A method for detecting the activity of gdnf protein in vitro

Abstract

The application relates to a method for detecting GDNF protein activity in vitro, comprising the following steps: a) contacting an adeno-associated virus (AAV) containing a nucleotide sequence encoding a GDNF protein with first cells expressing an adeno-associated virus receptor (AAVR), culturing the first cells, and collecting culture supernatant; b) incubating the culture supernatant in step a) with second cells, wherein the second cells are reporter gene cells; and c) detecting an indicating signal generated by the reporter gene, wherein the reporter gene cells contain a GFR alpha 1 expression element, a RET expression element, a transcription response module and a reporter gene module; the transcription response module contains a GAL4-ELK1 fusion protein and / or a nucleic acid encoding the fusion protein; and the reporter gene module contains a nucleic acid encoding a GAL4 response element and a reporter gene.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a method for detecting the activity of GDNF protein expressed by AAV-GDNF gene drugs. Background Technology

[0002] Glial cell line-derived neurotrophic factor (GDNF) is a representative member of the GDNF family of neurotrophic factors (GFLs), belonging to the transforming growth factor-β (TGF-β) superfamily, and possesses a highly conserved dimer structure. GDNF binds to GDNF family receptor α1 (GFRα1) and synergizes with RET receptor tyrosine kinase on the cell membrane to form a ternary ligand-receptor complex, thereby initiating an intracellular signal transduction cascade. GDNF binding to GFRα1 promotes RET receptor dimerization and autophosphorylation, subsequently activating multiple downstream signaling pathways, primarily including the MAPK / ERK pathway, the PI3K / Akt pathway, and the PLCγ pathway. Activation of these signaling axes induces a series of functional effects in the nervous system, such as promoting neuronal survival, inducing axonal regeneration, inhibiting apoptosis, and regulating synaptic plasticity and inflammatory responses.

[0003] GDNF has demonstrated significant neuroprotective and functional recovery effects in various neurological disease models, particularly in Parkinson's disease, where its selective protective effect on dopaminergic neurons has attracted widespread attention. Studies have shown that GDNF can improve motor function in Parkinson's disease models, increase dopamine metabolism levels in the substantia nigra-striatal pathway, and slow the neurodegenerative process. Besides Parkinson's disease, GDNF has also shown broad potential therapeutic value in various other diseases, including motor neuron disease, peripheral nerve injury, spinal cord injury, chronic pain, hearing impairment, and depression.

[0004] With the ongoing development of GDNF gene therapy products (especially AAV-GDNF), several AAV-GDNF drugs have entered clinical trials. There is an urgent need to establish a scientific, stable, and easily standardized in vitro GDNF functional activity detection method for AAV-GDNF gene drugs to support their pharmaceutical research, pharmacodynamic evaluation, and clinical product release. This in vitro GDNF functional activity detection method has become one of the key technological bottlenecks in the development of such products.

[0005] Currently, the publicly available technologies for evaluating the in vitro activity of AAV-GDNF products mainly fall into two categories:

[0006] 1) Determination of GNDF protein expression level (e.g., CN118525098A). However, as a highly function-dependent protein, the efficacy of GDNF depends not only on concentration but also on conformational correctness, ligand-receptor binding capacity, and signal transduction efficiency. Therefore, it is difficult to comprehensively assess its biological effects solely through protein content or purity indicators; an in vitro detection system capable of quantifying its functional activity is required.

[0007] 2) Indirect activity assessment methods for detecting the protective effect (survival rate) of AAV-GDNF drugs on nerve cells (e.g., CN117018231B). However, these methods generally suffer from drawbacks such as narrow response windows, low throughput, complex operation, or large fluctuations in results. In particular, the signal response fold for cell proliferation or viability assays is limited, and background noise is high, making it difficult to meet the requirements for high-precision, batch-to-batch comparable quantification. These problems are especially prominent when GDNF enters the productization stage (e.g., batch release testing, stability verification).

[0008] For AAV-GDNF gene therapy products, there is still a need to establish direct, high dynamic range, high specificity and good reproducibility activity detection methods to meet the evaluation requirements of different vector configurations, titers and expression intensities. Summary of the Invention

[0009] This application provides a reporter gene detection method for directly and efficiently detecting the activity of GDNF protein expressed by AAV-GDNF viral particles. This method features rapid response (6–24 hours), significant signal amplification, a wide detection window, and EC50. 50 This method offers stable and simple operation, allowing direct detection of GDNF protein activity in the supernatant of AAVR-overexpressing cells infected with AAV-GDNF viral particles. It is suitable for multiple key stages, including AAV-GDNF drug screening, structure optimization, process development, quality control, and biopharmaceutical release. It addresses the shortcomings of existing in vitro activity detection techniques for AAV-GDNF-derived GDNF proteins, such as insufficient sensitivity, narrow detection window, and difficulties in standardization. This provides an activity detection platform with a clearly defined pathway mechanism, stable and reliable results, and suitability for industrial applications, demonstrating significant application and promotion value.

[0010] On the one hand, this application provides a method for detecting GDNF protein activity, the method comprising the following steps:

[0011] a) Contact adeno-associated virus (AAV) containing a nucleotide sequence encoding the GDNF protein with a first cell expressing an adeno-associated virus receptor (AAVR), culture the first cell, and collect the culture supernatant;

[0012] b) Incubate the culture supernatant from step a) with the second cell, wherein the second cell is a reporter gene cell; and

[0013] c) Detect the indicator signal generated by the reporter gene.

[0014] The reporter gene cell comprises a GFRα1 expression element, a RET expression element, a transcriptional response module, and a reporter gene module; the transcriptional response module comprises a GAL4-ELK1 fusion protein and / or nucleic acid encoding the fusion protein; the reporter gene module comprises nucleic acid encoding a GAL4 response element and a reporter gene.

[0015] On the other hand, this application provides a method for detecting GDNF protein activity, the method comprising the following steps:

[0016] a) Contact adeno-associated virus (AAV) containing a nucleotide sequence encoding the GDNF protein with a first cell expressing an adeno-associated virus receptor (AAVR), culture the first cell, and collect the culture supernatant;

[0017] b) Incubate the culture supernatant from step a) with the second cell, wherein the second cell is a reporter gene cell; and

[0018] c) Detect the indicator signal generated by the reporter gene.

[0019] The reporter gene cell contains a GFRα1 expression element, a RET expression element, a transcriptional response module, and a reporter gene module; the transcriptional response module contains a GAL4-ELK1 fusion protein; and the reporter gene module contains nucleic acids encoding a GAL4 response element and a reporter gene.

[0020] In some embodiments, the reporter gene is a nucleic acid encoding luciferase.

[0021] In some embodiments, the nucleic acid encoding luciferase comprises the nucleotide sequence shown in SEQ ID NO: 19. In some embodiments, the nucleic acid encoding luciferase expresses a luciferase protein, wherein the luciferase protein comprises the amino acid sequence shown in SEQ ID NO: 18.

[0022] In some embodiments, the GAL4-ELK1 fusion protein, the DNA-binding domain derived from the GAL4 protein, and the transcriptional activation domain derived from the ELK1 protein are described.

[0023] In some embodiments, the DNA-binding domain derived from the GAL4 protein is amino acid residues 1-147 of the GAL4 protein, and the transcriptional activation domain derived from the ELK1 protein is amino acid residues 307-428 of the ELK-1 protein, wherein the amino acid sequence of the GAL4 protein is as shown in SEQ ID NO: 1, and the amino acid sequence of the ELK-1 protein is as shown in SEQ ID NO: 4.

[0024] In some embodiments, the GAL4-ELK1 fusion protein comprises an amino acid sequence as shown in SEQ ID NO: 7.

[0025] In some embodiments, the transcriptional response module further comprises a nucleic acid encoding the GAL4-ELK1 fusion protein. In some embodiments, the nucleic acid encoding the NLS-GAL4-ELK1 fusion protein comprises a nucleotide sequence as shown in SEQ ID NO: 12.

[0026] In some embodiments, the GFRα1 expression element comprises the GFRα1 protein, and the GFRα1 protein comprises the amino acid sequence shown in SEQ ID NO: 13.

[0027] In some embodiments, the GFRα1 expression element further comprises a nucleic acid encoding the GFRα1 protein, the nucleic acid encoding GFRα1 comprising a nucleotide sequence as shown in SEQ ID NO: 14.

[0028] In some embodiments, the RET expression element comprises a RET protein, and the RET protein comprises an amino acid sequence as shown in SEQ ID NO: 15.

[0029] In some embodiments, the RET expression element further comprises a nucleic acid encoding the RET protein, the nucleic acid encoding RET comprising a nucleotide sequence as shown in SEQ ID NO: 16.

[0030] In some embodiments, the GAL4-ELK1 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 7; the GFRα1 expression element comprises the GFRα1 protein, and the GFRα1 protein comprises the amino acid sequence shown in SEQ ID NO: 13; the RET expression element comprises the RET protein, and the RET protein comprises the amino acid sequence shown in SEQ ID NO: 15.

[0031] In some embodiments, the GAL4-ELK1 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 7; the GFRα1 expression element comprises the GFRα1 protein and / or nucleic acid encoding the GFRα1 protein, and the GFRα1 protein comprises the amino acid sequence shown in SEQ ID NO: 13; the RET expression element comprises the RET protein and / or nucleic acid encoding the RET protein, and the RET protein comprises the amino acid sequence shown in SEQ ID NO: 15.

[0032] In some embodiments, the GAL4-responsive element comprises four or five GAL4 binding sites arranged in series.

[0033] In some embodiments, the encoding of the GAL4 response element comprises a nucleotide sequence as shown in SEQ ID NO: 17.

[0034] In some embodiments, the module encoding the reporter gene comprises a nucleotide sequence as shown in SEQ ID NO: 20.

[0035] In some embodiments, the GFRα1 expression element further comprises a nucleic acid encoding the GFRα1 protein, and the nucleic acid encoding the GFRα1 protein comprises a nucleotide sequence as shown in SEQ ID NO: 14; the RET expression element further comprises a nucleic acid encoding the RET protein, and the nucleic acid encoding the RET protein comprises a nucleotide sequence as shown in SEQ ID NO: 16; the transcription response module further comprises a nucleic acid encoding the GAL4-ELK1 fusion protein, and the nucleic acid encoding the GAL4-ELK1 fusion protein comprises a nucleotide sequence as shown in SEQ ID NO: 8; the nucleic acid sequence encoding luciferase is shown in SEQ ID NO: 19; and the nucleic acid encoding the GAL4 response element comprises a nucleotide sequence as shown in SEQ ID NO: 17.

[0036] In some embodiments, the nucleic acid encoding the NLS-GAL4-ELK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 12; the nucleic acid encoding the GFRα1 protein comprises the nucleotide sequence shown in SEQ ID NO: 14; the nucleic acid encoding the RET protein comprises the nucleotide sequence shown in SEQ ID NO: 16; the nucleic acid encoding the luciferase comprises the nucleotide sequence shown in SEQ ID NO: 19; and the nucleic acid encoding the GAL4 response element comprises the nucleotide sequence shown in SEQ ID NO: 17.

[0037] In some embodiments, the transcription response module further comprises a nuclear localization signal (NLS) or a nucleic acid encoding the nuclear localization signal (NLS). In some embodiments, the nuclear localization signal NLS comprises the amino acid sequence shown in SEQ ID NO:9. In some embodiments, the nucleic acid encoding the nuclear localization signal (NLS) comprises a nucleotide sequence as shown in SEQ ID NO:10.

[0038] In some embodiments, the transcriptional response module comprises an NLS-GAL4-ELK1 fusion protein or a nucleic acid encoding an NLS-GAL4-ELK1 fusion protein.

[0039] In some embodiments, the NLS-GAL4-ELK1 fusion protein comprises an amino acid sequence as shown in SEQ ID NO: 11.

[0040] In some embodiments, the nucleic acid encoding the NLS-GAL4-ELK1 fusion protein comprises a nucleotide sequence as shown in SEQ ID NO: 12.

[0041] In some embodiments, the first cell is a HEK293 cell, and the reporter gene cell is a HEK293 cell.

[0042] In some embodiments, the indicator signal generated by the reporter gene is detected before, during, and / or after incubating the culture supernatant with the reporter gene cells.

[0043] In some embodiments, it further includes comparing the culture supernatant with reporter gene indicator signals generated before, during, and / or after incubation with reporter gene cells to determine the activity of the GDNF protein to be detected.

[0044] In some embodiments, it further includes comparing the indicator signal of the reporter gene with a reporter gene indicator signal generated from a control sample, wherein the control sample is selected from one or more of the following:

[0045] a) Samples containing exogenous GDNF protein;

[0046] b) Culture supernatant obtained after infecting first-cell cells with AAV that does not encode GDNF; and

[0047] c) Culture supernatant obtained after infecting first cells with AAV containing GDNF.

[0048] In some embodiments, the AAV containing a nucleotide sequence encoding GDNF is selected from one or more of the following groups: AAV encoding different GDNF variants, AAV from different production batches, AAV with different viral titers, AAV with different capsid serotypes, and AAV with different promoters.

[0049] In some embodiments, the comparison includes: comparing a reporter gene indicator signal generated from the culture supernatant obtained after infecting the first cells with different AAVs with a reporter gene indicator signal generated from the control sample, and normalizing the indicator signal to represent a relative GDNF protein activity value relative to the control sample.

[0050] On the other hand, this application provides an use for evaluating the biological activity of an AAV formulation encoding a GDNF protein, wherein the use includes determining the activity of the GDNF protein by the method.

[0051] On the other hand, this application provides the use of the method described herein in the preparation of gene therapy products, comprising:

[0052] (a) Provides an AAV formulation comprising a GDNF viral vector, wherein the GDNF viral vector comprises a nucleotide encoding one or more AADC polypeptides;

[0053] (b) Using the method described above, the relative GDNF protein activity value of the GDNF viral vector in the AAV formulation was determined;

[0054] (c) Compare the relative GDNF protein activity value with a threshold value for the relative GDNF protein activity value; and

[0055] (d) If the relative GDNF protein activity value is greater than or equal to the threshold, the corresponding batch of AAV formulation is dispensed into the formulation container.

[0056] On the other hand, this application provides a detection kit for detecting GDNF protein activity, wherein the kit is adapted to implement the method for detecting GDNF protein activity.

[0057] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0058] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0059] Figure 1 Flowchart of reporter gene cell construction

[0060] Figure 2 Flow cytometry validation results of GFRα1 receptor expression rate in reporter genes cells

[0061] Figure 3 Validation of reporter gene-mediated cell response to GDNF (dose-response relationship)

[0062] Figure 4 Results of GDNF activity detection by cell proliferation method

[0063] Figure 5 Response results of reporter gene cells to AAV-GDNF expression supernatant

[0064] Figure 6 Figure 1 shows the results of relative biological activity assay of AAV-GDNF test sample (TA). Detailed Implementation

[0065] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0066] Terminology Definition

[0067] In this application, the term "transcriptional response module" generally refers to a molecule or combination of molecules that has the function of regulating gene transcription. The transcriptional response module may be derived from naturally occurring transcriptional regulation-related domains, or may be the full-length form of the natural domain or a truncated form retaining its transcriptional regulatory function. The transcriptional response module may comprise one or more transcription factors, transcriptional regulatory domains, transcriptional activation domains, transcriptional repression domains, DNA-binding domains, nuclear localization signals (NLS), signal transduction-related domains, or any combination of the above domains. The transcriptional response module may exist in the form of nucleic acids, such as the nucleotide sequence encoding the transcriptional response module; it may also exist in the form of proteins, and the transcriptional response module may be a single protein or a fusion protein composed of two or more domains. In some embodiments, the transcriptional response module comprises a nuclear localization signal (NLS), a DNA-binding domain derived from the GAL4 protein, and a transcriptional activation domain derived from the ELK1 protein. In some embodiments, the DNA-binding domain derived from the GAL4 protein is amino acid residues 1-147 of the GAL4 protein, and the transcriptional activation domain derived from the ELK1 protein is amino acid residues 307-428 of the ELK-1 protein, wherein the amino acid sequence of the GAL4 protein is SEQ ID NO: 1, and the amino acid sequence of the ELK-1 protein is SEQ ID NO: 4. In some embodiments, the transcriptional response module is an NLS-GAL4-ELK1 fusion protein, and the amino acid sequence of the fusion protein is SEQ ID NO: 11. In some embodiments, the transcriptional response module comprises nucleic acid encoding the NLS-GAL4-ELK1 fusion protein, and the sequence of the nucleic acid is shown in SEQ ID NO: 12.

[0068] In this application, the term "reporter gene module" generally encompasses reporter gene-related components for generating detectable indicator signals, including nucleic acid constructs containing a reporter gene and its regulatory sequences, and / or protein products or functional derivatives thereof produced by the expression of the reporter gene. The regulatory sequences may include promoters, enhancers, response elements (e.g., GAL4 response elements), terminators, and / or other transcriptional or translational regulatory elements, and the reporter gene module may be directly or indirectly linked to a transcriptional response module, such that the expression of the reporter gene is regulated by the transcriptional response module. The detectable indicator signal may be an optical signal, a fluorescence signal, an electrochemical signal, an enzyme activity signal, or a combination thereof. Exemplary examples of reporter genes include, but are not limited to, luciferase, NanoLuc, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), mCHerry, alkaline phosphatase (SEAP), β-galactosidase (β-gal), or a luciferase / fluorescent protein combined reporter system. In some implementations, the reporter gene module may include a GAL4 response element and a Luciferase gene. When the GAL4 response element is activated by a transcription factor, it can drive the downstream Luciferase gene to express the Luciferase protein, generating a quantifiable fluorescence signal.

[0069] In this application, the term "GAL4 response element" generally refers to a nucleic acid sequence containing one or more binding sites capable of specifically binding to the GAL4 protein or its DNA-binding domain, and mediating transcriptional regulation of downstream genes. The one or more GAL4 binding sites can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or equivalently described as 1×UAS, 5×UAS, or 10×UAS), and these binding sites can be tandem, repeated, or arranged in any order. The GAL4 response element can be derived from the natural GAL4 upstream activating sequence (AUS) or is an artificially designed, modified, or variant sequence. The GAL4 protein or its DNA-binding domain also includes variants or fragments functionally equivalent to the GAL4 DNA-binding domain. The GAL4 response element can functionally link to a promoter or other transcriptional regulatory sequence and drive the expression of any type of reporter gene.

[0070] In this application, the term "GFRα1 expression element" generally encompasses components associated with GFRα1 expression, including (i) nucleic acid constructs containing a nucleotide sequence encoding a GFRα1 protein; and / or (ii) the GFRα1 protein or a functional variant thereof expressed by said nucleotide sequence. In this application, the term "GFRα1 protein" generally refers to glial cell line-derived neurotrophic factor family receptor alpha 1, a receptor protein capable of binding to glial cell line-derived neurotrophic factor (GDNF), typically located on the cell membrane surface, and participating as a co-receptor in GDNF-mediated signal transduction processes. The GFRα1 protein includes the full-length protein in its natural or recombinant form, as well as its biologically functional fragments, truncated forms, variants, or homologs; the GFRα1 protein may be derived from human sources (e.g., it can be equivalently described as hGFRα1 or H_GFRA1) or other species, and may be in a post-translational modified form, a form fused with other proteins or peptides, or in a free or complex state, as long as it retains the biological functions associated with GFRα1.

[0071] In this application, the term "RET expression element" generally encompasses components associated with RET expression, including (i) nucleic acid constructs containing a nucleotide sequence encoding a RET protein; and / or (ii) a RET protein or a functional variant thereof expressed from said nucleotide sequence. In this application, the term "RET protein" refers to a rearranged-during-transfection receptor tyrosine kinase, a transmembrane receptor tyrosine kinase typically located on the cell membrane surface, which mediates downstream signal transduction upon binding to a ligand-co-receptor complex. In this application, the RET protein includes full-length proteins in natural or recombinant forms, as well as fragments, truncated versions, variants, or homologs thereof with biological function; the RET protein may be derived from human sources (e.g., can be equivalently described as hRET or H_RET) or other species, and may be in a post-translational modified form, fused with other proteins or peptides, or in an inactive or activated state, as long as it retains its RET-related biological function.

[0072] In this application, the term "GDNF" or "GNDF protein" generally refers to glial cell line-derived neurotrophic factor, a secretible growth factor protein belonging to the GDNF family. In this application, the GDNF protein includes the full-length protein in its natural or recombinant form, as well as biologically active fragments, variants, homologs, or post-translational modified forms. Further, the GDNF protein can be obtained by expression in cells using a vector containing a nucleotide sequence encoding GDNF, including but not limited to adeno-associated virus (AAV). In some embodiments, the GDNF protein can be expressed and secreted by cells infected with the vector. In some embodiments, the GDNF protein can exist in a free form in a culture system, including but not limited to being present in cell culture supernatants, or in the form of recombinant protein formulations, purified proteins, or mixtures thereof. In this application, the biological activity of GDNF can be detected through its mediated signal transduction processes. For example, the activity of GDNF can be characterized by detecting its activated downstream signaling pathways, such as by using the method for detecting GDNF protein activity described in this application.

[0073] In this application, the term "luciferase" refers to a coding product that can be used as a reporter gene, which, upon expression, generates a detectable luminescent signal under appropriate substrate and reaction conditions, thereby serving as an indicator signal to reflect the expression status of a target gene, protein activity, or the activation state of a cellular signaling pathway. When used as a reporter gene, the luciferase can be expressed alone or constructed into a fusion reporter system with other proteins, peptides, or regulatory elements. In this application, the luminescent signal generated by the luciferase can be compared to quantitatively or qualitatively characterize the activity of the GDNF protein.

[0074] In this application, the term "AAV vector" or "AAV" generally refers to adenovirus itself or a derivative thereof. Adeno-associated virus (AAV) generally refers to a class of single-stranded DNA viruses belonging to the family Parvoviridae and the genus Dependent Virus. An AAV genome may contain inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs). The ORFs may include rep and cap. rep consists of multiple overlapping sequences encoding the Rep protein required for the AAV life cycle, and cap contains overlapping nucleotide sequences encoding capsid proteins, which may include VP1, VP2, and VP3. The capsid proteins interact to form a capsid. In the absence of a helper virus, AAV can integrate its genome into a specific site on human chromosome 19 (AAVS site) until a helper virus rescues it from its latent state (Kotin et al., 1990). AAV is generally considered to be predominantly in an unintegrated form. AAVs, with their site-specific integration capability, broad host range, high safety, low immunogenicity, stable expression, and stable physical properties, have been widely used in basic research and clinical trials, and have become one of the most commonly used gene therapy vectors in the world. AAVs have many common serotypes and over 100 viral variants. In this application, the AAV capsid, ITR, and other selected AAV components are selected from any AAV, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, and AAVAnc80, any known or mentioned variants of AAVs, or undiscovered AAVs or their variants or mixtures.

[0075] The term "adeno-associated virus receptor" or "AAVR" is the universal major receptor for most adeno-associated virus (AAV) serotypes (such as AAV1, AAV2, AAV5, AAV8, AAV9, etc.) to enter cells. The function of AAVR is to mediate the binding of AAV to host cells and promote viral internalization through clathrin or caveolin-mediated endocytosis, thereby determining the tissue tropism and transduction efficiency of the virus. Studies have found that removing AAVR significantly reduces the infectivity of AAV, indicating its crucial role in AAV gene therapy. AAVR, also known as KIAA0319L, is a multi-transmembrane protein, currently classified in the literature into four major structural / functional domains: signal peptide region (SP, amino acids 1-28), extracellular region (amino acids 29-739), single transmembrane region (TM, amino acids 740-760), and cytoplasmic tail region (C-tail, amino acids 761-835). The extracellular region consists of three tandem carboxypeptidase-like domains (CP1, CP2, and CP3), containing amino acids 29-264, 265-492, and 493-739, respectively. CP1 is the core for AAV binding. The three domains together form a catalytic pocket, but enzyme activity is not essential for the receptor function of AAVR. The single transmembrane region anchors the protein to the cell membrane; deletion or mutation in this region leads to decreased efficiency of AAV entry into cells expressing AAVR. The cytoplasmic tail region participates in downstream signal transduction and protein stability regulation; deletion or mutation in this region also leads to decreased efficiency of AAV entry into cells expressing AAVR. It is important to note that the amino acid site counts are based on the wild-type AAVR protein site count; regardless of any mutations or deletions in AAVR compared to the wild-type, the amino acid site count remains unchanged.

[0076] The expression level of AAVR in wild-type cells is related to cell type, and the expression level varies significantly among different cell types. Therefore, it should be noted that the phrase "cells expressing AAVR" and similar descriptions in this application should be understood as referring to cells with low AAVR expression levels, specifically "cells overexpressing AAVR" or "cells expressing exogenous AAVR." For cells with high AAVR expression levels, this can refer to wild-type cells themselves, or to "cells overexpressing AAVR" or "cells expressing exogenous AAVR." In short, "cells expressing AAVR" should be understood as cells with high AAVR expression levels, regardless of whether they are wild-type cells or transformed cells. The expression level can be obtained by flow cytometry, and "high expression level" refers to an AAVR expression rate greater than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%. The expression rate can specifically be the proportion of reporter gene expression in transformed cells, such as the expression proportion of fluorescent protein or drug resistance. More specifically, the expression rate refers to the proportion of cells expressing the reporter gene after transformation out of all transformed cells. A high expression level can also be characterized by a fold change in the mRNA or protein encoding AAVR (e.g., a fold change greater than 2, 3, 4, 5, 10, 20, 30, 40, 50, or 100 times).

[0077] In this application, the term "control sample" generally refers to a sample used as a reference standard when detecting, comparing, or evaluating a target sample. The "control sample" can be used to provide a background level, reference level, or standardized benchmark for relative or quantitative analysis of the detection results generated by the target sample. In this application, the control sample may contain components that are known or expected to lack target activity, have known target activity, or have a predetermined expression level, and its forms include, but are not limited to, cell culture supernatant, exogenous protein formulations, samples obtained through vector expression, untreated samples, or combinations thereof. In some embodiments, the control sample may be a culture supernatant obtained after infecting cells with a vector that does not contain the target protein, a sample containing the exogenous target protein, or a reference sample obtained from different constructs or conditions.

[0078] In this application, the term "normalization" generally refers to processing one or more test results relative to a reference standard. Normalization can be used to eliminate or reduce inter-sample differences, differences in experimental conditions, or systematic biases, enabling different test results to be compared or analyzed on the same scale. In this application, normalization can be based on a control sample, a reference signal, a background signal, an internal reference signal, or a combination thereof, and can be achieved through proportional conversion, standardization, background subtraction, relative value calculation, or other equivalent methods. In some embodiments, the normalization method includes fitting the dose-response data of the test sample and the reference standard respectively using a four-parameter logistic regression (4PL) model to obtain their respective half-maximum effective concentrations (EC50) while ensuring comparability of curve shapes. 50 ), and by testing the EC of the sample and the reference standard. 50 The ratio is normalized to obtain the relative bioactivity value. For example, relative bioactivity can be obtained by measuring the EC50 of the test sample. 50 EC with reference standard 50 The ratio is obtained by calculation.

[0079] In this application, the term "human embryonic kidney 293 cells" or "HEK293 cells" generally refers to a cell line derived from human embryonic kidney cells. HEK293 cells are characterized by their ease of culture and high transfection efficiency, and are a commonly used cell line in the art for studying endogenous or exogenous genes. Uses of HEK293 cells in the art include, but are not limited to, their use as nucleic acid vectors, for the production of various viruses, for the production of nucleic acids, and for the production of recombinant proteins.

[0080] The term "incubation" or "co-incubation" refers to the process of placing two or more systems together in the same container for a period of time. Typically, both systems are liquid, although there are exceptions.

[0081] In the context of this disclosure, unless otherwise indicated, the words “comprise” and its variations such as “comprises” and “comprising” will be understood to imply inclusion of the stated elements (e.g., amino acid sequences, nucleotide sequences, properties, steps, or groups thereof), but do not exclude any other elements (e.g., amino acid sequences, nucleotide sequences, properties, and steps). When used herein, the term “comprise” or any variation thereof may be replaced by the terms “containing,” “comprising,” or sometimes “having,” or equivalent variations thereof. In some embodiments, the word “comprise” also includes the case of “consisting of.”

[0082] In this application, the term “selected from” generally refers to the selection of objects and all combinations thereof. For example, “selected from A, B and C” means all combinations of A, B and C, such as A, B, C, A+B, A+C, B+C, or A+B+C.

[0083] As used herein (including the appended claims), unless the context clearly indicates otherwise, singular terms such as “a”, “an” and “the” include their corresponding plural counterparts. Invention Details

[0085] Method

[0086] On the one hand, this application provides a method for detecting GDNF protein activity, the method comprising the following steps:

[0087] a) Contact adeno-associated virus (AAV) containing a nucleotide sequence encoding the GDNF protein with a first cell expressing an adeno-associated virus receptor (AAVR), culture the first cell, and collect the culture supernatant;

[0088] b) Incubate the culture supernatant from step a) with the second cell, wherein the second cell is a reporter gene cell; and

[0089] c) Detect the indicator signal generated by the reporter gene.

[0090] The reporter gene cell comprises a GFRα1 expression element, a RET expression element, a transcriptional response module, and a reporter gene module; the transcriptional response module comprises a GAL4-ELK1 fusion protein and / or nucleic acid encoding the fusion protein; the reporter gene module comprises nucleic acid encoding a GAL4 response element and a reporter gene.

[0091] First cell

[0092] In some embodiments, the adeno-associated virus receptor (AAR) expressed by the first cell may include any AAVR of any vertebrate origin, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). Preferably, the AAVR is human. Specifically, the AAVR may be a human protein numbered Q8IZA0 on the Uniprot website.

[0093] In some embodiments, the AAVR is a wild-type AAVR. In some embodiments, the AAVR is a mutated AAVR, where the mutation can be the deletion, substitution, and / or insertion of one or more amino acids. In some embodiments, the AAVR is an AAVR with the deletion or insertion of an entire amino acid fragment. In some embodiments, the AAVR is an AAVR with a domain deletion. In some embodiments, the AAVR is an AAVR with a domain insertion.

[0094] In a more specific embodiment, the amino acid sequence of the AAVR is shown in SEQ ID NO: 21. In a more specific embodiment, the nucleotide sequence encoding the AAVR is shown in SEQ ID NO: 22.

[0095] In some embodiments, the first cell may be a primary cell or a cell line. In a preferred embodiment, the cell may be a cell that is easily transformed. For example, the cell may be selected from one or more of the following: HEK293, 293T, 293F, HeLa, COS-7, CHO-K1, CHO-S, NIH / 3T3, Jurkat, K562, HCT116, A549, MEF, HBEC, HMEC, and RK3E. In a more specific embodiment, the first cell is a HEK293 cell expressing AAVR.

[0096] In some embodiments, the method for causing the first cell to express AAVR includes, but is not limited to: (1) introducing a foreign nucleic acid containing a nucleotide sequence encoding AAVR, for example, the foreign nucleic acid may be a plasmid; (2) delivering the nucleotide encoding AAVR into the cell using a viral vector, for example, the viral vector includes, but is not limited to, lentivirus, adenovirus or adeno-associated virus; (3) delivering mRNA encoding AAVR protein or modifying mRNA into the cell; (4) causing the cell to overexpress AAVR by regulating the expression level of the endogenous AAVR gene in the cell.

[0097] In some implementations, the first cell may transiently or continuously express AAVR stably.

[0098] In some embodiments, the first cell has higher AAV infection or transduction efficiency. In some embodiments, the first cell is used to contact an AAV containing an AAV encoding a target sample protein (e.g., a GDNF protein for which the activity is to be analyzed), the contact including infection and / or transduction.

[0099] Step a)

[0100] In some embodiments, the first cell is contacted with adeno-associated virus (AAV) containing a nucleotide sequence encoding the GDNF protein, the first cell is cultured, and the culture supernatant is collected.

[0101] In some embodiments, the culture lasts for about 12 to about 96 hours, preferably about 24 to about 84 hours, more preferably about 36 to about 84 hours, even more preferably about 48 to about 76 hours, and most preferably about 72 hours, for example 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, and 76 hours.

[0102] In some embodiments, the adeno-associated virus (AAV) containing a nucleotide sequence encoding the GDNF protein that the first cell contacts infects the cell with a multiple of infection (MOI) of about 1E3 to about 1E6, preferably about 1E2 to about 5E6, more preferably about 9E2 to about 5E5, for example, about 5E2, about 6E2, about 7E2, 8E2, about 9E2, about 1E3, about 2E3, 3E3, etc. Approximately 4E3, approximately 5E3, approximately 6E3, approximately 7E3, approximately 8E3, approximately 9E3, approximately 1E4, approximately 2E4, approximately 3E4, approximately 4E4, approximately 5E4, approximately 6E4, approximately 7E4, approximately 8E4, approximately 9E4, approximately 1E5, approximately 2E5, approximately 3E5, approximately 4E5, approximately 5E5, approximately 6E5, approximately 7E5, approximately 8E5, approximately 9E5, approximately 1E6, approximately 2E6, approximately 3E6, approximately 4E6, approximately 5E6.

[0103] In some embodiments, the culture is carried out under conventional or regulated culture conditions, including but not limited to variations in temperature, gas conditions, culture medium composition, or combinations thereof.

[0104] In some embodiments, the culture supernatant is obtained by separating the cells from the liquid components in the culture system. The separation methods include, but are not limited to, centrifugation, settling and collecting the supernatant, filtration, or other methods capable of removing cells or cell debris.

[0105] In some embodiments, after collecting the culture supernatant, the supernatant may be further processed to obtain a sample suitable for subsequent detection or analysis. Such processing includes, but is not limited to: removing cell debris or impurities; concentration or dilution; changing the buffer system or culture medium; transferring to a new detection container; preservation, cryopreservation, or thawing.

[0106] In some embodiments, the culture supernatant contains a target protein expressed and secreted by the first cell, which may exist in free form or coexist with culture medium components, carrier residues, or other cell-derived components. In this application, whether the culture supernatant has undergone purification is not a limitation.

[0107] Second cell

[0108] In some embodiments, the second cell is a reporter gene cell. In some embodiments, the reporter gene cell includes a GFRα1 expression element, a RET expression element, a transcriptional response module, and a reporter gene module.

[0109] In some embodiments, the reporter gene is a nucleic acid encoding luciferase.

[0110] In some embodiments, the nucleic acid encoding luciferase comprises the nucleotide sequence shown in SEQ ID NO: 19. In some embodiments, the nucleic acid encoding luciferase expresses a luciferase protein, wherein the amino acid sequence of the luciferase protein is SEQ ID NO: 18.

[0111] In some embodiments, the transcriptional response module comprises a GAL4-ELK1 fusion protein.

[0112] In some embodiments, the GAL4-ELK1 fusion protein package is derived from the DNA-binding domain of the GAL4 protein and the transcriptional activation domain of the ELK1 protein.

[0113] In some embodiments, the transcriptional response module comprises the NLS-GAL4-ELK1 fusion protein.

[0114] In some embodiments, the NLS-GAL4-ELK1 fusion protein includes a nuclear localization signal (NLS), a DNA-binding domain derived from the GAL4 protein, and a transcriptional activation domain derived from the ELK1 protein.

[0115] In some embodiments, the DNA-binding domain derived from the GAL4 protein is amino acid residues 1-147 of the GAL4 protein, and the transcriptional activation domain derived from the ELK1 protein is amino acid residues 307-428 of the ELK-1 protein, wherein the amino acid sequence of the GAL4 protein is as shown in SEQ ID NO: 1, and the amino acid sequence of the ELK-1 protein is as shown in SEQ ID NO: 4.

[0116] In some embodiments, the GAL4-ELK1 fusion protein comprises an amino acid sequence as shown in SEQ ID NO: 7.

[0117] In some embodiments, the transcriptional response module comprises a nucleic acid encoding the GAL4-ELK1 fusion protein. In some embodiments, the nucleic acid encoding the GAL4-ELK1 fusion protein comprises a nucleotide sequence as shown in SEQ ID NO: 8.

[0118] In some embodiments, the NLS-GAL4-ELK1 fusion protein comprises an amino acid sequence as shown in SEQ ID NO: 11.

[0119] In some embodiments, the transcriptional response module comprises a nucleic acid encoding the NLS-GAL4-ELK1 fusion protein. In some embodiments, the nucleic acid encoding the NLS-GAL4-ELK1 fusion protein comprises a nucleotide sequence as shown in SEQ ID NO: 12.

[0120] In some embodiments, the GFRα1 expression element comprises the GFRα1 protein, and the GFRα1 protein comprises the amino acid sequence shown in SEQ ID NO: 13.

[0121] In some embodiments, the GFRα1 expression element comprises a nucleic acid encoding the GFRα1 protein, the nucleic acid encoding GFRα1 comprising a nucleotide sequence as shown in SEQ ID NO: 14.

[0122] In some embodiments, the RET expression element comprises a RET protein, and the RET protein comprises an amino acid sequence as shown in SEQ ID NO: 15.

[0123] In some embodiments, the RET expression element comprises a nucleic acid encoding the RET protein, wherein the nucleic acid encoding GFRα1 comprises a nucleotide sequence as shown in SEQ ID NO: 16.

[0124] In some embodiments, the NLS-GAL4-ELK1 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 11; the GFRα1 expression element comprises the GFRα1 protein, and the GFRα1 protein comprises the amino acid sequence shown in SEQ ID NO: 13; the RET expression element comprises the RET protein, and the RET protein comprises the amino acid sequence shown in SEQ ID NO: 15.

[0125] In some implementations, the reporter gene module comprises nucleic acids encoding a GAL4 response element and a reporter gene.

[0126] In some embodiments, the GAL4 responsive element comprises 1 to 10 GAL4 binding sites arranged in series, for example, it may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GAL4 binding sites arranged in series.

[0127] In some embodiments, the encoding of the GAL4 response element comprises a nucleotide sequence as shown in SEQ ID NO: 17.

[0128] In some embodiments, the module encoding the reporter gene comprises a nucleotide sequence as shown in SEQ ID NO: 20.

[0129] In some embodiments, the GFRα1 expression element comprises a nucleic acid encoding the GFRα1 protein, and the nucleic acid encoding the GFRα1 protein comprises a nucleotide sequence as shown in SEQ ID NO: 14; the RET expression element further comprises a nucleic acid encoding the RET protein, and the nucleic acid encoding the RET protein comprises a nucleotide sequence as shown in SEQ ID NO: 16; the transcription response module further comprises a nucleic acid encoding the NLS-GAL4-ELK1 fusion protein, and the nucleic acid encoding the NLS-GAL4-ELK1 fusion protein comprises a nucleotide sequence as shown in SEQ ID NO: 12; the nucleic acid encoding luciferase comprises a nucleotide sequence as shown in SEQ ID NO: 19; and the nucleic acid encoding the GAL4 response element comprises a nucleotide sequence as shown in SEQ ID NO: 17.

[0130] In some embodiments, the reporter gene cell can be a primary cell or a cell line. In a preferred embodiment, the cell can be a cell that is easily transformed. For example, the cell is selected from one or more of the following: HEK293, 293T, 293F, HeLa, COS-7, CHO-K1, CHO-S, NIH / 3T3, Jurkat, K562, HCT116, A549, MEF, HBEC, HMEC, and RK3E. In a more specific embodiment, the reporter gene cell is HEK293 cell.

[0131] In some embodiments, the first cell is a HEK293 cell, and the reporter gene cell is a HEK293 cell.

[0132] Step b)

[0133] In some embodiments, the method involves incubating the culture supernatant from step a) with a second cell, the second cell being a reporter gene cell.

[0134] In some embodiments, the culture supernatant can be brought into contact with reporter gene cells by direct addition, replacement of the culture medium, proportional mixing, or gradual addition.

[0135] In some embodiments, the culture supernatant may be untreated raw supernatant, or supernatant that has been diluted, concentrated, filtered, or replaced with buffer solution.

[0136] In some embodiments, the culture supernatant may be incubated with reporter cells at different volume ratios or different equivalent concentrations to create a dose gradient or concentration gradient. In some embodiments, the culture supernatant may be serially diluted at a predetermined ratio for use in dose-response analysis.

[0137] In some embodiments, the incubation duration can be several minutes, several hours, or longer, depending on the target protein's functional characteristics, signaling pathway activation kinetics, or detection requirements. In some embodiments, the incubation duration can be about 1-48 hours, about 2-24 hours, about 3-23 hours, about 4-22 hours, about 5-21 hours, about 6-20 hours, about 8-20 hours, or about 12-20 hours. In some embodiments, the incubation duration can be about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, or about 48 hours.

[0138] In some embodiments, the incubation conditions may be the same as the standard culture conditions for reporter gene cells, or may be adjusted according to the detection purpose. In some embodiments, the incubation conditions include: a culture temperature of 37°C and a carbon dioxide (CO2) content of 5%.

[0139] In some implementations, the reporter gene cells may be washed, have their culture medium changed, or be further cultured before detection.

[0140] Step c)

[0141] In some implementations, the method includes detecting an indicator signal generated by the reporter gene.

[0142] In some embodiments, the indicator signal is a detectable signal generated directly or indirectly by the reporter gene expression product. The signal includes, but is not limited to: luminescence signals, fluorescence signals, absorbance changes, enzyme activity signals, electrochemical signals, or combinations of the above.

[0143] In some implementations, the indication signal is obtained by optical detection, imaging detection, spectral detection, or plate reading detection.

[0144] In some embodiments, the indicator signal originates from the reporter gene expression product itself. In other embodiments, the indicator signal originates from the reaction product catalyzed or mediated by the reporter gene expression product. In some embodiments, the detection is performed at the cellular level or the well plate level.

[0145] In some embodiments, a substrate, cofactor, or chromogenic reagent for generating or enhancing an indicator signal may be added to the system before or during detection. In some embodiments, the detection is performed without lysing cells or after cell lysis.

[0146] In some embodiments, the reporter gene is a luciferase and / or a nucleic acid encoding a luciferase. In some embodiments, the luciferase can be a single luciferase or a reporter system composed of multiple luciferases. In some embodiments, the luciferase reporter gene can be used in conjunction with other reporter genes or internal controls.

[0147] In some embodiments, the luminescent signal is generated by the reaction of luciferase with its corresponding substrate. The substrate may be added before, during, or at the start of the detection step. In some embodiments, the luminescent signal is a transient luminescent signal, a stable luminescent signal, or a combination of both.

[0148] In some embodiments, the detection is used to obtain a quantitative signal value for subsequent comparison, normalization, or activity calculation. In some embodiments, the detection is used to qualitatively or semi-quantitatively assess the presence or trend of an indicator signal.

[0149] Detection time point

[0150] In some implementations, the detection may be performed at a single time point or repeated at multiple time points to obtain time-related signal changes.

[0151] In some embodiments, the indicator signal generated by the reporter gene is detected before, during, and / or after incubating the culture supernatant with the reporter gene cells.

[0152] In some implementations, the detection of the indicator signal generated by the reporter gene can be performed before, during, and / or after incubation of the culture supernatant with the reporter gene cells to obtain background signals, dynamic signals, and / or endpoint signals.

[0153] In some embodiments, it further includes comparing the culture supernatant with reporter gene indicator signals generated before, during, and / or after incubation with reporter gene cells to determine the activity of the GDNF protein to be detected.

[0154] Detection object

[0155] In some embodiments, it further includes comparing the indicator signal of the reporter gene with a reporter gene indicator signal generated from a control sample, wherein the control sample is selected from one or more of the following:

[0156] a) Samples containing exogenous GDNF protein;

[0157] b) Culture supernatant obtained after infecting first-cell cells with AAV that does not encode GDNF; and

[0158] c) Culture supernatant obtained after infecting first cells with AAV containing GDNF.

[0159] In some embodiments, the AAV containing a nucleotide sequence encoding GDNF is selected from one or more of the following groups: AAV encoding different GDNF variants, AAV from different production batches, AAV with different viral titers, AAV with different capsid serotypes, and AAV with different promoters.

[0160] In some embodiments, the AAV comprising a nucleotide sequence encoding a GDNF protein, wherein the nucleotide sequence may encode different GDNF protein variants. The GDNF variants include, but are not limited to: full-length GDNF protein, truncated, fragmented, or fused forms of GDNF, GDNF variants with amino acid substitutions, deletions, or insertions, and nucleotide sequences that have been codon-optimized or sequence-modified but encode functionally equivalent GDNF proteins. In some embodiments, the relative biological activity of different GDNF variants can be assessed by comparing the indicator signals induced by different AAV-expressed GDNF variants.

[0161] In some embodiments, the comparison includes: comparing a reporter gene indicator signal generated from the culture supernatant obtained after infecting first cells with different AAVs with a reporter gene indicator signal generated from the control sample, and normalizing the indicator signal to represent a relative GDNF protein activity value relative to the control sample.

[0162] In some embodiments, the AAV may originate from different production batches. These different production batches of AAV may differ in preparation time, production conditions, purification processes, or storage conditions. In some embodiments, the indicator signals induced by GDNF protein from different batches of AAV can be compared to assess the consistency of AAV preparation processes or batch-to-batch consistency, compare the ability of different batches of AAV to express active GDNF protein, or detect the relative biological activity of GDNF protein expressed by different batches of AAV.

[0163] In some embodiments, the expression level of GDNF protein can be modulated by contacting the first cell with AAVs of different viral titers, thereby establishing a dose gradient. In some embodiments, the different viral titers of AAVs are used to establish a dose-response relationship and, in conjunction with the detection of reporter gene indicator signals, to assess changes in GDNF protein activity.

[0164] In this application, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence that enables the transcription of a specific gene. Promoters can be recognized by RNA polymerase, which initiates transcription to synthesize RNA. During RNA synthesis, promoters can interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription). A promoter comprises a core promoter region and a regulatory region, located in the regulatory sequence controlling gene expression, upstream of the gene transcription start site (at the 5' direction of the DNA antisense strand), and does not have a coding function itself. Based on their mode of action and function, promoters are classified into three categories: constitutive promoters (maintaining continuous activity in most or all tissues), specific promoters (tissue-specific or developmental stage-specific), and inducible promoters (regulated by external chemical or physical signals).

[0165] In some embodiments, the AAV described in this application has different promoters, wherein the promoters may comprise cell- or tissue-specific promoters or constitutive promoters. The promoters may also comprise mammalian β-actin promoters or viral promoters. The promoters may also comprise CAG promoters (hybrid CMV early enhancer / chicken β-actin promoter, also known as CAGGS promoters, CB promoters, or CBA promoters), human β-actin promoters, small CBA (smCBA) promoters, CBS promoters or CBh promoters, elongation factor 1α short (EFS) promoters, elongation factor 1α (EF-1α) promoters, CMV promoters, PGK promoters, UBC promoters, GUSB promoters, UCOE promoters, VMD2 (also known as BEST1) promoters, OPEFS promoters, RPE65 promoters, or hybrids or derivatives thereof. For example, the promoter may be a CAG promoter.

[0166] In some embodiments, the capsid may be derived from any AAV serotype known in the art or to be characterized in the future. The capsid and ITR may be derived from the same serotype of AAV or from different serotypes of AAV. Preferably, the capsid is suitable for ocular delivery, such as subretinal, intravitreal, or intraocular delivery. In specific embodiments, the AAV vector comprises a capsid of serotype AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2.7m8, or AAVAnc80L65, or a variant thereof.

[0167] In some embodiments, the normalization may be based on the control sample and is used to convert reporter gene indicator signals obtained under different AAV conditions into directly comparable relative values. Through this normalization process, the reporter gene indicator signal is expressed as a relative GDNF protein activity value relative to the control sample.

[0168] In some embodiments, the normalization is based on signal intensity, background subtraction, or a reference signal. In some embodiments, the normalization is based on parameters of the dose-response relationship. For example, in some embodiments, the dose-response data of the test sample and the control sample can be curve-fitted separately, and the reporter gene indicator signal can be normalized based on the obtained characteristic parameters (such as the half-maximum effective concentration EC50) to obtain results reflecting the relative biological activity of the GDNF protein.

[0169] Use

[0170] On the other hand, this application provides an use for evaluating the biological activity of an AAV formulation encoding a GDNF protein, wherein the use includes determining the activity of the GDNF protein by the method.

[0171] In this application, the term "AAV formulation" refers to a product form containing an adeno-associated virus (AAV) vector, which is a composition suitable for storage, transport, detection, or use. In this application, the AAV formulation may be an intermediate, semi-finished product, or final product in the manufacturing process, and may be in liquid, lyophilized, or other suitable form. Whether the AAV formulation is used for administration, research, or detection is not a limitation. In some embodiments, the AAV formulation may also contain buffer solutions, stabilizers, excipients, preservatives, diluents, or combinations thereof.

[0172] In some embodiments, the use may be part of the quality control or potency characterization of the AAV formulation. In some embodiments, the results of the bioactivity assessment may be used to characterize the expression capacity, functional integrity, or relative potency of the GDNF protein in the AAV formulation, without limitation to any specific clinical use or route of administration.

[0173] On the other hand, this application provides the use of the method described herein in the preparation of gene therapy products, comprising:

[0174] (a) Provides an AAV formulation comprising a GDNF viral vector, wherein the GDNF viral vector comprises a nucleotide encoding one or more AADC polypeptides;

[0175] (b) Using the method described above, the relative GDNF protein activity value of the GDNF viral vector in the AAV formulation was determined;

[0176] (c) Compare the relative GDNF protein activity value with a threshold value for the relative GDNF protein activity value; and

[0177] (d) If the relative GDNF protein activity value is greater than or equal to the threshold, the AAV formulation is dispensed into a formulation container.

[0178] In some embodiments, the method may also be used in the preparation of gene therapy products as a detection step for critical quality attributes (CQA) during the manufacturing process, as part of release testing or intermediate release testing, or as an evaluation tool in process optimization, batch screening, or stability studies.

[0179] On the other hand, this application provides a detection kit for detecting GDNF protein activity, wherein the kit is adapted to implement the method for detecting GDNF protein activity.

[0180] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the fusion protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0181] Example

[0182] Example 1: Construction and validation of reporter gene cell lines

[0183] 1) Reporter gene cell construction process

[0184] The reporter gene cell line is obtained by first delivering a reporter gene plasmid (which contains H_RET, GAL4 response element, and luciferase reporter gene) to the HEK293 cell line via lentivirus, and then delivering the GFRα1 gene and the signaling pathway fusion protein (NLS-GAL4(1–147)-ELK1(307–428)) expression cassette to the cell line. After drug resistance selection, a cell line stably expressing GFRα1, the signaling pathway, and luciferase is obtained. The process is briefly described as follows: First, four lentiviral expression plasmids (GFRα1 gene plasmid, GAL4-ELK1 pathway plasmid, RET pathway plasmid, and Luciferase reporter gene plasmid) were cloned and packaged into lentiviruses. Wild-type HEK293 cells were first infected with RET pathway and Luciferase reporter gene lentiviruses to obtain a cell pool. Then, the cell pool was infected with lentiviruses containing the GFRα1 gene and GAL4-ELK1 pathway. After infection, blastcin, bleomycin, hygromycin, and puromycin were added simultaneously for positive cell selection. Two weeks after drug screening, the cell pool was continuously expanded and cryopreserved. Then, one cell line was thawed and subjected to flow cytometry and GDNF activity assessment to confirm successful expression of GFRα1, the pathway, and the reporter gene in the cell line. Then, single-clone selection was performed. The flowchart of the above reporter gene cell construction is attached. Figure 1The coding information of the four plasmids is as follows: (1) GFRα1 gene plasmid: After the human GFRα1 gene is synthesized with nucleic acid, the coding fragment is inserted into the lentiviral vector plasmid to form the lenti-CMV-H_GFRA1-PGK-Zeo expression plasmid. The main elements of this plasmid include long terminal repeats (LTRs), packaging signal (Ψ), CMW promoter, GFRα1 CDS region, WPRE element, PGK promoter and resistance selection gene Bleomycin. (2) GAL4-ELK1 pathway plasmid: After the NLS gene, human GAL4(1-147) gene and ELK1(307-428) gene are synthesized with nucleic acid, the coding fragment is inserted into the lentiviral vector plasmid to form the PGMLV-CMV-NLS-H_GAL4(1-147)-H_ELK1(307-428)-PGK-Puro expression plasmid. The main components of this plasmid include LTRs, packaging signal (Ψ), CMW promoter, NLS, GAL4 (1-147) and ELK1 (307-428) CDS regions, WPRE element, PGK promoter and the resistance selection gene Puromycin. (3) RET plasmid: After the RET gene is synthesized with nucleic acid, the coding fragment is inserted into the lentiviral vector plasmid to form CMV-NLS-H_RET-PGK-BlaS expression plasmid. The main components of this plasmid include LTRs, packaging signal (Ψ), CMW promoter, RET CDS region, WPRE element, PGK promoter and the resistance selection gene Blasticidin. (4) Luciferase reporter gene plasmid: After the Luciferase gene is synthesized with nucleic acid, the coding fragment is inserted into the lentiviral vector plasmid to form CMV-Luciferase-PGK-HY expression plasmid. The main components of this plasmid include LTRs, packaging signals (Ψ), the CMW promoter, the Luciferase CDS region, the WPRE element, the PGK promoter, and the resistance selection gene Hygromycin.

[0185] The nucleotide sequence encoding the H_RET is shown in SEQ ID NO: 16, the nucleotide sequence encoding the GAL4 response element is shown in SEQ ID NO: 17, the nucleotide sequence encoding the luciferase reporter gene is shown in SEQ ID NO: 20, the nucleotide sequence encoding the NLS-GAL4-ELK1 fusion protein is shown in SEQ ID NO: 12, and the nucleotide sequence encoding the GFRα1 is shown in SEQ ID NO: 14.

[0186] 2) Resistance screening and monoclonal selection methods

[0187] Complete culture medium preparation process: Take 36 mL DMEM basic (1×), add 4 mL FBS and 400 μL Penicillin-Streptomycin, mix well and store at 4℃. Shelf life is one month.

[0188] Complete culture medium for drug administration: 90% DMEM medium + 10% FBS + 1% PS + 4ug / ml blastomycin + 150ug / ml bleomycin + 125ug / ml hygromycin + 0.75ug / ml puromycin.

[0189] Monoclonal screening was performed using a combination of limiting dilution and Clone Select Imager (brand: Molecular Devices, model: CSI8154).

[0190] Screening process:

[0191] a. Cell digestion: When the cell confluence reaches 80%-90%, transfer the T25 culture flask containing cells to a biosafety cabinet and discard the old culture medium. Rinse with 2 mL of complete culture medium and discard the complete culture medium. Add 2 mL of TrypLE and digest for 4-5 min. Add 2 mL of complete culture medium to stop digestion, and repeatedly pipette the cells until a single-cell suspension is formed. Transfer the entire cell suspension to a 15 mL centrifuge tube and centrifuge at 300g for 5 min.

[0192] b. Counting: Discard the supernatant and gently resuspend the cells in 1 mL of complete culture medium. Mix the cell suspension with trypan blue at a 1:1 ratio before counting.

[0193] c. Cell dilution and plating: Based on the cell count results, dilute the cell suspension to 10 cells / mL with complete culture medium. Add 100 µL of cell suspension to each well of a 96-well plate. On day 7, replenish with 100 µL of complete culture medium containing double the amount of the drug.

[0194] d. Recording: After the plate is laid up, use Clone Select Imager to take pictures and check the number of cells and cell status in each well.

[0195] e. Selecting Single Clones: Select single clones based on the results of Clone Select Imager.

[0196] f. Single-clonal expansion: After culturing for 14-16 days, when the confluence is >40%, digest and collect the cells. Then, culture the cells in complete medium with added drug, seed the cells into 24-well plates, and incubate at 37°C with 5% CO2. When the confluence is >80%, expand to 6-well plates. When the confluence is >80%, further expand to T25 culture flasks. The single clone is named HEK293-GFRα1-Luciferase.

[0197] g. Cell bank construction: Once the confluence is >80%, expand the culture to T75 culture flasks. Once the confluence is >80%, expand the culture to T225 culture flasks. Then, cryopreserve the cells.

[0198] Example 2: Validation of reporter gene cell lines

[0199] 2.1 Flow verification of GFNRα1 expression

[0200] To assess the binding of GDNF to cells with and without the GFRα1 receptor, we labeled recombinant GDNF protein (Sino Biological, catalog number: 10561-HNCH) with biotin and then incubated it with monoclonal cells and wild-type HEK293 cells, respectively. Subsequently, we added FITC-labeled streptavidin (Solepro, catalog number: SF068) for incubation and finally used flow cytometry to detect the binding.

[0201] Cells and culture conditions: HEK293 cells and HEK293-GFRα1-Luciferase cells, incubated at 37°C in a 5% CO2 incubator. The culture medium preparation in this example is the same as in Example 1.

[0202] The operation steps in this embodiment are as follows:

[0203] 1) Preparation of cell suspension

[0204] Remove the cell culture dish, remove the culture medium, and wash twice with 1×PBS; add TrypLE TM After incubating with ExpressEnzyme solution at 37°C for approximately 5 minutes, the reaction was terminated by adding 1×PBS. All solutions were transferred to centrifuge tubes and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in an appropriate amount of culture medium and counted. After counting, 100,000 cells were placed in 1.5 mL EP tubes, 1 mL of PBS was added, and the cells were centrifuged at 400 g for 5 minutes at room temperature. The supernatant was discarded, and the cells were resuspended in 50 μL of PBS to obtain a cell suspension.

[0205] 2) Cell staining

[0206] Add 1 µL of Biotin-GDNF to each tube and incubate at room temperature in the dark for 30 min. Then add 1 mL of PBS, centrifuge at 400g for 5 min at room temperature, and discard the supernatant. Resuspend the cells in 50 μL of PBS, add 1 µL of FITC-labeled streptavidin to each tube, centrifuge at 400g for 5 min at room temperature, and discard the supernatant.

[0207] 3) After resuspending the cells in 50 μL PBS, they were detected by flow cytometry.

[0208] The expression rate of GFRα1 receptor in wild-type HEK293 cells and HEK293-GFRα1-Luciferase cells was detected using Biotin-GDNF. With wild-type HEK293 as a control, the positive rate of GFRα1 receptor in HEK293-GFRα1-Luciferase cells was 98.57%. The results indicate that the expression rate of GFRα1 receptor in HEK293-GFRα1-Luciferase cells was significantly higher than that in wild-type HEK293 (see attached image). Figure 2 ).

[0209] 2.2 Report gene cell response verification to GDNF (dose-response relationship)

[0210] To evaluate the response of the HEK293-GFRα1-Luciferase cell line to GDNF, we co-incubated the HEK293-GFRα1-Luciferase cell line with serially diluted recombinant GDNF protein (brand: Sino Biological, catalog number: 10561-HNCH) for 16 h and then detected the fluorescence signal.

[0211] Cells and culture conditions: HEK293-GFRα1-Luciferase cells, incubated at 37°C in a 5% CO2 incubator. The culture medium preparation in this example is the same as in Example 1.

[0212] The operation steps in this embodiment are as follows:

[0213] a. Preparation of cell suspension

[0214] Remove the cell culture dish, remove the culture medium, and wash twice with 1×PBS; add TrypLE TM Incubate with ExpressEnzyme solution at 37°C for approximately 5 minutes, then add 1×PBS to terminate the reaction. Transfer all solutions to centrifuge tubes and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in an appropriate amount of complete culture medium, and count the cells. After counting, dilute the cells to 1E5 / ml and add 100 μL of the diluted cells to each well of a 96-well plate (10,000 cells / well). Incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0215] b. GDNF stimulation

[0216] Discard the culture medium from the 96-well plate and add 100 μL of diluted drug solution (concentration shown in Table 1). Incubate at 37℃ in a 5% CO2 incubator for 16 h.

[0217] c. Add 100 μL of the detection reagent (name: GMOne-Step 2.0, brand: Jiman Biotechnology, catalog number: GM-040513A) to each well and read the fluorescence value using an ELISA reader.

[0218] The signaling pathway and Luciferase expression in HEK293-GFRα1-Luciferase cells were validated using recombinant GDNF protein. The results showed that stimulation of HEK293-GFRα1-Luciferase cells with different concentrations of GDNF protein exhibited a significant dose-response relationship, with a window of 88.75-fold. Detailed results are attached. Figure 3 And Table 1. This indicates that the signaling pathway and Luciferase gene expression were successful, and this cell line can be used for GDNF activity detection.

[0219] Table 1. Verification results of GDNF response

[0220]

[0221] Example 3: Comparison of detection window between reporter gene cell method and other methods

[0222] We also developed a method to assess the biological activity of GDNF and AAV-GDNF using the percentage of cell proliferation. Cells and culture conditions: U87 and SH-SY5Y cells, 37°C, 5% CO2 incubator.

[0223] Complete medium 1: Take 36 mL of DMEM basic (1×), add 4 mL of FBS and 400 μL of Penicillin-Streptomycin, mix well, and store at 4℃. Shelf life is one month. Used for U87 cell culture.

[0224] Complete medium 2: Take 36 mL of DMEM / F12 (1:1) (1×), add 4 mL of FBS and 400 μL of Penicillin-Streptomycin, mix well, and store at 4℃. It has a shelf life of one month. Used for the culture of SH-SY5Y cells.

[0225] The operation steps in this embodiment are as follows:

[0226] a. Cell plating

[0227] Remove the frozen SH-SY5Y cells and thaw them rapidly in a 37°C water bath. In a sterile laminar flow hood, gently pipette to mix the cells. Transfer the cell suspension to a sterile centrifuge tube containing 6 mL of complete culture medium. Centrifuge at 900 rpm for 5 min, discard the supernatant, resuspend the cells in 5 mL of complete culture medium, and transfer the suspension to a T75 Flask containing 15 mL of complete culture medium 2. Incubate the cells at 37°C in a 5% CO2 incubator. When cell confluence is greater than 80% and viability is ≥ 90%, digest the cells with TrypLe, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cells in an appropriate amount of complete culture medium 2, count the cells using a cell counter, adjust the cell density to 3E4 / mL, and seed 50 μL per well.

[0228] b. Co-incubation of samples and cells

[0229] AAV-GDNF: 50 μL of AAV-GDNF supernatant was added to each microplate seeded with SH-SY5Y cells 3 days after transduction. An equal volume of supernatant from U87 cells not transduced with AAV-GDNF (MOI 0 vg / cell) was used as a negative control. Then, 10 μL of GDNF protein ligand solution (400 ng / mL) was added, and the cells were transferred to a 37°C, 5% CO2 incubator for 6 days.

[0230] Recombinant GDNF protein: 50 μL of diluted recombinant GDNF protein was added to each well of a microplate seeded with SH-SY5Y cells, with the culture medium serving as a negative control. Then, 10 μL of GDNF protein ligand solution (400 ng / mL) was added, and the cells were transferred to a 37°C, 5% CO2 incubator for 6 days.

[0231] c. Cell viability assay

[0232] Remove the cultured SH-SY5Y cells and add 10 μL of Alarma Blue (Solarbio, catalog number: A7631) solution to each well. Incubate at 37°C with 5% CO2 for 2 h. Read the fluorescence values ​​using a microplate reader (Excitation 540 nm, Emission 590 nm). Export the data using Softmax software, format in Excel, and calculate the proliferation percentage.

[0233] Proliferation percentage (Proliferation%) = (RLU of Sx / RLU of M' - 1) × 100% (Sx is the sample, M' is the culture medium or supernatant of untransduced U87 cells)

[0234] The activity of recombinant GDNF protein and AAV-GDNF was detected using a cell proliferation assay. U87 cells were transfected with two batches of AAV-GDNF (batch A and batch D), and the percentage of SH-SY5Y cell proliferation caused by the GDNF protein expressed in the cell supernatant was measured. The amino acid sequence and nucleotide sequence encoding the GDNF protein are those disclosed in PCT Publication No. WO2023202637, the entire contents of which are incorporated herein by reference. The percentage of SH-SY5Y cell proliferation caused by different concentrations of recombinant GDNF protein was also measured. The results showed that at the highest MOI, batch A of AAV-GDNF had the largest window, with a proliferation percentage of 116.93% relative to the negative control, which only increased the proliferation percentage of SH-SY5Y cells by approximately 1 time. Furthermore, the intra-batch and inter-batch results showed very large differences, with a CV% as high as 59.21% between six replicates. The recombinant GDNF protein at concentrations of 40 ng / mL and 1000 ng / mL promoted the proliferation of SH-SY5Y cells by 36.38% and 62.00%, respectively, with cross-sectional values ​​(CVs) of 80.65% and 38.56% across six replicates. See attached figure for detailed results. Figure 4 And Table 2.

[0235] In summary, this method has a small window, is complex to operate, and produces extremely unstable results, making it unsuitable for detecting the biological activity of GDNF.

[0236] Table 2 Results of GDNF activity detection by cell proliferation method

[0237]

[0238] Example 4 Functional activity assay of AAV-GDNF expression supernatant

[0239] To evaluate the response of the supernatant from AAV-GDNF transfection of HEK293-AAVR cells to the HEK293-GFRα1-Luciferase cell line, we transfected the HEK293-AAVR cell line with AAV-GDNF at different MOIs and co-incubated for 72 h. Then, we stimulated the HEK293-GFRα1-Luciferase cell line with the cell supernatant and detected the fluorescence signal.

[0240] Cells and culture conditions: HEK293-GFRα1-Luciferase cells, 37℃, 5% CO2 incubator.

[0241] The culture medium was prepared in the same way as in Example 1.

[0242] The operation steps in this embodiment are as follows:

[0243] A. Construction process of HEK293-AAVR cells

[0244] (1) HEK293-AAVR cell construction process

[0245] A stable AAVR-expressing cell line was obtained by delivering an AAVR expression cassette to HEK293 wild-type cells via lentivirus and selecting for drug resistance. The process is as follows: First, the lentiviral expression plasmid was cloned. After the human AAVR gene was synthesized, the coding fragment was inserted into the lentiviral vector plasmid pBMLY007, forming the pBMLY007-Lenti-AAVR expression plasmid. The main components of this plasmid include LTRs, packaging signal (Ψ), CMV promoter, AAVR CDS region, WPRE element, PGK promoter, and the drug resistance selection gene Blasticidin. The nucleic acid sequence of the human AAVR gene is shown in SEQ ID NO: 22, and the amino acid sequence of the AAVR protein is shown in SEQ ID NO: 21. Next, lentivirus preparation was carried out. Transfection-grade plasmids were prepared and lentiviruses were packaged by Genewiz Biotechnology Co., Ltd. Then, the cell line was constructed. Wild-type HEK293 cells were infected with the above lentivirus, and AAVR-positive cells were selected by adding methimazole S two days after infection. Two weeks after drug screening, the cell pool was continuously expanded and cryopreserved. Finally, a single cell line was thawed and subjected to monoclonal screening.

[0246] (2) Resistance screening and monoclonal selection methods

[0247] Complete culture medium preparation process: Take 36 mL DMEM (1×) + GlutaMAX-I, add 4 mL FBS and 400 μL Penicillin-Streptomycin, mix well and store at 4℃. It is valid for one month.

[0248] Complete culture medium for drug administration: Add 50 mL of FBS and 1.5 mg of Blasticidin S to 450 mL of DMEM (1X) + GlutaMAX-I medium, mix by inversion, store at 5℃±3℃ and use within 1 month.

[0249] Monoclonal screening was performed using a combination of limiting dilution and Clone Select Imager (brand: Molecular Devices, model: CSI8154).

[0250] Screening process:

[0251] 1) Cell digestion: When the cell confluence reaches 80%-90%, transfer the T25 culture flask containing cells to a biosafety cabinet and discard the old culture medium. Rinse with 2 mL of complete culture medium and discard the complete culture medium. Add 2 mL of TrypLE and digest for 4-5 min. Add 2 mL of complete culture medium to stop digestion, and repeatedly pipette the cells until a single-cell suspension is formed. Transfer the entire cell suspension to a 15 mL centrifuge tube and centrifuge at 300g for 5 min.

[0252] 2) Counting: Discard the supernatant and gently resuspend the cells in 1 mL of complete culture medium. Mix the cell suspension with trypan blue at a 1:1 ratio before counting.

[0253] 3) Cell dilution and plating: Based on the cell count results, dilute the cell suspension to 10 cells / mL with complete culture medium. Add 100 µL of cell suspension to each well of a 96-well plate. On day 7, add 100 µL of complete culture medium containing double the amount of drug.

[0254] 4) Record: After the plate is laid up, use Clone Select Imager to take pictures to check the number of cells and cell status in each well.

[0255] 5) Selecting Single Clones: Select single clones based on the results of Clone Select Imager.

[0256] 6) Single-clonal expansion: After culturing for 14-16 days, when the confluence is >40%, digest and collect the cells. Then, culture the cells in complete medium with added drugs, seed them into 24-well plates, and incubate at 37°C with 5% CO2. When the confluence is >80%, expand to 6-well plates. When the confluence is again >80%, expand to T25 culture flasks. The single clone is named HEK293-AAVR.

[0257] 7) Cell bank construction: Once the confluence is >80%, expand the culture to T75 culture flasks. Once the confluence is >80%, expand the culture to T225 culture flasks. Then, cryopreserve the cells.

[0258] B. HEK293-AAVR cell plating

[0259] Remove the HEK293-AAVR cell culture dish, remove the culture medium, and wash twice with 1×PBS; add TrypLE TMIncubate with Express Enzyme solution at 37°C for approximately 5 minutes, then add 1×PBS to terminate the reaction. Transfer all solutions to centrifuge tubes and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in an appropriate amount of complete culture medium, and count the cells. After counting, dilute the cells to 1E5 / ml and add 100 μL of the diluted cells to each well of a 96-well plate (10,000 cells / well). Incubate at 37°C in a 5% CO2 incubator for 18–24 hours.

[0260] C. Add AAV-GDNF

[0261] Add 10 μL of diluted AAV-GDNF virus to each well (concentrations are shown in Table 3). Incubate at 37℃ and 5% CO2 for 72 h ± 4 h.

[0262] D. HEK293-GFRα1-Luciferase cell plating

[0263] Remove the HEK293-GFRα1-Luciferase cell culture dish, remove the culture medium, and wash twice with 1×PBS; add TrypLE TM Incubate with Express Enzyme solution at 37°C for approximately 5 minutes, then add 1×PBS to terminate the reaction. Transfer all solutions to centrifuge tubes and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in an appropriate amount of complete culture medium, and count the cells. After counting, dilute the cells to 2E5 / ml and add 50 μL of the diluted cells to each well of a 96-well plate (10,000 cells / well). Incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0264] E. Stimulation

[0265] The supernatant from AAV-GDNF transfection of HEK293-AAVR cells was transferred to a new 96-well plate, mixed thoroughly by pipetting, and then 50 μL was added to a 96-well plate seeded with HEK293-GFRα1-Luciferase cells. The plate was incubated at 37°C with 5% CO2 for 18 hours.

[0266] F. Reading

[0267] Remove the 96-well plate containing HEK293-GFRα1-Luciferase cells and allow it to equilibrate to room temperature for 20-30 minutes. Add 100 μL of the assay reagent (name: GMOne-Step 2.0, brand: Jiman Biotechnology, catalog number: GM-040513A) equilibrated to room temperature to each well and read the fluorescence value using an ELISA reader.

[0268] HEK293-AAVR cells were transfected with AAV-GDNF (from batch A) at different MOIs, and the response of the cell supernatant to stimulation of the HEK293-GFRα1-Luciferase cell line was measured. The results showed that this method had a 73.26-fold window, and the 4-parameter fitting produced a complete S-shaped curve with R0. 2 The value is 0.9894. See the detailed results below. Figure 5 And Table 3.

[0269] Table 3 Response results of AAV-GDNF expression supernatant

[0270]

[0271] Example 5: Detection of relative biological activity of AAV-GDNF test sample (TA)

[0272] To evaluate the relative biological activity of AAV-GDNF test samples (TA), we transfected HEK293-AAVR cell lines with AAV-GDNF test samples (TA) and reference standards (RS) at different MOIs. After co-incubation for 72 h, we stimulated HEK293-GFRα1-Luciferase cell lines with cell supernatant for 18 h and detected fluorescence signals.

[0273] Cells and culture conditions: HEK293-AAVR and HEK293-GFRα1-Luciferase cells, incubated at 37°C in a 5% CO2 incubator.

[0274] The preparation of the culture medium in this embodiment is the same as in Example 1.

[0275] The operation steps in this embodiment are as follows:

[0276] a) HEK293-AAVR cell plating

[0277] Remove the HEK293-AAVR cell culture dish, remove the culture medium, and wash twice with 1×PBS; add TrypLE TM Incubate with Express Enzyme solution at 37°C for approximately 5 minutes, then add 1×PBS to terminate the reaction. Transfer all solutions to centrifuge tubes and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in an appropriate amount of complete culture medium, and count the cells. After counting, dilute the cells to 1E5 / ml and add 100 μL of the diluted cells to each well of a 96-well plate (10,000 cells / well). Incubate at 37°C in a 5% CO2 incubator for 18–24 hours.

[0278] b. Add AAV-GDNF

[0279] Add 10 μL of diluted AAV-GDNF virus to each well (concentrations are shown in Table 4). Incubate at 37℃ and 5% CO2 for 72 h ± 4 h.

[0280] c. HEK293-GFRα1-Luciferase cell plating

[0281] Remove the HEK293-GFRα1-Luciferase cell culture dish, remove the culture medium, and wash twice with 1×PBS; add TrypLE TM Incubate with Express Enzyme solution at 37°C for approximately 5 minutes, then add 1×PBS to terminate the reaction. Transfer all solutions to centrifuge tubes and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in an appropriate amount of complete culture medium, and count the cells. After counting, dilute the cells to 2E5 / ml and add 50 μL of the diluted cells to each well of a 96-well plate (10,000 cells / well). Incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0282] d. Stimulation

[0283] The supernatant from AAV-GDNF transfection of HEK293-AAVR cells was transferred to a new 96-well plate, mixed thoroughly by pipetting, and then 50 μL was added to a 96-well plate seeded with HEK293-GFRα1-Luciferase cells. The plate was incubated at 37°C with 5% CO2 for 18 hours.

[0284] e. Reading

[0285] Remove the 96-well plate containing HEK293-GFRα1-Luciferase cells and allow it to equilibrate to room temperature for 20-30 minutes. Add 100 μL of the assay reagent (name: GMOne-Step 2.0, brand: Jiman Biotechnology, catalog number: GM-040513A) equilibrated to room temperature to each well and read the fluorescence value using an ELISA reader.

[0286] f. Use four-parameter logistic regression (4PL) to fit the response curves of TA and RS respectively, ensuring that the curves have similar shapes (slope consistent with Emax, deviation from parallel terms > 0.01). Calculate EC under the premise of satisfying parallelism. 50 Ratio. Relative biological activity (%RP) is calculated using the following formula:

[0287]

[0288] Three different batches of AAV-GDN (GDNF protein, amino acid sequence, and nucleotide sequence encoding GDNF protein are those disclosed in PCT Publication No. WO2023202637, the entire contents of which are incorporated herein by reference) were selected. Batch A was used as the reference standard (RS), and the relative biological activities of the test samples batches B (TA1) and C (TA2) were detected. The results showed that the three batches of AAV-GDNF, after being fitted with four parameters, met the criteria for an S-shaped curve, R... 2 The CV% of replicates were 0.996, 0.993, and 0.978, respectively, and the CV% could be controlled within 30%. Batch B (TA1) and batch C (TA2) underwent deviation from parallelism tests compared to batch A, and the results were all >0.01, meeting the acceptance criteria. The EC50 values ​​of these three batches were 3.11E+04, 2.78E+04, and 2.92E+04, respectively, and the biological activities of batches B and C relative to batch A were 111.93% and 106.65%, respectively. See details below. Figure 6 And Table 4.

[0289] In summary, the use of the HEK293-GFRα1-Luciferase cell line to detect the biological activity of AAV-GDNF [is effective / effective]. 2 The parameters, including CV% and parallel item test, all met the acceptance criteria. This strategy complies with the general requirements of ICH Q6B, the Chinese Pharmacopoeia, and USP for functional testing of protein and gene therapy drugs.

[0290] Table 4. Results of relative biological activity assays for AAV-GDNF test samples (TA).

[0291]

Claims

1. A method for detecting GDNF protein activity, the method comprising the following steps: a) Contact adeno-associated virus (AAV) containing a nucleotide sequence encoding the GDNF protein with a first cell expressing an adeno-associated virus receptor (AAVR), culture the first cell, and collect the culture supernatant; b) Incubate the culture supernatant described in step a) with the second cell, wherein the second cell is a reporter gene cell; and c) Detect the indicator signal generated by the reporter gene. The reporter gene cell comprises a GFRα1 expression element, a RET expression element, a transcriptional response module, and a reporter gene module; the transcriptional response module comprises a GAL4-ELK1 fusion protein and / or nucleic acid encoding the fusion protein; the reporter gene module comprises nucleic acid encoding a GAL4 response element and a reporter gene.

2. The method according to claim 1, wherein the reporter gene is a nucleic acid encoding luciferase.

3. The method according to any one of claims 1-2, wherein the amino acid sequence of the GAL4-ELK1 fusion protein is as shown in SEQ ID NO: 7; wherein the GFRα1 expression element comprises the GFRα1 protein and / or nucleic acid encoding the GFRα1 protein, and the GFRα1 protein comprises the amino acid sequence shown in SEQ ID NO: 13; wherein the RET expression element comprises the RET protein and / or nucleic acid encoding the RET protein, and the RET protein comprises the amino acid sequence shown in SEQ ID NO:

15.

4. The method according to any one of claims 2-3, wherein the nucleic acid encoding the GAL4-ELK1 fusion protein comprises the nucleotide sequence shown in SEQ ID NO: 8; the nucleic acid encoding the GFRα1 protein comprises the nucleotide sequence shown in SEQ ID NO: 14; the nucleic acid encoding the RET protein comprises the nucleotide sequence shown in SEQ ID NO: 16; the nucleic acid encoding the luciferase comprises the nucleotide sequence shown in SEQ ID NO: 19; and the nucleic acid encoding the GAL4 response element comprises the nucleotide sequence shown in SEQ ID NO:

17.

5. The method according to any one of claims 1-4, wherein the first cell is a HEK293 cell, and the reporter gene cell is a HEK293 cell.

6. The method according to any one of claims 1-5, further comprising comparing the culture supernatant with a reporter gene indicator signal generated before, simultaneously with and / or after incubation with reporter gene cells to determine the activity of the GDNF protein to be detected.

7. The method according to any one of claims 1–6, further comprising: The indicator signal of the reporter gene is compared with the indicator signal of the reporter gene generated by a control sample, wherein the control sample is selected from one or more of the following: a) Samples containing exogenous GDNF protein; b) Culture supernatant obtained after infecting first-cell cells with AAV that does not encode GDNF; and c) Culture supernatant obtained after infecting first cells with AAV containing GDNF.

8. The method of claim 7, wherein the AAV comprising a nucleotide sequence encoding GDNF is selected from one or more of the following: AAV encoding different GDNF variants, AAV from different production batches, AAV from different production processes, AAV from different purification processes, AAV with different viral titers, AAV with different capsid serotypes, and AAV with different promoters.

9. The method according to any one of claims 6-8, wherein, The comparison includes: comparing the reporter gene indicator signal generated from the culture supernatant obtained after infecting the first cell with different AAVs with the reporter gene indicator signal generated from the control sample, and normalizing the indicator signal to express it as a relative GDNF protein activity value relative to the control sample; or determining the half-maximal effective concentration (EC50) of the reporter gene indicator signal generated from the culture supernatant obtained after infecting the first cell with different AAVs and the control sample, respectively. 50 ), and by comparing the EC 50 The value determines the relative GDNF protein activity of the culture supernatant relative to the control sample.

10. Use for evaluating the biological activity of an AAV formulation encoding GDNF protein, wherein, The uses include determining the activity of the GDNF protein by the method described in claims 1-9.

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