, ten or more, or even more delivery to a subject. Without wishing to be bound by theory, polypeptides. In some cases, one polypeptide is encoded by the liposomes of the composition can bind to and penetrate one replicon molecule. In other cases, more than one poly the columnar epithelia of the intestines and release the RNA peptide is encoded by a single replicon molecule. In some replicon “payload' into the cytoplasm. In some embodi embodiments, the oral composition is a quadrivalent influ ments, the replicon is then translated into antigen polypep enza vaccine comprising a plurality of replicon molecules tides to evoke an immune response. encoding hemagglutinin (HA) derived from four different 0129. In some cases, the compositions comprise Small influenza virus subtypes or strains. However, this disclosure nucleic acid lipid particles (SNALPs). In some embodi is not limited to delivering vaccines. In some embodiments, ments, SNALPs are microscopic particles (~120 nm in a replicon encoding essentially any polypeptide as described diameter) that are used to deliver nucleic acids therapeuti herein is used in oral compositions (e.g., for gene therapy). cally to subjects. In some embodiments, SNALPs comprise 0.125. In some cases, the oral composition comprises viral any mix of cationic and fusogenic lipids. In some embodi vectors. In some embodiments, viral vectors are used to ments, SNALPs are coated with diffusible polyethylene deliver an RNA or DNA sequence that encodes an antigen. glycol (PEG). In some embodiments, SNALPs are used to In some embodiments, viral vectors are derived from a encapsulate the polynucleotides of the present disclosure variety of viruses, of which non-limiting examples include and deliver the polynucleotides to a subject. In some lentiviruses, retroviruses, adenoviruses, adeno-associated embodiments, a SNALP is a liposome. viruses (AAV), rhabdoviruses (e.g., vesicular stomatitis 0.130. In some embodiments, compositions as described virus), poxviruses (e.g., fowlpox virus, avian poxvirus, or herein comprise a Solid formulation. In some embodiments, vaccinia), alphavirus es (e.g., VEE. Sindbis, or SFV), and non-limiting examples of solid formulations include a tablet, the like. In some embodiments, the viral vector is derived a capsule, or a pill. In some embodiments, Solid formulations from vesicular stomatitis virus (VSV). In some embodi are Suitable for oral administration of the composition to a ments, viral vectors are derived from attenuated viruses such subject in need thereof. In some embodiments, slow release that the viral vector is replication defective. In some embodi formulations for oral administration are prepared in order to ments, the viral vector exhibits tropism such that specific achieve a controlled release of the polynucleotides in contact tissues or cell types Support the growth of the virus and other with the body fluids in the gastrointestinal tract, and to tissues or cell types do not support the growth of the virus. provide a substantial constant and effective level of the In some embodiments, the viral vector is modified to expand polynucleotides in the blood plasma. In some embodiments, the tropism of the virus. In some embodiments, the viral the polynucleotides are embedded for this purpose in a vector is modified to limit the tropism of the virus. In some polymer matrix of a biological degradable polymer, a water embodiments, the tropism is a particular species (e.g., a soluble polymer or a mixture of both, and optionally suitable virus that can only infectavian cells). In some embodiments, Surfactants. In some embodiments, embedding is the incor the tropism of the virus is matched to the vaccine and poration of micro-particles in a matrix of polymers. In some consideration is made as to the species being vaccinated and embodiments, controlled release formulations are obtained the desired target tissue or cell type. through encapsulation of dispersed micro-particles or emul sified micro-droplets via known dispersion or emulsion Formulations of Oral Compositions coating technologies. In some embodiments, the polynucle 0126. In some embodiments, oral compositions are otide of the composition is formulated as a capsule. In some designed to be compatible with the environment of the embodiments, the polynucleotide of the composition is a gastrointestinal system. In some embodiments, oral compo Solid or liquid formulation encapsulated by the capsule. sitions are limited by a number of factors that affect oral 0.131. In some embodiments, the formulation of the com bioavailability, including poor solubility, low permeability, position is enteric-coated. In some cases, a dried polynucle instability, and rapid metabolism. In some embodiments, otide composition is formulated as an enteric-coated cap these factors, among others, are considered when producing Sule. In some embodiments, a dried polynucleotide an oral composition of the disclosure. composition is formulated as an enteric-coated tablet. With 0127. In some embodiments, oral compositions of the out wishing to be bound by theory, enteric-coating can disclosure comprise any polynucleotide as disclosed herein. protect the composition from the acidic gastric juices of the In some embodiments, polynucleotides are “naked’ (i.e., stomach, can prevent irritation of the gastric mucosa, can Substantially free of other bioactive agents, excipients, and delay the onset of action of the composition, and can target the like). In some embodiments, an oral composition com the release of the composition to the Small intestine. In some prises naked RNA replicons. In some embodiments, the embodiments, the enteric-coated formulation is a coated naked polynucleotides are in a Solid formulation. In some tablet, a Sugar-coated tablet, a soft gelatin capsule, a hard embodiments, the naked polynucleotides are precipitated gelatin capsule, a granulate, or a pellet. Non-limiting (e.g., with PEG) and dehydrated to form a dry powder. In examples of enteric coatings suitable for use include: enteric Some embodiments, the naked polynucleotides are in a film formers, for example, polymethacrylates (e.g., meth liquid formulation. In some embodiments, the polynucle acrylic acid ethacrylate poly (MA1 -EA 1), methacrylic acid otides are complexed with dendrimers, e.g., G5 and G9 methyl methacrylate poly (MA 1 -IMMA 1) and poly (MA dendrimers (Dendritech). Any suitable dendrimer, such as 1 -MMA2)), cellulose-based polymers (e.g., cellulose those described herein, are contemplated. acetate phthalate, cellulose acetate trimellitate, cellulose 0128. In some embodiments, the polynucleotides are acetate Succinate, hydroxypropylmethylcellulose phthalate, encapsulated in liposomes. In some embodiments, methods hydroxypropylmethylcellulose acetate Succinate), polyvinyl of encapsulating polynucleotides in liposomes are described derivatives (e.g., polyvinyl acetate phthalate), half esters of herein. In some embodiments, the composition comprises the copolymerisate of styrene and maleic acid, half esters of US 2017/O 196966 A1 Jul. 13, 2017 28 the copolymerisate of vinyl ether and maleic acid, copoly mg/kg, about 900 mg/kg, about 920 mg/kg, about 940 merisate of vinyl acetate and crotonic acid; plasticizers, for mg/kg, about 960 mg/kg, about 980 mg/kg, about 1000 example, alkyl esters of citric, tartaric and sebacic acids mg/kg, or greater than 1000 mg/kg. (e.g., diethyl sebacate, triethylcitrate, tributyl citrate, acety I0135) In some embodiments, the composition is admin itriethylcitrate, acetyltributyl citrate, dibutyl tartrate), esters istered orally once or more than once. In some cases, the of phthalic acid (e.g., dimethyl phthalate, diethyl phthalate, composition is administered orally as a single dose. In other clibutyl phthalate, dioctyl phthalate, ethylphthaloyl- and cases, the composition is administered orally as two, three, butylphthaloyl ethyl glycolate), glycerol esters (e.g., castor four, five, six, seven, eight, nine, ten, or ten or more doses. oil, sesame oil, acetylated fatty acid glycerides, glycerol In some embodiments, the doses are administered orally diacetate, glycerol triacetate), higher alcohols (e.g., glycerol, once a day, twice a day, three times a day, four times a day, 1.2-propylene glycol), polyethers (e.g., polyethylene glycols five times a day, six times a day, seven times a day, eight and polyoxyethyiene polyoxypropylene block copolymers), times a day, nine times a day, ten times a day, or more than surfactants (e.g., PEG-400 stearate, PEG sorbitane ten times a day. In some cases, the doses are administered monooleate, Sorbitane monooleate); anti-adhesion agents once a day for one day, two days, three days, four days, five (e.g., talcum, magnesium Stearate, micronized amorphous days, six days, seven days, eight days, nine days, ten days, silicic acid, kaolin); colorants and pigments (e.g., titanium or more than ten days. In some embodiments, the doses are oxide, iron oxide pigments); and other additives. administered consecutively (i.e., on consecutive days) or are 0.132. In some embodiments, the compositions of the separated by days in which no dose is administered. In some present disclosure further comprise any number of excipi cases, the doses are administered once a week, once every ents. In some embodiments, excipients include any and all two weeks, once every three weeks, once every month, once Solvents, coatings, flavorings, colorings, lubricants, disinte every two months, once every three months, once every four grants, preservatives, Sweeteners, binders, diluents, and months, once every five months, once every six months, vehicles. In some embodiments, the excipient is compatible once every seven months, once every eight months, once with the therapeutic compositions of the present disclosure. every nine months, once every ten months, once every In some embodiments, the excipient comprises vitamin B12, eleven months, once every year, or once more than every folate, or a combination thereof to facilitate uptake of the year. composition from the Small intestine. In some cases, the 0.136. In some embodiments, the compositions are excipient comprises polyethylene glycol (PEG). In some administered as a “prime-boost regimen. In some embodi cases, the excipient comprises PEG derivatized with vitamin ments, a “prime-boost regimen as defined herein is any B12 (PEG/B12). In some embodiments, PEG and PEG/B12 vaccination regimen in which the immune system is is used to precipitate the polynucleotides of the disclosure. “primed to a target antigen and the immunity is selectively In some embodiments, the use of excipients in pharmaceu boosted by re-administering the target antigen. In some tical compositions is well known in the art. embodiments, the prime-boost is heterologous (i.e., a first vector is used to administer the target antigen (“prime') and Dosage and Administration a second, distinct vector is used to administer the target 0133. In some embodiments, suitable doses of formula antigen (“boost)). In some embodiments, the prime-boost is tions of the disclosure are administered orally to a subject in homologous (i.e., the first and second vector are the same). need thereof. In some embodiments, the composition is In some embodiments, the “boost requires multiple doses administered with food. In some cases, a subject is in need of any number of distinct vectors. In some embodiments, the or want of the formulation. In some embodiments, a subject prime-boost regimen comprises a “prime' with a first vector, in need or want of the formulation is a subject in need or followed by a “boost with a second vector, followed by want of a vaccine. another "boost with a third vector. 0134. In some embodiments, a therapeutically effective 0.137 In some embodiments, the heterologous prime amount of a polynucleotide of the disclosure is expressed as boost comprises two or more distinct vectors. In some mg of the polynucleotide per kg of Subject body mass. In embodiments, the prime-boost comprises an alphavirus rep Some instances, a therapeutically effective amount is about licon encoding a target antigen to prime the immune system, 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, followed by a VSV viral vector encoding the same target about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 antigen to boost the immunity. Any combinations of distinct mg/kg, about 9 mg/kg, about 10 mg/kg, about 20 mg/kg, vectors, including any described herein, are contemplated about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 for use in a heterologous prime-boost strategy. In some mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, embodiments, the target antigens are the same. In some about 100 mg/kg, about 120 mg/kg, about 140 mg/kg, about cases, the target antigens are different. In some cases, both 160 mg/kg, about 180 mg/kg, about 200 mg/kg, about 220 vectors are administered by the same route of administra mg/kg, about 240 mg/kg, about 260 mg/kg, about 280 tion. In some embodiments, a first vaccine and a second mg/kg, about 300 mg/kg, about 320 mg/kg, about 340 vaccine are both delivered orally. In some embodiments, the mg/kg, about 360 mg/kg, about 380 mg/kg, about 400 first vaccine and second vaccines are administered by dif mg/kg, about 420 mg/kg, about 440 mg/kg, about 460 ferent routes of administration. In some embodiments, the mg/kg, about 480 mg/kg, about 500 mg/kg, about 520 first vaccine is administered orally to prime the immune mg/kg, about 540 mg/kg, about 560 mg/kg, about 580 system, followed by microneedle injection of the second mg/kg, about 600 mg/kg, about 620 mg/kg, about 640 vaccine to selectively boost the immunity. In some embodi mg/kg, about 660 mg/kg, about 680 mg/kg, about 700 ments, the first vaccine is administered by microneedle mg/kg, about 720 mg/kg, about 740 mg/kg, about 760 injection followed by oral administration of the second mg/kg, about 780 mg/kg, about 800 mg/kg, about 820 vaccine. Non-limiting examples of administration routes mg/kg, about 840 mg/kg, about 860 mg/kg, about 880 suitable to perform the methods of the disclosure include US 2017/O 196966 A1 Jul. 13, 2017 29

Subcutaneous injection, intravenous injection, intramuscular cases, the affinity-tagged HA and/or NA protein standards injection, intradermal injection, intraperitoneal injection, are used as antigens to rapidly produce HA and/or NA oral administration, intranasal administration, and infusion. antibody standards. Methods of producing polyclonal anti In some cases, the first and second vectors are the same but bodies in animals (e.g., rabbits) are well known in the art. are delivered by different routes of administration. In other 0.141. In some embodiments, the protein and antibody cases, the first and second vectors are different but are standards described herein are further utilized in an enzyme delivered by the same route of administration. In yet other linked immunosorbent assay (ELISA). Methods of produc cases, the first and second vectors are different and are ing and performing ELISA assays are commonly known in delivered by different routes of administration. the art. In some embodiments, the ELISA assays are used to 0.138. In some embodiments, prime-boost regimens quantify the amount of HA and/or NA antigen synthesized in encompass a period of time between delivery of the first cell culture in response to an influenza vaccine of the present vaccine and delivery of the second vaccine. In some embodi disclosure. In some embodiments, other methods of quan ments, delivery of the first vaccine and delivery of the tifying antigens and/or antibodies are known in the art and second vaccine occur on the same day (e.g., at the same include, without limitation, single radial immunodiffusion hospital visit). In some embodiments, the second vaccine is assay (SRID), Surface plasmon resonance detection, and delivered on the same day or one day, two days, three days, Titer-on-Chip (Flu-ToC). four days, five days, six days, one week, two weeks, three 0142. In some embodiments, the protein and antibody weeks, one month, two months, three months, four months, standards described herein, in some cases, are used to five months, six months, seven months, eight months, nine develop a hemagglutination inhibition assay. In some months, ten months, eleven months, one year, two year, embodiments, the ability of a specific strain of influenza three years, four years, five years, ten years, or more than ten virus to agglutinate red blood cells is tested. In some years after delivery of the first vaccine. embodiments, the influenza virus is added to a microwell plate comprising a plurality of red blood cells. Without Methods for Producing Influenza Vaccines wishing to be bound by theory, hemagglutinin (HA) present on the surface of the influenza virus can bind to N-acetyl 0139 Disclosed herein, in some embodiments, are meth neuraminic acid on the Surface of the red blood cells causing ods for producing influenza virus vaccines. Current stan the virus to stick to the cells and form a lattice-like structure. dards in influenza virus vaccine production require months In some cases, an HA antibody generated by any antigen or to manufacture an influenza vaccine. In some embodiments, vaccine disclosed herein is used to prevent binding of the the methods described herein reduce the time required to influenza virus to the red blood cells (and prevent aggluti manufacture an influenza vaccine. In some cases, the nation). In some embodiments, the minimum concentration improved methods require only two or three weeks to of HA antibody needed to inhibit binding of the virus to the manufacture a new influenza vaccine. In some embodi cells determines the titer of the virus. In some embodiments, ments, one rate-limiting step in the manufacture of an the HA antibody is any HA antibody produced by the influenza vaccine is the time required for antibody standards methods of the present disclosure. In some cases, the HA showing strain-specific hemagglutinin (HA) immunoreac antibody is derived from blood serum from a subject inocu tivity to be produced. lated with a composition of the present disclosure (i.e., an 0140. In some aspects, the disclosure provides an HA antigen). improved method for producing strain-specific HA or 0143. In some aspects, the disclosure provides for meth neuraminidase (NA) protein standards. In some cases, the ods of gene profiling. In some embodiments, gene profiling HA and/or NA protein standards are produced in an in vitro is used to identify biomarkers of an immune response in a coupled transcription-translation system. In vitro transcrip Subject. In some embodiments, a Subject undergoing vacci tion-translation systems are known in the art and can be nation with a composition of the present disclosure is purchased as kits commercially (e.g., from Promega). In screened for biomarkers indicative of an immune response. some embodiments, the strain-specific HA and/or NA pro In some embodiments, gene profiling is performed on tein standards are produced with an affinity tag at the peripheral blood cells prior to vaccination as well as post C-terminal end of the protein. In some embodiments, affinity vaccination. In some embodiments, gene profiling is per tags are known in the art and include, without limitation, formed on peripheral blood cells prior to vaccination, 1-day chitin binding protein (CBP), maltose binding protein post-vaccination, 3-days post-vaccination, and 7-days post (MBP), glutathione S-transferase (GST), and poly Histidine tag (HIS). In some embodiments, the affinity tag is used to vaccination. In some cases, gene profiling is performed on a purify the HA and/or NA protein standards. In some embodi Subject inoculated with a composition of the present disclo ments, the HA and/or NA proteins comprise a HIS tag Sure. In some embodiments, the Subject is a human. In some appended to the C-terminus. In some embodiments, the cases, the gene profiling is performed to assess the efficacy HIS-tagged HA and/or NA proteins are purified by binding of a new influenza vaccine as part of e.g., a clinical trial. the HIS-tagged proteins to e.g., a nickel-affinity column or nickel magnetic agarose beads. In some embodiments, Kits recombinant affinity-tagged HA and/or NA proteins are 0144. Disclosed herein, in some embodiments, are kits produced by any protein engineering methods commonly for producing and/or administering a bioactive agent, Such known in the art. In some cases, the recombinant affinity as a recombinant alphavirus replicon encoding an exog tagged HA and/or NA proteins are produced in vitro, e.g., in enous polypeptide. In some embodiments, a kit comprises bacterial cells. In some embodiments, the HA and/or NA one or more of any of the compositions disclosed herein, in protein standards are used to quantitate the amount of HA any Suitable combination. In some embodiments, the kit and/or NA antigen synthesized in cell culture in response to comprises materials for packaging the bioactive agent into an influenza vaccine of the present disclosure. In some or on a microneedle, materials for generating RNA nano US 2017/O 196966 A1 Jul. 13, 2017 30 particles (e.g., dendrimers), materials for dehydrating the GeneBank. Recombinant alphavirus replicons that encode bioactive agent (before or after application to a an exogenous polypeptide were designed as illustrated in microneedle), and/or materials for encapsulating the FIG. 7. Briefly, DNA sequences that encode for either alphavirus replicon in liposomes. In some embodiments, the enhanced green fluorescent protein (EGFP), influenza kit comprises a composition in accordance with an embodi hemagglutinin protein (HA-see, e.g., GenBank Accession ment of the disclosure in combination with instructions for No.: KFO09554.1), or hepatitis B virus surface antigen administering the composition to a subject. In some embodi (HBSAg—see, e.g., GenBank Accession No.: KP659247.1; ments, a kit comprises a tool for administering the compo 155-835) were fused downstream (i.e., in the 3' direction) of sition to a Subject. In some embodiments, the composition is an alphavirus replicon cassette containing the four alphavi provided in any Suitable form, such as forms ready for immediate use or forms that require reconstitution by mix rus non-structural proteins (nsP1, nsP2, nsP3, and nsP4). An ture with other materials (whether supplied in the kit or by empty replicon cassette (i.e., containing only the alphavirus the user). non-structural proteins and no gene-of-interest) was also 0145. In some embodiments, materials suitable for pro designed. 5' and 3' untranslated regions (5' UTR and 3' UTR ducing an alphavirus replicon include, without limitation, respectively) were also included in the replicon constructs to any combination of the following: a synthesized template facilitate expression in host cells. The recombinant alphavi DNA molecule comprising a gene encoding an exogenous rus replicons were inserted into the puC57-Kan-T7 vector, polypeptide, an alphavirus replicon derived from any Suit as shown in FIG. 3 (pUC57-Kan-T7-VEEV-EGFP), FIG. 4 able alphavirus strain, an RNA-dependent RNA polymerase (pUC57-Kan-T7-VEEV-HA), FIG. 5 (pUC57-Kan-T7 (e.g., T7 RNA Polymerase), buffers, primers, NTPs, restric VEEV-HBs.Ag), and FIG. 6 (pUC57-Kan-T7-VEEV). tion enzymes, RNA ligases, ribonuclease inhibitors, and the 0150 DNA was synthesized and cloned by Genewiz like. (South Plainfield, N.J.) or DNA 2.0 (Newark, Calif.). DNA 0146 In some embodiments, material suitable for pack MiniPreps were prepared using the ZyppyTM Plasmid Mini aging the alphavirus replicon into or on microneedles include, without limitation, any of the following: a Prep Kit Cat #D4036 (Zymo Research, Irvine, Calif.). DNA microneedle or materials to produce a microneedle (e.g., Maxipreps were performed using the PureLinkTM Hi Pure polymer, molds, and the like), materials to dehydrate a Plasmid Maxiprep Kit (Invitrogen Cat #8002708). DNA replicon composition, and the like. sequencing was performed by the UC Davis DNA sequenc 0147 In some embodiments, kits contemplated herein ing facility using the ABI Prism R 3730 Genetic Analyzer include kits which include a composition comprising an and BigDyeR Terminator v. 3.1 Cycle Sequencing Kit with alphavirus replicon encoding an exogenous polypeptide to a Gel Company Better Buffer. Any suitable gene-of-interest Subject, wherein the composition is administered orally, and can be designed and synthesized as a recombinant alphavi instructions for use of the same. In some embodiments, kits rus replicon as described above. include a first composition comprising an alphavirus repli con encoding an exogenous polypeptide by a first route of Example 2 administration and a second composition comprising an alphavirus replicon encoding an exogenous polypeptide by a second route of administration, as well as instructions for In Vitro Synthesis of Recombinant Alphavirus use of the same. In some embodiments, the first and second Replicon RNA routes are the same. In some embodiments, the first and second routes are different. In some embodiments, the first 0151. Plasmid DNA described in Example 1 was linear route of administration is oral administration and the second ized 3' to the insert with Ndel or Mlul (New England route of administration is intradermal administration. In BioLabs). RNA was synthesized in vitro from the linearized Some embodiments, kits include compositions packaged in DNA template using the MEGAScript(R) Kit according to a microneedle when intradermal administration is contem manufacturers instructions (Ambion Cat it AM1333). RNA plated. was purified according to manufacturers instructions with the MEGAclearTM Kit for purification of large scale tran EXAMPLES scription reactions (Ambion Cat #1908). A 5' cap was added to the RNA using the Vaccinia Capping System (New 0148. The following examples are given for the purpose England BioLabs Cat #M20805). RNA was purified again of illustrating various embodiments of the invention and are using the MEGAclearTM Kit according to manufacturers not meant to limit the present invention in any fashion. The instructions. In some instances, Trillink (San Diego, Calif.) present examples, along with the methods described herein synthesized replicon RNA constructs from the appropriate are presently representative of preferred embodiments, are DNA template. Various methods for in vitro transcription of exemplary, and are not intended as limitations on the scope RNA from plasmid DNA are available and any suitable of the invention. Changes therein and other uses which are methodology can be employed to generate the recombinant encompassed within the spirit of the invention as defined by alphavirus replicons described herein. the scope of the claims will occur to those skilled in the art. 0152 1 ug of replicon RNA was diluted to 0.2g/uL and Example 1 further diluted with an equal Volume of sample loading dye, and run on a 1.0% agarose gel until the RNA bands were Recombinant Alphavirus DNA Constructs fully resolved. RNA from the T7-VEEV replicon (nsP1-4 only), T7-VEEV-EGFP replicon, and T7-VEEV-HA repli 0149 DNA constructs were designed using DNASTAR con (influenza A strain A/California/7/2009 H1N1) are Lasergene Software (Madison, Wis.) with sequences from shown in FIG.8. US 2017/O 196966 A1 Jul. 13, 2017

Example 3 timepoints. Native EGFP (produced by the transfected EGFP replicons described above) is a 238 amino acid, 26.9 Transfected Replicon RNA Induces Protein kDa protein while Biovision commercial EGFP (positive Expression. In Vitro control, lane 1, FIG.9) is 293 amino acid, 32.7 kDa protein. The 24 and 48 hour lysis buffer only samples (negative Tissue Culture Methods control, lanes 2 and 5. FIG.9) did not contain any detectable EGFP bands. 0153. HEK-293T cells were provided by Dr. Peter Barry 0158 HEK-293T cells transfected with either 1 ug or 2 (UC Davis). Cells were grown in 5% CO at 37° C. using ug (data not shown) of influenza A HA-replicon expressed Dulbecco's High Glucose MEM (Hyclone Cat #5H30022. readily detectable amounts of HA protein at both the 24 hour 01) with 10% Fetal Calf Serum (Gibco Cat #2140-087) and and 48 hour timepoints. As shown in FIG. 10, a full length 50 U/ml Penicillin and 50 lug/ml Streptomycin. Cells were 72 kDa HA protein band (positive control, lane 2, FIG. 10) transfected with the HA or EGFP RNA replicons described is clearly visible. While the anti-HA antibody exhibits some in Example 2 using the StemfectTM RNA Transfection Kit cross-reactivity, there is no corresponding 72 kDa band seen according to manufacturers instructions (Stemgent, Cam in the buffer only negative control (lane 3, FIG.10). Two bridge, Mass.). different lysis buffers were tested on HA-replicon trans fected samples (lanes 3-6, FIG. 10) and, as indicated by the Western Blots arrow in FIG. 10, all samples analyzed had the 72 kDa HA 0154) HEK-293T cells were transfected in a 6-well plate band present. with 1 lug or 2g of HA-replicon or EGFP replicon and incubated for 24 hours or 48 hours at 37° C. in 5% CO. EGFP Fluorescence Assay After the incubation period, cells were washed carefully 0159 HEK-293T cells were seeded in a 6-well plate and with warm HBSS and harvested in 250 ul 1x lysis buffer. allowed to come to -70% confluency after 24 hours. Media Lysates were incubated on ice for 20 min, sonicated for 1 was then replaced and cells were transfected using Stem min, and then centrifuged for 10 min at 14,000 rpm. The fectTM with either 0, 0.5, 1, 2, or 4 pg of an EGFP RNA Supernatant was transferred to clean tubes and the protein replicon as described in Example 2. The remaining well was concentration determined using the Quant-iTTM Protein transfected with lug EGFP mRNA as a positive control. Assay Kit (Invitrogen) according to manufacturers instruc After a 24 or 48 hour incubation period, media was removed tions. and 0.5 mL of Cell Lysis Buffer was added to lyse cells. Cell 0155 Samples for gel electrophoresis were prepared in lysates were analyzed for fluorescence using the Cubit two concentrations, 20 ug/15ul and 40 g/15ul. For sample fluorimeter according to manufacturers instructions. preparation, Supernatant aliquots were diluted with ddH2O (0160. As seen in FIGS. 11 and 12, transfection of the and mixed with 4x Laemmli loading buffer, followed by T7-VEEV-EGFP replicon leads to robust expression of heating for 20 minutes at 60° C. for denaturation. The EGFP. When the fluorescence of similar amounts of samples (15ul per lane) and a ladder (5ul) were then loaded T7-VEEV-EGFP RNA and EGFP mRNA are compared, the onto a 4-20% TGX gradient gel (Biorad) and run for 30 T7-VEEV-EGFP replicon produces roughly 3.5 times as minutes at 200V. After electrophoresis, the separated pro much EGFP compared to an EGFP mRNA (FIG. 12). At 48 teins were blotted onto a nitrocellulose membrane by tank hours post transfection, cells transfected with the T7-VEEV (wet) electrotransfer. The gel, filter paper, and membrane EGFP replicon showed increased EGFP production suggest were first equilibrated in transfer buffer and then were placed ing mRNA was still being synthesized (FIG. 11). Cells in the “transfer sandwich filter paper-gel-membrane-filter transfected with EGFP mRNA show similar levels of EGFP paper, cushioned by pads, and pressed together by a Support fluorescence at 24 and 48 hours (FIG. 11). grid. The Supported gel sandwich was placed vertically in a tank between stainless steel/platinum wire electrodes and Example 4 filled with transfer buffer. The transfer was performed at 100V for 1 hour. After transfer, the nitrocellulose membrane Coating and Elution of EGFP mRNA from BioDot was stained with Ponceau S to confirm transfer. Printed Microneedle 0156 After destaining in ddH2O, the membrane was (0161 EGFP mRNA was transferred onto 5x5 blocked for 1 hour in 5% non-fat milk powder in 1XTBS, microneedle arrays using two methods: (1) dipping; and (2) followed by incubation with mouse-anti-HA antibody using the microfluidic dispensing BioDot printer. (1:1000) or mouse anti-EGFP antibody in 0.5% nonfat milk powder in 1x TBS for 1 hour at room temperature on a Dipping Method rocking shaker. The membrane was then washed three times for 5 minutes each in 1x TBS/0.05% Tween-20 and then 0162 Arrays were first sonicated for ten minutes fol incubated with HRP-conjugated anti-mouse antibody lowed by baking at 450° C. for one hour. Following steril (1:5000) in 0.5% nonfat milk powder in 1x TBS for 1 hour ization, 3 arrays were floated (dipped) on a 100LL globule of at room temperature on a rocking shaker, followed by three 0.1 mg/mL EGFP mRNA in DEPC treated ddHO on washes. Finally, the membrane was incubated with 1-Step parafilm for 30 minutes. Following coating, arrays were TMB-Blotting substrate (Thermo Scientific) for 30 minutes allowed to dry at ambient temperature. Three more arrays at room temperature. were coated using the BioDot printer. O157. As shown in FIG. 9, HEK-293T cells transfected with either lug or 2g of EGFP-replicon expressed readily BioDot Printing detectable amounts of EGFP protein at both the 24 hour 0163 Microneedle arrays were first cleaned by sonica (lanes 3 and 4, FIGS. 9) and 48 hour (lanes 6 and 7, FIG.9) tion at room temperature for 11 minutes at power setting 9 US 2017/O 196966 A1 Jul. 13, 2017 32 in a Steris Reliance Ultrasonic Cleaning System. Following glycero-3-phosphoethanolamine-Nimethoxy(polyethylene sonication, arrays are either heat sterilized for 1 hour at 171° glycol)-2000 (Avanti Polar Lipids) were combined in etha C. or siliconized. For siliconization, clean microneedle nol. RNA was diluted with DNase/RNase-free, endotoxin arrays are incubated for 20 seconds in a solution of 0.1% free water (Invitrogen) and sterile 100 mM (pH 3.0) QB Dow Corning MDX4/2.5% Stoddard solvent/97.5% Isopro Citrate Buffer (Teknova) or 100 mM. Na Acetate (pH4.0) to pyl alcohol, removed from the solution, dried for 1 hour at a final citrate oracetate concentration of 10 mM. The ethanol room temperature, and cured over night at 60° C. Following and aqueous streams were loaded into a Nano Assemblr siliconization, arrays are heat sterilized as described previ Microfluidic Cartridge and mixed in a 1:3 volumetric ratio ously and are now ready for printing. with a combined flow rate of 5.0 ml/minute to produce (0164. The BioDot (AD1520) system allows for the fab nanoparticles. Nanoparticles were also prepared with poly rication of mRNA microneedles with high levels of repro ethleneimine (PEI, Sigma Aldrich) in the ethanol phase. ducibility due to the accurate printing Volumes. The instru Nanoparticles were dialyzed against PBS using 20,000 ment utilizes a moveable stage holding a ceramic needle molecular weight cut-off Slide-A-Lyzer G2 dialysis cas (orifice 75-190 um) which accurately picks up pre-deter settes. Dialyzed nanoparticles were concentrated with Ami mined Volumes of reagent (mRNA) and then ejects (prints) con spin filters and sterile-filtered using 0.2 Lum poly(ether nanoliter Volumes on microneedle arrays positioned on a sulfone) filters (Genesee Scientific). Nanoparticles were printing table. characterized with a Zetasizer NanoZS (Malvern); see Lipid 0.165. The BioDot undergoes a wash step prior to mRNA Nano Particle and Lipid Nano Particle #2 in FIGS. 14 and printing including a number of aspiration and elution steps 15. with 0.01% PBS to ensure removal of any air bubbles in the needle. Then, 5ul of mRNA is aspirated into the ceramic Example 6 needle and printed on microneedle arrays by repeatedly ejecting 5 nL drops on each microneedle, with a drying time Formulation of RNA Replicon Dendrimer of 60-120 seconds between prints. Following printing and Nanoparticles drying, the microneedles are bent upwards in a 90° angle and are ready for use. A total of 40 nL of 1.0 mg/mL EGFP (0168 G5 and G9 NH, PAMAM dendrimers were from mRNA in Tris Buffer was coated onto each needle of the 5x5 Dendritech. EGFP-replicon dendriplexes were formed at an array 5nL at a time (1 lug total mRNA for the entire array). N/P ratio of 20 (N from the dendrimer, P from the replicon Arrays were allowed to dry at ambient temperature. RNA). Size (FIGS. 13 and 14) and polydispersity index RNA Elution from Coated Microneedles (PDI) (FIG. 15) was determined for naked RNA, the den 0166. After coating and drying, arrays were floated on a drimer (G5 and G9 respectively), and the corresponding globule of 100LL DEPC treated ddHO on parafilm for 5, dendrimer nanoparticle (G5+replicon RNA and G9+replicon 15, and 30 minutes with mild shaking. Samples were col RNA). As shown in FIGS. 13 and 14, both G5 and G9 lected from the arrays at each interval and kept on ice for dendrimers dramatically reduced the size of RNA molecule. further processing. After all samples were collected, an aliquot of each sample was tested for the presence of RNA Example 7 using the cubit fluorimeter. The remaining samples were utilized for quantitative RT-PCR analysis. As seen in Table In Vivo Detection of EGFP Fluorescence in Mice 1 and FIG. 16, recovery of EGFP mRNA from BioDot Treated with an EGFP Protein Coated Microneedle printed samples, was consistent and robust throughout the Device three elution times measured. 0169 Microneedle arrays were prepared as described in Example 8. EGFP protein was coated onto microneedle TABLE 1. array using the BioDot microfluidic dispensing device as EGFP mRNA Recovery from Dipped vs. BioDot descried in Example 4. The dorsal skin hair of Balb/c mice Printed Microneedles was removed as described in Example 8, and the EGFP protein coated microneedle patch was applied to the exposed Coating RT-PCR skin. After a 20 minute incubation period, the presence of Sample Method Time (min) Cubit (ng. IL) (ng LL) EGFP protein was visualized by fluorescence. Localization 1 BioDot 5 2.52 7.9 of EGFP from an array patch applied to a mouse is exem 2 BioDot 15 2.04 7.75 3 BioDot 30 2.04 7.54 plified in FIG. 21. 4 Dip 5 6.12 24.75 5 Dip 15 4.46 17.18 Example 8 6 Dip 30 1.6 7.95 In Vivo Production of Anti-EGFP Antibodies in Mice Treated with a Replicon-RNA Coated Example 5 Microneedle Device Microfluidic Formulation of RNA Nanoparticles 0170 Microneedle arrays were made from stainless steel foil (SS304, 75um thick). The microneedle arrays contained 0167 Nanoparticles were also formulated using the 25 microneedles in a 5x5 grid pattern in a 1 cm. Needles Nano Assemblr microfluidic mixing device (Precision Nano and the array are illustrated in FIGS. 17 and 18 respectively. systems) using a staggered herringbone micromixer chip. Microneedles were manufactured by photolithography by Briefly, polyamidoamine (PAMAM) C12 dendrimers (Den Kemac, Azusa, Calif. Wells and hinge were half-etch 37 um dritech cat #53,685-7 or 53,687-3) and 1,2-dimyristoyl-sn deep. US 2017/O 196966 A1 Jul. 13, 2017

(0171 G5 and G9 NH, PAMAM dendrimers were from Screen for EGFP-Antibodies Dendritech (Midland, MI). EGFP-replicon dendriplexes as described in Examples 1 and 2 were formed at an N/P ratio (0175 Mouse sera from the day 28 blood draw were tested of 20 (N from the dendrimer, Pfrom the mRNA). Reactions for EGFP-antibodies. An ELISA plate was coated with were carried out at 100LL as per table below at room EGFP protein (2 ug/ml) in carbonate buffer over night at 4 temperature (RT) for 30 minutes. In brief, dendrimers were C. The plate was washed 3x with TBST (20mM Tris-HC1 diluted in nuclease-free water and Hepes buffer (final con pH 7.5, 500 mM. NaCl, 0.05% Tween 20) and blocked with centration 10 mM; pH 7.4) and replicon RNA was added. 5% BSA (bovine serum albumin) in TBS for 1 hour at room After 30 minutes, samples were placed in the BioDot AD temperature. After washing, mouse sera (1:100-1: 12500) 1520 printer loading tray and 5x5 welled microneedle arrays and positive control (1:500-1:12.500; anti-GFP-antibody, were printed 8 times with 5nL/well (0.6 ug of mRNA/array). Cell Signaling) in 1% BSA/TBST were added and incubated The printing solution was mixed by gentle pipetting between for 2 hours at room temperature, followed by washing. Next, arrays. anti-rabbit secondary antibody (for control) or anti-mouse secondary antibody (for sera) at 1:5000 in 1% BSA/TBST TABLE 2 was added for 1 hour at room temperature. The plate was washed again and then incubated with anti-SA (1:200) in 1% Replicon Dendrimer Reaction BSA/TBST for 20 minutes at room temperature. After Rep washing, substrate was added and incubated for 30 minutes Final licon at room temperature. The reaction was stopped by addition Treat- NP 19. Wol RNA 100 mM of 50u1 2N Sulfuric acid. ment Den Ratio mRNA (IL) L Den HEPES daHO (0176 A minor color reaction was visible at the 1:100 G5 GS 20.OO 6O.OO 100 6O.O. 10.1 1O.O 19.9 dilution of mouse antisera in most samples (including sera G9 G9, 20.OO 6O.OO 100 6O.O 8 1O.O 21.7 from untreated animals) and can be considered background binding from serum contents. Mouse #36 (EGFP-Rep/G5 0172 Immediately after each array was printed, needles dendrimer, N.P 20:1) showed a color reaction more than were bent to a 90° angle by hand using a probe and jig. Thus, double the background and mouse #39 (EGFP-Rep/G9 the needles are now in the Z plane at right angles to the SS dendrimer, N.P 20:1) showed a color reaction four-fold over foil sheet (FIG. 19). The completed microneedle arrays background. Thus the titer of EGFP antibodies by ELISA of containing replicon RNA with needles in the Z plane were mouse 36 was 1:200 and that of mouse 39 was 1:400 as placed on 2.5 cm diameter adhesive bandage (Curad) with shown in FIG. 22. EGFP-replicon RNA without dendrimers out pads, sealed in foil bags, and stored on dry ice until (“EGFP-Rep (3.2ug) Trilink”) resulted in no titer as shown applied to mice at the UC Davis Mouse Biology Program in FIG. 22. The positive control was a commercial anti (MBP). EGFP antibody (FIG. 22). 0173 6-8 week old female Balb/c mice were acquired from The Jackson Laboratory. After one week of habituation Example 9 to the vivarium, mice were lightly anesthetized with isoflu rane and hair removed over an area of the dorsal skin for patch application (lumbosacral and upper hind limb region) In Vivo Production of Anti-Influenza HA a minimum of 24 hours prior to the study. Hair removal Antibodies in Mice Treated with a Replicon-RNA consisted of removing thicker hair with electronic clippers Coated Microneedle Device followed by application of a depilatory cream (Nair Sensi tive Hair Remover Cream). The depilatory cream was 0177 Microneedle arrays are manufactured as described applied to the skin using a cotton Swab and left to set for in Example 8. Influenza HA-replicon RNA is prepared as 10-15 seconds before being wiped away with gauze. Nair described in Examples 1 and 2. G5 and G9 NH, PAMAM was not left on for longer than 15 seconds as it may cause dendrimers are from Dendritech (Midland, MI). HA-repli serious chemical burns. If additional applications were nec con dendriplexes are formed at a N/Pratio of 20 (N from the essary, Nair was applied only to the haired area and removed dendrimer, P from the RNA), as described in Example 8. after 5-10 seconds. After application of Nair, the entire skin HA-replicon RNA is then printed onto 5x5 welled area was washed with Saline to ensure that no residue microneedle arrays using a BioDot AD 1520 printer as remained on the skin to prevent irritation. Following hair described in Example 8. The completed microneedle arrays removal and recovery, array patches were applied and gently containing replicon RNA is placed on 2.5 cm diameter pressed on to the hairless area for 20 minutes (FIG. 20). The adhesive bandage without pads, sealed in foil bags, and animals were then single housed in duplex cages during the stored on dry ice until applied to mice. time of patch exposure and watched carefully to prevent the 0.178 The dorsal skin hair is removed from 6-8 week old animal from removing the patch. All animals were then female Balb/c mice as described in Example 8, and returned to group housing once the patch was removed. microneedle patches are applied to the hairless area for 20 0.174 Blood was collected from the lateral saphenous minutes. As a positive control, mice will be vaccinated using vein and sera were isolated and stored at -80° C. at 7, 14, a commercially available influenza vaccine (Flulaval(R) QIV and 21 days after application of the patch. Blood was 1X 5ML MDV 2016-2017 Season- GlaxoSmithKline). collected from alternate hind limbs each week to allow Blood is collected from the lateral saphenous vein and sera Sufficient healing and to maintain vessel integrity. At 28 days were isolated and stored at -80° C. at 7, 14, and 21 days after after patch application, mice were euthanized, blood was patch application of the patch. At 28 days after patch collected transcardially, and Sera was isolated and stored at application, mice are euthanized, blood is collected trans -80° C. cardially, and sera is isolated and stored at -80° C. The US 2017/O 196966 A1 Jul. 13, 2017 34 presence of anti-HA antibodies in the sera of microneedle Example 11 and control HA mice is measured by ELISA as described in Generating and Administering an Oral Influenza Example 8. Vaccine 0182 An oral vaccine designed to immunize a human Example 10 Subject against influenza virus is described. Briefly, a plu rality of alphavirus replicons are produced, each expressing a different hemagglutinin (HA); HAs derived from: an Generating and Administering a Quadrivalent influenza A virus H1N1 strain, an influenza A virus H3N2 Alphavirus Replicon Vaccine for Influenza Virus strain, and two separate influenza B virus lineages. The strains used will depend upon the predicted dominant influ 0179 A vaccine designed to immunize a human subject enza virus strains for that given season. HA replicon RNA against influenza virus is described. Briefly, a plurality of sequences are generated as described in Examples 1 and 2. alphavirus replicons are produced, each expressing a dif Each HA replicon is then encapsulated in liposomes and ferent hemagglutinin (HA); HAs derived from: an influenza lyophilized. Alternatively, the HA replicon could be encap A virus H1N1 strain, an influenza A virus H3N2 strain, and sulated in SNALPs. The lyophilized and encapsulated rep two separate influenza B virus lineages. The strains used will licon is packaged into an enteric-coated capsule for oral depend upon the predicted dominant influenza virus strains administration. The vaccine is administered to a Subject Such for that given season. HA replicon RNA sequences are that the replicon is delivered to the small intestine. generated as described in Examples 1 and 2. Optionally, Example 12 replicons are formulated with G5 or G9 NH, PAMAM dendrimer nanoparticles using a microfluidic mixing device A Heterologous Prime-Boost Regimen for Influenza as described in Examples 5 and 6. If G5 or G9 NH, PAMAM Vaccination dendrimers are utilized, they are optionally modified by 0183. A dosing regimen for vaccinating a human Subject fluorination. with an influenza virus vaccine is described. Briefly, a capsule comprising an alphavirus replicon encoding hemag 0180 Replicons are then packaged onto the microneedle glutinin (HA) derived from influenza A virus strain H1N1 is array using a microfluidic dispensing device (e.g., BioDot). administered orally to a human Subject to prime the Subjects Optionally, replicons or replicon-dendrimer nanoparticles immune system. Two weeks later, the alphavirus replicon are packaged (e.g., embedded) into a microneedle array. If encoding the HA is re-administered intradermally to the replicons are packaged into a microneedle array, a polymer subject with a microneedle, thereby selectively boosting the is chosen such that it is dissolvable upon contact with the Subject’s immune response. interstitial fluid of the dermis. The replicon is mixed with the 0.184 While preferred embodiments of the present inven polymer prior to polymerization. The polymer mixture is tion have been shown and described herein, it will be poured into a mold and polymerized. obvious to those skilled in the art that such embodiments are 0181. The microneedle is then packaged and shipped to a provided by way of example only. Numerous variations, medical facility at room temperature. The vaccine is applied changes, and Substitutions will now occur to those skilled in to an individual. The microneedle is applied to the dermal the art without departing from the invention. It should be surface of the individuals arm such that the microneedle understood that various alternatives to the embodiments of pierces the dermal surface of the skin. The microneedle is the invention described herein may be employed in practic applied for five minutes. If replicons are packaged into a ing the invention. It is intended that the following claims dissolvable microneedle array, the polymer mixture contain define the scope of the invention and that methods and ing the replicon is dissolved within the five minutes, such structures within the scope of these claims and their equiva that the replicon is delivered to the immune cells of the skin. lents be covered thereby.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 1

<21 Os SEQ ID NO 1 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OOs SEQUENCE: 1

taatacgact cactataggg US 2017/O 196966 A1 Jul. 13, 2017

1. A microneedle device for administering an RNA mol (b) an HA polypeptide from a viral strain of an influenza ecule, comprising: A virus H3 subtype: (a) a Substrate comprising a plurality of microneedles; and (c) an HA polypeptide from a viral strain of an influenza (b) a composition comprising an RNA encoding an exog B virus Yamagata lineage; or enous polypeptide coated onto or embedded into the (d) an HA polypeptide from a viral strain of an influenza plurality of microneedles. B virus Victoria lineage. 2. The microneedle device of claim 1, wherein the RNA molecule is a recombinant alphavirus replicon. 19. The microneedle device of claim 18, wherein each of 3. The microneedle device of claim 1, wherein the RNA the exogenous polypeptides are encoded on a single recom molecule is dehydrated. binant alphavirus replicon. 4. (canceled) 20. The microneedle device of claim 18, wherein the 5. (canceled) exogenous polypeptides are encoded on different recombi 6. (canceled) nant alphavirus replicons. 7. The microneedle device of claim 2, wherein the exog 21. The microneedle device of claim 1, wherein the enous polypeptide is an antigen associated with an infectious exogenous polypeptide is a hepatitis B virus Surface antigen agent. (HBSAg). 8. The microneedle device of claim 2, wherein the recom 22.-34. (canceled) binant alphavirus replicon is present in an amount effective 35. The microneedle device of claim 2, wherein the to induce an immune response to the exogenous polypep recombinant alphavirus replicon is formulated as a den tide. drimer-replicon nanoparticle. 9. The microneedle device of claim 1, wherein the exog enous polypeptide is an influenza virus HA or NA polypep 36. The microneedle device of claim 35, wherein the tide. dendrimer is a PAMAM dendrimer. 10. The microneedle device of claim 9, wherein the 37. The microneedle device of claim 36, wherein the influenza virus HA polypeptide is an influenza A virus HA PAMAM dendrimer comprises amino surface reactive polypeptide or an influenza B virus HA polypeptide. groups. 11. The microneedle device of claim 10, wherein the 38. The microneedle device of claim 37, wherein the influenza virus HA polypeptide is from a viral strain of a PAMAM dendrimer is a G5 or G9 PAMAM dendrimer group 1 influenza A virus subtype selected from H1, H2, H5, comprising amino Surface reactive groups. H6, H8, H9, H11, H12, H13, H16, H17, or H18. 39. The microneedle device of claim 37, wherein the 12. The microneedle device of claim 10, wherein the PAMAM dendrimer comprises modified amino surface reac influenza virus HA polypeptide is from a viral strain of a tive groups. group 2 influenza A virus subtype selected from H3, H4. H7. 40. The microneedle device of claim 39, wherein the H10, H14, or H15. modified amino Surface reactive groups are modified with a 13. The microneedle device of claim 10, wherein the fluorinating agent, an N-hydroxysuccinimide ester, or an influenza virus HA polypeptide is from a viral strain of an amino acid. influenza B virus. 14. The microneedle device of claim 11, wherein the 41. The microneedle device of claim 40, wherein the influenza virus HA polypeptide is from a viral strain of an N-hydroxysuccinimide ester is a N-hydroxysuccinimide influenza A virus H1 subtype. ester of PEG or an N-hydroxysuccinimide ester of a cell 15. The microneedle device of claim 12, wherein the penetrating peptide. influenza virus HA polypeptide is from a viral strain of an 42. The microneedle of clam 40, wherein the fluorinating influenza A virus H3 subtype. agent is heptafluorobutyric acid anhydride. 16. The microneedle device of claim 13, wherein the 43. The microneedle of clam 40, wherein the amino acid influenza virus HA polypeptide is from a viral strain of an is arginine or histidine. influenza B virus Yamagata or Victoria lineage. 44. The microneedle device of claim 35, wherein the 17. The microneedle device of claim 2, wherein the dendrimer-replicon nanoparticle is formulated by a micro recombinant alphavirus replicon encodes an exogenous polypeptide comprising: fluidic mixing device. (a) an HA polypeptide from a viral strain of an influenza 45. The microneedle device of claim 2, wherein the A virus H1 subtype: recombinant alphavirus replicon is coated onto the plurality (b) an HA polypeptide from a viral strain of an influenza of microneedles using a microfluidic dispensing device. A virus H3 subtype: 46. A microneedle device for administering an RNA (c) an HA polypeptide from a viral strain of an influenza molecule, comprising: B virus Yamagata lineage; (a) a Substrate comprising a plurality of microneedles; and (d) an HA polypeptide from a viral strain of an influenza (b) a pharmaceutical composition comprising a recombi B virus Victoria lineage; or nant alphavirus replicon encoding an exogenous poly (e) any combinations thereof. peptide and a pharmaceutically acceptable carrier or 18. The microneedle device of claim 17, wherein the excipient coated onto or embedded into the plurality of recombinant alphavirus replicon encodes at least two exog enous polypeptides comprising: microneedles. (a) an HA polypeptide from a viral strain of an influenza 47-97. (canceled) A virus H1 subtype: