Materials and methods for characterizing potency

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 a Z1 transcription factor binding site. Also provided is a cell comprising the nucleic acid. In various aspects, the cells stably overexpress adeno-associated virus receptors. The present disclosure further provides a method of 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 samples containing AAV vectors.

[0002] Materials Submitted Electronically and Incorporated by Reference

[0003] The computer-readable nucleotide / amino acid sequence listing filed herewith and identified as follows is hereby incorporated by reference in its entirety: File Name: "55328A_SeqListing.XML"; 126,534 bytes; created on December 28, 2023. Background Art

[0004] Regulatory-approved drugs and biopharmaceuticals must be accompanied by analytical assays to confirm that product batches meet defined standards (purity, safety, potency, etc.) that are considered suitable for approval for human use. Potency assays and related tests are used to ensure consistent quality between product batches and to ensure the identity, purity, strength (potency), and stability of the product during research. Analytical assays have long been used to characterize small molecule drug products and protein-based therapeutics such as monoclonal antibodies. However, the nature of gene therapy products complicates the development of potency assays. Several characteristics of gene and cell therapies present significant challenges to the development of 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. As the number of gene therapy candidates increases, the industry continues to work on developing assays that can reliably quantify the potency of samples of gene therapy products. Summary of the Invention

[0005] The present disclosure provides materials and methods for measuring the potency of an expression vector delivery agent. For example, the present 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 a 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 having 1, 2, or 3 nucleotide differences from SEQ ID NO:1. 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 luminescent proteins or enzymes that produce bioluminescence, such as luciferase; fluorescent proteins, such as green fluorescent protein (GFP), enhanced GFP (EGFP), and mCherry; and colored proteins or enzymes that produce colored products, such as β-galactosidase. In some embodiments, the nucleic acid may comprise a second reporter nucleotide sequence operably linked to a constitutive promoter.

[0006] Also provided is a cell comprising the nucleic acid. For example, the present disclosure provides a cell comprising a nucleic acid comprising a response element (e.g., the response element comprises 2 to 10 copies (e.g., 3 to 8 or 3 to 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 virus receptor (AAVR), such as wild-type AVR. Optionally, the response element comprises one or more TF binding sites capable of being bound by a TF. In various aspects, 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 bound by an exogenous TF. In various aspects, the exogenous TF comprises an engineered DNA binding domain specific for the TF binding site, such as an engineered DNA binding site comprising 2 to 10 zinc fingers.

[0007] The present disclosure further provides a method for determining the potency of a sample comprising an AAV vector. The method comprises contacting a cell comprising a response element operably linked to a reporter nucleotide sequence with all or a portion of the sample. The AAV vector encodes a regulatory factor that directly or indirectly regulates the expression of the reporter nucleotide sequence through the response element. The method further comprises measuring the expression of the reporter nucleotide sequence in the cell. The method further optionally comprises determining the potency of the sample based on the measured expression level of the reporter nucleic acid sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figures 1A - 1D is a schematic diagram of a nucleic acid comprising a response element operably linked to a reporter nucleotide sequence.

[0009] Figure 2 Shows reporter gene expression (measured in relative light units (RLU; y-axis)) of various response element constructs in the presence and absence of a regulatory factor (SEQ ID NO: 34). For each response element construct, the presence of the reporter gene is represented only by the bar on the upper left; for each response element construct, reporter gene + activator is represented by the bar on the upper right.

[0010] Figure 3 Shows reporter gene expression (measured in relative light units (RLU; y-axis)) of various response element constructs in the presence of increasing amounts of a regulatory factor (SEQ ID NO: 34; 0 ng, 0.01 ng, 0.1 ng, 1 ng, 10 ng, 30 ng, 50 ng, or 70 ng).

[0011] Figure 4 Shows the results of Example 3. Figure 4 Shows reporter gene expression (measured in relative light units (RLU; y-axis)) observed from a control expression cassette with a TET reporter sequence instead of a DNA binding sequence (P-1) and a cassette (P-5) comprising a Z1-based response element operably linked to a reporter nucleic acid sequence.

[0012] Figure 5 is a schematic diagram of a nucleic acid comprising a response element operably linked to a reporter nucleotide sequence and further comprising, in some cases, a second reporter nucleic acid operably linked to a constitutive promoter.

[0013] Figure 6A and 6B show AAV9 infection (%) ( Figure 6A ) or mean fluorescence intensity (Figure 6B )(y-axis).

[0014] Figure 7A and 7B show AAV9 infection (%) of unmodified HEK293 cells, HEK293 cells overexpressing AAVR, unmodified CHO cells, and CHO cells overexpressing AAVR( Figure 7A ) or mean fluorescence intensity( Figure 7B )(y-axis).

[0015] Figure 8A and 8B show green fluorescent protein expression (%) of subclones of HEK cells modified to overexpress AAVR( Figure 8A ) or mean fluorescence intensity( Figure 8B )(y-axis). All subclones tested showed improved infection rates (measured by transgene expression) compared to unmodified cells.

[0016] Figure 9A and 9B are graphs showing fold change of VP64 in HEK293 cells and HeLa cells modified to overexpress AAVR at various multiplicities of infection (MOI; viral genomes / cell).

[0017] Figures 10A - 10D is a bar graph showing AAV infection (%) of various AAV serotypes in HEK293 cells modified to overexpress AAVR( Figure 10A ) or HeLa cells modified to overexpress AAVR( Figure 10B ), and mean fluorescence intensity observed from the same AAV vectors in HEK293 cells modified to overexpress AAVR( Figure 10C ) or HeLa cells modified to overexpress AAVR( Figure 10D ).

[0018] Figure 11 shows AAV9 transduction in the HeRC32 cell line compared to the HerRC32-AAVR clone cell line.

[0019] Figure 12 shows relative bioluminescence (RLU) in cells measured 48 hours after transduction. Conditions of plasmid transfection or AAV9 transduction at MOI (0, 10 4 , 10 5 , 10 6 ) are indicated on the x-axis. Each bar represents the mean of triplicate wells, and error bars indicate standard deviation.

[0020] Figure 13Schematic of the nucleic acid used in the study described in Example 6, the nucleic acid comprising a response element operably linked to a reporter nucleotide sequence and a lentiviral backbone.

[0021] Figure 14A and 14B Graph showing reporter gene expression (measured in relative light units, y-axis) in cells engineered to integrate a response element-reporter gene construct into the cell genome in response to various multiplicities of infection (MOI; viral genomes / cell) of an AAV vector encoding a regulator.

[0022] Figures 15A - 15C Corresponds to the data for the response element-reporter gene construct P-6 described in Example 6. Figure 15A and 15B Graph showing reporter gene expression (measured in relative light units, y-axis) in cells engineered to integrate a response element-reporter nucleic acid into the cell genome in response to various multiplicities of infection (MOI; viral genomes / cell) of an AAV vector encoding a regulator. Figure 15A Corresponds to luciferase expression mediated by the response element; Figure 15B Corresponds to firefly luciferase expression mediated by a constitutive promoter. Figure 15C Schematic of the P-6 construct.

[0023] Figure 16 Shows dose-dependent induction of luciferase reporter gene activity by a regulator (here SEQ ID NO:84) in a clonal cell line containing a response element-reporter gene construct stably integrated into the cell genome and engineered to stably express AAVR. Relative potency measurements are represented by parallel line analysis. The full range of MOIs for the experiment is shown.

[0024] Figure 17 Shows dose-dependent induction of luciferase reporter gene activity by a regulator (here SEQ ID NO:84) in a clonal cell line containing a response element-reporter gene construct stably integrated into the cell genome and engineered to stably express AAVR. Relative potency measurements are represented by parallel line analysis. Although Figure 16 includes the full range of MOIs for the experiment, Figure 17 the relative potency analysis is limited to four dose points within the optimal linear dose range for each cell line.

[0025] Figure 18A Shows eTF and control eTF DNA-binding proteins Δz1 eTF with mutations in the DNA-binding α-helical zinc finger domain.

[0026] Figure 18B Shows the dose response to increasing MOI in AAV variants expressing eTF (triangle, AAV9-CBA-z1-eTF) or expressing control Δz1 eTF (square, AAV9-CBA-Δz1-eTF, SEQ ID NO:96). The reference standard (RS, SEQ ID NO:94) is shown as circles.

[0027] Figure 19 Shows the assay specificity for the transgene expressing eTF in a matched control sample. Dose response to increasing MOI in the assay control (AC, red, SEQ ID NO:94) or reference standard sample (green, CBA-Δz1-eTF, SEQ ID NO:96). The matched control AAV sample expressing Δz1 eTF (blue, S20-1052 (bottom line)) does not activate reporter gene expression. DETAILED DESCRIPTION

[0028] The present disclosure provides materials and methods for characterizing the potency of AAV gene therapy formulations. Previous methods for estimating the potency of gene therapy vectors required measuring viral genomes or empty viral capsids. While these methods are useful for characterizing the amount of nucleic acid in a formulation, they do not accurately inform about the biological activity associated with the formulation. Gene therapy vectors exert their therapeutic effect by delivering a payload to a cell, which then exerts a biological effect (i.e., the payload itself mediates the biological effect or encodes a protein or nucleic acid that mediates the biological effect). Quantifying only the vector genome or empty capsids does not adequately capture these features that contribute to the activity and function of gene therapy vectors, i.e., vector potency. The systems and methods described herein provide an accurate and concise method for measuring potency, thereby making the consistency between product batches and accurate patient dosing more reliable.

[0029] On the one hand, 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. A transcription factor is generally a protein that controls the rate of DNA transcription by binding to a specific nucleotide sequence (e.g., a response element) and regulating expression, for example, by promoting or hindering the recruitment of RNA polymerase or other expression cofactors. A transcription factor typically contains at least one DNA-binding domain and a transcriptional regulatory domain, the binding domain of which can bind to a transcription factor binding site in the target DNA, and the transcriptional regulatory domain contains binding sites for other proteins that promote or repress the expression of the target nucleic acid sequence. A transcription factor can act through any 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 histones, and recruiting coactivators or corepressors to the transcription factor-DNA complex. In various aspects, the transcription factor is a transcriptional activator (i.e., it promotes the transcription of a nucleic acid sequence). In alternative aspects, the transcription factor is a transcriptional repressor (i.e., it reduces or blocks the transcription of a nucleic acid sequence). A transcription factor (TF) can be endogenous (i.e., a factor naturally expressed by the host cell) or can be exogenous (i.e., recombinantly produced in the host cell and optionally engineered to contain one or more modifications compared to the wild-type transcription factor). The TF can be naturally occurring, modified from a naturally occurring TF, or can be a non-naturally occurring synthetic TF.

[0030] Examples of the structure of a TF DNA binding domain include, but are not limited to, helix-turn-helix, zinc finger, leucine zipper (e.g., bZIP), helix-loop-helix, and β-scaffold. The TF can be engineered such that, for example, the DNA binding domain is operably linked to a transcriptional regulatory domain, where the DNA binding domain and the transcriptional regulatory domain are not naturally linked (e.g., derived from different transcription factors or 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 additionally, the TF can comprise an engineered DNA binding domain that is specific for a TF binding site of interest. In this regard, the TF can comprise multiple copies of the same DNA binding domain, or can comprise multiple DNA binding domains of different sequences. For example, aspects of the present disclosure provide TFs having an engineered DNA binding site comprising 2 to 10 DNA binding domains, such as zinc fingers (e.g., 3 to 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 NO: 85-90. See also International Patent Publication No. WO 2020 / 243651, which is incorporated herein by reference in its entirety.

[0031] Transcription factors may be active in most cell types. Transcription factors can also be tissue-specific, such as transcription factors from muscle cells (e.g., MyoD and myocyte enhancer factor 2 (MEF2)) or from 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 binds to a ligand. The activity of a ligand-dependent transcription factor may depend on whether it binds 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 transcription factors that regulate metal (iron, zinc, or copper) homeostasis. The transcription factors of the present disclosure can 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). In response to heavy metals such as copper, MTF-1 induces the expression of metallothionein and other genes involved in metal homeostasis. MTF-1 binds to a transcription factor binding site that contains a DNA sequence motif called a metal-responsive element (MRE) with a core consensus sequence 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. February 2004; 3(1):1-13; and Wang et al., Biol Chem. July 2004; 385(7):623-32.

[0032] A transcriptional regulatory domain (TMD) is the region of a TF that contains binding sites for other proteins that promote or repress transcription of a target nucleic acid sequence. The TMD can contact the transcriptional machinery (e.g., RNA polymerase) directly or through other proteins (referred to as coactivators or coregulators). The TMD and the DNA binding domain (DBD) can be derived from different proteins. Engineered TFs can contain more than one TMD, and two or more TMDs can be derived from (e.g., isolated from) different proteins. In various aspects, 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. WO2019 / 109051, which is incorporated herein by reference in its entirety and particularly with respect to the disclosure regarding transactivation domain sequences. In some aspects, the TMD is a repression domain that reduces or blocks expression.

[0033] The DBD and the TMD can be directly linked, for example without an intervening amino acid sequence. Alternatively, the DBD and the TMD can be linked by a peptide linker. In various embodiments, the DBD is conjugated to the TMD by a linker having 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, 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 the TMD are conjugated by naturally occurring intervening residues present in the naturally occurring protein from which the domain is derived or from another naturally occurring transcription factor. In other embodiments, the DBD and the TMD are conjugated by a synthetic or exogenous linker sequence. Suitable linkers can be flexible, cleavable, non-cleavable, hydrophilic and / or hydrophobic. In certain embodiments, the DBD and the TMD can be fused together by a linker comprising 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 equal to or greater than 1. In various aspects, the linker for conjugating the DBD and the TAD is GGSGGGSG (SEQ ID NO:7). In various embodiments, when the 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 directly conjugated to the DBD (e.g., without an intervening linker sequence), or both TMDs can be directly conjugated to the DBD (e.g., without an intervening linker sequence).

[0034] Examples of transcription factors include, but are not limited to, 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-like 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, nuclear orphan receptor transcription factor, estrogen receptor transcription factor, P53 transcription factor, PAX transcription factor, PC4 transcription factor, POU transcription factor, PPAR receptor transcription factor, PREB transcription factor, progesterone receptor transcription factor, Prox1 transcription factor, retinoic acid receptor transcription factor, RFX transcription factor, RHD transcription factor, ROR receptor transcription factor, Runt transcription factor, SAND transcription factor, SPZ1 transcription factor, SRF transcription factor, STAT transcription factor, T-box transcription factor, TEA transcription factor, TF-bZIP transcription factor, TF-Otx transcription factor, THAP transcription factor, thyroid hormone receptor transcription factor, TSC22 transcription factor, Tub transcription factor, ZBTB transcription factor, zf-BED transcription factor, zf-C2H2 transcription factor, zf-C2HC transcription factor, zf-GATA transcription factor, zf-LITAF-like transcription factor, zf-MIZ transcription factor, and zf-NF-X1 transcription factor.

[0035] The nucleic acids of the present disclosure comprise response elements that are recognized and bound by transcription factors. The response elements comprise one or more TF binding sites, which comprise target nucleic acid sequences to which the DNA binding domain of the TF can bind. The binding site motifs of many transcription factors found in the genome have been identified and characterized. See, for example, Inukai et al., Current Opinion in Genetics & Development 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 sites 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.

[0036] The response elements of the present disclosure can comprise a single TF binding site or can comprise multiple TF binding sites. For example, the response element can comprise 2 to 10 copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies) of a TF binding site. In cases where multiple TF binding sites are found in the response element, the multiple TF binding sites can all be the same (i.e., multiple copies of the same TF binding site sequence) or can be different (i.e., two or more TF binding sites having different nucleic acid sequences). Optionally, in cases where there are multiple different TF binding sites, the different TF binding sites can be recognized by different TFs or the same TF.

[0037] In various aspects, the response element comprises 2 to 10 copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies) of a 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 should be understood that a TF can recognize a large number of DNA binding site sequences. See, e.g., Siggers et al., Nucleic Acids Res. 2014;42:2099-2111. Thus, the TF binding site can comprise 1, 2, or 3 nucleotide differences from SEQ ID NO:1 (i.e., the TF binding site can comprise SEQ ID NO:1 with a substitution at 1, 2, or 3 nucleotide positions within SEQ ID NO:1). Thus, in various aspects of the present disclosure, 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 having 1, 2, or 3 nucleotide differences from SEQ ID NO:1. In this regard, each copy can comprise the sequence of SEQID NO:1, a subset of the copies can comprise SEQ ID NO:1, and the other copies can comprise SEQ ID NO:1 including one or more substitutions, or each copy can comprise SEQ ID NO:1 having 1, 2, or 3 substitutions. In some aspects, the response element can comprise a sequence having at least 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:1.

[0038] In various aspects, the response element comprises TF binding sites bound by an endogenous metal-dependent TF, such as a copper-dependent TF. For example, the response element can comprise 1 to 10 copies or 2 to 10 copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies) of TF binding sites bound by an endogenous metal-dependent TF. In various aspects, 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 to 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 1, 2, or 3 nucleotide differences from SEQ ID NO:8 (i.e., the TF binding site can comprise SEQ ID NO:8 with substitutions at 1, 2, or 3 nucleotide positions within SEQ ID NO:8). Thus, in various aspects 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 having 1, 2, or 3 nucleotide differences from SEQ ID NO:8. In this regard, each copy can comprise the sequence of SEQ ID NO:8, a subset of the copies can comprise SEQ ID NO:8, and other copies can comprise SEQ ID NO:8 including one or more substitutions, or each copy can comprise SEQ ID NO:8 having 1, 2, or 3 substitutions. In some aspects, the response element can comprise a sequence having at least 80%, 85%, 90%, 95%, or 98% sequence identity (e.g., 100% identity) to SEQ ID NO:8.

[0039] If desired, a response element comprising multiple TF binding sites can comprise a spacer sequence between two or more TF binding sites. The spacer can be of any length as long as TF binding and regulation of gene expression are not abrogated. In some embodiments, the response element comprises 2 to 10 copies of the Z1 TF binding site and further comprises a spacer sequence between at least two copies 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 comprise a sequence having at least 80%, 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO:2 or 3.

[0040] In various aspects of the present disclosure, a nucleic acid comprising a response element comprises a sequence of any one of SEQ ID NO: 10-15, SEQ ID NO: 17-23, and SEQ ID NO: 25. The present disclosure further contemplates nucleic acids having 1, 2, or 3 nucleotide differences relative to the sequences of any one of SEQ ID NO: 10-15, SEQ ID NO: 17-23, and SEQ ID NO: 25. In some aspects, a response element comprising a nucleic acid may comprise a sequence having at least 80%, 85%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NO: 10-15, SEQ ID NO: 17-23, and SEQ ID NO: 25.

[0041] Optionally, the response element further comprises a promoter that further drives expression in a host cell. In various aspects, the response element comprises two promoters.

[0042] A promoter can be natural or non-natural with respect to the nucleic acid sequence operably linked thereto, and can be natural or non-natural with respect to a particular host cell. In various aspects, the 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 the estrogen gene promoter). Another example of an inducible promoter is the tet promoter responsive to tetracycline. An example of a tissue-specific promoter is a liver-specific promoter, such as the HLP promoter. Other 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, April 25, 2013, 121(17):3335-44; LPL: Nathwani et al., Blood, April 1, 2006, 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 U S A., March 30, 1999, 96(7):3906-3910.

[0043] In some aspects, the promoter is a minimal promoter. Minimal promoters are those that generally cannot drive expression in the absence of additional regulatory elements. An example of a minimal promoter suitable for the context of the present disclosure is minP (SEQ ID NO:32). Other minimal promoters include, but are not limited to, the CMV-minimal promoter, the hsp70 minimal promoter, the minimal promoter included in the tetracycline response element, and the MinTk minimal promoter.

[0044] The nucleic acids of the present disclosure include a response element operably linked to a reporter nucleotide sequence. "Operably linked" refers to a functional linkage between nucleic acid sequences or amino acid sequences. For example, "operably linked" means that a control sequence is in the correct position and orientation relative to another nucleic acid sequence to effect its function on the nucleic acid sequence (e.g., initiation of transcription). Here, the response element is operably linked to the reporter nucleotide sequence such that the response element can regulate the expression rate or abundance of the encoded reporter gene. Generally, operably linked DNA sequences are contiguous and, where necessary, in the same reading frame (although this is not always required).

[0045] "Reporter gene" refers to any biomolecule (e.g., protein) that directly or indirectly produces a detectable signal. In various aspects, the reporter nucleotide sequence encodes a luminescent protein or an enzyme that produces bioluminescence. Examples of reporter genes include, but are not limited to, green fluorescent protein (GFP), green fluorescent protein variant (GFP10), enhanced GFP (eGFP), TurboGFP, GFPS65T, TagGFP2, mUKGEmerald GFP, superfolder GFP, GFPuv, unstable EGFP (dEGFP), AzamiGreen, mWasabi, Clover, mClover3, mNeonGreen, NowGFP, skyblue, T-skyblue, mAmetrine, photoactivatable GFP (PA-GFP), Kaede, Kikume, mKikGR, tdEos, Dendra2, mEosFP2, Dronpa, blue fluorescent protein (BFP), eBFP2, shijin BFP, mTagBFP, mKalamal, mTagBFP2, shBFP, cyan fluorescent protein (CFP), eCFP, Cerulian CFP, SCFP3A, unstable ECFP (dECFP), CyPet, mTurquoise, mTurquoise2, mTFPI, photoswitchable CFP2 (PS-CFP2), TagCFP, mTFP1, mMidoriishi-Cyan, viridian, mKeima, mBeRFP, LSS-mKate2, LSS-mKatel, LSS-mOrange, CyOFP1, Sandercyanin, red fluorescent protein (RFP), eRFP, mRaspberry, mRuby, mApple, mCardinal, mStable, mMaroonl, mGarnet2, tdTomato, mTangerine, mStrawberry, TagRFP, TagRFP657, TagRFP675, mKate2, HcRed, t-HcRed, HcRed-Tandem, mPlum, mNeptune, NirFP, Kindling, far-red fluorescent protein, yellow fluorescent protein (YFP), eYFP, unstable EYFP (dEYFP), TagYFP, Topaz, Venus, SYFP2, mCherry, PA-mCherry, lemon yellow, mCitrine, Ypet, IANRFP-AS83, mPapayal, mCyRFP1, mHoneydew, mBanana, mOrange, KusabiraOrange, Kusabira Orange 2, mKusabiraOrange, 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, BV 605, BV 510, BV 711, BV786, PerCP, PerCP / Cy5.5, DsRed, DsRed2, mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP or phycobiliprotein, luciferase, LacZ, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), β-galactosidase, and β-glucuronidase (GUS), and biologically active variants and fragments of the foregoing. In various aspects, the reporter nucleic acid encodes GFP, EGFP, mCherry, or luciferase.

[0046] If desired, the nucleic acid can include a second reporter nucleotide sequence that is not operably linked to the response element. Any reporter gene, including any of the reporter genes described herein, is suitable for use as the second reporter gene. 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 encoding an enzyme that produces bioluminescence (e.g., luciferase). A second reporter gene can be selected that has a signal complementary to the first reporter gene. As an example, if the first reporter gene is a fluorescent reporter gene, the second reporter gene can be a fluorescent reporter gene having a different excitation and / or emission wavelength. In another example, if the first reporter gene is luciferase, the second reporter gene can be Renilla, or if the first reporter gene is alkaline phosphatase, the second reporter gene can be LacZ. In various aspects, the second reporter nucleic acid is operably linked to a separate promoter (i.e., a promoter different from the promoter that is part of the response element, if the response element includes a promoter). Suitable promoters include, but are not limited to, the promoters 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 (SV), murine mammary tumor virus (MMTV), Ad E1A, and cytomegalovirus (CMV) promoters.

[0047] In various aspects, the nucleic acids provided herein include 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, stabilizing elements, WPRE sequences, Kozak consensus sequences, post-translational response elements, microRNA binding sites, polyadenylation (polyA) signal sequences, or combinations thereof. Regulatory elements can regulate gene expression during the transcriptional, post-transcriptional, or translational phases of gene expression. At the RNA level, regulation can occur at the level of translation (e.g., a stability element that stabilizes the mRNA for translation), RNA cleavage, RNA splicing, and / or transcription termination.

[0048] In certain embodiments, the nucleic acid further includes a polyA signal sequence. Suitable polyA signal sequences include, for example, artificial polyA (PA75) of about 75 bp in length (see, e.g., International Patent Publication No. WO 2018 / 126116), bovine growth hormone polyA, SV40 early polyA signal, SV40 late polyA signal, rabbit β-globin polyA, HSV thymidine kinase polyA, protamine gene polyA, adenovirus 5E1b polyA, growth hormone polyA, or PBGD polyA. In an exemplary aspect, the polyA sequence suitable for the expression cassette provided herein is hGH polyA (SEQ ID NO:27) or synthetic polyA (SEQ ID NO:28 or SEQ ID NO:91). In various aspects, the polyA comprises the nucleic acid sequence of SEQ ID NO:91. Generally, the polyA signal sequence is operably linked to the reporter nucleic acid sequence.

[0049] The present disclosure further provides a cell comprising the nucleic acid described herein. The nucleic acid can be stably integrated into the cell genome or can be present in a separate expression vector construct. The cell can be a cell from any organism (e.g., prokaryotic cell, eukaryotic cell, bacterial cell, plant cell, algal cell, fungal cell (e.g., yeast cell), mammalian cell, animal cell (human or non-human), etc.). Mammalian cells include cells isolated from or derived from, for example, humans, non-human primates (such as apes, chimpanzees, monkeys, and orangutans), domesticated 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, frontal cortex cell, glial cell, microglial cell, or striatal cell. Examples of cells include but are not limited to Chinese hamster ovary (CHO) cells and their derivatives (e.g., CHO-K1, CHO pro-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), African green 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, human adenocarcinoma alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, mouse embryonic fibroblasts NIH 3T3, mouse fibroblasts 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 hepatoma cells Hep G2, mouse B myeloma cells J558L, and baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263 (2013)).

[0050] In some aspects, cells have been modified with exogenous nucleic acid comprising a nucleotide sequence encoding a receptor that enhances the transduction efficiency of an expression vector of interest. In this aspect, the present disclosure provides cells that have been modified with exogenous nucleic acid encoding an adeno-associated virus receptor (AAVR). The cells are optionally engineered to stably overexpress AAVR. "Overexpress" means an increase in the total amount of AAVR in the cell (i.e., the cell produces more AAVR than an unmodified matched cell). Prior to modification, the cell may or may not naturally express AAVR. The AAVR can be wild-type AAVR or modified AAVR. Expression of AAVR enhances AAV infection of the host cell, for example, by increasing the number of receptors on the cell surface, enhancing the affinity of AAVR for the AAV capsid protein, or facilitating entry of AAV into the cell.

[0051] Wild-type AAVR is a predicted type I transmembrane protein. The protein comprises a signal peptide, a MANSC domain (a motif with seven cysteines at the N-terminus), and five Ig-like domains (polycystic kidney disease (PKD) domains 1-5). The transmembrane domain is located at the C-terminus of the MANSC and PKD domains and is followed by a cytoplasmic tail. The structure of AAVR is further characterized by, for example, Summerford et al., Molecular Therapy, 24(4):663 (2016); Meyer et al., eLife 8:e44707 (2019); and International Patent Publication No. WO 2017 / 083423 (incorporated herein by reference in its entirety and particularly with respect to the disclosure of AAVR structure, AAVR sequences, and variant AAVRs). The AAVR can be from any species, such as mammalian AAVR proteins, such as rodent AAVR proteins, primate AAVR proteins, rat AAVR proteins, mouse AAVR proteins, porcine AAVR proteins, bovine AAVR proteins, ovine AAVR proteins, rabbit AAVR proteins, canine AAVR proteins, or human AAVR proteins. Preferably, the AAVR is human AAVR. The wild-type human AAVR amino acid sequence provided herein is 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. WO 2017 / 083423 (which is hereby incorporated by reference).

[0052] Accordingly, the present disclosure provides a cell comprising a nucleic acid comprising a response element as described herein operably linked to a reporter nucleotide sequence, wherein the cell is engineered to stably overexpress AAVR, such as wild-type AAVR. The response element can comprise from 2 to 10 copies (e.g., from 3 to 8 copies or from 3 to 6 copies) of a transcription factor (TF) binding site, although one copy of the TF binding site is also contemplated. As described above, the TF binding site can comprise a sequence that is bound by an endogenous TF or an exogenous TF. 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, which recognizes a binding comprising SEQ ID NO:8). In various aspects, the TF binding site comprises a sequence that is bound by an exogenous TF, such as an engineered TF comprising a modified DNA binding site having 2 to 10 zinc fingers. In various aspects, the TF binding site comprises one or more Z1 TF binding sites. In this aspect, in various embodiments, the TF binding site comprises SEQ ID NO:1. Alternatively or additionally, 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 DNA binding domains (such as a peptide comprising the amino acid sequence of SEQ ID NO:93).

[0053] In various aspects, the present disclosure provides a composition comprising a cell comprising a response element (optionally engineered to stably express AAVR) and a TF that binds to the response element. In this aspect, the cell can comprise any one or more of the TFs described herein. The cell can naturally produce the TF (i.e., the TF is endogenous), or the TF can be exogenous relative to the host cell (i.e., expressed from 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, representative transcription factors bind to the Z1 TF binding site. In this aspect, the TF can comprise an engineered Z1 binding domain, such as a Z1 binding domain comprising SEQ ID NO:1. Alternatively, the TF binds 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.

[0054] Any nucleic acid described herein, including a nucleic acid comprising a response element operably linked to a reporter nucleic acid, a nucleic acid encoding a TF, etc., can be provided in an expression vector. An "expression vector" is any molecule or part that transports, transduces, or otherwise serves as a vehicle for a heterologous polynucleotide. The vector can be an integrating or non-integrating vector, referring to the ability of the vector to integrate the nucleic acid into the genome of the host cell. Examples of expression vectors include, but are not limited to, (a) non-viral vectors such as nucleic acid vectors including 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. The viral vector can be obtained by deleting all or part of the coding region from the viral genome while retaining those sequences (e.g., terminal repeats) that may be necessary or beneficial for functions such as packaging the vector genome into the viral capsid.

[0055] The viral vectors of the present invention can be recombinantly produced and can be based on an adeno-associated virus (AAV) parental or reference sequence. AAV is a small, replication-defective, non-enveloped animal virus that infects humans and some other primates. AAV vectors can also infect dividing and quiescent cells without integrating into the host cell genome. The AAV genome consists of 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 ITR consists of a nucleotide sequence (5' ITR) containing a palindromic sequence at the 5' end and a nucleotide sequence (3' ITR) at the 3' end. The ITR acts in cis by folding using complementary base pairing to form a T-shaped hairpin structure that serves as a primer during DNA replication initiation of the second-strand synthesis. The two open reading frames encode the rep and cap genes involved in virion replication and packaging. In an exemplary aspect, the AAV vectors provided herein do not contain the rep or cap genes. It should be understood that AAV-based vectors are generally packaged into viral particles capable of infecting host cells. Thus, as used herein, "AAV vector" encompasses AAV viral particles that contain at least one AAV capsid protein and a capsidated AAV polynucleotide containing a transgene.

[0056] Serotypes that can be used 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, AAV 12, AAV13, AAVrh8, AAVrhlO, AAV-DJ, and AAV-DJ8. Serotypes typically differ in their tropism or the cell types they infect. An AAV can contain genomes and capsids from multiple serotypes (e.g., pseudotyped). For example, an AAV can contain the genome (e.g., ITR) of serotype 2 packaged in a capsid from serotype 5 or serotype 9. Pseudotyped vectors can exhibit improved transduction efficiency and altered tropism. In some cases, AAV serotypes that can cross the blood-brain barrier or infect cells of the CNS are preferred. An AAV can be self-complementary AAV (scAAV). See, e.g., Raj et al., Expert Rev Hematol. October 2011; 4(5):539-549. In certain aspects, the expression vector is an AAV vector comprising a 5' ITR and a 3' ITR. In some aspects, the expression vector is an AAV vector comprising a 5' ITR, a promoter, a nucleic acid encoding a regulatory factor (such as a TF), and a 3' ITR. In some aspects, the expression vector is an AAV vector comprising a 5' ITR, an enhancer, a promoter, a nucleic acid encoding a regulatory factor (such as a TF), a polyA sequence, and a 3' ITR. In some aspects, the AAV vector contains a nucleic acid comprising a response element operably linked to a reporter nucleic acid. In various aspects, the AAV vector is an AAV9 vector or an scAAV9 vector. In various aspects 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.

[0057] In some aspects, the present disclosure provides a composition comprising (i) a cell comprising a response element, wherein the cell is optionally engineered to stably express AAVR, and (ii) a sample comprising an AAV. In one example, the composition comprises (i) a HeRC32 cell comprising a response element, wherein the response element comprises a TF binding sequence comprising SEQ ID NO:1 and a reporter gene, and (ii) a sample comprising an AAV that comprises a nucleic acid sequence encoding a TF that binds to the SEQ ID NO:1 sequence. In some examples, HeRC32 stably expresses AAVR of SEQ ID NO:92. In some examples, the response element comprises a sequence of any of SEQ ID NO:10-15 and SEQ ID NO:17-23. In some cases, the sample comprises an AAV that comprises the nucleic acid sequence of SEQ ID NO:94.

[0058] The present disclosure further provides a method for determining the potency of a sample comprising an AAV vector. The AAV vector is as described above. The method comprises contacting all or a portion of the sample with the cells described herein. The cells comprise a response element operably linked to a reporter nucleotide sequence and are engineered to stably overexpress AAVR. The AAV vector encodes a regulatory factor that directly or indirectly modulates the expression of the reporter nucleotide sequence through the response element. The method further comprises measuring the expression of the reporter nucleotide sequence in the cells. In various aspects, the method then comprises determining the potency of the sample based on the measured expression level of the reporter nucleic acid sequence.

[0059] A "sample" comprising an AAV vector of interest can be any type of sample suitable for characterizing potency, typically an amount of AAV vector in a formulation 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 gene. For example, the sample can comprise at least about 1×10 2 per mL of the composition, 1×10 3 per mL of the composition, 1×10 4 per mL of the composition, 1×10 5 per mL of the composition, 1×10 6 per mL of the composition, 1×10 7 per mL of the composition, 1×10 8 per mL of the composition, 1×10 9 per mL of the composition, 1×10 10 per mL of the composition, 1×10 11 per mL of the composition, 1×10 12 per mL of the composition, 1×10 13 per mL of the composition, 1×10 14 per mL of the composition, 1×10 15 per mL of the composition, or 1×10 16 AAV vectors per mL of the composition. In various aspects, the sample is a composition of AAV vectors collected at one or more points during vector manufacture or purification. The sample can be a composition of AAV vectors extracted from a product batch prior to shipment. The methods of the present disclosure can comprise other additional steps that can further increase the purity of the AAV and remove other unwanted components and / or concentrate fractions for testing. The methods of the present disclosure are suitable for, for example, confirming the safety of an AAV vector product batch, characterizing the dose of an AAV vector, evaluating the activity of a vector composition, evaluating the stability of a vector composition (wherein the method is performed, for example, at different time points of the same sample) to show comparability of manufacturing variations, and / or determining the consistency between AAV product samples.

[0060] The AAV vector encodes a regulator that directly or indirectly regulates the expression of the reporter nucleotide sequence through the response element. In various aspects, the regulator comprises a DNA binding domain (DBD) that binds to the TF binding domain in the response element and activates or inhibits the expression of the reporter nucleotide sequence by virtue of the binding of the response element itself or the recruitment of other proteins that affect expression. In some embodiments, the regulator is a transcription factor that binds to the TF binding site in the response element. Transcription factors are further described above. In an exemplary embodiment, the TF is an engineered TF comprising a zinc finger domain that binds to a Z1 binding domain operably linked to a VP64 TMD.

[0061] The transcription factor can be any transcription factor disclosed herein or can comprise components of any of the transcription factors mentioned herein (e.g., the DNA binding domain or the transcriptional regulatory domain of the mentioned transcription factor). In an exemplary aspect 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 an exemplary aspect of the present disclosure, the engineered transcription factor comprises a DNA binding domain that comprises 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 from 1 to 15, such as 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., recognition helix) capable of binding three bp of the target sequence. In an exemplary embodiment, n is 3, 6, or 9. In a particularly preferred embodiment, n is 6. In various embodiments, each X can independently have the same amino acid sequence or a different amino acid sequence compared to other X sequences in the DNA binding domain. In an exemplary embodiment, each X is a sequence comprising seven amino acids that is designed to interact with three bp of the target binding site of interest using the Scripps Zinger Finger design tool located at scripps.edu / barbas / zfdesign / zfdesignhome.php on the World Wide Web. The engineered transcription factor optionally further comprises a VP64 transcriptional regulatory domain. In some cases, the transcription factor can have a sequence having 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.

[0062] In alternative aspects 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 can be recombinantly produced by the cell. In various aspects, the regulator is ATP7B, which is a copper-transporting P-type ATPase. The ATP7B protein is located in the trans-Golgi network of the liver and brain and balances the 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 the copper levels in the cell, thereby indirectly regulating reporter gene expression through the metal-responsive TF. When the copper level is high, the TF (e.g., MTF-1) drives the expression of the reporter gene by binding to the response element (e.g., a response element containing one or more MTF-1 binding sites). When the copper level is low, the reporter gene expression also decreases. Although the above description describes the method in the context of copper metabolism, it should be understood that the method is not limited thereto; the method can be used in combination with any ligand-dependent TF (including other metal-responsive TFs) and other regulators that regulate the amount of ligand in the cell.

[0063] The reporter nucleic acid and its encoded protein are as described above. Methods for measuring the expression of the reporter nucleotide sequence in a cell 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 gene expression is characterized by measuring the activity or property of the protein encoded by the reporter nucleotide sequence, i.e., measuring the luminescence of a luminescent protein or measuring the luminescence mediated by an enzyme that produces bioluminescence. The luminescence activity can be determined by any suitable method (such as the method described in, for example, Inouye, S. and Shimomura, O. (1977) Biochem. Biophys. Res. Commun. 233, 349-353). In fact, fluorescence can be detected and quantified using, for example, flow cytometry, and / or the reporter mRNA level can be quantified by RT-qPCR. The amount of the signal or expression product produced by the reporter gene can be used to obtain quantifiable absolute or relative data regarding the potency of the test vector preparation.

[0064] In various aspects, the method includes determining the potency of a sample based on the measured expression level of a reporter nucleic acid sequence or expression product. In this regard, the method optionally includes comparing the measured expression level to a standard potency curve of the AAV vector. The standard curve is generated by evaluating the reporter signal for a specific amount of the AAV vector (i.e., vector concentrations at different dilutions) in multiple replicates. Alternatively, the standard curve can be generated by evaluating the measured signal for a specific amount of the reporter nucleic acid sequence DNA, mRNA, or expression product.

[0065] The present disclosure further provides a system or kit comprising any combination of the components described herein. In an exemplary embodiment, the system or kit comprises one, two, or all of the following: (A) a nucleic acid currently disclosed comprising a response element operably linked to a reporter nucleic acid sequence, (B) a cell overexpressing AAVR, and / or (C) a regulator that directly or indirectly affects reporter gene expression through the response element. As described herein, the regulator can directly contact the response element to activate or repress transcription of the reporter nucleic acid. Alternatively, the regulator can indirectly affect transcription of the reporter nucleic acid through the response element, for example, by regulating the amount of a ligand within the cell, thereby regulating the activity of a ligand-dependent transcription factor that binds to the response element of (A). For example, the regulator can comprise a protein or a nucleic acid comprising a sequence encoding a protein. Accordingly, the present disclosure provides a kit comprising a cell 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 Z1TF binding site). The kit can contain instructions for using the components in a method (e.g., a method according to the present disclosure). Ancillary materials for assisting or enabling performance of such methods can be included in the kits of the present disclosure.

[0066] Examples

[0067] The following examples are given only to illustrate the invention and do not limit its scope in any way.

[0068] Example 1

[0069] This example describes the construction and testing of different nucleic acid constructs comprising a response element operably linked to a reporter nucleic acid.

[0070] Using standard molecular biology techniques, several reporter gene constructs encoding N-luciferase ("Nluc") operably linked to different response elements were constructed. A summary of the components of each reporter gene construct is provided in Table 1, and the structures of the different reporter gene constructs are shown in Figure 1. The description of the response element (RE) includes the number of repeats of a specific TF binding domain (e.g., the Z1 TF binding domain) (e.g., "8x" is eight copies).

[0071] Figure 1A The cascade response element design found in class I synthetic reporter gene constructs is detailed. The Z1 response element is activated by VP64 and / or CITED4 binding found in class I synthetic reporter gene constructs. Construct P1 contains the Tet-responsive promoter PTight consisting of seven tet operator sequences, followed by the minimal CMV promoter. The second class of response elements (class II) contains multiple Z1 sequences inserted into the tetracycline response element (TRE) scaffold. Figure 1B The response element: minPro design found in some (i.e., P13, P12, and P22) of the class II synthetic reporter gene constructs is detailed. Figure 1C The response element: endogenous SCN1A target / promoter domain found in the genomic reporter gene constructs is detailed. Figure 1D The hybrid design found in some reporter gene constructs (i.e., P15, P16, and P14) is detailed.

[0072] Table 1

[0073]

[0074]

[0075] The function of each reporter gene construct was tested as follows: HEK293T cells were placed in individual wells of a 96-well plate at approximately 10,000 cells per well. Using HD transfection reagent (Promega, Madison, WI), cells in each well were transfected with three plasmids at a total amount of 100 ng. Each cell well was triple-transfected with (a) a constitutive transfection control plasmid (20 ng), (b) a reporter plasmid described in Table 1 or shown in Figure 1 (40 ng), and (c) an activator plasmid or a control filler plasmid (40 ng) (a total of 100 ng of plasmids). (a) The constitutive transfection control plasmid contains a plasmid that expresses firefly luciferase under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter element (TK-firefly). (b) The reporter gene construct contains one of the Z1-based response elements described in Table 1 and Figure 1, or lacks the Z1 response element (designated by "Δ" in the name of the response element in Table 1). If present, the Z1 TF binding domain (SEQ ID NO:1) is present in 1 to 8 tandem copies (represented by "1X", "3X", "6X", "8X"). (c) The activator plasmid encodes an artificial transcription factor activator that binds to the Z1 TF binding domain (amino acid sequence of SEQ ID NO:84) of the reporter gene construct. The control filler plasmid does not contain the artificial transcription factor activator but encodes a non-bioluminescent enhanced green fluorescent protein (EGFP). If the cells are successfully transfected, it is expected that each well will exhibit a baseline fluorescence signal, which is attributed to the constitutive expression of firefly luciferase from this transfection control plasmid. Any fluorescence signal above the baseline is attributed to the N-luciferase of the reporter gene construct. An additional luciferase signal is expected to be produced only when the reporter gene construct contains the Z1 response element and the activator plasmid is co-transfected into the cells. If the reporter gene construct does not contain the Z1 response element (element with "ΔZ1"), or if the control filler plasmid (which does not encode a transcription factor activator) is co-transfected, it is expected that the cells will not exhibit a fluorescence signal above the baseline.

[0076] After transfection (24 hours post-transfection), the plates were lysed and diluted 1:100 in ONE-Glo EX luciferase assay buffer (Promega). Firefly and NanoLuc luminescence of the diluted lysates were measured according to the manufacturer's protocol (Nano-Glo Dual-Luciferase Reporter Assay System, Promega), and relative light units (RLU) were measured on a bioluminescence plate reader (GloMax, Promega). Each transfection was performed in triplicate, and the data reflect the mean of triplicate wells (mean ± standard deviation).

[0077] The results are shown in Figure 2 Due to the expression of the firefly luciferase gene, all cells exhibited similar baseline fluorescence signals ( Figure 2The left bar of each construct in demonstrated successful transfection of the cells. When cells were co-transfected with a reporter gene construct containing a Z1-based response element and an activator plasmid, a fluorescence signal above baseline was observed ( Figure 2 to the right bar of the construct in ). The strongest Nluc reporter gene induction was observed in class I and II synthetic variants, where genomic and hybrid elements showed less upregulation from activator co-transfection ( Figure 4 ). Higher baseline and induction activities were generally observed in class I synthetic variants compared to class II variants. Based on this preliminary screening, a subset of synthetic variants was selected for further screening: P-5, P-9, P-10, P-11, P-12, and P-13.

[0078] These data demonstrated activator-dependent luciferase reporter gene induction in a plasmid transfection system by a reporter gene construct containing a Z1 response element.

[0079] Example 2

[0080] This example demonstrated dose-dependent luciferase reporter gene induction in a nucleic acid containing a response element operably linked to a reporter nucleic acid in a transient co-transfection system.

[0081] The leading reporter gene constructs from Example 1 were selected for further study. In one experiment, the sensitivity of class I and II synthetic reporter gene constructs to the amount of activator plasmid was measured. Briefly, HEK293T cells were aliquoted into wells of a 96-well plate and then transfected with three types of plasmids as described in Example 1. However, unlike Example 1, different amounts of activator plasmid were used. The cells in each well received 20 ng of a constitutive transfection control plasmid, 10 ng of a reporter gene construct, and a combination of a total of 70 ng of activator plasmid and control filler plasmid. To evaluate the sensitivity of the reporter gene construct to different 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 filler plasmid such that a total of 70 ng of activator and filler plasmid was used in the transfection. The cells were processed after transfection, and the luminescence levels were measured as described in Example 1. Each transfection was performed in triplicate; the data reflect the mean of triplicate wells (mean ± standard deviation).

[0082] The results are shown in Figure 3Shown in. Each reporter gene construct exhibited sensitivity to different amounts of activator, showing 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 wider range of signal induction was also observed with increasing copies of the z1 element. Therefore, lead candidate reporter gene variants with the maximum number of z1 element copies were selected from each response element class, P-5 and P-13.

[0083] Example 3

[0084] This example demonstrates that induction of reporter gene expression in the nucleic acids of the present disclosure requires an activator binding response element in a transient co-transfection system.

[0085] HEK293T cells were cultured by standard methods and each well of a 96-well plate was co-transfected (a total of 100 ng plasmid) by (FugeneHD): constitutive transfection control plasmid (20 ng), reporter plasmid (40 ng), and 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 included a tetracycline response element (TRE) upstream of the Nluc open reading frame (“Δ reporter gene”). The Z1-based reporter plasmid contained a Z1 response element (7XZ1-TRE, P-5) upstream of the Nluc open reading frame. In this element, multiple Z1 sequences were inserted into the tetracycline response element (TRE) scaffold (Gossen and Bujard 1992). The activator plasmid expressed an artificial transcription factor activator targeting the Z1 18-bp DNA element (Z1 eTF, SEQ ID NO:84) or a transcription factor activator not targeting the Z1 18-bp DNA element (“ΔZ1 eTF”, SEQ ID NO:95). Twenty-four hours after transfection, the cells were lysed and diluted 1:1000 in ONE-Glo EX luciferase assay buffer (Promega Corporation). Firefly and NanoLuc luminescence of the diluted lysate was measured according to the manufacturer's protocol (Nano-Glo Dual-Luciferase Reporter Assay System, Promega Corporation), and relative light units (RLU) were measured on a bioluminescence plate reader (GloMax, Promega Corporation). Each transfection was performed in triplicate, and the data reflect the mean of triplicate wells (mean ± standard deviation).

[0086] Figure 4It was shown that no significant signal was observed in the case of a single reporter gene, a reporter gene and a non-targeted transcription factor (reporter gene + ΔZ1 eTF), or a TRE response element and a Z1-targeted transcriptional activator (Δreporter gene + Z1 eTF). A strong response was only observed in the condition containing a reporter gene (reporter gene + Z1 eTF) containing a Z1 element and a Z1-targeted transcriptional activator, indicating a specific interaction between the Z1 eTF and the Z1 element.

[0087] Example 4

[0088] This example describes the generation of reporter gene constructs containing Z1-based response elements, which constructs have a single reporter gene (enhanced green fluorescent protein (EGFP) coding sequence) and a dual reporter gene system.

[0089] A single reporter gene construct containing a single enhanced GFP coding cassette (sPA = polyA signal sequence) and a dual reporter gene construct containing an EGFP coding cassette and an mCherry coding cassette were prepared using standard techniques. Figure 5 Schematic diagrams of different single constructs and dual constructs are shown.

[0090] The single reporter gene construct was assayed as follows: HEK293T cells were cultured by standard methods and each well of a 96-well plate was triple-transfected (FugeneHD) with a reporter plasmid (50 ng), an activator plasmid (50 ng), or a non-fluorescent plasmid filler (50 ng) (100 ng total plasmid). The Z1-based reporter plasmid contained a Z1 response element (6Xz1-mPro) upstream of the EGFP open reading frame (P-47). This 6XZ1-mPro element contains six 18-bp Z1 target DNA sites and a 5-bp spacer sequence, cascaded 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 (Δ6XZ1-mPro). Twenty-four hours after transfection, the plates were imaged for EGFP and mCherry fluorescence (ImageXpress DLR, Molecular Devices). Fluorescence was detected only in cells transfected with the Z1-based reporter plasmid (6XZ1-mPro) operably linked to the EGFP open reading frame (P-47). No fluorescence was observed using the control plasmid lacking the intact Z1 response element.

[0091] As described above for single reporter gene constructs, dual reporter gene constructs were assayed. In addition to the Z1 reporter gene and control plasmid, the dual reporter gene constructs also contained a second expression cassette that contained an mCherry element under the control of the constitutive EF1a short (EFS) promoter. To allow for independent expression, this constitutive expression cassette was separated from the reporter gene expression cassette by an insulator sequence containing the human β-globin transcriptional termination sequence (hACTB) and the chicken β-globin insulator (cHS4). Detection of mCherry-based fluorescence in the samples being assayed demonstrated that the second reporter nucleic acid under the control of a separate promoter was functional in the context of the system of the present disclosure. As in the single reporter gene assay, EGFP-based 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 EGFP fluorescence was observed using a control plasmid lacking the complete Z1 response element.

[0092] Example 5

[0093] This example describes the generation and characterization of cells stably expressing the wild-type AAV receptor (AAVR). The in vitro transduction efficiency of AAV9 in immortalized cell lines is low. To address this, cell lines were engineered with AAVR to significantly enhance AAV9 transduction.

[0094] 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), and were grown in a humidified incubator with 5% CO2 at 37°C. Purified and titrated stock solutions of adeno-associated virus (AAV) serotypes 1, 3, 5, 6, 8, 9, and DJ were either prepared in-house or purchased from Vector Biolabs. All AAV stock solutions were ssDNA·AAV vectors encoding the reporter gene (GFP).

[0095] A stable cell line expressing AAVR was generated using a lentiviral AAVR vector. A recombinant lentiviral vector containing the AAVR coding sequence was generated 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 for chimeric bioengineering in HEK293T cells. The vector was harvested from the cell supernatant 48 hours after transfection and placed in the corresponding cell line to generate a heterologous stable cell population overexpressing AAVR. Puromycin selection was used to isolate lentivirus-positive cells. Once western blot confirmed overexpression of AAVR in the heterologous population, the cells were sorted into 96-well plates as single cells using BD FACSAriaII. The resulting single subclones were grown for over 14 days and screened for AAV transduction efficiency using AAV9-CBA-GFP. The top clones of each cell line were grown and frozen.

[0096] Cells stably expressing AAVR were seeded at 10,000 cells / well (96-well plate) or 100,000 cells (24-well plate) overnight. The cells were then infected with the AAV stock solution at a multiplicity of infection (MOI) of specified viral genomes / cell in complete DMEM. Viral infectivity was determined 48 hours after infection by measuring transgene expression by flow cytometry (fluorescence) or at the mRNA level (RT-qPCR).

[0097] Flow cytometry: To measure GFP expression after AAV-GFP infection, the cells were trypsinized 48 hours after infection and BD FACS Melody was performed to detect fluorescent cells. Uninfected cells were used as a negative control. Two parameters were evaluated, namely the percentage of GFP-positive cells (infection %) and the mean fluorescence intensity (mean fluorescence per cell).

[0098] RNA extraction and qPCR: RNA was extracted from the corresponding cell pellet using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions and then used to synthesize cDNA with SuperScript IV Reverse Transcriptase (Invitrogen). Quantitative PCR was performed using a lamin A / C, lamin A, or lamin C-specific primer set to evaluate the expression level of the corresponding gene.

[0099] Western blot: 2×10 6The cell pellet of cells was boiled at 95 °C for 10 minutes. The lysate was separated by SDS-PAGE on a 4-15% polyacrylamide gradient gel (Bio-Rad) using the Mini-Protean system (Bio-Rad). In a semi-wet preparation, proteins were transferred to a nitrocellulose membrane (Bio-Rad) using the Transblot protein transfer system from Bio-Rad. The membrane was blocked by incubating with 1× PBS buffer containing 5% non-fat milk at room temperature (RT) for 1 hour. Subsequently, the membrane was incubated overnight at 4 °C with primary antibodies diluted at 1:1000 (anti-KIAA0319L antibody) or 1:2000 (anti-GAPDH antibody) in the blocking buffer. The membrane was washed three times for 5 minutes using the wash buffer (1× PBS buffer containing 0.1% Tween-20) and further incubated for 1 hour at RT in an HRP-conjugated secondary antibody (anti-mouse and anti-rabbit-1:5000 in the blocking buffer) (GeneTex). After another set of three washes, antibody-bound AAVR (bands expected at 150 kD) was visualized on a chemiluminescence reader.

[0100] RNA extraction, cDNA generation, and duplex qPCR: RNA was extracted from the respective infected cells (24-well format) using the RNAeasy Mini Kit (Qiagen) according to the manufacturer's instructions, and then used to synthesize cDNA with the SuperScript TM VILO TM cDNA Synthesis Kit with ezDNase TM enzyme (Invitrogen). Duplex quantitative PCR was performed using TaqMan Fast Advanced Mastermix and VP64 or GAPDH (housekeeping) probes. Calculate 2 -ddCt to determine the relative fold change.

[0101] Results

[0102] Figure 6A and Figure 6B show the results of AAVR overexpression in HEK293T cells and HeLa-RC32 cells. AAV9-EF1a-GFP-KASH was provided at a multiplicity of infection (MOI) of 50,000. Cells were harvested 48 hours after infection, and the samples assayed contained 30,000 cells. Overexpression of AAVR increased GFP-positive cells (i.e., AAV+-transduced cells) 8- to 10-fold ( Figure 6A ), and also increased the expression of GFP by approximately three-fold ( Figure 6B ; MFI = mean fluorescence intensity). Figure 7A and Figure 7BShows the results of overexpression of AAVR in HEK293T cells and CHO-Lec2 cells. AAV9-EF1a-GFP-KASH was provided at a multiplicity of infection (MOI) of 10,000. Cells were harvested 48 hours after infection, and the samples assayed contained 30,000 cells. Overexpression of AAVR quadrupled the number of GFP-positive CHO-Lec2 cells, while engineered HEK-293T cells showed a 35-fold higher effect at this higher MOI and using the CAG promoter to drive GFP expression. The cells expressed significantly more GFP on average. Subclones of the engineered cells stably expressing AAVR showed enhanced AAV9 transduction. See, for example, Figure 8A and 8B . Similar results were observed for HeLa-RC32 and CHO-Lec2 subclones. Enhanced infectivity of multiple AAV serotypes was demonstrated. See Figures 10A - 10D . HEK293T cells and HeLa-RC32 cells stably overexpressing wild-type AAVR were exposed to different serotype AAV vectors encoding GFP, and the AAV infection rate was detected. When AAVR was stably expressed, the infectivity of AAV-1, AAV-3, AAV-5, AAV-8, AAV-9, and AAV-DJ was enhanced in both cell types assayed, while AAV-3, AAV-8, and AAV-9 showed approximately 10-fold improvement in transduction. Mean fluorescence intensity data supplemented the transduction data ( Figures 10C - 10D ), showing a correlation between AAV infection rate and transgene expression.

[0103] Example 6

[0104] 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.

[0105] Specifically, the HeRC32-AAVR cell line was generated by stably transducing the HeRC32 cell line (ATCC, catalog number CRL-2972) with a lentivirus overexpressing the human AAVR gene under the EFS promoter (P-64). Positive transduced cells were selected using puromycin, and the resulting heterogeneous population was sorted by single cell sorting to isolate a clonal population overexpressing AAVR. These clones were screened based on AAV9 transduction efficiency. The corresponding cell lines were infected with AAV9-CBA-GFP at an MOI of 100,000 for 48 hours, and GFP expression was analyzed by flow cytometry. The percentage of GFP-positive cells was used to measure AAV transduction. As Figure 11As seen, the selected clones exhibited more than fifty-fold transduction compared to the parental HeRC32 cell line.

[0106] Candidate reporter cell lines were then generated by stable lentiviral transduction of HeRC32-AAVR cells for each of three reporter transgenes: 1) P-6, including a 7X Cascade Z1 response element in a V1 dual reporter gene format, 2) P-8, including a 7X Cascade Z1 response element in a V2 single reporter gene format, or 3) P-13, including a Z1 substituted TRE response element in a V2 single reporter gene format. The V1 dual reporter gene 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 that allows internal control. The resulting heterologous stable cell lines (designated HeRC32-AAVR-P-6, HeRC32-AAVR-P-8, HeRC32-AAVR-P-13) were then further evaluated for reporter gene expression and assay feasibility.

[0107] Preliminary assays were performed in the HeRC32-AAVR-P-8 heterologous cell line. These cells were plated 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 at a 3-point MOI series from 1E4 to 1E6 genomic copies / cell. As a positive control condition, separate reporter cell lines were transfected with either the individual activator (eTF, SEQ ID NO:84) plasmid or co-transfected reporter plasmids in the original screening vector (P-5) or lentiviral packaging plasmid (P-8).

[0108] Cells were assayed 48 hours after transduction with the eTF activator (SEQ ID NO:84). Luciferase assays were performed as previously described and the cell lysates were not diluted to address the overall signal reduction due to reduced reporter gene copy number and AAV transduction.

[0109] As Figure 12 seen, strong reporter gene induction above baseline was observed under both reporter plasmid transfection conditions (P-5 or P-8) and 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.

[0110] Example 7

[0111] 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. Specifically, the nucleic acids of the present disclosure were integrated into the genome of HeLa - AAVR cells that constitutively express the AAVR gene using a lentiviral vector, resulting in the generation of a stable cell line (AAVR - reporter gene HeLa) that expresses a luciferase reporter gene.

[0112] As Figure 13 shown therein, different lentiviral constructs were prepared. One backbone (V2) contained a response element - reporter gene cassette, while the other backbone (V1) contained a response element - reporter gene cassette and an additional reporter gene 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, which contained the same response element as P - 8 (7Xz1 - TRE - Nluc), but further contained a second expression cassette that included a firefly luciferase element under the control of a constitutive EF1a short (EFS) promoter. The response element - reporter gene 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. With respect to the response element - reporter gene cassette P - 6, the constitutive expression cassette encoding firefly luciferase was separated from the response element - Nluc cassette by an insulator sequence containing the human β - globin transcriptional termination sequence (hACTB) and the chicken β - globin insulator (cHS4).

[0113] The lentivirally transduced AAVR - reporter gene HeLa cells were cultured as a heterogeneous population by standard methods. The cells were transduced with AAV expressing a transcription factor targeting Z1 (SEQ ID NO:94) (as a reference standard (RS)) at different multiplicities of infection (MOI) of 1×10 4 、3×10 4 、1×10 5 、3×10 5 、1×10 6 、3×10 6 . To test samples of known potency, the same vector was repeated as an assay control (AC), or diluted to 70% and 40% before performing the same dilution series. After 48 hours, the cells were lysed and the firefly and NanoLuc luminescence of the cells were measured according to the manufacturer's protocol (Nano - Glo Dual - Luciferase Reporter Assay System, Promega Corporation). Relative luminescence (RLU) was measured on a bioluminescence plate reader (GloMax, Promega Corporation). Each transfection was performed in triplicate; the data reflect the mean of the triplicate wells (mean ± standard deviation).

[0114] The results are shown in Figures 14A - 14B and Figures 15A - 15B . As expected, dose-dependent Nluc expression was observed in all three cell lines for the eTF activator (SEQ ID NO:94). The dual reporter line HeRC32-AAVR-P-6 produced constitutive expression of firefly luciferase, which showed only minimal response to the eTF activator (SEQ ID NO:94) dose. To evaluate whether these dose responses could be used to assess relative potency, dose responses under different dilution conditions, linear regression was applied to each dose response curve, and preliminary parallel line analysis was performed on the three test samples against a reference standard using log10 MOI and log10 RLU values ( Figure 16 and 17 ). Variability was typically observed in the cell lines and in the linearity of the dose responses; however, the responses in each cell line could be described by a linear relationship between Log10 MOI and Log10 RLU. Table 2 summarizes the results of the parallel line analysis of relative potency, providing proof of concept for the feasibility of relative potency measurements in the reporter cell lines.

[0115] Table 2

[0116]

[0117] Table 2: Summary of the relative potencies of samples tested in candidate cell lines obtained by parallel line analysis (PLA): across two independent experiments, the control (AC), 40% relative potency sample (40%), 70% relative potency sample (70%) were assayed.

[0118] To confirm the specificity of the reporter gene for AAV in the final HeRC32-AAVR reporter cell line, the reporter gene response was compared with AAV9 and a control AAV (AAV9-CBA-Δz1-eTF) carrying eTF targeting z1 under the control of a strong ubiquitous CBA promoter, which is identical except for a sequence mutation in the DNA-binding domain of eTF targeting z1. The AAV9-CBA-z1-eTF construct should produce a robust dose response as it expresses the eTF transgenic targeting Z1. In contrast, the DNA-binding mutant vector (AAV9-CBA-Δz1-eTF) should not produce reporter gene activity as the reporter gene response element cassette contains the specific z1 eTF target sequence. Additionally, a reference standard control sample was included, which contains eTF targeting z1 expressed under the control of a GABA-selective promoter element (SEQ ID NO:94). Due to the strength of the CBA promoter compared to the GABA-selective promoter, the AAV9-CBA-z1eTF construct was expected to produce a stronger induction. Therefore, to capture the linear range of all samples, a complete 10-point dose curve was included. A robust dose-dependent reporter gene activation was observed in response to AAV9-CBA-z1-eTF, see Figure 16 B (triangle). Due to the stronger CBA promoter, the dose response showed a leftward shift compared to the reference standard (circle). In contrast, the control AAV9-CBA-Δz1-eTF sample (square) did not activate reporter gene activity. Collectively, these data demonstrate the assay specificity for the eTF activator and the requirement for sequence-dependent eTF-DNA target interactions for reporter gene activation.

[0119] Additionally, to further confirm the assay specificity, a control AAV9 vector was generated using the same promoter and vector design as SEQ ID NO:94, which is identical to SEQ ID NO:94 except for a mutant sequence in the six 7-amino acid zinc finger DNA-binding domains that define the 18bp target DNA sequence. This control sample did not activate reporter gene activity compared to the reference standard or the assay control (AC) vector, see Figure 17 . Therefore, this assay is specific for the eTF of SEQ ID NO:84.

[0120] All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference to the extent that each reference is specifically and individually indicated to be incorporated by reference and is set forth in its entirety herein.

[0121] Unless otherwise indicated herein or clearly contradicted by context, the terms "a / an" and "the" and similar referential terms used in the context of describing the present disclosure (especially in the context of the following claims) shall be construed to cover both the singular and the plural; the terms "a" (or "an"), "one or more", and "at least one" may be used interchangeably herein. Unless the context clearly requires otherwise, the term "or" shall be understood to cover alternative or conjunctive items. The term "and / or" shall be understood to cover each item in the list (individually), any combination of items in the list, and all items in the list together. Unless otherwise stated, the terms "comprising", "having", "including", and "containing" are construed as open-ended terms (i.e., meaning "including but not limited to"). The present disclosure contemplates embodiments described as "comprising" a feature to include embodiments "consisting of" or "consisting essentially of" the feature.

[0122] Unless otherwise indicated herein, the recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value and each endpoint falling within the range, and each separate value and endpoint is incorporated into the specification as if recited individually herein.

[0123] Unless otherwise indicated herein or clearly contradicted by context, all method steps described herein may be performed in any suitable order. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the present disclosure and does not limit the scope of the present disclosure. No language in this specification should be construed as indicating any non-claimed element as essential to practicing the present disclosure.

[0124] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the present disclosure. After reading the foregoing description, variations of those preferred embodiments may become apparent to those of ordinary skill in the art. Accordingly, to the extent permitted by applicable law, the present disclosure includes all modifications and equivalents of the subject matter recited in the appended claims. In addition, unless otherwise indicated herein or clearly contradicted by context, the present disclosure covers any combination of the above elements in all possible variations thereof. Indeed, the features of the invention described herein may be recombined into additional embodiments that are also intended to be aspects of the invention, regardless of whether the combination of features is designated as an aspect or embodiment of the invention. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of the features described herein (even if described in separate sections) are also contemplated, even if the combinations of features do not coexist in the same sentence or paragraph or section of this document.

[0125] Sequence

[0126] <OO00365>

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] Note: There seems to be a typo in the original text where "OO00365" should probably be " ". This has been corrected in the translation.

[0142]

[0143]

[0144]

Claims

1. 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 Z1 transcription factor (TF) binding site.

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

3. The nucleic acid according to claim 1 or claim 2, 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 having 1, 2, or 3 nucleotide differences from SEQ ID NO:

1.

4. The nucleic acid according to 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 according to 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 the sequence of any one of SEQ ID NO:10 - 15, SEQ ID NO:17 - 23, and SEQ ID NO:

25.

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

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

9. The nucleic acid according to claim 8, wherein the minimal promoter is any one of the following: CMV minimal promoter, hsp70 minimal promoter, the minimal promoter included in the tetracycline response element, or MinTk minimal promoter.

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

32.

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

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

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

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

91.

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

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

20. The cell according to claim 19, wherein the AAVR is wild-type AAVR.

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

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

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

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

27. The cell according to 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, wherein the cell is further engineered to stably overexpress an adeno-associated virus receptor (AAVR).

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

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

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

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

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

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

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

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

8.

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

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

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

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

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

1.

42. The cell according to claim 41, wherein the engineered DNA binding domain is selected from the group consisting of SEQ ID NO: 76-83.

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

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

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

46. The cell according to claim 45, wherein the promoter is a minimal promoter.

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

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

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

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

91.

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

52. A method for determining the potency of a sample comprising an AAV vector, the method comprising: (a) contacting a cell according to any one of claims 28 to 51 with all or part of the sample, wherein the expression vector encodes a regulator that directly or indirectly regulates the expression of the reporter nucleotide sequence through 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. The method according to claim 52, wherein the regulator is a transcription factor that binds to the TF binding site in the response element.

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

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

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

57. The nucleic acid according to claim 16, wherein the second reporter nucleotide sequence encodes a luminescent protein or an enzyme that produces bioluminescence.

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

59. The nucleic acid according to claim 57, wherein the enzyme is luciferase.

60. The nucleic acid according to 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, wherein the response element comprises a TF binding site bound by an endogenous metal-dependent transcription factor (TF).

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

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

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

8.

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

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

67. The nucleic acid according to claim 66, wherein the minimal promoter is any one of the following: CMV minimal promoter, hsp70 minimal promoter, the minimal promoter included in the tetracycline response element, or MinTk minimal promoter.

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

32.

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

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

71. The nucleic acid according to claim 69, wherein the enzyme is luciferase.

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

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

91.

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

75. The nucleic acid according to claim 74, wherein the second reporter nucleotide sequence encodes a luminescent protein or an enzyme that produces bioluminescence.

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

77. The nucleic acid according to claim 75, wherein the enzyme is luciferase.

78. The nucleic acid according to 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, an Ad E1A promoter or a cytomegalovirus (CMV) promoter.

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

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

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

82. The cell according to claim 81, wherein the AAVR is wild-type AAVR.

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

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

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

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

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

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