Trans-amplifying RNAS having microrna target sites

Abstract

Provided herein are trans amplifying ribonucleic acids (taRNA) comprising replicase constructs and / or trans replicon constructs (trRNA) with target sites for microRNA (e.g., cell- type enriched microRNA), for example, in a 3' untranslated region (UTR) of the replicase construct and / or trRNA, and methods of use thereof. These taRNA may be useful for mitigating taRNA- specific immune responses or obstacles to expression in subjects.

Description

[0001] TRANS-AMPLIFYING RNAS HAVING MICRORNA TARGET SITES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of US Provisional Application No. 63 / 708,235, filed October 10, 2024, entitled “TRANS-AMPLIFYING RNAS HAVING MICRORNA TARGET SITES”, the content of which is hereby incorporated by reference herein in its entirety for all purposes.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (A141470010WO00-SEQ-ARM.xml; Size: 135,297 bytes; and Date of Creation: October 15, 2025) are herein incorporated by reference in their entirety.

[0006] BACKGROUND

[0007] Presence and expression of non-self-nucleic acids (e.g., viral or synthetic polynucleotides) in mammalian cells often leads to host immune responses and can cause undesired secondary effects, posing several major obstacles for ribonucleic acid (RNA)-based therapies.

[0008] SUMMARY

[0009] MicroRNAs are abundantly expressed in mammalian cells and are an important component of post-transcriptional regulation of gene expression. Engineered polynucleotides comprising microRNA target sites may be useful for evading certain host immune responses.

[0010] Provided herein are compositions and methods useful for, in some aspects, selectively expressing taRNA in a given cell type (e.g., cell types expressing certain microRNAs).

[0011] In some aspects, provided herein are trans amplifying ribonucleic acid (RNA) (taRNA) comprising a first RNA polynucleotide (e.g., a replicase construct), comprising (i) a nucleic acid encoding a replicase, and (ii) a 3’ untranslated region (UTR); and a second RNA polynucleotide (e.g., a trans replicon (trRNA)), comprising: (i) a nucleic acid payload or a nucleic acid encoding a payload, and (ii) a 3’ UTR, wherein the 3’ UTR of the first RNA polynucleotide and / or the 3’ UTR of the second RNA polynucleotide further comprises a target site for a microRNA.

[0012] In some embodiments, the microRNA is a cell type-enriched microRNA. In some embodiments, the cell type-enriched microRNA is an immune cell-enriched microRNA. In some

[0013] #14495043v1 embodiments, the immune cell-enriched microRNA is miR-142 or miR-223. In some embodiments, the cell type-enriched microRNA is a hepatocyte-enriched microRNA. In some embodiments, the hepatocyte-enriched microRNA is miR-122. In some embodiments, the cell type-enriched microRNA is an endothelial cell-enriched microRNA. In some embodiments, the endothelial cell-enriched microRNA is miR-126. In some embodiments, the cell type-enriched microRNA is a skeletal muscle cell-enriched microRNA. In some embodiments, the skeletal muscle cell-enriched microRNA is miR-206.

[0014] In some embodiments, the 3’ UTR of the first RNA polynucleotide (e.g., the replicase construct) and / or the 3’ UTR of the second RNA polynucleotide (e.g., the trRNA) is a 3’ UTR of an alphavirus. In some embodiments, the 3’ UTR of the first RNA polynucleotide (e.g., the replicase construct) comprises the target site for the microRNA. In some embodiments, the 3’ UTR of the second RNA polynucleotide comprises the target site for the microRNA. In some embodiments, the 3’ UTR of the first RNA polynucleotide (e.g., the replicase construct) comprises a target site for a microRNA and wherein the 3’ UTR of the second RNA polynucleotide (e.g., the trRNA) comprises a target site for a microRNA.

[0015] In some embodiments, the 3’ UTR of the first RNA polynucleotide (e.g., the replicase construct) comprises: (i) a first target site for a first microRNA; and (ii) a second target site for a second microRNA. In some embodiments, the 3’ UTR of the first RNA polynucleotide (e.g., the replicase construct) comprises: (i) a first target site for a first microRNA; (ii) a second target site for a second microRNA; and (iii) a third target site for a third microRNA.

[0016] In some embodiments, the 3’ UTR of the second RNA polynucleotide (e.g., the trRNA) comprises: (i) a first target site for a first microRNA; and (ii) a second target site for a second microRNA. In some embodiments, the 3’ UTR of the second RNA polynucleotide (e.g., the trRNA) comprises: (i) a first target site for a first microRNA; (ii) a second target site for a second microRNA; and (iii) a third target site for a third microRNA.

[0017] In some embodiments, the first target site and the second target site of a 3’ UTR of an RNA polynucleotide are target sites for different microRNA. In some embodiments, the first target site is a target site for miR-142 and the second target site is a target site for miR-122.

[0018] In some embodiments, the first target site and the second target site of a 3’ UTR of an RNA polynucleotide are target sites for the same microRNA. In some embodiments, the first target site and the second target site are target sites for miR-122.

[0019] In some embodiments, the first target site, the second target site, and the third target site of a 3’ UTR of an RNA polynucleotide are target sites for at least two different microRNA. In

[0020] #14495043v1 some embodiments, the first target site is a target site for miR-142, the second target site is a target site for miR-122 and the third target site is a target site for miR-122. In some embodiments, the first target site is a target site for miR-142, the second target site is a target site for miR-142, and the third target site is a target site for miR-122.

[0021] In some embodiments, the first target site, the second target site, and the third target site of a 3’ UTR of an RNA polynucleotide are target sites for the same microRNA.

[0022] In some embodiments, a 3’ UTR of an RNA polynucleotide comprising at least two target sites (e.g., a first target site and a second target site) further comprises a spacer sequence between two or more of the target sites. In some embodiments, the spacer sequence comprises UUUAAA.

[0023] In some embodiments, the second RNA polynucleotide (e.g., trRNA) of the taRNA comprises a conserved sequence element (CSE) cognate to the replicase encoded by the first RNA polynucleotide (e.g., the replicase construct) of the taRNA. In some embodiments, the replicase is a Semliki Forest Virus (SFV) replicase, a Venezuelan Equine Encephalitis Virus (VEEV), a Chikungunya virus (CHIKV), or a Sindbis virus (SINV) replicase.

[0024] In some aspects, provided herein are taRNA comprising: (a) a first RNA polynucleotide (e.g., a replicase construct), comprising: (i) a nucleic acid encoding a replicase, and (ii) a 3’ untranslated region (UTR), comprising: a target site for miR-122, and a target site for miR-142; and (b) a second RNA polynucleotide (e.g., a trRNA), comprising: (i) a nucleic acid payload or a nucleic acid encoding a payload; and (ii) a conserved sequence element (CSE) cognate to the replicase encoded by the nucleic acid of (a)(i).

[0025] In some aspects, provided herein are taRNA comprising: (a) a first RNA polynucleotide (e.g., a replicase construct), comprising: (i) a nucleic acid encoding a replicase; and (ii) a 3’ untranslated region (UTR), comprising: a first target site for miR-122; and a second target site for miR-122; and (b) a second RNA polynucleotide (e.g., a trRNA), comprising: (i) a nucleic acid payload or a nucleic acid encoding a payload; and (ii) a conserved sequence element (CSE) cognate to the replicase encoded by the nucleic acid of (a)(i).

[0026] In some aspects, provided herein are taRNA comprising: (a) a first RNA polynucleotide (e.g., a replicase construct), comprising: (i) a nucleic acid encoding a replicase; and (ii) a 3’ untranslated region (UTR), comprising: a first target site for miR-142; and a second target site

[0027] #14495043v1 for miR-142; and (b) a second RNA polynucleotide (e.g., a trRNA), comprising: (i) a nucleic acid payload or a nucleic acid encoding a payload; and (ii) a conserved sequence element (CSE) cognate to the replicase encoded by the nucleic acid of (a)(i).

[0028] In some aspects, provided herein are taRNA comprising: (a) a first RNA polynucleotide (e.g., a replicase construct), comprising (i) a nucleic acid encoding a replicase; and (ii) a 3’ untranslated region (UTR), comprising: a first target site for miR-122; a second target site for miR-122; and a third target site for miR-142; and (b) a second RNA polynucleotide (e.g., a trRNA), comprising: (i) a nucleic acid payload or a nucleic acid encoding a payload; and (ii) a conserved sequence element (CSE) cognate to the replicase encoded by the nucleic acid of (a)(i).

[0029] In some aspects, provided herein are taRNA comprising: (a) a first RNA polynucleotide (e.g., a replicase construct), comprising: (i) a nucleic acid encoding a replicase; and (ii) a 3’ untranslated region (UTR), comprising: a first target site for miR-122; a second target site for miR-142; and a third target site for miR-142; and (b) a second RNA polynucleotide (e.g., a trRNA), comprising: (i) a nucleic acid payload or a nucleic acid encoding a payload; and (ii) a conserved sequence element (CSE) cognate to the replicase encoded by the nucleic acid of (a)(i).

[0030] Also provided herein, in some aspects, are methods for selectively expressing a taRNA in a first cell, the method comprising: contacting a first cell and a second cell with a taRNA comprising: (a) a first RNA polynucleotide (e.g., a replicase construct), comprising: (i) a nucleic acid encoding a replicase, (ii) a 3’ untranslated region (UTR); and (b) a second RNA polynucleotide (e.g., a trRNA), comprising: (i) a nucleic acid payload or a nucleic acid encoding a payload, (ii) a 3’ UTR; and (iii) a conserved sequence element (CSE) cognate to the replicase of (a)(i), wherein the 3’ UTR of the first RNA polynucleotide (e.g., the replicase construct) and / or the 3’ UTR of the second RNA polynucleotide (e.g., the trRNA) comprise a target site for a microRNA expressed by the second cell and not expressed by the first cell.

[0031] In some embodiments, the second cell is a liver cell. In some embodiments, the microRNA target site is a target site for miR-122. In some embodiments, the second cell is an immune cell. In some embodiments, the microRNA target site is a target site for miR-142.

[0032] In some embodiments, the replicase is an SFV replicase.

[0033] Also provided herein, in some aspects, are methods for reducing the immunogenicity of a taRNA in a subject, the method comprising: administering a taRNA to the subject, the taRNA comprising: (a) a first RNA polynucleotide (e.g., a replicase construct), comprising: (i) a nucleic acid encoding a replicase, and (ii) a 3’ untranslated region (UTR) ; and (b) a second RNA

[0034] #14495043v1 polynucleotide (e.g., a trRNA), comprising: (i) a nucleic acid payload or a nucleic acid encoding a payload, (ii) a 3’ UTR; and (iii) a conserved sequence element (CSE) cognate to the replicase of (a)(i), wherein the 3’ UTR of the first RNA polynucleotide and / or the 3’ UTR of the second RNA polynucleotide comprise a target site for a microRNA, and wherein the microRNA is not expressed by a target cell type of the subject and is expressed by at least one non-target cell type of the subject.

[0035] In some embodiments, the taRNA non-target cell is a liver cell. In some embodiments, the microRNA target site is a target site for miR-122. In some embodiments, the taRNA non- target cell is an immune cell. In some embodiments, the microRNA target site is a target site for miR-142.

[0036] In some embodiments, reducing the immunogenicity of the taRNA comprises reducing expression of one or more interferon (IFN), chemokine, and / or interleukin that are associated with an immune response of the cell to the taRNA. In some embodiments, the IFN is IFN-a, IFN-P, or IFN-y. In some embodiments, the chemokine is a keratinocyte chemoattractant (KCj / human growth-regulated oncogene (GRO) chemokine, monocyte chemoattractant protein- 1 (MCP-1), interferon gamma-induced protein 10 (IP- 10), or macrophage inflammatory protein la (MIP-la). In some embodiments, the interleukin is IL-6. In some embodiments, reducing the immunogenicity of the taRNA does not comprise significantly decreasing the expression of IFN- Y-

[0037] In some embodiments, the replicase is an SFV replicase.

[0038] BRIEF DESCRIPTION OF DRAWINGS

[0039] FIGs. 1A-1B show luciferase (Flue) expression in adult wildtype mice (C57BL / 6) after intramuscular (IM) injection of compositions of ALC-315 LNPs and 5pg total RNA of either mRNA (mRNA-LNP) or taRNA including trRNA encoding luciferase and absent of any target sites for microRNAs and a replicase construct encoding Semliki Forest Virus (SFV) replicase (taRNA-LNP). FIG. 1A shows representative images of 2D fluorescence imaging in live animals. FIG. IB shows quantification of luciferase activity (p / s = photons / second) in the whole body, the hind leg (injection site) and the liver; each circle represents a separate mouse.

[0040] FIGs. 2A-2C show effects of intramuscular (IM) vs intravenous (IV) administration on SEAP expression for compositions of ALC-315 LNPs and 5pg total RNA of either mRNA (mRNA-LNP) or taRNA including trRNA encoding luciferase and absent of any target sites for microRNAs (mRNA-LNP) and a replicase construct encoding SFV replicase, absent of any

[0041] #14495043v1 target sites for microRNAs (taRNA-LNP). FIG. 2A shows in vivo expression of SEAP in adult wildtype mice (C57BL / 6) injected with mRNA-LNP or taRNA-LNP via intramuscular (IM) or intravenous (IV) administration. Expression is shown as relative luminance units (RLU) * hours (area under curve (AUC)). FIG. 2B shows changes to body weight (% body weight) in mice injected with mRNA-LNP, taRNA-LNP, or saline via IM vs IV across 168 hours. FIG. 2C shows expression of interferon gamma induced protein 10 (IP- 10, also known as CXCL10; pg / mL) at 6 hours, 24 hours, or 72 hours after injection (IM or IV) with mRNA-LNP, taRNA- LNP, or saline.

[0042] FIGs. 3A-3B show induction of IP- 10 expression in mice treated with compositions of ALC-315 LNPs and 5pg of either mRNA or taRNA encoding SEAP. FIG. 3A shows IP- 10 expression (pg / mL) at 6 hours or 24 hours in wildtype mice (C57BL / 6) mice injected (IM) with mRNA encoding SEAP (mRNA-LNP), taRNA consisting of a trRNA encoding SEAP and a SFV replicase construct (taRNA-LNP); an SFV replicase construct only (no trRNA; Replicase- LNP); or a trRNA encoding SEAP only (no replicase construct; trRNA-LNP). FIG. 3B shows induction of IP-10 expression (pg / mL) in wildtype mice (C57BL / 6), wildtype mice treated with 2mg / kg dexamethasone, or interferon A receptor (IFNAR) knockout mice, after administration of saline, mRNA-LNP, taRNA-LNP, or Replicase-LNP only.

[0043] FIGs. 4A-4B show in vitro effects on payload expression mediated by taRNAs with microRNA target site insertions in the 3’ UTR of replicase constructs. FIG. 4A shows expression of nano Luciferase (nLuc) (normalized to control) in Vero cells transfected with one of two taRNAs: a taRNA having a trRNA comprising a SINV 5’ UTR, a nucleic acid encoding nLuc, and a replicase construct having 2 target sites for miR-142 (2xmiR-142ts); or a taRNA having a trRNA comprising a SINV 5’ UTR, a nucleic acid encoding nLuc, and a replicase construct having 2 target sites for miR-122 (2xmiR-122ts). Cells were transfected with microRNA mimics: miR-142 or miR-122. FIG. 4B shows fold-expression of nLuc in RAW cells transfected with taRNA having a trRNA comprising an nLuc encoding sequence only or an nLuc encoding sequence and having two miR-142 target sites (miR142ts); and a replicase construct encoding a replicase or a replicase construct encoding a replicase and having a microRNA target site.

[0044] FIGs. 5A-5B show cytokine expression and liver enzyme levels in the blood of wildtype mice (C57BL / 6) administered ALC-315 LNPs comprising mRNAs encoding luciferase (Flue) (mRNA-LNP), or taRNAs having a trRNA encoding Flue and a replicase construct encoding SFV replicase (taRNA-LNP) with or without microRNA target sites. FIG. 5A shows fold-

[0045] #14495043v1 expression of cytokines in wildtype mice at 24 hours post IM administration of Ipg of: mRNA encoding luciferase (Flue); taRNA having a trRNA with an oeSTR 5’ UTR and encoding luciferase, and a replicase construct encoding a wildtype Semliki Forest Virus (WT SFV) replicase; a taRNA having a trRNA with an oeSTR 5’ UTR and encoding luciferase, and a replicase construct encoding a WT SFV replicase and having a target site (ts) for miR-142 (miR- 142ts); a taRNA having a trRNA with an oeSTR 5’ UTR and encoding luciferase, and a replicase construct encoding a WT SFV replicase and having a miR-122ts; a taRNA having a trRNA with an oeSTR 5’ UTR and encoding luciferase, and a replicase construct encoding a WT SFV replicase and having a miR-126ts. Expression of tumor necrosis factor (TNF)-alpha (TNF- a), interferon gamma (IFNg), interferon beta (IFNb), keratinocyte chemoattractant (KC) / human growth regulated oncogene (GRO) (KC / GRO), monocyte chemoattractant protein-1 (MCP-1), interferon gamma induced protein 10 (IP- 10), macrophage inflammatory protein 1 alpha (MIP- la; also known as CCL3), and interleukin 6 (IL6) is shown as fold change relative to saline. FIG. 5B shows levels of two liver enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), at 24 hours after administration of mRNA or taRNA as in FIG. 5A.

[0046] DETAILED DESCRIPTION OF INVENTION

[0047] In some aspects, this disclosure describes a trans- amplifying ribonucleic acid (RNA) (taRNA) comprising (a) a first RNA polynucleotide comprising (i) a nucleic acid encoding a replicase; and (b) a second RNA polynucleotide comprising (i) a nucleic acid payload or a nucleic acid encoding a payload; and wherein the first RNA polynucleotide and / or the second RNA polynucleotide further comprises: (ii) a target site for a microRNA.

[0048] A “trans- amplifying RNA,” hereinafter referred to as “taRNA,” comprises a first and second RNA polynucleotide, wherein the first RNA polynucleotide encodes a replicase, the second RNA polynucleotide comprises a nucleic acid payload or nucleic acids encoding a payload, and wherein the second RNA polynucleotide can be replicated by the encoded replicase in trans. A “polynucleotide” refers to a polymer of nucleotides. A polynucleotide is generally composed of nucleotides that are naturally found in DNA or RNA (e.g., adenosine / deoxyadenosine (A), uridine (U) / deoxythymidine (T), guanosine / deoxyguanosine (G), cytidine / deoxycytidine (C)) and joined by phosphodiester bonds. However, the term polynucleotide may also refer to polynucleotides comprising nucleotides or nucleotide analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not

[0049] #14495043v1 these modifications are found in naturally occurring nucleic acids; indeed, such molecules may be preferred for certain applications. In some sequences described herein, T / U is used to denote a particular nucleotide may be a T or U depending on whether the polynucleotide is an RNA polynucleotide (U) or a DNA polynucleotide (T). The first and second RNA polynucleotides of a taRNA are separate polynucleotides (i.e., separate molecules which are not a single continuous strand of RNA). The terms “replicase construct” and “trans replicon” (trRNA) construct are used synonymously herein to refer to the first and second RNA polynucleotides of a taRNA, respectively. As used herein, a “construct” refers to an artificial (i.e., not naturally occurring) polynucleotide.

[0050] As demonstrated in the section entitled “Examples,” administration of taRNA to a subject (e.g., IM administration) results in different cytokine induction and transfection / expression patterns across various organ systems compared to mRNA. For example, intramuscular (IM) administration of taRNA leads to increased muscle transfection, decreased liver transfection, increased liver enzyme levels, and unique cytokine expression relative to IM administration of mRNA, while intravenous administration (IV) administration of taRNA is poorly expressive, but induces similar cytokine expression. Without wishing to be bound by theory, taRNA-induced cytokine expression is thought to be due, in part, to accumulation of replicase constructs in the liver, and to specific immune responses induced by taRNA components. Accordingly, the inventors have recognized that reduced expression of components of a taRNA (e.g., replicase construct) in certain cell types (e.g., in certain tissues), but not in others, may be uniquely useful for enhancing expression and / or reducing immunogenicity of taRNA in a subject. taRNA (e.g., a replicase construct of a taRNA, a trRNA of a taRNA) described herein comprise one or more target sites for microRNA. As detailed in the section entitled “MicroRNA Target Sites,” microRNA can be useful for selectively reducing expression of taRNA (e.g., in specific cell types), which may be useful to alter expression and / or transfection of taRNA for certain applications.

[0051] Trans Replicon (trRNA) Constructs

[0052] A “trans replicon” (trRNA) refers to a polynucleotide (e.g., RNA polynucleotide) comprising a nucleic acid encoding a payload operably linked to a conserved sequence element (CSE), wherein the polynucleotide does not encode a replicase cognate to the CSE. A “polynucleotide” refers to a polymer of nucleotides. A polynucleotide is generally composed of nucleotides that are naturally found in DNA or RNA (e.g., adenosine / deoxyadenosine (A),

[0053] #14495043v1 thymidine / deoxythymidine (T), guanosine / deoxyguanosine (G), cytidine / deoxycytidine (C) and uridine (U) joined by phosphodiester bonds. However, the term polynucleotide may also refer to polynucleotides comprising nucleotides or nucleotide analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not these modifications are found in naturally occurring nucleic acids; indeed, such molecules may be preferred for certain applications. trRNAs are capable of being replicated by cognate replicases.

[0054] Payloads trRNAs comprise at least a payload-encoding sequence and one or more CSEs. A trRNA does not comprise the replicase that can amplify the trRNA. Instead, the simultaneous presence of a trRNA in a cell and the expression of a cognate replicase can result in amplification of the trRNA and its encoded payload. As used herein, a “payload” refers to one or more gene products of interest for delivery to or expression by an organism. A payload may be a functional nucleic acid (e.g., RNA), a protein, a peptide or protein fragment, or a fusion protein.

[0055] In some embodiments, a payload is a selectable marker. As used herein, a “selectable marker” is a peptide or protein that can be used to screen cells by artificial selection. Nonlimiting examples of selectable markers include antibiotic resistance proteins (e.g., ampicillin, puromycin) and negative selection markers (e.g., thymidine kinase).

[0056] In some embodiments, a payload is a reporter. A “reporter” is a peptide or protein which alters the appearance of a cell such that cells can be visually or optically screened for presence or absence of the peptide or protein. In some embodiments, a reporter is an enzyme which alters the appearance of a cell, such as beta-galactosidase. In some embodiments, a reporter is a peptide or peptide fragment (e.g., secreted embryonic alkaline phosphatase (SEAP)) which can be detected in combination with additional reagents (e.g., assay-specific media). In some embodiments, a reporter is a fluorophore, such as, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), or any derivative thereof.

[0057] In some embodiments, a payload is “therapeutic payload,” here referring to a gene product useful for treating or preventing a disease or disorder. A “gene” refers to a nucleic acid (e.g., DNA, RNA) encoding a polypeptide (e.g., a vaccine antigen or a replicase). In some embodiments, a gene is a naturally occurring gene. In some embodiments, a gene is a transgene (e.g., derived from a different organism). In some embodiments, a therapeutic payload knocks down, knocks in, increases, inhibits, or otherwise modulates gene expression. In some embodiments, a therapeutic payload replaces or edits an endogenous gene or gene product. In

[0058] #14495043v1 some embodiments, a therapeutic payload is a functional RNA; non-limiting examples of include short hairpin RNA (shRNA), microRNA (miRNA), artificial microRNA (amiRNA), small interfering RNA (siRNA), or circular RNA (circRNA).

[0059] In some embodiments, a therapeutic payload is a protein. A therapeutic protein may replace or interfere with activity of deficient or absent endogenous proteins; augment activity of existing metabolic or synthetic pathways; provide a novel function or activity; or interfere with the activity of a pathogen or toxic molecule. Non-limiting examples of therapeutic proteins suitable for use as payloads include membrane proteins, membrane-associated proteins, secreted proteins, intracellular proteins, antigens, antibodies, or fragments thereof.

[0060] In some embodiments, a therapeutic protein is an antigen. An “antigen” refers to a protein or protein fragment of a pathogen. In some embodiments, an antigen is a protein or peptide that can be recognized by the immune system of a host organism. In some embodiments, an antigen, when introduced into a host, induces an immune response in the host (e.g., antibody production against the antigen). In some embodiments, an antigen is a nucleic acid (e.g., aptamer), polysaccharide, polypeptide, oligosaccharide, lipid, particulate antigen, or cancer antigen (e.g., tumor antigen). Preferably, an antigen is a protein or a fragment thereof (e.g., a surface protein). In some embodiments, an antigen is a protein derived from a pathogen. In some embodiments, the antigen is a viral antigen or bacterial antigen.

[0061] Conserved Sequence Elements (CSEs) trRNAs comprise one or more conserved sequence elements (CSEs). A “conserved sequence element” (CSE) refers to a recognition site for an alphavirus replicase. Typically, a CSE functions as a core promoter or enhancer for initiation of replication of a downstream sequence, such that a 5 ’-CSE may initiate synthesis of a plus-strand and a 3 ’-CSE may initiate synthesis of a minus-strand. An RNA polynucleotide may comprise one or more 5 ’-CSEs and / or 3’-CSEs. CSEs may be comprised in a UTR, for example a 5’-UTR and / or a 3’ UTR. In some embodiments, a CSE forms one or more secondary structure(s), such as one or more stem-loops. Non-limiting examples of CSEs include CSE1, CSE2, CSE3, CSE4, and variants or derivatives thereof. In some embodiments, the CSE is a CSE derived from SFV, SINV, VEEV, or CHIKV alphavirus. CSEs are known in the art, e.g., as described in Hyde JL, Virus Res. 405 Aug 3;45:99-107.

[0062] #14495043v1 Replicase Constructs

[0063] A “replicase construct” (i.e., the first RNA polynucleotide) refers to mRNA which comprises a nucleic acid encoding a replicase and does not comprise nucleic acids encoding an antigen. In some embodiments, a replicase construct is a non-replicating mRNA. As used herein, the term “non-replicating mRNA” refers to an mRNA which is processed for translation into a gene product or else degraded, and which does not self-replicate. Once introduced to an environment comprising translational machinery (such as a cell), replicase constructs can be translated to generate the encoded replicase.

[0064] A “replicase” is an RNA-dependent RNA polymerase capable of transcribing (i.e., reading) an RNA template to produce an RNA (e.g., trRNA). A replicase construct may encode a modified replicase from an RNA virus, for example, an alphavirus. The term “alphavirus” refers to an RNA virus belonging to the Togaviridae family. In some embodiments, a replicase of the replicase construct comprises one or more mutations. In some embodiments, the alphavirus comprises a single-stranded RNA genome encoding at least nsPl, nsP2, nsP3, nsP4, El, E2, E3, 6K / TF and capsid proteins. In some embodiments, a replicase construct of a taRNA encodes a replicase derived from a Semliki Forest virus (SFV) (SFV replicase). In some embodiments, a wildtype SFV replicase comprises an amino acid sequence of SEQ ID NO: 46. In some embodiments, the SFV replicase is a REE replicase. An REE replicase is an SFV replicase variant comprising (i) an arginine at a position corresponding to A144 of SEQ ID NO: 46; (ii) a leucine at a position corresponding to D145 of SEQ ID NO: 46; and (iii) a glutamic acid at a position corresponding to A1207 of SEQ ID NO: 46. In some embodiments, the RLE replicase comprises an amino acid sequence of SEQ ID NO: 47.

[0065] In some embodiments, a replicase construct of a taRNA encodes a replicase derived from a Sindbis virus (SINV) (SINV replicase). In some embodiments, a wildtype SINV replicase comprises an amino acid sequence of SEQ ID NO: 49. In some embodiments, a replicase construct of a taRNA encodes a replicase derived from a Venezuelan equine encephalitis virus (VEEV) (VEEV replicase). In some embodiments, a wildtype VEEV replicase comprises an amino acid sequence of SEQ ID NO: 50. In some embodiments, a replicase construct of a taRNA encodes a replicase derived from a Chikungunya virus (CHIKV) (CHIKV replicase). In some embodiments, a wildtype CHIKV replicase comprises an amino acid sequence of SEQ ID NO: 51.

[0066] In some embodiments, a taRNA comprises a replicase construct encoding an alphavirus replicase. Typically, an alphaviral replicase comprises a complex formed by the non-structural

[0067] #14495043v1 proteins nsPl, nsP2, nsP3, and nsP4. Thus, a nucleic acid encoding an alphaviral replicase (also referred to herein as a “replicase-coding sequence”) is understood to encode at least nsPl, nsP2, nsP3, nsP4, and variants thereof. Once expressed, alphaviral replicase may interact with a RNA polynucleotide comprising one or more CSEs which are cognate to the replicase and generate mirrored copies of the RNA polynucleotide, which can be subsequently translated (e.g., by a host cell). Replicase constructs of taRNA necessarily do not comprise a CSE; thus, once the replicase of a replicase construct is translated, the encoded replicase cannot replicate the replicase construct.

[0068] A replicase that is “cognate” to a CSE refers to a replicase that is the capable of transcribing a portion of a polynucleotide comprising the CSE (e.g., capable of transcribing a polynucleotide comprising or encoding a payload). A replicase construct and trRNA construct are considered “compatible” or “cognate” when a trRNA comprises a CSE to which the replicase encoded by the replicase construct can bind, such that the trans replicon is replicated. In some embodiments, a compatible replicase construct and CSE are derived from the same alphavirus. In some embodiments, a replicase and CSE from the same species are cognate; for example, an SFV replicase is assumed to be cognate to a CSE derived from an SFV. In some embodiments, a replicase construct encodes a replicase derived from an SFV (SFV replicase). In some embodiments, a replicase derived from a SFV is capable of binding to a trRNA comprising a CSE from an SFV. In some embodiments, a replicase construct encodes a replicase derived from a SINV (SINV replicase). In some embodiments, a replicase derived from a SINV is capable of binding to a trRNA comprising a CSE from an SINV. In some embodiments, a compatible replicase construct and CSE are derived from different alphaviruses. In some embodiments, a replicase derived from a SFV is capable of binding to a trRNA comprising a CSE from a SINV. In some embodiments, a replicase derived from a SINV is capable of binding to a trRNA comprising a CSE from an SFV.

[0069] Untranslated Regions (UTRs)

[0070] In some embodiments, a trRNA and / or replicase construct comprise one or more untranslated regions (UTRs). UTRs may act as stabilizing elements and / or provide regulation of transcription of a gene (e.g., a nucleic acid encoding an antigen). Typically, UTRs are found upstream and / or downstream of a gene or transgene. A UTR located directly upstream of a start codon and operably linked to a gene is referred to herein as a 5 ’-UTR. As a skilled artisan will understand, 5 ’-UTRs may comprise sequence elements which play roles in regulation of

[0071] #14495043v1 expression (e.g., Kozak sequences) or structural elements which alter stability of the molecule (e.g., 5’ cap structures). A UTR located directly downstream of a stop codon operably linked to a gene or transgene is referred to herein as a 3 ’-UTR. 3’-UTRs may comprise structural elements which alter the stability of a construct and / or provide transcriptional control, including, but not limited to AU-rich elements and polyA tails. A variety of 5’-UTRs and a 3’-UTRs are known to those of ordinary skill in the art. UTRs may be naturally occurring or synthetic.

[0072] In some embodiments, a trRNA comprises a UTR. In some embodiments, a trRNA and / or replicase construct comprise a UTR derived from an alphavirus. In some embodiments, an alphavirus UTR comprises a CSE. In some embodiments, a trRNA comprises a 5 ’-UTR of an alphavirus (e.g., an alphavirus 5 ’-UTR). In some embodiments, a trRNA comprises a 3 ’-UTR of an alphavirus (e.g., an alphavirus 3’-UTR). Alphavirus 5’-UTR and alphavirus 3’-UTR sequences are described in the art, e.g., by Hyde JL et al., Virus Res. 2015 Aug 3;45:99-107. In some embodiments, a trRNA comprises a 5 ’-UTR and / or 3 ’-UTR derived from a Semliki Forest Virus (SFV), hereinafter referred to as a “SFV-UTR”. In some embodiments, the RNA polynucleotide comprises a 5 ’-UTR and / or 3 ’-UTR derived from a Sindbis virus (SINV), hereinafter referred to as a “SINV-UTR”. An exemplary wildtype 5’-SINV-UTR is provided in SEQ ID NO: 25. In some embodiments, an alphavirus 5’-UTR comprises one or more mutations relative to a wildtype alphavirus 5 ’-UTR. In some embodiments, an alphavirus 3’- UTR comprises one or more mutations relative to a wildtype alphavirus 3 ’-UTR. In some embodiments, the RNA polynucleotide comprises a 5 ’-UTR and 3 ’-UTR from the same virus. Non-limiting examples include an RNA polynucleotide comprising a 5’-UTR derived from a SINV (5 ’-SINV-UTR), and a 3’-UTR derived from a SINV (3’-SINV-UTR) or the RNA polynucleotide comprising a 5 ’-UTR derived from a SFV (5 ’-SFV-UTR), and a 3 ’-UTR derived from a SFV (3’-SFV-UTR). In some embodiments, the RNA polynucleotide comprises a 5’- UTR and 3 ’-UTR from different alphaviruses.

[0073] In some embodiments, a trRNA comprises one or more CSEs, wherein the CSEs are present in one or more UTRs. In some embodiments, a trRNA comprises a 5’-UTR having one or more CSEs. In some embodiments, a trRNA comprises a 3 ’-UTR having one or more CSEs. In some embodiments, a trRNA comprises a 5’-UTR having one or more CSEs, a nucleic acid encoding a payload, and a 3’-UTR having one or more CSEs. In some embodiments, a trRNA comprises a 5 ’-UTR having one or more CSEs, a nucleic acid encoding a payload, and a 3 ’-UTR having one or more CSEs. In some embodiments, a trRNA comprises UTRs having one or more CSEs, wherein the UTRs are derived from one or more alphaviruses. A skilled artisan will

[0074] #14495043v1 appreciate that UTRs derived from alphaviruses comprise one or more CSEs unless stated otherwise. In some embodiments, a trRNA comprises a 5’-UTR derived from a first alphavirus and a 3’-UTR derived from a second alphavirus. In some embodiments, a trRNA comprises a 3’- UTR comprising one or more repeat sequence elements (RSE).

[0075] In some embodiments, a trRNA comprises a 5’-UTR having an “oeSTR” extension. An oeSTR extension is a sequence of 9 nucleotides in length (AGAAGAUGG) inserted into the 5’ terminal of a 5’ UTR. The term “5’ terminal of a 5’-UTR” refers to nucleotides at the 5’ end of the 5 ’-UTR. The 5’ terminal of the wildtype 5’ SINV UTR comprises six conserved nucleotides AUCGGC. The 5’ extension described herein is inserted between the AU and CGGC (e.g., AUAGAAGAUGGCGGC (SEQ ID NO: 52), as shown in SEQ ID NO: 27).

[0076] In some embodiments, a replicase construct comprises a 5 ’-UTR and / or a 3 ’-UTR. In some embodiments, a replicase construct comprises a 5’-UTR. In some embodiments, a replicase construct comprises a 3 ’-UTR. In some embodiments, a replicase construct comprises a 5 ’-UTR and a 3 ’-UTR. In some embodiments, a replicase construct comprises a 5 ’-UTR derived from human alpha-globin (5’-HAG-UTR). An exemplary wildtype 5’-HAG-UTR is provided in SEQ ID NO: 24. In some embodiments, a replicase construct comprises a 3’-UTR derived from human alpha-globin (3’-HAG-UTR). An exemplary wildtype 3’-HAG-UTR is provided in SEQ ID NO: 28. In some embodiments, a replicase construct comprises a 5’-HAG- UTR and a 3’-HAG-UTR. In some embodiments, a replicase construct comprises a 5’-HAG- UTR, a SFV replicase-encoding sequence, and a 3’-HAG-UTR (5’-HAG-UTR-SFV replicase- 3’-HAG-UTR).

[0077] MicroRNA Target Sites

[0078] In some embodiments, a trRNA and / or replicase construct of a taRNA comprises one or more microRNA (miRNA) target sites. A microRNA “target site” as used herein, refers to a sequence of nucleotides within an RNA polynucleotide that can be recognized by a microRNA. In some embodiments, the microRNA target site comprises 6 to 8 nucleotides that are complementary to a miRNA seed region. miRNA target sites are known in the art, e.g., as described in Yang, Tzu-Hsien, et al. Journal of Chemical Information and Modeling. 2024 64.7 (413): 2445-2453 and Huang HY, Nucleic Acids Res. 2022 Jan 7;50(Dl):D222-D230. As used herein, the term “microRNA” refers to an RNA polynucleotide that comprises a seed region, and comprises a guide strand having a length of between about 18 to 25 ribonucleotides. A “seed region” refers to a region of a microRNA having high complementarity (e.g., at least 90%

[0079] #14495043v1 complementarity, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to a cognate microRNA target site in a target RNA polynucleotide. Typically, a seed region is within positions 2-8 (e.g., 2-7) of a guide strand of a microRNA and has 100% complementarity to a cognate microRNA target site. A target site and a microRNA are considered “cognate” if the target site and the microRNA comprise sequences with sufficient complementarity to hybridize (e.g., by traditional Watson-Crick hybridization rules or by any stable association of the microRNA with the target RNA polynucleotide at or adjacent to the microRNA target site).

[0080] Typically, a microRNA capable of recognizing (e.g., binding to) a target RNA polynucleotide is a mature microRNA (e.g., a microRNA guide strand comprising a seed region, and complexed with RISC). However, mature microRNA capable of binding to a target RNA polynucleotide can be recognized in any precursor form, for example, a pri-microRNA, a pre- microRNA, or a microRNA duplex. As used herein, “pri-microRNA” and “pre-microRNA” both refer to single- stranded RNA duplexes comprising a guide strand and a passenger strand, wherein the guide strand and passenger strand are comprised in the same continuous RNA polynucleotide and connected by means of a hairpin. A pri-microRNA, while similar to a pre- microRNA, is the primary transcript of a nucleic acid (e.g., gene, transgene) encoding a microRNA, while the pre-microRNA is the product resulting from cleavage of pri-microRNA by additional enzymes (e.g., Drosha and DGCR8). While not capable of binding directly to a microRNA target site, the cognate target site for a pri-microRNA or pre-microRNA can be determined using methods known to those of ordinary skill in the art, e.g., by identifying the seed region. Pre-microRNAs are further processed by additional enzymes (e.g., Dicer) to produce an intermediate “microRNA duplex.” A “microRNA duplex” refers to a two-stranded RNA duplex comprising a guide strand and a passenger strand, wherein the guide strand and passenger strand are not comprised in a single continuous RNA polynucleotide (e.g., are not connected by a hairpin). microRNA duplexes can be incorporated into RNA-induced silencing complex (RISC). Once incorporated into RISC, the passenger strand of the microRNA duplex is ejected, leaving a mature microRNA (e.g., a microRNA guide strand complexed with RISC). Once a mature microRNA recognizes and binds to a cognate microRNA target site in a target RNA polynucleotide, the target RNA polynucleotide can be degraded by various means (e.g., deadenylation complexes, decapping factors, degradation factors, translational inhibition, dissociation of initiation factors, etc.)

[0081] #14495043v1 Cell-type enriched microRNA / microRNA target sites

[0082] By inserting microRNA target sites into trRNA and / or replicase constructs, a trRNA and / or replicase construct can be made into a target RNA polynucleotide (e.g., a target trRNA, a target replicase construct) for a cognate microRNA. Accordingly, expression of the target trRNA (e.g., expression of the payload encoded by the target trRNA) and / or of a target replicase construct can be reduced (e.g., inhibited) in environments in which the cognate microRNA is enriched. Reduction of expression of a target trRNA and / or replicase construct may be useful for a variety of applications, e.g., to reduce expression of the taRNA (e.g., by reducing expression of the trRNA and / or replicase construct) in one or more cell types, to selectively express the taRNA in a first cell type and not a second cell type, and / or to reduce immunogenicity of the taRNA in a subject (see: “Methods of Use”).

[0083] In some embodiments, a trRNA and / or replicase construct of a taRNA comprises one or more target sites for a cell-type enriched microRNA. In some embodiments, a cell-type enriched microRNA is a microRNA which is enriched in a given tissue (e.g., tissue of an organ). In some embodiments, a cell-type enriched microRNA is enriched in a subpopulation of cells in a tissue (e.g., a population of cells within a tissue). A cell-enriched microRNA is any microRNA which is abundantly expressed in a given cell type relative to a majority of other cell types, such that the microRNA is not considered to ubiquitously expressed. The skilled artisan will understand that microRNAs expressed abundantly or exclusively in desired cell types can be identified using gene sequencing and analysis methods known in the art (see: Londin et al. Proceedings of the National Academy of Sciences. 2015 Mar 10; 112(10) :E1106-15 and Keller et al. Nucleic Acids Research. 2022 Jan 7; 5O(D1):D44-D221).

[0084] In some embodiments, a cell-type enriched microRNA is a hepatocyte-enriched microRNA. Hepatocyte-enriched microRNAs are any microRNAs which are abundantly expressed in hepatocytes relative to a majority of other cell types (e.g., non-liver cells), such that they are not considered ubiquitously expressed. Hepatocyte-enriched microRNAs are known in the art, for example, as described in Musaddaq G, et al. Biomarkers. 2019 Feb 17;24(2): 103-9. In some embodiments, a hepatocyte-enriched microRNA is miR-122, miR-451, miR-26a, miR- 192, miR-21, miR-22, miR-143, miR-199, miR-29, miR-16, miR-24, miR-126, miR-144, miR- 23, miR-194, miR-101, miR-27, miR-191, miR-23, miR-193, miR-130, miR-378, miR-99, miR- 148, miR-145, miR-15, miR-195, miR-34, miR-92, miR-103, miR-181, miR-20, miR-320, miR- 30, miR-142, miR-151, miR-214, miR-19, miR-106, miR-146, miR-15, miR-365, miR-28, or miR-17. In some embodiments, a hepatocyte-enriched microRNA is miR-122. In some

[0085] #14495043v1 embodiments, miR-122 comprises the sequence set forth in SEQ ID NO: 2. In some embodiments, a trRNA and / or a replicase construct of a taRNA comprises a target site for miR- 122. In some embodiments, a target site for miR-122 comprises the sequence set forth in SEQ ID NO: 2.

[0086] In some embodiments, a cell-type enriched microRNA is an endothelial cell-enriched microRNA. Endothelial cell-enriched microRNAs are any microRNAs which are abundantly expressed in endothelial cells relative to a majority of other cell types (e.g., non-endothelial cells), such that they are not considered ubiquitously expressed. Endothelial cell-enriched microRNAs are known in the art, for example, as described in Fernandez-Hernando and Suarez. Curr Opin Hematol. 2018 May 25(3): 227-236. In some embodiments, an endothelial cell- enriched microRNA is miR-126, miR-149, miR-218, miR-17~92, miR-23-24-27, miR-132, miR-221 / 222, miR-150, miR-210, miR-424, miR-217, miR-146, miR-216, miR-125, miR-147, miR-155, miR-133, miR-125, miR-31, miR-17, miR-181, miR-10, or miR-365. In some embodiments, an endothelial cell-enriched microRNA is miR-126. In some embodiments, miR- 126 comprises the sequence set forth in SEQ ID NO: 3. In some embodiments, a trRNA and / or a replicase construct of a taRNA comprises a target site for miR-126. In some embodiments, a target site for miR-126 comprises the sequence set forth in SEQ ID NO: 4.

[0087] In some embodiments, a cell-type enriched microRNA is an immune cell enriched microRNA. Immune cell-enriched microRNAs are any microRNAs which are abundantly expressed in immune cells (e.g., immune cell progenitors, cells in the hematopoietic system) relative to a majority of other cell types (e.g., non-immune cells), such that they are not considered ubiquitously expressed. Immune cell-enriched microRNAs are known in the art, for example, as described in D. Baltimore et al. Nature Immunology. 2008 August:9(8):839-845. In some embodiments, an immune cell-enriched microRNA is miR-155, miR-146, miR-652, miR- 17-92, miR-424, miR-106a-363, miR-221, miR-222, miR-223, miR-142, miR-451, miR-150, miR-130, miR-10, or miR-181a. In some embodiments, the immune cell-enriched microRNA is miR-142 or miR-223. In some embodiments, miR-142 comprises the sequence set forth in SEQ ID NO: 5. In some embodiments, a trRNA and / or a replicase construct of a taRNA comprises a target site for miR-142. In some embodiments, a target site for miR-142 comprises the sequence set forth in SEQ ID NO: 6. In some embodiments, miR-223 comprises the sequence set forth in SEQ ID NO: 7. In some embodiments, a trRNA and / or a replicase construct of a taRNA comprises a target site for miR-223. In some embodiments, a target site for miR-223 comprises the sequence set forth in SEQ ID NO: 8.

[0088] #14495043v1 In some embodiments, a cell-type enriched microRNA is a skeletal muscle cell-enriched microRNA. Skeletal muscle-enriched microRNAs are any microRNAs which are abundantly expressed in skeletal muscle cells relative to a majority of other cell types (e.g., non-skeletal muscle cells), such that they are not considered ubiquitously expressed. Skeletal muscle cell- enriched microRNAs are known in the art, for example, as described in M. Horak et al. Developmental Biology. 2016 Feb:410(l):l-13. In some embodiments, a skeletal muscle cell- enriched microRNA is miR-1, miR-133, miR-206, miR-208, miR-486, and miR-499. In some embodiments, a skeletal muscle-enriched microRNA is miR-206. In some embodiments, miR- 206 comprises the sequence set forth in SEQ ID NO: 9. In some embodiments, a trRNA and / or a replicase construct of a taRNA comprises a target site for miR-206. In some embodiments, a target site for miR-206 comprises the sequence set forth in SEQ ID NO: 10.

[0089] Additional cell-enriched and / or tissue-enriched miRNA and corresponding target sites are available on the world wide web at ccb-web.cs.uni-saarland.de / tissueatlas2 / tissues; mircarta.cs.uni-saarland.de; and mirbase.org. microRNA target sites in UTRs

[0090] In some embodiments, a trRNA and / or a replicase construct comprises one or more UTRs (e.g., a 3’ UTR and / or a 5’ UTR) and one or more microRNA target sites (e.g., one or more cell-type enriched microRNA target sites). In some embodiments, a trRNA and / or a replicase construct comprises one or more UTRs (e.g., a 3’ UTR and / or a 5’ UTR) and 2 or more microRNA target sites (e.g., two or more cell-type enriched microRNA target sites). In some embodiments, a trRNA and / or a replicase construct comprises one or more UTRs (e.g., a 3’ UTR and / or a 5’ UTR) 3 or more microRNA target sites (e.g., three or more cell-type enriched microRNA target sites). In some embodiments, a trRNA and / or a replicase construct comprises a 3’ UTR and one or more (e.g., 1, 2, 3, or more) microRNA target sites (e.g., cell-type enriched microRNA target sites). In some embodiments, a trRNA and / or a replicase construct comprises a 5’ UTR and one or more (e.g., 1, 2, 3, or more) microRNA target sites (e.g., one or more celltype enriched microRNA target sites). In some embodiments, a trRNA and / or a replicase construct comprises a 3’ UTR, a 5’ UTR, and one or more (e.g., 1, 2, 3, or more) microRNA target sites (e.g., cell-type enriched microRNA target sites).

[0091] In some embodiments, a trRNA and / or a replicase construct comprises a 3’ UTR and one or more (e.g., 2 or more) microRNA target sites (e.g., cell-type enriched microRNA target sites), wherein the one or more microRNA target sites are comprised in the 3’ UTR. In some embodiments, a trRNA and / or a replicase construct comprises a 3’ UTR comprising one or more

[0092] #14495043v1 (e.g., 2 or more) microRNA target sites (e.g., cell-type enriched microRNA target sites), wherein the one or more microRNA target sites are comprised in the 3’ UTR. By inserting one or more (e.g., 2 or more) microRNA target sites (e.g., one or more cell-type enriched microRNA target sites) in a 3’ UTR of an RNA polynucleotide (e.g., a trRNA and / or a replicase construct), translation and / or replication of the RNA polynucleotide can be reduced in an environment in which a cognate microRNA is present. In some embodiments, translation and / or replication of the RNA polynucleotide is reduced but not eliminated.

[0093] This disclosure describes, in some aspects, trRNA and replicase constructs comprising a 3’ UTR having one or more insertions of target sites for a microRNA relative to a given 3’ UTR sequence (e.g., a given wildtype 3’ UTR sequence). In this context, one or more insertions of targets sites may be in any location of the given 3’ UTR sequence, for example, 5’ of (i.e., “upstream of’) the first nucleotide of a given 3’ UTR sequence.

[0094] In some embodiments, a 3’ UTR comprises one or more insertions of target sites for microRNA relative to a wildtype 3’ UTR sequence. In some embodiments, the 3’ UTR comprises one or more insertions of target sites for microRNA (e.g., cell-type enriched microRNA) relative to a wildtype 3’ HAG UTR (e.g., a 3’ HAG UTR of SEQ ID NO: 28). In some embodiments, a 3’ UTR comprises one or more insertions of target sites for microRNA (e.g., a cell-type enriched microRNA) relative to SEQ ID NO: 28. In some embodiments, a 3’ UTR comprises one or more insertions of target sites for microRNA (e.g., a cell-type enriched microRNA) relative to a wildtype 3’ SINV UTR (e.g., a 3’ SINV UTR of SEQ ID NO: 32). In some embodiments, a 3’ UTR comprises one or more insertions of target sites for microRNA (e.g., a cell-type enriched microRNA) relative to SEQ ID NO: 32. In some embodiments, the 3’ UTR comprises one or more insertions of target sites for microRNA (e.g., a cell-type enriched microRNA) relative to a wildtype 3’ SFV UTR (e.g., a 3’ SFV UTR of SEQ ID NO: 36). In some embodiments, a 3’ UTR comprises one or more insertions of target sites for microRNA (e.g., a cell-type enriched microRNA) relative to SEQ ID NO: 36. In some embodiments, a 3’ UTR comprises an insertion of a target sits for microRNA, wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37).

[0095] In some embodiments, a 3’ UTR comprises a plurality of target sites for microRNA. In some embodiments, a 3’ UTR comprises two or more target sites for microRNA (e.g., two or more insertions of target sites for microRNA). In some embodiments, a 3’ UTR comprises a first target site for a first microRNA and a second target site for a second microRNA. In some

[0096] #14495043v1 embodiments, a 3’ UTR comprises a first target site for a first microRNA, a second target site for a second microRNA, and a third target site for a third microRNA. In some embodiments, each target site of a plurality (e.g., the first target site, the second target site, and / or the third target site) of target sites is a target site for a different microRNA. In some embodiments, the plurality of target sites (e.g., the two or more target sites) are target sites for the same microRNA. In some embodiments, one or more of the target sites of the plurality (e.g., two or more) of target sites is a different target site for the same microRNA. Preferably, a 3’ UTR comprises two target sites for a same microRNA. For example, in some embodiments, a 3’ UTR comprises two target sites for a first microRNA and a target site for a second microRNA, wherein the first microRNA and the second microRNA are different microRNAs, and wherein the two target sites for the first microRNA are identical target sites. In some embodiments, the first microRNA and the second microRNA are enriched in different cell types. In some embodiments, the first microRNA and the second microRNA are different microRNAs enriched in the same cell types.

[0097] In some embodiments, a 3’ UTR comprising a plurality of microRNA target sites comprises a spacer sequence between each microRNA target site. As used herein, the term “spacer sequence” refers to a short sequence (e.g., fewer than 10 nucleotides) of non-coding nucleotides which are not part of a microRNA target site. In some embodiments, a spacer sequence comprises 5’-UUUAAA-3’. In some embodiments, a 3’ UTR comprises two or more insertions of target sites wherein a spacer sequence is present between each target site. In some embodiments, a 3’ UTR comprises three or more insertions of target sites wherein a spacer sequence is present between each target site. In some embodiments, a 3’ UTR comprises two or more target sites wherein no spacer sequence is present between each target site.

[0098] In some embodiments, a 3’ UTR comprises two target sites for a cell-type enriched microRNA. In some embodiments, a 3’ UTR comprises two target sites for a hepatocyte- enriched microRNA (e.g., miR-122). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for a hepatocyte-enriched microRNA (e.g., miR-122) relative to a wildtype 3’ HAG UTR (e.g., a 3’ HAG UTR of SEQ ID NO: 28). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for a hepatocyte-enriched microRNA (e.g., miR-122) relative to SEQ ID NO: 28. In some embodiments, a 3’ UTR comprises two or more insertions of target sites for a hepatocyte-enriched microRNA (e.g., miR-122) relative to a wildtype 3’ SINV UTR (e.g., a 3’ SINV UTR of SEQ ID NO: 32). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for a hepatocyte-enriched microRNA (e.g.,

[0099] #14495043v1 miR-122) relative to SEQ ID NO: 32. In some embodiments, a 3’ UTR comprises two or more insertions of target sites for a hepatocyte-enriched microRNA (e.g., miR-122) relative to a wildtype 3’ SFV UTR (e.g., a 3’ SFV UTR of SEQ ID NO: 36). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for a hepatocyte-enriched microRNA (e.g., miR-122) relative to SEQ ID NO: 36. In some embodiments, a 3’ UTR comprises two target sites for a hepatocyte-enriched microRNA and a spacer sequence, wherein the target sites and a spacer sequence are inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 30), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 34), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 38).

[0100] In some embodiments, a 3’ UTR comprises two target sites for an endothelial cell- enriched microRNA (e.g., miR-126). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for an endothelial cell-enriched microRNA (e.g., miR-126) relative to a wildtype 3’ HAG UTR (e.g., a 3’ HAG UTR of SEQ ID NO: 28). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for an endothelial cell microRNA (e.g., miR-126) relative to SEQ ID NO: 28. In some embodiments, a 3’ UTR comprises two or more insertions of target sites for an endothelial cell-enriched microRNA (e.g., miR-126) relative to a wildtype 3’ SINV UTR (e.g., a 3’ SINV UTR of SEQ ID NO: 32). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for an endothelial cell-enriched microRNA (e.g., miR-126) relative to SEQ ID NO: 32. In some embodiments, a 3’ UTR comprises two or more insertions of target sites for an endothelial cell-enriched microRNA (e.g., miR-126) relative to a wildtype 3’ SFV UTR (e.g., a 3’ SFV UTR of SEQ ID NO: 36). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for an endothelial cell- enriched microRNA (e.g., miR-126) relative to SEQ ID NO: 36. In some embodiments, a 3’ UTR comprises two target sites for an endothelial cell-enriched microRNA (e.g., miR-126) and a spacer sequence, wherein the target sites and the spacer sequences are inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 31), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 35), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 39).

[0101] In some embodiments, a 3’ UTR comprises two target sites for a skeletal muscle cell- enriched microRNA (e.g., miR-206). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for a skeletal muscle cell-enriched microRNA (e.g., miR-206) relative to a wildtype 3’ HAG UTR (e.g., a 3’ HAG UTR of SEQ ID NO: 28). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for a skeletal muscle cell-enriched microRNA (e.g., miR-206) relative to SEQ ID NO: 28. In some embodiments, a 3’ UTR comprises two or more insertions of target sites for a skeletal muscle cell-enriched microRNA

[0102] #14495043v1 (e.g., miR-206) relative to a wildtype 3’ SINV UTR (e.g., a 3’ SINV UTR of SEQ ID NO: 32). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for a skeletal muscle cell-enriched microRNA (e.g., miR-206) relative to SEQ ID NO: 32. In some embodiments, a 3’ UTR comprises two or more insertions of target sites for a skeletal muscle cell-enriched microRNA (e.g., miR-206) relative to a wildtype 3’ SFV UTR (e.g., a 3’ SFV UTR of SEQ ID NO: 36). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for a skeletal muscle cell-enriched microRNA (e.g., miR-206) relative to SEQ ID NO: 36. In some embodiments, a 3’ UTR comprises two target sites for a skeletal muscle cell- enriched microRNA (e.g., miR-206) and a spacer sequence, wherein the target sites and the spacer sequence are inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 30), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 34), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 38).

[0103] In some embodiments, a 3’ UTR comprises two target sites for an immune cell-enriched microRNA (e.g., miR-142, miR-223). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for an immune cell-enriched microRNA (e.g., miR-142, miR-223) relative to a wildtype 3’ HAG UTR (e.g., a 3’ HAG UTR of SEQ ID NO: 28). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for an immune cell- enriched microRNA (e.g., miR-142, miR-223) relative to SEQ ID NO: 28. In some embodiments, a 3’ UTR comprises two or more insertions of target sites for an immune cell- enriched microRNA (e.g., miR-142, miR-223) relative to a wildtype 3’ SINV UTR (e.g., a 3’ SINV UTR of SEQ ID NO: 32). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for an immune cell-enriched microRNA (e.g., miR-142, miR-223) relative to SEQ ID NO: 32. In some embodiments, a 3’ UTR comprises two or more insertions of target sites for an immune cell-enriched microRNA (e.g., miR-142, miR-223) relative to a wildtype 3’ SFV UTR (e.g., a 3’ SFV UTR of SEQ ID NO: 36). In some embodiments, a 3’ UTR comprises two or more insertions of target sites for an immune cell-enriched microRNA (e.g., miR-142, miR-223) relative to SEQ ID NO: 36. In some embodiments, a 3’ UTR comprises two target sites for an immune cell-enriched microRNA (e.g., miR-142, miR-223) and a spacer sequence, wherein the target sites and the spacer sequence are inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 30), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 34), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 38).

[0104] In some embodiments, a 3’ UTR comprises a target site for a hepatocyte-enriched microRNA (e.g., miR-122) and a target site for an endothelial cell-enriched microRNA (e.g., miR-126). In some embodiments, a 3’ UTR comprises a target site for a hepatocyte-enriched

[0105] #14495043v1 microRNA (e.g., miR-122) and a target site for an immune cell-enriched microRNA (e.g., miR- 142, miR-223, miR-652). In some embodiments, a 3’ UTR comprises a target site for a hepatocyte-enriched microRNA (e.g., miR-122) and a target site for a skeletal muscle cell- enriched microRNA (e.g., miR-206). In some embodiments, a 3’ UTR comprises a target site for an endothelial cell-enriched microRNA (e.g., miR-126) and a target site for an immune cell- enriched microRNA (e.g., miR-142, miR-223, miR-652). In some embodiments, a 3’ UTR comprises a target site for an endothelial cell-enriched microRNA (e.g., miR-126) and a target site for a skeletal muscle cell-enriched microRNA (e.g., miR-206, miR-1). In some embodiments, a 3’ UTR comprises a target site for an immune cell-enriched microRNA (e.g., miR-142, miR-223, miR-652) and a target site for a skeletal muscle cell-enriched microRNA (e.g., miR-206, miR-1). In some embodiments, the target sites are separated by a spacer sequence.

[0106] In some embodiments, a 3’ UTR comprises two target sites for a hepatocyte-enriched microRNA (e.g., miR-122) and a target site for an endothelial cell-enriched microRNA (e.g., miR-126). In some embodiments, a 3’ UTR comprises two target sites for a hepatocyte-enriched microRNA (e.g., miR-122) and a target site for an immune cell-enriched microRNA (e.g., miR- 142, miR-223). In some embodiments, a 3’ UTR comprises two target sites for a hepatocyte- enriched microRNA (e.g., miR-122) and a target site for a skeletal muscle cell-enriched microRNA (e.g., miR-206). In some embodiments, a 3’ UTR comprises two target sites for an endothelial cell-enriched microRNA (e.g., miR-126) and a target site for an immune cell- enriched microRNA (e.g., miR-142, miR-223). In some embodiments, a 3’ UTR comprises two target sites for an endothelial cell-enriched microRNA (e.g., miR-126) and a target site for a skeletal muscle cell-enriched microRNA (e.g., miR-206). In some embodiments, a 3’ UTR comprises two target sites for an immune cell-enriched microRNA (e.g., miR-142, miR-223) and a target site for a skeletal muscle cell-enriched microRNA (e.g., miR-206). In some embodiments, the target sites are separated by spacer sequences.

[0107] In some embodiments, a 3’ UTR comprises a target site for miR-142 and a target site for miR-223. In some embodiments, a 3’ UTR comprises a target site for miR-142 and a target site for miR-652. In some embodiments, a 3’ UTR comprises a target site for miR-142 and a target site for miR-126. In some embodiments, a 3’ UTR comprises a target site for miR-142 and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-142 and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-223 and a target site for miR-652. In some embodiments, a 3’ UTR comprises a target site for miR-

[0108] #14495043v1 223 and a target site for miR-126. In some embodiments, a 3’ UTR comprises a target site for miR-223 and a target site for miR-122. In some embodiments, a 3’ UTR comprises a target site for miR-223 and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-223 and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-652 and a target site for miR-126. In some embodiments, a 3’ UTR comprises a target site for miR-652 and a target site for miR-122. In some embodiments, a 3’ UTR comprises a target site for miR-652 and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-652 and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-126 and a target site for miR-122. In some embodiments, a 3’ UTR comprises a target site for miR-126 and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-126 and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-122 and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-122 and a target site for miR-1.

[0109] In some embodiments, the target sites are separated by spacer sequences.

[0110] In some embodiments, a 3’ UTR comprises three or more target sites for microRNA (e.g., three or more insertions of target sites for microRNA). In some embodiments, a 3’ UTR comprises three target sites for a cell-type enriched microRNA. In some embodiments, the target sites are separated by spacer sequences. In some embodiments, the 3’ UTR comprises a first microRNA target site, a second microRNA target site and a third microRNA target site. In some embodiments, the first microRNA target site, the second microRNA target site and the third microRNA target site are the same microRNA target site. In some embodiments, the first microRNA target site, the second microRNA target site and the third microRNA target site are different microRNA target sites. In some embodiments, the first microRNA target site and the second microRNA target site are the same microRNA target site, and the third microRNA target site is a different microRNA target site.

[0111] In some embodiments, a 3’ UTR comprises a target site for a hepatocyte-enriched microRNA (e.g., miR-122), a target site for an endothelial cell-enriched microRNA (e.g., miR- 126), and a target site for an immune cell-enriched microRNA (e.g., miR-142, miR-223, miR- 652). In some embodiments, a 3’ UTR comprises a target site for a hepatocyte-enriched microRNA (e.g., miR-122), a target site for an endothelial cell-enriched microRNA (e.g., miR- 126), and a target site for a skeletal muscle cell-enriched microRNA (e.g., miR-206, miR-1). In some embodiments, a 3’ UTR comprises a target site for a hepatocyte-enriched enriched microRNA (e.g., miR-122), a target site for an immune cell-enriched microRNA (e.g., miR-142,

[0112] #14495043v1 miR-223, miR-652), and a target site for a skeletal muscle cell-enriched microRNA (e.g., miR- 206, miR-1). In some embodiments, a 3’ UTR comprises a target site for an endothelial cell- enriched microRNA (e.g., miR-126), a target site for an immune cell-enriched microRNA (e.g., miR-142, miR-223, miR-652), and a target site for a skeletal muscle cell-enriched microRNA (e.g., miR-206, miR-1). In some embodiments, the target sites are separated by spacer sequences.

[0113] In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-126, and miR-142. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-126, and miR-223. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-126, and miR-652. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-126, and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-126, and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-142, and a target site for miR-223. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-142, and a target site for miR-652. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-142, and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-142, and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-223, and a target site for miR-652. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-223, and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-223, and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-652, and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-652, and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-122, a target site for miR-206, and a target site for miR-1.

[0114] In some embodiments, a 3’ UTR comprises a target site for miR-126, a target site for miR-142, and a target site for miR-223. In some embodiments, a 3’ UTR comprises a target site for miR-126, a target site for miR-142, and a target site for miR-652. In some embodiments, a 3’ UTR comprises a target site for miR-126, a target site for miR-142, and a target site for miR- 206. In some embodiments, a 3’ UTR comprises a target site for miR-126, a target site for miR- 142, and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR- 126, a target site for miR-223, and a target site for miR-652. In some embodiments, a 3’ UTR

[0115] #14495043v1 comprises a target site for miR-126, a target site for miR-223, and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-126, a target site for miR-223, and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-126, a target site for miR-652, and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-126, a target site for miR-652, and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-126, a target site for miR-206, and a target site for miR-1.

[0116] In some embodiments, a 3’ UTR comprises a target site for miR-142, a target site for miR-223, and a target site for miR-652. In some embodiments, a 3’ UTR comprises a target site for miR-142, a target site for miR-223, and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-142, a target site for miR-223, and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-142, a target site for miR-652, and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR- 142, a target site for miR-652, and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-142, a target site for miR-206, and a target site for miR-1.

[0117] In some embodiments, a 3’ UTR comprises a target site for miR-223, a target site for miR-652, and a target site for miR-206. In some embodiments, a 3’ UTR comprises a target site for miR-223, a target site for miR-652, and a target site for miR-1. In some embodiments, a 3’ UTR comprises a target site for miR-223, a target site for miR-206, and a target site for miR-1.

[0118] In some embodiments, the target sites are separated by spacer sequences.

[0119] Exemplary 3 ’ UTR Sequences

[0120] In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising one or more target sites for a cell-type enriched microRNA. In some embodiments, the 3’ UTR comprises one or more insertions of target sites for cell-type enriched microRNA relative to a wildtype 3’ HAG UTR (e.g., a 3’ HAG UTR of SEQ ID NO: 28). In some embodiments, a 3’ UTR comprises one or more insertions of target sites for cell-type enriched microRNA relative to SEQ ID NO: 28. In some embodiments, a 3’ UTR comprises one or more insertions of target sites for cell-type enriched microRNA relative to a wildtype 3’ SINV UTR (e.g., a 3’ SINV UTR encoded by SEQ ID NO: 32). In some embodiments, a 3’ UTR comprises one or more insertions of target sites for cell-type enriched microRNA relative to SEQ ID NO: 32. In some embodiments, the 3’ UTR comprises one or more insertions of target sites for celltype enriched microRNA relative to a wildtype 3’ SFV UTR (e.g., a 3’ SFV UTR encoded by

[0121] #14495043v1 SEQ ID NO: 36). In some embodiments, a 3’ UTR comprises one or more insertions of target sites for cell-type enriched microRNA relative to SEQ ID NO: 36.

[0122] In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising one or more target sites for hepatocyte-enriched microRNA. In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising one or more insertions of target sites for hepatocyte-enriched microRNA. In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising one or more insertions of target sites for hepatocyte-enriched microRNA, wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising a target site for miR-122 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 1). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-122 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 2). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR- 122 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 1), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-122 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 2), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37).

[0123] In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising a target site for an endothelial cell-enriched microRNA. In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising one or more insertions of target sites for an endothelial cell-enriched microRNA. In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising one or more insertions of target sites for an endothelial cell-enriched microRNA, wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising a target site for miR-126 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 3). In some embodiments, a replicase

[0124] #14495043v1 construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR- 126 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 4). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-126 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 3), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-126 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 4), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37).

[0125] In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising one or more target sites for an immune cell-enriched microRNA. In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising one or more insertions of target sites for an immune cell-enriched microRNA. In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising one or more insertions of target sites for an immune cell-enriched microRNA, wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising a target site for miR-142 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 5). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-142 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 6). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-142 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 5), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-142 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 6), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising a target site for miR-223 (e.g., a microRNA comprising

[0126] #14495043v1 the sequence set forth in SEQ ID NO: 7). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-223 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 8). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR- 223 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 7), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-223 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 8), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37).

[0127] In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising one or more target sites for a skeletal muscle-enriched microRNA. In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising one or more insertions of target sites for a skeletal muscle-enriched microRNA. In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising one or more insertions of target sites for a skeletal muscle-enriched microRNA, wherein the one or more target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising a target site for miR-206 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 9). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-206 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 10). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-206 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 9), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-206 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 10), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37).

[0128] #14495043v1 In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising a target site for a cell-type enriched microRNA. In some embodiments, the 3’ UTR comprises an insertion of a target site for a cell-type enriched microRNA relative to a wildtype 3’ HAG UTR (e.g., a 3’ HAG UTR encoded by SEQ ID NO: 28). In some embodiments, a 3’ UTR comprises an insertion of a target site for a cell-type enriched microRNA relative to SEQ ID NO: 28. In some embodiments, a 3’ UTR comprises an insertion of a target site for a cell-type enriched microRNA relative to a wildtype 3’ SINV UTR (e.g., a 3’ SINV UTR encoded by SEQ ID NO: 32). In some embodiments, a 3’ UTR comprises an insertion of a target site for a cell-type enriched microRNA relative to SEQ ID NO: 36. In some embodiments, the 3’ UTR comprises an insertion of a target site for a cell-type enriched microRNA relative to a wildtype 3’ SFV UTR (e.g., a 3’ SFV UTR encoded by SEQ ID NO: 36). In some embodiments, a 3’ UTR comprises an insertion of a target site for a cell-type enriched microRNA relative to SEQ ID NO: 36.

[0129] In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising a target site for a hepatocyte-enriched microRNA. In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for a hepatocyte-enriched microRNA. In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for a hepatocyte-enriched microRNA, wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising a target site for miR-122 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 1). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-122 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 2). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-122 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 1), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-122 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 2), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37).

[0130] #14495043v1 In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising a target site for an endothelial cell-enriched microRNA. In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for an endothelial cell- enriched microRNA. In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for an endothelial cell-enriched microRNA, wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising a target site for miR-126 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 3). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-126 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 4). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-126 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 3), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-126 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 4), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37).

[0131] In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising a target site for an immune cell-enriched microRNA. In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for an immune cell-enriched microRNA. In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for an immune cell-enriched microRNA, wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising a target site for miR- 142 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 5). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-142 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 6). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-142 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 5),

[0132] #14495043v1 wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-142 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 6), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising a target site for miR-223 (e.g., a micro RNA comprising the sequence set forth in SEQ ID NO: 7). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-223 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 8). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-223 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 7), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-223 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 8), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37).

[0133] In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising a target site for a skeletal muscle-enriched microRNA. In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for a skeletal muscle-enriched microRNA. In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for a skeletal muscle-enriched microRNA, wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising a target site for miR- 206 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 9). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-206 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 10). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-206 (e.g., a microRNA comprising the sequence set forth in SEQ ID NO: 9), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’

[0134] #14495043v1 SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37). In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for miR-206 (e.g., a target site comprising the sequence set forth in SEQ ID NO: 10), wherein the target site is inserted into a 3’ HAG UTR (e.g., as shown in SEQ ID NO: 29), a 3’ SINV UTR (e.g., as shown in SEQ ID NO: 33), or a 3’ SFV UTR (e.g., as shown in SEQ ID NO: 37).

[0135] In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising an insertion of a target site for a first cell-type enriched microRNA and an insertion of a target site for a second cell-type enriched microRNA. In some embodiments, a trRNA disclosed herein comprises a 3’ UTR comprising a target site for a first cell-type enriched microRNA and a target site for a second cell-type enriched microRNA.

[0136] In some embodiments, the first cell-type enriched microRNA is a hepatocyte-enriched microRNA and the second cell-type enriched microRNA is an immune cell-enriched microRNA. In some embodiments, the 3’ UTR comprises one target site for a hepatocyte- enriched microRNA and one target site for an immune cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two identical target sites for a hepatocyte-enriched microRNA and one target site for an immune cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two different target sites for a hepatocyte-enriched microRNA and one target site for an immune cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two target sites for a hepatocyte-enriched microRNA and two target sites for an immune cell-enriched microRNA. In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR-142. In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR-142, wherein the target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR-142, wherein the two target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2 and SEQ ID NO: 6. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2, a spacer sequence comprising UUUAAA, and SEQ ID NO: 6. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2, a first spacer sequence comprising UUUAAA, SEQ ID NO: 2, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 6. In some embodiments, the 3’ UTR comprises SEQ ID NO: 6, a first spacer sequence comprising UUUAAA, SEQ ID NO: 6, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 2. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2 and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2, a

[0137] #14495043v1 spacer sequence comprising UUUAAA, and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2, a first spacer sequence comprising UUUAAA, SEQ ID NO: 2, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 4, a first spacer sequence comprising UUUAAA, SEQ ID NO: 4, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 2. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2, a first spacer sequence comprising UUUAAA, SEQ ID NO: 6, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 4.

[0138] In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR-223. In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR-223, wherein the target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR- 223, wherein the two target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2 and SEQ ID NO: 8. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2, a spacer sequence comprising UUUAAA, and SEQ ID NO: 8. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2, a first spacer sequence comprising UUUAAA, SEQ ID NO: 2, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 8. In some embodiments, the 3’ UTR comprises SEQ ID NO: 6, a first spacer sequence comprising UUUAAA, SEQ ID NO: 8, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 2.

[0139] In some embodiments, the first cell-type enriched microRNA is a hepatocyte-enriched microRNA and the second cell-type enriched microRNA is an endothelial cell-enriched microRNA. In some embodiments, the 3’ UTR comprises one target site for a hepatocyte- enriched microRNA and one target site for an endothelial cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two identical target sites for a hepatocyte-enriched microRNA and one target site for an endothelial cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two different target sites for a hepatocyte-enriched microRNA and one target site for an endothelial cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two target sites for a hepatocyte-enriched microRNA and two target sites for an endothelial cell-enriched microRNA. In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR-126. In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR-126, wherein the

[0140] #14495043v1 target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR-126, wherein the two target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2 and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2, a spacer sequence comprising UUUAAA, and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2, a first spacer sequence comprising UUUAAA, SEQ ID NO: 2, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 4, a first spacer sequence comprising UUUAAA, SEQ ID NO: 4, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 2.

[0141] In some embodiments, the first cell-type enriched microRNA is a hepatocyte-enriched microRNA and the second cell-type enriched microRNA is a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises one target site for a hepatocyte- enriched microRNA and one target site for a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two identical target sites for a hepatocyte-enriched microRNA and one target site for a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two different target sites for a hepatocyte-enriched microRNA and one target site for a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two target sites for a hepatocyte-enriched microRNA and two target sites for a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR-206. In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR- 206, wherein the target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR-206, wherein the two target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2 and SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2, a spacer sequence comprising UUUAAA, and SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises SEQ ID NO: 2, a first spacer sequence comprising UUUAAA, SEQ ID NO: 2, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises SEQ ID NO: 10, a first spacer sequence comprising UUUAAA, SEQ ID NO: 10, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 2.

[0142] In some embodiments, the first cell-type enriched microRNA is an immune cell-enriched microRNA and the second cell-type enriched microRNA is an endothelial cell-enriched microRNA. In some embodiments, the 3’ UTR comprises one target site for an immune cell-

[0143] #14495043v1 enriched microRNA and one target site for an endothelial cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two identical target sites for an immune cell-enriched microRNA and one target site for an endothelial cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two different target sites for an immune cell-enriched microRNA and one target site for an endothelial cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two target sites for an immune cell-enriched microRNA and two target sites for an endothelial cell-enriched microRNA. In some embodiments, the 3’ UTR comprises one target site for miR-142 and one target site for miR-126. In some embodiments, the 3’ UTR comprises one target site for miR-142 and one target site for miR- 126, wherein the target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises one target site for miR-142 and one target site for miR-126, wherein the two target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises SEQ ID NO: 6 and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 6, a spacer sequence comprising UUUAAA, and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 6, a first spacer sequence comprising UUUAAA, SEQ ID NO: 6, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 4, a first spacer sequence comprising UUUAAA, SEQ ID NO: 4, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 6. In some embodiments, the 3’ UTR comprises one target site for miR-223 and one target site for miR-126. In some embodiments, the 3’ UTR comprises one target site for miR-223 and one target site for miR- 126, wherein the target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises one target site for miR-223 and one target site for miR-126, wherein the two target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises SEQ ID NO: 8 and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 8, a spacer sequence comprising UUUAAA, and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 8, a first spacer sequence comprising UUUAAA, SEQ ID NO: 8, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 4. In some embodiments, the 3’ UTR comprises SEQ ID NO: 4, a first spacer sequence comprising UUUAAA, SEQ ID NO: 4, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 8.

[0144] In some embodiments, the first cell-type enriched microRNA is an immune cell-enriched microRNA and the second cell-type enriched microRNA is a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises one target site for an immune cell- enriched microRNA and one target site for a skeletal muscle cell-enriched microRNA. In some

[0145] #14495043v1 embodiments, the 3’ UTR comprises two identical target sites for an immune cell-enriched microRNA and one target site for a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two different target sites for an immune cell-enriched microRNA and one target site for a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two target sites for an immune cell-enriched microRNA and two target sites for a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises one target site for miR-122 and one target site for miR-206. In some embodiments, the 3’ UTR comprises one target site for miR-142 and one target site for miR- 206, wherein the target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises one target site for miR-124 and one target site for miR-206, wherein the two target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises SEQ ID NO: 6 and SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises SEQ ID NO: 6, a spacer sequence comprising UUUAAA, and SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises SEQ ID NO: 6, a first spacer sequence comprising UUUAAA, SEQ ID NO: 6, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises SEQ ID NO: 10, a first spacer sequence comprising UUUAAA, SEQ ID NO: 10, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 6. In some embodiments, the 3’ UTR comprises one target site for miR-223 and one target site for miR-206. In some embodiments, the 3’ UTR comprises one target site for miR-223 and one target site for miR- 206, wherein the target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises one target site for miR-223 and one target site for miR-206, wherein the two target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises SEQ ID NO: 8 and SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises SEQ ID NO: 8, a spacer sequence comprising UUUAAA, and SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises SEQ ID NO: 8, a first spacer sequence comprising UUUAAA, SEQ ID NO: 8, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises SEQ ID NO: 10, a first spacer sequence comprising UUUAAA, SEQ ID NO: 4, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 8.

[0146] In some embodiments, the first cell-type enriched microRNA is an endothelial cell- enriched microRNA and the second cell-type enriched microRNA is a skeletal muscle cell- enriched microRNA. In some embodiments, the 3’ UTR comprises one target site for an endothelial cell-enriched microRNA and one target site for a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two identical target sites for an

[0147] #14495043v1 endothelial cell microRNA and one target site for a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two different target sites for an endothelial cell microRNA and one target site for a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises two target sites for an endothelial cell microRNA and two target sites for a skeletal muscle cell-enriched microRNA. In some embodiments, the 3’ UTR comprises one target site for miR-126 and one target site for miR-206. In some embodiments, the 3’ UTR comprises one target site for miR-126 and one target site for miR-206, wherein the target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises one target site for miR-126 and one target site for miR-206, wherein the two target sites are separated by a spacer sequence. In some embodiments, the 3’ UTR comprises SEQ ID NO: 4 and SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises SEQ ID NO: 4, a spacer sequence comprising UUUAAA, and SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises SEQ ID NO: 4, a first spacer sequence comprising UUUAAA, SEQ ID NO: 4, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 10. In some embodiments, the 3’ UTR comprises SEQ ID NO: 10, a first spacer sequence comprising UUUAAA, SEQ ID NO: 10, a second spacer sequence comprising UUUAAA, and SEQ ID NO: 4.

[0148] In some embodiments, a replicase construct disclosed herein comprises a 3’ UTR comprising the sequence set forth in any one of the sequences shown in Table 3 (e.g., any one of SEQ ID NOs: 28-39).

[0149] Methods of Use

[0150] By engineering microRNA target sequences into a UTR of trRNAs and / or replicase constructs described herein, taRNAs (e.g., trRNA of a taRNA, replicase construct of a taRNA) can be selectively targeted for degradation and / or reduced expression in environments (e.g., cells, tissues) in which a cognate microRNA is present. As used herein, the terms “selective” and “selectively” refer to the ability of a method or composition to produce an effect (e.g., reduce expression of a taRNA) compared to a reference method or composition. For example, expression of a taRNA comprising a target site for a microRNA can be considered to be “selectively” reduced if its expression is reduced to a degree that is distinguishable from expression of a reference taRNA (not comprising a target site for a microRNA) in a similar context (e.g., similar environment and similar conditions). In another example, a taRNA comprising a target site for a microRNA can be considered to be “selectively targeted” for

[0151] #14495043v1 degradation if it is degraded to a degree that is distinguishable from expression of a reference taRNA in a similar context.

[0152] In some aspects, this disclosure relates to methods for selectively expressing a taRNA in a first cell relative to a second cell. In some embodiments, the method comprises contacting a first cell and a second cell with a taRNA comprising: a replicase construct comprising a nucleic acid encoding a replicase and a target site for microRNA; and a trRNA comprising a nucleic acid encoding a payload, and a conserved sequence element (CSE) cognate to the replicase; wherein the microRNA is expressed by the second cell and not expressed by the first cell. In some embodiments, the method comprises contacting a first cell and a second cell with a taRNA comprising: a replicase construct comprising a nucleic acid encoding a replicase; and a trRNA comprising a nucleic acid encoding a payload, a conserved sequence element (CSE) cognate to the replicase, and a target site for a microRNA; wherein the microRNA is expressed by the second cell and not expressed by the first cell. In some embodiments, the second cell is a hepatocyte. In some embodiments, the second cell is an endothelial cell. In some embodiments, the second cell is an immune cell. In some embodiments, the second cell is a skeletal muscle cell.

[0153] In some embodiments, expression of an RNA polynucleotide described herein (e.g., a trRNA and / or a replicase construct comprising a UTR comprising one or more microRNA target sites) is selectively reduced in one or more environments (e.g., one or more cell types) in which a cognate microRNA is present. In some embodiments, expression of a trRNA and / or a replicase construct comprising one or more microRNA target sites is reduced by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least

[0154] 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least

[0155] 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in an environment in which a cognate microRNA is present, compared to a reference trRNA and / or replicase construct in which the microRNA target site is absent or in much lower abundance (e.g., at least 100-fold less) than the first environment. In some embodiments, expression of a trRNA and / or a replicase construct comprising a 3’ UTR comprising one or more microRNA target sites is reduced by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in an environment in which a cognate microRNA is present, compared to a reference trRNA and / or replicase construct in which the UTR is absent.

[0156] #14495043v1 In some aspects, this disclosure relates to methods for reducing the immunogenicity of a taRNA in a subject. In some embodiments, the method comprising administering a taRNA to the subject, the taRNA comprising: a replicase construct comprising a nucleic acid encoding a replicase and a target site for microRNA; and a trRNA comprising a nucleic acid encoding a payload, and a conserved sequence element (CSE) cognate to the replicase; wherein the microRNA is not expressed by a target cell of the subject and is expressed by at least one nontarget cell of the subject. In some embodiments, the method comprising administering a taRNA to the subject, the taRNA comprising: a replicase construct comprising a nucleic acid encoding a replicase; and a trRNA comprising a nucleic acid encoding a payload, a conserved sequence element (CSE) cognate to the replicase, and a target site for microRNA; wherein the microRNA is not expressed by a target cell of the subject and is expressed by at least one non-target cell of the subject. In some embodiments, the non-target cell is a hepatocyte. In some embodiments, the non-target cell is an endothelial cell. In some embodiments, the non-target cell is an immune cell. In some embodiments, the non-target cell is a skeletal muscle cell.

[0157] In some embodiments, reducing the immunogenicity of the taRNA comprises reducing expression of one or more interferon (IFN), chemokine, and / or interleukin that are associated with an immune response of the cell (e.g., the non-target cell) and / or the subject to the taRNA.

[0158] Interferons (IFNs) are molecules which interfere with viral replication in mammalian cells. Once non-self-nucleic acids are recognized in a cell (e.g., by pattern recognition receptors), activated IFN can act as a signal for further gene transcription (e.g., of interferon stimulated genes (ISGs)) and for responses by neighboring cells. IFN-mediated signaling can lead to degradation of non-self-nucleic acids (e.g., of taRNA in a mammalian cell), as well as other immune and / or inflammatory responses. In some embodiments, reducing the immunogenicity of the taRNA comprises reducing expression of one or more IFNs. In some embodiments, the interferon is IFN-a, IFN-P, or IFN-y. In some embodiments, reducing the immunogenicity comprises significantly decreasing expression of IFN-a. In some embodiments, reducing the immunogenicity comprises significantly decreasing expression of IFN- p. In some embodiments, reducing the immunogenicity does not comprise significantly decreasing expression of IFN-y. In some embodiments, reducing the immunogenicity comprises significantly decreasing expression of IFN-a and / or IFN-P, but not IFN-y.

[0159] Chemokines are a type of cytokine that stimulates movement of immune cells (e.g., towards sites of inflammation). Recruitment of immune cells towards sites in which non-self- nucleic acids are present (e.g., injection sites, transfection sites) can lead to degradation of non-

[0160] #14495043v1 self-nucleic acids (e.g., of taRNA in a mammalian cell), as well as other immune and / or inflammatory responses. In some embodiments, reducing the immunogenicity of the taRNA comprises reducing expression of one or more chemokines. In some embodiments, the chemokine is keratinocyte chemoattractant (KC) / human growth-regulated oncogene (GRO) chemokine, monocyte chemoattractant protein- 1 (MCP-1), interferon gamma-induced protein 10 (IP- 10), or macrophage inflammatory protein la (MIP-la).

[0161] Interleukins are a type of cytokine that are expressed and secreted by immune cells; many play important roles in the differentiation, maturation, and induction of immune cells (e.g., B cells, T cells). In some embodiments, the interleukin is IL-6. IL-6, also known as B-cell stimulatory factor-2 (BSF-2) and interferon beta-2, is critical for differentiation of B cells and several other cell types, and acute phase reactants in hepatocytes.

[0162] In some embodiments, immunogenicity of an RNA polynucleotide described herein (e.g., a trRNA and / or a replicase construct comprising a UTR comprising one or more microRNA target sites) is reduced in a subject. In some embodiments, immunogenicity of a trRNA and / or a replicase construct is reduced in a subject to whom the trRNA and / or replicase construct have been administered by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% compared to a subject to whom a reference trRNA and / or replicase construct is administered.

[0163] In some embodiments, this disclosure describes a cell comprising one or more of any of the taRNAs described herein. In some aspects, the taRNAs described herein may be used to express a payload in a mammalian cell. Mammalian cells may be derived from any mammal, including, but not limited to, mice, hamsters, pigs, cows, sheep, goats, horses, and primates, including humans. In some embodiments, the taRNA described herein are used to express payloads in isolated cells from established cell lines generally known in the art, such as 3T3, A549, BHK21, C127, CHO, HeLa, HEK, HT-1080, Huh7, Jurkat, NS0, PER.C6, Sp2 / 0, Vero, and derivatives thereof. In some embodiments, the taRNA described herein are used to express payloads in cells isolated from a subject (e.g., immune cells).

[0164] In some embodiments, the taRNAs described herein are transfected into a cell. The term “transfection” refers to the process by which cells uptake foreign polynucleotides into the cytoplasm, in the absence of viral vectors. Non-limiting examples of transfection include electroporation, heat shock, liposome-mediated delivery, nanoparticle-mediated delivery,

[0165] #14495043v1 microinjections, sonoporation, photoporation, magnetofection, hydroporation, biolistics, continuous infusion, impalefection, and any technique known to those of ordinary skill in the art. In some embodiments, a cell transfected with a taRNA is transfected with the replicase construct and trans replicon construct concurrently (i.e., at the same time). In some embodiments, a cell transfected with a taRNA is transfected with the replicase construct and trans replicon construct sequentially; for example, the replicase construct may be transfected at a first time point and a trans replicon construct transfected at a second, later, time point. Once one or more foreign polynucleotides have entered the cytoplasm of a cell, the polynucleotides may be expressed by the cell. In some embodiments, a cell transfected with a taRNA expresses the replicase encoded by the replicase construct. In some embodiments, a cell transfected with a taRNA expresses (i.e., translates) the payload encoded by the trans replicon construct or its replicants.

[0166] In some aspects, the taRNAs described herein may be used to express a payload in a cell (e.g., a cell of subject). taRNAs described herein may be administered to a subject using any method known to those of ordinary skill in the art, such as injection (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous). In some embodiments, the taRNAs described herein are formulated as a pharmaceutical composition. A “pharmaceutical composition” refers to a composition comprising taRNAs formulated with one or more pharmaceutically acceptable excipients. In some embodiments, taRNAs are formulated in a carrier (e.g., lipidoid, liposome, lipid nanoparticle, polymer, lipoplex, ligand). In some embodiments, one or more cells transfected with taRNAs may be administered to the subject.

[0167] In some embodiments, taRNAs are administered to mammalian subjects. Non-limiting examples of mammalian subjects include mice, hamsters, pigs, cows, sheep, goats, horses, and primates (e.g., humans, non-human primates).

[0168] In some embodiments, taRNAs are administered to human subjects.

[0169] In some embodiments, taRNAs are expressed in a cell-free system, for example, using in vitro transcription.

[0170] EXEMPLARY SEQUENCES

[0171] Table 1. Exemplary MicroRNA and MicroRNA Target Site Sequences

[0172] #14495043v1

[0173] Table 2, Exemplary 5’ UTR Sequences

[0174] #14495043v1

[0175] Table 3. Exemplary 3’ UTR Sequences

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[0177] Table 4, Exemplary Replicase Sequences

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[0203] EXAMPLES

[0204] Example 1: Systemic transfection and expression patterns resulting from taRNA administration in vivo are unique relative to mRNA administration In this example, differences between transfection and expression of payloads encoded by mRNA and taRNA were assessed in mice. Adult mice (N = 5 per group) received intramuscular (IM) injections of mRNA-lipid nanoparticle (LNP) (mRNA-LNP) compositions or taRNA-LNP compositions of ALC-315 LNPs and 5pg of either mRNA or taRNA (lacking any target sites for microRNA) encoding luciferase (Flue), respectively. Flue expression was assessed in anesthetized mice (IP: 90mg / kg Ketamine, lOmg / kg Xylazine) and full body expression of luciferase was obtained using 2D fluorescence imaging (FIG. 1A). As shown in FIG. IB, expression of luciferase in mice injected with mRNA-LNPs and taRNA-LNPs was similar when averaged across the whole body; however, when sampled in the injection site (hind leg), taRNA- mediated luciferase expression was significantly higher than mRNA-mediated luciferase

[0205] #14495043v1 expression. Luciferase expression also varied in the liver, where taRNA-mediated decreased expression was significantly reduced relative to mRNA.

[0206] In a similar experiment, 5pg mRNAs or taRNAs (lacking any target sites for microRNA) encoding secreted embryonic alkaline phosphatase (SEAP) and formulated with LNPs (as mRNA-LNPs or taRNA-LNPs) were administered into mice via IM or intravenous (IV) injection. As shown in FIG. 2A, taRNA-LNPs were highly expressive when administered via IM injection, but poorly expressive after IV injection. Mice receiving IV taRNA-LNPs also exhibited dramatic losses of bodyweight at 24 and 72 hours compared to mice injected with mRNA vaccines (FIG. 2B), suggesting an administration route-specific cytotoxic effect. Indeed, cytokine and chemokine levels in the sera of IV taRNA-LNP-injected mice were significantly elevated at 24 hours compared to levels in mice receiving any IM administration, or IV mRNA- LNP administration (FIG. 2C).

[0207] To determine possible causes for these expression and transfection patterns, mice were injected with compositions of ALC-315 LNPs and one of the following: mRNAs encoding SEAP only (mRNA-LNP)s, taRNAs consisting of a trRNA encoding SEAP and a replicase construct encoding WT SFV replicase (taRNA-LNPs); trRNA encoding SEAP only (no replicase; trRNA-LNPs), or replicase constructs only (no trRNA or mRNA encoding SEAP; replicase-LNP). Induction of interferon gamma induced protein 10 (IP- 10; also known as CXCL10) expression was assessed after 6 hours and 24 hours. Notably, mice injected with replicase-LNPs induced similar levels of IP- 10 expression at 6 hours and 24 hours as mice injected with taRNA-LNPs (FIG. 3A). These findings suggest that the cytotoxic and immunogenic effects of taRNA may be due to the replicase construct.

[0208] A similar experiment was conducted on wildtype (C57BL / 6) mice treated with 2mg / kg dexamethasone, a glucocorticoid with anti-inflammatory effects, and interferon A receptor (IFNAR) knockout (IFNAR KO) mice, which are expected to have a reduced IFN-mediated immune response. Surprisingly, despite decreasing mRNA-induced IP- 10 expression, dexamethasone did not decrease taRNA-induced cytokine induction at 24 hours (FIG. 3B); similarly, replicase constructs induced significant IP- 10 expression even in IFNAR KO mice, while no IP-10 expression was observed in IFNAR KO mice treated with mRNA.

[0209] Overall, these findings indicate that taRNA and mRNA differ in transfection / expression patterns across multiple tissues (e.g., skeletal muscle, liver), and that replicase constructs of taRNA induce unique cytokine expression relative to mRNA.

[0210] #14495043v1 Example 2: Replicase construct-induced cytokine expression is reduced when cell-type enriched microRNA target sites are present in 3’ UTRs in vitro

[0211] In this Example, microRNA target sites were inserted into 3’ untranslated regions (UTRs) of replicase constructs of taRNAs encoding luciferase. taRNAs had a trRNA having a 5’ SINV UTR and a nucleic acid encoding luciferase and a replicase construct having a 3’ UTR having insertions of two identical target sites for either miR-142ts (an immune cell-enriched microRNA; 2xmiR-142ts) or miR-122ts (a hepatocyte-enriched microRNA; 2xmiR-122ts). Insertion of each microRNA target site was expected to decrease expression by >95% in cells containing cognate microRNA.

[0212] In a first experiment, Vero cells were transfected with Ipg taRNAs (75ng replicase, 15ng trRNA) and with microRNA mimics corresponding to the taRNAs. Surprisingly, insertion of two microRNA target sites into the 3’ UTR of the replicase constructs only reduced luciferase expression by 50-70% in cells with a cognate microRNA (FIG. 4A). These results may indicate that mimic-microRNA are less effective than endogenous microRNA; however, as the relationship between replicase expression and trRNA-encoded payload expression is not necessarily linear given the characteristic amplification of taRNA, these results may indicate that even very low levels of replicase construct (e.g., predicted to be <5%) may still be sufficient to amplify payload expression.

[0213] In a second experiment, RAW cells were transfected with 33ng of taRNAs having a replicase construct encoding an SFV replicase or a trRNA encoding luciferase. Control replicase constructs and control trRNA did not include any microRNA target sites, while test replicase constructs and test trRNA included 2 target sites for miR-142 in their respective 3’ UTRs. Combinations of control replicase constructs, test replicase constructs, control trRNAs, and test trRNAs were transfected into RAW cells. As shown in FIG. 4B, insertion of any microRNA target sites in replicase constructs or trRNAs reduced expression by at least 94%, while insertion of microRNA target sites in both replicase constructs and trRNAs reduced expression by about 99%.

[0214] Overall, these findings indicate that microRNA target sites can be inserted into taRNAs (e.g., replicase constructs and / or trRNAs) to restrict expression at variable levels in specific cells. Certain combinations of microRNA target sites in taRNA components may thus be useful for a variety of applications.

[0215] #14495043v1 Example 3: Replicase construct-induced cytokine expression is reduced when cell-type enriched microRNA target sites are present in 3’ UTRs in vivo

[0216] In this Example, the in vivo applicability of microRNA target sites in 3’ UTRs of taRNAs was assessed. Compositions of ALC-315 and taRNAs having a replicase construct encoding a wildtype Semliki Forest Virus (SFV) replicase (and a trRNA having a 5’ SINV UTR with an oeSTR extension (SEQ ID NO: 52) and a nucleic acid encoding luciferase were prepared. Two identical target sites for one of miR-142ts (an immune cell-enriched microRNA) (2xmiR142ts-taRNA-ENP), miR-122ts (a hepatocyte-enriched microRNA) (2xmiR122ts- taRNA-ENP), or miR-126ts (an endothelial cell-enriched microRNA) (2xmiR126ts-taRNA- ENP) were inserted into the 3’ UTR of the replicase constructs.

[0217] Adult wildtype mice were injected with Ipg of each taRNA-ENP or an AEC-315 LNP formulated with mRNA encoding luciferase only (mRNA-ENP). 24 hours after injection, expression of each of a panel of cytokines tumor necrosis factor (TNF)-alpha (TNF-a), interferon gamma (IFNg), interferon beta (IFNb), keratinocyte chemoattractant (KC) / human growth regulated oncogene (GRO) (KC / GRO), monocyte chemoattractant protein-1 (MCP-1), interferon gamma induced protein 10 (IP- 10), macrophage inflammatory protein 1 alpha (MIP- la; also known as CCL3), and interleukin 6 (IL6)) was assessed.

[0218] Insertion of each microRNA target site was expected to decrease expression by >95% in cells containing cognate microRNA. However, as shown in FIG. 5A, each taRNA reduced expression of some, but not all, cytokines. For example, 2xmiR122ts-taRNA-ENPs significantly reduced expression of all cytokines except IFNy (IFNb decreased by 99%, MCP-1 decreased by 93%, IP-10 decreased by 58%, MIP-la decreased by 78%, and IL-6 decreased by 84%), while 2xmiR-142ts-taRNA-LNPs significantly reduced expression of several cytokines, including IFNy (decreased by 61%) and IFNP (decreased by 30%). Changes to sera levels of liver enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), at 24 hours after administration of mRNA or taRNA was also assessed. Liver enzymes were elevated in mice treated with taRNA with WT SFV replicase constructs (no microRNA target sites in 3’ UTR; taRNA-LNP), and in mice treated with 2xmiR126ts-taRNA-LNPs or 2xmiR142ts-taRNA- LNPs (FIG. 5B). Notably, mice injected with 2xmiR122ts-taRNA-LNPs exhibited significantly decreased liver enzymes, consistent with cell-specific inhibition of replicase constructs.

[0219] Overall, these findings indicate that replicase construct presence in the liver can cause system- wide cytotoxicity and immune responses to taRNA in a subject, and that this can be mitigated by inserting liver-specific microRNA target sites into the 3’ UTR of replicase

[0220] #14495043v1 constructs. Other microRNA target sites can reduce expression of certain cytokines at differing levels; accordingly, insertion of these target sites into taRNA (e.g., replicase constructs) may be useful for a variety of purposes.

[0221] #14495043v1

Claims

CLAIMSWhat is claimed is:

1. A trans amplifying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide, comprising:(i) a nucleic acid encoding a replicase, and(ii) a 3’ untranslated region (UTR); and(b) a second RNA polynucleotide, comprising:(i) a nucleic acid payload or a nucleic acid encoding a payload, and(ii) a 3’ UTR, wherein the 3’ UTR of the first RNA polynucleotide and / or the 3’ UTR of the second RNA polynucleotide further comprises a target site for a microRNA.

2. The taRNA of claim 1, wherein the microRNA is a cell type-enriched microRNA.

3. The taRNA of claim 2, wherein the cell type-enriched microRNA is an immune cell- enriched microRNA.

4. The taRNA of claim 3, wherein the immune cell-enriched microRNA is miR-142 or miR-223.

5. The taRNA of claim 2, wherein the cell type-enriched microRNA is a hepatocyte- enriched microRNA.

6. The taRNA of claim 5, wherein the hepatocyte-enriched microRNA is miR-122.

7. The taRNA of claim 2, wherein the cell type-enriched microRNA is an endothelial cell- enriched microRNA.

8. The taRNA of claim 7, wherein the endothelial cell-enriched microRNA is miR-126.

9. The taRNA of claim 2, wherein the cell type-enriched microRNA is a skeletal muscle cell-enriched microRNA.#14495043v110. The taRNA of claim 9, wherein the skeletal muscle cell-enriched microRNA is miR- 206.

11. The taRNA of any one of claims 1-10, wherein the 3’ UTR of the first RNA polynucleotide and / or the 3’ UTR of the second RNA polynucleotide is a 3’ UTR of an alphavirus.

12. The taRNA of any one of claims 1-11, wherein the 3’ UTR of the first RNA polynucleotide comprises the target site for the microRNA.

13. The taRNA of any one of claims 1-12, wherein the 3’ UTR of the second RNA polynucleotide comprises the target site for the microRNA.

14. The taRNA of any one of claims 1-13, wherein the 3’ UTR of the first RNA polynucleotide comprises a target site for a microRNA and wherein the 3’ UTR of the second RNA polynucleotide comprises a target site for a microRNA.

15. The taRNA of claim 1, wherein the 3’ UTR of the first RNA polynucleotide comprises:(i) a first target site for a first microRNA; and(ii) a second target site for a second microRNA.

16. The taRNA of claim 1, wherein the 3’ UTR of the first RNA polynucleotide comprises:(i) a first target site for a first microRNA;(ii) a second target site for a second microRNA; and(iii) a third target site for a third microRNA.

17. The taRNA of claim 1, wherein the 3’ UTR of the second RNA polynucleotide comprises:(i) a first target site for a first microRNA; and(ii) a second target site for a second microRNA.#14495043v118. The taRNA of claim 1, wherein the 3’ UTR of the second RNA polynucleotide comprises:(i) a first target site for a first microRNA;(ii) a second target site for a second microRNA; and(iii) a third target site for a third microRNA.

19. The taRNA of claim 12-18, wherein the first target site and the second target site are target sites for different microRNA.

20. The taRNA of claim 12-18, wherein the first target site and the second target site are target sites for the same microRNA.

21. The taRNA of claim 20, wherein the first target site and the second target site are target sites for miR-122.

22. The taRNA of claim 18, wherein the first target site is a target site for miR-142 and the second target site is a target site for miR-122.

23. The taRNA of claim 16 or claim 18, wherein the first target site is a target site for miR- 142, the second target site is a target site for miR-122 and the third target site is a target site for miR-122.

24. The taRNA of claim 16 or claim 18, wherein the first target site is a target site for miR- 142, the second target site is a target site for miR-142, and the third target site is a target site for miR-12225. The taRNA of any one of claims 16-24, further comprising a spacer sequence between two or more of the target sites.

26. The taRNA of claim 25, wherein the spacer sequence comprises UUUAAA.#14495043v127. The taRNA of any one of claims 1-26, wherein the second RNA polynucleotide comprises a conserved sequence element (CSE) cognate to the replicase encoded by the first RNA polynucleotide.

28. The taRNA of any one of claims 1-27, wherein the replicase is a Semliki Forest Virus (SFV) replicase, a Venezuelan Equine Encephalitis Virus (VEEV), a Chikungunya virus (CHIKV), or a Sindbis virus (SINV) replicase.

29. A trans amplifying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide, comprising:(i) a nucleic acid encoding a replicase; and(ii) a 3’ untranslated region (UTR), comprising: a target site for miR-122; and a target site for miR-142; and(b) a second RNA polynucleotide, comprising:(i) a nucleic acid payload or a nucleic acid encoding a payload; and(ii) a conserved sequence element (CSE) cognate to the replicase encoded by the nucleic acid of (a)(i).

30. A trans amplifying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide, comprising:(i) a nucleic acid encoding a replicase; and(ii) a 3’ untranslated region (UTR), comprising: a first target site for miR-122; and a second target site for miR-122; and(b) a second RNA polynucleotide, comprising:(i) a nucleic acid payload or a nucleic acid encoding a payload; and(ii) a conserved sequence element (CSE) cognate to the replicase encoded by the nucleic acid of (a)(i).

31. A trans amplifying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide, comprising:(i) a nucleic acid encoding a replicase; and(ii) a 3’ untranslated region (UTR), comprising: a first target site for miR-142; and a second target site for miR-142; and#14495043v1(b) a second RNA polynucleotide, comprising:(i) a nucleic acid payload or a nucleic acid encoding a payload; and(ii) a conserved sequence element (CSE) cognate to the replicase encoded by the nucleic acid of (a)(i).

32. A trans amplifying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide, comprising:(i) a nucleic acid encoding a replicase; and(ii) a 3’ untranslated region (UTR), comprising: a first target site for miR-122; a second target site for miR-122; and a third target site for miR-142; and(b) a second RNA polynucleotide, comprising:(i) a nucleic acid payload or a nucleic acid encoding a payload; and(ii) a conserved sequence element (CSE) cognate to the replicase encoded by the nucleic acid of (a)(i).

33. A trans amplifying ribonucleic acid (RNA) (taRNA) comprising:(a) a first RNA polynucleotide, comprising:(i) a nucleic acid encoding a replicase; and(ii) a 3’ untranslated region (UTR), comprising: a first target site for miR-122; a second target site for miR-142; and a third target site for miR-142; and(b) a second RNA polynucleotide, comprising:(i) a nucleic acid payload or a nucleic acid encoding a payload; and(ii) a conserved sequence element (CSE) cognate to the replicase encoded by the nucleic acid of (a)(i).

34. A method for selectively expressing a trans amplifying ribonucleic acid (taRNA) in a first cell, the method comprising: contacting a first cell and a second cell with a taRNA comprising:(a) a first RNA polynucleotide, comprising:(i) a nucleic acid encoding a replicase,(ii) a 3’ untranslated region (UTR); and(b) a second RNA polynucleotide, comprising:(i) a nucleic acid payload or a nucleic acid encoding a payload,#14495043v1(ii) a 3’ UTR; and(iii) a conserved sequence element (CSE) cognate to the replicase of (a)(i), wherein the 3’ UTR of the first RNA polynucleotide and / or the 3’ UTR of the secondRNA polynucleotide comprise a target site for a microRNA expressed by the second cell and not expressed by the first cell.

35. The method of claim 34, wherein the second cell is a liver cell.

36. The method of claim 35, wherein the microRNA target site is a target site for miR-122.

37. The method of claim 34, wherein the second cell is an immune cell.

38. The method of claim 37, wherein the microRNA target site is a target site for miR-142.

39. The method of any one of claims 34 to 38, wherein the replicase is an SFV replicase.

40. A method of reducing the immunogenicity of a trans amplifying ribonucleic acid (taRNA) in a subject, the method comprising: administering a taRNA to the subject, the taRNA comprising:(a) a first RNA polynucleotide, comprising:(i) a nucleic acid encoding a replicase, and(ii) a 3’ untranslated region (UTR) ; and(b) a second RNA polynucleotide, comprising:(i) a nucleic acid payload or a nucleic acid encoding a payload,(ii) a 3’ UTR; and(iii) a conserved sequence element (CSE) cognate to the replicase of (a)(i), wherein the 3’ UTR of the first RNA polynucleotide and / or the 3’ UTR of the secondRNA polynucleotide comprise a target site for a microRNA, and wherein the microRNA is not expressed by a target cell type of the subject and is expressed by at least one non-target cell type of the subject.

41. The method of claim 40, wherein the taRNA non-target cell is a liver cell.#14495043v142. The method of claim 41, wherein the microRNA target site is a target site for miR-122.

43. The method of claim 40, wherein the taRNA non-target cell is an immune cell.

44. The method of claim 43, wherein the microRNA target site is a target site for miR-142.

45. The method of claim 41, wherein reducing the immunogenicity of the taRNA comprises reducing expression of one or more interferon (IFN), chemokine, and / or interleukin that are associated with an immune response of the cell to the taRNA.

46. The method of claim 45, wherein the IFN is IFN-a, IFN-P, or IFN-y.

47. The method of claim 45, wherein the chemokine is a keratinocyte chemoattractant (KC) / human growth-regulated oncogene (GRO) chemokine, monocyte chemoattractant protein- 1 (MCP-1), interferon gamma-induced protein 10 (IP- 10), or macrophage inflammatory protein la (MIP-la).

48. The method of any one of claims 45 to 47, wherein the interleukin is IL-6.

49. The method of claim 40 to 48, wherein reducing the immunogenicity of the taRNA does not comprise significantly decreasing the expression of IFN-y.

50. The method of any one of claims 40 to 49, wherein the replicase is an SFV replicase.#14495043v1

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