Cells and composition of cells comprising nucleic acid construct

Abstract

Provided herein are cells and compositions of cells comprising one or more nucleic acid constructs for use in assays that evaluate the delivery and potency of RNA silencing conjugates in cells.

Description

Attorney Docket No.: 31006_WQCELLS AND COMPOSITION OF CELLS COMPRISING NUCLEIC ACID CONSTRUCTSEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled “31006_WO” created September 14, 2025, and is 20,059 bytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates to genetically engineered cells, composition thereof, and methods of using the same to evaluate RNA silencing conjugates.BACKGROUND

[0003] Genetic diseases are caused by abnormalities in an individual’s DNA. Aberrant gene expression is a common feature of many genetic diseases, where, for example, overexpression of a particular gene may drive the disease progression and / or the disease phenotype. Similarly, mutations in genes may result in the misexpression of a gene, or in the production of nonfunctional proteins, which can contribute to and drive disease. Thus, modalities that restore proper expression of genes, such as the silencing of a particular gene that is overexpressed, are of particular interest and emphasis in the medical community as our understanding of genetic diseases has increased in recent years.

[0004] Gene knockdown is a temporary reduction of expression of a gene within a cell. Gene knockdown can be induced through various mechanisms, including through RNA silencing and antisense oligonucleotides. RNA silencing, also referred to as RNA interference, is a process that regulates gene expression in a cell. Small interfering RNA (siRNA)-mediated RNA interference holds great promise as a strategy for treating a variety of diseases, such as neurodegenerative diseases, cancer, and metabolic diseases. siRNA is currently being developed as a new class of therapeutics to influence gene expression at the messenger RNA levels. Similar to siRNA,Attorney Docket No.: 31006_WO antisense oligonucleotide (ASO) molecules are short, synthetic nucleic acid sequences that bind to a target sequence and subsequently modify expression of the target sequence. Due to the unique pharmacological properties of siRNAs and other nucleic acid-based technologies, like ASOs, achieving efficient and specific delivery of such RNA silencing therapeutics to target specific tissues has been highly challenging. Several clinically proven strategies have been developed to tackle this problem, including conjugation of tissue specific ligands (including N- acetylgalactosamine (GalNAc), lipids, peptides, or antibodies) and the design of novel delivery vehicles.

[0005] Thus, although different RNA silencing mechanisms have been known for at least the past two decades, challenges still exist regarding the identification of efficacious constructs, and the delivery of these constructs into cells. Moreover, determining knockdown of gene transcripts after delivering RNA silencing therapies using traditional RT-PCR or similar methods is cumbersome, time-consuming, expensive, and requires specialized equipment. There is a need for assays that quickly and efficiently measure (i) delivery of an RNA silencing moiety into a cell, and (ii) the potency ( / .e., efficacy) of an RNA silencing moiety to allow for more rapid screening of candidate molecules.SUMMARY OF INVENTION

[0006] The present disclosure provides a cell for measuring potency of or efficiency of delivering into the cell an RNA silencing conjugate comprising a celltargeting moiety and an RNA silencing moiety, the cell comprising a nucleic acid comprising a promoter operably linked to a sequence encoding a reporter gene and a target sequence complementary to the RNA silencing moiety, wherein the cell expresses a transmembrane receptor capable of binding the cell-targeting moiety. In some embodiments, the cell-targeting moiety is a protein, a peptide, a carbohydrate, a small molecule, or a lipid. In some embodiments, the RNA silencing moiety is a siRNA, an ASO, a miRNA, or a piRNA. In some embodiments, the reporter gene is a photoprotein or a fluorescent protein. In some embodiments, the reporter gene is luciferase. In some embodiments, the transmembrane receptor is human transferrin receptor 1 (TfR), natriuretic peptide receptor C (NPR-C), CD19, or CD40. In some embodiments, the cell has been engineered to overexpress the transmembraneAttorney Docket No.: 31006_WO receptor. In some embodiments, the transmembrane receptor is human transferrin receptor 1 (TfR). In some embodiments, the nucleic acid further comprises a polyA signal. In some embodiments, the nucleic acid comprises, in 5' to 3' orientation, (a) a promoter, (b) a sequence encoding luciferase, (c) a target sequence complementary to the RNA silencing moiety, and (d) a polyA signal. In some embodiments, the promoter is a CMV promoter. In some embodiments, the CMV promoter comprises SEQ ID NO: 1. In some embodiments, the promoter is an inducible promoter, e.g., a cumate-inducible promoter. In some embodiments, the promoter is a cumate-inducible promoter that comprises a mutated CMV promoter and the cumate operator sequence. In some embodiments, the inducible promoter comprises SEQ ID NO: 10. In some embodiments, the polyA signal is a SV40 polyA signal. In some embodiments, the polyA signal comprises SEQ ID NO: 5.

[0007] In some embodiments, the target sequence is 10-5000 nucleotides in length. In some embodiments, the target sequence is 15-5000 nucleotides in length.In some embodiments, the target sequence is 18-4000 nucleotides in length. In some embodiments, the target sequence is 20-4000 nucleotides in length. In some embodiments, the target sequence is 100-3500 nucleotides in length. In some embodiments, the target sequence is 119-3500 nucleotides in length. In some embodiments, the target sequence is a portion of an alpha synuclein (SNCA), microtubule-associated protein tau (MAPT), amyloid-beta precursor protein (APP), sterile alpha and TIR motif containing 1 (SARM1 ), superoxide dismutase 1 (SOD1 ), ataxin 2 (ATXN2), ataxin 3 (ATXN3), apolipoprotein E (APOE), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1 ), fragile X messenger ribonucleoprotein 1 (FMR1 ), leucine-rich repeat kinase 2 (LRRK2), huntingtin (HTT), sodium voltagegated channel alpha subunit 10 (SCN10A), sodium voltage-gated channel alpha subunit 9 (SCN9A), calcium voltage-gated channel subunit alpha 1 B (CACNA1 B), prion protein (PRNP), phosphoinositide kinase, activin A receptor type 2A (ACVR2a), activin A receptor type 2A (ACVR2b), perilipin-1 (PLIN1 ), or hypoxanthine phosphoribosyltransferase 1 (HPRT) gene sequence. In some embodiments, the target sequence is a portion of the SNCA gene sequence. In some embodiments, the target sequence comprises SEQ ID NO: 6. In some embodiments, the target sequence is a portion of the MAPT gene sequence. In some embodiments, the target sequence comprises SEQ ID NO: 7. In some embodiments, the target sequence is a portion ofAttorney Docket No.: 31006_WQ the APP gene sequence. In some embodiments, the target sequence comprises SEQ ID NO: 8. In some embodiments, the target sequence is a portion of the Navi .8 gene sequence. In some embodiments, the target sequence comprises SEQ ID NO: 9. In some embodiments, the target sequence is a portion of the PLIN1 gene sequence. In some embodiments, the target sequence comprises SEQ ID NO: 11. In some embodiments, the sequence encoding luciferase comprises SEQ ID NO: 2, 3, or 4. In some embodiments, the nucleic acid is a plasmid.

[0008] In some embodiments, the cell further comprises a second nucleic acid comprising a second promoter operably linked to a second sequence encoding a second reporter gene and a second target sequence. In some embodiments, the second nucleic acid is a plasmid. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a CHO, EFO21 , HEK293, or 3T3 cell. In some embodiments, the present disclosure provides a composition comprising a plurality of the cells as described herein.

[0009] The present disclosure also provides a method of measuring potency or efficiency of delivering into a cell an RNA silencing conjugate comprising a celltargeting moiety and an RNA silencing moiety, the method comprising: contacting the cell or the composition of cells described herein with the RNA silencing conjugate to form a mixture; incubating the mixture for a period of time; and measuring expression of the reporter gene in the cell after the incubating.

[0010] The present disclosure further provides a method of screening RNA silencing conjugates comprising a cell-targeting moiety and an RNA silencing moiety, the method comprising: contacting the cell or the composition of cells as described herein with a first RNA silencing conjugate to form a first mixture; contacting the cell or the composition of cells as described herein with a second RNA silencing conjugate to form a second mixture; incubating the first and second mixtures for a period of time; measuring luciferase expression in the first and second mixtures after the incubating; and comparing expression of the reporter gene in the first mixture with expression of the reporter gene in the second mixture. In some embodiments, the method further comprises selecting the RNA silencing conjugate achieving lower reporter gene expression.

[0011] In some embodiments the period of time for incubation is up to 10 days. In some embodiments, the period of time for incubation is 15 minutes to 10 days. InAttorney Docket No.: 31006_WQ some embodiments, the period of time for incubation is 3 days to 7 days. In some embodiments, the period of time for incubation is 6 days. In some embodiments, the mixture comprises 2,000 cells to 100,000 cells. In some embodiments, the mixture comprises 10,000 cells. In some embodiments, the mixture further comprises a cell culture media. In some embodiments, the cell culture media comprises 10% FBS.

[0012] The present disclosure also provides a cell for measuring potency or efficiency of delivering into the cell an RNA silencing conjugate comprising a celltargeting moiety and an RNA silencing moiety, the cell comprising a nucleic acid, the nucleic acid comprising, in 5' to 3' orientation, (a) a promoter comprising SEQ ID NO: 1 or 10; (b) a sequence encoding luciferase comprising SEQ ID NO: 2, 3, or 4; (c) a target sequence comprising SEQ ID NO: 6, 7, 8, 9 or 11 ; and (d) a polyA signal comprising SEQ ID NO: 5, wherein the cell expresses a transmembrane receptor that is human transferrin receptor 1 (TfR), natriuretic peptide receptor C (NPR-C), CD19, or CD40, and wherein the RNA silencing moiety is complementary to the target sequence.BRIEF DESCRIPTION OF THE FIGURES

[0013] Figures 1A-1 E depict the plasmids that were used in the Examples to assess gene knockdown efficiency for MAPT (FIG. 1 A), SNCA (FIG. 1 B), Navi .8 (FIG. 1 C), APP (FIG. 1 D) and PLIN1 (FIG. 1 E).

[0014] Figure 2A shows the cell-based assay utilizes luciferase as a surrogate marker. In the absence of antibody siRNA conjugate (ARC), high luciferase activity is observed, whereas the addition of ARC results in reduced luciferase activity. Figure 2B illustrates the benefits and improvements of an inducible promoter system. S / N means signal to noise ratio. Figure 2C illustrates the cumate-inducible system. Without cumate, the repressor binds to the CuO sequence, inhibiting the promoter and preventing gene expression. In the presence of cumate, cumate binds to the repressor, causing its release from the CuO sequence, thereby allowing gene expression.

[0015] Figures 3A and 3B illustrate a cumate-inducible cell line using the Piggybac transposon plasmid and transposase.

[0016] Figure 4A shows cumate successfully induces GFP expression in a stable cumate-inducible GFP cell line. Figure 4B shows cumate successfully inducesAttorney Docket No.: 31006_WOLuciferase expression in a stable cumate-inducible luciferase cell line. Figure 4C shows the cumate-inducible luciferase system is titratable, with different cumate doses generating varying signal levels. The heatmap graph shows signal intensity from low to high, color from black to white. Figure 4D shows the cumate-inducible luciferase system is reversible. Figure 4E shows identification of optimal cumate concentration in a 3-day assay.

[0017] Figure 5A shows the workflow of the cell-based assay using a cumate- inducible system. Figure 5B shows the time-course results based on the workflow in Figure 5A.DETAILED DESCRIPTION

[0018] As used herein, the term “antibody” refers to a molecule that binds to an antigen through amino acid sequences called complementarity determining regions (CDRs). Embodiments of an antibody include, but are not limited to, a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, heterodimeric antibody, bispecific or multispecific antibody, conjugated antibody, a variable new antigen receptor (VNAR), a camelid antibody, and other antibody formats, including non-traditional antibody formats. The antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA), and any subclass (e.g., lgG1 , lgG2, lgG3, lgG4).

[0019] As used herein, the term “antibody fragment” refers to a molecule that comprises at least a portion of an antibody retaining the ability to bind to an antigen or an epitope of an antigen. Embodiments of an antibody fragment include, but are not limited to, a Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fvs (sdFv), and a Fd fragment.

[0020] As used herein, “antisense” refers to a nucleic acid sequence that is capable of binding to a region of a target nucleic acid sequence through complementary base pairing.

[0021] As used herein, the term “antisense oligonucleotide” or“ASO” refers to single-stranded nucleic acid sequences that are capable of binding to a target mRNA. Once bound to a target mRNA, expression of the mRNA is altered through a variety of mechanisms, including ribonuclease H-mediated degradation, steric blockages, and splicing site binding on pre-mRNA.Attorney Docket No.: 31006_WO

[0022] As used herein, the term “complementary” refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acid strands, or on opposing regions of a single nucleic acid strand such as a hairpin) that permits the two nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid strand that is complementary to a pyrimidine nucleotide of a different nucleic acid strand may base pair together by forming hydrogen bonds with one another. Complementary nucleotides may base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. Likewise, two nucleic acid sequences may have regions of nucleotides that are complementary with each other that allow for the binding of the two nucleic acid sequences, as described herein.

[0023] As used herein, the term “detectable signal” refers to a signal that is capable of being measured over the background noise of a detection system. For example, luciferase produces a detectable signal in the form of visible light when it emits a photon during the oxidation of its substrate. As a further example, a fluorescent protein produces a detectable signal in the form of fluorescent light when illuminated with light of a certain wavelength (e.g., ultraviolet light).

[0024] As used herein, the term “duplex” refers to a structure formed through complementary base pairing of two antiparallel sequences of nucleotides ( / .e., in opposite directions).

[0025] As used herein, the term “enhancer” or “enhancer element” refers to a cis-acting transcriptional regulatory element, i.e. , cis-element, which confers an aspect of the overall expression pattern, but is usually insufficient alone to drive transcription, of an operably linked nucleic acid sequence. Unlike promoters, enhancer elements do not usually include a transcription start site (TSS) or TATA box or an equivalent sequence. A promoter may naturally comprise one or more enhancer elements that affect the transcription of an operably linked nucleic acid sequence. An isolated enhancer element may also be fused to a promoter. A promoter may comprise one or more enhancer elements that affect the transcription of operably linked nucleic acid molecules. Enhancers may be positioned upstream or downstream of an operably linked nucleic acid molecule.Attorney Docket No.: 31006_WO

[0026] As used herein, an “inducible promoter” is a promoter which initiates transcription only when the cell is exposed to some particular external stimulus, as distinguished from constitutive promoters.

[0027] As used herein, the term “knockdown” or “expression knockdown” refers to reduced mRNA or protein expression of a gene after treatment of a reagent.

[0028] As used herein, the term “miRNA” refers to microRNA. miRNA bind to and degrades a target sequence through a well-characterized process that involves Dicer and the RNA-lnduced Silencing Complex (RISC). miRNA are small, singlestranded RNA sequences that regulate gene expression by either (i) cleaving the target mRNA, (ii) destabilizing mRNA by shortening the polyA tail, or (iii) inhibiting translation of the mRNA into protein.

[0029] As used herein, “modified internucleotide linkage” refers to an internucleotide linkage having one or more chemical modifications when compared with a reference internucleotide linkage having a phosphodiester bond. A modified internucleotide linkage can be a non-naturally occurring linkage. In some embodiments, the modified internucleotide linkage is phosphorothioate linkage.

[0030] As used herein, “modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide, including, but not limited to: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide can have, for example, one or more chemical modification(s) in its sugar, nucleobase, and / or phosphate group(s). Additionally, or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to a corresponding reference nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl modified nucleotide. In some embodiments, the modified nucleotide has a phosphate analog, e.g., 5’-vinylphosphonate. In some embodiments, the modified nucleotide has an abasic moiety or an inverted abasic moiety.

[0031] As used herein, “nucleic acid” refers to a polymeric compound including covalently linked nucleotides comprising natural subunits (e.g., purine or pyrimidine bases) or modified subunits. In some embodiments, a nucleic acid molecule is aAttorney Docket No.: 31006_WO polynucleotide molecule. Nucleic acid molecules include ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).

[0032] As used herein, the term “nucleotide” means an organic compound having a nucleoside (a nucleobase, e.g., adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar, e.g., ribose or 2'-deoxyribose) linked to a phosphate group. A nucleotide can serve as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0033] As used herein, the term “operably linked” refers to a first molecule joined to a second molecule, wherein the molecules are so arranged that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, in some embodiments, a promoter is operably linked to a nucleic acid molecule when the promoter is capable of modulating transcription of the nucleic acid molecule in a cell.

[0034] As used herein, the term “photoprotein” refers to a protein that produces light when exposed to oxygen, hydrogen peroxide, or another oxidizing agent.

[0035] As used herein, the term “piRNA” refers to piwi-interacting RNA. piRNA are small, single-stranded RNA sequences that regulate gene expression, including transposons, by forming a complex with RISC and binding to a target sequence.

[0036] As used herein, the term “potency” refers to the ability of an RNA silencing moiety to exert its function on a target. In some embodiments, for example, the potency of an siRNA refers to the ability of a siRNA construct to silence its target gene (i.e. , how effective the siRNA is at silencing the target mRNA).

[0037] As used herein, the term “promoter” refers generally to a DNA molecule that is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

[0038] As used herein, the term “protein” refers to a polymer of amino acid residues. The term applies to polymers comprising naturally occurring amino acids and polymers comprising one or more non-naturally occurring amino acids. Proteins include, but are not limited to, antibodies, hormones, neurotransmitters, growth factors, polypeptides, viral proteins, and receptor ligands.Attorney Docket No.: 31006_WO

[0039] As used herein, the term “siRNA” refers to small interfering RNA. siRNA are double-stranded RNA that regulate gene expression by binding to and subsequently degrading a specific messenger RNA (mRNA) sequence. siRNA bind to and degrade a target sequence through a well-characterized process that involves Dicer and RISC. Briefly, once an siRNA enters a cell and has been processed by Dicer, it forms a complex with RISC, the siRNA binds to a target sequence (e.g., mRNA), and the RISC complex then induces cleavage of the target sequence. The cleaved target sequence is then degraded by the cell, thereby inhibiting translation of the target sequence into a protein.

[0040] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and / or “consisting essentially of” are also provided. In this disclosure, “comprises,” “comprising,” “containing,” and “having” and the like can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics as described herein of that which is recited are not changed by the presence of more than that which is recited, but excludes prior art aspects.

[0041] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.RNA Silencing Conjugate

[0042] In some embodiments, the present disclosure provides an RNA silencing conjugate, comprising a cell targeting moiety and an RNA silencing moiety. In some embodiments, the RNA silencing conjugate is used to deliver an RNA silencing moiety into a cell. In some embodiments, the cell-targeting moiety is conjugated to the RNA silencing moiety in a manner such that the cell-targeting moiety facilities entry of the RNA silencing moiety into a cell. In some embodiments, once in a cell, the RNA silencing moiety binds to a target sequence, silencing the target sequence and anything that may be linked to the target sequence, such as the reporter gene as described herein.

[0043] In some embodiments, the RNA silencing moiety is a siRNA, miRNA, piRNA, or ASO. In some embodiments, the RNA silencing moiety is a small interfering RNA (siRNA). In some embodiments, the RNA silencing moiety is a micro RNA (miRNA). In some embodiments, the RNA silencing moiety is a piwi-interacting RNAAttorney Docket No.: 31006_WO(piRNA). In some embodiments, the RNA silencing moiety is an antisense oligonucleotide (ASO).

[0044] In some embodiments, the RNA silencing moiety comprises natural nucleotides. In some embodiments, the RNA silencing moiety comprises modified nucleotides. In some embodiments, the modified nucleic acid molecules improve delivery to tissues and / or prolong the half-life of the molecule. In some embodiments, the RNA silencing moiety comprises modified internucleotide linkages. In some embodiments, the modified internucleotide linkages improve delivery to issues and / or prolong the half-life of the molecule.

[0045] In some embodiments, the cell targeting moiety comprises a protein, a peptide, a carbohydrate, a small molecule or a lipid. In some embodiments, the cell targeting moiety recognizes and binds to a target on a cell surface, such that it facilitates delivery of the RNA silencing moiety into a cell of interest. The target for the cell targeting moiety may include any molecule that is present on the surface of the cell. In some embodiments, the cell targeting moiety facilitates delivery of the RNA silencing moiety through endocytosis, which includes but is not limited to, phagocytosis, pinocytosis, lipid-mediated endocytosis, lipid rafts, Mincle-mediated endocytosis, and receptor-mediated endocytosis. In some embodiments, the cell targeting moiety is a protein, a carbohydrate, or a lipid. In some embodiments, the cell targeting moiety is a protein. In some embodiments, the protein is an antibody, a hormone, a neurotransmitter, a growth factor, a polypeptide. In some embodiments, the cell targeting moiety is a peptide. In some embodiments, the cell targeting moiety is a small molecule. In some embodiments, the cell targeting moiety is a carbohydrate. In some embodiments, the cell targeting moiety is a lipid.

[0046] Exemplary RNA silencing conjugates include any of the molecules described herein, and additional RNA silencing conjugates as described, for example, in PCT / US2023 / 071755, filed August 7, 2023, the contents of which are incorporated by reference herein in its entirety.Nucleic acid

[0047] In some embodiments, the present disclosure provides a nucleic acid encoding a reporter gene that provides a detectable signal. In some embodiments, the reporter gene is a photoprotein. In some embodiments, the photoprotein is luciferase. In some embodiments, the reporter gene encodes luciferase and comprises SEQ IDAttorney Docket No.: 31006_WQNO: 2, 3, or 4. In some embodiments, the reporter gene encodes luciferase and comprises SEQ ID NO: 2. In some embodiments, the reporter gene encodes luciferase and comprises SEQ ID NO: 3. In some embodiments, the reporter gene encodes luciferase and comprises SEQ ID NO: 4. Luciferase is a protein that has enzymatic activity, and when luciferase is in the presence of adenosine triphosphate (ATP), oxygen, and a substrate, which includes, but is not limited to, luciferin, the substrate is oxidized and a photon is emitted. Other photoproteins that can be encoded by the nucleic acid include, but are not limited to, Renilla luciferase, pholasin, TurboLuc, aequorin, and berovin. In some embodiments, the reporter gene is a fluorescent protein. Fluorescent proteins that can be encoded by the nucleic acid include, but are not limited to, green fluorescent protein (GFP), yellow fluorescent protein (YFP), mCherry, and DsRed.

[0048] In some embodiments, the nucleic acid further encodes a promoter that is operably connected to the reporter gene, such that the promoter drives expression of the reporter gene. In some embodiments, the promoter is positioned upstream of the reporter gene. Suitable promoters include, but are not limited to, a CMV promoter, a EF1 a promoter, an SV40 promoter, a CAG promoter, and a PGK promoter. In some embodiments, the promoter is a CMV promoter comprising SEQ ID NO: 1. In some embodiments, the promoter is an inducible promoter, e.g., a cumate-inducible promoter. In some embodiments, the promoter is a cumate-inducible promoter that comprises a mutated CMV promoter and the cumate operator sequence. In some embodiments, the inducible promoter comprises SEQ ID NO: 10. Other examples of suitable inducible promoters include, but are not limited to, tetracycline-regulated promoter, steroid-regulated promoter (e.g., estrogen receptor-regulated promoter), metal ion-regulated promoter (e.g., promoters derived from metallothionein), temperature / heat-inducible promoters (e.g., heat shock promoters).

[0049] The promoters described herein may be operably linked to one or more additional regulatory elements described herein or known in the art. In some embodiments, the nucleic acid further comprises an enhancer. In some embodiments,Attorney Docket No.: 31006_WO the enhancer is positioned upstream of the reporter gene. In some embodiments, the enhancer is positioned downstream of the reporter gene. In some embodiments, the enhancer is an untranslated region (UTR). In some embodiments, a promoter as described herein, is operably linked to a 5' UTR. In some embodiments, the nucleic acid further comprises a 3' UTR.

[0050] In some embodiments, the nucleic acid further comprises a nucleic acid sequence encoding a target sequence. In some embodiments, the target sequence is connected to the reporter gene by way of a linker. It is understood than any suitable linker for connecting two nucleic acid sequences can be used to link the reporter gene to the target sequence. In some embodiments, the target sequence is positioned downstream of the reporter gene. In some embodiments, the target sequence is complementary to an RNA silencing moiety, such that the RNA silencing moiety is capable of binding to and / or hybridizing with the target sequence. In some embodiments, the target sequence is complementary to a siRNA. In some embodiments, the target sequence is complementary to a miRNA. In some embodiments, the target sequence is complementary to a piRNA. In some embodiments, the target sequence is complementary to an ASO.

[0051] In some embodiments, the target sequence comprises a portion of a nucleic acid sequence of interest. It is appreciated that the target sequence may comprise any nucleic acid sequence that is capable of binding to an RNA silencing moiety as described herein. For example, it is appreciated that the target sequence may comprise a portion of any mRNA that has been identified for RNA silencing. In some embodiments, the target sequence includes, but is not limited to, a portion of an alpha synuclein (SNCA), microtubule-associated protein tau (MAPT), amyloid-beta precursor protein (APP), sterile alpha and TIR motif containing 1 (SARM1 ), superoxide dismutase 1 (SOD1 ), ataxin 2 (ATXN2), ataxin 3 (ATXN3), apolipoprotein E (APOE), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1 ), fragile X messenger ribonucleoprotein 1 (FMR1 ), leucine-rich repeat kinase 2 (LRRK2), huntingtin (HTT), sodium voltage-gated channel alpha subunit 10 (SCN10A), sodium voltage-gated channel alpha subunit 9 (SCN9A), calcium voltage-gated channel subunit alpha 1 B (CACNA1 B), prion protein (PRNP), phosphoinositide kinase, activin A receptor type 2A (ACVR2a), activin A receptor type 2A (ACVR2b), or hypoxanthine phosphoribosyltransferase 1 (HPRT) transcript.Attorney Docket No.: 31006_WQ

[0052] In some embodiments, the target sequence comprises a portion of the SNCA gene sequence. In some embodiments, the target sequence comprises SEQ ID NO: 6. In some embodiments, the target sequence comprises a portion of the MAPT gene sequence. In some embodiments, the target sequence comprises SEQ ID NO: 7. In some embodiments, the target sequence comprises a portion of the APP gene sequence. In some embodiments, the target sequence comprises SEQ ID NO: 8. In some embodiments, the target sequence comprises a portion of the Nav1.8 gene sequence. In some embodiments, the target sequence comprises SEQ ID NO: 9.

[0053] In some embodiments, the target sequence is 10 base pairs to 5000 base pairs. In some embodiments, the target sequence is 15 base pairs to 5000 base pairs in length. In some embodiments, the target sequence is 18 base pairs to 4000 base pairs in length. In some embodiments, the target sequence is 20 base pairs to 4000 base pairs in length. In some embodiments, the target sequence is 50 base pairs to 4000 base pairs in length. In some embodiments, the target sequence is 100 base pairs to 3500 base pairs in length. In some embodiments, the target sequence is 119 base pairs to 3500 base pairs in length. In some embodiments, the target sequence is 150 base pairs to 3000 base pairs in length. In some embodiments, the target sequence is 200 base pairs to 2500 base pairs in length. In some embodiments, the target sequence is 300 base pairs to 2000 base pairs in length. In some embodiments, the target sequence is 350 base pairs to 1500 base pairs in length. In some embodiments, the target sequence is 375 base pairs to 1250 base pairs in length. In some embodiments, the target sequence is 450 base pairs to 1000 base pairs in length. In some embodiments, the target sequence is 500 base pairs to 750 base pairs in length.

[0054] In some embodiments, the target sequence is 10 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 10 base pairs to 190 base pairs in length. In some embodiments, the target sequence is 10 base pairs to 180 base pairs in length. In some embodiments, the target sequence is 10 base pairs to 170 base pairs in length. In some embodiments, the target sequence is 10 base pairs to 160 base pairs in length. In some embodiments, the target sequence is 10 base pairs to 150 base pairs in length. In some embodiments, the target sequence is 10 base pairs to 140 base pairs in length. In some embodiments, the target sequence is 10 base pairs to 130 base pairs in length. In some embodiments, theAttorney Docket No.: 31006_WO target sequence is 10 base pairs to 120 base pairs in length. In some embodiments, the target sequence is 10 base pairs to 110 base pairs in length. In some embodiments, the target sequence is 10 base pairs to 100 base pairs in length. In some embodiments, the target sequence is 10 base pairs to 90 base pairs in length. In some embodiments, the target sequence is 10 base pairs to 80 base pairs in length.In some embodiments, the target sequence is 10 base pairs to 70 base pairs in length.In some embodiments, the target sequence is 10 base pairs to 60 base pairs in length.In some embodiments, the target sequence is 10 base pairs to 50 base pairs in length.In some embodiments, the target sequence is 10 base pairs to 40 base pairs in length.In some embodiments, the target sequence is 10 base pairs to 30 base pairs in length.In some embodiments, the target sequence is 10 base pairs to 20 base pairs in length.In some embodiments, the target sequence is 20 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 30 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 40 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 50 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 60 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 70 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 80 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 90 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 100 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 120 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 130 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 140 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 150 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 160 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 170 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 180 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 190 base pairs to 200 base pairs in length. In some embodiments, the target sequence is 20 base pairs to 190 base pairs in length. In some embodiments, the target sequence is 30 base pairs to 180 base pairs in length. In some embodiments, the target sequence is 40 base pairs to 170 base pairs in length. In some embodiments, the target sequence is 50 base pairsAttorney Docket No.: 31006_WO to 160 base pairs in length. In some embodiments, the target sequence is 60 base pairs to 150 base pairs in length. In some embodiments, the target sequence is 70 base pairs to 140 base pairs in length. In some embodiments, the target sequence is 80 base pairs to 130 base pairs in length. In some embodiments, the target sequence is 90 base pairs to 120 base pairs in length. In some embodiments, the target sequence is 100 base pairs to 110 base pairs in length.

[0055] In some embodiments, the target sequence comprises a flanking sequence. In some embodiments, the target sequence comprises a flanking sequence that is positioned upstream of the target sequence. In some embodiments, the target sequence comprises a flanking sequence that is positioned downstream of the target sequence. In some embodiments, the target sequence comprises a flanking sequence that is positioned upstream of the target sequence and a flanking sequence that is positioned downstream of the target sequence..

[0056] In some embodiments the flanking sequence is 10 base pairs to 100 base pairs in length. In some embodiments the flanking sequence is 20 base pairs to 100 base pairs in length. In some embodiments the flanking sequence is 30 base pairs to 100 base pairs in length. In some embodiments the flanking sequence is 40 base pairs to 100 base pairs in length. In some embodiments the flanking sequence is 50 base pairs to 100 base pairs in length. In some embodiments the flanking sequence is 60 base pairs to 100 base pairs in length. In some embodiments the flanking sequence is 70 base pairs to 100 base pairs in length. In some embodiments the flanking sequence is 80 base pairs to 100 base pairs in length. In some embodiments the flanking sequence is 90 base pairs to 100 base pairs in length. In some embodiments the flanking sequence is 10 base pairs to 90 base pairs in length. In some embodiments the flanking sequence is 10 base pairs to 80 base pairs in length. In some embodiments the flanking sequence is 10 base pairs to 70 base pairs in length. In some embodiments the flanking sequence is 10 base pairs to 60 base pairs in length. In some embodiments the flanking sequence is 10 base pairs to 50 base pairs in length. In some embodiments the flanking sequence is 10 base pairs to 40 base pairs in length. In some embodiments the flanking sequence is 10 base pairs to 30 base pairs in length. In some embodiments the flanking sequence is 10 base pairs to 20 base pairs in length. In some embodiments the flanking sequence is 20 base pairs to 90 base pairs in length. In some embodiments the flanking sequence isAttorney Docket No.: 31006_WQ30 base pairs to 80 base pairs in length. In some embodiments the flanking sequence is 40 base pairs to 70 base pairs in length. In some embodiments the flanking sequence is 50 base pairs to 60 base pairs in length. In some embodiments the flanking sequence is 10 base pairs in length. In some embodiments the flanking sequence is 10 base pairs in length. In some embodiments the flanking sequence is 15 base pairs in length. In some embodiments the flanking sequence is 20 base pairs in length. In some embodiments the flanking sequence is 25 base pairs in length. In some embodiments the flanking sequence is 30 base pairs in length. In some embodiments the flanking sequence is 35 base pairs in length. In some embodiments the flanking sequence is 40 base pairs in length. In some embodiments the flanking sequence is 45 base pairs in length. In some embodiments the flanking sequence is 50 base pairs in length. In some embodiments the flanking sequence is 55 base pairs in length. In some embodiments the flanking sequence is 60 base pairs in length. In some embodiments the flanking sequence is 65 base pairs in length. In some embodiments the flanking sequence is 70 base pairs in length. In some embodiments the flanking sequence is 75 base pairs in length. In some embodiments the flanking sequence is 80 base pairs in length. In some embodiments the flanking sequence is 85 base pairs in length. In some embodiments the flanking sequence is 90 base pairs in length. In some embodiments the flanking sequence is 95 base pairs in length. In some embodiments the flanking sequence is 100 base pairs in length.

[0057] In some embodiments, the nucleic acid further comprises a sequence encoding a polyA signal. In some embodiments, the polyA signal includes, but is not limited to, a hGH polyA signal, a BGH polyA signal, a rbGlob polyA signal, and a SV40 polyA signal. In some embodiments, the polyA signal is a SV40 polyA signal comprising SEQ ID NO: 5.

[0058] In some embodiments, the nucleic acid comprises, in 5' to 3' orientation: a promoter; a sequence encoding a reporter gene; a target sequence complementary to an RNA silencing moiety; and a polyA signal. In some embodiments, the nucleic acid comprises, in 5' to 3' orientation: a promoter; a sequence encoding luciferase; a target sequence complementary to an RNA silencing moiety; and a polyA signal. In some embodiments, the nucleic acid comprises, in 5' to 3' orientation: a promoter comprising SEQ ID NO: 1 or 10; a sequence encoding luciferase comprising SEQ ID NO: 2, 3, or 4; a target sequence comprising SEQ IDAttorney Docket No.: 31006_WONO: 6, 7, 8, 9 or 11 that is complementary to an RNA silencing moiety; and a polyA signal comprising SEQ ID NO: 5. In some embodiments, the nucleic acid is a plasmid. Cells

[0059] In some embodiments, the present disclosure provides a cell comprising a nucleic acid as described herein, wherein the cell expresses a transmembrane receptor capable of binding a cell-targeting moiety as described herein. The nucleic acid described herein can be delivered to the cell by any suitable means, including but not limited to, transfection, transduction, and electroporation. In some embodiments, the cell comprises more than one nucleic acid encoding a reporter gene and a target sequence. In some embodiments, the cell expresses second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more nucleic acids encoding a reporter gene and a target sequence. A cell comprising more than one nucleic acid as described herein can be used to assay more than one different RNA silencing conjugate, and can provide for the testing of potential therapeutics that incorporate more than one RNA silencing moiety.

[0060] In some embodiments, the cell overexpresses a transmembrane receptor capable of binding a cell-targeting moiety as described herein. In some embodiments, overexpression of a transmembrane receptor is induced by transfection of a cell with an expression plasmid construct that comprises a nucleic acid sequence encoding the transmembrane receptor. In some embodiments, the nucleic acid sequence encoding the transmembrane receptor is operably linked to a promoter and / or enhancers that drive constitutive expression and / or overexpression of the nucleic acid sequence encoding the transmembrane receptor. It is appreciated that any transmembrane receptor capable of binding a cell-targeting moiety may be used in the embodiments described herein. In some embodiments, transmembrane receptors include, but are not limited to, G protein-coupled receptors (GPCRs), ion channel receptors, hormone receptors, and enzyme-linked receptors. In some embodiments, the transmembrane receptor is a transferrin receptor (TfR).

[0061] It is appreciated that any cell type capable of being transfected and / or transduced with a nucleic acid as described herein may be used in the methods described herein. In some embodiments, the cell is a primary cell. In some embodiments, the cell is from a cell line. In some embodiments, the cell is anAttorney Docket No.: 31006_WO immortalized cell. In some embodiments, the cell is a mammalian cell. In some embodiments, suitable cells include, but are not limited to, IMR-32, HeLa, and CHO.

[0062] In some embodiments, the present disclosure provides a composition of the cells as described herein, such that the composition comprises a plurality of the cells. In some embodiments, the composition of cells comprises at least 2,000 cells, at least 5,000 cells, at least 10,000 cells, at least 20,000 cells, at least 30,000 cells, at least 40,000 cells, at least 50,000 cells, at least 60,000 cells, at least 70,000 cells, at least 80,000 cells, at least 90,000 cells, or at least 100,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 3,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 4,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 5,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 6,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 7,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 8,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 9,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 10,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 20,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 30,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 40,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 50,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 60,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 70,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 80,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 90,000 cells to 100,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 90,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 80,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 70,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 60,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 50,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 40,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 30,000 cells. In some embodiments, the compositionAttorney Docket No.: 31006_WO of cells comprises 2,000 cells to 20,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 10,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 9,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 8,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 7,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 6,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 5,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 4,000 cells. In some embodiments, the composition of cells comprises 2,000 cells to 3,000 cells.

[0063] In some embodiments, the cell or composition of cells described herein can be stored for later use. Such storage typically consists of cryopreservation at - 70°C to approximately -90°C, as well as liquid nitrogen storage. In some embodiments, the cell or composition of cells described herein is provided in a kit for testing the potency and / or efficacy of an RNA silencing conjugate.Methods of Using Cells

[0064] In some embodiments, the present disclosure provides methods of using the cell or composition of cells described herein in an assay. In some embodiments, the cell or composition of cells is used to assess the delivery rate of an RNA silencing conjugate as described herein ( / .e., comprising a cell targeting moiety and an RNA silencing moiety) into a cell and / or the potency of an RNA silencing conjugate. In some embodiments, the cell comprises a nucleic acid comprising a promoter operably linked to a sequence encoding a reporter gene and a target sequence complementary to the RNA silencing moiety, and the cell expresses a transmembrane receptor capable of binding the cell-targeting moiety. In some embodiments, the cell or composition of cells is contacted with the RNA silencing conjugate to form a mixture, and the mixture is incubated. In some embodiments, the mixture is incubated for a period of time from less than 1 day to 10 days. After the incubation period, reporter gene expression in the mixture is measured using any known technique for measuring reporter gene expression. It is appreciated that if a different reporter gene is used in the methods, the appropriate techniques for measuring expression of the reporter gene will be used. The efficiency of delivery into a cell, and / or the potency of an RNA silencing conjugate can be assessed by comparing the level of reporter gene expression (e.g., comparing reporter geneAttorney Docket No.: 31006_WO expression for cells contacted with an RNA silencing conjugate relative to control cells that were not contacted with an RNA silencing conjugate). In some embodiments, the cell or composition of cells is used to screen RNA silencing conjugates, in that the effect of one RNA silencing conjugate is compared to the effect of another, where the RNA silencing conjugate with the greatest knockdown effect may be desirable as a more efficacious construct.

[0065] In some embodiments, the present disclosure provides methods of using the cell or composition of cells described herein in an assay. In some embodiments, the cell or composition of cells is used to assess the delivery rate of an RNA silencing moiety into a cell and / or the potency of an RNA silencing moiety. In some embodiments, the cell comprises a nucleic acid comprising a promoter operably linked to a sequence encoding a reporter gene and a target sequence complementary to the RNA silencing moiety, and the cell expresses a transmembrane receptor capable of binding the cell-targeting moiety. In some embodiments, the cell or composition of cells is contacted with the RNA silencing moiety to form a mixture, and the mixture is incubated. In some embodiments, the RNA silencing moiety is delivered into the cell by any suitable method, including those described herein such as electroporation, transfection, and transduction. In some embodiments, the mixture is incubated for a period of time from less than 1 day to 10 days. After the incubation period, reporter gene expression in the mixture is measured using any known technique for measuring reporter gene expression. It is appreciated that if a different reporter gene is used in the methods, the appropriate techniques for measuring expression of the reporter gene will be used. The efficiency of delivery into a cell, and / or the potency of an RNA silencing moiety can be assessed by comparing the level of reporter gene expression (e.g., comparing reporter gene expression for cells contacted with an RNA silencing moiety relative to control cells that were not contacted with an RNA silencing moiety). In some embodiments, the cell or composition of cells is used to screen RNA silencing moieties, in that the effect of one RNA silencing moiety is compared to the effect of another, where the RNA silencing moiety with the greatest knockdown effect may be desirable as a more efficacious construct.

[0066] In some embodiments, the mixture of a cell or composition of cells and an RNA silencing conjugate as described herein comprises cell culture media that is suitable for the culturing of the cell or composition of cells. In some embodiments, theAttorney Docket No.: 31006_WO cell culture media includes suitable supplements (e.gr, 10% FBS). In some embodiments, the mixture of a cell or composition of cells and an RNA silencing conjugate as described herein is incubated for a period of time from less than 1 day to 10 days. In some embodiments, the mixture is incubated for a period of time from less than 1 day to 9 days. In some embodiments, the mixture is incubated for a period of time from less than 1 day to 8 days. In some embodiments, the mixture is incubated for a period of time from less than 1 day to 7 days. In some embodiments, the mixture is incubated for a period of time from less than 1 day to 6 days. In some embodiments, the mixture is incubated for a period of time from less than 1 day to 5 days. In some embodiments, the mixture is incubated for a period of time from less than 1 day to 4 days. In some embodiments, the mixture is incubated for a period of time from less than 1 day to 3 days. In some embodiments, the mixture is incubated for a period of time from 1 day to 9 days. In some embodiments, the mixture is incubated for a period of time from 2 days to 8 days. In some embodiments, the mixture is incubated for a period of time from 3 days to 7 days. In some embodiments, the mixture is incubated for a period of time from 4 days to 6 days.

[0067] In some embodiments, the mixture is incubated for 15 minutes. In some embodiments, the mixture is incubated for 30 minutes. In some embodiments, the mixture is incubated for 45 minutes. In some embodiments, the mixture is incubated for 1 hour. In some embodiments, the mixture is incubated for 2 hours. In some embodiments, the mixture is incubated for 4 hours. In some embodiments, the mixture is incubated for 6 hours. In some embodiments, the mixture is incubated for 8 hours. In some embodiments, the mixture is incubated for 10 hours. In some embodiments, the mixture is incubated for 12 hours. In some embodiments, the mixture is incubated for 1 day. In some embodiments, the mixture is incubated for 2 days. In some embodiments, the mixture is incubated for 3 days. In some embodiments, the mixture is incubated for 4 days. In some embodiments, the mixture is incubated for 5 days. In some embodiments, the mixture is incubated for 6 days. In some embodiments, the mixture is incubated for 7 days. In some embodiments, the mixture is incubated for 8 days. In some embodiments, the mixture is incubated for 9 days. In some embodiments, the mixture is incubated for 10 days.

[0068] In some embodiments, the cell or composition of cells are plated in a multi-well plate. In some embodiments, a cell or composition of cells comprisesAttorney Docket No.: 31006_WO multiple nucleic acids (i.e., multiple, different target sequences) and in the methods described herein, the cell or composition of cells is contacted with multiple different RNA silencing conjugates such that multiple RNA silencing conjugates can be evaluated within a given cell or composition. In some embodiments, each different RNA silencing conjugate uses the same reporter gene (e.g., luciferin). In some embodiments, each different RNA silencing conjugate uses a different reporter gene. It is appreciated that different reporter genes can be linked to the different target sequences, such that the effect of each distinct RNA silencing conjugate on its respective target sequence can be ascertained.

[0069] The following examples provide illustrative embodiments of the disclosure. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The examples provided do not in any way limit the disclosure.EXAMPLESExample 1 : In vitro evaluation of siRNA delivery to target cells1.1 - Methods

[0070] Generation of CHO-Transferrin receptor (TfR) overexpressing clonal cell line: CHO-K1 cells were cultured in DMEM / F12 growth media with 10% FBS and penicillin / streptomycin antibiotics (“complete growth media”). CHO-K1 cells were detached with trypsin, collected in complete growth media, counted, plated at 3x106cells per plate, and incubated overnight at 37°C, 5% CO2. The next day, the CHO-K1 cells were transfected with FuGENE HD Transfection Reagent according to the manufacturer’s instructions. Specifically, the TfR expression plasmid construct was prepared in Opti-MEM along with FuGENE reagent and then added to the CHO- K1 plates. After 48 hours, hygromycin was added at 1 mg / mL to select for transfected cells. After the selected cells had grown for 10 days, the cells were incubated with an anti-CD71 / PE antibody and a single cell FACS sort was performed using a Sony MA900 Cell Sorter to select a high expressing clone with about 300-fold of TfR expression over the parental CHO-K1 cell. This clone, designated as clone B7, was further grown out and expression was confirmed by flow cytometry.Attorney Docket No.: 31006_WO

[0071] Generation of CHO-TfR target / Luciferase clonal cell lines: Clone B7 was transfected with the MAPT-Luciferase, SNCA-Luciferase, APP-Luciferase, or Navi ,8-Luciferase constructs depicted in FIGs. 1 A-1 D following the same FuGene HD Transfection Reagent protocol as described above. Once the selected cells had grown for 10-14 days after selection with 20 pg / mL puromycin, single cell clones were plated by diluting cells to 0.5 cells per 200 pL and plating 200 pL per well of a 96-well plate. After the clones had grown out, the clones were further expanded for functional screening. Briefly, 10,000 cells of the individual clones were plated per well in complete growth media (no selection) in triplicate for each experimental condition. The following day, the respective conjugate was prepared at 2-fold concentration in Accell siRNA Delivery Media with between 0.5-10% FBS (“assay media”). The complete growth media was aspirated from the plates and replaced with 50 pL of assay media followed by the addition of 50 pL of the prepared conjugate. The plates were incubated for 3 days at 37°C, 5% CO2 before addition of BioGio according to manufacturer’s instructions. The plates were then read on a BioTek Neo2 plate reader, and top clones were selected by signal window of conjugate knockdown and raw signal strength. Once top clones were selected, full dose titrations with conjugate and assay optimization were done for each as described below.

[0072] Treatment of CHO-TfR target / Luciferase clonal cell lines: CHO-TfR MAPT-Luc (clone 3), CHO-TfR SNCA-Luc (clone 7), CHO-TfR APP-Luc (clone 2-2), or CHO-TfR Nav1.8-Luc (clone 22) clonal lines were cultured in complete growth media with the following selection agents: 1 mg / mL hygromycin and 20 pg / mL puromycin. Plates were set up in duplicate for both assessment of luciferase knockdown and viability following conjugate treatment. The day before the experiment, cells were detached with trypsin, collected in complete growth media, counted, and plated at 2,000-100,000 cells per well in complete growth media (no selection) overnight at 37°C, 5% CO2. The following day, media was aspirated and replaced with 50pL of assay media. Conjugate treatments (i.e., RNA silencing conjugate as described herein) were prepared at 2-fold concentrations and serially diluted in assay media. Untreated wells (receiving only the assay media) were included on each plate in triplicate. Conjugates were added at 50pL per well for a final volume of 100 pL per well in triplicate. Plates were incubated at 37°C, 5% CO2 for 3-7 days depending on the cell line. After incubation, plates were removed from the incubator and equilibratedAttorney Docket No.: 31006_WO at room temperature. Either BioGio or CellTiter-Glo were added to the duplicate plates, according to the manufacturer’s instructions and plates were then read for luminescence on a BioTek Neo2 plate reader. BioGio was used for luciferase knockdown, whereas CellTiter-Glo was used for cell viability.

[0073] BioGio luciferase knockdown data was reported as percent (%) knockdown using the average untreated RLU value on each plate as the 0% knockdown reference. CellTiter-Glo data was reported as percent (%) inhibition using the average untreated RLU value on each plate as the 0% inhibition reference. GraphPad Prism was used to generate ICso values for knockdown as well as to plot RLU values for viability.

[0074] The assay adapted for each cell line with respect to percentage of PBS in assay media, incubation time, and cell count using a Design of Experiments (DOE) generated and analyzed using JMP software.

[0075] Generation of CHO-NPRC (natriuretic peptide receptor C) overexpressing clonal cell line: CHO-K1 cells were cultured in DMEM / F12 growth media with 10% FBS and penicillin / streptomycin antibiotics (“complete growth media”). CHO-K1 cells were detached with trypsin, collected in complete growth media, counted, plated at 3x106cells per plate, and incubated overnight at 37°C, 5% CO2. The next day, the CHO-K1 cells were transfected with FuGENE HD Transfection Reagent according to the manufacturer’s instructions. Specifically, the NPRC expression plasmid construct was prepared in Opti-MEM along with FuGENE reagent and then added to the CHO-K1 plates. After 48 hours, hygromycin was added at 1 mg / mL to select for transfected cells. After the selected cells had grown for 10 days, the cells were incubated with an anti-NPRC / PE antibody and a single cell FACS sort was performed using a Sony MA900 Cell Sorter to select a high expressing clone. This clone, designated as clone G3, was further grown out and expression was confirmed by flow cytometry.

[0076] Generation of CHO-NPRC target / Luciferase clonal cell line: Clone G3 was transfected with the PLIN1 -Luciferase construct depicted in FIG. 1 E following the same FuGene HD Transfection Reagent protocol as described above. Once the selected cells had grown for 18 days after selection with 20 pg / mL puromycin, single cell clones were plated by diluting cells to 0.5 cells per 200 pL and plating 200 pL per well of a 96-well plate. After the clones had grown out, the clones were furtherAttorney Docket No.: 31006_WO expanded for functional screening. Briefly, 10,000 cells of the individual clones were plated per well in complete growth media (no selection) in triplicate for each experimental condition. The following day, the respective conjugate was prepared at 2-fold concentration in Accell siRNA Delivery Media with 2% FBS (“assay media”). The complete growth media was aspirated from the plates and replaced with 50 pL of assay media followed by the addition of 50 pL of the prepared conjugate. The plates were incubated for 3 days at 37°C, 5% CO2 before addition of BioGio according to manufacturer’s instructions. The plates were then read on a BioTek Neo2 plate reader, and top clones were selected by signal window of conjugate knockdown and raw signal strength. Once top clones were selected, full dose titrations with conjugate and assay optimization were done for each as described below.

[0077] Treatment of CHO-NPRC target / Luc if erase clonal cell line: CHO- NPRC-Luc (clone 3) was cultured in complete growth media with the following selection agents: 1 mg / mL hygromycin and 20 pg / mL puromycin. The day before the experiment, cells were detached with trypsin, collected in complete growth media, counted, and plated at 2,000-100,000 cells per well in complete growth media (no selection) overnight at 37°C, 5% CO2. The following day, media was aspirated and replaced with 50pL of assay media. Conjugate treatments (i.e., RNA silencing conjugate as described herein) were prepared at 2-fold concentrations and serially diluted in assay media. Untreated wells (receiving only the assay media) were included on each plate in triplicate. Conjugates were added at 50pL per well for a final volume of 100 pL per well in triplicate. Plates were incubated at 37°C, 5% CO2 for 3 days. After incubation, plates were removed from the incubator and equilibrated at room temperature. BioGio was added according to the manufacturer’s instructions and plates were then read for luminescence on a BioTek Neo2 plate reader.

[0078] BioGio luciferase knockdown data was reported as percent (%) knockdown using the average untreated RLU value on each plate as the 0% knockdown reference. CellTiter-Glo data was reported as percent (%) inhibition using the average untreated RLU value on each plate as the 0% inhibition reference. GraphPad Prism was used to generate IC50 values for knockdown as well as to plot RLU values for viability.

[0079] Materials used in Example 1 are described in Table 1.Table 1. Materials used in Example 1.Attorney Docket No.: 31006_WOAttorney Docket No.: 31006_WO7.2 - Results

[0080] CHO-TfR Clone B7 and CHO-NPRC Clone G3 were successfully generated, and Transferrin Receptor or NPRC expression was confirmed by flow cytometry, respectively. These clones were used to generate pools of CHO TfR MAPT-Luc, CHO TfR SNCA-Luc, CHO TfR APP-Luc, CHO TfR Nav1.8-Luc, and CHO-NPRC-PLIN1-Luc as indicated. Clones were identified from these pools based on their percent (%) knockdown and these clones were used for full dose titration curves with conjugates to demonstrate activity and potency of the conjugate.

[0081] Conjugate treatment achieved between 67.9% and 91.9% knockdown of BioGio luciferase signal depending on the cell line, with ICso values between 1.60 nM and 20.6 nM (Table 2). Conjugate treatment did not reduce viability of the clonal cell lines.Table 2. BioGio IC50 and Knockdown Efficiency.Example 2: Improved cell-based assay with an inducible promoter system Design of the Cumate-lnducible system

[0073] Robust cell-based assays (CBA) are essential, particularly in late-stage development, to evaluate the potency and ensure quality standards of antibody- oligonucleotide conjugates such as antibody-siRNA conjugates (ARC). However, current CBA face challenges such as prolonged incubation times, limited throughput, and susceptibility to inconsistencies such as edge effects. Furthermore, traditionalAttorney Docket No.: 31006_WO promoters used in these assays often produce either excessively high or insufficiently low expression levels, complicating assay optimization (Figure 2A).

[0074] To enhance CBA performance, an inducible promoter with reduced background expression was used to mitigate the long incubation times and edge effects caused by strong promoters (Figure 2B). One such inducible promoter is a cumate-inducible promoter system. It consists of the CymR repressor, the cumate operator (CuO) sequence, and cumate. The CymR repressor binds to the CuO sequence, inhibiting the promoter and keeping the gene "off." When cumate is added, it binds to the CymR repressor, causing its release from the CuO sequence. This allows the promoter to activate, turning the gene "on" and enabling protein production (Figure 2C). An exemplary cumate-inducible promoter is provided in SEQ ID NO: 10.

[0075] YS001 , a cumate-inducible system was designed for use in the CBA testing method described in Example 1. YS001 integrates cumate-inducible components and Piggybac transposon (see Figures 3A and 3B), along with the Kozak sequence, luciferase gene, and ARC target sequence. The Piggybac transposon components include specific inverted repeat sequences (ITR) that are recognized by the transposase enzyme, facilitating precise gene insertion into the target genome. The cumate-inducible system consists of a mutated CMV promoter (CMV5), the cumate operator sequence (CuO) which binds to the cumate repressor, the EF1 promoter, the cumate repressor (CymR), and the Puromycin-resistant gene (PuroR). The Kozak sequence is strategically placed before the luciferase gene to enhance translation efficiency.Generation of the Cumate-inducible Cell Lines

[0076] Two cumate-inducible cell lines were generated from CHO-TfR B7 parental cell lines described in Example 1. The parental cell lines were cultured in DMEM / F12 growth media supplemented with 10% FBS, 1x penicillin / streptomycin antibiotics, and 1 mg / mL Hygromycin. Transfection was performed using Lipofectamine 2000 Transfection Reagent according to the manufacturer's instructions. Specifically, 1 pg of the Piggybac transposon (either PBQM812A-1 orAttorney Docket No.: 31006_WOYS001 ) was combined with 0.4 pg of transposase (Catalog PB210PA-1 , S B I ) in a 2.5:1 ratio in 50 pL of OptiMEM for 5 minutes at room temperature. The plasmid to Lipofectamine ratio is 1 :2 (1 pg plasmid: 2 pL Lipofectamine). Therefore, 2.8 pL of Lipofectamine 2000 was diluted in 50 pL of OptiMEM for 5 minutes at room temperature. The plasmid mixture and diluted Lipofectamine 2000 were then combined and incubated at room temperature for 20 minutes.

[0077] CHO-TfR B7 parental cells were detached with trypsin, collected in growth media, counted, and plated at 5x10A5 cells per well in a 6-well plate with 2 mL growth media per well, and incubated overnight at 37°C, 5% CO2. The next day, 1 pg / mL of puromycin was added to select for transfected cells. After 4 days, 20 pg / mL of puromycin was added to further select transfected cells for another 4 days. The selected cumate-inducible GFP or luciferase cells were pooled and cultured until sufficient cells were available for cryopreservation, then saved for future experiments.Testing Cumate Inducible System with GFP and Luciferase Cell Lines

[0078] Cumate-inducible GFP and luciferase cells were cultured in DMEM / F12 growth media supplemented with 10% FBS, 1x penicillin / streptomycin antibiotics, 1 mg / mL Hygromycin, and 20 pg / mL Puromycin. The cumate working concentration was initially set to 1x by diluting a stock concentration of 10000x using assay media. The assay media comprised ACCELL siRNA Delivery media supplemented with 10% FBS and 1x penicillin / streptomycin antibiotics.

[0079] To test GFP activation, cumate-inducible GFP cells were treated with assay media containing either 1x cumate or no cumate for 6 days in 24-well plates. After treatment, the cells were detached with trypsin, collected in growth media, counted, and plated at 1.5x10A4 cells per well in a 96-well plate with 100 pL of assay media per well. The cells were incubated for half a day at 37°C, 5% CO2 to allow them to settle and attach to the bottom of the plates. The cells were then prepared for imaging. Images were acquired using a BioTek Lionheart FX Automated Microscope equipped with a 20x objective and GFP and brightfield channels.Attorney Docket No.: 31006_WO

[0080] To test luciferase activation, cumate-inducible luciferase cells were detached with trypsin, collected in growth media, counted, and plated at 1x10A4 cells per well in a 96-well plate with 100 pL of 1x cumate assay media per well. The cells were incubated for 3 days at 37°C, 5% CO2. Following incubation, plates were removed from the incubator and equilibrated at room temperature. The cells were treated with 100 pL of BrightGlo per well according to the manufacturer's instructions. The plates were read on a SpectraMaxL plate reader. Negative control groups included a blank group (no cells, just PBS), a parental cell group (CHO-TfR B7 parental cell lines in assay media with or without cumate for 3 days), and cumate- inducible Luciferase cells in assay media without cumate for 3 days.Testing Titratable Property of Cumate-inducible Luciferase Cell Line

[0081] To evaluate the effect of different cumate doses on luciferase expression, a titration experiment was designed using cumate concentrations ranging from 0.25x to 10x. The aim was to determine the sensitivity of luciferase expression in response to varying cumate concentrations. The cumate working concentration was diluted to various concentrations from a stock concentration of 10000x using assay media. The assay media comprised ACCELL siRNA Delivery media supplemented with 10% FBS and 1x penicillin / streptomycin antibiotics.

[0082] Cumate-inducible luciferase cells were cultured in DMEM / F12 growth media supplemented with 10% FBS, 1x penicillin / streptomycin antibiotics, 1 mg / mL Hygromycin, and 20 pg / mL Puromycin. Then these cells were detached with trypsin, collected in growth media, counted, and plated at 1x10A4 cells per well in a 96-well plate with 100 pL of assay media containing varying cumate concentrations per well. The cells were incubated for 1 day at 37°C, 5% CO2, to allow for cumate-induced luciferase expression. Following incubation, plates were removed from the incubator and equilibrated at room temperature. The cells were treated with 100 pL of BrightGlo per well according to the manufacturer's instructions. The luminescence was measured using a SpectraMaxL plate reader. Negative control groups included cumate-inducible luciferase cells in assay media without cumate for 1 day to ensure that any observed luminescence was specific to luciferase expression induced byAttorney Docket No.: 31006_WO cumate. The results were displayed in a heatmap graph, with signal intensity greyscale-coded from low to high, ranging from black to white.Testing Reversible Property of Cumate-lnducible Luciferase Cell Line

[0083] To test whether the removal of cumate can turn off luciferase expression and how quickly this occurs, a reversible experiment was designed by removing cumate for different durations to observe signal reduction. The cumate working concentration was set to 1x, diluted from a stock concentration of 10000x using assay media. The assay media comprised ACCELL siRNA Delivery media supplemented with 10% FBS and 1x penicillin / streptomycin antibiotics.

[0084] Cumate-inducible luciferase cells were cultured in DMEM / F12 growth media supplemented with 10% FBS, 1x penicillin / streptomycin antibiotics, 1 mg / mL Hygromycin, and 20 pg / mL Puromycin. Then these cells were treated with assay media containing either 1x cumate or no cumate for 4 days in 24-well plates. Cumate-inducible luciferase cells were detached with trypsin, collected in growth media, counted, and plated at 1x10A4 cells per well in a 96-well plate. For the positive group, cells were treated with 1x cumate continuously. The negative group was treated without cumate throughout the experiment. For the -1 d, -2d, and -3d cumate groups, 1x cumate assay media was switched to no cumate assay media on different days accordingly. Thus, these cells were incubated for an additional 4 days at 37°C, 5% CO2. Following incubation, plates were removed from the incubator and equilibrated at room temperature. The cells were treated with 100 pL of BrightGlo per well according to the manufacturer's instructions. The luminescence was measured using a SpectraMaxL plate reader. The results were displayed in a bar graph, with the y-axis representing the relative light unit (RLU) and the x-axis representing different groups. The relative luciferase change was normalized to the negative group, which is shown at the top of the bar as 1x.Identification of Optimal Cumate Dose for ARC Cell Based Assay

[0085] As ARC CBA typically involves a 3-day assay, various cumate concentrations were tested over 1 to 3 days to find the optimal concentration. The experiment was designed with cumate concentrations ranging from 0.25x to 10x andAttorney Docket No.: 31006_WO incubation times varying from 1 to 3 days. The cumate working concentration was diluted to various concentrations from a stock concentration of 10000x using assay media. The assay media comprised ACCELL siRNA Delivery media supplemented with 10% FBS and 1x penicillin / streptomycin antibiotics.

[0086] Cumate-inducible luciferase cells were cultured in DMEM / F12 growth media supplemented with 10% FBS, 1x penicillin / streptomycin antibiotics, 1 mg / mL Hygromycin, and 20 pg / mL Puromycin. Then these cells were detached with trypsin, collected in growth media, counted, and plated at 1x10A4 cells per well in a 96-well plate with 100 pL of varying cumate doses. The cells were incubated for 1 to 3 days at 37°C, 5% CO2. Following incubation, plates were removed from the incubator and equilibrated at room temperature. The cells were treated with 100 pL of BrightGlo per well according to the manufacturer's instructions. The luminescence was measured using a SpectraMaxL plate reader. The results were displayed in a bar graph, with the x-axis representing different doses of cumate over 1 to 3 days, and the y-axis representing the relative luciferase change normalized to the negative group, which is shown as 1 .Time-Course Testing of Cell-Based Assay in Cumate-inducible Cell Line for ARC

[0087] To evaluate luciferase-MAPT target knockdown at different time points following ARC treatment, 1x cumate was added for 1 day, then switched to no cumate assay media with ARC, and incubated for 24-72 hours.

[0088] Cumate-inducible luciferase cells were cultured in DMEM / F12 growth media supplemented with 10% FBS, 1x penicillin / streptomycin antibiotics, 1 mg / mL Hygromycin, and 20 pg / mL Puromycin. The cumate working concentration was set to 1x, diluted from a stock concentration of 10000x using assay media. The assay media comprised ACCELL siRNA Delivery media supplemented with 10% FBS and 1x penicillin / streptomycin antibiotics.

[0089] Plates were set up in triplicates. The day before the experiment, cells were detached with trypsin, collected in growth media, counted, and plated at 1x10A4 cells per well in 100 pL of assay media containing 1x cumate overnight at 37°C, 5% CO2. The following day, the media was aspirated and replaced with 100 pL of assayAttorney Docket No.: 31006_WO media without cumate. ARC treatments were prepared at 3-fold concentrations and serially diluted in assay media. ARC was added at 50 pL per well for a final volume of 150 pL per well in triplicate. Plates were incubated at 37°C, 5% CO2 for 1-3 days. After incubation, plates were removed from the incubator and equilibrated at room temperature. The cells were treated with 100 pL of BrightGlo per well according to the manufacturer's instructions. The luminescence was measured using a SpectraMaxL plate reader and analyzed using SoftMax Pro software.Results

[0090] Cumate-inducible GFP and luciferase cells were successfully generated. In both the GFP system (Figure 4A) and the luciferase system (Figure 4B), cumate effectively induced expression of the reporter protein. Notably, the luciferase system demonstrated quantitative strength with a low background and a high signal-to-noise ratio (S / N) of 10x.

[0091] Figure 4C shows a heatmap illustrating the value of the relative light unit (RLU) from negative control to 10x cumate group. The RLU ranges from 2.6x10A5 to 3.9x10A6. When normalized by the negative control, the relative luciferase activity changes from 1x to 15x. This positive trend in luciferase activity corresponds with the increase in cumate concentrations after a 1-day incubation, demonstrating concentration-dependent activation. Figure 4D indicates a decrease in luciferase activity based on RLU values, ranging from 5.0x10A6 in the positive group, 3.1x10A5 in the negative, 6.1 x10A5 to 3.8x10A5 in the cumate removing groups. In relative luciferase activity, the signal also significantly decreased from 16x to 2x after 1 day of cumate removal, and by 2 to 3 days, it approached negative control levels at 1x, suggesting the system is reversible.

[0092] To identify the optimal cumate concentration in a 3-day assay, Figure 4E shows that concentrations above 1x resulted in a decrease in luciferase signal, indicating suboptimal treatment and not recommended for ARC CBA. Concentrations between 0.25x and 1x resulted in a gradual signal increase over time. Consequently, 1x cumate was used for the following ARC treatment experiment.Attorney Docket No.: 31006_WO

[0093] Time-course results based on Figure 5B show that a 48-hour incubation can generate a nice dose-response curve, which is shorter than the standard 72-hour incubation.Attorney Docket No.: 31006_WOSEQUENCE LISTINGAttorney Docket No.: 31006_WOAttorney Docket No.: 31006_WOAttorney Docket No.: 31006_WOAttorney Docket No.: 31006_WOAttorney Docket No.: 31006_WO

Claims

Attorney Docket No.: 31006_WOCLAIMS1. A cell for measuring potency of or efficiency of delivering into the cell an RNA silencing conjugate comprising a cell-targeting moiety and an RNA silencing moiety, the cell comprising a nucleic acid comprising a promoter operably linked to a sequence encoding a reporter gene and a target sequence complementary to the RNA silencing moiety, wherein the cell expresses a transmembrane receptor capable of binding the celltargeting moiety.

2. The cell of claim 1 , wherein the cell-targeting moiety is a protein, a peptide, a carbohydrate, a small molecule, or a lipid.

3. The cell of claim 1 or 2, wherein the reporter gene is a photoprotein or a fluorescent protein.

4. The cell of claim 3, wherein the reporter gene is luciferase or GFP.

5. The cell of any one of claims 1-4, wherein the RNA silencing moiety is a siRNA, an ASO, a miRNA, or a piRNA.

6. The cell of any one of claims 1 -5, wherein the transmembrane receptor is human transferrin receptor 1 (TfR), natriuretic peptide receptor C (NPR-C), CD19, or CD40.

7. The cell of any one of claims 1 -6, wherein the cell has been engineered to overexpress the transmembrane receptor.

8. The cell of any one of claims 1 -7, wherein the transmembrane receptor is human transferrin receptor 1 (TfR) or natriuretic peptide receptor C (NPR-C).Attorney Docket No.: 31006_WO9. The cell of any one of claims 1 -8, wherein the nucleic acid further comprises a polyA signal.

10. The cell of any one of claims 1-9, wherein the nucleic acid comprises, in 5' to 3' orientation,(a) the promoter;(b) the sequence encoding a reporter gene;(c) the target sequence complementary to the RNA silencing moiety; and(d) a polyA signal.11 . The cell of any one of claims 1 -10, wherein the promoter is a CMV promoter.

12. The cell of claim 11 , wherein the CMV promoter comprises SEQ ID NO: 1 .

13. The cell of any one of claims 1 -10, wherein the promoter is an inducible promoter.

14. The cell of claim 13, wherein the inducible promoter is a cumate-inducible promoter.

15. The cell of claim 14, wherein the cumate-inducible promoter comprises a mutated CMV promoter and the cumate operator sequence.

16. The cell of any one of claims 13-15, wherein the inducible promoter comprises SEQ ID NO: 10.

17. The cell of any one of claims 9-16, wherein the polyA signal is a SV40 polyA signal.

18. The cell of any one of claims 9-17, wherein the polyA signal comprises SEQ ID NO: 5.Attorney Docket No.: 31006_WO19. The cell of any one of claims 1-18, wherein the target sequence is 10-5000 nucleotides in length.

20. The cell of any one of claims 1-19, wherein the target sequence is 20-4000 nucleotides in length.

21. The cell of any one of claims 1-20, wherein the target sequence is 100-3500 nucleotides in length.

22. The cell of any one of claims 1 -21 , wherein the target sequence is a portion of an alpha synuclein (SNCA), microtubule-associated protein tau (MAPT), amyloid-beta precursor protein (APP), sterile alpha and TIR motif containing 1 (SARM1 ), superoxide dismutase 1 (SOD1 ), ataxin 2 (ATXN2), ataxin 3 (ATXN3), apolipoprotein E (APOE), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1 ), fragile X messenger ribonucleoprotein 1 (FMR1 ), leucine-rich repeat kinase 2 (LRRK2), huntingtin (HTT), sodium voltage-gated channel alpha subunit 10 (SCN10A), sodium voltage-gated channel alpha subunit 9 (SCN9A), calcium voltage-gated channel subunit alpha 1 B (CACNA1 B), prion protein (PRNP), phosphoinositide kinase, activin A receptor type 2A (ACVR2a), activin A receptor type 2A (ACVR2b), perilipin-1 (PLIN1 ), or hypoxanthine phosphoribosyltransferase 1 (HPRT) gene sequence.

23. The cell of claim 22, wherein the target sequence is a portion of the SNCA gene sequence.

24. The cell of claim 23, wherein the target sequence comprises SEQ ID NO: 6.

25. The cell of claim 22, wherein the target sequence is a portion of the MAPT gene sequence.

26. The cell of claim 25, wherein the target sequence comprises SEQ ID NO: 7.Attorney Docket No.: 31006_WQ 1. The cell of claim 22, wherein the target sequence is a portion of the APP gene sequence.

28. The cell of claim 27, wherein the target sequence comprises SEQ ID NO: 8.

29. The cell of claim 22, wherein the target sequence is a portion of the Navi .8 gene sequence.

30. The cell of claim 29, wherein the target sequence comprises SEQ ID NO: 9.

31. The cell of claim 22, wherein the target sequence is a portion of the PLIN1 gene sequence.

32. The cell of claim 31 , wherein the target sequence comprises SEQ ID NO: 11 .

33. The cell of any one of claims 1-32, wherein the sequence encoding luciferase comprises SEQ ID NO: 2, 3, or 4.

34. The cell of any one of claims 1 -33, wherein the nucleic acid is a plasmid.

35. The cell of any one of claims 1-34, wherein the cell further comprises a second nucleic acid comprising a second promoter operably linked to a second sequence encoding a second luciferase and a second target sequence.

36. The cell of claim 35, wherein the second nucleic acid is a plasmid.

37. The cell of any one of claims 1 -36, wherein the cell is a mammalian cell.Attorney Docket No.: 31006_WO38. The cell of any one of claims 1 -37, wherein the cell is a CHO, EFO21 , HEK293, or 3T3 cell.

39. A composition comprising a plurality of the cells of any one of claims 1 -38.

40. A method of measuring potency or efficiency of delivering into a cell an RNA silencing conjugate comprising a cell-targeting moiety and an RNA silencing moiety, the method comprising: contacting the cell of any one of claim 1 -38 or the composition of claim 39 with the RNA silencing conjugate to form a mixture; incubating the mixture for a period of time; and measuring luciferase expression in the cell after the incubating.

41. A method of screening RNA silencing conjugates comprising a cell-targeting moiety and an RNA silencing moiety, the method comprising: contacting the cell of any one of claim 1 -38 or the composition of claim 39 with a first RNA silencing conjugate to form a first mixture; contacting the cell of any one of claim 1 -38 or the composition of claim 39 with a second RNA silencing conjugate to form a second mixture; incubating the first and second mixtures for a period of time; measuring luciferase expression in the first and second mixtures after the incubating; and comparing luciferase expression of the first mixture with luciferase expression of the second mixture.

42. The method of claim 41 , wherein the method further comprises selecting the RNA silencing conjugate achieving lower luciferase expression.Attorney Docket No.: 31006_WO43. The method of any one of claims 40-42, wherein the period of time is 2 days to 7 days.

44. The method of any one of claims 40-43, wherein the period of time is 6 days.

45. The method of any one of claims 40-43, wherein the period of time is 2 days.

46. The method of any one of claims 40-45, wherein the mixture comprises 2,000 cells to 100,000 cells.

47. The method of any one of claims 40-46, wherein the mixture comprises 10,000 cells.

48. The method of any one of claims 40-47, wherein the mixture further comprises a cell culture media.

49. The method of claim 48, wherein the cell culture media comprises 10% FBS.

50. A cell for measuring potency or efficiency of delivering into the cell an RNA silencing conjugate comprising a cell-targeting moiety and an RNA silencing moiety, the cell comprising a nucleic acid, the nucleic acid comprising, in 5' to 3' orientation,(a) a promoter comprising SEQ ID NO: 1 or 10;(b) a sequence encoding luciferase comprising SEQ ID NO: 2, 3, or 4;(c) a target sequence comprising SEQ ID NO: 6, 7, 8, 9 or 11 ; and(d) a polyA signal comprising SEQ ID NO: 5, wherein the cell expresses a transmembrane receptor that is human transferrin receptor 1 (TfR), natriuretic peptide receptor C (NPR-C), CD19, or CD40, and wherein the RNA silencing moiety is complementary to the target sequence.

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