Assay for measuring the efficacy of gene therapy drugs

Abstract

Disclosed herein is a cell-based assay for determining the efficacy of a recombinant viral vector expressing a transgene.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 092,189, filed on 15 October 2020, the disclosures of which Provisional Patent Application are incorporated herein by reference in their entirety.

[0002] Description of electronically submitted text files The contents of the text file electronically submitted with this specification are incorporated herein by reference in their entirety: a computer-readable copy of the sequence listing (filename: PRVL_017_01WO_SeqList_ST25.txt, data recording date: October 15, 2021, file size approximately 7,474 bytes).

[0003] This disclosure broadly relates to the field of gene therapy. More specifically, this disclosure provides a cell-based assay for analyzing the potency of compositions comprising recombinant viral vectors expressing transgenes. [Background technology]

[0004] Recombinant viral vectors encoding glucocerebrosidase (GCase; encoded by the GBA1 gene) are useful for treating disorders such as Parkinson's disease and Gaucher disease. Assays are needed to measure the relative potency of recombinant viral compositions delivering GCase intended for use in gene therapy. [Overview of the project]

[0005] Provided herein is a method for measuring the relative potency of a test sample containing a first recombinant virus comprising a transgene encoding glucocerebrosidase (GCase), comprising: a) transducing a plurality of first cells in the test sample; b) incubating the transduced plurality of first cells under conditions sufficient to express GCase; c) collecting a first cell lysate from the transduced plurality of first cells; d) mixing the first cell lysate with resorufin-beta-D-glucopyranoside; and e) imaging the first cell lysate and obtaining a first fluorescence readout. The method comprises: f) transducing a second group of cells with a reference standard containing a second recombinant virus containing a transgene encoding GCase; g) incubating the transduced second group of cells under conditions sufficient to express GCase; h) collecting a second cell lysate from the transduced second group of cells; i) mixing the second cell lysate with resorphin-beta-D-glucopyranoside; j) imaging the second cell lysate to obtain a second fluorescence reading; and k) calculating the relative potency of the test sample by comparing the first fluorescence reading with the second fluorescence reading using parallel line analysis.

[0006] In some embodiments of the methods disclosed herein, the first recombinant virus and the second recombinant virus contain the same transgene encoding GCase.

[0007] In some embodiments of the methods disclosed herein, the first recombinant virus and / or the second recombinant virus is a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV comprises the AAV9 capsid protein. In some embodiments, the rAAV comprises the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 capsid protein, or a variant of any of these capsid proteins.

[0008] In some embodiments of the methods disclosed herein, the GCase comprises SEQ ID NO: 1. In some embodiments, the transgene encoding the GCase comprises a codon-optimized nucleotide sequence. In some embodiments, the codon-optimized nucleotide sequence comprises SEQ ID NO: 2.

[0009] In some embodiments of the methods disclosed herein, the first plurality of cells and / or the second plurality of cells are HEK-293T or HEK-293 cells.

[0010] In some embodiments of the methods disclosed herein, about 1.25 mM resorphine-beta-D-glucopyranoside is mixed with a first cell lysate and / or a second cell lysate.

[0011] In some embodiments of the methods disclosed herein, a first plurality of cells and a second plurality of cells are seeded in a multiwell plate. In some embodiments, the first plurality of cells and / or the second plurality of cells are seeded at approximately 20,000 cells per well.

[0012] In some embodiments of the methods disclosed herein, the test sample and / or reference standard are progressively diluted before transduction.

[0013] In some embodiments of the methods disclosed herein, a first plurality of cells and a second plurality of cells are incubated for about 68 to about 81 hours before cell lysate harvesting. In some embodiments, a first plurality of cells and a second plurality of cells are incubated for about 66 to about 78 hours after transduction and before cell lysate harvesting.

[0014] In some embodiments of the methods disclosed herein, a first plurality of cells are transduced by a test sample with at least two different modalities (MOIs) of infection with a first recombinant virus. In some embodiments, a second plurality of cells are transduced by a reference standard with at least two different modalities (MOIs) of infection with a second recombinant virus.

[0015] In some embodiments of the methods disclosed herein, the first fluorescence reading and / or the second fluorescence reading reflect the measured GCase activity. In some embodiments, the measured GCase activity is in relative fluorescence units (RFU) / hour.

[0016] In some embodiments of the methods disclosed herein, for each of the test sample and the reference standard, comparison step (k) includes performing a logarithmic transformation of the amount of recombinant virus and RFU / time, and plotting a standard curve of the logarithm of the amount of recombinant virus against the logarithm of RFU / time. In some embodiments, comparison step (k) includes calculating a linear regression of the logarithm of the amount of recombinant virus against the logarithm of RFU / time for each of the test sample and the reference standard, thereby deriving the slope for the test sample and the slope for the reference standard. In some embodiments, comparison step (k) includes calculating a linear regression with a common slope using the linear regressions obtained for each of the test sample and the reference standard. In some embodiments, relative potency is given by the formula: Relative potency (%) = 10^((bb reference It is calculated using () / A) × 100. In some embodiments, the ratio of the slope of the test sample to the common slope is about 0.60 to about 1.40. In some embodiments, the ratio of the slope of the reference standard to the common slope is about 0.60 to about 1.40.

[0017] In some embodiments, the method disclosed herein involves linear regression of the test sample and the reference standard R 2 This further includes calculating the value of the test sample and the reference standard R 2 The value is 0.9 or higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] [Figure 1] Figure 1 is a diagram of a PCR plate map for an rAAV potency assay. "RS" refers to "reference standard". "TS" refers to "test sample". [Figure 2] Figure 2 shows a line graph and calculated values of relative potency of several rAAV samples expressing GCase. MODE FOR CARRYING OUT THE INVENTION

[0019] The present disclosure relates to a cell-based transduction assay for measuring the relative potency of recombinant viral compositions that deliver a transgene encoding glucocerebrosidase (e.g., human glucocerebrosidase). Glucocerebrosidase (also called beta-glucocerebrosidase, lysosomal acid β-glucocerebrosidase, GCase, and GBA) is encoded by the GBA1 gene. In subjects having a mutation in only one allele of GBA1, the risk of Parkinson's disease is greatly increased. Subjects having mutations in both copies of GBA1 suffer from Gaucher disease. Viral compositions that deliver a transgene encoding GCase are useful for gene therapy for Parkinson's disease (e.g., Parkinson's disease with a GBA1 mutation) and Gaucher disease.

[0020] In some embodiments, the recombinant viral vector encoding GCase is a recombinant adeno-associated virus (rAAV) vector.

[0021] The method disclosed herein utilizes resorufin-β-D-glucopyranoside, a fluorogenic substrate that is catalyzed in the presence of GCase to form the fluorescent product resorufin. Resorufin production is monitored directly as the reaction progresses, and the rate of product formation is calculated. In the presence of excess resorufin-β-D-glucopyranoside substrate and under assay conditions, the rate of product formation is linearly proportional to the amount of GCase protein.

[0022] The term "recombinant virus" refers to a genetically modified virus, for example, by addition or insertion of a heterologous nucleic acid construct into a viral particle.

[0023] The term "heterologous" is used synonymously with the term "exogenous" herein, and refers to a substance derived from any source other than its natural source. For example, the terms "exogenous protein" or "exogenous gene" refer to a protein or gene from a non-AAV source that is artificially introduced into an AAV genome or an AAV particle.

[0024] The term "recombinant adeno-associated virus" or "rAAV" refers to an AAV particle or AAV virion comprising an rAAV vector encapsidated by one or more AAV capsid proteins.

[0025] The term "rAAV vector" refers to a nucleic acid, which is either single-stranded or double-stranded, comprising an AAV 5' inverted terminal repeat (ITR) sequence and an AAV 3' ITR flanking a protein coding sequence operably linked to a transcriptional regulatory element heterologous to the AAV viral genome, for example, one or more promoters and / or enhancers, and optionally comprising a polyadenylation sequence and / or one or more introns inserted between exons of the protein coding sequence.

[0026] The term "IU" refers to infectious unit.

[0027] The term "TCID50" refers to 50% tissue culture infectious dose.

[0028] The term "USP" refers to the United States Pharmacopeia.

[0029] The term "test sample" refers to a sample comprising an rAAV vector containing a sequence encoding an exogenous protein of interest (e.g., GCase), whose potency is unknown and is determined using the methods described herein.

[0030] The term "reference standard" refers to a composition that includes an rAAV vector encoding the exogenous protein of interest (e.g., GCase) and whose potency is known.

[0031] In some embodiments, provided herein is a method for measuring the relative potency of a test sample comprising a first recombinant virus comprising a transgene encoding glucocerebrosidase (GCase), comprising: a) transducing a plurality of first cells in the test sample; b) incubating the transduced plurality of first cells under conditions sufficient to express GCase; c) collecting a first cell lysate from the transduced plurality of first cells; d) mixing the first cell lysate with resorphin-beta-D-glucopyranoside; e) imaging the first cell lysate to obtain a first fluorescence reading; f) G The method comprises: g) transducing a second group of cells with a reference standard containing a second recombinant virus containing a transgene encoding Case; h) incubating the transduced second group of cells under conditions sufficient to express GCase; i) collecting a second cell lysate from the transduced second group of cells; j) mixing the second cell lysate with resorphin-beta-D-glucopyranoside; j) imaging the second cell lysate to obtain a second fluorescence reading; and k) calculating the relative potency of the test sample by comparing the first fluorescence reading with the second fluorescence reading using parallel line analysis.

[0032] In some embodiments, the first recombinant virus and the second recombinant virus are identical. In some embodiments, the first recombinant virus and the second recombinant virus are not identical. In some embodiments, the first recombinant virus and the second recombinant virus contain the same transgene encoding GCase, but are from different manufacturing lots or production lots. In some embodiments, GCase contains the amino acid sequence of SEQ ID NO: 1.

[0033] In some embodiments, the first plurality of cells and / or the second plurality of cells are HEK-293T or HEK-293 cells.

[0034] The methods disclosed herein may be carried out in multiwell plates. In some embodiments, the methods disclosed herein are carried out in 96-well plates. In some embodiments, a first plurality of cells and a second plurality of cells are seeded in the multiwell plate. In some embodiments, the first plurality of cells and / or the second plurality of cells are seeded at approximately 20,000 cells per well, respectively, before transduction of the test sample and reference standard. In some embodiments, the cells are allowed to adhere overnight at 37°C and 5% CO2.

[0035] In some embodiments, transduction is performed approximately 24 hours after cell seeding.

[0036] In some embodiments, the test sample and / or reference standard are progressively diluted before transduction. In some embodiments, the test sample is diluted to 50%, 100%, and 200% of the reference standard. In some embodiments, the progressive dilution results in the following total vector genome (vg) amounts per well: 5.00E+10vg / well, 3.33E+10vg / well, 2.22E+10vg / well, 1.48E+10vg / well, 9.88E+9vg / well, and 6.58E+9vg / well.

[0037] In some embodiments, a standard curve for purified recombinant GCase (rGBA, 0-333 ng / ml, R&D catalog number 7410-GHB-020, purity >95%) is run in parallel with the test sample.

[0038] In some embodiments, a first group of cells is transduced by a test sample with at least two different MOIs (Moritonatums of Infection) of a first recombinant virus. In some embodiments, a second group of cells is transduced by a reference standard with at least two different MOIs of a second recombinant virus.

[0039] In some embodiments, the first and second sets of cells are incubated for approximately 68 to 81 hours after transduction and before cell lysate collection. In some embodiments, the first and second sets of cells are incubated for approximately 2 to 2.5 hours before recovery medium (e.g., 10% FBS / DMEM / 1μM Hoechst 33342) is added to the cells. In some embodiments, the first and second sets of cells are incubated for approximately 72 ± 6 hours (e.g., approximately 66 to 78 hours) after transduction and before cell lysate collection. In some embodiments, incubation is performed at 37°C and 5% CO2.

[0040] In some embodiments, approximately 1.25 mM resorphine-beta-D-glucopyranoside is mixed with the first cell lysate in mixing step (d) and / or with the second cell lysate in mixing step (i).

[0041] In some embodiments, imaging steps (e) and / or (j) are performed using a plate reader.

[0042] In some embodiments, the first fluorescence reading and / or the second fluorescence reading reflect the measured GCase activity. In some embodiments, the measured GCase activity is in relative fluorescence units (RFU) per hour.

[0043] In some embodiments, comparison step (k) includes performing logarithmic transformations of the amount of recombinant virus and RFU / time for each of the test sample and the reference standard, and plotting a standard curve of the logarithm of the amount of recombinant virus against the logarithm of RFU / time. In some embodiments, comparison step (k) includes calculating a linear regression of the logarithm of the amount of recombinant virus against the logarithm of RFU / time for each of the test sample and the reference standard, thereby deriving the slope for the test sample and the slope for the reference standard. In some embodiments, comparison step (k) includes calculating a linear regression with a common slope using the linear regressions obtained for each of the test sample and the reference standard. In some embodiments, the relative potency of the test sample is given by the formula: Relative potency (%) = 10^((bb reference It is calculated using () / A) × 100. In some embodiments, the ratio of the slope of the test sample to the common slope is about 0.60 to about 1.40. In some embodiments, the ratio of the slope of the reference standard to the common slope is about 0.60 to about 1.40.

[0044] In some embodiments, a method for measuring the relative efficacy of a test sample involves linear regression of the test sample and a reference standard using R. 2 This further includes calculating the value of the test sample and the reference standard R 2 The value is 0.9 or greater. In some embodiments, the R of the test sample and the reference standard 2 The value is 0.96 or higher.

[0045] In some embodiments, the relative potency of the viral vector is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 100%, at least 110%, at least 120%, at least 130%, or at least 140% compared to a reference standard. In some embodiments, the relative potency of the viral vector is at least 90% compared to a reference standard.

[0046] The infectivity titer (also called functional titer) of an rAAV vector is the concentration of viral particles capable of infecting cells. In some embodiments, a cell transduction assay is used to determine the infectivity titer. In some embodiments, the infectivity titer of the viral vector is determined using the method presented in Example 1. In some embodiments, the infectivity titer of the compositions disclosed herein is about 8.0E+9IU / mL to about 1.2E+10IU / mL. In some embodiments, the infectivity titer of the compositions disclosed herein is about 8.0E+9IU / mL, about 8.15E+9IU / mL, about 8.5E+9IU / mL, about 9.0E+9IU / mL, about 9.5E+9IU / mL, about 9.99E+9IU / mL, about 1E+10IU / mL, about 1.12E+10IU / mL, or about 1.2E+10IU / mL. In some embodiments, the TCID50 of the compositions disclosed herein is about 4,500 vg / IU to about 10,000 vg / IU. In some embodiments, the TCID50 of the compositions disclosed herein is about 4,500 vg / IU, about 5,000 vg / IU, about 5,500 vg / IU, about 6,000 vg / IU, about 6,290 vg / IU, about 6,500 vg / IU, about 7,000 vg / IU, about 7,500 vg / IU, about 8,000 vg / IU, about 8,500 vg / IU, about 9,000 vg / IU, about 9,500 vg / IU, about 9,980 vg / IU, or about 10,000 vg / IU.

[0047] Examples of suitable rAAV vectors that can be used in the methods disclosed herein are disclosed in WO2019 / 070891, WO2019 / 070893, WO2019 / 070894, and WO2019 / 084068, the contents of which each of these disclosures is incorporated herein by reference in their entirety.

[0048] In some embodiments of the methods disclosed herein, the rAAV vector further comprises one or more of the following: a chicken beta-actin (CBA) promoter, a cytomegalovirus (CMV) enhancer, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a bovine growth hormone polyA signaling tail, an artificial intron, an artificial exon, and one or more of the following transcriptional regulatory activation sites within the promoter region: TATA, RBS, and YY1 (Francois et al., (2005) J. Virol. 79(17):11082-11094). The transcriptional regulatory activation sites of TATA, RBS, and YY1 may be located at the 5' end of the promoter region.

[0049] In some embodiments of the methods disclosed herein, the rAAV vector comprises a first AAV reverse terminal repeat (ITR) and a second ITR that flank a polynucleotide encoding the gene product of interest and associated regulatory sequences. In some embodiments, each ITR is a wild-type AAV2 ITR (SEQ ID NO: 3). In some embodiments, each ITR is derived from a wild-type AAV2 ITR.

[0050] In some embodiments of the methods disclosed herein, the rAAV vector comprises, in order, a first AAV reverse-terminal repeat (ITR), a cytomegalovirus (CMV) enhancer, a chicken beta-actin (CBA) promoter, a polynucleotide encoding a human GCase protein, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a bovine growth hormone polyA signaling tail, and a second AAV ITR. In some embodiments, the polynucleotide encoding the human GCase protein is codon-optimized (e.g., codon-optimized for expression in human cells). In some embodiments, the polynucleotide encoding the human GCase protein includes SEQ ID NO: 2. In some embodiments, an rAAV particle comprising an rAAV vector containing the polynucleotide including SEQ ID NO: 2 is referred to as "PR001".

[0051] In some embodiments of the methods disclosed herein, the rAAV vector is a self-complementary recombinant adeno-associated virus (scAAV) vector. For example, scAAV vectors are described in McCarty et al., Gene Ther. 2001;8(16):1248-54.

[0052] In some embodiments of the methods disclosed herein, the recombinant virus is AAV. In some embodiments of the methods disclosed herein, rAAV comprises the AAV9 capsid protein. In some embodiments of the methods disclosed herein, rAAV comprises the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 capsid protein, or a variant of any of these capsid proteins.

[0053] All publications, patents, and patent applications are incorporated herein by reference to the same extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference in its entirety.

[0054] The following examples are provided to give a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods described herein and claimed are prepared and evaluated, and are intended to be illustrative only and not intended to limit the scope of what the inventors consider to be their invention. [Examples]

[0055] Example 1: In vitro enzyme efficacy assay of rAAV encoding glucocerebrosidase. The objective of this assay is to measure the in vitro relative potency of AAV (e.g., AAV9) encapsulation vectors encoding glucocerebrosidase (GCase; encoded by the GBA1 gene) using a cell-based assay.

[0056] Laboratory testing methods The objective of this method is to measure the dose-response of an AAV inclusion vector encoding glucocerebrosidase (GCase; encoded by the GBA1 gene) in vitro using a cell-based functional assay. This assay method can be used for research purposes, such as comparing the responses of different AAV gene therapy product lots. [Table 1] [Table 2]

[0057] Background / Method Theory: PR001 is an exemplary rAAV expressing GBA1. Transduction assays involved introducing PR001 into HEK293T cells, resulting in GCase enzyme expression. Enzyme activity derived from transduction was assayed in cell lysates using the fluorescent substrate 4-methylumbelliferyl-β-D-glucopyranoside, which generates the fluorescent product resorphin by GCase catalysis. Relative potency between two or more rAAVs was calculated from the enzyme activity resulting from transduction of different amounts of PR001 using parallel line analysis. [Table 3]

[0058] Procedure: HEK293T cells were plated at 20,000 cells / well in 96-well plates and deposited overnight at 37°C and 5% CO2. Serial dilutions of AAV were prepared in the excipients shown in Table 4. [Table 4]

[0059] 10 μL of AAV dilution or vehicle was transferred to the wells according to the plate map in Figure 1. The resulting total vg was achieved (Table 5). [Table 5]

[0060] Cells were incubated at 37°C in a 5% CO2 incubator for 2–2.5 hours. After incubation, 100 μL of recovery medium was added to the cell / transduction medium in the wells to bring the total volume to 150 μL. Cells were incubated at 37°C in a 5% CO2 incubator for 72+6 hours to enable virus-derived GCase expression.

[0061] Cell lysates were collected. GCase activity was measured by adding 10 μL of a 1.25 mM resorphin-β-D glucopyranoside diluted standard solution to a clear, flat-bottomed black plate, followed by 40 μL of cell lysates. This plate was immediately read using a Varioskan plate reader at 37°C.

[0062] Analysis: A parallel analysis of the data to calculate relative efficacy was performed as follows: 1. Calculate the %CV for each vg / wellpoint. This must be ≤30%. To achieve this, you may discard up to one iterative experiment per vg / wellpoint if necessary. 2. Perform logarithmic transformation of viral load and GCase activity (relative fluorescence units (RFU) / hr). 3. Plot the response as Log(RFU / hr) vs. Log(virus). 4. Perform linear regression on each sample. 5. Perform a new linear regression using the common slope "A" (Y=AX+b). 6. Using the parameters obtained in Step 5, calculate the relative potency using the following formula: Relative efficacy (%) = 10^((bb reference ) / A)×100 7. Report the result compared to the reference standard as a percentage without decimals (for example, if the result is 100.50, report it as 101%). [Table 6]

[0063] Test method qualification assessment protocol Objective: The objective of this qualification scheme is to define a test method for measuring the relative potency of PR001 in vitro using a cell-based assay. This protocol demonstrates that the method produces reliable data and is suitable for the analysis of AAV samples for research and process development purposes (non-GXP). [Table 7]

[0064] Suitability Evaluation Plan: Validation shall be conducted in accordance with validation of analytical test methods set forth in International Conference on Harmonization (ICH) Q2(R1), USP<1032> and USP<1033>. The validation test consists of testing of AAV9-GBA DP at 50%, 100%, and 200% relative potency levels, as well as testing for specificity. Each level is tested by two analysts to evaluate the linearity, accuracy and precision (repeatability and intermediate precision) of the method. Relative potency from each assay is independent and is considered a single assay determination. Each plate contains one reference standard and up to two test samples. If a plate fails system suitability, the plate is repeated. If system suitability fails for a sample, only the failing sample is repeated. All samples must meet the assay acceptance criteria defined in the method and the validation criteria defined in this protocol. Specificity is also determined using an irrelevant AAV product that does not carry GBA1. Limit of detection and limit of quantitation are not included, as they are not relevant to the method for reporting relative potency described in USP<1032>. Table 8 summarizes the validation procedures and acceptance criteria used to evaluate the performance of the method. [Table 8-1] [Table 8-2]

[0065] Linearity: AAV9-GBA test samples are diluted to 50%, 100%, and 200% of the reference standard and tested in seven assays by two analysts. The mean (measured) relative potency is plotted against the expected relative potency and analyzed using linear regression. The resulting linearity equation and coefficient of determination (R 2) will be reported. Assay plates that fail system compatibility should not be used for analysis.

[0066] Accuracy: Accuracy is assessed by evaluating linearity of the data. The average recovery rate is calculated at each level using the following formula:

number

[0067] Parallel precision: Parallel precision is assessed by evaluating linearity data. Percentage relative standard deviation (%RSD) is calculated and reported for each level of each assay (i.e., same analyst and same week).

[0068] Intra-line accuracy: Parallel accuracy is assessed by evaluating linearity data. The overall %RSD is calculated and reported at each level.

[0069] Scope: The scope of the method is determined and reported using the lowest and highest efficacy tested, meeting experimental criteria for linearity, accuracy, and precision.

[0070] Specificity: A surrogate molecule (specificity sample) is tested by one analyst in a single assay. The specificity sample is diluted in the assay as if it were the AAV9-GBA test sample. The specificity sample is the surrogate molecule (AM): PR006.

[0071] Data Handling and Reporting: Raw data will be acquired using SkanIt RE 5.0 software, and parallel line analysis will be performed as described in the test method above. This data will be exported to a spreadsheet to calculate additional assay parameters (e.g., accuracy and precision). All result data, including experimental details such as materials, reagents, equipment used, and test conditions, will be recorded and verified by a second analyst.

[0072] Based on the results of all valid assay runs and all valid concentrations of the reference standard virus and the study virus, the overall average relative potency across all runs from the qualification is used to establish the nominal RP value of these samples for use in further assay runs.

[0073] Figure 2 shows an example of efficacy assay data from several PR001 samples. [Table 9-1] [Table 9-2]

[0074] Numbered Embodiments Notwithstanding the attached claims, this disclosure describes the following numbered embodiments:

[0075] 1. A method for measuring the relative potency of a test sample containing a first recombinant virus that includes a transgene encoding glucocerebrosidase (GCase), a) Transducing a first group of cells in the aforementioned test sample, b) Incubating the transduced first group of cells under conditions sufficient to express GCase, c) Collecting a first cell lysate from the first plurality of transfected cells, d) Mixing the first cell lysate with resorphin-beta-D-glucopyranoside, e) Imaging the first cell lysate and obtaining a first fluorescence reading. f) Transducing a second group of cells with a reference standard containing a second recombinant virus that includes a transgene encoding GCase, g) Incubate the transduced second group of cells under conditions sufficient to express GCase, h) Collecting a second cell lysate from the second plurality of transfected cells, i) Mixing the second cell lysate with resorphin-beta-D-glucopyranoside, j) Imaging the second cell lysate to obtain a second fluorescence reading, and k) Calculate the relative efficacy of the test sample by comparing the first fluorescence reading with the second fluorescence reading using parallel line analysis. The method, including the method described above.

[0076] 2. The method according to Embodiment 1, wherein the first recombinant virus and the second recombinant virus contain the same transgene encoding GCase.

[0077] 3. The method according to Embodiment 1 or 2, wherein the first recombinant virus and / or the second recombinant virus is a recombinant adeno-associated virus (rAAV).

[0078] 4. The method according to Embodiment 3, wherein the rAAV comprises the AAV9 capsid protein.

[0079] 5. The method according to Embodiment 3, wherein the rAAV comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 capsid protein, or a variant of any of these capsid proteins.

[0080] 6. The method according to any one of Embodiments 1 to 5, wherein the GCase includes Sequence ID No. 1.

[0081] 7. The method according to any one of Embodiments 1 to 6, wherein the transgene encoding GCase comprises a codon-optimized nucleotide sequence.

[0082] 8. The method according to Embodiment 7, wherein the codon-optimized nucleotide sequence includes SEQ ID NO: 2.

[0083] 9. The method according to any one of Embodiments 1 to 8, wherein the first plurality of cells and / or the second plurality of cells are HEK-293T or HEK-293 cells.

[0084] 10. The method according to any one of Embodiments 1 to 9, wherein approximately 1.25 mM resorphine-beta-D-glucopyranoside is mixed with the first cell lysate and / or the second cell lysate.

[0085] 11. The method according to any one of Embodiments 1 to 10, wherein the first plurality of cells and the second plurality of cells are seeded in a multiwell plate.

[0086] 12. The method according to Embodiment 11, wherein the first plurality of cells and / or the second plurality of cells are seeded at a rate of approximately 20,000 cells per well.

[0087] 13. The method according to any one of Embodiments 1 to 12, wherein the test sample and / or the reference standard are progressively diluted before transduction.

[0088] 14. The method according to any one of Embodiments 1 to 13, wherein the first plurality of cells and the second plurality of cells are incubated for about 68 to about 81 hours before cell lysate collection.

[0089] 15. The method according to any one of Embodiments 1 to 13, wherein the first plurality of cells and the second plurality of cells are incubated for about 66 to about 78 hours after transduction and before cell lysate collection.

[0090] 16. The method according to any one of Embodiments 1 to 15, wherein the first plurality of cells are transduced by the test sample at at least two different modalities (MOIs) of infection with the first recombinant virus.

[0091] 17. The method according to any one of Embodiments 1 to 16, wherein the second plurality of cells are transduced by the reference standard at at least two different multiplicities (MOIs) of infection with the second recombinant virus.

[0092] 18. The method according to any one of Embodiments 1 to 17, wherein the first fluorescence reading and / or the second fluorescence reading reflects a measured value of GCase activity.

[0093] 19. The method according to Embodiment 18, wherein the measured value of the GCase activity is in relative fluorescence units (RFU) / hour.

[0094] 20. The method according to Embodiment 19, wherein the comparison step (k) includes performing a logarithmic transformation of the amount of recombinant virus and RFU / time for each of the test sample and the reference standard, and plotting a standard curve of the logarithm of the amount of recombinant virus against the logarithm of RFU / time.

[0095] 21. The method according to Embodiment 20, wherein the comparison step (k) comprises calculating a linear regression of the logarithm of the amount of recombinant virus against the logarithm of the RFU / time for each of the test sample and the reference standard, thereby deriving the slope for the test sample and the slope for the reference standard.

[0096] 22. The method according to Embodiment 21, wherein the comparison step (k) includes calculating a linear regression with a common slope using the linear regressions obtained for each of the test sample and the reference standard.

[0097] 23. The relative efficacy is given by the formula: Relative efficacy (%) = 10^((bb reference The method according to Embodiment 22, calculated using ) / A × 100.

[0098] 24. The method according to Embodiment 22 or 23, wherein the ratio of the inclination of the test sample to the common inclination is about 0.60 to about 1.40.

[0099] 25. The method according to any one of embodiments 22 to 24, wherein the ratio of the slope of the reference standard to the common slope is about 0.60 to about 1.40.

[0100] 26. The R of the linear regression of the test sample and the reference standard 2 The method according to any one of embodiments 20 to 25, further comprising calculating a value.

[0101] 27. The R of the test sample and the reference standard 2 The method according to embodiment 26, wherein the value is 0.9 or greater.

Claims

1. A method for measuring the relative potency of a test sample containing a first recombinant virus that includes a transgene encoding glucocerebrosidase (GCase), a) Transducing a first group of cells in the aforementioned test sample, b) Incubating the transduced first group of cells under conditions sufficient to express GCase, c) Collecting a first cell lysate from the first plurality of transfected cells, d) Mixing the first cell lysate with resorphin-beta-D-glucopyranoside, e) Imaging the first cell lysate and obtaining a first fluorescence reading, f) Transducing a second group of cells with a known potency reference standard containing a second recombinant virus that includes a transgene encoding GCase, g) Incubating the transduced second group of cells under conditions sufficient to express GCase, h) Collecting a second cell lysate from the second plurality of transfected cells, i) Mixing the second cell lysate with resorphin-beta-D-glucopyranoside, j) Imaging the second cell lysate to obtain a second fluorescence reading, and k) Using parallel line analysis, compare the first fluorescence reading with the second fluorescence reading to calculate the relative efficacy of the test sample. Includes, The method wherein both the first plurality of cells and the second plurality of cells are HEK-293T or HEK-293 cells.

2. The method according to claim 1, wherein the first recombinant virus and the second recombinant virus each contain the same transgene encoding GCase.

3. The method according to claim 1 or 2, wherein the first recombinant virus and / or the second recombinant virus is a recombinant adeno-associated virus (rAAV).

4. The method according to claim 3, wherein the rAAV comprises the AAV9 capsid protein.

5. The method according to claim 3, wherein the rAAV comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 capsid protein, or a variant of any of these capsid proteins.

6. The method according to any one of claims 1 to 5, wherein the GCase includes sequence number 1.

7. The method according to any one of claims 1 to 6, wherein the transgene encoding GCase includes a codon-optimized nucleotide sequence.

8. The method according to claim 7, wherein the codon-optimized nucleotide sequence includes sequence number 2.

9. The method according to any one of claims 1 to 8, wherein approximately 1.25 mM resorphin-beta-D-glucopyranoside is mixed with the first cell lysate and / or the second cell lysate.

10. The method according to any one of claims 1 to 9, wherein the first plurality of cells and the second plurality of cells are seeded in a multiwell plate.

11. The method according to claim 10, wherein the first plurality of cells and / or the second plurality of cells are seeded at a rate of approximately 20,000 cells per well.

12. The method according to any one of claims 1 to 11, wherein the test sample and / or the reference standard are progressively diluted before transduction.

13. The method according to any one of claims 1 to 12, wherein the first plurality of cells and the second plurality of cells are incubated for about 68 hours to about 81 hours before cell lysate collection.

14. The method according to any one of claims 1 to 12, wherein the first plurality of cells and the second plurality of cells are incubated for about 66 to about 78 hours after transduction and before cell lysate collection.

15. The method according to any one of claims 1 to 14, wherein the first plurality of cells are transduced by the test sample at at least two different multiples of infection (MOI) of the first recombinant virus.

16. The method according to any one of claims 1 to 15, wherein the second plurality of cells are transduced by the reference standard at at least two different multiples of infection (MOI) of the second recombinant virus.

17. The method according to any one of claims 1 to 16, wherein the first fluorescence reading and / or the second fluorescence reading reflects a measured value of GCase activity.

18. The method according to claim 17, wherein the measured value of the GCase activity is in relative fluorescence units (RFU) per hour.

19. The method according to claim 18, wherein the comparison step (k) includes performing a logarithmic transformation of the amount of recombinant virus and RFU / time for each of the test sample and the reference standard, and plotting a standard curve of the logarithm of the amount of recombinant virus against the logarithm of the RFU / time.

20. The method according to claim 19, wherein the comparison step (k) includes calculating a linear regression of the logarithm of the amount of recombinant virus against the logarithm of the RFU / time for each of the test sample and the reference standard, thereby deriving the slope for the test sample and the slope for the reference standard.

21. The method according to claim 20, wherein the comparison step (k) includes calculating a linear regression with a common slope using the linear regressions obtained for each of the test sample and the reference standard.

22. The aforementioned relative effectiveness is given by the formula: Relative effectiveness (%) = 10^((b-b) ｒｅｆｅｒｅｎｃｅ The method according to claim 21, calculated using ) / A) × 100.

23. The method according to claim 21 or 22, wherein the ratio of the inclination of the test sample to the common inclination is about 0.60 to about 1.

40.

24. The method according to any one of claims 21 to 23, wherein the ratio of the slope of the reference standard to the common slope is about 0.60 to about 1.

40.

25. The R of the linear regression of the test sample and the reference standard ２ The method according to any one of claims 20 to 24, further comprising calculating a value.

26. The R of the aforementioned test sample and the aforementioned reference standard ２ The method according to claim 25, wherein the value is 0.9 or greater.

Images