Materials and methods for characterizing efficacy

Abstract

The present disclosure provides a nucleic acid comprising a response element operably linked to a reporter nucleotide sequence. In various embodiments, the response element comprises 2 to 10 copies of the Z1 transcription factor binding site. Cells comprising the nucleic acid are also provided. In various aspects, the cells stably overexpress an adeno-associated virus receptor. The present disclosure further provides a method for determining the potency of a sample comprising an AAV vector.

Description

[Technical Field]

[0001] The present disclosure relates to nucleic acids, cells, and methods for characterizing the potency of a sample containing an AAV vector.

[0002] Incorporation by Reference of Electronically Submitted Materials The computer-readable nucleotide / amino acid sequence listing identified by the file name "55328A_SeqListing.XML", which was filed concurrently herewith and created on December 28, 2023, and is 126,534 bytes, is incorporated by reference in its entirety. [Background technology]

[0003] Drugs and biopharmaceuticals approved by regulatory agencies must be accompanied by analytical assays to confirm that product lots meet defined standards (e.g., purity, safety, and potency) deemed suitable for therapeutics approved for human use. Potency assays and related tests are used to ensure consistent quality between product lots and to ensure product identity, purity, strength (potency), and stability during testing. Analytical assays have long been used to characterize protein-based therapeutics such as small molecule drugs and monoclonal antibodies. However, the nature of gene therapy products complicates the development of potency assays. Several characteristics of gene and cell therapies pose significant challenges in developing potency assays, including the inherent variability of starting materials, the lack of appropriate reference standards, complex mechanisms of action, and the in vivo fate of the product. With an increasing number of gene therapy candidates, the industry continues to struggle to develop assays that reliably quantify the potency of gene therapy product samples. Summary of the Invention

[0004] The present disclosure provides materials and methods for measuring the efficacy of expression vector delivery vehicles. For example, the disclosure provides a nucleic acid comprising a response element (RE) operably linked to a reporter nucleotide sequence. In various embodiments, the response element comprises 2 to 10 copies (e.g., 3 to 8 or 3 to 6 copies) of the Z1 transcription factor (TF) binding site. Optionally, the Z1 TF binding site has the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence that differs from SEQ ID NO: 1 by 1, 2, or 3 nucleotides. In various embodiments, the response element comprises a spacer sequence (e.g., the sequence of SEQ ID NO: 2 or SEQ ID NO: 3) between at least two copies of the Z1 TF binding site. The response element may further comprise a promoter, such as a minimal promoter (e.g., SEQ ID NO: 32) and / or a polyA signal sequence. In various aspects of the present disclosure, the reporter nucleotide sequence encodes a reporter protein that can be identified and measured. Examples of reporter proteins include photoproteins or enzymes that generate bioluminescence, such as luciferase, fluorescent proteins, such as green fluorescent protein (GFP), enhanced GFP (EGFP), and mCherry, and colored proteins or enzymes that generate colored products, such as beta-galactosidase. The nucleic acid, in some embodiments, can include a second reporter nucleotide sequence operably linked to a constitutive promoter.

[0005] Cells comprising the nucleic acid are also provided. For example, the present disclosure provides cells comprising a nucleic acid comprising a response element (e.g., the response element comprises 2-10 copies (e.g., 3-8 or 3-6 copies) of a transcription factor (TF) binding site) operably linked to a reporter nucleotide sequence, wherein the cell is further engineered to stably overexpress an adeno-associated viral receptor (AAVR), such as a wild-type AAVR. Optionally, the response element comprises one or more TF binding sites capable of binding by the TF. In various embodiments, the TF is a ligand-dependent TF, such as a metal (e.g., copper)-dependent TF. Alternatively, the response element comprises one or more TF binding sites that are bound by an exogenous TF. In various embodiments, the exogenous TF comprises an engineered DNA-binding domain specific for a TF binding site, such as an engineered DNA-binding site comprising 2-10 zinc fingers.

[0006] The present disclosure further provides a method for determining the efficacy of a sample containing an AAV vector. The method includes contacting all or a portion of the sample with a cell containing a response element operably linked to a reporter nucleotide sequence. The AAV vector encodes a regulatory factor that directly or indirectly regulates expression of the reporter nucleotide sequence via the response element. The method further includes measuring expression of the reporter nucleotide sequence in the cell. Optionally, the method further includes determining the efficacy of the sample based on the measured expression level of the reporter nucleic acid sequence. [Brief explanation of the drawings]

[0007] [Figures 1A-1D] FIG. 1 is a schematic diagram of a nucleic acid comprising a response element operably linked to a reporter nucleotide sequence. [Figure 2] Reporter expression (measured as relative light units (RLU, y-axis)) of various response element constructs in the presence and absence of a regulator (SEQ ID NO: 34) is shown. The presence of reporter alone is represented by the left bar for each response element construct, and reporter plus activator is represented by the right bar for each response element construct. [Figure 3] Reporter expression (measured as relative light units (RLU, y-axis)) of various response element constructs in the presence of increasing amounts of regulator (SEQ ID NO: 34; 0 ng, 0.01 ng, 0.1 ng, 1 ng, 10 ng, 30 ng, 50 ng, or 70 ng) is shown. [Figure 4] Figure 4 shows the results of Example 3. Figure 4 shows the reporter expression (measured as relative light units (RLU, y-axis)) observed from a control expression cassette (P-1) with a TET reporter sequence in place of the DNA binding sequence, and a cassette (P-5) containing a Z1-based response element operably linked to a reporter nucleic acid sequence. [Figure 5] 1 is a schematic diagram of a nucleic acid comprising a response element operably linked to a reporter nucleotide sequence, and in some cases further comprising a second reporter nucleic acid operably linked to a constitutive promoter. [Figures 6A-6B] AAV9 infection (%) (Figure 6A) or mean fluorescence intensity (Figure 6B) (y-axis) is shown for unmodified HEK293 cells, HEK293 cells overexpressing AAVR, unmodified HeLa cells, and HeLa cells overexpressing AAVR. [Figures 7A-7B] AAV9 infection (%) (Figure 7A) or mean fluorescence intensity (Figure 7B) (y-axis) is shown for unmodified HEK293 cells, HEK293 cells overexpressing AAVR, unmodified CHO cells, and CHO cells overexpressing AAVR. [Figure 8A-8B] Green fluorescent protein expression (%) (Figure 8A) or mean fluorescence intensity (Figure 8B) (y-axis) are shown for subclones of HEK cells engineered to overexpress AAVR. All subclones tested demonstrated improved infection rates (measured as transgene expression) compared to unmodified cells. [Figure 9A-9B] Graph showing fold change of VP64 in HEK293 and HeLa cells engineered to overexpress AAVR at various multiplicities of infection (MOI, viral genomes / cell). [Figures 10A-10D]10A and 10B are bar graphs showing AAV infection (%) for various AAV serotypes in HEK293 cells modified to overexpress AAVR (FIG. 10A) or HeLa cells modified to overexpress AAVR (FIG. 10B), and the mean fluorescence intensity observed from the same AAV vectors in HEK293 cells modified to overexpress AAVR (FIG. 10C) or HeLa cells modified to overexpress AAVR (FIG. 10D). [Figure 11] 1 shows AAV9 transduction in the HeRC32 cell line compared to the HerRC32-AAVR clone cell line. [Figure 12] Relative bioluminescence (RLU) in cells assayed 48 hours after transduction is shown. Plasmid or AAV9 transduction conditions at MOI (0, 10, 10, 10) are indicated on the x-axis. Each bar represents the average of triplicate wells, and error bars indicate standard deviation. [Figure 13] FIG. 1 is a schematic diagram of the nucleic acid comprising the response element operably linked to a reporter nucleotide sequence and the lentiviral backbone used in the studies described in Example 6. [Figures 14A-14B] Graph showing reporter expression (measured as relative light units, y-axis) in cells engineered to integrate a response element-reporter construct into the cellular genome in response to various multiplicities of infection (MOI, viral genomes / cell) of AAV vectors encoding regulatory factors. [Figures 15A-15C] Figures 15A and 15B correspond to data described in Example 6 for response element-reporter construct P-6. Figures 15A and 15B are graphs showing reporter expression (measured as relative light units, y-axis) in cells engineered to integrate a response element-reporter nucleic acid into the cellular genome in response to various multiplicities of infection (MOI, viral genomes / cell) of AAV vectors encoding regulatory factors. Figure 15A corresponds to luciferase expression mediated by the response element, and Figure 15B corresponds to firefly luciferase expression mediated by a constitutive promoter. Figure 15C is a schematic diagram of the P-6 construct. [Figure 16]This figure shows the dose-dependent induction of luciferase reporter activity by the regulator (here, SEQ ID NO: 84) in a clonal cell line that contains a response element-reporter construct stably integrated into the cell genome and is engineered to stably express AAVR. Relative efficacy measurements are presented by parallel line analysis. The full MOI range from the experiment is represented. [Figure 17] Figure 17 shows the dose-dependent induction of luciferase reporter activity by a regulator (here, SEQ ID NO: 84) in a clonal cell line containing a response element-reporter construct stably integrated into the cellular genome and engineered to stably express AAVR. Relative potency measurements are presented by parallel line analysis. Figure 16 includes the full MOI range from the experiment, while the relative potency analysis in Figure 17 was limited to four dose points in the best linear dose range for each cell line. [Figure 18A] eTF and a control eTF DNA-binding protein, Δz1 eTF, mutated in the DNA-binding alpha-helical zinc finger domain are shown. [Figure 18B] Dose response for increasing MOIs of eTF-expressing (triangles, AAV9-CBA-z1-eTF) or control Δz1 eTF-expressing (squares, AAV9-CBA-Δz1-eTF, SEQ ID NO: 96) AAV variants is shown. The reference standard (RS, SEQ ID NO: 94) is indicated by a circle. [Figure 19] Assay specificity for eTF expressing transgenes in matched control samples. Dose response for increasing MOI in assay control (AC, red, SEQ ID NO: 94) or reference standard sample (green, CBA-Δz1-eTF, SEQ ID NO: 96). Matched control Δz1 eTF-expressing AAV sample (blue, S20-1052 (underlined)) does not activate reporter expression. DETAILED DESCRIPTION OF THE INVENTION

[0008] The present disclosure provides materials and methods useful for characterizing the efficacy of AAV gene therapy preparations. Previous methods for estimating the efficacy of gene therapy vectors involved measuring viral genomes or empty viral capsids. While these methods are useful for characterizing the amount of nucleic acid in a preparation, they cannot accurately inform about the biological activity associated with the preparation. Gene therapy vectors exert their therapeutic effect by delivering a payload to cells, which then exert a biological effect (i.e., the payload either mediates the biological effect itself or encodes a protein or nucleic acid that mediates the biological effect). Simply quantitating vector genomes or empty capsids does not adequately capture gene therapy vector activity and function, i.e., these features that contribute to vector efficacy. The systems and methods described herein provide an accurate and elegant way to measure efficacy, thus enabling greater confidence in consistency between product batches and accurate patient dosing.

[0009] In one aspect, the present disclosure provides a nucleic acid comprising a response element operably linked to a reporter nucleotide sequence. A response element is a nucleic acid sequence that can be recognized and bound by a transcription factor. Transcription factors are generally proteins that control the rate of DNA transcription by binding to specific nucleotide sequences (e.g., response elements) and modulating expression, for example, by promoting or inhibiting the recruitment of RNA polymerase or other expression cofactors. Transcription factors typically contain at least one DNA-binding domain capable of binding to a transcription factor binding site in target DNA and a transcriptional regulatory domain containing binding sites for other proteins that promote or repress expression of the target nucleic acid sequence. Transcription factors can act through any one of a variety of mechanisms, including, but not limited to, stabilizing or blocking the binding of RNA polymerase to DNA, catalyzing the acetylation or deacetylation of histone proteins, or recruiting coactivator or corepressor proteins to the transcription factor-DNA complex. In various aspects, the transcription factor is a transcriptional activator (i.e., it promotes transcription of a nucleic acid sequence). In alternative aspects, the transcription factor is a transcriptional repressor (i.e., it reduces or blocks transcription of a nucleic acid sequence). Transcription factors (TFs) can be endogenous (i.e., those naturally expressed by the host cell) or exogenous (i.e., those recombinantly produced within the host cell and, optionally, engineered to contain one or more modifications compared to a wild-type transcription factor). TFs can be naturally occurring, modified from naturally occurring TFs, or non-naturally occurring synthetic TFs.

[0010] Examples of TF DNA-binding domain structures include, but are not limited to, helix-turn-helix, zinc finger, leucine zipper (e.g., bZIP), helix-loop-helix, and beta-backbone. TFs can be engineered to operably link a DNA-binding domain to a transcriptional regulatory domain to which it is not naturally linked (e.g., derived from a different transcription factor or a different species). For example, a zinc finger DNA-binding domain or a transcription factor-like effector DNA-binding domain can be fused to a transcriptional regulatory domain (e.g., VP16 or VP64). Alternatively, or in addition, a TF can contain an engineered DNA-binding domain specific for a TF binding site of interest. In this regard, a TF can contain multiple copies of the same DNA-binding domain or multiple DNA-binding domains of different sequences. For example, various embodiments of the present disclosure provide TFs with engineered DNA-binding sites comprising 2-10 DNA-binding domains, such as zinc fingers (e.g., 3-8 zinc fingers, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zinc fingers). Examples of engineered DNA-binding domains are provided as SEQ ID NOs: 85-90. See also International Patent Publication No. WO 2020 / 243651, which is incorporated herein by reference in its entirety.

[0011] Transcription factors can be active in most cell types. Transcription factors can also be tissue-specific, such as those from muscle cells (e.g., MyoD and muscle enhancer factor 2 (MEF2)) or neuronal cells (e.g., nuclear factor 1C (NF1C), nuclear factor 1X (NF1X), brain-1 (Brn-1), or brain-2 (Brn-2)). Transcription factors can also be ligand-dependent. Ligand-dependent transcription factors contain an additional domain that is bound by a ligand. The activity of a ligand-dependent transcription factor can depend on whether it is bound to its ligand. For example, a ligand-dependent binding factor can be a transcriptional repressor in the absence of a ligand and a transcriptional activator in the presence of a ligand. Steroid hormone receptors and nuclear receptors are examples of ligand-dependent transcription factors. Other examples of ligand-dependent transcription factors include metal-responsive transcription factors, such as those that regulate metal (iron, zinc, or copper) homeostasis. The transcription factors of the present disclosure may be ligand-dependent transcription factors, where the ligand is a metal such as iron, zinc, nickel, manganese, magnesium, potassium, sodium, molybdate, or copper. In one embodiment, the ligand of the ligand-dependent transcription factor described herein is copper. Metal-responsive transcription factors include, but are not limited to, Aft1, Aft2, Fep1, SREA, Urbs1, Ace1, Amt1, Srf1, Mac1, Cuf1, GRISEA, Crr1, Zap1, and metal-responsive element-binding transcription factor-1 (MTF-1). MTF-1 induces the expression of metallothionein and other genes involved in metal homeostasis in response to heavy metals such as copper. MTF-1 binds to transcription factor binding sites containing a DNA sequence motif known as a metal-responsive element (MRE), which has the core consensus TGCRCNC, where R is any purine (A or G) and N is any base (SEQ ID NO: 33). See, for example, Rutherford and Bird, Eukaryot Cell. 2004 Feb;3(1):1-13, and Wang et al., Biol Chem. 2004 Jul;385(7):623-32.

[0012] A transcriptional regulatory domain (TMD) is a region of a TF that contains binding sites for other proteins that promote or repress transcription of a target nucleic acid sequence. A TMD can contact the transcription machinery (e.g., RNA polymerase) directly or through other proteins (known as coactivators or comodulators). The TMD and DNA-binding domain (DBD) can be derived from different proteins. An engineered TF can contain two or more TMDs, and the two or more TMDs can be derived from (e.g., isolated from) different proteins. In various embodiments, the TMD is a transactivation domain that enhances or upregulates expression. Examples of transactivation domains include, for example, VP64 (SEQ ID NO: 76), VPR (SEQ ID NO: 77), VP16, VP128, p65, p300, CBP / p300-interacting transactivator 2 (CITED2) (SEQ ID NO: 78 or 79), CBP / p300-interacting transactivator 4 (CITED4) (SEQ ID NO: 80 or 81), EGR1 (SEQ ID NO: 82), or EGR3 (SEQ ID NO: 83). See also International Patent Publication No. WO 2019 / 109051, incorporated herein by reference in its entirety, particularly for disclosures related to transactivation domain sequences. In some aspects, the TMD is a repression domain that reduces or inhibits expression.

[0013] The DBD and TMD can be directly linked, for example, without an intervening amino acid sequence. Alternatively, the DBD and TMD can be linked via a peptide linker. In various embodiments, the DBD can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 75, 80, 90, or 100 amino acids, or 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 1-75, The DBD is conjugated to the TMD via a linker having 1-100, 5-10, 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 10-20, 10-30, 10-40, 10-50, 10-75, 10-100, 20-30, 20-40, 20-50, 20-75, or 20-100 amino acids. In some cases, the DBD and TMD are conjugated via naturally occurring intervening residues found in the naturally occurring protein from which the domain is derived or from another naturally occurring transcription factor. In other embodiments, the DBD and TMD are conjugated via a synthetic or exogenous linker sequence. Suitable linkers can be flexible, cleavable, non-cleavable, hydrophilic, and / or hydrophobic. In certain embodiments, the DBD and TMD can be fused together via a linker containing multiple glycine and / or serine residues. Examples of glycine / serine peptide linkers include [GS]n, [GGGS]n (SEQ ID NO:4), [GGGGS]n (SEQ ID NO:5), or [GGSG]n (SEQ ID NO:6), where n is an integer greater than or equal to 1. In various aspects, a linker useful for conjugating a DBD and a TAD is GGSGGGSG (SEQ ID NO:7). In various embodiments, when a DBD is conjugated to two TMDs, the first and second TMDs can be conjugated to the DBD with the same or different linkers, or one TMD can be conjugated to the DBD with a linker and the other TMD can be conjugated directly to the DBD (e.g., without an intervening linker sequence), or both TMDs can be conjugated directly to the DBD (e.g., without an intervening linker sequence).

[0014] Examples of transcription factors include AF-4 transcription factor, androgen receptor transcription factor, AP-2 transcription factor, ARID transcription factor, bHLH transcription factor, C / EBP transcription factor, CBF transcription factor, CG-1 transcription factor, COE transcription factor, COUP transcription factor, CP2 transcription factor, CSD transcription factor, CSL transcription factor, CTF / NFI transcription factor, CUT transcription factor, DM transcription factor, E2F transcription factor, EAF2 transcription factor, Ecdystd receptor transcription factor, ETS transcription factor, forkhead transcription factor, GCM transcription factor, GCR transcription factor, GTF2I transcription factor, HMG transcription factor, HMGI / HMGY transcription factor, homeobox transcription factor, HSF transcription factor, HTH transcription factor, IRF transcription factor, MBD transcription factor, MH1 transcription factor, MYB transcription factor, NDT80 / PhoG transcription factor, NF-YA transcription factor, NF-YB / C transcription factor, Nrf1 transcription factor, and nuclear orphan receptor transcription factor , estrogen receptor transcription factors, P53 transcription factors, PAX transcription factors, PC4 transcription factors, POU transcription factors, PPAR receptor transcription factors, PREB transcription factors, progesterone receptor transcription factors, Prox1 transcription factors, retinoic acid receptor transcription factors, RFX transcription factors, RHD transcription factors, ROR receptor transcription factors, Runt transcription factors, SAND transcription factors, SPZ1 transcription factors, SRF transcription factors, STAT transcription factors, T-box transcription factors, TEA transcription factors, TF-bZIP transcription factors, TF-Otx transcription factors, THAP transcription factors, thyroid hormone receptor transcription factors, TSC22 transcription factors, Tub transcription factors, ZBTB transcription factors, zf-BED transcription factors, zf-C2H2 transcription factors, zf-C2HC transcription factors, zf-GATA transcription factors, zf-LITAF-like transcription factors, zf-MIZ transcription factors, and zf-NF-X1 transcription factors.

[0015] The nucleic acids of the present disclosure contain response elements that are recognized and bound by transcription factors. Response elements contain one or more TF binding sites, which contain target nucleic acid sequences to which the DNA-binding domain of a TF can bind. Binding site motifs found within the genome for many transcription factors have been identified and characterized. See, e.g., Inukai et al., Curr Opin Genet Dev. 2017 Apr;43:110-119 (doi:10.1016 / j.gde.2017.02.007); Weirauch et al., "HOCOMOCO: expansion and enhancement of the collection of transcription factor binding site models." Cell. 2014;158:1431-1443; Kulakovskiy et al., "JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles." Nucleic Acids Res. 2016;44:D116-125; and Wingender et al., "The TRANSFAC project as an example of framework technology that supports the analysis of genomic regulation." Brief Bioinform. 2008;9:326-332.

[0016] A response element of the present disclosure may comprise a single TF binding site, or may comprise multiple TF binding sites. For example, a response element may comprise 2 to 10 copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies) of a TF binding site. When multiple TF binding sites are found within a response element, the multiple TF binding sites may all be the same (i.e., multiple copies of the same TF binding site sequence) or may be different (i.e., two or more of the TF binding sites have different nucleic acid sequences). Optionally, when multiple different TF binding sites are present, the different TF binding sites may be recognized by different TFs or the same TF.

[0017] In various embodiments, the response element comprises 2 to 10 copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies) of the Z1 transcription factor (TF) binding site. For example, the response element optionally comprises 3 to 8 copies of the Z1 TF binding site. The nucleic acid sequence of the Z1 TF binding site is provided as SEQ ID NO: 1. It is understood that TFs can recognize multiple DNA binding site sequences. See, e.g., Siggers et al., Nucleic Acids Res. 2014;42:2099-2111. Thus, the TF binding site can comprise one, two, or three nucleotide differences from SEQ ID NO: 1 (i.e., the TF binding site can comprise SEQ ID NO: 1 with substitutions at one, two, or three nucleotide positions within SEQ ID NO: 1). Thus, in various embodiments of the present disclosure, each copy of the Z1 TF binding site has the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence that has one, two, or three nucleotide differences from SEQ ID NO: 1. In this regard, each of the copies may comprise the sequence of SEQ ID NO: 1, a subset of the copies may comprise SEQ ID NO: 1, other copies may comprise SEQ ID NO: 1 with one or more substitutions, or each of the copies may comprise SEQ ID NO: 1 with one, two, or three substitutions. In some embodiments, the response element may comprise a sequence having at least 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 1.

[0018] In various embodiments, the response element comprises a TF binding site that is bound by an endogenous metal-dependent TF, such as a copper-dependent TF. For example, the response element can comprise 1-10 copies or 2-10 copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies) of a TF binding site that is bound by an endogenous metal-dependent TF. In various embodiments, the response element comprises one or more (e.g., two or more) copies of the MTF-1 TF binding site. For example, the response element optionally comprises 3-8 copies of the MTF-1 TF binding site. The nucleic acid sequence of the MTF-1 TF binding site is provided as SEQ ID NO:8. The TF binding site can comprise one, two, or three nucleotide differences from SEQ ID NO:8 (i.e., the TF binding site can comprise SEQ ID NO:8 with substitutions at one, two, or three nucleotide positions within SEQ ID NO:8). Thus, in various embodiments of the present disclosure, each of the copies of the MTF-1 TF binding site has the nucleic acid sequence of SEQ ID NO: 8, or a nucleic acid sequence that differs by 1, 2, or 3 nucleotides from SEQ ID NO: 8. In this regard, each of the copies may comprise the sequence of SEQ ID NO: 8, a subset of the copies may comprise SEQ ID NO: 8, other copies may comprise SEQ ID NO: 8 with one or more substitutions, or each of the copies may comprise SEQ ID NO: 8 with 1, 2, or 3 substitutions. In some embodiments, the response element may comprise a sequence having at least 80%, 85%, 90%, 95%, or 98% sequence identity (e.g., 100% identity) to SEQ ID NO: 8.

[0019] If desired, a response element containing multiple TF binding sites can include a spacer sequence between two or more of the TF binding sites. The spacer can be of any length, as long as TF binding and regulation of gene expression are not abolished. In some embodiments, the response element contains 2 to 10 copies of the Z1 TF binding site and further includes a spacer sequence between at least two of the Z1 TF binding site copies (optionally between each copy of the Z1 TF binding site). Examples of spacer sequences include the nucleic acid sequences of SEQ ID NO: 2 (CTCGAT) and SEQ ID NO: 3 (GATAGGGAGTAAACTCGA). In some aspects, the spacer sequence can include a sequence having at least 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 2 or 3.

[0020] In various aspects of the present disclosure, the response element-constituting nucleic acid comprises the sequence of any one of SEQ ID NOs: 10-15, 17-23, and 25. The present disclosure further contemplates nucleic acids that contain one, two, or three nucleotide differences relative to the sequence of any one of SEQ ID NOs: 10-15, 17-23, and 25. In some aspects, the response element comprising a nucleic acid can comprise a sequence having at least 80%, 85%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 10-15, 17-23, and 25.

[0021] Optionally, the response element further comprises a promoter that further drives expression in the host cell. In various aspects, the response element comprises two promoters.

[0022] A promoter can be native or non-native to the nucleic acid sequence to which it is operably linked, and can be native or non-native to a particular host cell. In various embodiments, a promoter can be a constitutive promoter, a tissue-specific promoter, or an inducible promoter. Examples of constitutive promoters include herpes simplex virus (HSV), thymidine kinase (TK), Rous sarcoma virus (RSV), simian virus 40 (SV40), mouse mammary tumor virus (MMTV), Ad E1A, and cytomegalovirus (CMV) promoters. Examples of inducible promoters include, but are not limited to, those from genes such as cytochrome P450 genes, heat shock protein genes, metallothionein genes, and hormone-inducible genes such as estrogen gene promoters. Another example of an inducible promoter is the tet promoter, which is responsive to tetracycline. An example of a tissue-specific promoter is a liver-specific promoter such as the HLP promoter. Additional examples of promoters include, but are not limited to, the LPL, HCR-hAAT, ApoE-hAAT, and LSP promoters. These promoters are described in more detail in the following references: HLP: Mcintosh J, et al, Blood 2013 Apr 25, 121(17):3335-44; LPL: Nathwani et al, Blood. 2006 April 1, 107(7):2653-2661; HCR-hAAT: Miao et al, Mol Ther. 2000; 1:522-532; ApoE-hAAT: Okuyama et al, Human Gene Therapy, 7, 637-645(1996); and LSP: Wang et al, Proc Natl Acad Sci US A. 1999 March 30, 96(7):3906-3910.

[0023] In some embodiments, the promoter is a minimal promoter. A minimal promoter is typically one that cannot drive expression without the presence of additional regulatory elements. An example of a minimal promoter suitable for use in the context of the present disclosure is minP (SEQ ID NO: 32). Other minimal promoters include, but are not limited to, a CMV minimal promoter, an hsp70 minimal promoter, a minimal promoter contained in a tetracycline response element, and a MinTk minimal promoter.

[0024] The nucleic acids of the present disclosure comprise a response element operably linked to a reporter nucleotide sequence. "Operably linked" refers to a functional linkage between nucleic acid or amino acid sequences. For example, "operably linked" means that a regulatory sequence is in the correct position and orientation relative to another nucleic acid sequence to exert its effect on the nucleic acid sequence (e.g., initiation of transcription). In this regard, the response element is operably linked to the reporter nucleotide sequence such that the response element can regulate the rate of expression or abundance of the encoded reporter. Generally, operably linked DNA sequences are contiguous and, optionally, but not necessarily, in the same reading frame.

[0025] "Reporter" refers to any biomolecule (e.g., a protein) that directly or indirectly produces a detectable signal. In various embodiments, the reporter nucleotide sequence encodes a photoprotein or an enzyme that produces bioluminescence. Examples of reporters include green fluorescent protein (GFP), green fluorescent protein variant (GFP10), enhanced GFP (eGFP), TurboGFP, GFPS65T, TagGFP2, mUKGEmerald GFP, superfolder GFP, GFPuv, destabilized EGFP (dEGFP), Azami Green, mWasabi, Clover, mClover3, mNeonGreen, NowGFP, Sapphire, T-Sapphire, mAmetrine, photoactivatable GFP (PA-GFP), Kaede, Kikume, mKikGR, tdEos, Dendra2, mEosFP2, Dronpa, blue fluorescent protein (BFP), eBFP2, azurite BFP, mTagBFP, mKalamal, mTagBFP2, shBFP, cyan fluorescent protein (CFP), eCFP, and Cerulian.CFP, SCFP3A, destabilized ECFP (dECFP), CyPet, mTurquoise, mTurquoise2, mTFPI, photoswitchable CFP2 (PS-CFP2), TagCFP, mTFP1, mMidoriishi-Cyan, aquamarine, mKeima, mBeRFP, LSS-mKate2, LSS-mKatel, LSS-mOrange, CyOFP1, Sandercyanin, red fluorescent protein (RFP), eRFP, mRaspberry, mRuby, mApple, mCardinal, mStable, mMaroonl, mGarnet2, tdTomato, mTangerine, mSt rawberry, TagRFP, TagRFP657, TagRFP675, mKate2, HcRed, t-HcRed, HcRed-Tandem, mPlum, mNeptune, NirFP, Kindling, far-red fluorescent protein, yellow fluorescent protein (YFP), eYFP, destabilized EYFP (dEYFP), TagYFP, Topaz, Venus, SYFP2, mCherry, PA-mCherry, Citrine, mCitrine, Ypet, IANRFP-AS83, mPapayal, mCyRFP1, mHoneydew, mBanana, mOrange, Kusabira Orange, Kusabira Orange 2, mKusabira Orange, mOrange2, mKOK, mKO2, mGrapel, mGrape2, zsYellow, eqFP611, Sirius, Sandercyanin, shBFP-N158S / L173I, near-infrared protein, iFP1.4, iRFP713, iRFP670, iRFP682, iRFP702, iRFP720, iFP2.0, mIFP, TDsmURFP, miRFP670, Brilliant Violet (BV) 421, BV605, BV510, BV711, BV786, PerCP, PerCP / Cy5.5, DsRed, DsRed2, mRFP1, Pocilloporin, Renilla GFP, MonsterReporter nucleic acids include, but are not limited to, GFP, paGFP, or phycobiliproteins, luciferase, LacZ, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, and beta-glucuronidase (GUS), as well as biologically active variants and fragments of the above. In various embodiments, the reporter nucleic acid encodes GFP, EGFP, mCherry, or luciferase.

[0026] Optionally, the nucleic acid may include a second reporter nucleotide sequence that is not operably linked to a response element. Any reporter, including any of the reporters described herein, is suitable for use as a second reporter. Examples of second reporter nucleotide sequences include, but are not limited to, nucleotide sequences encoding a luminescent protein (e.g., green fluorescent protein (GFP), enhanced GFP (EGFP), or mCherry) or an enzyme that generates bioluminescence (e.g., luciferase). The second reporter may be selected to have a signal that is complementary to the first reporter. By way of example, if the first reporter is a fluorescent reporter, the second reporter may be a fluorescent reporter with different excitation and / or emission wavelengths. In another example, if the first reporter is luciferase, the second reporter may be Renilla, or if the first reporter is alkaline phosphatase, the second reporter may be LacZ. The second reporter nucleic acid, in various aspects, is operably linked to a separate promoter (i.e., a promoter that is different from the promoter that is part of the response element, if the response element comprises a promoter). Suitable promoters include, but are not limited to, those described herein. Optionally, the separate promoter is a constitutive promoter. Examples of constitutive promoters include herpes simplex virus (HSV), thymidine kinase (TK), Rous sarcoma virus (RSV), simian virus 40 (SV40), mouse mammary tumor virus (MMTV), Ad E1A, and cytomegalovirus (CMV) promoters.

[0027] In various embodiments, the nucleic acids provided herein contain one or more additional regulatory elements (optionally in addition to a promoter), such as sequences associated with transcription initiation or termination, enhancer sequences, and efficient RNA processing signals. Exemplary regulatory elements include, for example, introns, enhancers, UTRs, stability elements, WPRE sequences, Kozak consensus sequences, post-translational response elements, microRNA binding sites, polyadenylation (polyA) signal sequences, or combinations thereof. Regulatory elements can function to regulate gene expression at the transcriptional, post-transcriptional, or translational stages of gene expression. At the RNA level, regulation can occur at the level of translation (e.g., stability elements that stabilize mRNA for translation), RNA cleavage, RNA splicing, and / or transcription termination.

[0028] In certain embodiments, the nucleic acid further comprises a polyA signal sequence. Suitable polyA signal sequences include, for example, an artificial polyA (PA75) having a length of approximately 75 bp (see, e.g., International Patent Publication No. 2018 / 126116), bovine growth hormone polyA, SV40 early polyA signal, SV40 late polyA signal, rabbit beta globin polyA, HSV thymidine kinase polyA, protamine gene polyA, adenovirus 5 Elb polyA, growth hormone polyA, or PBGD polyA. In exemplary embodiments, a polyA sequence suitable for use in the expression cassettes provided herein is hGH polyA (SEQ ID NO:27) or synthetic polyA (SEQ ID NO:28 or SEQ ID NO:91). In various embodiments, the polyA comprises the nucleic acid sequence of SEQ ID NO:91. Typically, the polyA signal sequence is operably linked to a reporter nucleic acid sequence.

[0029] The present disclosure further provides a cell comprising a nucleic acid described herein. The nucleic acid can be stably integrated into the genome of the cell or can be present on a separate expression vector construct. The cell can be from any organism (e.g., a prokaryotic cell, a eukaryotic cell, a bacterial cell, a plant cell, an algae cell, a fungal cell (e.g., a yeast cell), a mammalian cell, an animal cell (human or non-human), etc.). Mammalian cells include those isolated from or derived from, for example, humans, non-human primates (such as apes, chimpanzees, monkeys, and orangutans), companion animals (including dogs and cats), livestock (such as horses, cows, pigs, sheep, and goats), or other mammalian species, including, but not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc. The cell can also be isolated from or derived from any tissue. In various aspects, the cell is a central nervous system cell, a frontal cortex cell, a glial cell, a microglial cell, or a striatal cell. Examples of cells include Chinese hamster ovary (CHO) cells and their derivatives (e.g., CHO-K1, CHOpro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), human embryonic kidney 293 (HEK293) cells or their derivatives (e.g., HEK293T, HEK293-EBNA), green African monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa and derivatives such as HeRC32), human osteosarcoma epithelial cells U2-OS, and adenocarcinoma cells. Examples of such cells include, but are not limited to, human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, mouse embryonic fibroblast cells NIH3T3, mouse fibroblast cells L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human neuroblastoma cells SH-SY5Y, human liver cancer cells HepG2, mouse B-myeloma cells J558L, and baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0030] In some aspects, the cells are modified with an exogenous nucleic acid comprising a nucleotide sequence encoding a receptor that improves the transduction efficiency of an expression vector of interest. In this regard, the present disclosure provides cells modified with an exogenous nucleic acid encoding an adeno-associated virus receptor (AAVR). The cells are optionally engineered to stably overexpress AAVR. "Overexpression" refers to increasing the total amount of AAVR in the cell (i.e., the cell produces more AAVR than a matched, unmodified cell). The cells may or may not naturally express AAVR before modification. The AAVR may be wild-type AAVR or modified AAVR. Expression of AAVR enhances AAV infection of host cells, for example, by increasing the number of receptors on the cell surface, presenting AAVR with enhanced affinity for AAV coat proteins, or facilitating AAV entry into cells.

[0031] Wild-type AAVR is a predicted type I transmembrane protein. The protein contains a signal peptide, a MANSC domain (an N-terminal motif with seven cysteines), and five Ig-like domains (polycystic kidney disease (PKD) domains 1-5). The transmembrane domain is located C-terminal to the MANSC and PKD domains, followed by a cytoplasmic tail. For the structure of AAVR, see, for example, Summerford et al., Molecular Therapy, 24(4):663 (2016); Meyer et al., eLife 8:e44707 (2019); and International Patent Publication No. 2017 / 083423 (incorporated herein by reference in its entirety, particularly for its disclosure of AAVR structure, AAVR sequence, and variant AAVRs). The AAVR can be from any species, for example, a mammalian AAVR protein such as a rodent AAVR protein, a primate AAVR protein, a rat AAVR protein, a mouse AAVR protein, a pig AAVR protein, a bovine AAVR protein, a sheep AAVR protein, a rabbit AAVR protein, a dog AAVR protein, or a human AAVR protein. Preferably, the AAVR is human AAVR. The wild-type human AAVR amino acid sequence is provided herein as SEQ ID NO: 92. The AAVR can also be a variant AAVR, such as any of the variant AAVRs described in International Patent Publication No. 2017 / 083423, which is incorporated herein by reference.

[0032] Accordingly, the present disclosure provides cells comprising a nucleic acid comprising a response element described herein operably linked to a reporter nucleotide sequence, wherein the cells are engineered to stably overexpress AAVR, such as wild-type AAVR. The response element can comprise 2-10 copies of transcription factor (TF) binding sites (e.g., 3-8 copies or 3-6 copies of TF binding sites), although a single copy of a TF binding site is also contemplated. The TF binding site can comprise a sequence bound by an endogenous TF or an exogenous TF, as described above. In various aspects, the TF binding site is bound by a ligand-dependent TF, such as a metal-responsive TF (e.g., a copper-responsive TF, such as MTF-1, that recognizes a bond comprising SEQ ID NO: 8). In various aspects, the TF binding site comprises a sequence bound by an exogenous TF, such as an engineered TF comprising a modified DNA-binding site with 2-10 zinc fingers. In various aspects, the TF binding site comprises one or more Z1 TF binding sites. In this regard, the TF binding site, in various embodiments, comprises SEQ ID NO: 1. Alternatively, or in addition, the TF binding site is optionally recognized by an engineered DNA binding domain comprising a sequence selected from the group consisting of SEQ ID NOs: 85-90, and / or a peptide comprising one or more of the DNA binding domains (such as a peptide comprising the amino acid sequence of SEQ ID NO: 93).

[0033] In various aspects, the disclosure provides compositions comprising cells comprising a response element (optionally engineered to stably express AAVR) and a TF that binds to the response element. In this regard, the cells can comprise any one or more of the TFs described herein. The cells can naturally produce the TF (i.e., the TF is endogenous), or the TF can be exogenous to the host cell (i.e., expressed from an exogenous nucleic acid introduced into the cell). In various aspects, the TF is a transcriptional activator, although transcriptional repressors are also contemplated. As described above, exemplary transcription factors bind to the Z1 TF binding site. In this regard, the TF can comprise an engineered Z1 binding domain, such as a Z1 binding domain comprising SEQ ID NO: 1. Alternatively, the TF can bind to a TF binding site recognized by MTF-1. The TF can be a metal-responsive TF (e.g., a copper-responsive TF such as MTF-1). Optionally, the TF is MTF-1 or comprises the DBD of MTF-1.

[0034] Any of the nucleic acids described herein, including nucleic acids comprising a response element operably linked to a reporter nucleic acid, nucleic acids encoding a TF, etc., can be provided in an expression vector. An "expression vector" is any molecule or moiety that transports, transduces, or otherwise acts as a carrier for a heterologous polynucleotide. A vector can be an integrative or non-integrative vector, referring to the vector's ability to integrate a nucleic acid into the genome of a host cell. Examples of expression vectors include, but are not limited to, (a) non-viral vectors, such as nucleic acid vectors comprising linear oligonucleotides and circular plasmids; artificial chromosomes, such as human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs or PACs); episomal vectors; and transposons (e.g., PiggyBac); and (b) viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. In various embodiments, the expression vector is a viral vector. Viral vectors can be obtained by deleting all or part of the coding region from the viral genome, while leaving intact those sequences (e.g., terminal repeats) that may be necessary or advantageous for functions such as packaging the vector genome into a viral capsid.

[0035] The viral vectors of the present invention can be recombinantly produced and based on an adeno-associated virus (AAV) parent or reference sequence. AAV is a small, replication-deficient, non-enveloped animal virus that infects humans and several other primate species. AAV vectors can also infect both dividing and resting cells without integrating into the host cell genome. The AAV genome consists of a linear, single-stranded DNA approximately 4.7 kb in length. The genome consists of two open reading frames (ORFs) flanked by inverted terminal repeat (ITR) sequences approximately 145 bp in length. The ITRs consist of a nucleotide sequence at the 5' end (5' ITR) and a nucleotide sequence located at the 3' end (3' ITR) that contains a palindromic sequence. The ITRs function in cis by folding to form a T-shaped hairpin structure through complementary base pairing, which serves as a primer during the initiation of DNA replication for second-strand synthesis. The two open reading frames encode the rep and cap genes, which are involved in virion replication and packaging. In exemplary embodiments, the AAV vectors provided herein do not contain rep or cap genes. It should be understood that AAV-based vectors are typically packaged into viral particles that can infect host cells. Thus, as used herein, "AAV vector" encompasses an AAV viral particle that contains at least one AAV capsid protein and an encapsidated AAV polynucleotide containing a transgene.

[0036] Serotypes that may be useful in the context of the present disclosure include any of those derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrhlO, AAV-DJ, and AAV-DJ8. Serotypes generally differ in their tropism, or the cell type they infect. AAV may contain genomes and capsids from multiple serotypes (e.g., pseudotypes). For example, an AAV may contain a serotype 2 genome (e.g., ITRs) packaged in a capsid from serotype 5 or serotype 9. Pseudotype vectors may demonstrate improved transduction efficiency as well as altered tropism. In some cases, AAV serotypes that can cross the blood-brain barrier or infect cells of the CNS are preferred. The AAV can be a self-complementary AAV (scAAV). See, e.g., Raj et al., Expert Rev Hematol. 2011 Oct;4(5):539-549. In certain embodiments, the expression vector is an AAV vector comprising a 5' ITR and a 3' ITR. In some embodiments, the expression vector is an AAV vector comprising a 5' ITR, a promoter, a nucleic acid encoding a regulatory factor (e.g., a TF), and a 3' ITR. In some embodiments, the expression vector is an AAV vector comprising a 5' ITR, an enhancer, a promoter, a nucleic acid encoding a regulatory factor (e.g., a TF), a polyA sequence, and a 3' ITR. In some embodiments, the AAV vector comprises a nucleic acid comprising a response element operably linked to a reporter nucleic acid. In various embodiments, the AAV vector is an AAV9 vector or a scAAV9 vector. In various embodiments of the present disclosure, the AAV vector is an AAV3 vector. In various aspects of the present disclosure, the AAV vector is an AAV8 vector.

[0037] In some aspects, the disclosure provides a composition comprising (i) a cell comprising a response element, optionally engineered to stably express an AAVR, and (ii) a sample comprising an AAV. In some examples, the composition comprises (i) a HeRC32 cell comprising a response element, the response element comprising a TF-binding sequence comprising SEQ ID NO: 1 and a reporter, and (ii) a sample comprising an AAV comprising a nucleic acid sequence encoding a TF that binds to the sequence of SEQ ID NO: 1. In some examples, the HeRC32 stably expresses the AAVR of SEQ ID NO: 92. In some examples, the response element comprises the sequence of any one of SEQ ID NOs: 10-15 and 17-23. In some cases, the sample comprises an AAV comprising the nucleic acid sequence of SEQ ID NO: 94.

[0038] The present disclosure further provides a method for determining the efficacy of a sample containing an AAV vector. The AAV vector is described above. The method includes contacting a cell described herein with all or a portion of the sample. The cell includes a response element operably linked to a reporter nucleotide sequence and is engineered to stably overexpress AAVR. The AAV vector encodes a regulatory factor that directly or indirectly regulates expression of the reporter nucleotide sequence via the response element. The method further includes measuring expression of the reporter nucleotide sequence in the cell. In various embodiments, the method then includes determining the efficacy of the sample based on the measured expression level of the reporter nucleic acid sequence.

[0039] A "sample" containing an AAV vector of interest can be any type of sample suitable for characterizing the potency, typically the amount of AAV vector in a preparation suitable for vector manufacture, storage, or administration to a patient. The sample can contain any amount of AAV vector suitable for transducing cells to obtain a detectable signal from the reporter. For example, the sample can contain at least about 1 x 10 per mL of composition. 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6, 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , or 1×10 16 The sample may contain 100 AAV vectors. In various aspects, the sample is a composition of AAV vectors collected at one or more time points during the vector manufacturing or purification process. The sample may be a composition of AAV vectors harvested from a product batch prior to shipping. The disclosed methods may include other additional steps that may further increase the purity of the AAV, remove other undesirable components, and / or concentrate the fraction for testing. The disclosed methods are suitable, for example, for verifying the safety of AAV vector product lots, characterizing the dose of AAV vectors, assessing the activity of vector compositions, and assessing the stability of vector compositions (e.g., methods performed on the same sample at different time points) to demonstrate comparability of manufacturing variations and / or determining consistency between AAV product samples.

[0040] The AAV vector encodes a regulatory factor that directly or indirectly regulates expression of the reporter nucleotide sequence via a response element. In various aspects, the regulatory factor comprises a DNA-binding domain (DBD) that binds to a TF-binding domain within the response element and activates or inhibits expression of the reporter nucleotide sequence, either by binding the response element itself or by recruiting other proteins that affect expression. In some embodiments, the regulatory factor is a transcription factor that binds to a TF-binding site within the response element. Transcription factors are further described above. In an exemplary embodiment, the TF is an engineered TF that comprises a zinc finger domain that binds to a Z1-binding domain operably linked to a VP64 TMD.

[0041] The transcription factor can be any of the transcription factors disclosed herein or can include any component of the reference transcription factors referenced herein (e.g., the DNA-binding domain or transcription regulatory domain of the reference transcription factor). In exemplary aspects of the present disclosure, the heterologous nucleic acid encodes a transcription factor that upregulates SCN1A production and is any of the engineered transcription factors described in International Patent Publication No. WO 2020 / 243651 (incorporated herein by reference in its entirety). For example, in exemplary aspects of the present disclosure, an engineered transcription factor comprises a DNA-binding domain comprising a zinc finger motif having the following structure: LEPGEKP-[YKCPECGKSFS X HQRTH TGEKP]n-YKCPECGKSFS X HQRTH-TGKKTS (SEQ ID NO: 29), where n is an integer between 1 and 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, and each X is independently a recognition sequence (e.g., a recognition helix) capable of binding to 3 bp of a target sequence. In exemplary embodiments, n is 3, 6, or 9. In particularly preferred embodiments, n is 6. In various embodiments, each X can independently have the same or a different amino acid sequence compared to other X sequences within the DNA-binding domain. In exemplary embodiments, each X is a sequence comprising 7 amino acids that is designed to interact with 3 bp of a target binding site of interest using the Zinger Finger Design Tool from Scripps on the World Wide Web (scripps.edu / barbas / zfdesign / zfdesignhome.php). The engineered transcription factor optionally further comprises a VP64 transcriptional regulatory domain. In some cases, the transcription factor may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 35-75.

[0042] In alternative embodiments of the present disclosure, the regulator is a protein that regulates metal metabolism, and the cell contains a metal-responsive TF that binds to the response element. Exemplary metal-responsive transcription factors include, for example, Aft1, Aft2, Fep1, SREA, Urbs1, Ace1, Amt1, Srf1, Mac1, Cuf1, GRISEA, Crr1, Zap1, and MTF-1. The metal-responsive transcription factor can be endogenous to the cell or recombinantly produced by the cell. In various embodiments, the regulator is ATP7B, a copper-transporting P-type ATPase. The ATP7B protein is located in the trans-Golgi network in the liver and brain and balances copper levels in the body by excreting excess copper into bile and plasma. The amino acid sequence of ATP7B is provided as SEQ ID NO: 30. When expressed, ATP7B regulates intracellular copper levels, thereby indirectly regulating reporter gene expression via the metal-responsive TF. When copper levels are high, the TF (e.g., MTF-1) drives reporter expression by binding to a response element (e.g., a response element comprising one or more MTF-1 binding sites). When copper levels are low, reporter expression is also reduced. While the above description describes the method in the context of copper metabolism, it should be understood that the method is not so limited and can be used in connection with any ligand-dependent TF (including other metal-responsive TFs) and other regulatory factors that mediate the amount of ligand in a cell.

[0043] Reporter nucleic acids and their encoded proteins are described above. Methods for measuring the expression of reporter nucleotide sequences in cells are well known in the art. The amount of reporter RNA can be determined, or the amount of the resulting protein can be determined. Generally, reporter expression is characterized by measuring the activity or characteristics of the protein encoded by the reporter nucleotide sequence, i.e., by measuring the luminescence of a luminescent protein or measuring the luminescence mediated by an enzyme that generates bioluminescence. Luminescence activity can be determined by any suitable method, such as the method described in Inouye, S. & Shimomura, O. (1977) Biochem. Biophys. Res. Commun. 233, 349-353. In practice, fluorescence can be detected and quantified, for example, using flow cytometry, and / or reporter mRNA levels can be quantified via RT-qPCR. The amount of signal or expression product generated by the reporter can be used to derive quantifiable absolute or relative data regarding the efficacy of the vector preparation being tested.

[0044] In various aspects, the method includes determining the titer of the sample based on the measured expression level of the reporter nucleic acid sequence or expression product. In this regard, the method optionally includes comparing the measured expression level to a standard potency curve for the AAV vector. The standard curve is generated by evaluating the reporter signal for a specific amount of AAV vector (i.e., different dilutions of vector concentration) using multiple replicates. Alternatively, the standard curve can be generated by evaluating the signal measured for a specific amount of reporter nucleic acid sequence DNA, mRNA, or expression product.

[0045] The present disclosure further provides systems or kits comprising any combination of the components described herein. In exemplary embodiments, the systems or kits comprise one, two, or all of: (A) a nucleic acid of the present disclosure comprising a response element operably linked to a reporter nucleic acid sequence; (B) cells overexpressing AAVR; and / or (C) a regulatory factor that directly or indirectly affects reporter expression via the response element. As described herein, a regulatory factor may directly contact the response element to activate or repress transcription of the reporter nucleic acid. Alternatively, a regulatory factor may indirectly affect transcription of the reporter nucleic acid via the response element, for example, by regulating the amount of a ligand in the cell, thereby regulating the activity of a ligand-dependent transcription factor that binds to the response element in (A). The regulatory factor may comprise, for example, a protein or a nucleic acid comprising a sequence encoding a protein. Accordingly, the present disclosure provides a kit comprising cells overexpressing AAVR as described herein and a nucleic acid comprising a response element operably linked to a reporter nucleotide sequence, wherein the response element comprises 2 to 10 copies of a TF binding site (e.g., a Z1 TF binding site). The kits can include instructions for use of the components in a method, e.g., a method according to the present disclosure. Ancillary materials that aid in or enable such methods to be performed can be included in the kits of the present disclosure. [Example]

[0046] The following examples are presented merely to illustrate the present invention and are not intended to limit its scope.

[0047] Example 1 This example describes the construction and testing of different nucleic acid constructs that contain a response element operably linked to a reporter nucleic acid.

[0048] Several reporter constructs encoding N-luciferase ("Nluc") operably linked to different response elements were constructed using standard molecular biology techniques. A summary of the components of each reporter construct is provided in Table 1, and the structures of the different reporter constructs are shown in Figure 1. The response element (RE) description includes the number of repeats (e.g., "8x" is 8 copies) of a particular TF binding domain (e.g., the Z1 TF binding domain).

[0049] Figure 1A details the linked response element design found in the class I synthetic reporter constructs. The Z1 response element is activated by VP64 and / or CITED4 binding found in the class I synthetic reporter constructs. Construct P1 contains the Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by a minimal CMV promoter. The second class of response elements (class II) consisted of multiple Z1 sequences inserted into a tetracycline-responsive element (TRE) scaffold. Figure 1B details the response element:minPro design found in several of the class II synthetic reporter constructs, namely, P13, P12, and P22. Figure 1C details the response element:endogenous SCN1A target / promoter domain found in the genomic reporter constructs. Figure 1D details the hybrid design found in several reporter constructs, namely, P15, P16, and P14.

[0050] [Table 1]

[0051] The functionality of each reporter construct was tested as follows. HEK293T cells were seeded at approximately 10,000 cells per well into individual wells of a 96-well plate. Using FuGENE® HD Transfection Reagent (Promega, Madison, WI), each well of cells was transfected with three plasmids totaling 100 ng. Each well of cells was triply transfected with (a) a constitutive transfection control plasmid (20 ng), (b) a reporter plasmid (40 ng) listed in Table 1 or shown in Figure 1, and (c) an activator plasmid or control filler plasmid (40 ng) (total of 100 ng of plasmid). (a) The constitutive transfection control plasmid included a plasmid expressing firefly luciferase under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter element (TK-Firefly). (b) The reporter constructs contained one of the Z1-based response elements listed in Table 1 and Figure 1 or lacked a Z1 response element (indicated by "delta" in the name of the response element in Table 1). When present, the Z1 TF-binding domain (SEQ ID NO: 1) was present in one to eight tandem copies (represented by "1x," "3x," "6x," or "8x"). (c) The activator plasmid encoded an artificial transcription factor activator that bound to the Z1 TF-binding domain (amino acid sequence SEQ ID NO: 84) of the reporter construct. The control filler plasmid did not contain the artificial transcription factor activator but encoded the non-bioluminescent enhanced green fluorescent protein (EGFP). Each well was expected to show a baseline fluorescence signal due to the constitutive expression of firefly luciferase in this transfection control plasmid if the cells were successfully transfected. Any fluorescence signal above the baseline was due to N-luciferase in the reporter construct. An additional luciferase signal was expected only if the reporter construct contained a Z1 response element and the activator plasmid was cotransfected into the cells.If the reporter construct did not contain a Z1 response element (those with "delta Z1") or was cotransfected with a control filler plasmid (which did not encode a transcription factor activator), cells were not expected to show a fluorescence signal above baseline.

[0052] After transfection (24 hours post-transfection), plates were lysed and diluted 1:100 in ONE-Glo EX Luciferase Assay Buffer (Promega). Diluted lysates were assayed for firefly and NanoLuc luminescence according to the manufacturer's protocol (Nano-Glo Dual Luciferase Reporter Assay system, Promega), and relative light units (RLU) were measured using a bioluminescence plate reader (GloMax, Promega). Each transfection was performed in triplicate, and data reflect the average of triplicate wells (mean ± standard deviation).

[0053] The results are shown in Figure 2. All cells exhibited similar baseline fluorescence signals due to expression of the firefly luciferase gene (left bars for each construct in Figure 2), indicating successful transfection of the cells. When cells were cotransfected with a reporter construct containing a Z1-based response element and an activator plasmid, fluorescence signals above baseline were observed (right bars for the reach construct in Figure 2). The strongest Nluc reporter induction was observed with the class I and class II synthetic variants, while the genomic and hybrid elements showed less upregulation from activator cotransfection (Figure 4). Higher baseline and induction activity was generally observed in the class I synthetic variants compared to the class II variants. Based on this initial screening, a subset of synthetic variants was selected for further screening: P-5, P-9, P-10, P-11, P-12, and P-13.

[0054] These data demonstrated activator-dependent luciferase reporter induction of a reporter construct containing a Z1 response element in a plasmid transfection system.

[0055] Example 2 This example demonstrates dose-dependent luciferase reporter induction in a nucleic acid containing a response element operably linked to a reporter nucleic acid in a transient co-transfection system.

[0056] The lead reporter construct from Example 1 was selected for further testing. In one experiment, the sensitivity of class I and class II synthetic reporter constructs to the amount of activator plasmid was assayed. Briefly, HEK293T cells were aliquoted into wells of a 96-well plate and subsequently transfected with three types of plasmids as described in Example 1. However, unlike Example 1, various amounts of activator plasmid were used. Each well of cells received 20 ng of constitutive transfection control plasmid, 10 ng of reporter construct, and a total of 70 ng of the combination of activator plasmid and control filler plasmid. To evaluate the sensitivity of the reporter construct to various amounts of activator plasmid, different amounts of activator plasmid (0, 0.01 ng, 0.1 ng, 1 ng, 10 ng, 30 ng, 50 ng, or 70 ng) were titrated into samples containing the filler plasmid, such that a total of 70 ng of activator and filler plasmid was used for transfection. Cells were treated after transfection, and luminescence levels were measured as described in Example 1. Each transfection was performed in triplicate and data reflect the average of triplicate wells (mean±standard deviation).

[0057] The results are shown in Figure 3. Each reporter construct exhibited sensitivity to different amounts of activator, demonstrating a dose-response result, with higher activity in response to higher amounts of activator plasmid. Similar to previous observations, class I response elements produced stronger expression levels than class II elements, and a broader range in signal induction was also observed with increasing copies of the z1 element. Therefore, lead candidate reporter variants with the highest number of z1 element copies were selected from each response element class, P-5 and P-13.

[0058] Example 3 This example demonstrates that induction of reporter expression in nucleic acids of the disclosure requires binding of a response element by an activator in a transient co-transfection system.

[0059] HEK293T cells were cultured using standard methods, and each well of a 96-well plate was triple-transfected (Fugene HD) with a total of 100 ng of plasmid: a constitutive transfection control plasmid (20 ng), a reporter plasmid (40 ng), and an activator plasmid (40 ng). The constitutive transfection control was a plasmid expressing firefly luciferase under the control of the HSV thymidine kinase (TK) promoter element (TK-Firefly). The control reporter plasmid contained a tetracycline-responsive element (TRE) upstream of the Nluc open reading frame ("Δreporter"). The Z1-based reporter plasmid contained a Z1-responsive element upstream of the Nluc open reading frame (7xZ1-TRE, P-5). In this element, multiple Z1 sequences were inserted into a tetracycline-responsive element (TRE) scaffold (Gossen and Bujard 1992). The activator plasmids expressed either an artificial transcription factor activator targeting the Z1 18-bp DNA element (Z1 eTF, SEQ ID NO: 84) or a transcription factor activator that does not target the Z1 18-bp DNA element ("ΔZ1 eTF," SEQ ID NO: 95). Twenty-four hours after transfection, cells were lysed and diluted 1:1000 in ONE-Glo EX luciferase assay buffer (Promega). The diluted lysates were assayed for firefly and NanoLuc luminescence according to the manufacturer's protocol (Nano-Glo Dual Luciferase Reporter Assay system, Promega), and relative light units (RLU) were measured using a bioluminescence plate reader (GloMax, Promega). Each transfection was performed in triplicate, and data reflect the average of triplicate wells (mean ± standard deviation).

[0060] Figure 4 shows that no significant signal was observed with either the reporter alone, the reporter and a non-targeted transcription factor (reporter + ΔZ1 eTF), or the TRE response element and a Z1-targeted transcriptional activator (ΔReporter + Z1 eTF). A strong response was observed only in the condition containing the Z1 element-containing reporter and the Z1-targeted transcriptional activator (reporter + Z1 eTF), indicating a specific interaction between the Z1 eTF and the Z1 element.

[0061] Example 4 This example describes the generation of reporter constructs containing a Z1-based response element with a single reporter (enhanced green fluorescent protein (EGFP) coding sequence) and a dual reporter system.

[0062] Single reporter constructs containing a single enhanced GFP-encoding cassette (sPA = polyA signal sequence) and dual reporter constructs containing EGFP-encoding cassettes and mCherry-encoding cassettes were generated using standard techniques. Schematics of the different single and dual constructs are shown in Figure 5.

[0063] Single reporter constructs were assayed as follows: HEK293T cells were cultured using standard methods, and each well of a 96-well plate was triply transfected with reporter plasmid (50 ng) and either activator plasmid (50 ng) or non-fluorescent plasmid filler (50 ng) (100 ng total plasmid) (FugeneHD). The Z1-based reporter plasmid (P-47) contained a Z1 response element (6×Z1-mPro) upstream of the EGFP open reading frame. This 6×Z1-mPro element contained six 18-bp Z1 target DNA sites with a 5-bp spacer sequence linked upstream of a minimal promoter element (mPro). The control reporter plasmid (P-48) was identical to P-47, except that the 18-bp Z1 target element was replaced with an alternative 18-bp sequence (Δ6×Z1-mPro). Twenty-four hours after transfection, plates were imaged for EGFP and mCherry fluorescence (ImageXpress DLR, Molecular Devices). Fluorescence was detected only in cells transfected with a Z1-based reporter plasmid (6xZ1-mPro) operably linked to the EGFP open reading frame (P-47). No fluorescence was observed using a control plasmid lacking an intact Z1 response element.

[0064] The dual reporter construct was assayed similarly as described above for the single reporter construct, except that the Z1 reporter and control plasmids also contained a second expression cassette containing an mCherry element under the control of the constitutive EF1a short (EFS) promoter. To enable independent expression, this constitutive expression cassette was separated from the reporter expression cassette by an insulator sequence containing the human beta-globin transcription termination sequence (hACTB) and the chicken beta-globin insulator (cHS4). mCherry-based fluorescence was detected in the samples tested, demonstrating that the second reporter nucleic acid under the control of a separate promoter functions in the context of the disclosed system. As with the single reporter assay, EGFP-based fluorescence was detected only in cells (P-47) transfected with the Z1-based reporter plasmid (6xZ1-mPro) operably linked to the EGFP open reading frame. EGFP fluorescence was not observed using a control plasmid lacking an intact Z1 response element.

[0065] Example 5 This example describes the generation and characterization of cells stably expressing the wild-type AAV receptor (AAVR). The in vitro transduction efficiency of AAV9 is low in immortalized cell lines. To address this, cell lines were engineered using AAVR to significantly enhance AAV9 transduction.

[0066] HEK-293T (ATCC), HeLa-RC32 cells (ATCC), CHO-Lec2 (ATCC), and all derivatives were grown in medium supplemented with 10% fetal calf serum (FCS) (Sigma, St. Louis), 3% L-alanyl-L-glutamine (Corning), and 1% NEAA (Corning) in a humidified incubator at 37°C with 5% CO. Purified and titrated stocks of adeno-associated virus (AAV) serotypes 1, 3, 5, 6, 8, 9, and DJ were produced in-house or purchased from Vector Biolabs. All AAV stocks were ssDNA AAV vectors encoding a reporter (GFP).

[0067] Stable AAVR-expressing cell lines were generated using lentiviral AAVR vectors. Recombinant lentiviral vectors containing the AAVR coding sequence were generated in HEK293T cells using a construct containing the nucleic acid sequence of SEQ ID NO: 31 and lentiviral packaging plasmids (pMD2.G and psPAX2) according to the protocol from Chimera Bioengineering. The vector was collected in the cell supernatant 48 hours after transfection and seeded with each cell line to generate stable heterogeneous populations of AAVR-overexpressing cells. Lentivirus-positive cells were isolated using puromycin selection. Once AAVR overexpression in the heterogeneous population was confirmed by Western blot, the cells were single-cell sorted into 96-well plates using a BD FACS Aria II. The resulting single subclones were grown for 14 days and screened for AAV transduction efficiency using AAV9-CBA-GFP. The top clones from each cell line were expanded and frozen.

[0068] Cells stably expressing AAVR were seeded overnight at 10,000 cells / well (96-well plates) or 100,000 cells / well (24-well plates). Cells were then infected with AAV stocks at a multiplicity of infection (MOI) of the specified viral genome / cell in complete DMEM. Viral infectivity was determined 48 hours postinfection by measuring transgene expression via either flow cytometry (fluorescence) or mRNA levels (RT-qPCR).

[0069] Flow cytometry: To measure GFP expression after AAV-GFP infection, cells were trypsinized 48 hours postinfection and passed through a BD FACS Melody to detect fluorescent cells. Uninfected cells were used as a negative control. Two parameters were assessed: the percentage of cells that were GFP-positive (% infected) and the mean fluorescence intensity (mean fluorescence per cell).

[0070] RNA extraction and qPCR: RNA was extracted from each cell pellet using the RNAeasy Mini Kit (Qiagen) according to the manufacturer's instructions and then used to synthesize cDNA using SuperScript IV Reverse Transcriptase (Invitrogen). Quantitative PCR was performed using lamin A / C, lamin A, or lamin C-specific primer sets to assess the expression levels of each gene.

[0071] Western blot: 2 x 10 cells 6Cell pellets were lysed in Laemmli SDS sample buffer containing 5% β-mercaptoethanol and boiled at 95°C for 10 min. Lysates were separated by SDS-PAGE using a Mini-Protean system (Bio-Rad) on a 4-15% polyacrylamide gradient gel (Bio-Rad). Proteins were transferred to nitrocellulose membranes (Bio-Rad) in semi-wet preparations using a Bio-Rad Transblot protein transfer system. The membranes were blocked by incubating with 1x PBS buffer containing 5% nonfat milk at room temperature (RT) for 1 h. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies diluted 1:1000 (anti-KIAA0319L antibody) or 1:2000 (anti-GAPDH antibody) in blocking buffer. The membrane was washed three times for 5 min with wash buffer (1x PBS buffer supplemented with 0.1% Tween-20) and further incubated in HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit - 1:5000 in blocking buffer) (GeneTex) for 1 h at RT. After an additional series of three washes, antibody-bound AAVR (a band at 150 kD is expected) was visualized using a chemiluminescence reader.

[0072] RNA extraction, cDNA generation, and two-color qPCR: RNA was extracted from each infected cell (24-well format) using the RNAeasy Mini Kit (Qiagen) according to the manufacturer's instructions and then used to synthesize cDNA using the SuperScript™ VILO™ cDNA Synthesis Kit with ezDNase™ enzyme (Invitrogen). Two-color quantitative PCR was performed using TaqMan Fast Advanced Mastermix and a VP64 or GAPDH (housekeeping) probe. -ddCt was calculated to determine the relative fold change.

[0073] result Figures 6A and 6B show the results of AAVR overexpression in HEK293T cells and HeLa-RC32 cells. AAV9-EF1a-GFP-KASH was delivered at a multiplicity of infection (MOI) of 50,000. Cells were harvested 48 hours postinfection, and the tested samples contained 30,000 cells. AAVR overexpression resulted in an 8- to 10-fold increase in GFP-positive cells (i.e., AAV+-transduced cells) (Figure 6A) and an approximately 3-fold increase in GFP expression (Figure 6B, MFI = mean fluorescence intensity). Figures 7A and 7B show the results of AAVR overexpression in HEK293T cells and CHO-Lec2 cells. AAV9-EF1a-GFP-KASH was delivered at a multiplicity of infection (MOI) of 10,000. Cells were harvested 48 hours postinfection, and the tested samples contained 30,000 cells. While AAVR overexpression resulted in a 4-fold increase in GFP-positive CHO-Lec2 cells, engineered HEK-293T cells showed a 35-fold higher effect at this higher MOI, using the CAG promoter to drive GFP expression. Cells expressed significantly higher GFP on average. Subclones of engineered cells that stably expressed AAVR demonstrated enhanced AAV9 transduction. See, for example, Figures 8A and 8B for HEK293T cell subclones. Similar results were observed for HeLa-RC32 and CHO-Lec2 subclones. Enhanced infectivity was demonstrated for multiple AAV serotypes. See Figures 10A–10D. HEK293T and HeLa-RC32 cells stably overexpressing wild-type AAVR were exposed to different serotype AAV vectors encoding GFP, and AAV infection rates were examined. Infectivity was enhanced with AAV-1, AAV-3, AAV-5, AAV-8, AAV-9, and AAV-DJ in both cell types when stably expressing AAVR, with AAV-3, AAV-8, and AAV-9 demonstrating approximately a 10-fold improvement in transduction. Mean fluorescence intensity data complemented the transduction data (Figures 10C-10D) and demonstrated a correlation between AAV infection rates and transduced gene expression.

[0074] Example 6 This example demonstrates the generation of cells stably overexpressing AAVR. HeRC32-AAVR was engineered to significantly enhance AAV9 transduction. This cell line stably expresses AAVR, a cellular receptor identified as an essential host factor for AAV transduction.

[0075] Specifically, the HeRC32-AAVR cell line was generated by stable transduction of the HeRC32 cell line (ATCC, catalog number CRL-2972) with a lentivirus (P-64) overexpressing the human AAVR gene under the EFS promoter. Positively transduced cells were selected using puromycin, and the resulting heterogeneous population was single-cell sorted to isolate clonal populations overexpressing AAVR. These clones were screened based on AAV9 transduction efficiency. Each cell line was infected with AAV9-CBA-GFP at an MOI of 100,000 for 48 hours and analyzed for GFP expression by flow cytometry. The percentage of GFP-positive cells was used to measure AAV transduction. As shown in Figure 11, the selected clones demonstrated greater than 50-fold transduction compared to the parental HeRC32 cell line.

[0076] Candidate reporter cell lines were then generated by stable transduction of HeRC32-AAVR cells with lentiviruses containing three reporter transgenes: 1) P-6, which contains 7x concatenated Z1 response elements in a V1 dual reporter format; 2) P-8, which contains 7x concatenated Z1 response elements in a V2 standalone reporter format; or 3) P-13, which contains a Z1-substituted TRE response element in a V2 standalone reporter format. The V1 dual reporter format contains a dual expression vector in which Nluc is expressed under the control of the Z1 response element and firefly luciferase is expressed under the control of an independent ubiquitous promoter element, allowing for internal regulation. The resulting heterologous stable cell lines (designated HeRC32-AAVR-P-6, HeRC32-AAVR-P-8, and HeRC32-AAVR-P-13) were then further evaluated for reporter expression and assay viability.

[0077] Initial assays were performed with the HeRC32-AAVR-P-8 heterologous cell line. These cells were seeded in 96-well plates at an initial density of 10,000 cells / well, and AAV encoding the eTF activator (SEQ ID NO: 84) was added over a three-point MOI series of 1E4 to 1E6 genome copies / cell. As a positive control condition, the reporter cell line alone was transfected with the activator (eTF, SEQ ID NO: 84) plasmid alone or co-transfected with the reporter plasmid in either the initial screening vector (P-5) or the lentiviral packaging plasmid (P-8).

[0078] Cells were assayed 48 hours after eTF activator (SEQ ID NO: 84) transduction. Luciferase assays were performed as described above, and cell lysates were not diluted to account for the lower overall signal due to the reduced reporter copy number and AAV transduction.

[0079] As shown in Figure 12, strong reporter induction above baseline was observed in the reporter plasmid transfection conditions (P-5 or P-8) and the activator (eTF) plasmid transfection conditions, and as expected, the response to infection with AAV encoding the eTF activator (SEQ ID NO: 94) increased with increasing MOI.

[0080] Example 7 This example demonstrates the generation of cells that stably overexpress AAVR and contain a response element-reporter nucleic acid stably integrated into the cellular genome. In particular, a luciferase reporter-expressing stable cell line (AAVR-reporter HeLa) was generated using a lentiviral vector to integrate a nucleic acid of the present disclosure into the genome of HeLa-AAVR cells, which constitutively express the AAVR gene.

[0081] Different lentiviral constructs were generated as shown in Figure 13. One backbone (V2) contained the response element-reporter cassette, and another backbone (V1) contained the response element-reporter cassette along with an additional reporter cassette. The constructs contained a Z1-based response element (6xz1-mPro or 7xz1-TRE (P-8)) upstream of the luciferase open reading frame. A third Z1-based response element (P-6) was constructed containing the same response element as P-8 (7xz1-TRE-Nluc) but also contained a second expression cassette containing a firefly luciferase element under the control of the constitutive EF1a short (EFS) promoter. The response element-reporter cassette was flanked by cHS4 insulator elements, and this entire cassette was expressed in the lentiviral expression cassette in the reverse orientation, such that it was antisense to the lentiviral cassette. For response element-reporter cassette P-6, a constitutive expression cassette encoding firefly luciferase was separated from the response element-Nluc cassette by insulator sequences including the human beta-globin transcription termination sequence (hACTB) and the chicken beta-globin insulator (cHS4).

[0082] Lentivirally transduced AAVR-reporter HeLa cells were cultured as a heterogeneous population by standard methods. Cells were transduced at 1 x 10 6 cells / ml with an AAV expressing the Z1-targeted transcription factor (SEQ ID NO: 94) as a reference standard (RS). 4 , 3×10 4 , 1×10 5 , 3×10 5 , 1×10 6 , 3×10 6Cells were transduced at various multiplicities of infection (MOI) of 1000-150 ...

[0083] The results are illustrated in Figures 14A-14B and 15A-15B. As expected, eTF activator (SEQ ID NO: 94) dose-dependent Nluc expression was observed across all three cell lines. The dual reporter line, HeRC32-AAVR-P-6, produces constitutive expression of firefly luciferase, which showed only a minimal response to eTF activator (SEQ ID NO: 94) dose. To examine whether these dose responses could be used to assess the relative potency of dose responses under different dilution conditions, linear regression was applied to each dose-response curve, and exploratory parallel line analysis was performed across the three test samples against a reference standard using log10 MOI and log10 RLU values ​​(Figures 16 and 17). Generally, variability was observed in the linearity of the dose response across cell lines and experiments, but the response in each cell line can be described by a linear relationship between Log10 MOI and Log10 RLU. Table 2 summarizes the relative potency results of the parallel line analysis and provides proof of concept for the feasibility of relative potency measurements in reporter cell lines.

[0084] [Table 2]

[0085] Table 2: Summary of relative potency in samples tested in candidate cell lines by parallel line analysis (PLA): assay control (AC), 40% relative potency sample (40%), 70% relative potency sample (70%) across two independent experiments.

[0086] To confirm reporter specificity for AAV in the final HeRC32-AAVR-reporter cell line, reporter responses were compared between AAV9 carrying a z1-targeting eTF under the control of the strong, ubiquitous CBA promoter and a control AAV (AAV9-CBA-Δz1-eTF) that is identical except for a sequence mutation in the z1-targeting DNA-binding domain of the eTF. The AAV9-CBA-z1-eTF construct should produce a robust dose response because it expresses the Z1-targeting eTF transgene. In contrast, the DNA-binding mutant vector (AAV9-CBA-Δz1-eTF) should not produce reporter activity because the reporter response element cassette contains the specific z1 eTF target sequence. In addition, a reference standard control sample containing a z1-targeting eTF expressed under the control of a GABA-selective promoter element (SEQ ID NO: 94) was included. Due to the strength of the CBA promoter compared to GABA-selective promoters, the AAV9-CBA-z1-eTF construct was expected to produce stronger induction. Therefore, a full 10-point dose curve was included to capture the linear range of all samples. Robust dose-dependent reporter activation was observed in response to AAV9-CBA-z1-eTF (see Figure 16B (triangles)). The dose response showed a leftward shift compared to the reference standard (circles), due to the much stronger CBA promoter. In contrast, the control AAV9-CBA-Δz1-eTF sample (squares) did not activate reporter activity. Together, these data demonstrate assay specificity for eTF activators and the requirement for sequence-dependent eTF-DNA target interactions for reporter activation.

[0087] Additionally, to further confirm assay specificity, a control AAV9 vector identical to SEQ ID NO: 94 was generated using the same promoter and vector design as SEQ ID NO: 94, except for mutated sequences in the six 7-amino acid zinc finger DNA-binding domains that define the 18-bp target DNA sequence. In contrast to the reference standard or assay control (AC) vector, this control sample did not activate reporter activity (see Figure 17). Thus, this assay is specific for the eTF of SEQ ID NO: 84.

[0088] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0089] In the context of describing the present disclosure (particularly in the context of the claims that follow), the use of the terms "a," "an," and "the" and similar referents should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context, and the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The term "or" should be understood to include items in the alternative or together unless the context clearly requires otherwise. The term "and / or" should be understood to include each item in a list (individually), any combination of items in a list, and all items in a list together. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise stated. The present disclosure contemplates embodiments described as "comprising" a feature to include embodiments "consisting of" or "consisting essentially of" the feature.

[0090] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value that falls within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated herein as if it were individually recited herein.

[0091] The steps of all methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0092] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the present disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Indeed, features of the invention described herein, regardless of whether a combination of features is designated as an aspect or embodiment of the invention, can be recombined into additional embodiments that are also contemplated as aspects of the invention. The entire document is intended to be linked as a unified disclosure, and it should be understood that all combinations of features described herein (even if described in separate sections) are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of the specification.

[0093] [Table 3-1]

[0094] Table 3-2

[0095] Table 3-3

[0096] Table 3-4

[0097] Table 3-5

[0098] Table 3-6

[0099] Table 3-7

[0100] Table 3-8

[0101] Table 3-9

[0102] Table 3-10

[0103] Table 3-11

[0104] Table 3-12

[0105] Table 3-13

[0106] Table 3-14

[0107] Table 3-15

[0108] Table 3-16

[0109] Table 3-17

[0110] Table 3-18

[0111] Table 3-19

[0112] Table 3-20

[0113] Table 3-21

Claims

1. A nucleic acid comprising a response element operably linked to a reporter nucleotide sequence, said response element comprising 2 to 10 copies of a Z1 transcription factor (TF) binding site.

2. The nucleic acid of claim 1, wherein the response element comprises 3 to 8 copies of the Z1 TF binding site.

3. 3. The nucleic acid of claim 1, wherein each of the copies of the Z1 TF binding site has the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence that differs from SEQ ID NO: 1 by 1, 2, or 3 nucleotides.

4. The nucleic acid of any one of claims 1 to 3, wherein the response element comprises a spacer sequence between at least two copies of the Z1 TF binding site.

5. The nucleic acid of claim 4, wherein the spacer sequence comprises the sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

6. The nucleic acid according to any one of claims 1 to 4, comprising any one of the sequences of SEQ ID NOs: 10 to 15, 17 to 23, and 25.

7. The nucleic acid of claim 1 or 2, wherein the response element further comprises a promoter.

8. The nucleic acid of claim 7 , wherein the promoter is a minimal promoter.

9. 9. The nucleic acid of claim 8, wherein the minimal promoter is any one of a CMV minimal promoter, an hsp70 minimal promoter, a minimal promoter contained in a tetracycline response element, or a MinTk minimal promoter.

10. The nucleic acid of claim 9, wherein the minimal promoter comprises the sequence of SEQ ID NO:

32.

11. The nucleic acid of any one of claims 1 to 10, wherein the reporter nucleotide sequence encodes a luminescent protein or an enzyme that produces bioluminescence.

12. The nucleic acid of claim 11 , wherein the luminescent protein is green fluorescent protein (GFP), enhanced GFP (EGFP), or mCherry.

13. The nucleic acid of claim 11 , wherein the enzyme is luciferase.

14. The nucleic acid of any one of claims 1 to 13, further comprising a polyA signal sequence operably linked to the reporter nucleic acid sequence.

15. 15. The nucleic acid of claim 14, wherein the polyA signal sequence comprises SEQ ID NO:

91.

16. 16. The nucleic acid of any one of claims 1 to 15, comprising a second reporter nucleotide sequence operably linked to a constitutive promoter.

17. A cell comprising the nucleic acid according to any one of claims 1 to 16.

18. 18. The cell of claim 17, wherein the nucleic acid is stably integrated into the genome of the cell.

19. 19. The cell of claim 17 or 18, wherein the cell is engineered to stably overexpress an adeno-associated virus receptor (AAVR).

20. The cell of claim 19, wherein the AAVR is a wild-type AAVR.

21. The cell of any one of claims 17 to 20, further comprising a transcription factor (TF) that binds to the Z1 TF binding site.

22. The cell of claim 21 , wherein the TF is expressed from an exogenous nucleic acid.

23. 23. The cell of claim 22, wherein the exogenous nucleic acid is present in an AAV vector.

24. The cell of claim 23, wherein the AAV vector is an AAV9 vector or an scAAV9 vector.

25. The cell according to any one of claims 21 to 24, wherein the TF is a transcriptional activator.

26. The cell of any one of claims 21 to 25, wherein the TF comprises an engineered Z1 binding domain.

27. 27. The cell of claim 26, wherein the Z1-binding domain comprises SEQ ID NO:

93.

28. A cell comprising a nucleic acid comprising a response element operably linked to a reporter nucleotide sequence, said cell being further engineered to stably overexpress an adeno-associated virus receptor (AAVR).

29. The cell of claim 28, wherein the response element comprises 2 to 10 copies of a transcription factor (TF) binding site.

30. 30. The cell of claim 29, wherein the response element comprises 3 to 8 copies of the TF binding site.

31. 30. The cell of claim 29, wherein the response element comprises 3 to 6 copies of the TF binding site.

32. The cell of any one of claims 29 to 31, wherein the TF binding site comprises a sequence bound by endogenous TF.

33. The cell of claim 32, wherein the endogenous TF is a ligand-dependent TF.

34. The cell of claim 33 , wherein the ligand is a metal.

35. 35. The cell of claim 34, wherein the metal is copper.

36. The cell of claim 35, wherein the TF binding site comprises SEQ ID NO:

8.

37. The cell of claim 36, wherein the endogenous TF is metal-responsive transcription factor 1 (MTF-1).

38. The cell of any one of claims 29 to 31, wherein the TF binding site comprises a sequence bound by exogenous TF.

39. 39. The cell of claim 38, wherein the exogenous TF comprises an engineered DNA-binding domain specific for the TF binding site.

40. 40. The cell of claim 39, wherein the engineered DNA-binding domain comprises 2 to 10 zinc fingers.

41. The cell of claim 40, wherein the TF binding site comprises the sequence of SEQ ID NO:

1.

42. 42. The cell of claim 41, wherein the engineered DNA binding domain is selected from the group consisting of SEQ ID NOs: 76-83.

43. The cell of any one of claims 38 to 42, wherein the exogenous TF is expressed in the cell from an expression vector.

44. 44. The cell of claim 43, wherein the expression vector is AAV.

45. The cell of any one of claims 28 to 44, wherein the response element further comprises a promoter.

46. 46. ​​The cell of claim 45, wherein the promoter is a minimal promoter.

47. 47. The cell of any one of claims 28 to 46, wherein the reporter nucleotide sequence encodes a photoprotein or an enzyme that produces bioluminescence.

48. 48. The cell of claim 47, wherein the luminescent protein is green fluorescent protein (GFP), enhanced GFR (EGFR), or mCherry.

49. 49. The cell of any one of claims 28 to 48, wherein the nucleic acid further comprises a polyA signal sequence operably linked to a reporter nucleic acid sequence.

50. 20. The cell of claim 19, wherein the polyA signal sequence comprises SEQ ID NO:

91.

51. The cell of any one of claims 28 to 50, wherein the AAVR is a wild-type AAVR.

52. 52. A method for determining the potency of a sample containing an AAV vector, the method comprising: (a) contacting a cell of any one of claims 28 to 51 with all or a portion of the sample, wherein the expression vector encodes a regulatory element that directly or indirectly regulates expression of the reporter nucleotide sequence via the response element; (b) measuring the expression of the reporter nucleotide sequence in the cell; and (c) determining the potency of the sample based on the measured expression level of the reporter nucleic acid sequence.

53. 53. The method of claim 52, wherein the regulatory factor is a transcription factor that binds to a TF binding site in the response element.

54. 53. The method of claim 52, wherein the regulator is a protein that regulates metal metabolism and the response element is a metal-responsive response element.

55. The method of any one of claims 52 to 54, wherein the AAV vector is an AAV9 vector or a scAAV9 vector.

56. 56. The method of any one of claims 52-55, wherein determining step (c) comprises comparing the measured expression level to a standard efficacy curve for the expression vector delivery vehicle.

57. 17. The nucleic acid of claim 16, wherein the second reporter nucleotide sequence encodes a photoprotein or an enzyme that produces bioluminescence.

58. 58. The nucleic acid of claim 57, wherein the luminescent protein is green fluorescent protein (GFP), enhanced GFP (EGFP), or mCherry.

59. 58. The nucleic acid of claim 57, wherein the enzyme is luciferase.

60. 60. The nucleic acid of any one of claims 16 and 57 to 59, wherein the constitutive promoter is a herpes simplex virus (HSV) promoter, a thymidine kinase (TK) promoter, a Rous sarcoma virus (RSV) promoter, a simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, an Ad E1A promoter, or a cytomegalovirus (CMV) promoter.

61. A nucleic acid comprising a response element operably linked to a reporter nucleotide sequence, said response element comprising a transcription factor (TF) binding site that is bound by an endogenous metal-dependent TF.

62. 62. The nucleic acid of claim 61, wherein the metal is copper.

63. 63. The nucleic acid of claim 61 or 62, wherein the metal-dependent TF is metal response element-binding transcription factor-1 (MTF-1).

64. 63. The nucleic acid of claim 61 or 62, wherein the TF binding site comprises SEQ ID NO:

8.

65. 66. The nucleic acid of any one of claims 61 to 65, wherein the response element further comprises a promoter.

66. 66. The nucleic acid of claim 65, wherein the promoter is a minimal promoter.

67. 67. The nucleic acid of claim 66, wherein the minimal promoter is any one of a CMV minimal promoter, an hsp70 minimal promoter, a minimal promoter contained in a tetracycline response element, or a MinTk minimal promoter.

68. 67. The nucleic acid of claim 66, wherein the minimal promoter comprises the sequence of SEQ ID NO:

32.

69. 69. The nucleic acid of any one of claims 61 to 68, wherein the reporter nucleotide sequence encodes a photoprotein or an enzyme that produces bioluminescence.

70. 70. The nucleic acid of claim 69, wherein the luminescent protein is green fluorescent protein (GFP), enhanced GFP (EGFP), or mCherry.

71. 70. The nucleic acid of claim 69, wherein the enzyme is luciferase.

72. 72. The nucleic acid of any one of claims 61 to 71, further comprising a polyA signal sequence operably linked to the reporter nucleic acid sequence.

73. 73. The nucleic acid of claim 72, wherein the polyA signal sequence comprises SEQ ID NO:

91.

74. 74. The nucleic acid of any one of claims 61 to 73, comprising a second reporter nucleotide sequence operably linked to a constitutive promoter.

75. 75. The nucleic acid of claim 74, wherein the second reporter nucleotide sequence encodes a photoprotein or an enzyme that produces bioluminescence.

76. 76. The nucleic acid of claim 75, wherein the luminescent protein is green fluorescent protein (GFP), enhanced GFP (EGFP), or mCherry.

77. 76. The nucleic acid of claim 75, wherein the enzyme is luciferase.

78. 78. The nucleic acid of any one of claims 74 to 77, wherein the constitutive promoter is a herpes simplex virus (HSV) promoter, a thymidine kinase (TK) promoter, a Rous sarcoma virus (RSV) promoter, a simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, aa Ad E1A promoter, or a cytomegalovirus (CMV) promoter.

79. A cell comprising a nucleic acid according to any one of claims 61 to 78.

80. 80. The cell of claim 79, wherein the nucleic acid is stably integrated into the genome of the cell.

81. 81. The cell of claim 79 or 80, wherein the cell is engineered to stably overexpress an adeno-associated virus receptor (AAVR).

82. The cell of claim 81, wherein the AAVR is a wild-type AAVR.

83. 83. The cell of any one of claims 79 to 82, further comprising a transcription factor (TF) that binds to the TF binding site.

84. 84. The cell of claim 83, wherein the TF is expressed from an exogenous nucleic acid.

85. 85. The cell of claim 84, wherein the exogenous nucleic acid is present in an AAV vector.

86. 86. The cell of claim 85, wherein the AAV vector is an AAV9 vector or a scAAV9 vector.

87. The cell of any one of claims 83 to 86, wherein the TF is MTF-1.

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