Multiplexed lipid nanoformulation compositions and methods

Abstract

Provided herein are methods of analyzing intracellular delivery of a heterogeneous lipid nanoformulation by use of barcoded self-amplifying RNA (saRNA).

Description

[0001] FAZ-40125

[0002] MULTIPLEXED LIPID NANOFORMULATION COMPOSITIONS AND METHODS CROSS-RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application Serial No.

[0004] 63 / 737,968, filed December 23, 2024. The entire contents of which are incorporated herein by this reference.

[0005] BACKGROUND

[0006] mRNA technologies offer several advantages for therapy due to their size, ability to induce protein expression without delivery to the nucleus (similar to DNA based therapies), and versatility for treating various diseases. However, delivery of these therapeutics poses challenges due to degradation and failure to deliver to tissues and cells of interest.

[0007] Use of mRNA therapeutics in vivo requires a delivery vehicle for cellular uptake; exemplary delivery platforms include lipid nanoparticles (LNPs), polyplexes, and cationic nanoemulsions. LNP formulations for delivering mRNA therapeutics have been developed for use in the clinic, however, identifying LNP formulations which can deliver mRNA therapeutics is an ongoing challenge.

[0008] SUMMARY

[0009] The present disclosure provides compositions and methods for high throughput screening of lipid nanoformulations in vivo. Provided herein are self-amplifying RNA (saRNA) comprising a unique barcode for identification. The barcoded saRNA are encapsulated in lipid nanoformulations which are subsequently administered to a subject. The intracellular delivery of these lipid nanoformulations and the encapsulated saRNA is then determined by sequencing of RNA from a sample. This screening platform enables determination of a plurality of lipid nanoformulations which successfully deliver cargo intracellularly.

[0010] In some aspects, the disclosure provides a method for screening delivery vehicle formulations, comprising:

[0011] (a) providing a pooled composition comprising a plurality of distinct delivery vehicles, each delivery vehicle encapsulating a self-amplifying RNA (saRNA) comprising a unique nucleotide barcode;

[0012] (b) administering the pooled composition to a subject;

[0013] (c) isolating RNA from one or more biological samples obtained from the subject; and (d) identifying barcode sequences present in the isolated RNA,FAZ-40125

[0014] wherein detection of a barcode indicates intracellular delivery of the corresponding delivery vehicle.

[0015] In some or any of the foregoing or related aspects, identifying barcode sequences comprises detecting negative-strand RNA generated from the saRNA.

[0016] In some or any of the foregoing or related aspects, the plurality of distinct delivery vehicles comprises a plurality of distinct lipid nanoparticles (LNPs) differing in lipid composition.

[0017] In some or any of the foregoing or related aspects, the LNPs differ in at least one of: ionizable lipid structure, helper lipid content, sterol content, or PEG-lipid content.

[0018] In some or any of the foregoing or related aspects, the saRNA encodes a reporter protein expressed from a subgenomic promoter.

[0019] In some or any of the foregoing or related aspects, identifying barcode sequences comprises performing next-generation sequencing. In some aspects, sequencing is performed following strand- specific reverse transcription using a primer comprising a purification moiety.

[0020] In some or any of the foregoing or related aspects, the purification moiety is biotin and the reverse transcription product is purified using a streptavidin solid support.

[0021] In some or any of the foregoing or related aspects, the pooled composition comprises at least 10 distinct delivery vehicles.

[0022] In some aspects, the disclosure provides a composition comprising a plurality of lipid nanoparticles (LNPs), each of the plurality encapsulating a self-amplifying RNA (saRNA) that (a) comprises a unique barcode sequence, and optionally (b) encodes a reporter protein expressed from a subgenomic promoter.

[0023] In some or any of the foregoing or related aspects, the plurality comprises at least ten. In some or any of the foregoing or related aspects, the composition is configured for pooled administration to a subject.

[0024] In some aspects, the disclosure provides a method of analyzing intracellular delivery of a delivery vehicle composition, the method comprising:

[0025] (i) providing a delivery vehicle composition having encapsulated therein a barcoded self-amplifying RNA (saRNA) to a subject,

[0026] (ii) identifying the barcode sequence of the saRNA in one or more samples of the subject, thereby analyzing the intracellular delivery of the delivery vehicle composition.

[0027] In some or any of the foregoing or related aspects, the delivery vehicle composition comprises a heterogeneous lipid nanoformulation composition.FAZ-40125

[0028] In some aspects, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0029] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a self-amplifying RNA (saRNA), wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation;

[0030] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0031] (iii) isolating and sequencing RNA from one or more samples of the subject; and, (iv) identifying the barcode sequence, thereby analyzing the intracellular delivery of each lipid nanoformulation of the heterogeneous lipid nanoformulation composition.

[0032] In some aspects, the disclosure provides a method of analyzing intracellular delivery of saRNA encapsulated in a lipid nanoformulation, the method comprising:

[0033] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a self-amplifying RNA (saRNA), wherein the saRNA generates a detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation; (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0034] (iii) sorting cells from one or more samples of the subject by measuring the detectable signal; and,

[0035] (iv) identifying the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of the saRNA.

[0036] In some or any of the foregoing or related aspects, the saRNA encodes at least one non-structural protein. In some aspects, the saRNA encodes four non- structural proteins. In some aspects, the saRNA encodes a viral non- structural protein 1 (nsPl), non-structural protein 2 (nsP2), non-structural protein 3 (nsP3), and non-structural protein 4 (nsP4). In some aspects, nsP4 is RNA-dependent RNA polymerases (RdRP).

[0037] In some or any of the foregoing or related aspects, one or more of nsPl, nsP2, nsP3, and nsP4 are derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV), or any combination thereof.FAZ-40125

[0038] In some or any of the foregoing or related aspects, the saRNA comprises a genomic promoter. In some aspects, the genomic promoter drives expression of one or more non-structural proteins.

[0039] In some or any of the foregoing or related aspects, the saRNA comprises a subgenomic promoter.

[0040] In some or any of the foregoing or related aspects, the saRNA comprises a poly(A) tail. In some or any of the foregoing or related aspects, the saRNA comprises a 5’ cap. In some aspects, the 5’ cap is an m7G cap.

[0041] In some or any of the foregoing or related aspects, the saRNA comprises a nucleic acid sequence encoding a reporter gene. In some aspects, the subgenomic promoter drives expression of the reporter gene. In some aspects, the reporter gene encodes a protein that produces the detectable signal. In some aspects, the protein is a fluorescent protein or an enzyme.

[0042] In some or any of the foregoing or related aspects, the detectable signal is produced by a protein selected from the group consisting of a luciferase, a horseradish peroxidase, an alkaline phosphatase, a P-galactosidase, and any combination thereof. In some aspects, the detectable signal is produced by luciferase. In some aspects, the luciferase is Gaussia luciferase.

[0043] In some or any of the foregoing or related aspects, the saRNA comprises a nucleic acid sequence encoding, from 5’ to 3’:

[0044] i) a 5’ cap;

[0045] ii) a 5’ UTR, including a genomic promoter;

[0046] iii) nsPl, nsP2, nsP3, and nsP4;

[0047] iv) a subgenomic promoter;

[0048] v) a subgenomic 5’ UTR;

[0049] vi) a reporter gene;

[0050] vii) a barcode;

[0051] viii) a 3 ’ UTR; and

[0052] ix) a poly(A) tail.

[0053] In some or any of the foregoing or related aspects, the saRNA comprises a nucleic acid sequence encoding, from 5’ to 3’:

[0054] i) a 5’ cap;

[0055] ii) a promoter;

[0056] iii) a 5’ UTR;

[0057] iv) nsPl, nsP2, nsP3, and nsP4;FAZ-40125

[0058] v) a subgenomic promoter;

[0059] vi) a subgenomic 5’ UTR;

[0060] vii) a reporter gene;

[0061] viii) a barcode;

[0062] ix) a 3 ’ UTR; and

[0063] x) a poly(A) tail.

[0064] In some or any of the foregoing or related aspects, the barcode is 8-12 nucleotides in length. In some aspects, the barcode is 3’ relative to the subgenomic promoter.

[0065] In some or any of the foregoing or related aspects, the saRNA comprises the nucleic acid sequence set forth in SEQ ID NO: 2.

[0066] In some or any of the foregoing or related aspects, the saRNA comprises at least one modified nucleic acid. In some aspects, the modified nucleic acid is 5-methylcytidine (5mC).

[0067] In some or any of the foregoing or related aspects, the saRNA is a positive strand RNA or a negative strand RNA.

[0068] In some or any of the foregoing or related aspects, the heterogeneous lipid nanoformulation composition comprises lipid nanoformulations generated using different lipid compositions. In some aspects, the lipid nanoformulations vary in the molar amount and / or structure of the ionizable lipid, the molar amount and / or structure of the helper lipid, the molar amount / or structure of PEG, and / or the molar amount of cholesterol.

[0069] In some or any of the foregoing or related aspects, the composition comprises greater than 10 different lipid nanoformulations. In some aspects, the composition comprises greater than 100 different lipid nanoformulations. In some aspects, the composition comprises greater than 200 different lipid nanoformulations.

[0070] In some or any of the foregoing or related aspects, the intracellular delivery is intracellular delivery. In some aspects, the intracellular delivery is cytoplasmic delivery.

[0071] In some or any of the foregoing or related aspects, upon delivery of the saRNA, nsPl, nsP2, nsP3, and nsP4 are translated and assemble into an RdRP.

[0072] In some or any of the foregoing or related aspects, identifying the barcode sequence comprises amplification and sequencing of the negative strand saRNA or positive strand saRNA.

[0073] In some or any of the foregoing or related aspects, the amplification comprises reverse transcription of the saRNA. In some aspects, reverse transcription comprises using a reverse transcription primer.FAZ-40125

[0074] In some or any of the foregoing or related aspects, the reverse transcription primer comprises a purification moiety.

[0075] In some or any of the foregoing or related aspects, the purification moiety is an amino modifier, a biotin modifier, or an alkyne modification. In some aspects, the amino modifier, biotin modifier, or alkyne modification is selected from the group comprising: 5'-C6, 5'-C12, 5'- C6 dT, 5'-Uni-Link™, 5'-biotin, 5'-biotin (azide), 5'-biotin dT, 5'-biotin-TEG, dual 5'-biotin, 5'-PC biotin, 5'-desthiobiotin-TEG, 5’ hexynyl and 5-Octadiynyl dU. In some aspects, the reverse transcription primer comprises a biotin modifier. In some aspects, the biotin modifier comprises 5 '-biotinylation.

[0076] In some or any of the foregoing or related aspects, the reverse transcription with the reverse transcription primer forms a reverse transcription product, and the reverse transcription primer comprises a means for purification of the reverse transcription product.

[0077] In some or any of the foregoing or related aspects, the means for purification of the reverse transcription product comprises an amino modifier, a biotin modifier, or an alkyne modification.

[0078] In some aspects, the amino modifier, biotin modifier, or alkyne modification is selected from the group comprising: 5'-C6, 5'-C12, 5'- C6 dT, 5'-Uni-Link™, 5'-biotin, 5'-biotin (azide), 5'-biotin dT, 5'-biotin-TEG, dual 5'-biotin, 5'-PC biotin, 5'-desthiobiotin-TEG, 5’ hexynyl and 5-Octadiynyl dU.

[0079] In some or any of the foregoing or related aspects, the reverse transcription primer comprises a unique molecular identifier (UMI). In some aspects, the UMI is 8-12 nucleotides in length.

[0080] In some or any of the foregoing or related aspects, the reverse transcription primer comprises a sequence set forth in SEQ ID NO: 7 or 10.

[0081] In some or any of the foregoing or related aspects, the method further comprises purifying the reverse transcription product. In some aspects, the purification comprises immobilizing the reverse transcription product on a solid surface. In some aspects, the solid surface is a magnetic bead or a coated plate. In some aspects, the purification method is performed using a biotin- streptavidin pulldown.

[0082] In some or any of the foregoing or related aspects, the purification results in a composition that is substantially free of unprimed positive strand reverse transcription product relative to a reverse transcription product produced without a purification step. In some or any of the foregoing or related aspects, the purification results in a composition that is enrichedFAZ-40125

[0083] with primed negative strand reverse transcription product relative to a reverse transcription product produced without a purification step.

[0084] In some or any of the foregoing or related aspects, the amplification comprises polymerase chain reaction (PCR) of the reverse transcription product. In some aspects, the PCR uses a forward primer comprising the sequence set forth in SEQ ID NO: 8. In some aspects, the PCR uses a reverse primer selected from the primers set forth in SEQ ID NOs: 9 and 11.

[0085] In some or any of the foregoing or related aspects, the sequencing is performed using Next Generation Sequencing.

[0086] In some aspects, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0087] (i) providing a heterogeneous lipid nanoformulation comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA), wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0088] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0089] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is transcribed into a negative strand saRNA;

[0090] (iv) wherein upon delivery of the saRNA, the nsP4 is translated assembles into an RdRp and the RdRp translates the reporter gene to the protein that produces the detectable signal;

[0091] (v) isolating RNA from one or more samples of the subject;

[0092] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence, thereby analyzing the intracellular delivery of each lipid nanoformulation of the heterogeneous lipid nanoformulation.

[0093] In some aspects, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0094] (i) providing a heterogeneous lipid nanoformulation comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA), wherein the saRNA comprisesFAZ-40125

[0095] a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0096] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0097] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is translated to form an RNA-dependent RNA polymerase (RdRp);

[0098] (iv) wherein upon delivery of the saRNA, the RdRp catalyzes the formation of negative and subgenomic saRNAs, and cellular machinery translates the protein that produces the detectable signal from the subgenomic RNA;

[0099] (v) isolating RNA one or more samples of the subject;

[0100] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence, thereby analyzing the intracellular delivery of each lipid nanoformulation of the heterogeneous lipid nanoformulation.

[0101] In some or any of the foregoing or related aspects, the subject is a mammal. In some aspects, the subject is a non-human mammal. In some aspects, the subject is a mouse.

[0102] In some or any of the foregoing or related aspects, the sample comprises a cell lysate, a tissue lysate, serum, plasma, saliva, urine, or a cerebral spinal fluid. In some aspects, the sample is tissue lysate.

[0103] In some or any of the foregoing or related aspects, the sample originates from heart, skeletal muscle, liver, lung, brain, kidney, pancreas, spleen, small intestine, colon, rectum, gallbladder, bladder, eye, or skin.

[0104] In some or any of the foregoing or related aspects, the lipid nanoformulation is administered intravenously, subcutaneously, intramuscularly, or intraperitoneally, to the subject.

[0105] In some or any of the foregoing or related aspects, the sample is collected from the subject about 1 to about 24 hours after administration of the heterogeneous lipid nanoformulation.

[0106] In some or any of the foregoing or related aspects, the sample is collected from the subject about 6 hours after administration of the heterogeneous lipid nanoformulation.

[0107] In some or any of the foregoing or related aspects, about 0.01 mg / kg to about 1 mg / kg of the heterogeneous lipid nanoformulation is administered to the subject. In some aspects,FAZ-40125

[0108] about 0.1 mg / kg of the lipid heterogeneous nanoformulation is administered to the subject. In some aspects, about 1 mg / kg of the heterogeneous lipid nanoformulation is administered to the subject.

[0109] In some aspects, the disclosure provides a method of making a heterogeneous lipid nanoformulation, the method comprising encapsulating saRNA in different lipid nanoformulations, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, and wherein each lipid nanoformulation varies in the molar amount and / or structure of the ionizable lipid, the molar amount and / or structure of the helper lipid, the molar amount / or structure of PEG, and / or the molar amount of cholesterol.

[0110] These and other aspects are addressed in more detail in the detailed description set forth below.

[0111] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic of a positive strand saRNA template comprising nucleic acid sequences, from 5’ to 3’, for a Capl T7 promoter, a 5’ UTR including a genomic promoter, nonstructural proteins nsPl, nsP2, nsP3, and nsP4, a subgenomic promoter, a subgenomic 5’UTR, Gaussia luciferase (hGluc), a barcode, a primer binding site, a 3 ’UTR, and a poly(A) tail.

[0112] FIG. 2A provides a schematic demonstrating insertion of barcoded sequences into the saRNA plasmid and subsequent subcloning to generate clones with unique barcode sequences.

[0113] FIGs. 2B-2C are Sanger sequencing chromatograms. FIG.2B shows the barcode region in an unusable double-transformant colony (top), and an acceptable single-transformant (bottom) as generated in FIG. 2A. FIG. 2C shows and poly-A screening of generated clones.

[0114] FIG. 3 provides a schematic of reverse transcription of positive (+) strand saRNA to generate cDNA, further second strand synthesis, and subsequent PCR amplification prior to Next Generation Sequencing (NGS). A reverse transcription primer comprising a study tag, a unique molecular identifier, and a sequencing by synthesis (SBS) sequence generates cDNA of the saRNA of interest. Forward and reverse primers comprising NGS adapter sequences and SBS sequences are then used for PCR amplification to generate a library for NGS. 5’ UTR= 5’ untranslated region; hGluc = humanized Gaussia luciferase; BC=barcode; PBS= primer binding site; 3’ UTR = 3’ untranslated region; pA= polyAtail; Tag= study tag; UMI=unique molecular identifier; SBS32 and SBS 12 =sequencing by synthesis site; P5= Illumina P5 adapter; P7= Illumina P7 adapter.FAZ-40125

[0115] FIGs. 4A-4B are schematics showing an in vivo multiplex lipid nanoparticle (LNP) screening method. FIG. 4A shows barcoded saRNA are inserted into LNPs using a microfluidic device. Libraries are then administered to mice and delivery of the LNPs is analyzed from tissues by NGS, luminescence, and / or RT-qPCR, as shown in FIG. 4B.

[0116] FIG. 5 is a graph showing luminescence measuring hGluc reporter expression in serum from mice administered barcoded saRNA encapsulated in LNPs. Serum was collected at 6 and 24 hours post dose of the LNPs.

[0117] FIGs. 6A-6B are graphs showing total copies of positive (+) and negative (-) saRNA from spleen (FIG. 6A) or serum (FIG. 6B) of mice administered barcoded saRNA encapsulated in LNPs. Spleen and serum were collected at 6 and 24 hours post dose of the LNPs. Quantification of saRNA copies was performed by RT-qPCR from total RNA.

[0118] FIGs. 7A-7B provide graphs showing reads of total unique molecular identifiers (UMI) and UMI fractions from mice administered a 2-LNP (FIG. 7A) or 9-LNP (FIG. 7B) library. Mice were administered LNPs at 1 mg / kg (mpk) or 0.1 mg / kg and spleen was collected 6 or 24 hours later. UMI counts were compared to sample retains (i.e., non-dosed sample control) to compare total UMI count after administration and UMI fraction for each administered LNP.

[0119] FIG. 8 is a schematic illustrating delivery of a saRNA encapsulated in lipid nanoparticles (LNPs) to a subject. A pool of LNP, each encapsulating saRNA with unique barcodes, is administered to a subject. Although some LNP are not delivered to a tissue, others will be delivered to cells and endocytosed. Upon endocytosis, some saRNA may escape the endosome and release into the cytoplasm. Other saRNA which do not escape the endosome will be degraded. For saRNA which are released into the cytoplasm, replication and amplification of the saRNA occurs.

[0120] FIG. 9 provides a schematic of a positive (+) strand saRNA comprising, from 5’ to 3’, a 5’ cap (m7G), genomic promoter, nonstructural proteins (nsP), a subgenomic promoter, an open reading frame (ORF) that encodes a reporter, a barcode (BC), and a poly A tail. saRNA is dosed as a positive (+) sense strand saRNA. Upon delivery to the cytoplasm, nsPl-4 are expressed from the positive strand saRNA and are translated into a viral RNA-dependent RNA polymerase (RdRP). RdRP catalyzes the formation of complimentary (-) strand saRNA. The resulting dsRNA complex is in turn transcribed by RdRP into full-length and subgenomic (+) strands. The reporter gene open reading from (ORF) is only expressed from the subgenomic strand. As the replication cycle proceeds, maturation of the RdRP complex leads to decreased (-) strand and increased subgenomic strand production.FAZ-40125

[0121] FIGs. 10A-10B provide schematics of magnetic bead-based purification of RT products for strand- specific NGS libraries. FIG. 10A show strand targeted RT-PCR primers do not produce strand- specific libraries. FIG. 10B shows bead-based purification improves specificity allowing specific PCR amplification of negative strand NGS libraries from bead-bound cDNA.

[0122] FIGs. 11A-11C provide graphs showing library preparation from in vitro pools of saRNA with or without biotinylated primers / streptavidin bead-based purification of RT products. FIG. 11A provides the input ratios of positive and negative strands. FIG. 11B provides total UMI counts for each barcoded saRNA using biotin purification or direct RT-PCR. FIG. 11C provides graphs showing the total fraction of each barcode based on biotin purification or direct RT-PCR.

[0123] FIGs. 11D-11F provides graphs showing library preparation from in vivo LNP delivery of saRNA and delivery to lung and spleen.

[0124] DEFINITIONS

[0125] As used herein, the term “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. The term “subject” may be used interchangeably herein with “patient.” In some embodiments, the patient or subject is a primate (e.g., non-human primate). In some embodiments, the patient or subject is a human. Non-limiting examples of human subjects are adults, juveniles, infants and fetuses.

[0126] Described herein are self-amplifying RNAs. As used herein, the terms “self-amplifying RNA” or “saRNA” or “self-replicating RNA” or “srRNA” are used interchangeably and refer to an RNA strand capable of undergoing replication activity that results in replicate strands from an original strand.

[0127] As used herein, the term “derived from” refers to the origin or source, and can include naturally occurring, recombinant, unpurified, or purified molecules, such as nucleic acids or polypeptides. In some embodiments, “derived from” includes mutation and / or maturation of any of the nucleic acids or polypeptides as described herein.

[0128] As used herein the term “intracellular delivery” refers to delivery within a cell. Delivery may be to any internal structure or component of a cell including, but not limited to, organelles, exosomes, the nucleus, and the cytoplasm. In some embodiments, intracellular delivery of an saRNA will lead to replication thereof.FAZ-40125

[0129] Any publications, patents, and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0130] DETAILED DESCRIPTION

[0131] I. Self-Amplifying RNA (saRNA)

[0132] saRNA is a type of RNA which has the ability to replicate and amplify itself, in situ. saRNA molecules may be produced by using replication elements derived from a virus or viruses, e.g., alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest. A self-amplifying RNA molecule is typically a positivestrand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a detectable signal or barcode, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the protein of interest, e.g., a detectable signal or barcode. The overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNAs.

[0133] In some embodiments, the self-amplifying RNA includes at least one or more viral genes selected from any one of viral replicases, viral proteases, viral helicases and other nonstructural viral proteins. In some embodiments, the self-amplifying RNA may also include 5'- and 3 '-end tractive replication sequences, and optionally an open reading frame (ORF) sequence that encodes a desired sequence (e.g., a detectable signal or barcode). A subgenomic promoter that directs expression of the ORFs sequence may be included in the self-amplifying RNA. Optionally, the ORF may be fused in frame to other coding regions in the self- amplifying RNA and / or may be under the control of an internal ribosome entry site (IRES).

[0134] In some embodiments, the self-amplifying RNA molecule is not encapsulated in a viruslike particle. Self-amplifying RNA molecules described herein may be designed so that the self-amplifying RNA molecule cannot induce production of infectious viral particles. This may be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary to produce viral particles in the self- amplifying RNA. For example, when the self-amplifying RNA molecule is based on an alphavirus, such as Sinbis virus (SIN), SemlikiFAZ-40125

[0135] forest virus and Venezuelan equine encephalitis virus (VEE), one or more genes encoding viral structural proteins, such as capsid and / or envelope glycoproteins, may be omitted.

[0136] In some embodiments, an saRNA described herein encodes both viral and non-viral components. In some embodiments, an saRNA molecule described herein encodes (i) an RNA-dependent RNA polymerase that may transcribe RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, e.g., a detectable signal or a barcode. In some embodiments, the polymerase may be an alphavirus replicase, e.g., including any one of alphavirus protein nsPl, nsP2, nsP3, nsP4, and any combination thereof.

[0137] Viral Components

[0138] In some embodiments, at least one component of the saRNA described herein is derived from at least one virus. The phrase “derived from at least one virus” can include any virus. Non-limiting examples of such viruses include: Venezuela Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV), Sindbis Virus (SIN), Chikungunya Virus (CHIKV), Eastern Equine Encephalitis Virus (EEEV), Mayaro Virus (MAYV), Getah Virus (GETV), Ross River Virus (RRV), Una Virus (UNAV), Middleburg Virus (MIDV), O'nyong nyong virus (ONNV), Barmah Forest Virus (BFV), Mucambo Virus (MUCV), Tonate Virus (TONV), Everglades Virus (EVEV), Rio Negro Virus (RNV), Turnip Rosette Virus (TROV), Highlands J Virus (HJV), Western Equine Encephalitis Virus (WEEV), Fig Mosaic Emaravirus (FMV), Aura Virus (AURAV), Kunjin Virus (KUN), Measles virus (MV), Coronavirus (CoV), Rabies virus (RABV), and Vesicular Stomatitis virus (VSV).

[0139] In some embodiments, at least one component of the saRNAs described herein are derived from at least one alphavirus. Non-limiting examples of such alphaviruses include: Aura Virus (AURAV), Barmah Forest Virus (BFV), Bebaru virus, Caaingua virus, Cabassou virus, Chikungunya Virus (CHIKV), Eastern Equine Encephalitis Virus (EEEV), Eliat virus, Everglades Virus (EVEV), Fort Morgan virus, Getah Virus (GETV), Highlands J Virus (HJV), Madariaga virus, Mayaro Virus (MAYV), Middleburg Virus (MIDV), Mosso das Pedras virus, Mucambo Virus (MUCV), Ndumu virus, O'nyong nyong virus (ONNV), Pixuna virus, Rio Negro Virus (RNV), Ross River Virus (RRV), Salmon pancreas disease virus, Semliki Forest Virus (SFV), Sindbis Virus (SIN), Southern elephant seal virus, Tonate Virus (TONV), Trocara virus, Una Virus (UNAV), Venezuela Equine Encephalitis Virus (VEEV), Western Equine Encephalitis Virus (WEEV), and Whataroa virus. In some embodiments, at least one component of the saRNAs described herein are derived from Venezuela Equine Encephalitis Virus (VEEV).FAZ-40125

[0140] In some embodiments, the virus or viral components are derived from Venezuelan Equine Encephalitis Virus (VEEV), Sindbis Virus (SINV), Semliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV) or any combination thereof. In some embodiments, the virus or viral components are derived from Venezuelan Equine Encephalitis Virus (VEEV). In some embodiments, the virus or viral components are derived from Sindbis Virus (SINV). In some embodiments, the virus or viral components are derived from Semliki Forest Virus (SFV). In some embodiments, the virus or viral components are derived from Classical Swine Fever Virus (CSFV). In some embodiments, the virus is a fusion of VEEV and SINV (VEE-SINV). In some embodiments, the virus or viral components are derived from a virus described in Blakney et al. An Update on Self- Amplifying mRNA Vaccine Development. Vaccines (Basel). 2021 Jan 28; 9(2).

[0141] Alphavirus is a genus of RNA viruses, the sole genus in the Togaviridae family.

[0142] Alphaviruses belong to group IV of the Baltimore classification of viruses, with a positivesense, single-stranded RNA genome. The alphaviruses are small, spherical, enveloped viruses with a genome of a single strand of positive-sense RNA. The total genome length ranges between 11,000 and 12,000 nucleotides, and the genome has a 5’ cap and a 3’ poly- A tail. The four non-structural protein genes are encoded in the 5' two-thirds of the genome, while the three structural proteins are translated from a subgenomic mRNA colinear with the 3' one-third of the genome. There are two open reading frames (ORFs) in the genome, nonstructural and structural. The first is non-structural and encodes proteins (nsPl-nsP4) necessary for transcription and replication of viral RNA. The second encodes three structural proteins: the core nucleocapsid protein C, and the envelope proteins P62 and El.

[0143] In some embodiments, the viral components include at least one non-structural protein. In some embodiments, the viral components include a subgenomic promoter. In some embodiments, the viral components include a 5’ untranslated region (5’ UTR). In some embodiments, the viral components include a 3’ untranslated region (3’ UTR). In some embodiments, the viral components include a polyA tail. In some embodiments, the viral components comprise at least one non-structural protein, a subgenomic promoter, 5’ untranslated region (5’ UTR), and / or a 3’ untranslated region (3’ UTR).

[0144] In some embodiments one or more of the viral components, for example nspl, nsp2, nsp3, nsp4, subgenomic promoter, 5’ UTR, and / or 3’ UTR, in an saRNA described herein are derived from the same virus. In some embodiments one or more of the viral components, for example, nspl, nsp2, nsp3, nsp4, subgenomic promoter, 5’ UTR, and / or 3’ UTR, in an saRNA described herein are derived from different viruses.FAZ-40125

[0145] As a non-limiting example, at least one of the non-coding conserved sequence elements (e.g., 5’ UTR, subgenomic promoter, 3’ UTR), each derived from at least one virus, can be mutated and / or matured compared to the wild-type sequence(s), e.g., to increase the replication rate of the RNA. For example, at least one of the non-coding conserved sequence elements (e.g., 5’ UTR) can be mutated or matured to comprise A-rich regions, which can accelerate RNA replication. See e.g., Li et al., Scientific Reports volume 9, Article number: 6932 (2019); Perkovic et al., Molecular Therapy Volume 31, Issue 6, P1636-1646 (2023); the contents of each of which are incorporated herein by reference in their entireties.

[0146] In some embodiments, the viral components derived from at least one virus, comprise sequences at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least at least about 98%, at least about 99%, or 100%, identical to the naturally occurring component.

[0147] In some embodiments, a viral component having less than 100% identity to the naturally occurring viral component maintains at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least at least about 98%, at least about 99%, or 100%, of activity as compared to the naturally occurring component of the virus.

[0148] Methods of determining sequence identity (e.g., percent identity) are known to those of skill in the art. The amino acid or nucleic acid sequence identity (e.g., percent identity) can be determined by any suitable method, such as using BLAST, BLAST-2, ALIGN, ALIGN-2, Clustal Omega, or Megalign software.

[0149] i. Non-Structural Proteins

[0150] In some embodiments, the saRNAs described herein comprise at least one non-structural protein derived from at least one virus. In some embodiments, the viral components include at least one non- structural protein. In some embodiments, the viral components include at least two non-structural proteins. In some embodiments, the viral components include at least three non-structural proteins. In some embodiments, the viral components include at least four non-structural proteins. In some embodiments the viral components include non-structural protein 1 (nspl), non-structural protein 2 (nsp2), non-structural protein 3 (nsp3), non-structural protein 4 (nsp4).

[0151] As a non-limiting example, at least one of the non-structural proteins (nspl-4), each derived from at least one virus, can be mutated and / or matured compared to the wild-typeFAZ-40125

[0152] sequence(s), e.g., to increase the amount and / or duration of cargo expression (i.e., a barcoded mRNA). Non-limiting examples of non- structural protein mutations include: nsP2 A1979G (nucleic acid), G656G (amino acid); nsP2 G3936C (nucleic acid), G1309R (amino acid); nsP3 A4311G (nucleic acid), K1434E (amino acid): nsP3 A4758G (nucleic acid), S1583G (amino acid); nsP3 G4796T (nucleic acid), E1595D (amino acid); and / or nsP3 G4944A (nucleic acid), V1645M (amino acid).

[0153] In some embodiments, the saRNAs described herein comprise at least one non-structural protein derived from at least one alphavirus. In some embodiments, the saRNAs described herein comprise at least one non-structural protein derived from VEEV. Alphavirus nonstructural proteins can be selected from the four nonstructural proteins (nsPl-4) which are produced as a single polyprotein and constitute the virus' replication machinery. For example, in VEEV and other alphaviruses, nspl is a methyl / guanylyltransferase involved with RNA capping; nsp2 is a cysteine protease, helicase, and NTPase; nsp3 is a polyADP-ribose hydroxylase; and nsp4 is an RNA-dependent RNA polymerase (RdRp). In some embodiments, the saRNAs described herein comprise at least one non-structural protein that functions as an RNA-dependent RNA polymerase (RdRp) and allows for replication of the saRNA (e.g., +RNA to -RNA to +RNA) and / or production of the subgenomic RNA (e.g., -RNA to sgRNA using the subgenomic promoter).

[0154] In some embodiments, the saRNAs described herein comprise an nsPl derived from an alphavirus, an nsP2 derived from an alphavirus, an nsP3 derived from an alphavirus, and / or an nsP4 derived from an alphavirus, or any combination thereof. As a non-limiting example, an saRNA described herein can comprise an nsPl derived from an alpha virus, an nsP2 derived from an alphavirus, an nsP3 derived from an alphavirus, and an nsP4 derived from an alphavirus, or any combination thereof. In some embodiments, an saRNA described herein comprises from 5’ to 3’: nsPl, nsP2, nsP3, and nsP4, each derived from a species of alphavirus, which each can be the same or different species of alphavirus. In some embodiments, the 5’ region of an saRNA described herein comprises from 5’ to 3’: nsPl, nsP2, nsP3, and nsP4, each derived from a species of alphavirus, which each can be the same or different species of alphavirus.

[0155] In some embodiments, the saRNAs described herein comprise an nsPl derived from a strain of VEEV, an nsP2 derived from a strain of VEEV, an nsP3 derived from a strain of VEEV, and / or an nsP4 derived from a strain of VEEV, or any combination thereof. As a non-limiting example, an saRNA described herein can comprise an nsPl derived from a strain of VEEV, an nsP2 derived from a strain of VEEV, an nsP3 derived from a strain of VEEV, and an nsP4FAZ-40125

[0156] derived from a strain of VEEV. In some embodiments, an saRNA described herein comprises from 5’ to 3’: nsPl, nsP2, nsP3, and nsP4, each derived from a strain of VEEV, which each can be the same or different strain of VEEV In some embodiments, the 5’ region of an saRNA described herein comprises from 5’ to 3’: nsPl, nsP2, nsP3, and nsP4, each derived from a strain of VEEV, which each can be the same or different strain of VEEV.

[0157] In some embodiments, saRNA described herein can comprise any combination of non-structural proteins, each derived from the same or different virus. In some embodiments, saRNA described herein can comprise any of the combinations of non- structural proteins shown, each derived from an alphavirus, which can be the same or different species of alphavirus. In some embodiments, the saRNA comprises an nsPl. In some embodiments, the saRNA comprises an nsP2. In some embodiments, the saRNA comprises an nsP3. In some embodiments, the saRNA comprises an nsP4. In some embodiments, the saRNA comprises an nsPl and nsP2. In some embodiments, the saRNA comprises an nsPl and nsP3. In some embodiments, the saRNA comprises an nsPl and nsP4. In some embodiments, the saRNA comprises an nsP2 and nsP3. In some embodiments, the saRNA comprises an nsPl, nsP2, and nsP3. In some embodiments, the saRNA comprises an nsP2 and nsP4. In some embodiments, the saRNA comprises an nsPl, nsP2, and nsP4. In some embodiments, the saRNA comprises an nsP3 and nsP4. In some embodiments, the saRNA comprises an nsP2, nsP3, and nsP4. In some embodiments, the saRNA comprises an nsPl, nsP2, nsP3, and nsP4.

[0158] ii. PolyA Tail

[0159] In some embodiments, described herein are saRNAs comprising a polyA tail, which is found on the 3' end of the saRNA molecule. As used herein, “polyA tail” refers to a stretch of consecutive adenine residues, which may be attached to the 3’ end of the RNA molecule. The poly-A tail may increase the half-life of the RNA molecule. PolyA tails may play key regulatory roles in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. In some embodiments, the polyA tail is derived from at least one virus. In some embodiments, the polyA tail is derived from at least one alphavirus. In some embodiments, the polyA tail is derived from VEEV.

[0160] In some embodiments, the polyA tail is 5-400 nucleotides in length. The polyA tail nucleotide length may be equal to any one of, at least any one of, at most any one of, or between any two of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, and 400. In some embodiments, the RNA molecule includes a polyA tail that includes about 25 to about 400 adenosine nucleotides, aFAZ-40125

[0161] sequence of about 50 to about 400 adenosine nucleotides, a sequence of about 50 to about 300 adenosine nucleotides, a sequence of about 50 to about 250 adenosine nucleotides, a sequence of about 60 to about 250 adenosine nucleotides, or a sequence of about 40 to about 100 adenosine nucleotides.

[0162] In some embodiments, the saRNA comprises a polyA signal (e.g., AAUAAA) that leads to cleaving of the 3' end of the RNA to free a 3' hydroxyl and recruitment of a poly-A polymerase to add a chain of adenine nucleotides to the RNA. In some embodiments, the polyA tail is encoded in the saRNA, such as by using a polyT sequence at the 5’ end of a negativestrand template for the saRNA.

[0163] Hi. Promoters

[0164] In some embodiments, the saRNAs described herein can self-replicate due to inclusion of conserved sequence elements derived from at least one virus, located on the 5' and 3' ends of the RNA in combination with protein machinery (RNA-dependent RNA polymerase or RdRp derived from at least one virus). In some embodiments, the saRNA can also include amplification of a sub-genomic RNA, which can encode a cargo of interest (e.g., a barcoded mRNA), from a subgenomic promoter derived from a virus that is recognized by the RdRp.

[0165] As used herein, the term “subgenomic promoter” refers to a nucleic acid sequence upstream (5') of a nucleic acid sequence (e.g. coding sequence), which controls transcription of the nucleic acid sequence by providing a recognition and binding site for RNA polymerase, typically RNA-dependent RNA polymerase (e.g., a functional alphavirus non- structural protein). The subgenomic promoter may include further recognition or binding sites for further factors. A subgenomic promoter is typically a genetic element of a positive strand RNA virus, such as an alphavirus. A subgenomic promoter of alphavirus is a nucleic acid sequence comprised in the viral genomic RNA. The subgenomic promoter is generally characterized in that it allows initiation of the transcription (RNA synthesis) in the presence of an RNA-dependent RNA polymerase, e.g. functional alphavirus non- structural protein. A RNA (-) strand, i.e. the complement of alphaviral genomic RNA, serves as a template for synthesis of a (+) strand subgenomic transcript, and synthesis of the (+) strand subgenomic transcript is typically initiated at or near the subgenomic promoter. The term “subgenomic promoter” as used herein, is not confined to any particular localization in a nucleic acid comprising such subgenomic promoter.

[0166] The subgenomic promoter (SGP) can be located at the end of nsp4 and contains sequences within and after the coding sequence of nsp4. The SGP can be dependent on viralFAZ-40125

[0167] machinery (e.g., nsPl-4; e.g., RdRP) to generate the subgenomic RNA (sgRNA) of the saRNA. The subgenomic promoter controls expression of sgRNA from anti-sense template RNA independently of its genomic length counterpart. In some embodiments, the strength of the subgenomic promoter (SGP) leads to more copies of the sgRNA compared to the full-length+RNA. In the context of alphaviruses, the higher concentration of the sgRNA compared to the full-length+RNA can allow for a higher concentration of translated structural proteins compared to translated non-structural proteins. In the context of the saRNAs described herein, the higher concentration of the sgRNA compared to the full-length+RNA can allow for a higher concentration of the at least one cargo (which can be a translated protein or a non-coding RNA, as described further herein) compared to translated non-structural proteins.

[0168] In some embodiments, the saRNA comprises a subgenomic promoter. In some embodiments, at least one open reading frame of the saRNA is under the control of a subgenomic promoter. In some embodiments, the saRNAs described herein comprise at least one subgenomic promoter derived from at least one virus. In some embodiments, the SGP is 3' (downstream) of the at least one nonstructural proteins and 5' (upstream) of the 5' UTR and at least one cargo.

[0169] In some embodiments, the saRNAs described herein comprise at least one subgenomic promoter derived from at least one alphavirus. In some embodiments, the saRNA comprises at least one subgenomic promoter derived from one or more of Venezuelan Equine Encephalitis Virus (VEEV), Sindbis Virus (SINV), Semliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), and a fusion of VEEV and SINV (VEE-SINV). In some embodiments, the saRNAs described herein comprise at least one subgenomic promoter derived from VEEV. In some embodiments, the saRNAs described herein comprise at least one subgenomic promoter derived from SINV. In some embodiments, the saRNAs described herein comprise at least one subgenomic promoter derived from SFV. In some embodiments, the saRNAs described herein comprise at least one subgenomic promoter derived from CSFV. In some embodiments, the saRNAs described herein comprise at least one subgenomic promoter derived from VEE-SINV. In some embodiments, the subgenomic promoter is derived from a virus described in Blakney et al. An Update on Self-Amplifying mRNA Vaccine Development. Vaccines (Basel). 2021 Jan 28; 9(2).

[0170] In some embodiments, the subgenomic promoter is recognized by an RNA-dependent RNA polymerase (RdRP) and / or RNA molecules encoding an RNA-dependent RNA polymerase (RdRP). Examples of such subgenomic promoters and RdRP include a Brome Mosaic Virus subgenomic promoter and RdRP (Siegal et al.1998, doi:FAZ-40125

[0171] 10.1073 / pnas.95.20.11613), barley yellow dwarf virus (BYDV) sgRNAl, sgRNA2, and sgRNA3 subgenomic promoters and RdRP (Koev and Miller; J Virol.2000 Jul;74(13):5988-96. Doi: 10.1128 / jvi.74.13.5988-5996.2000), Alternanthera mosaic virus (AltMV-MU) sgpl, sgp2, and sgp3 subgenomic promoters and RdRP (Putlyaev et al., Biochemistry (Mose). ; 80(8) :1039-46DOI: 10.1134 / S000629791508009X).

[0172] If functional non- structural protein, i.e. non-structural protein with replicase function, is encoded by a nucleic acid molecule according to the disclosure, it is preferable that the subgenomic promoter of the replicon, if present, is compatible with the replicase. Compatible in this context means that the replicase is capable of recognizing the subgenomic promoter. In some embodiments, this is achieved when the subgenomic promoter is native to the virus from which the replicase is derived, i.e. the natural origin of these sequences is the same virus. In some embodiments, the subgenomic promoter is not native to the virus from which the virus replicase is derived, provided that the virus replicase is capable of recognizing the subgenomic promoter. In other words, the replicase is compatible with the subgenomic promoter (cross-virus compatibility). Examples of cross-virus compatibility concerning subgenomic promoter and replicase originating from different alphaviruses are known in the art. Any combination of subgenomic promoter and replicase is possible as long as cross-virus compatibility exists. Cross-virus compatibility can readily be tested by the skilled person by incubating a replicase to be tested together with an RNA, wherein the RNA has a subgenomic promoter to be tested, at conditions suitable for RNA synthesis from the subgenomic promoter. If a subgenomic transcript is prepared, the subgenomic promoter and the replicase are determined to be compatible. Various examples of cross-virus compatibility are known (reviewed by Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562).

[0173] In some embodiments, such subgenomic promoters are placed either 5’ and / or 3’ to an RNA molecule comprising a 5’ RNA replication element, a cargo RNA, and a 3’ RNA replication element to permit production of either or both + and - strands of the RNA molecule when the RdRP is provided.

[0174] In some embodiments, subgenomic promoters are operably linked to a cargo RNA molecule and / or to any additional RNA element to permit production of the corresponding cargo and / or additional RNA when the RdRP is provided.

[0175] In some embodiments, the saRNAs described herein can self-replicate due to inclusion of a genomic promoter derived from at least one virus, located on the 5' end of the RNA in combination with protein machinery (RNA-dependent RNA polymerase or RdRp derived fromFAZ-40125

[0176] at least one virus). The term “genomic promoter” or “promoter region” refers to a nucleic acid sequence which controls synthesis of a plus-strand or negative-strand saRNA transcript, by providing a recognition and binding site for RNA polymerase. The promoter region may include recognition or binding sites for further factors involved in regulating transcription of the gene. In some embodiments, the genomic promoter is a viral promoter. In some embodiments, the genomic promoter is an alphavirus promoter. In some embodiments, the saRNA comprises at least one genomic promoter derived from one or more of Venezuelan Equine Encephalitis Virus (VEEV), Sindbis Virus (SINV), Semliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), and a fusion of VEEV and SINV (VEE-SINV). In some embodiments, the saRNAs described herein comprise at least one genomic promoter derived from VEEV. In some embodiments, the saRNAs described herein comprise at least one genomic promoter derived from SINV. In some embodiments, the saRNAs described herein comprise at least one genomic promoter derived from SFV. In some embodiments, the saRNAs described herein comprise at least one genomic promoter derived from CSFV. In some embodiments, the saRNAs described herein comprise at least one genomic promoter derived from VEE-SINV. In some embodiments, the genomic promoter is derived from a virus described in Blakney et al. An Update on Self-Amplifying mRNA Vaccine Development. Vaccines (Basel). 2021 Jan 28; 9(2).

[0177] iv. Caps and Initiating Nucleotides

[0178] In some embodiments, described herein are saRNAs comprising a 5’ cap, which is found on the 5' end of the saRNA molecule. In some embodiments, the 5’ cap is derived from at least one virus. In some embodiments, the 5’ cap is derived from at least one alphavirus. In some embodiments, the 5’ cap is selected from the group consisting of cap-0, cap-1, and cap-2. The 5’ cap can be involved in translation, nucleocytoplasmic transport, splicing, and / or stabilization of saRNA against 5' exonucleolytic degradation.

[0179] In eukaryotes, the 5' cap, referred to as cap-0, is found on the 5' end of an RNA molecule. Cap-0 consists of a guanine nucleotide connected to mRNA via a 5' to 5' triphosphate linkage. This guanosine is methylated on the 7 position directly after capping in vivo by a methyltransferase. Cap-0 can also be referred to as a 7-methylguanylate cap, abbreviated m7G. In some embodiments, an saRNA as described herein comprises cap-0. In some embodiments, an saRNA as described herein comprises a m7G 5’ cap. ARCA (Anti-Reverse Cap Analog) consisting of 3'-O-Me-m7G(5')ppp(5')G is a non-limiting example of a reagent for producing an saRNA comprising a 5’ CapO.FAZ-40125

[0180] In multicellular eukaryotes and some viruses, further 5’ cap modifications exist, including the methylation of the 2' hydroxy- groups of the first 2 ribose sugars of the 5' end of the RNA. Cap-1 has a methylated 2'-hydroxy group on the first ribose sugar, while cap-2 has methylated 2'-hydroxy groups on the first two ribose sugars. In some embodiments, the initiating nucleoside of an saRNA as described herein is methylated at the 2'0 position of the ribose (Capl). In some embodiments, Capl has the following chemical structure: m7GpppNm. Capl consisting of m7G(5')ppp(5')(2'OMeA)pU is a non- limiting example of a reagent for producing an saRNA comprising a 5’ Capl. In some embodiments, the initiating nucleotide and the subsequent nucleotide of an saRNA as described herein are both methylated at the 2'0 position of the ribose (Cap2). In some embodiments, Cap2 has the following chemical structure: m7GpppNmNm. In some embodiments, the 5’ cap is a Capl. In some embodiments, the 5’ cap modification comprises the structure set forth below:

[0181]

[0182] In some embodiments, the initiating nucleotide directly proximal to the 5’ cap in an saRNA as described herein comprises an adenosine or adenosine analog. In some embodiments, the initiating nucleotide directly proximal to the 5’ cap in an saRNA as described herein comprises a guanosine or guanosine analog. In some embodiments, the first two initiating nucleotides directly proximal to the 5’ cap in an saRNA as described herein comprises: in position 1 an adenosine or adenosine analog and in position 2 a uridine or a uridine analog. In some embodiments, the first three initiating nucleotides directly proximal to the 5’ cap in an saRNA as described herein comprises: in position 1 a guanosine or guanosine analog, in position 2 an adenosine or adenosine analog, and in position 3 a uridine or a uridine analog.

[0183] In some embodiments, the initiating nucleotide of an saRNA as described herein comprises an adenosine or adenosine analog, and the initiating nucleotide of the saRNA isFAZ-40125

[0184] methylated at the 2'0 position of the ribose (Capl). In some embodiments, the initiating nucleotide of an saRNA as described herein comprises an adenosine or adenosine analog, and the initiating nucleotide and the subsequent nucleotide of the saRNA are both methylated at the 2'0 position of the ribose (Cap2).

[0185] In some embodiments, the initiating nucleotide of an saRNA as described herein comprises a guanosine or guanosine analog, and wherein the initiating nucleotide of the saRNA is methylated at the 2'0 position of the ribose (Capl). In some embodiments, the initiating nucleotide of an saRNA as described herein comprises a guanosine or guanosine analog, and wherein the initiating nucleotide and the subsequent nucleotide of the saRNA are both methylated at the 2'0 position of the ribose (Cap2). In some embodiments, the initiating nucleotide of an saRNA as described herein comprises an adenosine or adenosine analog, and the 5’ cap is a m7G 5’ cap-0. In some embodiments, the initiating nucleotide of an saRNA as described herein comprises a guanosine or guanosine analog, and the 5’ cap is a m7G 5’ cap-0.

[0186] v. Nucleic Acid Modifications

[0187] In some embodiments, an saRNA as described herein comprises at least one nucleic acid modification, which can increase the efficacy of the saRNA. In some embodiments, an saRNA as described herein comprises modified nucleotides, e.g., at least 10%, at least 25%, or at least 50% modified nucleotides. In some embodiments, an saRNA as described herein comprises a specific combination of 5’ nucleotide(s) and initiating nucleotide(s). In some embodiments, an saRNA as described herein comprises end modifications, backbone modifications, and / or sugar modifications.

[0188] In some embodiments, described herein are saRNAs comprising modified nucleotides. The term “modified nucleotide” refers to any analog of cytidine, adenosine, guanosine, uridine, or pseudouridine. These analogs can include isomers of the nitrogenous base, as well as inclusion or exclusion of chemical groups, both natural occurring and synthetically introduced, on any aspect of the nitrogenous base. It is explicitly stated herein that modifications to the sugar-phosphate backbone are not included in the definition of the term “modified nucleotide.” This exception is not meant to exclude the methylation at the 2’0 position of the first and second initiating nucleotides, also referred to as Cap-1 and cap-2 structures.

[0189] In some embodiments, the modified nucleotides comprise a modified pyrimidine nucleoside phosphate. In some embodiments, the modified nucleotides comprise a pyrimidine nucleoside phosphate with a moiety on carbon 5 of the pyrimidine. In some embodiments, the moiety on carbon 5 of the pyrimidine is selected from the group consisting of methyl, ethyl,FAZ-40125

[0190] propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, and hydroxypropyl functional groups. In some embodiments, the pyrimidine comprises cytidine and / or uridine.

[0191] In some embodiments, the pyrimidine comprises cytidine. In some embodiments, the pyrimidine comprises cytidine, and the modified nucleotide comprises 5-methylcytidine. In some embodiments, the pyrimidine comprises cytidine, and the modified nucleotide comprises 5-hydroxymethylcytidine. In some embodiments, the pyrimidine comprises cytidine, and the modified nucleotide comprises 5-methylcytidine and 5-hydroxymethylcytidine.

[0192] In some embodiments, the pyrimidine comprises uridine. In some embodiments, the pyrimidine comprises uridine, and the modified nucleotide comprises 5-methyluridine. In some embodiments, the pyrimidine comprises uridine, and the modified nucleotide comprises 5-hydroxymethyluridine. In some embodiments, the pyrimidine comprises uridine, and the modified nucleotide comprises 5-methyluridine and 5-hydroxymethyluridine.

[0193] In some embodiments, the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine, or any combination thereof. In some embodiments, the modified nucleotides are selected from the group consisting of 5-methylcytidine, 5-methyluridine, and 5-hydroxymethylcytidine, or any combination thereof. In some embodiments, the modified nucleotides are selected from the group consisting of 5-methyluridine, 5-hydroxymethyluridine, and 5-hydroxymethylcytidine, or any combination thereof.

[0194] In some embodiments, an saRNA as described herein comprises no modified nucleotides. In some embodiments, an saRNA as described herein comprises at least 1% modified nucleotides and at most 100% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine; e.g., cytidine and / or uridine). In some embodiments, an saRNA as described herein comprises at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%,FAZ-40125

[0195] at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 99%, or 100% modified nucleotides, corresponding to at least one specific nucleotide (e.g., a pyrimidine; e.g., cytidine and / or uridine).

[0196] Subgenomic Cargo

[0197] In some embodiments, the saRNA comprises a subgenomic strand. In some embodiments, the subgenomic strand comprises a subgenomic promoter, an open reading from (ORF), and a barcode. In some embodiments, the saRNA comprises an open reading frame (ORF). In some embodiments, the ORF is operably linked to a 5’ or 3’ UTR of the saRNA.

[0198] In some embodiments, the saRNA described herein comprise an open reading frame (ORF) capable of being translated into a protein of interest. In some embodiments, the saRNA described herein comprise an open reading frame encoding a random barcode sequence. In some embodiments, the saRNA described herein comprise an open reading frame (i) encoding a protein of interest and (ii) comprising a random barcode sequence. In some embodiments, the open reading frame is under the control of a subgenomic promoter. In some embodiments, the ORF is located 3’ relative to the subgenomic promoter. In some embodiments, the ORF is located 5’ relative to the barcode. In some embodiments, the ORF is located 3’ relative to the subgenomic promoter and 5’ relative to the barcode.

[0199] In some embodiments, the open reading frame encodes a protein that produces a detectable signal. In some embodiments, the protein that produces the detectable signal is selected from a luciferase, a horseradish peroxidase, an alkaline phosphatase, a P-galactosidase, and any combination thereof. In some embodiments, the detectable signal is produced by luciferase. In some embodiments, the detectable signal is produced by Gaussia luciferase. In some embodiments, the detectable signal is produced by a codon optimized Gaussia luciferase. In some embodiments, the detectable signal is produced by a human codon optimized Gaussia luciferase. In some embodiments, the Gaussia luciferase contains silent G8064T and A8067G mutations, which introduce a BbvCI restriction site to enable subcloning of barcode sequences.

[0200] In some embodiments, the saRNA includes a barcode sequence. This sequence is a unique sequence which allows identification of the specific saRNA being tested or employed. The barcode sequence also allows for quantification of the in vitro transcribed saRNA during analysis by deep sequencing. The barcode can be designed to any length available usingFAZ-40125

[0201] synthesis technology, and the length of the barcode limits the number of formulations that may be tested simultaneously. For example, using the lObp barcode exemplified herein, there are a total of 1048576 possible combinations. Thus, the barcode sequence is, in some embodiments, between 5 nt to 100 nt in length. In some embodiments, the barcode sequence is between 8 nt to 12 nt in length. In some embodiments, the barcode sequence is between 10 nt to 20 nt in length. In some embodiments, the barcode is 8 nt in length. In some embodiments, the barcode is 9 nt in length. In some embodiments, the barcode is 10 nt in length. In some embodiments, the barcode is 11 nt in length. In some embodiments, the barcode is 12 nt in length. In some embodiments, the barcode is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nt in length.

[0202] In some embodiments, the subgenomic strand comprises the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, during synthesis of the subgenomic strand, a poly-A tail is added enzymatically. In some embodiments, the poly-A tail of the subgenomic strand is about 200 nucleotides in length.

[0203] SaRNA Exemplary Sequences

[0204] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0205] i) a 5’ cap;

[0206] ii) a promoter;

[0207] iii) a 5’ UTR;

[0208] iv) nsPl, nsP2, nsP3, and nsP4;

[0209] v) a subgenomic promoter;

[0210] vi) a subgenomic 5’ UTR;

[0211] vii) a barcode;

[0212] viii) a 3 ’ UTR; and

[0213] ix) a poly(A) tail.

[0214] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0215] i) a 5’ cap;

[0216] ii) a 5’ UTR, including a promoter;

[0217] iii) nsPl, nsP2, nsP3, and nsP4;

[0218] iv) a subgenomic promoter;

[0219] v) a subgenomic 5’ UTR;FAZ-40125

[0220] vi) a barcode;

[0221] vii) a 3 ’ UTR; and

[0222] viii) a poly(A) tail.

[0223] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0224] i) a 5’ cap;

[0225] ii) a promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0226] iii) a 5’ UTR;

[0227] iv) nsPl, nsP2, nsP3, and nsP4;

[0228] v) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0229] vi) a subgenomic 5’ UTR;

[0230] vii) a barcode;

[0231] viii) a 3 ’ UTR; and

[0232] ix) a poly(A) tail.

[0233] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0234] i) a 5’ cap;

[0235] ii) a 5’ UTR including a genomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0236] iii) nsPl, nsP2, nsP3, and nsP4;

[0237] iv) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0238] v) a subgenomic 5’ UTR;

[0239] vi) a barcode;

[0240] vii) a 3 ’ UTR; and

[0241] viii) a poly(A) tail.

[0242] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:FAZ-40125

[0243] i) a 5’ cap;

[0244] ii) a promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0245] iii) a 5’ UTR;

[0246] iv) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0247] v) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0248] vi) a subgenomic 5’ UTR;

[0249] vii) a barcode;

[0250] viii) a 3 ’ UTR; and

[0251] ix) a poly(A) tail.

[0252] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0253] i) a 5’ cap;

[0254] ii) a 5’ UTR including a genomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0255] iii) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0256] iv) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0257] v) a subgenomic 5’ UTR;

[0258] vi) a barcode;

[0259] vii) a 3 ’ UTR; and

[0260] viii) a poly(A) tail.

[0261] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0262] i) a 5’ cap;FAZ-40125

[0263] ii) a promoter;

[0264] iii) a 5’ UTR;

[0265] iv) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0266] v) a subgenomic promoter;

[0267] vi) a subgenomic 5’ UTR;

[0268] vii) a barcode;

[0269] viii) a 3 ’ UTR; and

[0270] ix) a poly(A) tail.

[0271] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0272] i) a 5’ cap;

[0273] ii) a 5’ UTR including a promoter;

[0274] iii) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0275] iv) a subgenomic promoter;

[0276] v) a subgenomic 5’ UTR;

[0277] vi) a barcode;

[0278] vii) a 3 ’ UTR; and

[0279] viii) a poly(A) tail. In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0280] i) a m7G cap;

[0281] ii) a promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0282] iii) a 5’ UTR;

[0283] iv) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0284] v) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);FAZ-40125

[0285] vi) a subgenomic 5’ UTR;

[0286] vii) a barcode;

[0287] viii) a 3 ’ UTR; and

[0288] ix) a poly(A) tail.

[0289] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0290] i) a m7G cap;

[0291] ii) a 5’ UTR including a genomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0292] iii) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0293] iv) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0294] v) a subgenomic 5’ UTR;

[0295] vi) a barcode;

[0296] vii) a 3 ’ UTR; and

[0297] viii) a poly(A) tail.

[0298] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0299] i) a m7G cap;

[0300] ii) a promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0301] iii) a 5’ UTR;

[0302] iv) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0303] v) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0304] vi) a subgenomic 5’ UTR;FAZ-40125

[0305] vii) a 10 nt barcode;

[0306] viii) a 3 ’ UTR; and

[0307] ix) a poly(A) tail.

[0308] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0309] i) a m7G cap;

[0310] ii) a 5’ UTR including a genomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0311] iii) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0312] iv) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0313] v) a subgenomic 5’ UTR;

[0314] vi) a 10 nt barcode;

[0315] vii) a 3 ’ UTR; and

[0316] viii) a poly(A) tail.

[0317] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0318] i) a 5’ cap;

[0319] ii) a promoter;

[0320] iii) a 5’ UTR;

[0321] iv) nsPl, nsP2, nsP3, and nsP4;

[0322] v) a subgenomic promoter;

[0323] vi) a subgenomic 5’ UTR;

[0324] vii) a reporter gene;

[0325] viii) a barcode;

[0326] ix) a 3 ’ UTR; and

[0327] x) a poly(A) tail.

[0328] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0329] i) a 5’ cap;FAZ-40125

[0330] ii) a 5’ UTR including a promoter;

[0331] iii) nsPl, nsP2, nsP3, and nsP4;

[0332] iv) a subgenomic promoter;

[0333] v) a subgenomic 5’ UTR;

[0334] vi) a reporter gene;

[0335] vii) a barcode;

[0336] viii) a 3 ’ UTR; and

[0337] ix) a poly(A) tail. In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0338] i) a 5’ cap;

[0339] ii) a promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0340] iii) a 5’ UTR;

[0341] iv) nsPl, nsP2, nsP3, and nsP4;

[0342] v) a subgenomic promoter;

[0343] vi) a subgenomic 5’ UTR;

[0344] vii) a reporter gene;

[0345] viii) a barcode;

[0346] ix) a 3 ’ UTR; and

[0347] x) a poly(A) tail.

[0348] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0349] i) a 5’ cap;

[0350] ii) a 5’ UTR including a genomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0351] iii) nsPl, nsP2, nsP3, and nsP4;

[0352] iv) a subgenomic promoter;

[0353] v) a subgenomic 5’ UTR;

[0354] vi) a reporter gene;

[0355] vii) a barcode;

[0356] viii) a 3 ’ UTR; and

[0357] ix) a poly(A) tail.FAZ-40125

[0358] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0359] i) a 5’ cap;

[0360] ii) a promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0361] iii) a 5’ UTR;

[0362] iv) nsPl, nsP2, nsP3, and nsP4;

[0363] v) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0364] vi) a subgenomic 5’ UTR;

[0365] vii) a reporter gene;

[0366] viii) a barcode;

[0367] ix) a 3 ’ UTR; and

[0368] x) a poly(A) tail.

[0369] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0370] i) a 5’ cap;

[0371] ii) a 5’ UTR including a genomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0372] iii) nsPl, nsP2, nsP3, and nsP4;

[0373] iv) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0374] v) a subgenomic 5’ UTR;

[0375] vi) a reporter gene;

[0376] vii) a barcode;

[0377] viii) a 3 ’ UTR; and

[0378] ix) a poly(A) tail.

[0379] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0380] i) a 5’ cap;FAZ-40125

[0381] ii) a promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0382] iii) a 5’ UTR;

[0383] iv) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0384] v) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0385] vi) a subgenomic 5’ UTR;

[0386] vii) a reporter gene;

[0387] viii) a barcode;

[0388] ix) a 3 ’ UTR; and

[0389] x) a poly(A) tail.

[0390] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0391] i) a 5’ cap;

[0392] ii) a 5’ UTR including a genomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0393] iii) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0394] iv) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0395] v) a subgenomic 5’ UTR;

[0396] vi) a reporter gene;

[0397] vii) a barcode;

[0398] viii) a 3 ’ UTR; and

[0399] ix) a poly(A) tail.

[0400] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:FAZ-40125

[0401] i) a 5’ cap;

[0402] ii) a promoter;

[0403] iii) a 5’ UTR;

[0404] iv) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0405] v) a subgenomic promoter;

[0406] vi) a subgenomic 5’ UTR;

[0407] vii) a reporter gene;

[0408] viii) a barcode;

[0409] ix) a 3 ’ UTR; and

[0410] x) a poly(A) tail.

[0411] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0412] i) a 5’ cap;

[0413] ii) a 5’ UTR including a promoter;

[0414] iii) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0415] iv) a subgenomic promoter;

[0416] v) a subgenomic 5’ UTR;

[0417] vi) a reporter gene;

[0418] vii) a barcode;

[0419] viii) a 3 ’ UTR; and

[0420] ix) a poly(A) tail.

[0421] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0422] i) a 5’ cap;

[0423] ii) a promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0424] iii) a 5’ UTR;FAZ-40125

[0425] iv) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0426] v) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0427] vi) a subgenomic 5’ UTR;

[0428] vii) a human gaussia luciferase;

[0429] viii) a barcode;

[0430] ix) a 3 ’ UTR; and

[0431] x) a poly(A) tail.

[0432] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0433] i) a 5’ cap;

[0434] ii) a 5’ UTR including a genomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0435] iii) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0436] iv) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0437] v) a subgenomic 5’ UTR;

[0438] vi) a humanized Gaussia luciferase;

[0439] vii) a barcode;

[0440] viii) a 3 ’ UTR; and

[0441] ix) a poly(A) tail.

[0442] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0443] i) a m7G;

[0444] ii) a promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);FAZ-40125

[0445] iii) a 5’ UTR;

[0446] iv) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0447] v) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0448] vi) a subgenomic 5’ UTR;

[0449] vii) a human gaussia luciferase;

[0450] viii) a barcode;

[0451] ix) a 3 ’ UTR; and

[0452] x) a poly(A) tail.

[0453] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0454] i) a m7G;

[0455] ii) a 5’ UTR including a genomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0456] iii) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0457] iv) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0458] v) a subgenomic 5’ UTR;

[0459] vi) a humanized Gaussia luciferase;

[0460] vii) a barcode;

[0461] viii) a 3 ’ UTR; and

[0462] ix) a poly(A) tail.

[0463] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0464] i) a m7G;FAZ-40125

[0465] ii) a promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0466] iii) a 5’ UTR;

[0467] iv) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0468] v) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0469] vi) a subgenomic 5’ UTR;

[0470] vii) a human gaussia luciferase;

[0471] viii) a 10 nt barcode;

[0472] ix) a 3 ’ UTR; and

[0473] x) a poly(A) tail.

[0474] In some embodiments, the saRNA described herein comprises a nucleic acid sequence encoding from 5’ to 3’:

[0475] i) a m7G;

[0476] ii) a 5’ UTR including a genomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0477] iii) nsPl, nsP2, nsP3, and nsP4 derived from one or more of Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV);

[0478] iv) a subgenomic promoter derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), or a fusion of VEEV and SINV (VEE-SINV);

[0479] v) a subgenomic 5’ UTR;

[0480] vi) a humanized Gaussia luciferase;

[0481] vii) a 10 nt barcode;

[0482] viii) a 3 ’ UTR; and

[0483] ix) a poly(A) tail.

[0484] In some embodiments, the saRNA described herein are synthesized from a plasmid. In some embodiments, the saRNA are synthesized from a plasmid comprising a Blue HeronFAZ-40125

[0485] pBR322 minusMCS vector, VEEV clone TC-83, human codon-optimized Gaussia luciferase, a barcode region, a custom-designed primer binding site, and a segmented poly-A tail as described in Nance, K and Meier, J, ACS Cent Sci. 7(5):748-756, April 6, 2021. In some embodiments, the TC-83 sequence contains a G3936C point mutation reported to increase subgenomic strand generation (see e.g., Li et al. Scientific Reports. 2019 May 6;9:6932).

[0486] In some embodiments, the saRNA is encoded by the nucleic acid sequence set forth in SEQ ID NO: 1. In some embodiments, the saRNA is encoded by a nucleic acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1.

[0487] In some embodiments, the positive (+) strand saRNA is encoded by the nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, the positive (+) strand saRNA is encoded by a nucleic acid sequence comprising at least 80%, at least 85%, 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% identity to SEQ ID NO: 2.

[0488] In some embodiments, the negative (-) strand saRNA is encoded by the nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments, the negative (-) strand saRNA is encoded by a nucleic acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 3.

[0489] In some embodiments, the subgenomic saRNA strand is encoded by the nucleic acid sequence set forth in SEQ ID NO: 4. In some embodiments, the subgenomic saRNA strand is encoded by a nucleic acid sequence comprising at least 80%, at least 85%, 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% identity to SEQ ID NO: 4.

[0490] Table 1. SaRNA Sequences

[0491]

[0492] FAZ-40125

[0493]

[0494] FAZ-40125

[0495]

[0496] FAZ-40125

[0497]

[0498] FAZ-40125

[0499]

[0500] FAZ-40125

[0501]

[0502] FAZ-40125

[0503]

[0504] FAZ-40125

[0505]

[0506] FAZ-40125

[0507]

[0508] FAZ-40125

[0509]

[0510] FAZ-40125

[0511]

[0512] FAZ-40125

[0513]

[0514] FAZ-40125

[0515]

[0516] FAZ-40125

[0517]

[0518] FAZ-40125

[0519]

[0520] FAZ-40125

[0521]

[0522] FAZ-40125

[0523]

[0524] FAZ-40125

[0525]

[0526] FAZ-40125

[0527]

[0528] FAZ-40125

[0529]

[0530] FAZ-40125

[0531]

[0532] II. Nanoparticle Compositions

[0533] Formulations

[0534] Provided herein are nanoparticle compositions. As used here, “nanoparticle composition” is used interchangeably with the terms “lipid-based carrier,” “lipid nanoformulation,” and “lipid nanoparticle.” In some embodiments, the lipid nanoformulation is a lipid nanoformulation known in the art.

[0535] In some embodiments, the nanoparticle composition comprises a lipid. In certain such embodiments, the lipid is a cationic, anionic, ionizable, or zwitterionic lipid.

[0536] In some embodiments, compounds described herein are formulated into a lipid-based carrier (or lipid nanoformulation). In some embodiments, the lipid-based carrier (or lipidFAZ-40125

[0537] nanoformulation) is a liposome or a lipid nanoparticle (LNP). In one embodiment, the lipid-based carrier is an LNP.

[0538] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid (e.g., an ionizable lipid), a non-cationic lipid (e.g., phospholipid), a structural lipid (e.g., cholesterol), and a PEG-modified lipid. In some embodiments, the lipid-based carrier (or lipid nanoformulation) contains one or more compounds described herein, or a pharmaceutically acceptable salt thereof.

[0539] As described herein, suitable compounds to be used in the lipid-based carrier (or lipid nanoformulation) include all the isomers and isotopes of the compounds described above, as well as all the pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystal forms, crystal form mixtures, and anhydrides or hydrates.

[0540] In addition to one or more compounds described herein, the lipid-based carrier (or lipid nanoformulation) may further include a second lipid. In some embodiments, the second lipid is a cationic lipid, a non-cationic (e.g., neutral, anionic, or zwitterionic) lipid, or an ionizable lipid.

[0541] One or more naturally occurring and / or synthetic lipid compounds may be used in the preparation of the lipid-based carrier (or lipid nanoformulation).

[0542] The lipid-based carrier (or lipid nanoformulation) may contain positively charged (cationic) lipids, neutral lipids, negatively charged (anionic) lipids, or a combination thereof.

[0543] In some embodiments, the lipid nanoparticle may be conjugated to a targeting moiety (e.g., an antibody or antigen-binding fragment thereof) through a linking group. Various linking groups known in the art may be used in the lipid nanoparticles described herein, and can comprise one or more of optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted alkenylene, optionally substituted heteroalkenylene, optionally substituted alkynylene, optionally substituted heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, a peptide moiety, a dipeptide moiety, -(C=O)-, a disulfide, a hydrazone, thioester, sulfone, sulfoxide, thiosulfinate, thiosulfonate, sulfate, sulfonate, sulfonylurea, ether, thioether, ester, amide, carbonate, carbamate, urea, sulfamide, succinimide, maleimide, phosphate, diphosphate, triazole, or a saccharide, or a combination thereof. Suitable linking groups are described, e.g., in WO 2024 / 015229, WO 2024 / 006272, and WO 2023 / 225359.

[0544] Cationic Lipids (Positively Charged} and Ionizable Lipids

[0545] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprisesFAZ-40125

[0546] one or more cationic lipids, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions.

[0547] Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Examples of positively charged (cationic) lipids include, but are not limited to, N,N'-dimethyl-N,N'-dioctacyl ammonium bromide (DDAB) and chloride DDAC), N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 3P-[N-(N',N'-dimethylaminoethyl)carbamoyl) cholesterol (DC-chol), l,2-dioleoyloxy-3-[trimethylammonio] -propane (DOTAP), l,2-dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP), and l,2-dioleoyloxypropyl-3-dimethyl-hydroxy ethyl ammonium chloride (DORI), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), l,2-Dioleoyl-3-Dimethylammonium-propane (DODAP), 1 ,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP), 1 ,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP), 3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis, cis-9',12'-octadecadienoxy)propane (CpLin DMA), N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA), and the cationic lipids described in e.g. Martin et al., Current Pharmaceutical Design, pages 1-394, which is herein incorporated by reference in its entirety. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises more than one cationic lipid.

[0548] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid having an effective pKa over 6.0. In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa) than the first cationic lipid.

[0549] In some embodiments, cationic lipids that can be used in the lipid-based carrier (or lipid nanoformulation) include, for example those described in Table 4 of WO 2019 / 217941, which is incorporated by reference.

[0550] In some embodiments, the cationic lipid is an ionizable lipid (e.g., a lipid that is protonated at low pH, but that remains neutral at physiological pH). In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise one or more additional ionizable lipids, different than the ionizable lipids described herein. Exemplary ionizable lipids include, but are not limited to,FAZ-40125

[0551]

[0552] 3- DMA),

[0553] (see WO 2017 / 004143A1, which is incorporated herein by reference in its entirety). In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more compounds described by WO 2021 / 113777 (e.g., a lipid of Formula (3) such as a lipid of Table 3 of WO 2021 / 113777), which is incorporated herein by referenceFAZ-40125

[0554] in its entirety.

[0555] In one embodiment, the ionizable lipid is a lipid disclosed in Hou, X., et al. Nat Rev Mater 6, 1078-1094 (2021). https: / / doi.org / 10.1038 / s41578-021-00358-0 (e.g., L319, C12-200, and DLin-MC3-DMA), (which is incorporated by reference herein in its entirety).

[0556] Examples of other ionizable lipids that can be used in lipid-based carrier (or lipid nanoformulation) include, without limitation, one or more of the following formulas: X of US 2016 / 0311759; I of US 20150376115 or in US 2016 / 0376224; Compound 5 or Compound 6 in US 2016 / 0376224; I, IA, or II of US 9,867,888; I, II or III of US 2016 / 0151284; I, IA, II, or IIA of US 2017 / 0210967; I-c of US 2015 / 0140070; A of US 2013 / 0178541 ; I of US 2013 / 0303587 or US 2013 / 0123338; I of US 2015 / 0141678; II, III, IV, or V of US 2015 / 0239926; I of US 2017 / 0119904; I or II of WO 2017 / 117528; A of US 2012 / 0149894; A of US 2015 / 0057373; A of WO 2013 / 116126; A of US 2013 / 0090372; A of US 2013 / 0274523; A of US 2013 / 0274504; A of US 2013 / 0053572; A of WO 2013 / 016058; A of WO 2012 / 162210; I of US 2008 / 042973; I, II, III, or IV of US 2012 / 01287670; I or II of US 2014 / 0200257; I, II, or III of US 2015 / 0203446; I or III of US 2015 / 0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US 2014 / 0308304; of US 2013 / 0338210; I, II, III, or IV of WO 2009 / 132131; A of US 2012 / 01011478; I or XXXV of US 2012 / 0027796; XIV or XVII of US 2012 / 0058144; of US 2013 / 0323269; I of US 2011 / 0117125; I, II, or III of US 2011 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US 2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US 2011 / 0076335; I or II of US 2006 / 008378; I of WO2015 / 074085 (e.g., ATX-002); I of US 2013 / 0123338; I or X-A-Y-Z of US 2015 / 0064242; XVI, XVII, or XVIII of US 2013 / 0022649; I, II, or III of US 2013 / 0116307; I, II, or III of US 2013 / 0116307; I or II of US 2010 / 0062967; I-X of US 2013 / 0189351; I of US 2014 / 0039032; V of US 2018 / 0028664; I of US 2016 / 0317458; I of US 2013 / 0195920; 5, 6, or 10 of US 10,221,127; III-3 of WO 2018 / 081480; 1-5 or 1-8 of WO 2020 / 081938; I of WO 2015 / 199952 (e.g., compound 6 or 22) and Table 1 therein; 18 or 25 of US 9,867,888; A of US 2019 / 0136231; II of WO 2020 / 219876; 1 of US 2012 / 0027803; OF-02 of US 2019 / 0240349; 23 of US 10,086,013; cKK-E12 / A6 of Miao et al (2020); C12-200 of WO 2010 / 053572; 7C1 of Dahlman et al (2017); 304-013 or 503-013 of Whitehead et al; TS-P4C2 of U S9, 708, 628; I of WO 2020 / 106946; I of WO 2020 / 106946; (1), (2), (3), or (4) of WO 2021 / 113777; and any one of Tables 1-16 of WO 2021 / 113777, all of which are incorporated herein by reference in their entirety.

[0557] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further includes biodegradable ionizable lipids, for instance, (9Z,12Z)-3-((4,4-FAZ-40125

[0558] bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate). See, e.g., lipids of WO 2019 / 067992, WO 2017 / 173054, WO 2015 / 095340, and WO 2014 / 136086, which are incorporated herein by reference in their entirety.

[0559] i. Non-Cationic Lipids (e.g., Phospholipids)

[0560] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more non-cationic lipids. In some embodiments, the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is a phospholipid substitute or replacement. In some embodiments, the non-cationic lipid is a negatively charged (anionic) lipid.

[0561] Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethylphosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl- sn-glycero-3-phosphocholine (DLPC), Sodium 1,2- ditetradecanoyl-sn-glycero-3-phosphate (DMPA), phosphatidylcholine (lecithin), phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acidsFAZ-40125

[0562] having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl.

[0563] Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, which is incorporated herein by reference. Such lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g., DGTS).

[0564] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise a combination of distearoylphosphatidylcholine / cholesterol, dipalmitoylphosphatidylcholine / cholesterol, dimyrystoylphosphatidylcholine / cholesterol, 1 ,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) / cholesterol, or egg sphingomyelin / cholesterol.

[0565] Other examples of suitable non-cationic lipids include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO 2017 / 099823 or US 2018 / 0028664, which are incorporated herein by reference in their entirety.

[0566] In one embodiment, the lipid-based carrier (or lipid nanoformulation) further comprises one or more non-cationic lipid that is oleic acid or a compound of Formula I, II, or IV of US 2018 / 0028664, which is incorporated herein by reference in its entirety.

[0567] The non-cationic lipid content can be, for example, 0-30% (mol) of the total lipid components present. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid components present.

[0568] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a neutral lipid, and the molar ratio of an ionizable lipid to a neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).

[0569] In some embodiments, the lipid-based carrier (or lipid nanoformulation) does not include any phospholipids.

[0570] In some embodiments, the lipid-based carrier (or lipid nanoformulation) can further include one or more phospholipids, and optionally one or more additional molecules of similar molecular shape and dimensions having both a hydrophobic moiety and a hydrophilic moiety (e.g., cholesterol).

[0571] Exemplary anionic lipids include dimyrystoyl-, dipalmitoyl-, and distearoyl-phasphatidylglycerol; dimyrystoyl-, dipalmitoyl-, and dipalmitoyl-phosphatidic acid;FAZ-40125

[0572] dimyrystoyl-, dipalmitoyl-, and dipalmitoyl-phosphatidylethanolamine; and their unsaturated diacyl and mixed acyl chain counterparts as well as cardiolipin.

[0573] Exemplary neutral lipids include DLPC (1,2-dilauroyl- sn-glycero-3-phosphocholine), DMPC (l,2-dimyristoyl-sn-glycero-3-phosphocholine), DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (l,2-distearoyl-sn-glycero-3- phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DMPA (Sodium 1,2- ditetradecanoyl-sn-glycero-3-phosphate), DPPE (l,2-Dipalmitoyl-sn-glycero-3- phosphoethanolamine), and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine).

[0574] Exemplary phospholipids include, but are not limited to, phosphatidylcholine (lecithin), lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, phosphatidylcholine, and dipalmitoylphosphatidylglycerol.

[0575] ii. Structural Lipids

[0576] The lipid-based carrier (or lipid nanoformulation) described herein may further comprise one or more structural lipids. As used herein, the term “structural lipid” refers to sterols (e.g., cholesterol and derivatives thereof) and to lipids containing sterol moieties.

[0577] Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipid in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol or cholesterol derivative, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alphatocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alphatocopherol.

[0578] In some embodiments, structural lipids may be incorporated into the lipid-based carrier at molar ratios ranging from about 0.1 to 1.0 (cholesterol phospholipid).

[0579] In some embodiments, sterols, when present, can include one or more of cholesterol or cholesterol derivatives, such as those described in WO 2009 / 127060 or US 2010 / 0130588, which are incorporated herein by reference in their entirety. Additional exemplary sterols include phytosterols, including those described in Eygeris et al. (2020), Nano Lett.

[0580] 2020;20(6):4543-4549, incorporated herein by reference.

[0581] In some embodiments, the structural lipid is a cholesterol derivative. Non-limitingFAZ-40125

[0582] examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 53-coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy) -butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue, e.g., cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in WO 2009 / 127060 and US 2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0583] In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises sterol in an amount of 0-50 mol% (e.g., 0-10 mol %, 10-20 mol %, 20-50 mol%, 20-30 mol %, 30-40 mol %, or 40-50 mol %) of the total lipid components.

[0584] Hi. Polymers and Polyethylene Glycol (PEG) Lipids

[0585] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may include one or more polymers or co-polymers, e.g., poly(lactic-co-glycolic acid) (PFAG) nanoparticles.

[0586] In some embodiments, the lipid-based carrier (or lipid nanoformulation) may include one or more polyethylene glycol (PEG) lipid (also referred to as a “PEGylated lipid”).

[0587] Examples of useful PEG-lipids include, but are not limited to, l,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-350] (mPEG 350 PE); 1,2-Diacyl-sn- Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-550] (mPEG 550 PE); 1,2- Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-750] (mPEG 750 PE); l,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-1000] (mPEG 1000 PE); l,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000] (mPEG 2000 PE); l,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy(Polyethylene glycol)-3000] (mPEG 3000 PE); 1,2-Diacyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-5000] (mPEG 5000 PE); N-Acyl- Sphingosine- 1- [Succinyl (Methoxy Polyethylene Glycol) 750] (mPEG 750 Ceramide); N-Acyl- Sphingosine- l-[Succinyl(Methoxy Polyethylene Glycol) 2000] (mPEG 2000 Ceramide); and N- Acyl-Sphingosine-l-[Succinyl(Methoxy Polyethylene Glycol) 5000] (mPEG 5000 Ceramide). In some embodiments, the PEG lipid is a polyethyleneglycoldiacylglycerol (i.e., polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-DMB) conjugate.

[0588] In some embodiments, the lipid-based carrier (or nanoformulation) includes one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO 2019 / 217941, which is incorporated herein by reference in itsFAZ-40125

[0589] entirety). In some embodiments, the one or more conjugated lipids is formulated with one or more ionic lipids (e.g., non-cationic lipid such as a neutral or anionic, or zwitterionic lipid); and one or more sterols (e.g., cholesterol).

[0590] The PEG conjugate can comprise a PEG-dilaurylglycerol (Cl 2), a PEG-dimyristylglycerol (C14), a PEG-dipalmitoylglycerol (C16), a PEG-disterylglycerol (C18), PEG-dilaurylglycamide (C12), PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16), and PEG-disterylglycamide (C18).

[0591] In some embodiments, conjugated lipids, when present, can include one or more of PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO 2019 / 051289 (which is herein incorporated by reference in its entirety), and combinations of the foregoing.

[0592] Additional exemplary PEG-lipid conjugates are described, for example, in US 5,885,613, US 6,287,591, US 2003 / 0077829, US 2003 / 0077829, US 2005 / 0175682, US 2008 / 0020058, US 2011 / 0117125, US 2010 / 0130588, US 2016 / 0376224, US 2017 / 0119904, US 2018 / 0028664, and WO 2017 / 099823, all of which are incorporated herein by reference in their entirety.

[0593] In some embodiments, the PEG-lipid is a compound of Formula III, III-a-I, III-a-2, III-b-1, III-b-2, or V of US 2018 / 0028664, which is incorporated herein by reference in its entirety. In some embodiments, the PEG-lipid is of Formula II of US 2015 / 0376115 or US 2016 / 0376224, both of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. In some embodiments, the PEG-lipid includes one of the following:FAZ-40125

[0594]

[0595] In some embodiments, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates,

[0596] polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid

[0597] (GPL) conjugates can be used in place of or in addition to the PEG-lipid.

[0598] Exemplary conjugated lipids, e.g., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids, include those described in Table 2 of WO

[0599] 2019 / 051289A9, which is incorporated herein by reference in its entirety.

[0600] In some embodiments, the conjugated lipid (e.g., the PEGylated lipid) can be present in an amount of 0-20 mol% of the total lipid components present in the lipid-based carrier (or lipid nanoformulation). In some embodiments, the conjugated lipid (e.g., the PEGylated

[0601] lipid) content is 0.5-10 mol% or 2-5 mol% of the total lipid components.

[0602] When needed, the lipid-based carrier (or lipid nanoformulation) described herein may be coated with a polymer layer to enhance stability in vivo (e.g., sterically stabilized LNPs).

[0603] Examples of suitable polymers include, but are not limited to, polyethylene glycol), which may form a hydrophilic surface layer that improves the circulation half-life of

[0604] liposomes and enhances the amount of lipid nanoformulations (e.g., liposomes or LNPs) that reach therapeutic targets. See, e.g., Working et al. J Pharmacol Exp Ther, 289: 1128-1133 (1999); Gabizon et al., J Controlled Release 53: 275-279 (1998); Adlakha Hutcheon et al., Nat Biotechnol 17: 775-779 (1999); and Koning et al., Biochim Biophys Acta 1420: 153-167 (1999), which are incorporated herein by reference in their entirety.

[0605] In certain embodiments, the nanoparticle composition further comprises a PEGylatedFAZ-40125

[0606] lipid, a sterol, a phospholipid, and / or a neutral lipid.

[0607] iv. Percentages of Lipid Nanoformulation Components

[0608] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises one or more of the compounds described herein, optionally a non-cationic lipid (e.g., a phospholipid), a sterol, a neutral lipid, and / or optionally conjugated lipid (e.g., a PEGylated lipid) that inhibits aggregation of particles. The relative amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the ionizable lipid including the lipid compounds described herein is present in an amount from about 20 mol% to about 100 mol% (e.g., 20-90 mol%, 20-80 mol%, 20-70 mol%, 25-100 mol%, 30-70 mol%, 30-60 mol%, 30-40 mol%, 40-50 mol%, or 50-90 mol%) of the total lipid and lipidoid components; a non-cationic lipid (e.g., phospholipid) is present in an amount from about 0 mol% to about 50 mol% (e.g., 0-40 mol%, 0-30 mol%, 5-50 mol%, 5-40 mol%, 5-30 mol%, or 5-10 mol%) of the total lipid and lipidoid components, a conjugated lipid (e.g., a PEGylated lipid) in an amount from about 0.5 mol% to about 20 mol% (e.g., 1-10 mol% or 5-10%) of the total lipid and lipidoid components, and a sterol in an amount from about 0 mol % to about 60 mol% (e.g., 0-50 mol%, 10-60 mol%, 10-50 mol%, 15-60 mol%, 15-50 mol%, 20-50 mol%, 20-40 mol%) of the total lipid and lipidoid components, provided that the total mol% of the lipid component does not exceed 100%.

[0609] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises about 25-100 mol% of the ionizable lipid including the lipid and lipidoid compounds described herein, about 0-50 mol% phospholipid, about 0-50 mol% sterol, and about 0-10 mol% PEGylated lipid.

[0610] In one embodiment, the lipid-based carrier (or lipid nanoformulation) comprises about 25-100 mol% of the ionizable lipid including the lipid and lipidoid compounds described herein; about 0-40 mol% phospholipid (e.g., DSPC), about 0-50 mol% sterol (e.g., cholesterol), and about 0-10 mol% PEGylated lipid.

[0611] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises about 30-60 mol% (e.g., about 35-55 mol%, or about 40-50 mol%) of the ionizable lipid including the lipid and lipidoid compounds described herein, about 0-30 mol% (e.g., 5-25 mol%, or 10-20 mol%) phospholipid, about 15-50 mol% (e.g., 18.5-48.5 mol%, or 30-40 mol%) sterol, and about 0-10 mol% (e.g., 1-5 mol%, or 1.5-2.5 mol%) PEGylated lipid.

[0612] In some embodiments, molar ratios of ionizable lipid / sterol / phospholipid (or another structural lipid) / PEG-lipid / additional components is varied in the following ranges: ionizable lipid (25-100%); phospholipid (DSPC) (0-40%); sterol (0-50%); and PEG lipid (0-5%).FAZ-40125

[0613] In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises, by mol% or wt% of the total lipid and lipidoid components, 50-75% ionizable lipid (including the lipid and lipidoid compounds as described herein), 20-40% sterol (e.g., cholesterol or derivative), 0 to 10% non-cationic-lipid, and 1-10% conjugated lipid (e.g., the PEGylated lipid).

[0614] In some embodiments, the lipidoid compound described herein is a component of the lipid-based carrier (or lipid nanoformulation, or nanoparticle composition) and comprises from 10 mol% to 95 mol%, from 10 mol% to 90 mol%, from 10 mol% to 80 mol%, from 10 mol% to 70 mol%, from 10 mol% to 60 mol%, from 20 mol% to 55 mol%, from 20 mol% to 45 mol%, 20 mol% to 40 mol%, from 25 mol% to 50 mol%, from 25 mol% to 45 mol%, from 30 mol% to 50 mol%, from 30 mol% to 45 mol%, from 30 mol% to 40 mol%, from 35 mol% to 45 mol%, or from 37 mol% to 42 mol% (or any fraction of these ranges) of the total lipid and lipidoid components.

[0615] In some embodiments, where the lipid-based carrier (or lipid nanoformulation) contains a mixture of phospholipid and sterol (e.g. cholesterol or derivative), the mixture may be present up to 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid and lipidoid components.

[0616] In some embodiments, the phospholipid component in the mixture may be present from 2 mol% to 20 mol%, from 2 mol% to 15 mol%, from 2 mol% to 12 mol%, from 4 mol% to 15 mol%, from 4 mol% to 10 mol%, from 5 mol% to 10 mol%, (or any fraction of these ranges) of the total lipid and lipidoid components. In some embodiments, the lipid-based carrier (or lipid nanoformulation or nanoparticle composition) is substantially free of a phospholipid. In certain embodiments, the lipid-based carrier (or lipid nanoformulation or nanoparticle composition) is substantially free of distearolyphosphatidycholine (DSPC).

[0617] In some embodiments, the sterol component (e.g. cholesterol or derivative) in the mixture may comprise from 25 mol% to 45 mol%, from 25 mol% to 40 mol%, from 25 mol% to 35 mol%, from 25 mol% to 30 mol%, from 30 mol% to 45 mol%, from 30 mol% to 40 mol%, from 30 mol% to 35 mol%, from 35 mol% to 40 mol%, from 27 mol% to 37 mol%, or from 27 mol% to 35 mol% (or any fraction of these ranges) of the total lipid and lipidoid components.

[0618] In some embodiments, where the lipid-based carrier (or lipid nanoformulation) is phospholipid-free, the sterol component (e.g. cholesterol or derivative) may be present up to 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid and lipidoid components. For instance, the sterol component (e.g. cholesterol orFAZ-40125

[0619] derivative) may be present from 25 mol% to 65 mol%, from 25 mol% to 60 mol%, from 25 mol% to 55 mol%, from 25 mol% to 50 mol%, from 25 mol% to 45 mol%, from 25 mol% to 40 mol%, from 30 mol% to 45 mol%, from 30 mol% to 40 mol%, from 35 mol% to 45 mol%, from 30 mol% to 35 mol%, or from 35 mol% to 40 mol% (or any fraction thereof or range therein) of the total lipid and lipidoid components.

[0620] In some embodiments, the non-ionizable lipid components in the lipid-based carrier (or lipid nanoformulation) may be present from 5 mol% to 90 mol%, from 10 mol% to 85 mol%, or from 20 mol% to 80 mol% (or any fraction of these ranges) of the total lipid and lipidoid components.

[0621] The ratio of total lipid components to the cargo (e.g., an encapsulated therapeutic agent such as a nucleic acid) can be varied as desired. For example, the total lipid components to the cargo (mass or weight) ratio can be from about 10:1 to about 30:1. In some embodiments, the total lipid and lipidoid components to the cargo ratio (mass / mass ratio; w / w ratio) can be in the range of from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of total lipid components and the cargo can be adjusted to provide a desired N / P ratio, for example, N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or higher. Generally, the lipid-based carrier (or lipid nanoformulation) ’s overall lipid content can range from about 5 mg / mL to about 30 mg / mL. Nitrogemphosphate ratios (N:P ratio) is evaluated at values between 0.1 and 100.

[0622] In some embodiments, the lipid-based carrier (or lipid nanoformulation) includes the ionizable lipid compound as described herein, phospholipid, cholesterol, and a PEGylated lipid in a molar ratio of 50:10:38.5:1.5. In some embodiments, the lipid-based carrier (or lipid nanoformulation) includes the ionizable lipid compound as described herein, cholesterol and a PEGylated lipid in a molar ratio of 60:38.5: 1.5.

[0623] In some embodiments of any of the aspects or embodiments herein, the lipid-based carrier (or lipid nanoformulation) further comprises a tissue targeting moiety. The tissue targeting moiety can be a peptide, oligosaccharide or the like, which can be used for the delivery of the lipid-based carrier (or lipid nanoformulation) to one or more specific tissues such as the liver. In some embodiments, the tissue targeting moiety is a ligand for liver specific receptors. In one embodiment, the ligand of liver specific receptors used for liver targeting is an oligosaccharide such as N-Acetylgalactosamine (GalNAc) which is covalently attached to a component of a lipid-based carrier (or lipid nanoformulation), e.g., PEG-lipidFAZ-40125

[0624] conjugates or the like. In some embodiments, the GalNAc is covalently attached to, for example, PEG-lipid conjugate. In some embodiments, the GalNAc is conjugated to DSPE-PEG2000. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.2% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.3% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.4% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.5% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.6% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.7% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.8% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 0.9% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 1.0% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of about 1.5% of the total lipid. In some embodiments, the GalNAc-PEG-lipid conjugate is present in the lipid-based carrier (or lipid nanoformulation) at a molar percentage of 2.0% of the total lipid.

[0625] v. Properties of Lipid Nanoformulations

[0626] In some embodiments, the average particle diameter of the lipid-based carrier (or lipid nanoformulation) may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average particle diameter of the lipid-based carrier (or lipid nanoformulation) ranges from about 1 mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm toFAZ-40125

[0627] about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, from about 38 mm to about 42 mm, from about 40 nm to about 150 nm (such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm), from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm.

[0628] The lipid-based carrier or lipid nanoformulation (e.g., liposome or LNP) may, in some instances, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a lipid nanoformulation (e.g., liposome or LNP), e.g., the particle size distribution of the liposome or LNP. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A lipid-based carrier or lipid nanoformulation (e.g., liposome or LNP) may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the lipid-based carrier or lipid nanoformulation (e.g., liposome or LNP) may be from about 0.10 to about 0.20.

[0629] The zeta potential of a lipid-based carrier or a lipid nanoformulation (e.g., liposome or LNP) may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of a liposome or LNP. Lipid nanoformulations (e.g., liposomes or LNP) with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a liposome or LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, orFAZ-40125

[0630] from about +5 mV to about +10 mV.

[0631] The efficiency of encapsulation of a cargo such as a protein and / or nucleic acid, describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with a lipid nanoformulation (e.g., liposome or LNP) after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., at least 70%. 80%. 90%. 95%, close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the liposome or LNP before and after breaking up the liposome or LNP with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in a solution. For the liposome or LNP described herein, the encapsulation efficiency of a protein and / or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0632] The lipid carrier or lipid nanoformulation may optionally include one or more coatings. In some embodiments, the lipid carrier or lipid nanoformulation (e.g., liposome or LNP) may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density.

[0633] Additional exemplary lipids, formulations, methods, and characterization of a lipid carrier or lipid nanoformulation (e.g., liposome or LNP) are taught by WO 2020 / 061457 and WO 2021 / 113777, which are incorporated herein by reference in their entirety. Further exemplary lipids, formulations, methods, and characterization of LNPs are taught by Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021). doi.org / 10.1038 / s41578-021-00358-0, which is incorporated herein by reference in its entirety (see, for example, exemplary lipids and lipid derivatives of Figure 2 of Hou et al.).

[0634] In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using the GenVoy lLM ionizable lipid mix (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[ 1,3] -dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of whichFAZ-40125

[0635] are taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), incorporated herein by reference in its entirety.

[0636] Lipid nanoformulations (e.g., liposome or LNP) optimized for the delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO 2019067992 and WO 2019067910, which are incorporated by reference in their entirety.

[0637] Additional specific lipid nanoformulations (e.g., liposome or LNP) useful for delivery of nucleic acid effector molecules are described in US 8158601 and US 8168775, which are incorporated by reference in their entirety.

[0638] A variety of methods can be used for preparing the lipid carrier or lipid nanoformulation (e.g., liposomes or LNPs) described herein. Such methods are known in the art or disclosed herein, for example, the methods described in Lichtenberg and Barenholz in Methods of Biochemical Analysis, 33:337-462 (1988), which is incorporated herein by reference in its entirety. See also Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); U.S. Patent Nos. 4,235,871; 4,501,728; and 4,837,028; Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1; and Hope, et al., Chem. Phys. Lip. 40:89 (1986), which are incorporated herein by reference in their entirety. Small unilamellar vesicles (SUV, size <100 nm) can be prepared by a combination of standard methods of thin-film hydration and repeated extrusion.

[0639] Techniques for sizing the lipid carrier or lipid nanoformulations (e.g., liposomes or LNPs) to a desired size are well-known to one skilled in the art. See, e.g., U.S. Patent No. 4,737,323, and Hope et al., Biochim. Biophys. Acta, 812: 55-65, which are incorporated by reference in their entirety. Sonicating a lipid nanoformulation (e.g., liposome or LNP) suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles less than about 50 nm in size. Homogenization or microfluidization are other methods which rely on shearing energy to fragment large lipid nanoformulations (e.g., liposomes or LNPs) into smaller ones. In a typical homogenization procedure, multilamellar vesicles are recirculated through a standard emulsion homogenizer until selected lipid nanoformulation (e.g., liposome or LNP) sizes, typically between about 100 and 500 nm, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size discrimination.

[0640] Extrusion of lipid nanoformulations (e.g., liposomes or LNPs) through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is a very effective method for reducing liposome or LNP sizes to a relatively well- defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome orFAZ-40125

[0641] LNP size distribution is achieved. The lipid-based carrier or lipid nanoformulations may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome or LNP size.

[0642] Any of the lipid-based carrier or lipid nanoformulations described herein can be analyzed by methods well-known to one skilled in the art to determine its physical and / or chemical features. For example, a phosphate assay can be used to determine the concentration of the lipid nanoformulations. One phosphate assay is based on the interaction between molybdate and malachite green dye. The main principle involves the reaction of inorganic phosphate with molybdate to form a colorless unreduced phosphomolybdate complex which is converted to a blue colored complex when reduced under acidic conditions. Phosphomolybdate gives 20 or 30 times more color when complexed with malachite green. The final product, reduced green soluble complex is measured by its absorbance at 620 nm and is a direct measure of inorganic phosphate in solution.

[0643] In some embodiments, the lipid-based carrier or lipid nanoformulations disclosed herein are tested for particle size, lipid concentration, and active agent encapsulation.

[0644] Delivery Vehicle Compositions

[0645] The disclosure provides delivery vehicle compositions comprising a lipid nanoformulation (e.g., an LNP) encapsulating an saRNA described herein. In some embodiments, the disclosure provides a heterogeneous delivery vehicle composition comprising a plurality of lipid nanoformulations. In some embodiments, each lipid nanoformulation of the delivery vehicle composition encapsulates a saRNA.

[0646] In some embodiments, the delivery vehicle comprises a mixture of ionizable and cationic lipids. In some embodiments, the delivery vehicle composition does not comprise a mixture of ionizable and cationic lipids.

[0647] In some embodiments, the delivery vehicle composition comprises at least one lipid nanoformulation (e.g., an LNP) encapsulating an saRNA described herein. In some embodiments, the delivery vehicle composition comprises a plurality of lipid nanoformulations. For example, in some embodiments, the delivery vehicle composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at leastFAZ-40125

[0648] 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 100 lipid nanoformulations encapsulating an saRNA, wherein the saRNA comprises a unique barcode sequence for identification of each lipid nanoformulation. In some embodiments, the delivery vehicle composition comprises at least two lipid nanoformulations encapsulating an saRNA, wherein the saRNA comprises a unique barcode sequence for identification of each lipid nanoformulation. In some embodiments, the delivery vehicle composition comprises at least five lipid nanoformulations encapsulating an saRNA, wherein the saRNA comprises a unique barcode sequence for identification of each lipid nanoformulation. In some embodiments, the delivery vehicle composition comprises at least ten lipid nanoformulations encapsulating an saRNA, wherein the saRNA comprises a unique barcode sequence for identification of each lipid nanoformulation. In some embodiments, the delivery vehicle composition comprises at least fifteen lipid nanoformulations encapsulating an saRNA, wherein the saRNA comprises a unique barcode sequence for identification of each lipid nanoformulation. In some embodiments, the delivery vehicle composition comprises at least twenty lipid nanoformulations encapsulating an saRNA, wherein the saRNA comprises a unique barcode sequence for identification of each lipid nanoformulation. In some embodiments, the delivery vehicle composition comprises at least thirty lipid nanoformulations encapsulating an saRNA, wherein the saRNA comprises a unique barcode sequence for identification of each lipid nanoformulation.

[0649] III. Screening Methods

[0650] The present disclosure provides methods for screening a plurality of lipid nanoformulations (e.g., from a heterogeneous lipid nanoformulation). The methods may be used, for example in multiplexed lipid nanoformulation screening to determine in vivo delivery of one or more of the lipid nanoformulations. These methods employ barcoded saRNAs (see e.g., FIG. 1) to facilitate the identification of each nanformulation in the screening process. Each saRNA contains a known barcode sequence and is encapsulated in a lipid nanoformulation, and one, or a plurality of nanoformulations associated with uniqueFAZ-40125

[0651] barcodes are administered to a subject. Intracellular vivo delivery (e.g., by endocytosis of the lipid nanoformulation as depicted in FIG. 8) of the lipid nanoformulations may be determined by sequencing of RNA from a sample isolated from the subject administered the lipid nanoformulation (i.e., identifying the known barcode sequence associated with a specific lipid nanoformulation). The sequencing may be of a positive strand (+) saRNA, or of a negative (-) strand saRNA which forms upon transcription of the positive strand when the saRNA is delivered intracellularly (see e.g., FIG. 9 and FIG. 3). In some embodiments, the sequencing is performed on a negative (-) strand saRNA.

[0652] The methods described herein comprise encapsulating a barcoded saRNA with a known barcode in a lipid nanoformulation and administering the lipid nanoformulation to a subject. Following administration, a tissue sample is collected from the subject and RNA sequencing is performed to measure intracellular delivery of the saRNA. In some embodiments, a plurality of lipid nanoformulations encapsulating saRNA with known barcodes are administered to a subject, wherein a tissue sample is collected following delivery and RNA sequencing is performed to measure intracellular delivery of the saRNA.

[0653] In some embodiments, the saRNA encapsulated by the lipid nanoformulation is a positive strand saRNA. In some embodiments, the sequencing of the saRNA is of the positive strand saRNA. In some embodiments, the sequencing if the saRNA is of a negative strand saRNA. Sequencing of a negative strand RNA is used to show delivery of the saRNA to the cytoplasm of a cell wherein the saRNA undergoes transcription to generate negative strand saRNA. Delivery of the saRNA and generation of negative strand saRNA is used to determine intracellular delivery or cytoplasmic delivery of the LNP and encapsulated saRNA. In some embodiments, delivery of an saRNA encapsulated by the lipid nanoformulation is a functional delivery. Functional delivery is defined herein as delivery of an saRNA encapsulated by a lipid nanoformulation to a cell (e.g., through endocytosis), wherein upon delivery to the cell, the saRNA is released into the cytoplasm enabling self-amplification of the saRNA.

[0654] In some embodiments, the disclosure provides methods for screening a plurality of different lipid nanoformulations, comprising: a) administering to a subject a plurality of nucleic acid molecules, wherein one nucleic acid molecule of the plurality of nucleic acid molecules encodes a reporter protein and a nucleic acid barcode; and b) detecting the reporter protein in a sample obtained from the subject; wherein the one nucleic acid molecule is encapsulated by a lipid nanoformulation of the plurality of different lipid nanoformulations, and wherein the peptidyl barcode is identifiable in an assay, therefore, identifying the lipid nanoformulations encapsulating the one nucleic acid molecule.FAZ-40125

[0655] In some embodiments, the method for screening a plurality of different lipid nanoformulations can identify or distinguish the lipid nanoformulations via the peptidyl barcode encoded by the nucleic acid sequence encapsulated by the lipid nanoformulation. In some embodiments, the method for screening a plurality of different lipid nanoformulations can identify or distinguish each lipid nanoformulation via the peptidyl barcode and the reporter protein encoded by the nucleic acid sequence encapsulated by the lipid nanoformulations. In some embodiments, the method can be used for high throughput screening of the lipid nanoformulations.

[0656] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a delivery vehicle composition, the method comprising:

[0657] (i) providing a delivery vehicle composition having encapsulated therein a barcoded self-amplifying RNA (saRNA) to a subject,

[0658] (ii) identifying the barcode sequence of the saRNA in one or more samples of the subject, thereby analyzing the intracellular delivery of the delivery vehicle composition.

[0659] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0660] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a selfamplifying RNA (saRNA), wherein the saRNA generates a detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation;

[0661] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0662] (iii) sorting cells from one or more samples of the subject by measuring the detectable signal; and,

[0663] (iv) identifying the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of each lipid nanoformulation of the lipid nanoformulation composition.

[0664] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of saRNA encapsulated in a lipid nanoformulation, the method comprising:

[0665] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a selfamplifying RNA (saRNA), wherein the saRNA generates a detectable signal upon intracellularFAZ-40125

[0666] delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation;

[0667] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0668] (iii) sorting cells from one or more samples of the subject by measuring the detectable signal; and,

[0669] (iv) identifying the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of the saRNA.

[0670] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0671] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA), wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0672] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0673] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is transcribed into a negative strand saRNA;

[0674] (iv) wherein upon delivery of the saRNA, the nsP4 is translated to an RdRp and the RdRp translates the reporter gene to the protein that produces the detectable signal;

[0675] (v) sorting cells from one or more samples of the subject by measuring the detectable signal;

[0676] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of each lipid nanoformulation of the heterogeneous lipid nanoformulation.

[0677] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0678] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA), wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces theFAZ-40125

[0679] detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0680] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0681] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is translated to form an RNA-dependent RNA polymerase (RdRp);

[0682] (iv) wherein upon delivery of the saRNA, the RdRp catalyzes the formation of negative and subgenomic saRNAs, and cellular machinery translates the protein that produces the detectable signal from the subgenomic RNA;

[0683] (v) sorting cells from one or more samples of the subject by measuring the detectable signal;

[0684] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of each lipid nanoformulation of the heterogeneous lipid nanoformulation.

[0685] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of saRNA encapsulated in a lipid nanoformulation, the method comprising:

[0686] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA), wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0687] (ii) administering the heterogeneous lipid nanoformulation to the subject;

[0688] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is transcribed into a negative strand saRNA;

[0689] (iv) wherein upon delivery of the saRNA, the nsP4 is translated to an RdRp and the RdRp translates the reporter gene to the protein that produces the detectable signal;

[0690] (v) sorting cells from one or more samples of the subject by measuring the detectable signal;

[0691] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of the saRNA.FAZ-40125

[0692] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of saRNA encapsulated in a lipid nanoformulation, the method comprising:

[0693] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA), wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0694] (ii) administering the heterogeneous lipid nanoformulation to the subject;

[0695] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is translated to form an RNA-dependent RNA polymerase (RdRp);

[0696] (iv) wherein upon delivery of the saRNA, the RdRp catalyzes the formation of negative and subgenomic saRNAs, and cellular machinery translates the protein that produces the detectable signal from the subgenomic RNA;

[0697] (v) sorting cells from one or more samples of the subject by measuring the detectable signal;

[0698] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of the saRNA.

[0699] In some embodiments, the method comprises administration of a catalytically dead saRNA. In some embodiments, the catalytically dead saRNA is a negative control saRNA which abolishes the catalytic activity of nsP4 preventing negative and subgenomic strand synthesis (Rubach, J. et al., Virology, 384(1): 201-208, Nov 25, 2008). In some embodiments the catalytically dead saRNA comprises one or more of mutations A7113C, T7114C, A7116C in the nsP4 region. As described in Rubach, J. et al., Virology, 384(1): 201-208, Nov 25, 2008, two aspartic acids are converted to alanines to eliminate terminal addition activity. In some embodiments, the catalytically dead saRNA comprises the sequence set forth in SEQ ID NO: 6. In some embodiments, the catalytically dead saRNA comprises a sequence 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 6. In some embodiments, the nucleic acid sequence encoding the catalytically dead saRNA comprises the nucleotide sequence set forth in SEQ ID NO: 5. In some embodiments, the nucleic acid sequence encoding the catalytically dead saRNA comprises a sequence 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO: 5.FAZ-40125

[0700] Delivery

[0701] In some embodiments, provided is a high throughput method for screening lipid nanoformulations in vivo. A mixture of different lipid nanoformulations can be mixed and administered into a subject in a single dose. In some embodiments, more than 2, more than 3, more than 5, more than 10, more than 13, more than 15, more than 17, more than 20, more than 23, more than 25, more than 30, more than 33, more than 35, more than 37, more than 40, more than 43, more than 45, more than 50, more than 53, more than 55, more than 57, more than 60, more than 63, more than 65, more than 70, more than 73, more than 75, more than 77, more than 80, more than 83, more than 85, more than 90, more than 93, more than 95, more than 97, more than 100 different lipid nanoformulations can be mixed and administered into the subject in a single dose.

[0702] In some embodiments, the method comprises screening about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 10 to about 100 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 100 to about 500 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 100 to about 1000 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 10 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 20 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 30 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 40 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 50 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 60 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 70 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 80 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 90 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 100 lipid nanoformulations in the same assay. In some embodiments, the methodFAZ-40125

[0703] comprises screening about 200 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 300 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 400 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 500 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 600 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 700 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 800 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 900 lipid nanoformulations in the same assay. In some embodiments, the method comprises screening about 1000 lipid nanoformulations in the same assay.

[0704] In some embodiments, the method comprises administering the lipid nanoformulation in a composition comprising a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other nontoxic compatible substances employed in pharmaceutical formulations.

[0705] In some embodiments, the method comprises administering the plurality of different lipid nanoformulations through any suitable routes comprising parenteral delivery (e.g., injections), such as intravenous, intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.

[0706] In some embodiments, the method provides potent delivery of the lipid nanoformulations to a plurality of different cell types, tissues, or organs of a subject. In someFAZ-40125

[0707] embodiments, the method comprises delivering the lipid nanoformulations to more than 5, more than 10, more than 15, more than 20, more than 25, more than 30 cell types, tissues, or organs of the subject.

[0708] In some embodiments, the saRNA molecules are present in the lipid nanoformulation formulation at a dose of about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1.5, about 1.0, about 0.5, about 0.2, about 0.1, about 0.05, about 0.02, about 0.01, about 0.005, about 0.002, or about 0.001 milligram per kilogram (mg / kg, or mpk) body weight, or of a range between (inclusive) any two of the foregoing values. In some embodiments, the saRNA molecules are present in the lipid nanoformulation at a dose of about 0.01 mg / kg to about 4 mg / kg. In some embodiments, the saRNA molecules are present in the lipid nanoformulation formulation at a dose of no more than about 10 milligram per kilogram (mg / kg, or mpk) body weight. In some embodiments, the saRNA molecules are present in the lipid nanoformulation formulation at a dose of no more than about 9 mg / kg, no more than about 8 mg / kg, no more than about 7 mg / kg, no more than about 6 mg / kg, no more than about 5 mg / kg, no more than about 4 mg / kg, no more than about 3 mg / kg, no more than about 2 mg / kg, no more than about 1 mg / kg, no more than about 0.5 mg / kg, no more than about 0.2 mg / kg, no more than about 0.1 mg / kg, no more than about 0.05 mg / kg, or no more than about 0.01 mg / kg. In some embodiments, the saRNA molecules are present in the lipid nanoformulation formulation at a concentration of no more than about 5 milligram per milliliter (mg / mL). In some embodiments, the saRNA molecules are present in the lipid nanoformulation formulation at a concentration of about 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 milligram per milliliter (mg / mL), or of a range between (inclusive) any two of the foregoing values. In some embodiments, the saRNA molecules are present in the lipid nanoformulation formulation at a concentration of no more than about 5 milligram per milliliter (mg / mL). In some embodiments, the saRNA molecules are present in the lipid nanoformulation formulation at a concentration of no more than about 2 milligram per milliliter (mg / mL). In some embodiments, the saRNA molecules are present in the lipid nanoformulation formulation at a concentration of no more than about 1 milligram per milliliter (mg / mL). In some embodiments, the saRNA molecules are present in the lipid nanoformulation formulation at a concentration of no more than about 0.5 milligram per milliliter (mg / mL). In some embodiments, the saRNA molecules are present in the lipid nanoformulation formulation at a concentration of no more than about 0.1 milligram per milliliter (mg / mL).

[0709] In some embodiments, the saRNA molecules (e.g., mRNA) is present in lipid nanoformulation formulations at a concentration of about 10, about 9, about 8, about 7, aboutFAZ-40125

[0710] 6, about 5, about 4, about 3, about 2, about 1, about 0.5, about 0.2, or about 0.1 microgram per milliliter (pg / mL), or of a range between (inclusive) any two of the foregoing values. In some embodiments, the saRNA molecules (e.g., mRNA) are present in lipid nanoformulation formulations at a concentration of no more than about 10, no more than about 9, no more than about 8, no more than about 7, no more than about 6, no more than about 5, no more than about 4, no more than about 3, no more than about 2, no more than about 1, no more than about 0.5, no more than about 0.2, no more than about 0.1 microgram per milliliter (pg / mL).

[0711] Any suitable dosage form of lipid nanoformulations can be prepared for delivery, for example, via oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0712] In some embodiments, the subject comprises a mammal. In some embodiments, the mammal comprises a rodent, a non-human primate, or human. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a mouse or a rat.

[0713] In some embodiments, provided herein is a method for delivery of the lipid nanoformulations to different cell types comprising contacting the cells with the lipid composition. In some embodiments of the method, the lipid nanoformulation comprises saRNA molecules assembled with a lipid composition as described in the present application, e.g., wherein the lipid composition comprises any of the head or tail groups disclosed herein. In some embodiments, the contacting is ex vivo. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0714] RNA Isolation

[0715] Following in vivo administration of the lipid nanoformulations and intracellular delivery of the saRNA, RNA is isolated from the subject. Isolation of the RNA is used to determine delivery of the lipid nanoformulations as measured by detecting and / or quantifying the barcoded saRNA encapsulated in the lipid nanoformulation.

[0716] In some embodiments, cells are isolated from a subject and are sorted by a surface marker encoded in the subgenomic RNA. In some embodiments, cells are isolated by sorting using an endogenous cell marker. In some embodiments, the RNA is extracted from a total RNA extraction from tissue homogenates. In some embodiments, the sample is from isolated cells. In some embodiments, the sample is from double stranded RNA (dsRNA) isolated from a whole RNA sample. In some embodiments, the sample is RNA isolated by size exclusion.FAZ-40125

[0717] In some embodiments, the RNA is isolated from a plurality of different cell types, tissues, or organs of a subject. In some embodiments, the method comprises isolation of RNA from 1, more than 1, more than 5, more than 10, more than 15, more than 20, more than 25, more than 30 cell types, tissues, or organs of the subject.

[0718] In some embodiments, the RNA is isolated from one or more of tissue homogenates of connective tissue, epithelial tissue, muscle tissue, and nervous tissue. In some embodiments, the RNA is isolated from one or more of the heart, kidney, lung, liver, brain, small intestine, large intestine, stomach, skin, bladder, bone, muscle, pancreas, adrenal gland, appendix, pharynx, thyroid, uterus, spleen, gallbladder, eyes, esophagus, spinal cord, and salivary glands.

[0719] Various methods of extraction are suitable for isolating saRNA described herein. In some embodiments, the isolation of nucleic acid involves lysis of tissue or cells. Methods of RNA isolation known to those skilled in the art may also be utilized. RNA can be isolated and prepared for hybridization by a variety of methods including, but not limited to, Trizol® and Guanidinium thiocyanate-phenol-chloroform extraction. The principle of RNA isolation is based on cell / tissue lysis, followed by extraction, precipitation, and washing. In some embodiments, RNA is isolated using a total RNA extraction kit. In other methods, mRNA can be extracted from patient biological samples (e.g. blood samples) using a commercial kit suitable for mRNA extraction, e.g. MagNA Pure LC mRNA HS kit and Mag NA Pure LC Instrument (Roche Diagnostics Corporation, Roche Applied Science, Indianapolis, Ind.).

[0720] Nucleic acid extracted from tissues, cells, plasma or serum can be amplified using nucleic acid amplification techniques well-known in the art. Many of these amplification methods can also be used to detect the presence of mutations simply by designing oligonucleotide primers or probes to interact with or hybridize to a particular target sequence in a specific manner (e.g., allele specific primers and / or probes or primers that flank target nucleic acids sequences). By way of example, but not by way of limitation, these techniques can include, but are not limited to, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), real-time PCR (qPCR), nested PCR, ligase chain reaction (LCA) (see Abravaya, K., et al., Nucleic Acids Research, 23:675-682, (1995)), branched DNA signal amplification (Urdea, M. S., et a .,AIDS, 7 (suppl 2):S11-S14, (1993)), amplifiable RNA reporters, Q-beta replication, transcription-based amplification system (TAS), boomerang DNA amplification, strand displacement activation (SDA), cycling probe technology, isothermal nucleic acid sequence based amplification (NASBA) (see Kievits, T. et al., J Virological Methods, 35:273-286, (1991)), Invader Technology, helicase dependentFAZ-40125

[0721] amplification (HD A) Amplification Refractory Mutation System (ARMS), and other sequence replication assays or signal amplification assays. Exemplary methods of amplification are described briefly below and are well-known in the art.

[0722] RNA Amplification and Sequencing

[0723] Methods of RNA amplification are known to those of skill in the art. In some embodiments, amplification of transcripts is by reverse transcription coupled with polymerase chain reaction. In some embodiments, any method of RNA amplification in the art may be used. This includes, but is not limited to:

[0724] i. Primers

[0725] In some embodiments, the disclosure provides primers for sequencing an saRNA strand sequence described herein. In some embodiments, the disclosure provides primers for sequencing a positive strand (+) saRNA sequence described herein. In some embodiments, the disclosure provides primers for sequencing a negative strand (-) saRNA sequence described herein.

[0726] In some embodiments, the disclosure provides primers for quantifying saRNA described herein. In some embodiments, the disclosure provides primers for quantifying positive strand (+) saRNA described herein. In some embodiments, the disclosure provides primers for quantifying negative strand (-) saRNA described herein. In some embodiments, the quantification of saRNA is by qRT-PCR.

[0727] In some embodiments, the qRT-PCR comprises use of a probe with the positive qPCR reaction. In some embodiments, the probe is the probe having the sequence set forth in SEQ ID NO: 18. The FAM and ZEN / Iowa Black quencher placements are indicated using standard Integrated DNA Technologies (IDT) notation and were made using IDTs PrimerQuest design tool. In some embodiments, the probe is a probe described in Plaskon et al. PloS One, Accurate Strand-Specific Quantification of Viral RNA; October 22, 2009.

[0728] In some embodiments, the disclosure provides primers for qRT-PCR analysis. Methods for qRT-PCR analysis are known to those of skill in the art. In some embodiments, the primer is a primer with a sequence set forth in any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, or any combination thereof. In some embodiments, the primer is a primer with one, two, or three base pair mismatches relative to a sequence set forth in any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, or any combination thereof. In some embodiments, the primer with the sequence set forth in SEQ ID NO: 13 is used for reverse transcription of a negative strand saRNA. In someFAZ-40125

[0729] embodiments, the primer with the sequence set forth in SEQ ID NO: 13 is used for reverse transcription of a negative strand saRNA with the sequence set forth in SEQ ID NO: 3. In some embodiments, a PCR described herein uses the forward primer with the sequence set forth in SEQ ID NO: 16. In some embodiments, a PCR described herein uses the reverse primer with the sequence set forth in SEQ ID NO: 17. In some embodiments, a PCR described herein uses the probe with the sequence set forth in SEQ ID NO: 18.

[0730] In some embodiments, the forward primer with the sequence set forth in SEQ ID NO: 16 and the reverse primer with the sequence set forth in SEQ ID NO: 17 are used for PCR amplification of a reverse transcription product produced by reverse transcription of the negative strand saRNA of SEQ ID NO: 3 using the primer with the sequence set forth in SEQ ID NO: 13. In some embodiments, the forward primer with the sequence set forth in SEQ ID NO: 16, the reverse primer with the sequence set forth in SEQ ID NO: 17, and the probe with the sequence set forth in SEQ ID NO: 18 are used for PCR amplification of a reverse transcription product produced by reverse transcription of the negative strand saRNA of SEQ ID NO: 3 using the primer with the sequence set forth in SEQ ID NO: 13.

[0731] In some embodiments, the primer with the sequence set forth in SEQ ID NO: 12 is used for reverse transcription of a positive strand saRNA. In some embodiments, the primer with the sequence set forth in SEQ ID NO: 12 is used for reverse transcription of a positive strand saRNA set forth in SEQ ID NO: 2 or 4. In some embodiments, the primer with the sequence set forth in SEQ ID NO: 12 is used for reverse transcription of a positive strand saRNA set forth in SEQ ID NO: 2. In some embodiments, the primer with the sequence set forth in SEQ ID NO: 12 is used for reverse transcription of a positive strand saRNA set forth in SEQ ID NO: 4. In some embodiments, a PCR described herein uses the forward primer with the sequence set forth in SEQ ID NO: 14. In some embodiments, a PCR described herein uses the reverse primer with the sequence set forth in SEQ ID NO: 15. In some embodiments, a PCR described herein uses the probe with the sequence set forth in SEQ ID NO: 18.

[0732] In some embodiments, the forward primer with the sequence set forth in SEQ ID NO: 14 and the reverse primer with the sequence set forth in SEQ ID NO: 15 are used for PCR amplification of a reverse transcription product produced by reverse transcription of the positive strand saRNA of SEQ ID NO: 2 using the primer with the sequence set forth in SEQ ID NO: 12. In some embodiments, the forward primer with the sequence set forth in SEQ ID NO: 14, the reverse primer with the sequence set forth in SEQ ID NO: 15, and the probe with the sequence set forth in SEQ ID NO: 18 are used for PCR amplification of a reverseFAZ-40125

[0733] transcription product produced by reverse transcription of the positive strand saRNA of SEQ ID NO: 2 using the primer with the sequence set forth in SEQ ID NO: 12.

[0734] In some embodiments, the forward primer with the sequence set forth in SEQ ID NO: 14 and the reverse primer with the sequence set forth in SEQ ID NO: 15 are used for PCR amplification of a reverse transcription product produced by reverse transcription of the positive strand saRNA of SEQ ID NO: 4 using the primer with the sequence set forth in SEQ ID NO: 12. In some embodiments, the forward primer with the sequence set forth in SEQ ID NO: 14, the reverse primer with the sequence set forth in SEQ ID NO: 15, and the probe with the sequence set forth in SEQ ID NO: 18 are used for PCR amplification of a reverse transcription product produced by reverse transcription of the positive strand saRNA of SEQ ID NO: 4 using the primer set with the sequence forth in SEQ ID NO: 12.

[0735] In some embodiments, the disclosure provides primers for sequencing saRNA described herein. In some embodiments, a reverse transcription primer for sequencing a positive (+) strand saRNA comprises a binding site for a Next Generation Sequencing (NGS) platform, a randomized unique molecular identifier (UMI), a study tag, and a primer binding site for the saRNA. In some embodiments, a reverse transcription primer for sequencing a negative (-) strand saRNA comprises a binding site for an NGS platform, a UMI, a study tag, and a binding site for the saRNA.

[0736] In some embodiments, a forward primer for use in sequencing cDNA generated by reverse transcription of an saRNA comprises an NGS platform adapter (e.g., a P5 adapter), a linker, and a a sequencing by synthesis (SBS) binding site. In some embodiments, a reverse primer for use in sequencing cDNA generated by reverse transcription of a positive strand saRNA comprises an index sequence. In some embodiments, a reverse primer for use in sequencing cDNA generated by reverse transcription of a negative strand saRNA comprises an index sequence.

[0737] In some embodiments, the primer includes a unique molecular identifier (UMI) to identify each individual saRNA. In some embodiments, the UMI is part of an adapter which is ligated to the saRNA. In some embodiments, the UMI is incorporated in a primer used during reverse transcription of the saRNA after extraction from a sample. In some embodiments, the UMI is ligated to the saRNA during second-strand synthesis.

[0738] UMI are randomly generated sequences which serve to avoid duplication during deep sequencing. Inclusion of these UMI in the first steps of sequencing library preparation offers several benefits. UMI create a distinct identity for each input molecule; this makes it possible to estimate the efficiency with which input molecules are sampled, identify sampling bias, andFAZ-40125

[0739] most importantly, identify and correct for the effects of PCR amplification bias. The UMI can be designed to any length available using synthesis technology.

[0740] The UMI is, in some embodiments, between 5 nt to 100 nt in length. In some embodiments, the UMI is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nt in length. In some embodiments, the UMI is between 10 nt to 20 nt in length. In some embodiments, the UMI is between 8 nt to 12 nt in length. In some embodiments, the UMI is 10 nt in length. Design of UMIs is known in the art, for example, Clement et al, AmpUMI: design and analysis of unique molecular identifiers for deep amplicon sequencing, Bioinformatics, Volume 34, Issue 13, 01 July 2018, Pages i202-i210 which is incorporated herein by reference.

[0741] In some embodiments, the primer comprises a study tag. A study tag is a nucleotide sequence used to identify material from a specific library preparation. In some embodiments, the study tag is a trinucleotide sequence. In some embodiments, the study tag is the trinucleotide sequence TAG. In some embodiments, the study tag is the trinucleotide sequence CTC. In some embodiments, the study tag is the trinucleotide sequence AGG. In some embodiments, the study tag is the trinucleotide sequence GCC. In some embodiments, the study tag is the trinucleotide sequence ACA.

[0742] In some embodiments, the primer comprises an index sequence. In some embodiments, the index sequence is an 8 nucleotide sequence. In some embodiments, the index sequence comprises a nucleotide sequence selected from SEQ ID NOs: 19-42.

[0743] In some embodiments, the disclosure provides primers for NGS analysis. Methods of NGS are known to those of skill in the art. In some embodiments, the primer is a primer with a sequence set forth in any one of SEQ ID NOs: 7, 8, 9, 10, 11, or any combination thereof. In some embodiments, the primer is a primer with one, two, or three base pair mismatches relative to a sequence set forth in any one of SEQ ID NOs: 7, 8, 9, 10, 11, or any combination thereof. In some embodiments, the primer with the sequence set forth in SEQ ID NO: 10 is used for reverse transcription of a negative strand saRNA. In some embodiments, the primer with the sequence set forth in SEQ ID NO: 10 is used for reverse transcription of a negative strand saRNA set forth in SEQ ID NO: 3.

[0744] In some embodiments, the primer with the sequence set forth in SEQ ID NO: 7 is used for reverse transcription of a positive strand saRNA. In some embodiments, the primer with the sequence set forth in SEQ ID NO: 7 is used for reverse transcription of a positive strand saRNA set forth in SEQ ID NO: 2 or 4. In some embodiments, the primer with the sequence set forth in SEQ ID NO: 7 is used for reverse transcription of a positive strand saRNA set forthFAZ-40125

[0745] in SEQ ID NO: 2. In some embodiments, the primer with the sequence set forth in SEQ ID NO: 7 is used for reverse transcription of a positive strand saRNA set forth in SEQ ID NO: 4.

[0746] In some embodiments the NGS comprises use of the forward primer with the sequence set forth in SEQ ID NO: 8. In some embodiments, the NGS comprises use of the reverse primer with the sequence set forth in SEQ ID NO: 11. In some embodiments, the NGS comprises use of the forward primer with the sequence set forth in SEQ ID NO: 8 and the reverse primer with the sequence set forth in SEQ ID NO: 11. In some embodiments, the NGS comprises use of the forward primer with the sequence set forth in SEQ ID NO: 8 and the reverse primer with the sequence set forth in SEQ ID NO: 11 for sequencing of a cDNA product of an saRNA described herein. In some embodiments, the forward primer with the sequence set forth in SEQ ID NO: 8 and the reverse primer with the sequence set forth in SEQ ID NO: 11 are used for NGS sequencing of a reverse transcription product produced by reverse transcription of the negative strand saRNA of SEQ ID NO: 3 using the primer with the sequence set forth in SEQ ID NO: 10.

[0747] In some embodiments the NGS comprises use of the forward primer with the sequence set forth in SEQ ID NO: 8. In some embodiments, the NGS comprises use of the reverse primer with the sequence set forth in SEQ ID NO: 9. In some embodiments, the NGS comprises use of the forward primer with the sequence set forth in SEQ ID NO: 8 and the reverse primer with the sequence set forth in SEQ ID NO: 9. In some embodiments, the NGS comprises use of the forward primer with the sequence set forth in SEQ ID NO: 8 and the reverse primer with the sequence set forth in SEQ ID NO: 9 for sequencing of a cDNA product of an saRNA described herein. In some embodiments, the forward primer with the sequence set forth in SEQ ID NO: 8 and the reverse primer with the sequence set forth in SEQ ID NO: 9 are used for NGS sequencing of a reverse transcription product produced by reverse transcription of the positive strand saRNA of SEQ ID NO: 2 using the primer with the sequence set forth in SEQ ID NO: 7. In some embodiments, the forward primer with the sequence set forth in SEQ ID NO: 8 and the reverse primer with the sequence set forth in SEQ ID NO: 9 are used for NGS sequencing of a reverse transcription product produced by reverse transcription of the positive strand saRNA of SEQ ID NO: 4 using the primer with the sequence set forth in SEQ ID NO: 7.

[0748] In some embodiments, a reverse transcription (RT) primer described herein comprises a modification for isolating a reverse transcription product. In some embodiments, the modification is for isolation of a negative strand reverse transcription product. In some embodiments, a reverse transcription primer (RT) comprises an amino modifier, a biotin modifier, or an alkyne modification. In some embodiments, an RT primer comprises an aminoFAZ-40125

[0749] modifier. In some embodiments, the amino modifier is selected from the group comprising a 5'-C6, 5'-C12, 5'- C6 dT, and 5'-Uni-Link™. In some embodiments, an amino modifier is capable of immobilization of the RT primer product by an NHS-ester or other amino-reactive resin. In some embodiments, the amino modified primer is coupled to a digoxigenin-NHS reagent. In some embodiments, the digoxigenin-linked primer is captured on beads comprising an anti-digoxigenin antibody. In some embodiments, the RT primer comprises a biotin modifier. In some embodiments, the biotin modifier is selected from the group comprising 5'-biotin, 5 '-biotin (azide), 5 '-biotin dT, 5'-biotin-TEG, dual 5 '-biotin, 5 '-PC biotin, and 5'-desthiobiotin-TEG. In some embodiments, the biotin modification is capable of immobilization of the RT primer product by coupling to a streptavidin magnetic bead. In some embodiments, the RT primer comprises an alkyne modification. In some embodiments, the alkyne modification is selected from the group comprising 5’ hexynyl and 5-Octadiynyl dU. In some embodiments, the alkyne modification is capable of immobilization of the RT primer product by coupling with an azide-modified resin.

[0750] In some embodiments, the amino modifier or biotin modifier is used to attach the RT primer to a solid surface. In some embodiments, the solid surface is a bead or a plate. In some embodiments, the solid surface is a magnetic bead. In some embodiments, the solid surface is a plate. In some embodiments, the solid surface is used to immobilize an RT primer product. Method of immobilization of protein or oligonucleotide products on a solid surfaces are known to those of ordinary skill in the art. In some embodiments, a immobilization of a RT product is performed using a method known in the art.

[0751] In some embodiments, a reverse transcription (RT) primer is 5 ’-biotinylated. Biotinylation of the RT primer permits removal of unprimed RT artifacts prior to qPCR amplification. In some embodiments, the biotinylation is performed using a method adapted from Boncristiani (Boncristiani, H et al., J Virol Methods, 161(1): 147-53, Oct 2009). In some embodiments, the 5 ’-biotinylated RT products are removed prior to qPCR purification using streptavidin-conjugated magnetic beads.

[0752] In some embodiments, the RT primer comprises a means for purification of an RT product. In some embodiments, the means for purification comprises an amino modifier, a biotin modifier, or an alkyne modification. In some embodiments, the means for purification of an RT product is selected from the group comprising: 5'-C6, 5'-C12, 5'- C6 dT, 5'-Uni-Link™, 5'-biotin, 5'-biotin (azide), 5'-biotin dT, 5'-biotin-TEG, dual 5'-biotin, 5'-PC biotin, 5'-desthiobiotin-TEG, 5’ hexynyl and 5-Octadiynyl dU.FAZ-40125

[0753] Table 2. Primers

[0754] ""

[0755]

[0756] FAZ-40125

[0757] "" "" ""

[0758]

[0759] FAZ-40125

[0760]

[0761] FAZ-40125

[0762]

[0763] ii. Sequencing

[0764] In some embodiments of the methods of determining nucleotide sequences and detecting sequence variants are known to those of skill in the art. In some embodiments, the nucleotide sequences are determined by next generation sequencing. As used herein “next- generation sequencing” or “NGS” refers to oligonucleotide sequencing technologies that have the capacity to sequence oligonucleotides at speeds above those possible with conventional sequencing methods (e.g. Sanger sequencing), due to performing and reading out thousands to millions of sequencing reactions in parallel. Non-limiting examples of next- generationFAZ-40125

[0765] sequencing methods / platforms include Massively Parallel Signature Sequencing (Lynx Therapeutics); 454 pyro- sequencing (454 Life Sciences / Roche Diagnostics); solid-phase, reversible dye-terminator sequencing (Solexa / Illumina): SOLiD technology (Applied Biosystems); Ion semiconductor sequencing (ION TORRENT™); DNA nanoball sequencing (Complete Genomics); and technologies available from Pacific Biosciences, Intelligen Biosystems, Oxford Nanopore Technologies, and Helicos Biosciences.

[0766] In some embodiments, an NGS library is prepared from in vivo samples collected from a subject administered a lipid nanoformulation having encapsulated therein a barcoded saRNA described herein. In some embodiments, an NGS library is prepared from in vivo samples collected from a subject administered a heterogeneous lipid nanoformulation having encapsulated therein barcoded saRNA described herein. In some embodiments, an NGS library is prepared from a RT-PCR product generated from an saRNA described herein.

[0767] In some embodiments, the sample is a whole tissue sample. In some embodiments, the sample is isolated from one or more of tissue homogenates of connective tissue, epithelial tissue, muscle tissue, and nervous tissue. In some embodiments, the sample is isolated from one or more of the heart, kidney, lung, liver, brain, small intestine, large intestine, stomach, skin, bladder, bone, muscle, pancreas, adrenal gland, appendix, pharynx, thyroid, uterus, spleen, gallbladder, eyes, esophagus, spinal cord, or salivary glands.

[0768] In some embodiments, the sample is from isolated cells. In some embodiments, the sample is from double stranded RNA (dsRNA) isolated from a whole RNA sample. In some embodiments, the sample is RNA isolated by size exclusion.

[0769] In some embodiments, the NGS library is prepared using methods known to those of skill in the art. In some embodiments, the NGS library is prepared by performing reverse transcription of saRNA described herein followed by PCR amplification of the resulting cDNA. In some embodiments, the reverse transcription comprises the positive strand primer set forth in SEQ ID NO: 7 and the negative strand primer set forth in SEQ ID NO: 10. In some embodiments, the reverse transcription comprises the positive strand primer set forth in SEQ ID NO: 7. In some embodiments, the reverse transcription comprises the negative strand primer set forth in SEQ ID NO: 10. In some embodiments, the reverse transcription product is PCR amplified using the negative saRNA strand forward and reverse primers set forth in SEQ ID NOs: 8 and 11, respectively. In some embodiments, the reverse transcription product is PCR amplified using the positive saRNA strand forward and reverse primers set forth in SEQ ID NOs: 8 and 9, respectively. In some embodiments, the reverse transcription primers are 5 ’-biotinylated.FAZ-40125

[0770] Following sequencing, analysis of the sequencing results is done using methods known to those in the art. In some embodiments, analysis if performed using publicly available analysis programs and pipelines. Sequencing analysis is used to determine the saRNA present in the sample. In some embodiments, barcodes identified in sequencing are uses to identify the lipid nanoformulation associated with the corresponding saRNA barcode.

[0771] IV. Exemplary Embodiments

[0772] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a delivery vehicle composition, the method comprising:

[0773] (i) providing a delivery vehicle composition having encapsulated therein a barcoded self-amplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2 to a subject,

[0774] (ii) identifying the barcode sequence of the saRNA in one or more samples of the subject, thereby analyzing the intracellular delivery of the delivery vehicle composition.

[0775] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0776] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a selfamplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA generates a detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation;

[0777] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0778] (iii) sorting cells from one or more samples of the subject by measuring the detectable signal; and,

[0779] (iv) identifying the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of each lipid nanoformulation of the lipid nanoformulation composition.

[0780] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of saRNA encapsulated in a lipid nanoformulation, the method comprising:

[0781] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a selfamplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein theFAZ-40125

[0782] saRNA generates a detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation;

[0783] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0784] (iii) sorting cells from one or more samples of the subject by measuring the detectable signal; and,

[0785] (iv) identifying the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of the saRNA.

[0786] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0787] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0788] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0789] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is transcribed into a negative strand saRNA;

[0790] (iv) wherein upon delivery of the saRNA, the nsP4 is translated to an RdRp and the RdRp translates the reporter gene to the protein that produces the detectable signal;

[0791] (v) sorting cells from one or more samples of the subject by measuring the detectable signal;

[0792] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of each lipid nanoformulation of the heterogeneous lipid nanoformulation.

[0793] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0794] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein theFAZ-40125

[0795] reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0796] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0797] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is translated to form an RNA-dependent RNA polymerase (RdRp);

[0798] (iv) wherein upon delivery of the saRNA, the RdRp catalyzes the formation of negative and subgenomic saRNAs, and cellular machinery translates the protein that produces the detectable signal from the subgenomic RNA;

[0799] (v) sorting cells from one or more samples of the subject by measuring the detectable signal;

[0800] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of each lipid nanoformulation of the heterogeneous lipid nanoformulation.

[0801] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of saRNA encapsulated in a lipid nanoformulation, the method comprising:

[0802] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0803] (ii) administering the heterogeneous lipid nanoformulation to the subject;

[0804] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is transcribed into a negative strand saRNA;

[0805] (iv) wherein upon delivery of the saRNA, the nsP4 is translated to an RdRp and the RdRp translates the reporter gene to the protein that produces the detectable signal;

[0806] (v) sorting cells from one or more samples of the subject by measuring the detectable signal;

[0807] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of the saRNA.FAZ-40125

[0808] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of saRNA encapsulated in a lipid nanoformulation, the method comprising:

[0809] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0810] (ii) administering the heterogeneous lipid nanoformulation to the subject;

[0811] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is translated to form an RNA-dependent RNA polymerase (RdRp);

[0812] (iv) wherein upon delivery of the saRNA, the RdRp catalyzes the formation of negative and subgenomic saRNAs, and cellular machinery translates the protein that produces the detectable signal from the subgenomic RNA;

[0813] (v) sorting cells from one or more samples of the subject by measuring the detectable signal;

[0814] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of the saRNA.

[0815] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0816] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a selfamplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA generates a detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation;

[0817] (ii) administering the heterogeneous lipid nanoformulation composition to the subject, wherein one or more of the lipid nanoformulations of the heterogenenous lipid nanoformulation are delivered intracellularly, wherein upon intracellular delivery, the saRNA is transcribed to a negative strand saRNA comprising the sequence set forth in SEQ ID NO: 3;

[0818] (iii) sorting cells from one or more samples of the subject by measuring the detectable signal; and,FAZ-40125

[0819] (iv) identifying the barcode sequence in the sorted cells, wherein the identification comprises next generation sequencing (NGS) of an NGS library prepared by performing reverse transcription followed by PCR amplification of the negative strand saRNA, thereby analyzing the intracellular delivery of each lipid nanoformulation of the lipid nanoformulation composition.

[0820] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0821] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a selfamplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA generates a detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation;

[0822] (ii) administering the heterogeneous lipid nanoformulation composition to the subject, wherein one or more of the lipid nanoformulations of the heterogenenous lipid nanoformulation are delivered intracellularly, wherein upon intracellular delivery, the saRNA is transcribed to a negative strand saRNA comprising the sequence set forth in SEQ ID NO: 3;

[0823] (iii) sorting cells from one or more samples of the subject by measuring the detectable signal; and,

[0824] (iv) identifying the barcode sequence in the sorted cells, wherein the identification comprises next generation sequencing (NGS) of an NGS library prepared by performing reverse transcription with 5 ’-biotinylated primers, followed by purification with streptavidin-conjugated magnetic beads and PCR amplification of the negative strand saRNA, thereby analyzing the intracellular delivery of each lipid nanoformulation of the lipid nanoformulation composition.

[0825] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0826] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a selfamplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA generates a detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation;

[0827] (ii) administering the heterogeneous lipid nanoformulation composition to the subject, wherein one or more of the lipid nanoformulations of the heterogenenous lipidFAZ-40125

[0828] nanoformulation are delivered intracellularly, wherein upon intracellular delivery, the saRNA is transcribed to a negative strand saRNA comprising the sequence set forth in SEQ ID NO: 3;

[0829] (iii) sorting cells from one or more samples of the subject by measuring the detectable signal; and,

[0830] (iv) identifying the barcode sequence in the sorted cells, wherein the identification comprises next generation sequencing (NGS) of an NGS library prepared by performing reverse transcription followed by PCR amplification of the negative strand saRNA, wherein the reverse transcription comprises the negative strand primer set forth in SEQ ID NO: 10 and the PCR amplification comprises the forward and reverse primers set forth in SEQ ID NOs: 8 and 11, respectively,

[0831] thereby analyzing the intracellular delivery of each lipid nanoformulation of the lipid nanoformulation composition.

[0832] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of saRNA encapsulated in a lipid nanoformulation, the method comprising:

[0833] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a selfamplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA generates a detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation;

[0834] (ii) administering the heterogeneous lipid nanoformulation composition to the subject, wherein upon intracellular delivery, the saRNA is transcribed to a negative strand saRNA comprising the sequence set forth in SEQ ID NO: 3;

[0835] (iii) sorting cells from one or more samples of the subject by measuring the detectable signal; and,

[0836] (iv) identifying the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of the saRNA.

[0837] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of saRNA encapsulated in a lipid nanoformulation, the method comprising:

[0838] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a selfamplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA generates a detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation;FAZ-40125

[0839] (ii) administering the heterogeneous lipid nanoformulation composition to the subject, wherein upon intracellular delivery, the saRNA is transcribed to a negative strand saRNA comprising the sequence set forth in SEQ ID NO: 3;

[0840] (iii) sorting cells from one or more samples of the subject by measuring the detectable signal; and,

[0841] (iv) identifying the barcode sequence in the sorted cells, wherein the identification comprises next generation sequencing (NGS) of an NGS library prepared by performing reverse transcription followed by PCR amplification of the negative strand saRNA, wherein the reverse transcription comprises the negative strand primer set forth in SEQ ID NO: 10 and the PCR amplification comprises the forward and reverse primers set forth in SEQ ID NOs: 8 and 11, respectively, thereby analyzing the intracellular delivery of the saRNA.

[0842] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:

[0843] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0844] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0845] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is transcribed into a negative strand saRNA comprising the sequence set forth in SEQ ID NO: 3;

[0846] (iv) wherein upon delivery of the saRNA, the nsP4 is translated to an RdRp and the RdRp translates the reporter gene to the protein that produces the detectable signal;

[0847] (v) sorting cells from one or more samples of the subject by measuring the detectable signal;

[0848] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of each lipid nanoformulation of the heterogeneous lipid nanoformulation.

[0849] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:FAZ-40125

[0850] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0851] (ii) administering the heterogeneous lipid nanoformulation composition to the subject;

[0852] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is transcribed into a negative strand saRNA comprising the sequence set forth in SEQ ID NO: 3;

[0853] (iv) wherein upon delivery of the saRNA, the nsP4 is translated to an RdRp and the RdRp translates the reporter gene to the protein that produces the detectable signal;

[0854] (v) sorting cells from one or more samples of the subject by measuring the detectable signal;

[0855] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence in the sorted cells, wherein the sequencing comprises next generation sequencing (NGS) of an NGS library prepared by performing reverse transcription followed by PCR amplification of the negative strand saRNA, wherein the reverse transcription comprises the negative strand primer set forth in SEQ ID NO: 10 and the PCR amplification comprises the forward and reverse primers set forth in SEQ ID NOs: 8 and 11, respectively, thereby analyzing the intracellular delivery of each lipid nanoformulation of the heterogeneous lipid nanoformulation.

[0856] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of saRNA encapsulated in a lipid nanoformulation, the method comprising:

[0857] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0858] (ii) administering the heterogeneous lipid nanoformulation to the subject;FAZ-40125

[0859] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is transcribed into a negative strand saRNA comprising the sequence set forth in SEQ ID NO: 3;

[0860] (iv) wherein upon delivery of the saRNA, the nsP4 is translated to an RdRp and the RdRp translates the reporter gene to the protein that produces the detectable signal;

[0861] (v) sorting cells from one or more samples of the subject by measuring the detectable signal;

[0862] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of the saRNA.

[0863] In some embodiments, the disclosure provides a method of analyzing intracellular delivery of saRNA encapsulated in a lipid nanoformulation, the method comprising:

[0864] (i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA) comprising the sequence set forth in SEQ ID NO: 2, wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;

[0865] (ii) administering the heterogeneous lipid nanoformulation to the subject;

[0866] (iii) wherein upon delivery of the saRNA to a cell, the saRNA is transcribed into a negative strand saRNA comprising the sequence set forth in SEQ ID NO: 3;

[0867] (iv) wherein upon delivery of the saRNA, the nsP4 is translated to an RdRp and the RdRp translates the reporter gene to the protein that produces the detectable signal;

[0868] (v) sorting cells from one or more samples of the subject by measuring the detectable signal;

[0869] (vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence in the sorted cells, wherein the sequencing comprises next generation sequencing (NGS) of an NGS library prepared by performing reverse transcription followed by PCR amplification of the negative strand saRNA, wherein the reverse transcription comprises the negative strand primer set forth in SEQ ID NO: 10 and the PCR amplification comprises the forward and reverse primers set forth in SEQ ID NOs: 8 and 11, respectively, thereby analyzing the intracellular delivery of the saRNA.

[0870] In some embodiments, the method comprises purifying the reverse transcription product. In some embodiments, purification of the reverse transcription product is done priorFAZ-40125

[0871] to PCR amplification. Methods of purifying proteins, polypeptides, and oligonucleotides are known in the art.

[0872] In some embodiments, purification of the reverse transcription product comprises immobilizing the reverse transcription product on a solid surface. In some embodiments, the solid surface is a magnetic bead or a coated plate. In some embodiments, the purification comprises immobilization of a reverse transcription product by a biotin- streptavidin pulldown. In some embodiments, the purification comprises immobilization of negative strand reverse transcription product by biotin- streptavidin coupling.

[0873] In some embodiments, purification results in a composition that is substantially free of unprimed positive strand reverse transcription product relative to a reverse transcription product produced without a purification step. In some embodiments, purification results in a composition that is enriched with primed negative strand reverse transcription product relative to a reverse transcription product produced without a purification step.

[0874] EXAMPLES

[0875] The Examples described below provide a non-limiting illustration of the methods described herein. For example, saRNA encapsulated in lipid nanoparticles (LNPs) are administered to a subject as depicted in FIG. 8. Following administration, LNPs may or may not be delivered to a tissue. LNPs which are delivered and are internalized via endocytosis may deliver the saRNA to the cell. Upon intracellular delivery, saRNA which are released into the cytoplasm, will replicate and amplify as depicted in FIG. 9. The amplified saRNA may then be isolated and sequenced to assess LNP delivery.

[0876] Example 1: Design of barcoded saRNA templates

[0877] saRNA template plasmids were designed to allow runoff IVT ( vitro transcription) of linearized plasmid DNA (pDNA). Nucleotide numbering reflects the number of the final construct (SEQ ID NO: 1) and begins at the start of the Capl T7 promoter. A schematic of the barcoded saRNA is provided in FIG. 1.

[0878] Barcoded saRNA templates were based on clone TC-83, an attenuated form of Venezuelan Equine Encephalitis Virus used frequently in saRNA therapeutic development (GenBank Ref # LO 1443.1 ; Bloom et al. Gene Then 28(3-4): 117-129, Oct 22, 2020). The TC-83 sequence was inserted at position 18, directly downstream of the Capl T7 promoter. The bl mutation (G3953C) from Li et al. was introduced to favor higher subgenomic RNA production and reporter expression (Li et al. Sci Rep. ,9:6932, May 6, 2019).FAZ-40125

[0879] The second TC-83 ORF (encoding VEEV structural proteins) was replaced with a human codon-optimized Gaussia luciferase (hGluc) ORF, occupying positions 7579 - 8133. Two silent mutations (G8064T, A8067G) were made in the hGluc ORF to introduce a BbvCI restriction site, facilitating subcloning of new barcodes. A triple stop codon was inserted at the 3’ end of the hGluc ORF (positions 8134-8142).

[0880] Barcode and NGS priming sequences were added immediately following the hGluc triple stop codon. The 12-nt barcode occupies positions 8143-8154, and the priming site occupies positions 8155-8174. These sequences were placed to allow barcode replication in positive, negative, and subgenomic saRNA transcripts, and to avoid disruption of TC-83 regulatory sequences. The primer binding site was designed by screening random DNA sequences using NCBI Primer-BLAST to identify good priming sites orthogonal to human, mouse, and rhesus macaque transcriptomes, followed by screening against the TC-83 and hGluc sequences. Candidate priming site sequences were folded in silico using mFold (UNAfold), in the context of the hGluc ORF and TC-833’ UTR, to find sites which did not disrupt TC-833’ UTR secondary structure. Sequences were also screened for microRNA target sites using miRDB. A segmented poly- A tail sequence was inserted at the end of the TC-833’ UTR (positions 8292-8401). A BspQI restriction site was inserted at positions 8402-8408 to allow linearization of IVT templates for runoff transcription.

[0881] A negative control saRNA was also designed, containing a unique barcode 002 and three mutations (A7113C, T7114C, A7116C) which abolish the catalytic activity of nsP4, preventing negative and subgenomic strand synthesis (Rubach, J. et al., Virology, 384(1): 201-208, Nov 25, 2008). Sequence design was performed using SnapGene version 8.0 and Benchling.

[0882] saRNA Template Barcode Library Expansion

[0883] The resulting barcoded saRNA cassettes (positions 1-8408) were assembled into the low copy number pBR322 minusMCS vector by Blue Heron Biotechnologies. This vector was selected to maintain stability of the long, homopolymer-rich insert in bacteria. Three active saRNAs (barcodes 001, 003, and 004) and one catalytic-dead saRNA (barcode 002) were constructed.

[0884] To generate new saRNA barcode sequences in the active construct, the barcode region was removed by digestion of the barcode 001 template plasmid using BbvCI and Mfel-HF (NEB #R0601, #R3589) (see FIG. 2A). 1 pg of plasmid was diluted in rCutSmart Buffer with 1 pL of each enzyme, in 50 pL. Reactions were incubated at 37°C for 30 min, followedFAZ-40125

[0885] by separation on a 1% agarose gel and extraction of the resulting upper band using a QIAquick Gel Extraction Kit (Qiagen #28704).

[0886] To generate inserts with novel barcode sequences a 121 nt Ultramer ssDNA oligo consisting of the saRNA template sequence from the BbvCI through the Mfel cut sites was ordered from Integrated DNA Technologies. This oligo contained 12 randomized nucleotides in place of a defined barcode sequence. This oligo was PCR-amplified using Phusion HS II polymerase (ThermoFisher #F549) with Buffer HF, according to the manufacturer’s instructions. Reactions were performed using primers which added 15, 25, or 40 bp homology arms up- and downstream of the BbvCI and Mfel cut sites to enable Gibson assembly into the above vector double-digest. PCR products were separated on a 2% agarose-TAE gel and extracted as described above.

[0887] Gel-extracted vector double-digests and insert PCR products were assembled at 3:1 insert-to-vector ratios using NEBuilder HiFI DNA Assembly Master Mix (NEB #E2621) according to the manufacturer’s instructions. Reaction products were transformed into One Shot Stbl3 Chemically Competent E. coli cells (ThermoFisher #C737303) according to the manufacturer’s instructions, followed by spreading onto carbenicillin-100 LB agar plates (Teknova #L1010) and growth at 30°C for 48 h (see e.g., FIG. 2A). Low transformation volumes were used to favor single-transformant colonies, with each containing a unique barcode sequence (Tomoiaga, D. et al., Sci Rep. 12:11515; July 7, 2022).

[0888] Colonies were picked using sterile pipet tips and inoculated into 5 mL starter cultures using Gibco Terrific Broth (ThermoFisher #A1374301) with 50 pg / mL carbenicillin (Sigma #C3416). Starter cultures were grown overnight at 30°C with orbital shaking at 250 rpm. The following day, starter cultures were inoculated into 50 mL of the same media and grown until turbid. 500 L of each culture was mixed with 500 pL of 50% glycerol to generate glycerol stocks, which were stored at -80°C. The remaining volume was then pelleted at 5000 x g for 15 min, followed by pDNA extraction using ZymoPURE II Plasmid Midiprep Kits (Zymo Research #D4201) according to the manufacturer’s instructions.

[0889] Midiprep pDNA samples were then sent to Eurofins Genomics for sequencing.

[0890] Whole-plasmid Nanopore sequencing was performed to validate correct assembly, and to detect any point mutations in the insert. Sanger sequencing of the barcode and poly-A regions was also performed in both the forward and reverse directions, using custom sequencing primers. For the barcode region, chromatograms were analyzed for single peaks at each barcode position to ensure the presence of one barcode per prep (FIG. 2B). For theFAZ-40125

[0891] poly-A region, forward- and reverse-reads were compared to ensure the presence of a full-length poly-A tail (FIG. 2C). Glycerol stocks and midipreps from colonies which failed any screening step were discarded. 22 additional barcodes were generated. Notably, 30-40% of colonies screened contained uncut parent (barcode 001) vector.

[0892] Due to the laborious and time-consuming nature of this method, isolation of further barcodes was outsourced to Neochromosome. Neochromosome accelerated colony screening by inserting a gene encoding a fluorophore between the BbvCI and Mfel sites such that colonies containing the parent vector could be identified visually and excluded from further screening. Using this method, Neochromosome generated 58 additional unique barcodes. 12 additional barcodes were also generated by GenScript using specified barcode sequences and standard cloning methods. Between synthesis at Mirai Bio, Blue Heron, Neochromosome, and GenScript, a total of 103 unique barcoded saRNA templates have been generated.

[0893] As a final screening step before saRNA synthesis and LNP formulation, Levenshtein distances between barcodes were calculated in Microsoft Excel using a VBA module.

[0894] Barcode sequences with a Levenshtein distance <4 were excluded from use in the same experiment to avoid ambiguous next- generation sequencing results. 101 of the 103 barcodes can be used simultaneously. (Barcodes 012 and 016 are separated by a Levenshtein distance of 2; barcodes 034 and 055 are separated by a Levenshtein distance of 3.)

[0895] Production and purification of barcoded saRNA templates

[0896] Validated saRNA barcode pDNA samples must be scaled up, linearized, and purified prior to in-vitro transcription (IVT).

[0897] Briefly, glycerol stocks of validated pDNA templates were briefly removed from -80°C and placed on dry ice. A sterile pipet tip was scraped on the surface of the stock, and then used to streak an LB-carbenicillin plate. Glycerol stocks were immediately returned to -80°C following plate streaking. Alternatively, plates may be obtained by retransforming purified pDNA into chemically-competent E. coli as described above. Plates were incubated at 30°C until colonies are visible. Colonies were picked and used to inoculate overnight 5 mL starter cultures as described above. Overnight cultures were inoculated into 2 L of TB-Carbenicillin and grown to turbidity at 30°C. 2 L cultures were split into 500 mL aliquots and pelleted at 5000 x g. Pellets were resuspended in buffer and pDNA extracted using ZymoPURE II Plasmid Gigaprep Kits (Zymo Research #D4204) according to the manufacturer’s instructions.FAZ-40125

[0898] Following pDNA extraction, templates were linearized to enable runoff IVT. Purified pDNA was diluted to 0.5 |lg / |lL in IX NEBuffer 3.1 (NEB #B6003) and digested using 1 U / pg BspQI (NEB #R0712) for 2-4h at 50°C. Digestion progress was monitored by analyzing reaction aliquots on an E-Gel EX 1 Agarose Gel (ThermoFisher #G401001), with electrophoresis performed on an E-Gel Power Snap System (ThermoFisher #G8100).

[0899] Digestion products were precipitated using LiCl, resuspended, and normalized to 1 mg / mL.

[0900] Production and purification of barcoded saRNA

[0901] Linearized pDNA templates were transcribed using a saRNA-optimized IVT method. Cytidine was substituted with 5-methylcytidine in these reactions. Following IVT, reactions were treated with TURBO DNase (ThermoFisher #AM2238) according to the manufacturer’s instructions to remove residual template pDNA, followed by LiCl precipitation to remove protein, DNA fragments, and residual nucleotide triphosphates. Precipitates were resuspended and purified on oligo-dT resin, then cellulose resin, using a dedicated AKTA pure chromatography system (Cytiva). Purified saRNA was normalized to 1 mg / mL for LNP formulation. An exemplary schematic of positive (+) strand saRNA is provided in FIG. 1.

[0902] Formulation and pooling of barcoded saRNA LNPs

[0903] Initial experiments were performed using LNPs generated using a NanoAssemblr (Precision Nanosystems according to the manufacturer’s instructions). Subsequent LNP formulation was performed using a proprietary, multiplexed microfluidic device developed at Mirai Bio by Beth Lally, Brie Skinner, Charles Warren, and Stephanie Knapp. A minimum of 500 pg saRNA is required for each formulation. LNP storage / dosing buffer consists of 20 mM HEPES (pH 7.8), 7.5% sucrose, and 25 mM NaCl. Individual formulations are pooled, diluted to the final dosing concentration, and stored at -80°C prior to dosing.

[0904] Example 2: Dosing of saRNA Encapsulated in Lipid Nanoformulation and Tissue Collection

[0905] In vivo experiments were performed using female C57B1 / 6 mice (Jackson Labs). 1 or 0.1 mpk LNPs, as well as phosphate-buffered saline (PBS) vehicle controls, were deliveredFAZ-40125

[0906] by tail vein injection. Animals were sacrificed using isofluorane at 6h, 24h, and 48h timepoints.

[0907] Following sacrifice blood, liver, spleen, and lung samples were collected. Blood samples were collected by cheek bleed, spun down to yield serum, and aliquoted in sterile, 96-well plates. Two 30 pL serum samples per animal were collected for luminescence and RT-qPCR analysis, while one 50 pL serum sample was collected for cytokine analysis. Liver and lung samples were collected as 3 x 3 mm tissue punches, while spleens were cut into thirds. Tissue samples were immediately immersed in RNAlater Stabilization Solution (ThermoFisher #AM7021) in sterile, 24- well plates. All sample plates were immediately sealed using Adhesive PCR Plate Foils (ThermoFisher #AB0626) and stored at -80°C.

[0908] Serum luminometry

[0909] For serum luminometry, one plate of 30 pL / well serum samples was thawed on ice. Plates were spun down and the sealing foil carefully removed. 3 x 10 pL aliquots of each serum sample were transferred to a flat white assay plate (Corning #3912) and placed on ice. Flash luminometry was performed on a Tecan Spark Multimode Microplate reader using Quanti-Luc 4 Lucia / Gaussia reagent (InvivoGen #QLCA-45-01). The assay reagent was diluted to IX concentration in MilliQ water, without stabilizer, and stored in the dark and on ice until the assay was performed. The reagent injector was primed with 3 x 500 pL of the assay solution prior to measurements. Assays were performed well-by-well using the following conditions: 100 pL of assay reagent was dispensed, followed by immediate 1000 ms integration with no attenuation. Results were exported to Microsoft Excel and graphed using GrapPad Prism 10.

[0910] Extraction of total RNAfrom in vivo tissue samples

[0911] Plates containing solid tissue samples in RNAlater were thawed overnight at 4°C prior to extraction. Tissue extractions were performed using MagMAX mirVana Total RNA Isolation Kits (ThermoFisher #A27828). Tissue samples were removed from RNAlater using sterile forceps and placed in 2.0 mL DNA-Lobind tubes (Eppendorf #022431048) containing 600 pL of kit lysis buffer supplemented with 4.2 pL of P-mercaptoethanol and a steel bead (Qiagen #69975). Sample tubes were inserted into clean tube cassettes, placed on a Qiagen Tissuelyser II instrument, and shaken at 30 cycles / s for 60 sec. This process was repeated until samples were completely homogenized. 100 pL aliquots of homogenates wereFAZ-40125

[0912] distributed into six 96-well plates such that each plate contained a full representation of all experimental animals. Plates were covered with sealing film and kept on ice. Plates were extracted serially on a Kingfisher Apex System (Thermo Scientific) using the MagMAX mirVana Total RNA Isolation Kit, according to the manufacturer’s instructions. Samples were eluted into 96-well plates in 100 pL, sealed, and stored at -80°C. The Kingfisher instrument was wiped down and UV-treated between extractions.

[0913] RT-qPCR quantitation of barcoded saRNA biodistribution

[0914] The barcoded saRNA content of both serum and tissue samples was analyzed by absolute quantitation against a standard curve, while tissue sample saRNA was also quantified by relative quantitation against the mouse Ppib housekeeping gene mRNA.

[0915] Separate assays were targeted to positive strands, negative strands, and Ppib.

[0916] To generate positive strand standards for absolute quantification an aliquot of barcode 001 saRNA was thawed on ice and its concentration determined using a Nanodrop Eight Spectrophotometer (ThermoFisher #NDE-GL). The stock was adjusted to IO10copies / pL (44.02 ng / pL) using nuclease-free water, followed by 10-fold serial dilutions to generate a standard curve ranging from 109- 102copies / pL.

[0917] To generate negative strand standards, a negative strand IVT template was generated by PCR. A barcode 001 template pDNA prep was PCR- amplified using Phusion HS II DNA polymerase and HF buffer, according to the manufacturer’s instructions. The forward primer was targeted at the sense strand downstream of the 5’ T7 transcription site, eliminating it from PCR products. The reverse primer targeted the 3’ end of the TC-833’ UTR and introduced a T7 start site to the bottom strand. PCR products were gel-purified on a 1% agarose-TAE gel to remove residual PCR template, as described above. Primer sequences are provided in Table 3.

[0918] Table 3. Negative Strand IVT Primers

[0919]

[0920] FAZ-40125

[0921] PCR products were then used as templates for the MEGAscript T7 Transcription Kit (ThermoFisher #AM1334) in 20 pL according to the manufacturer’s instructions. Following IVT reactions were treated with 2 U of TURBO DNase at 37°C for 30 min. Negative strand RNA was purified on an RNeasy Mini Kit (Qiagen #74104), adjusted to IO10copies / pL (41.05 ng / pE), and diluted to 109- 102copies / pL as described above.

[0922] Two-step RT-qPCR was performed using a stranded RT-qPCR assay adapted from Skidmore et al. (Plaskon, N et al., PLOS One, 4(1); e7468, Oct 2009; Skidmore, A, et al. Antiviral Res. 174:104674, December 6, 2019). It was determined that strand-targeted RT-PCR primers alone do not produce strand- specific NGS libraries. Reverse transcription using a negative strand-targeted primer also produces unprimed positive strand cDNA, possibly due to RT priming by structured RNA elements in the positive strand 3’ UTR. Incomplete Exol digestion of RT primers allows amplification of positive strand cDNA during NGS library prep PCR, producing NGS libraries with ambiguous mixes of positive and negative strand sequences (FIG. 10A). Reverse transcription primers were 5 ’-biotinylated to permit removal of unprimed RT artifacts prior to qPCR amplification using a method adapted from Boncristiani et al. (Chaves, RL et al., Nucleic Acids Res., 22(10): 1919-1920, May 25 1994; Boncristiani, H et al., J Virol Methods, 161(1): 147-53, Oct 2009) (FIG. 10B). cDNA synthesis was performed in duplicate for each sample, standard, and a water no-template control using Induro Reverse Transcriptase (NEB #M0681), without heat inactivation, according to the manufacturer’s instructions (using the primers set forth in SEQ ID NOs: 12 and 13). 2 pL of sample, standard, or control was used as a template per 20 pL reaction, in 96-well PCR plates. Following cDNA synthesis, 1 pL of Thermolabile Exonuclease I (NEB #M0568S) was added to each reaction to remove residual RT primer. Plates were incubated at 37°C for 4 min . Finally, 1 pL of Thermolabile Proteinase K (NEB #P8111) was added to each reaction and plates were incubated at 37°C for 15 min, followed by heat inactivation at 55°C for 10 min.

[0923] Off-target RT products were removed prior to qPCR by purification on streptavidin-conjugated magnetic beads (SA beads, Thermo #65001). 5 pL of SA beads and 50 pL of B / W buffer (5 mM Tris-HCl pH 7.5, 500 pM EDTA pH 8.0, IM NaCl, 0.05% Tween-20, 0.22 pm filtered) were added to each well of a 96-well plate, washed once in 1 mL B / W buffer, and resuspended in 20 pL B / W buffer. The entire RT volume (22 pL) was added to the resuspended beads and incubated for Ih. Beads were then subjected to five 10 min washes inFAZ-40125

[0924] 1 mL B / W buffer and deposited in 100 pF RNase-free water. To prevent beads for occluding the fluorescence detector during qPCR, washed beads were next transferred to a fresh well containing 100 pF RNase-free water, cDNA eluted at 70°C for 10 min, and beads quickly removed (Holmberg, A et al., Electrophoresis. 26(3):501-10, Feb 2006). Overall, bead-based purification of RT products improves NGS library specificity. Addition of a 5’ biotin modification to RT primers enables specific pulldown of primed, negative strand cDNA on streptavidin magnetic beads. Stringent washing removes unprimed positive strand RT products, allowing specific PCR amplification of negative strand NGS libraries from beadbound cDNA.

[0925] For the second step, positive and negative qPCR master mixes were assembled using TaqMan Fast Advanced Master Mix for qPCR (ThermoFisher #4444556), as well as positive or negative strand primer sets and a universal probe (see SEQ ID NOs: 14-18) derived from Skidmore et al. Primers were added for a final reaction concentration of 900 nM, while probe was added to a final concentration of 250 nM. 18 pF of master mix was added to each reaction well, followed by 2 pF of exonuclease-treated SA bead-purified RT product. qPCR was performed on a Quantstudio 6 or Quantstudio 7 qPCR System (Applied Biosystems) with the following conditions: an initial hold at 95°C for 20 sec, followed by 45 cycles of 95°C for 1 sec and 60°C for 30 sec.

[0926] For Ppib quantitation, a premade one-step RT-qPCR assay was used (ThermoFisher #Mm00478295_ml). Reactions were assembled using PrimeTime One-Step RT-qPCR Master Mix (Integrated DNA Technologies #10007066) supplemented with reference dye according to the manufacturer’s instructions. Assay and reaction mixes were combined with 2 pF of extracted total RNA, or a water non-template control, in 20 pF. RT-qPCR was performed on a Quantstudio 6 or Quantstudio 7 using the following conditions: 50°C for 5 min, 95°C for 10 min, and then 45 cycles of 95°C for 15 sec and 60°C for 60 sec.

[0927] Absolute quantitation analysis was performed using the Standard Curve module in the Design and Analysis program (Applied Biosystems). For relative quantitation, Cqvalues for positive / negative strand and Ppib were exported from Design and Analysis and the AACqmethod was applied using Microsoft Excel. RT-qPCR results were graphed using GraphPad Prism 10.

[0928] Example 3: Expression and Biodistribution of Barcoded saRNAFAZ-40125

[0929] An in vivo multiplex lipid nanoparticle (LNP) screen was conducted to determine expression and biodistribution of barcoded saRNA delivered by LNPs with unique formulations (FIGs. 4A and 4B). Individual formulations were quality-controlled for size distribution, encapsulation efficiency, and surface potential prior to pooling each LNP for multiplexing. Retained injection material was sequenced to determine input saRNA ratios for sequencing controls.

[0930] Following administration, serums was collected from mice at 6 and 24 hours. Serum luminescence was used to measure hGluc reporter expression from each sample. Catalytic dead saRNA (catdead) showed expression comparable to PBS-treated serum or blank buffer, while pooled samples showed higher expression compared to single-LNP groups (FIG. 5).

[0931] Biodistribution of the barcoded saRNA was measured by a stranded RT-qPCR approach adapted from Skidmore et al. by quantifying (+) and (-) strand saRNA levels in serum and spleen total RNA (FIG. 6A and 6B).

[0932] To determine biodistribution of each unique LNP formulation, NGS libraries were generated by an RT-PCR method (FIG. 3). RNA was extracted from spleens of mice at 6 or 24 hours post administration of multiplex LNP compositions comprising 2 LNPs or 9 LNPs administered at Img / kg or 0.1 mg / kg (mpk). Input (retain) material for each study group was diluted 1000-fold in water. RNA samples were then subjected to library generation RT-PCR as described in Example 3. RT primers targeted to either (+) or (-) strand installed a UMI, study tag, and a PCR priming site at the 5’ end of product cDNA. Following RT, PCR was performed with primers targeted toward the installed tag and a site downstream of each strand’s respective RT primer. PCR primers install the remaining elements required for NGS. Negative strand sequencing results of the 2-LNP screen from spleen tissue are provided in FIG. 7A. The screen demonstrates 5% DSG-PEG LNP inhibited accumulation in spleen compared to control. Successful negative strand amplification of retain material also indicates (+) strand bleedthrough, similar to results in Figure 3C. Negative strand sequencing results of the 9-LNP screen from spleen tissue are provided in FIG. 7B. For both screens, the total UMI count decreases after administration indicating clearance of dosed material (retain versus dose samples). Depletion of samples with 5% DSG-PEG is in the 9-LNP screen showed similar inhibited accumulation in the screen to that in the 2-LNP screen.

[0933] Example 4: NGS library preparation and sequencingFAZ-40125

[0934] NGS libraries were prepared from in vivo samples and undosed sample retains by RT-PCR (see exemplary schematic in FIG. 3). Both RT and PCR primers were designed in an attempt to obtain libraries derived from the positive or the negative strand only. Plates of total RNA extracts were thawed on ice prior to library amplification, while LNP sample retains (i.e., samples which have not been injected in a subject and are used to calculate barcode ratios) were diluted 1000X in water and serum samples were used directly. All RT and PCR reactions were performed in a 96-well format.

[0935] Reverse transcription and excess primer digestion were performed on 2 pL of template RNA using Induro Reverse Transcriptase and biotinylated RT primers as described above. Positive strand RT primers (SEQ ID NO: 7) were designed to hybridize to the engineered primer binding site, while negative strand RT primers (SEQ ID NO: 10) targeted the 3’ end of the hGluc ORF, such that RT primer extension began with the barcode region of each respective strand. RT primers contained each 5’ overhang sequences which installed a 3 nt study tag, a 10 nt randomized UMI sequence, and a fragment of the Illumina SB S3 sequence to serve as a PCR primer binding site. Excess RT primers were removed using Thermolabile Exonuclease I and enzymes degraded using Proteinase K as described above. RT products were bound to SA resin, washed, and deposited in 100 uL RNase-free water as described above, but without cDNA elution at 70°C.

[0936] Because fluorescence detection is not required for NGS library prep, all RT purification beads were collected and transferred directly to PCRs to maximize reaction yield.

[0937] 2 pL of RT product was then PCR amplified using Phusion HS II DNA polymerase with 500 nM each primer and HF buffer in 50 pL. The following cycling conditions were used: an initial hold of 98°C for 30s, followed by 35 cycles of 98° for 10 sec, 65°C for 30 sec, and 72°C for 30 sec, followed by a final hold of 72°C for 10 min. PCR primers installed the remainder of SBS3, SBS12, and the P5 and P7 adapter sites required for NGS on a MiniSeq instrument (Illumina) (see SEQ ID NOs: 8, 9, and 11). Unique, indexed reverse primers were used for each sample to enable multiplex NGS. PCR products were 245 bp for positive strand libraries and 284 bp for negative strand libraries. 25 pL aliquots of PCR products were brought to 100 pL with water and purified on a Kingfisher Apex instrument using a Mag JET NGS Cleanup and Size Selection Kit (ThermoFisher #K2821), following the manufacturer’s instructions for adapter removal. Purified library preps were eluted in 50 uLFAZ-40125

[0938] of kit elution buffer and quantified using a Qubit Flex Fluorometer (ThermoFisher #Q33327) and Qubit IX dsDNA HS Assay Kit (ThermoFisher #Q33231).

[0939] 30 ng each of up to 24 indexed sample library preps were pooled for multiplex NGS using a MiniSeq High Output Kit (150 cycles) (Illumina #FC-420-1002). Pools were quantified by Qubit as described above, diluted to 1 nM using RSB (Teknova #T7724), and denatured according to sequencing kit instructions. 1 pM PhiX Control v3 (Illumina #FC-110-3001) was denatured according to manufacturer instructions and spiked into 1 pM denatured library pools at 20%. Sequencing was performed on 500 pL pooled, spiked libraries according to the manufacturer’s instructions. Run results were uploaded to the BaseSpace Sequencing Hub (Illumina), where demultiplexing and .fastq file generation were performed.

[0940] To evaluate the effect of biotinylation on recovery of transcription product, both biotinylated and direct RT-PCR samples were prepared for library preparation. Specifically, three barcoded negative- strand saRNAs were purified, quantified by nanodrop, and pooled at specific mass ratios. Four barcoded positive-strand saRNAs were also pooled at equal mass ratios using the same methods (FIG. HA). Positive and negative strand saRNA pools were mixed at varying copy number ratios. Strand- specific NGS library prep RT-PCRs were performed with or without biotinylated primers / streptavidin bead-based purification of RT products. NGS was used to measure UMI counts for each barcoded saRNA. Both methods produced high UMI counts for positive strand. Only the RT-purification method produced high, dose-dependent negative strand UMI counts. Direct, positive strand-targeted RT-PCR of the 109-copy positive pool + 105-copy negative pool condition failed to yield viable NGS library DNA. Barcode UMI ratios were calculated for the data shown in panel FIG. 11B. RT purification generated more accurate negative strand UMI ratios at lower negative strand doses. No-template controls produced extremely low UMI counts and have their UMI ratios grayed out. (FIG. 11C)

[0941] In vivo validation was then conducted. Specifically, three barcoded, positive-strand saRNAs were formulated individually in LNPs. One barcoded saRNA (LNP1 nonrep.

[0942] Cargo), had a catalytic-dead nsP4 and was incapable of replication, including negative strand synthesis (FIG. HD). LNP1 and LNP2 represent formulations with distinct ionizable lipids. Barcoded LNPs were pooled and the pool’s barcode UMI ratio measured by positive strand-targeted NGS (FIG. HD). The LNP pool shown in FIG. HD was administered to mice intravenously and organs harvested at 6 or 24h. Total RNA was extracted and subjected toFAZ-40125

[0943] strand- specific NGS RT-PCR, including bead-based purification of biotinylated cDNA (FIG.

[0944] HE). Positive and negative strand UMI counts are shown for lung and spleen total RNA. Higher UMI counts suggest better biodistribution (positive strand) and functional delivery (negative strand) to spleen. Each bar represents results from a single animal. UMI ratios from FIG. HE were calculated (FIG. HF). Depletion of the nonreplicating cargo barcode in negative strand results reflects its inability to generate negative strand RNA following successful delivery to the cytosol. Low UMI counts from lung resulted in more variable UMI ratios compared to spleen.

[0945] NGS analysis

[0946] NGS analysis was performed using a pipeline composed of publicly available analysis programs, as well as a custom reference genome.

[0947] The analysis pipeline consisted of the following steps. Demultiplexed sample .fastq file quality reports were generated using FastQC. Reads were trimmed to remove study tags and irrelevant 3’ sequence, followed by elimination of reads with a quality score < 30, using Trimmomatic . UMI sequences were extracted from reads to headers using umi-tools , followed by a final 5’ trimming step with Trimmomatic. Bowtie was used to generate an artificial genome composed of saRNA barcode sequences plus flanking regions, with chromosome numbering corresponding to barcode numbering. Reads were mapped to this artificial genome (also using bowtie) and mapped reads were converted to BAM format, sorted, and indexed using samtools . UMIs were then deduplicated and files converted back to SAM format using umi-tools. UMI counts were then obtained and exported as .csv files. Raw read and UMI counts were converted to ratios in Microsoft Excel and graphed using GraphPad Prism 10.

Claims

FAZ-40125CLAIMSWhat is claimed is:

1. A method for screening delivery vehicle formulations, comprising:(a) providing a pooled composition comprising a plurality of distinct delivery vehicles, each delivery vehicle encapsulating a self-amplifying RNA (saRNA) comprising a unique nucleotide barcode;(b) administering the pooled composition to a subject;(c) isolating RNA from one or more biological samples obtained from the subject; and (d) identifying barcode sequences present in the isolated RNA,wherein detection of a barcode indicates intracellular delivery of the corresponding delivery vehicle.

2. The method of claim 1, wherein identifying barcode sequences comprises detecting negative- strand RNA generated from the saRNA.

3. The method of claim 1 or claim 2, wherein the plurality of distinct delivery vehicles comprises a plurality of distinct lipid nanoparticles (LNPs) differing in lipid composition.

4. The method of any one of claims 1-3, wherein the LNPs differ in at least one of: ionizable lipid structure, helper lipid content, sterol content, or PEG-lipid content.

5. The method of any one of claims 1-4, wherein the saRNA encodes a reporter protein expressed from a subgenomic promoter.

6. The method of any one of claims 1-5, wherein identifying barcode sequences comprises performing next- generation sequencing.

7. The method of claim 6, wherein sequencing is performed following strand- specific reverse transcription using a primer comprising a purification moiety.

8. The method of claim 7, wherein the purification moiety is biotin and the reverse transcription product is purified using a streptavidin solid support.FAZ-401259. The method of any one of claims 1-8, wherein the pooled composition comprises at least 10 distinct delivery vehicles.

10. A composition comprising a plurality of lipid nanoparticles (LNPs), each of the plurality encapsulating a self-amplifying RNA (saRNA) that (a) comprises a unique barcode sequence, and optionally (b) encodes a reporter protein expressed from a subgenomic promoter.

11. The composition of claim 10, wherein the plurality comprises at least ten.

12. The composition of claim 10, wherein the composition is configured for pooled administration to a subject.

13. A method of analyzing intracellular delivery of a delivery vehicle composition, the method comprising:(i) providing a delivery vehicle composition having encapsulated therein a barcoded self-amplifying RNA (saRNA) to a subject,(ii) identifying the barcode sequence of the saRNA in one or more samples of the subject, thereby analyzing the intracellular delivery of the delivery vehicle composition.

14. The method of claim 13, wherein the delivery vehicle composition comprises a heterogeneous lipid nanoformulation composition.

15. A method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:(i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a self-amplifying RNA (saRNA), wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation;(ii) administering the heterogeneous lipid nanoformulation composition to the subject;(iii) isolating and sequencing RNA from one or more samples of the subject; and,FAZ-40125(iv) identifying the barcode sequence, thereby analyzing the intracellular delivery of each lipid nanoformulation of the heterogeneous lipid nanoformulation composition.

16. A method of analyzing intracellular delivery of saRNA encapsulated in a lipid nanoformulation, the method comprising:(i) providing a heterogeneous lipid nanoformulation composition comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a self-amplifying RNA (saRNA), wherein the saRNA generates a detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation; (ii) administering the heterogeneous lipid nanoformulation composition to the subject;(iii) sorting cells from one or more samples of the subject by measuring the detectable signal; and,(iv) identifying the barcode sequence in the sorted cells, thereby analyzing the intracellular delivery of the saRNA.

17. The method of any one of claims 13-16, wherein the saRNA encodes at least one non-structural protein.

18. The method of any one of claims 13-17, wherein the saRNAencodes four non- structural proteins.

19. The method of any one of claims 13-18, wherein the saRNA encodes a viral non-structural protein 1 (nsPl), non-structural protein 2 (nsP2), non- structural protein 3 (nsP3), and non- structural protein 4 (nsP4).

20. The method of claim 19, wherein nsP4 is RNA-dependent RNA polymerases (RdRP).

21. The method of claim 19 or claim 20, wherein one or more of nsPl, nsP2, nsP3, and nsP4 are derived from Venezuelan equine encephalitis virus (VEEV), Sindbis Virus (SINV), Smliki Forest Virus (SFV), Classical Swine Fever Virus (CSFV), a fusion of VEEV and SINV (VEE-SINV), or any combination thereof.FAZ-4012522. The method of any one of claims 13-21, wherein the saRNA comprises a genomic promoter.

23. The method of claim 22, wherein the genomic promoter drives expression of one or more non- structural proteins.

24. The method of any one of claims 13-23, wherein the saRNA comprises a subgenomic promoter.

25. The method of any one of claims 13-24, wherein the saRNA comprises a poly(A) tail.

26. The method of any one of claims 13-25, wherein the saRNA comprises a 5’ cap.

27. The method of claim 26, wherein the 5’ cap is an m7G cap.

28. The method of any one of claims 13-27, wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene.

29. The method of claim 28, wherein the subgenomic promoter drives expression of the reporter gene.

30. The method of claim 28 or claim 29, wherein the reporter gene encodes a protein that produces the detectable signal.

31. The method of any one of claims 13-30, wherein the protein is a fluorescent protein or an enzyme.

32. The method of any one of claims 13-31, wherein the detectable signal is produced by a protein selected from the group consisting of a luciferase, a horseradish peroxidase, an alkaline phosphatase, a P-galactosidase, and any combination thereof.FAZ-4012533. The method of any one of claims 13-32, wherein the detectable signal is produced by luciferase.

34. The method of claim 33, wherein the luciferase is Gaussia luciferase.

35. The method of any one of claims 13-16, wherein the saRNA comprises a nucleic acid sequence encoding, from 5’ to 3’:i) a 5’ cap;ii) a 5’ UTR including a promoter;iii) nsPl, nsP2, nsP3, and nsP4;iv) a subgenomic promoter;v) a subgenomic 5’ UTR;vi) a reporter gene;vii) a barcode;viii) a 3 ’ UTR; andix) a poly(A) tail.

36. The method of any one of claims 3-35, wherein the barcode is 8-12 nucleotides in length.

37. The method of any one of claims 21-36, wherein the barcode is 3’ relative to the subgenomic promoter.

38. The method of any one of claims 13-37, wherein the saRNA comprises the nucleic acid sequence set forth in SEQ ID NO: 2.

39. The method of any one of claims 13-38, wherein the saRNA comprises at least one modified nucleic acid.

40. The method of claim 39, wherein the modified nucleic acid is 5 -methylcytidine (5mC).

41. The method of any one of claims 13-40, wherein the saRNA is a positive strand RNA or a negative strand RNA.FAZ-4012542. The method of any one of claims 14-41, wherein the heterogeneous lipid nanoformulation composition comprises lipid nanoformulations generated using different lipid compositions.

43. The method of claim 42, wherein the lipid nanoformulations vary in the molar amount and / or structure of the ionizable lipid, the molar amount and / or structure of the helper lipid, the molar amount / or structure of PEG, and / or the molar amount of cholesterol.

44. The method of any one of claims 14-43, wherein the composition comprises greater than 10 different lipid nanoformulations.

45. The method of any one of claims 14-43, wherein the composition comprises greater than 100 different lipid nanoformulations.

46. The method of any one of claims 14-43, wherein the composition comprises greater than 200 different lipid nanoformulations.

47. The method of any one of claims 13-46, wherein the intracellular delivery is intracellular delivery.

48. The method of any one of claims 13-46, wherein the intracellular delivery is cytoplasmic delivery.

49. The method of any one of claims 13-48, wherein upon delivery of the saRNA, nsPl, nsP2, nsP3, and nsP4 are translated and assemble into an RdRP.

50. The method of any one of claims 41-49, wherein identifying the barcode sequence comprises amplification and sequencing of the negative strand saRNA or positive strand saRNA.

51. The method of claim 50, wherein the amplification comprises reverse transcription of the saRNA.FAZ-4012552. The method of claim 51, wherein reverse transcription comprises using a reverse transcription primer.

53. The method of claim 52, wherein the reverse transcription primer comprises a purification moiety.

54. The method of claim 53, wherein the purification moiety is an amino modifier, a biotin modifier, or an alkyne modification.

55. The method of claim 53, wherein the amino modifier, biotin modifier, or alkyne modification is selected from the group comprising: 5'-C6, 5'-C12, 5'- C6 dT, 5'-Uni-Link™, 5 '-biotin, 5 '-biotin (azide), 5 '-biotin dT, 5'-biotin-TEG, dual 5 '-biotin, 5 '-PC biotin, 5'-desthiobiotin-TEG, 5’ hexynyl and 5-Octadiynyl dU.

56. The method of claim 52, wherein the reverse transcription primer comprises a biotin modifier.

57. The method of claim 56, wherein the biotin modifier comprises 5 '-biotinylation.

58. The method of claim 52, wherein the reverse transcription with the reverse transcription primer forms a reverse transcription product, and the reverse transcription primer comprises a means for purification of the reverse transcription product.

59. The method of claim 58, wherein the means for purification of the reverse transcription product comprises an amino modifier, a biotin modifier, or an alkyne modification.

60. The method of claim 59, wherein the amino modifier, biotin modifier, or alkyne modification is selected from the group comprising: 5'-C6, 5'-C12, 5'- C6 dT, 5'-Uni-Link™, 5 '-biotin, 5 '-biotin (azide), 5 '-biotin dT, 5'-biotin-TEG, dual 5 '-biotin, 5 '-PC biotin, 5'-desthiobiotin-TEG, 5’ hexynyl and 5-Octadiynyl dU.

61. The method of any one of claims 52-60, wherein the reverse transcription primer comprises a unique molecular identifier (UMI).FAZ-4012562. The method of claim 61, wherein the UMI is 8-12 nucleotides in length.

63. The method of any one of claims 52-62, wherein the reverse transcription primer comprises a sequence set forth in SEQ ID NO: 7 or 10.

64. The method of any one of claims 52-63, wherein the method further comprises purifying the reverse transcription product.

65. The method of claim 64, wherein the purification comprises immobilizing the reverse transcription product on a solid surface.

66. The method of claim 65, wherein the solid surface is a magnetic bead or a coated plate.

67. The method of claim 65 or claim 66, wherein the purification method is performed using a biotin- streptavidin pulldown.

68. The method of any one of claims 64-67, wherein the purification results in a composition that is substantially free of unprimed positive strand reverse transcription product relative to a reverse transcription product produced without a purification step.

69. The method of any one of claims 64-68, wherein the purification results in a composition that is enriched with primed negative strand reverse transcription product relative to a reverse transcription product produced without a purification step.

70. The method of any one of claims 50-69, wherein the amplification comprises polymerase chain reaction (PCR) of the reverse transcription product.

71. The method of claim 70, wherein the PCR uses a forward primer comprising the sequence set forth in SEQ ID NO: 8.

72. The method of any one of claims 59-71 , wherein the PCR uses a reverse primer selected from the primers set forth in SEQ ID NOs: 9 and 11.

73. The method of any one of claims 50-72, wherein the sequencing is performed using Next Generation Sequencing.FAZ-4012574. A method of analyzing intracellular delivery of a heterogeneous lipid nanoformulation composition, the method comprising:(i) providing a heterogeneous lipid nanoformulation comprising a plurality of lipid nanoformulations, wherein each lipid nanoformulation encapsulates a positive strand self-amplifying RNA (saRNA), wherein the saRNA comprises a nucleic acid sequence encoding a reporter gene, wherein the reporter gene encodes a protein that produces the detectable signal upon intracellular delivery in a subject, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, wherein the saRNA comprises a viral nsPl, nsP2, nsP3, and nsP4;(ii) administering the heterogeneous lipid nanoformulation composition to the subject;(iii) wherein upon delivery of the saRNA to a cell, the saRNA is translated to form an RNA-dependent RNA polymerase (RdRp);(iv) wherein upon delivery of the saRNA, the RdRp catalyzes the formation of negative and subgenomic saRNAs, and cellular machinery translates the protein that produces the detectable signal from the subgenomic RNA;(v) isolating RNA one or more samples of the subject;(vi) amplifying and sequencing the negative strand saRNA to identify the barcode sequence, thereby analyzing the intracellular delivery of each lipid nanoformulation of the heterogeneous lipid nanoformulation.

75. The method of any one of claims 13-74, wherein the subject is a mammal.

76. The method of any one of claims 13-75, wherein the subject is a non-human mammal.

77. The method of any one of claims 13-76, wherein the subject is a mouse.

78. The method of any one of claims 13-77, wherein the sample comprises a cell lysate, a tissue lysate, serum, plasma, saliva, urine, or a cerebral spinal fluid.

79. The method of any one of claims 13-78, wherein the sample is tissue lysate.FAZ-4012580. The method of any one of claims 13-79, wherein the sample originates from heart, skeletal muscle, liver, lung, brain, kidney, pancreas, spleen, small intestine, colon, rectum, gallbladder, bladder, eye, or skin.

81. The method of any one of claims 14-80, wherein the lipid nanoformulation is administered intravenously, subcutaneously, intramuscularly, or intraperitoneally, to the subject.

82. The method of any one of claims 14-81, wherein the sample is collected from the subject about 1 to about 24 hours after administration of the heterogeneous lipid nanoformulation.

83. The method of any one of claims 14-82, wherein the sample is collected from the subject about 6 hours after administration of the heterogeneous lipid nanoformulation.

84. The method of any one of claims 14-83, wherein about 0.01 mg / kg to about 1 mg / kg of the heterogeneous lipid nanoformulation is administered to the subject.

85. The method of any one of claims 14-83, wherein about 0.1 mg / kg of the lipid heterogeneous nanoformulation is administered to the subject.

86. The method of any one of claims 14-83, wherein about 1 mg / kg of the heterogeneous lipid nanoformulation is administered to the subject.

87. A method of making a heterogeneous lipid nanoformulation, the method comprising encapsulating saRNA in different lipid nanoformulations, wherein the saRNA comprises a barcode sequence for identification of each lipid nanoformulation, and wherein each lipid nanoformulation varies in the molar amount and / or structure of the ionizable lipid, the molar amount and / or structure of the helper lipid, the molar amount / or structure of PEG, and / or the molar amount of cholesterol.

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