Potency assay of progranulin

Abstract

Developed herein are methods, quantitative functional potency assays, and kits for measurement of progranulin (PGRN) secreted from cells transduced with adeno-associated viral genome vector comprising progranulin (AAV: GRN).

Description

POTENCY ASSAY OF PROGRANULIN1. SEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML copy, created on June 5, 2025, is named 63444WO_sequencelisting.xml, and is 6,019 bytes in size.2. BACKGROUND

[0002] Mutations in the progranulin gene (GRN) have been identified as a major contributor to neurodegenerative diseases, such as Frontotemporal dementia (FTD). Gene therapies for treating the neurodegenerative diseases by delivering a healthy copy of the GRN gene have been developed to restore the production of progranulin. However, several challenges remain including: the lack of a robust method to assess the potency and functionality of the progranulin produced from the gene therapy. Developing such a method is critical for evaluating the effectiveness of this treatment strategy for the neurodegenerative diseases.3. SUMMARY

[0003] In one aspect, described herein are methods of measuring potency of progranulins (PGRN), the method comprising:(a) contacting a test sample comprising PGRN with a recombinant human prosaposin (rhPSAP) in a condition that allows binding between an active form of PGRN and rhPSAP, thereby forming a PGRN:rhPSAP complex;(b) measuring bound PGRN in the PGRN:rhPSAP complex; and(c) determining potency of PGRN in the test sample based on the measurement of the bound PGRN in the PGRN:rhPSAP complex.

[0004] In some embodiments, the test sample has been obtained from a cell culture transduced with a gene therapy vector expressing PGRN.

[0005] In some embodiments, the gene therapy vector is a recombinant adeno-associated virus (rAAV) comprising a coding sequence of GRN.

[0006] In accordance with any of the embodiments, the test sample comprises a supernatant of a cell culture transduced with a recombinant adeno-associated virus (rAAV) comprising a coding sequence of PGRN.

[0007] In some embodiments, the rAAV comprising a coding sequence of GRN is infected with Multiplicity of Infection (MOI) between 102and 5xl04GC / cell, between 102and 104GC / cell, between 2xl02and 8xl03GC / cell, between 4xl02and 8xl03GC / cell, between 6xl02and 8xl03GC / cell, between 8xl02and 7xl03GC / cell, or between 4.12 x 102to about 1.25 x 104.

[0008] In accordance with any of the embodiments, the method further comprises a preceding step of preparing the sample comprising PGRN by harvesting, obtaining, and / or filtering a supernatant of a cell culture infected with a recombinant adeno-associated virus (rAAV) comprising a coding sequence of GRN.

[0009] In accordance with any of the embodiments, in step (a), the rhPSAP is coated on a solid support.

[0010] In accordance with any of the embodiments, in step (b), the PGRN:rhPSAP complex is measured by a method comprising: applying an anti-progranulin (anti-PGRN) antibody and detecting the anti-PGRN antibody bound to the PGRN:rhPSAP complex.

[0011] In some embodiments, the anti-PGRN antibody primary detection is labeled with biotin and the PGRN:rhPSAP complex is detected by the biotinylated primary antibody.

[0012] In some embodiments, the method further comprises applying a Streptavidin-HRP (SA-HRP) conjugated secondary antibody specifically binds to the biotinylated anti-PGRN antibody.

[0013] In accordance with any of the embodiments, the method further comprises applying an enzyme substrate comprising 3,3’, 5,5 ’-tetramethylbenzidine (TMB) to detect and quantify the PGRN:rhPSAP complex.

[0014] In some embodiments, the PGRN:rhPSAP complex is detected and quantified by colorimetric intensity.

[0015] In accordance with any of the embodiments, the rhPSAP comprises a BC linker of a human prosaposin.

[0016] In accordance with any of the embodiments, the active form of transduced PGRN comprises granulin motifs D and E.

[0017] In accordance with any of the embodiments, in step (c), the potency of PGRN is determined by comparing the measurement of the PGRN:rhPSAP complex against a standard curve.

[0018] In some embodiments, the standard curve is generated by measuring binding between rhPSAP and serial dilutions of a reference sample comprising PGRN.

[0019] In accordance with any of the embodiments, the method further comprises determining a relative potency of the bound PGRN in the sample by calculating: the potency measured in step (c) / expected potency x 100%.

[0020] In accordance with any of the embodiments, the potency of PGRN is determined by plotting a linear regression curve of the measured absorbance of the PGRN:rhPSAP complex against serial MOI doses of AAV.GRN or a reference standard.

[0021] In some embodiments, the method further comprises determining a relative potency of PGRN in the sample by (a) evaluating the two linear regression curves for parallelism, and (b) calculating the relative potency of AAV.GRN against the reference standard.

[0022] In some embodiments, determining the potency of PGRN-rhPSAP binding comprises determining the relative potency in the test sample based on the measurement of the bound PGRN in comparison to that in the reference standard.

[0023] In one aspect, described herein are methods for assessing functional potency of secreted progranulins (PGRNs) from a cell transduced with a recombinant adeno-associated virus (rAAV) capsid comprising a progranulin (AAV:GRN) vector genome packaged therein, the method comprising: contacting supernatant of a cell culture after transduction with the rAAV capsid comprising the (AAV:GRN) genome vector packaged therein with a solid support, wherein the supernatant comprises the active progranulins (PGRNs) and the solid support comprises one or more recombinant human prosaposins (rhPSAPs) being coated on the surface of the solid support, wherein the contacting of the supernatant and the solid support allows active PGRNs to bind to the rhPSAPs to form a PGRN:rhPSAP complex, thereby immobilizing the active PGRNs;applying an anti-progranulin (anti-PGRN) antibody targeting the active PGRNs of thePGRN:rhPSAP complex; and detecting and quantifying the immobilized active PGRNs.

[0024] In some embodiments, the rhPSAP is a polyhistidine -tagged recombinant rhPSAP (His-rhPSAP).

[0025] In accordance with any of the embodiments, the anti-PGRN antibody is a biotinylated anti-PGRN antibody.

[0026] In accordance with any of the embodiments, the method further comprising, prior to the contacting, providing or culturing cells with the rAAV capsid comprising the AAV:GRN packaged therein.

[0027] In accordance with any of the embodiments, the cell culture has a multiplicity of infection (MOI) range between about 4.12 x 102to about 1.25 x 104GC / cell.

[0028] In accordance with any of the embodiments, the method further comprising, prior to the contacting, diluting the supernatant at a six point 1 : 1.5 serial dilution scheme.

[0029] In accordance with any of the embodiments, detecting and qualifying the immobilized active PGRNs comprises applying a Streptavidin-HRP (SA-HRP) conjugated secondary antibody specifically binds to the biotinylated anti-PGRN antibody.

[0030] In accordance with any of the embodiments, the method further comprising applying an enzyme substrate comprising 3,3’, 5,5 ’-tetramethylbenzidine (TMB) to detect and quantify the immobilized active PGRNs.

[0031] In accordance with any of the embodiments, the bound active PGRNs are detected and quantified by colorimetric intensity.

[0032] In accordance with any of the embodiments, the colorimetric intensity is proportional to the amount of the active PGRNs binding to the rhPSAPs, thereby indicating the amount of the transduced PGRNs secreted from the cells.

[0033] In accordance with any of the embodiments, the detectable functional potency range is between about 50% to about 200%.

[0034] In accordance with any of the embodiments, the active PGRNs is suitable for delivery to a subject in need thereof via intracerebroventricular delivery, intraparenchymal delivery, and / or intra-ci sterna magna delivery.

[0035] In some embodiments, the active PGRNs is suitable for administration to the subject at one or more doses, each dose comprising about 1 x IO10GC / g brain mass to about 3.33 x 1011GC / g brain mass.

[0036] In accordance with any of the embodiments, the rAAV capsid is derived from an AAV capsid selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.rh79, AAV.RHM4-1, AAV.hu37, AAVhu68, AAV.Anc80, AAV.Anc80L65. AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGs. 1A and IB show PGRN and PSAP expression in supernatant of HEK-293 T cells infected with or without AAV:GRN. His-r hPGRN: His-tagged recombinant human progranulin; His-rhPSAP: His tagged recombinant human prosaposin.

[0038] FIGs. 2A and 2B depict PGRN and PSAP interaction in supernatant of HEK-293 T cells infected with or without AAV:GRN. 293T: HEK293T Cells.

[0039] FIG. 3A shows the measured concentrations of PSAP and PGRN in the AAV.GRN- transduced supernatant, along with their concentration ratios for each AAV:G7?N MOI. FIG. 3B demonstrates correlation between secreted PSAP and transduced PGRN. Scatterplot demonstrating correlation between two variables: total PSAP and total PGRN concentration in the supernatant measured in ng / mL. A linear regression was estimated within the 95% confidence ellipse.

[0040] FIG. 4 is a schematic diagram of an ELISA assay format demonstrating transducedPGRN binding to recombinant human PSAP coated on the plate. TMB: 3,3', 5,5'- tetramethylbenzidine; SA-HRP: Horseradish Peroxidase conjugated streptavidin; Sup.:supernatant; a-PGRN: anti-progranulin antibody; His-rhPSAP: His-tagged recombinant human prosaposin.

[0041] FIG. 5 shows dose-dependent response curves for PGRN-PSAP binding activity. AAV:GRN dose-dependent linear regression curves were plotted for samples at 100%, 50%, and 200% potency levels. Both MOI (x-axis) and Absorbance (y-axis) were loglO- transformed. RS: reference standard; SI : sample 1; S2: sample 2; Assay Ctrl: assay control; MOI: Multiplicity of Infection; GC: genome copy.

[0042] FIG. 6 shows results of PGRN-PSAP binding is specific to AAN.GRN, non-relevant AAV product (AA .GLB ) and empty AAV capsid do not exhibit PSAP binding responses (FIG. 6A). The PGRN-PSAP binding specificity is also demonstrated by antibody blocking experiments. AAV:GRN dose-dependent linear regression curves were plotted for each antibody-blocking condition along with non-blocked control (FIG. 6B). FIG. 6C shows relative PSAP binding potency reduction (%) of various PSAP and PGRN antibodies compared to unblocked control.

[0043] FIG. 7 shows comparison of PGRN binding to rhPSAP vs anti -PSAP coated ELISA plates. rhPSAP protein were coated on the ELISA plate at Ipg / mL (closed circles). Anti- PSAP antibody (open squares) was coated at Ipg / mL to replace the rhPSAP protein and capture the endogenous PSAP complexed with PGRN. The PGRN binding curve on the anti- PSAP coated plate (open squares) was compared to the curve generated with the rhPSAP coated plate (closed circles). Both MOI (x-axis) and Absorbance (y-axis) were loglO- transformed.

[0044] FIGs. 8A and 8B show stability indicating characteristic with AAG'GRN vector at 40°C for 72 hours. Potency was measured for untreated and heat-treated sample for (FIG. 8A) PSAP -PGRN binding and (FIG. 8B) PGRN expression. Both MOI (x-axis) and Absorbance (y-axis) were loglO-transformed.

[0045] FIGs. 9 A, 9B and 9C show stability indicating characteristic with AAV:GRN vector transduced supernatant at 60°C, 80°C and 100°C for 1.5 hours. Potency was measured for untreated and heat-treated samples for (FIG. 9A) PSAP -PGRN binding and (FIG. 9B) PGRN expression. Both MOI (x-axis) and Absorbance (y-axis) were loglO-transformed. FIG. 9C is a summary of relative PSAP binding potency reduction and relative PGRN expression potencyreduction at different treatment conditions: 60 °C, 1.5 hours; 80 °C, 1.5 hours; 100 °C, 1.5 hours.

[0046] FIG. 10 shows characterization of the heat-treated supernatant at 60°C, 80°C and 100°C for 1.5 hours by Western Blot. After infection, supernatants were collected and heat- treated at 60°C, 80°C or 100°C. Four groups (untreated, lane 1-2; 80°C 1.5hrs-treated, lane 3- 4; 80°C 1.5hrs-treated, lane 5-6; 100°C 1.5hrs-treated, lane 7-8) were run side-by-side, each containing two different MOIs (6.25xl03GC / Cell in lanes 1,3, 5, 7; 4.17xl03GC / Cell in lanes 2,4,6, 8). Sup.: supernatant.

[0047] FIG. 11 shows interaction of PGRN and prosaposin PSAP through the BC linker of PSAP and granulin motifs (primarily granulin D and E) to regulate each other's trafficking and function.5. DETAILED DESCRIPTION

[0048] Developed herein are methods and potency assays for measuring the functional biological activity of progranulin (PGRN). With this assay, biological activity is measured as a specific interaction between PGRN and one of its primary endogenous binding partners, prosaposin (PSAP). The protein binding assay can augment the established protein expression-based drug substance (DS) and drug product (DP) potency assay. Together, these two assays measure both transgene protein expression and transgene functional biological activity as part of an assay matrix to determine potency for lot release, stability and comparability studies. In some embodiments, the PGRN-PSAP binding assay (a) assesses a biologically relevant functionality potentially suitable as a critical quality attribute (CQA) that is informative of the mechanism of action (MO A) of PGRN and (b) complements the existing PGRN expression assay to yield an appropriate 2-component potency matrix. In Some embodiments, such method ensures manufacturing lot-to-lot consistency and better understanding of potency- related critical quality attributes (CQA).

[0049] PGRN and PSAP form complex and reciprocally facilitate each other’s transportation from the extracellular matrix to the lysosome by virtue of PGRN interaction with sortilin receptors or through PSAP binding to M6PR / LRP1 receptors on the cell-surface and lysosomal membranes. Once delivered to the lysosome, PGRN and PSAP are processed into granulins and saposins, respectively.

[0050] In some embodiments, the protein-binding bioassay described herein entails 2 parts: first, HEK-293 cells are transduced with serial dilutions of AAV: GRN vector to allow PGRN protein expression; second, after an incubation period, a dilution of cell-culture supernatant is used in a protein-binding bioassay that measures secreted PGRN binding activity to recombinant human PSAP (rhPSAP) coated on ELISA plates. PGRN-PSAP binding is detected with a PGRN-targeted antibody and quantified by means of colorimetric detection to measure PGRN-PSAP binding interaction. The initial assessments of specificity, linearity, accuracy and precision indicate the assay is fit-for-purpose and can be qualified and validated in a phase-appropriate manner. Furthermore, the assay has been found to be stability indicating for AAV: GRN product.6. EXAMPLES6.1. Assay development

[0051] To pursue the PGRN binding partners in an in-vitro cell culture model, the Applicant first examined the protein expressions in the supernatant from the HEK293T cell culture. The Applicant studied the proteins interacting with PGRN using immunoprecipitation and western blot technologies. The Applicant then developed a quantitative method to measure the protein binding between PGRN and its binding partner using an ELISA format.The preliminary characterization section describes the finding of transduced PGRN and endogenous PSAP protein-protein interaction in the cell culture (e.g., HEK-293) supernatant.The analytical procedures section describes the development of a quantitative ELISA method to measure the binding of PGRN to its binding partner, PSAP, evaluate the method's capability of reporting the relative potency of NNN.GRN, and proposed preliminary acceptance criteria.The preliminary assessment of pre-qualification parameters section provides data supporting binding specificity between PGRN and PSAP, verification of the method performance (linearity, range, accuracy, and precision) and stability indicating characteristics.The additional assay characterization section explores PSAP -PGRN binding specificity and the capacity of the assay to detect heat-stress induced changes to PGRN.6.2. Preliminary characterization6.2.1. In vitro measurement of transduced PGRN expression (current potency assay)

[0052] The current relative potency assay has been qualified to confirm the expression of PGRN protein in HEK-293 cells. This cell-based assay requires the vector to bind to the cell surface receptor, internalize, release the transgene, and express the PGRN protein. The PGRN protein is secreted and measured in cell supernatants by ELISA with a commercial kit. The amount of protein expression correlates to the amount of vector used to transduce target cells and is expressed as potency relative to a reference vector.6.2.2. Exploration of protein interactions of transduced PGRN in HEK293 T cell model

[0053] The interaction between PGRN and PSAP was investigated using cultured HEK-293 T cells. Cells were transduced with or without AAV.GRN in T-25 culture flasks. Forty-eight hours after the AAV:GWmfection, the supernatant was harvested, and the protein expression for PGRN and PSAP was examined by western blotting (WB).

[0054] In this model, both transduced PGRN and endogenous PSAP are secreted proteins that are detected in the supernatant by WB (FIGs. 1 A and IB), both are minimally expressed without AAV:GWmfection. FIGs. 1 A and IB show PGRN and PSAP expression in supernatant of HEK-293 T cells infected with or without AAV.GRN. Western blotting used to detect PGRN and PSAP in the supernatant with or without AAV:G7Winfection at IxlO5GC / cell for 48 hours. A recombinant human His-tagged PGRN protein and His-tagged PSAP protein were run in Lane 1 and Lane 4, respectively, as the positive control. (FIG. 1 A) Immunoblot (IB) with anti-PGRN antibody; (FIG. IB) Immunoblot with anti-PSAP antibody. The results indicate that the increasing PSAP secretion / expression in supernatant is associated with the increased PGRN expression (Lane 6 and 3, respectively).

[0055] The PGRN-PSAP interaction was established by co-immunoprecipitation with transduced PGRN (AAVGWmfection) and secreted endogenous PSAP in the supernatant of HEK293T cells. Immunoblots showed that co-immunoprecipitation of transduced supernatant with an anti-PGRN antibody led to detection of secreted PSAP protein on western blot Immunoprecipitation using 2 different anti-PGRN antibodies led to detection of secreted PSAP protein on western blot and demonstrated the presence of PSAP protein in AJW.GRN- transduced supernatants (FIG. 2A), and likewise, co-immunoprecipitation with an anti-PSAPantibody led to detection of PGRN protein on western blot (FIG. 2B). As a replication of the results presented in FIGs.2A and 2B, both PSAP and PGRN were detected in the supernatants of transduced cells (i.e., without the co-IP step; FIG.2B, Lanes 2 and 9, respectively); conversely, minimal levels of endogenous PSAP and PGRN were detected in the supernatant of non-transduced cells (FIGs. 2 A and 2B, Lanes 1 and 6, respectively).

[0056] FIGs. 2A and 2B depict PGRN and PSAP interaction in supernatant of HEK-293 T cells infected with or without AAV.GRN. Interaction between PGRN and PSAP secreted in the 293 T cell supernatant was demonstrated with co-immunoprecipitation (co-IP) and western blot. (FIG. 2A) 293T cells were infected with or without AAVGWmfection at IxlO5GC / cell. Supernatant was harvested 48 hours after infection. 1-mL supernatant was preincubated with lOpg of either anti -PGRN antibody polyclonal or monoclonal at 4°C for 2 hours before adding 40pL of Protein G magnetic beads. After incubation at 4°C overnight, the beads were washed, and the bound proteins were eluted with 2x Laemmli sample buffer with reducing reagent Dithiothreitol (DTT). Anti-PSAP antibody was used for immunoblotting to detect the bound PSAP. (FIG. 2B) Similar IP was prepared as described in (FIG. 2A) with the differences of IP conditions: supernatant volume at 200pL, anti-PSAP antibody of Ipg, and Protein G beads volume at lOpL. Anti -PGRN was as used for immunoblotting to detect the bound PGRN.

[0057] To understand the PSAP secretion in response to PGRN transduction, ELISA format was applied to measure the secreted PSAP and PGRN expression in HEK293T cell supernatant.

[0058] Briefly, HEK293T cells were infected with AJW.GRN at four concentrations between 3.13 x 103~1 x 105GC / Cell with duplicate per concentration. Forty-eight hours after infection, HEK293T cell supernatant was harvested and quantitated for PSAP and PGRN protein expression using a commercial kit or commercially available reagents.

[0059] The measured PSAP and PGRN concentrations of harvested supernatant are shown in FIG. 3A. Both PGRN and PSAP exhibit a dose-dependent relationship with PBFT02 multiplicity of infection (MOI). However, the levels of secreted PSAP are significantly lower than the levels of expressed PGRN at all MOIs tested. Minimal PSAP was detected in cell lysate in any condition tested. The correlation between the mean PSAP concentrations (y- axis) and mean PGRN concentrations (x- axis) (duplicate of each of the four AAV.GRNdoses) was plotted with a bivariate linear regression at a 95% confidence ellipse. A strong positive correlation (r=0.99) between the total secreted PSAP and total secreted PGRN was observed (FIG. 3), indicating that increased PSAP secretion / expression is associated with increased PGRN protein in the culture supernatant. This confirmed the observation and inference made in the WB data (FIGs. 1 A and IB).6.2.3. Analytical procedures

[0060] The functional potency assay uses the HEK293 cell line for AAV .'GAW transduction and measures binding activity of secreted PGRN to recombinant human PSAP (rhPSAP) coated on ELISA plates.

[0061] Based on the characterization of the extracellular interaction of PGRN with PSAP, PSAP binding is considered a quality attribute of PGRN, which reflects A V:GRN function. Due to the low endogenous PSAP expression level (~14 ng / mL or less) secreted in the supernatant (FIG. 3), the Applicant has developed a functional potency assay evaluating the transduced PGRN binding to exogenous PSAP recombinant protein (rhPSAP) using an ELISA format. The interference of the endogenously secreted PSAP in the supernatant and its impact on the rhPSAP binding was assessed and discussed herein.

[0062] This section describes the rhPSAP binding method details, data analysis for potency calculation, and proposed acceptance criteria.

[0063] Briefly, HEK293 cells, seeded overnight in 96-well plates, are pre-infected with wildtype adenovirus H5 (Ad H5) at 40 GC / cell MOI (multiplicity of infection), then transduced with AAV .’GAW using a starting MOI of 6.25 x 103GC / cell and a six-point 1 :1.5 serial dilution scheme. In all experiments, the reference standard (AAV: GAW RS, was used for preliminary method development) is deemed as a 100% potency dose-response curve. In parallel, cells are infected with two test samples per plate following the same infection scheme.

[0064] Each assay includes a plate blank, cells infected with Ad H5 only, and an assay control, cells infected with a qualified AAV / GAW lot at 100% potency.

[0065] Two days post-transduction, supernatant is harvested and assayed for PGRN-PSAP binding activity by ELISA. Briefly, the culture supernatant transduced with PBFT02 was diluted and then incubated on an ELISA plate coated with rhPSAP. PSAP -bound PGRN isdetected with biotinylated a-PGRN antibody followed by a Streptavidin-HRP conjugated secondary antibody. After the TMB (3,3', 5,5 '-tetramethylbenzidine) substrate is added, the color development can be measured at 450 nm Absorbance. The measured absorbance is plotted as a function of the vector MOI. The colorimetric intensity is proportional to the amount of active PGRN binding to PSAP (FIG. 4). FIG. 4 is a schematic diagram of an ELISA assay format demonstrating transduced PGRN binding to recombinant human PSAP coated on the plate.

[0066] The linear regression curves with an MOI range of 4.12 x 102- 1.25 x 104GC / cell are optimized for a predicted potency range between 50 -200% (FIG. 5). FIG. 5 shows dosedependent response curves for PGRN-PSAP binding activity. A V:GRN dose-dependent linear regression curves were plotted for samples at 100%, 50%, and 200% potency levels. Both MOI (x-axis) and Absorbance (y-axis) were loglO-transformed.

[0067] The assay validity is assessed with the following parameters and should meet the suitability criteria based on experience with current potency assay:• The individual RS and assay control curve fitting parameters.• The parallelism parameters of assay control.• The % potency range of the assay control.

[0068] The sample validity is assessed with the following parameters and should meet the following suitability criteria based on experience with current potency assay:• The individual sample curve fitting parameters.• The parallelism parameters of each sample.• The reportable potency range of samples on each plate.

[0069] For each sample testing, independent triplicate plates are performed, and the valid relative potency result from each plate is reported. The final reportable potency of the sample is calculated as the average of the valid relative potency results from triplicate plates.

[0070] In conclusion, the preliminary evaluation of the method performance shows the assay is within the historical trend for in-house potency assays and is fit -for purpose. The proposed acceptance criteria for the functional potency established is based on preliminarily data andwould be refined over time with additional work that will be performed during method qualification and additional manufacturing experience.6.2.4. Preliminary assessment of pre-qualification characteristics6.2.4.1 Specificity with antibody blocking

[0071] It was reported that PGRN and PSAP interact through the BC linker (274-310 aa) of PSAP and Grn D / E region of PGRN. Based on this information, antibody blocking experiments were designed to probe the specificity of the PGRN-PSAP binding by assay including an antibody specific to hPSAP (PSAP Ab2) to mask the BC linker region (immunogen peptide aa 1-320 aa). A different antibody (a-PSAP Abl), which does not recognize the BC linker, was used as a control. The immunogen peptide for a-PSAP Ablis 325-524 aa, which recognizes a section of PSAP closer to the C-terminus that is outside of the BC linker region. Table 1 summarizes the immunogen information for each antibody used in this study.Ag: Antigen; aa: amino acids; Gm: granulin

[0072] FIGs. 6A-6C show results of PGRN-PSAP binding specificity by antibody blocking. Various antibodies specific to PSAP or PGRN were used for pre-blocking. a-PSAP Abl, a- PSAP Ab2, and mouse isotype control at 8 pg / mL were applied to the PSAP (1 pg / mL)coated plate. The pre-incubation was at 37°C for 2 hours before loading samples of harvested supernatant (at 1 :5 dilution) from 441 :G7 Mnfection. The 1 :5 diluted harvested supernatant was also incubated with a-PGRN Abland a-PGRN Ab2 at a final concentration of 8pg / mL at 37°C for 2 hours before loading directly to the corresponding wells in an ELISA plate coated with rhPSAP (1 pg / mL).

[0073] The potency reduction outlined in Table 1 is also depicted in FIG. 6B. The rhPSAP - coated plate, blocked with the antibody specific to the BC linker, showed a 55% reduction in potency compared to the unblocked rhPSAP-coated plate (closed circles vs. open triangles, FIG. 6B) while the rhPSAP-coated plate blocked with the control anti-PSAP antibody that does not recognize the BC linker demonstrated a lesser (21%) reduction in potency (closed circles vs. closed squares, FIG. 6B; see also FIG. 6C). An anti-PGRN antibody specific to the Grn DZE motif showed a 57% reduction in potency (closed circles vs. open squares, FIG. 6B) while the control anti-PGRN antibody that does not recognize the Gm DZE motif had a lesser (18%) reduction in potency (closed circles vs. closed triangles, FIG. 6B; see also FIG. 6C). A non-specific isotype control Ab, non-specific to rhPSAP or PGRN, showed a 20% reduction in potency (closed circles vs. open circles, FIG. 6B; see also FIG. 6C). It's important to note that the concentration of blocking antibodies used to block the rhPSAP coat or PGRN in the supernatant was 8 pg / mL, which was 8 times higher than the rhPSAP used for plate coating (1 pg / m) or the highest MOI-transduced PGRN (~1.1 pg / mL PGRN level measured in the 6.25 x 103MOI infected supernatant as shown in FIG. 3 A. The potency differences observed with the control anti-PSAP or anti-PGRN antibodies may be due to steric hindrance secondary to binding to areas outside the binding interface or to non-specific blockage, similar to the 20% potency reduction seen with the IgG isotype control.

[0074] In summary, both the anti-PSAP antibody, recognizing the BC linker, and the anti- PGRN antibody, recognizing the Gm DZE motif, effectively blocked the binding between PSAP and PGRN, resulting in a 55% and 57% reduction in potency, respectively. These antibody-blocking experiments indicate that PGRN binding to PSAP is specific to the BC linker of PSAP and the Grn DZE motif of PGRN.6.2.4.1 Interference of endogenous PSAP secreted by the HEK293 cells (specificity)

[0075] As noted above, endogenous PSAP is secreted into the culture supernatant fromHEK-293 cells along with the transduced PGRN (FIGs. 1 A and IB). Even though the datasummarized in FIG. 3 A demonstrate that the level of secreted PSAP is significantly lower than that of PGRN in the AAV.GRN transduced supernatant, it is still possible that the relatively small amount of endogenous PSAP secreted into the culture media of transduced cells could interfere with the PGRN-rhPSAP binding measured in the ELISA assay. To assess the potential for interference, a PSAP capture antibody was coated on the plate instead of the rhPSAP. This antibody captures the secreted endogenous PSAP complex along with any PGRN bound to the PSAP. PGRN was measured using the same biotinylated anti-PGRN detection antibody as used in experiments described above.

[0076] As shown in FIG. 7, the optical density (OD) values of the plate wells coated with the anti-PSAP capture antibody (open squares) are either at or below the mean OD value of the plate blank (cells without AAV.GRN transduction, closed triangle) for plate wells coated with rhPSAP. This data indicates that endogenously produced PSAP, which can bind in solution to PGRN and potentially prevent PGRN interaction with rhPSAP, is unlikely to meaningfully interfere with PGRN binding to rhPSAP in the proposed potency assay.6.1.4.3 Linearity, range, accuracy and precision

[0077] A preliminary study evaluating assay performance was conducted. Three potency levels at 50%, 100% and 200% were evaluated per plate and in five independent replicate plates. A development process engineering lot of AAV: GRN was used for sample preparation at all potency levels. Relative potency results and %recovery (measured potency / expected potency x 100%) from each potency level obtained from five independent replicated plates were summarized and assessed.

[0078] The pre-qualification summary of the functional potency assay is presented in Table2.* Acceptance criteria were set based on prior experience with potency assay performance, and aligned with ICH Q2R2, and USP <1033>.6.1.4.4 Stability indicating properties

[0079] To test the ability of the assay to detect degraded AAV.GRN product, heat stress with elevated temperature was directly applied to AAV.GRN vector or to the harvested supernatant following vector infection. The condition for the heat stress of the AAV.GRN vector was at 40°C for 72 hrs. The conditions for the heat stress of the PGRN-containing supernatant, which was infected by an untreated AJW.GRN vector lot, were at 60°C, 80°C or 100°C for 1.5 hours. In a separate assay design, rhPSAP was also under heat stress at 100°C for 2 hours before being coated on the ELISA plate. A summary of all heat stress conditions and measured potency results is shown in Table 3. The 40°C heat-treated AAV.GRN vector resulted in a -50% PSAP binding compared to the untreated control (closed circles and squares FIG. 8A). The PGRN expression was also measured in the same supernatant. The 40°C heat-treated AAV: GRN vector showed a similar reduction compared to the untreated control (closed circles and squares FIG. 8B), indicating that the PSAP binding is PGRN-dose- dependent and consistent with the existing potency method by PGRN expression. FIGs. 8A and 8B show stability indicating characteristic with AAVPGRN vector) at 40°C for 72 hours. Potency was measured for untreated and heat-treated sample for (FIG. 8A) PSAP -PGRN binding and (FIG. 8B) PGRN expression. Both MOI (x-axis) and Absorbance (y-axis) were loglO-transformed. The heat-treated AAVPGRN vector results support the stabilityindicating characteristic of the functional potency assay.* %Potency for stress samples was measured against the same AAV.GRN lot or the same harvested supernatant without treatment; Potency values were reported here for comparative information only because some stress conditions may fail the sample suitability.

[0080] Heat treatment at 60°C, 80°C and 100°C for 1.5 hours was applied to the freshly harvested supernatant, which was infected by an untreated AAV.GRN vector lot . A near complete potency loss was observed in the supernatant treated at 100°C for 1.5 hours (closed circles vs. triangles, FIG. 9A), whereas -61% reduction in PSAP binding activity was observed in the supernatant treated at 80°C for 1.5 hours compared to the untreated control supernatant (closed circles vs. squares FIG. 9A). Similar to PSAP binding, PGRN expression in the 100°C, 1.5 hours treated supernatant also had a near complete potency loss (closed circles vs. Triangles, FIG. 9B). Interestingly, unlike the PSAP binding, the 80°C, 1.5 hours treated supernatant had only a -30% potency reduction (closed circles vs. squares FIG. 9B). The potency discrepancy between the two methods indicates that the PSAP binding feature of PGRN is more sensitive to detect protein degradation.

[0081] To understand the difference between the two potency methods for heat-treated supernatant, the heat-treated supernatant expressing transduced PGRN protein was further characterized by WB for PGRN degradation. Supernatant from the top two AAV:G7 V MOI- infected wells (4.17 x 103and 6.25 x 103GC / Cell) was used in the western blot. Supernatants from three groups, untreated, treated 60°C, 80°C and 100°C for 1.5 hrs, respectively were compared side-by-side. Confirmed by the western blot, a near complete loss of full-length protein was observed in the supernatant treated at 100°C for 1.5 hours (FIG. 10, lanes 7-8), consistent with the data shown in the ELISA formats in Figure 10. Reduced full-lengthPGRN band density was observed in the supernatant treated at 80°C for 1.5 hours on the immunoblot (FIG. 10, lanes 5-6). A semi-quantitation of the average density of the full-length PGRN (measured by Image Lab software) for 80°C 1.5 hrs treated group showed -66% reduction in density compared to the untreated group per MOI consistent with the potency levels measured by PGRN-PSAP binding (FIG. 9A). No significant changes in full-length PGRN band density in the supernatant treated at 60°C for 1.5 hours (lanes 3-4) compared to the untreated group (lanes 1-2). Immunoblot could not detect small fragments below 25kDa as they migrated out of the gel.

[0082] It’s possible that the PGRN capture antibody used in the PGRN expression ELISA could also capture the aggregates / fragments, resulting in higher PGRN expression in the 80°C 1.5-hrs treated supernatant. However, in the PSAP binding ELISA principle, only the presence of the intact Granulin D-E region can bind, whereas the improper folding and tertiary structure changes in aggregates / fragments may lead to inaccessibility to the PSAP binding site. This explains why PSAP binding assay is more sensitive to degradation than PGRN expression assay, indicating that PSAP binding assay truly measures the presence of PGRN with an active PSAP binding site.

[0083] The described potency assay utilized a full-length recombinant PSAP protein, for which the BC linker region is specific for the Grn DZE binding motif of PGRN. Without being bound by any theories, the Grn DZE binding motif may be occluded by improper folding and tertiary structure changes in degraded PGRN protein caused by heat stress, allowing the proposed potency assay to be more sensitive to detecting heat-induced stress than the existing potency assay. The described potency assay provides a sensitive measure of the presence of intact PGRN with an active PSAP binding site.

[0084] By comparison, the current potency expression assay, which utilizes a different anti- PGRN capture antibody, detects PGRN in a fashion less sensitive to PGRN degradation and conformational changes caused by heat stress.

[0085] Although heat denaturation of PGRN is not expected to occur in the course of DP manufacture or storage, the ability of this assay to detect defective protein highlights its ability to detect various potential impacts on PGRN protein integrity such as misfolding due to cellular stress, improper glycosylation, proteasomal degradation, etc. .

[0086] In some embodiments, the assay outcomes can be dependent on the structural integrity of PGRN expressed by AAV.GRN. In some embodiments, the assay measures an important aspect of the biological activity of PGRN potentially suitable as a CQAthat is informative of the mechanism of action (MO A) of A V.GRN.6.1.4.5 Summary of method assessment

[0087] Data presented herein describe an in vitro AAV: GRN Functional potency assay developed to use the transduced PGRN binding capability to rhPSAP in an easy-to-quantitate ELISA format. The PGRN-PSAP binding response is proportional to the transduced AAV:G / ?A’M0I and can be controlled by predefined system and sample suitability. Further, the quantitative functional potency method carries the following features:- Antibody blocking experiments indicate that the method is specific to active PGRN binding to PSAP through Gm D / E motif of PGRN and BC linker of PSAP. The endogenously secreted PSAP has minimum impact on the PGRN binding to the rhPSAP coat on the plate.- Preliminary method evaluation demonstrates linearity, accuracy, and precision within the 50-200% range.- The heat-stress studies demonstrate the new method’s stability -indicating property and superior sensitivity to the stressed PGRN protein over the previous PGRN expression assay. This indicates the assay is suitable for future manufacturing lot release and stability testing.7. CONCLUSION

[0088] The applicant has developed a quantitative functional potency assay to support the A AV: G W late clinical phase development. The new potency method is deemed suitable for its intended use.

[0089] The ongoing efforts in method optimization, robustness, and qualification are crucial for ensuring the effectiveness and reliability of this potency method. Once qualified, the method can be used to support its comparability to the existing potency method measuring PGRN expression and aid in the process development to understand the critical quality attributes (CQAs).. SEQUENCES

Claims

WHAT IS CLAIMED IS1. A method of measuring potency of progranulins (PGRN), comprising: a. contacting a test sample comprising PGRN with a recombinant human prosaposin (rhPSAP) in a condition that allows binding between an active form of PGRN and rhPSAP, thereby forming a PGRN:rhPSAP complex; b. measuring bound PGRN in the PGRN:rhPSAP complex; and c. determining potency of PGRN in the test sample based on the measurement of the bound PGRN in the PGRN:rhPSAP complex.

2. The method of claim 1, wherein the test sample has been obtained from a cell culture transduced with a gene therapy vector expressing PGRN.

3. The method of claim 2, wherein the gene therapy vector is a recombinant adeno- associated virus (rAAV) comprising a coding sequence of GRN.

4. The method of any one of claims 1-3, wherein the test sample comprises a supernatant of a cell culture transduced with a recombinant adeno-associated virus (rAAV) comprising a coding sequence of PGRN.

5. The method of claim 4, wherein the rAAV comprising a coding sequence of GRN is infected with Multiplicity of Infection (MOI) between 102and 5xl04GC / cell, between 102and 104GC / cell, between 2xl02and 8xl03GC / cell, between 4xl02and 8xl03GC / cell, between 6xl02and 8xl03GC / cell, between 8xl02and 7xl03GC / cell, or between 4.12 x 102to about 1.25 x 104.

6. The method of any one of claims 1-5, further comprising a preceding step of preparing the sample comprising PGRN by harvesting, obtaining, and / or filtering a supernatant of a cell culture infected with a recombinant adeno-associated virus (rAAV) comprising a coding sequence of GRN.

7. The method of any one of claims 1-6, wherein in step (a), the rhPSAP is coated on a solid support.

8. The method of any one of claims 1-7, wherein in step (b), the PGRN:rhPSAP complex is measured by a method comprising: applying an anti-progranulin (anti-PGRN) antibody and detecting the anti-PGRN antibody bound to the PGRN:rhPSAP complex.

9. The method of claim 8, wherein the anti-PGRN antibody primary detection is labeled with biotin and the PGRN:rhPSAP complex is detected by the biotinylated primary antibody.

10. The method of claim 8 or 9, further comprising applying a Streptavidin-HRP (SA- HRP) conjugated secondary antibody specifically binds to the biotinylated anti-PGRN antibody.

11. The method of any one of claims 8-10, further comprising applying an enzyme substrate comprising 3,3’, 5,5 ’-tetramethylbenzidine (TMB) to detect and quantify the PGRN:rhPSAP complex.

12. The method of claim 11, wherein the PGRN:rhPSAP complex is detected and quantified by colorimetric intensity.

13. The method of any one of claims 1-12, wherein the rhPSAP comprises a BC linker of a human prosaposin.

14. The method of any one of claims 1-13, wherein the active form of transduced PGRN comprises granulin motifs D and E.

15. The method of any one of claims 1-14, wherein in step (c), the potency of PGRN is determined by comparing the measurement of the PGRN:rhPSAP complex against a standard curve.

16. The method of claim 15, wherein the standard curve is generated by measuring binding between rhPSAP and serial dilutions of a reference sample comprising PGRN.

17. The method of any one of claims 1-16, further comprising determining a relative potency of PGRN in the sample by calculating: the potency measured in step (c) / expected potency x 100%.

18. The method of any one of claims 1-17, wherein the potency of PGRN is determined by plotting a linear regression curve of the measured absorbance of the PGRN:rhPSAP complex against serial MOI doses of AJW.GRN or a reference standard.

19. The method of claim 18, further comprising determining a relative potency of PGRN in the sample by (a) evaluating the two linear regression curves for parallelism, and (b) calculating the relative potency of AAV.GRN against the reference standard.

20. The method of any one of claims 1-19, further comprising determining a relative potency of the bound PGRN in the sample by calculating: the potency measured in step (c) / expected potency x 100%.

21. The method of any one of claims 1-20, wherein determining the potency of PGRN- rhPSAP binding comprises determining the relative potency in the test sample based on the measurement of the bound PGRN in comparison to that in the reference standard.

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