Cell-based assays for measuring the potency of drug products

Abstract

The present invention relates to a cell-based in vitro quantitative assay that uses a primary mouse cell model system permissive for viral vector infection and a high-content image-based quantification system to determine the potency of a transgene-expressing viral vector drug product for batch handling.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 682,263, filed on June 8, 2018, the contents of which are incorporated herein by reference in their entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] This application contains a sequence listing, which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on June 4, 2019 is named AVEX-004001WO_ST25.txt and is 25 kilobytes in size. Background Art

[0005] Adeno-associated virus (AAV) is a member of the parvoviridae family. The AAV genome consists of a linear single-stranded DNA molecule containing approximately 4.7 kilobases (kb) and consisting of two major open reading frames encoding nonstructural Rep (replication) and structural Cap (capsid) proteins. Flanking the AAV coding region are two cis-acting nucleotide inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with interrupted palindromic sequences that can fold into a hairpin structure that functions as a primer during the initiation of DNA replication. In addition to their role in DNA replication, ITR sequences have been shown to be essential for viral integration, rescue from the host genome, and encapsidation of viral nucleic acids into mature virions.

[0006] Vectors derived from AAV are particularly attractive for delivering genetic material because (i) they are able to infect (transduce) a wide variety of non-dividing and dividing cell types, including muscle fibers and neurons; (ii) they lack viral structural genes, thereby eliminating natural host cell responses to viral infection, such as interferon-mediated responses; (iii) the wild-type virus has never been associated with any pathology in humans; (iv) in contrast to wild-type AAV, which is able to integrate into the host cell genome, replication-defective AAV vectors typically persist as episomes, thus limiting the risk of insertional mutagenesis or activation of oncogenes; and (v) in contrast to other vector systems, AAV vectors do not trigger a significant immune response (see ii), thus allowing long-term expression of therapeutic transgenes (provided that their gene products are not rejected).

[0007] Self-complementary adeno-associated virus (scAAV) is a viral vector engineered from a naturally occurring adeno-associated virus (AAV) for use in gene therapy. scAAV is called "self-complementary" because the coding region has been designed to form an intramolecular double-stranded DNA template. The rate-limiting step in gene expression of the standard single-stranded AAV genome involves second-strand synthesis because the typical AAV genome is a single-stranded DNA template. However, this is not the case for the scAAV genome. After infection, the two complementary halves of the scAAV will associate instead of waiting for cell-mediated synthesis of the second chain to form a double-stranded DNA (dsDNA) unit ready for immediate replication and transcription.

[0008] Spinal muscular atrophy (SMA) is a severe neuromuscular disease caused by a genetic defect in the SMN1 gene, which leads to loss of motor neurons and progressive muscle weakness and paralysis. SMA is divided into subtypes - SMA types 1, 2, 3 and 4 - based on disease onset and severity, which are often associated with levels of the survival motor neuron (SMN) protein.

[0009] Due to the monogenic nature of SMA (meaning that it is caused by the deletion or mutation of a single gene), gene therapy via the use of viral vectors (as delivery vehicles) is a well-suited approach to treating the disease. AAV9 has previously been identified as a viral vector suitable for SMA gene therapy, where it has been used for SMA Type 1 and SMA Type 2. This viral vector has been shown to deliver a fully functional human SMN gene to target motor neuron cells, produce SMN protein at levels sufficient to improve motor neuron function, and provide a rapid onset of action in addition to sustained SMN protein expression.

[0010] However, there remains a need to develop robust, quantitative, cell-based in vitro assays for determining relative potency intended for batch handling of AAV9 drug products. The development of robust, quantitative, cell-based in vitro potency assays has been hampered by the fact that none of the transformed or primary cells (human or murine) tested to date, including the HeLa RC32 cell line commonly used in infectious titer testing with AAV9-based viral vectors, have been shown to be permissive for AAV9 vectors.

[0011] In the present disclosure, terminally differentiated non-dividing cells derived from neural progenitor cells in an SMN1- / - genetic background (terminally differentiated cells derived from NPCs, hereinafter referred to as mTD-NPC-Δ7) are provided for the first time, which can be efficiently transduced by non-replicating AAV9 vectors. More importantly, these cells are used in an in vitro cell model system to develop a cell-based quantitative assay to measure the dose-dependent increase in the expression of a target protein after transduction of an AAV9 vector at increasing multiplicity of infection (MOI) using a monoclonal antibody specific for the target protein by a high-content imaging system. Summary of the invention

[0012] In one aspect, the disclosure provides a method for measuring transgene expression, the method comprising the steps of: (a) culturing a plurality of cells, wherein the cells comprise a viral vector, wherein the viral vector comprises a transgene, wherein the culturing is under conditions sufficient to express a protein of interest from the transgene; (b) incubating the plurality of cells to allow transgenic expression of the protein of interest to ensu; (c) contacting the plurality of cells with a molecule specific for the protein of interest; (d) imaging the cells to obtain an integrated fluorescence intensity / cell (IFI-C) assay readout; and, (e) assaying expression of the transgene based on the IFI-C readout.

[0013] In a related aspect, the disclosure provides a method of measuring or quantifying viral infection titers in a plurality of cells, the method comprising the steps of: (a) culturing a plurality of cells, wherein the cells comprise a viral vector, wherein the viral vector comprises a transgene, wherein the culturing is under conditions sufficient to express a protein of interest from the transgene; (b) incubating the plurality of cells to allow transgene expression of the protein of interest to ensu; (c) contacting the plurality of cells with a molecule specific for the protein of interest; (d) imaging the cells to obtain an integrated fluorescence intensity / cell (IFI-C) assay readout; and, (e) assaying expression of the transgene based on the IFI-C readout.

[0014] In another aspect, the present disclosure provides a method for measuring transgene expression, the method comprising: (a) providing a first plurality of terminally differentiated neural progenitor cells (NPCs); (b) transducing the first plurality of terminally differentiated NPCs with a test sample comprising a viral vector comprising a sequence encoding a protein of interest; (c) incubating the transduced first plurality of terminally differentiated NPCs under conditions sufficient to express the protein of interest; (d) contacting the first plurality of terminally differentiated NPCs from (c) with a molecule specific for the protein of interest; (e) imaging the first plurality of terminally differentiated NPCs to obtain an integrated fluorescence intensity / cell (IFI-C) assay readout; and (f) assaying expression of the protein of interest based on the IFI-C readout.

[0015] In some aspects of the methods of the present disclosure, the first plurality of terminally differentiated NPCs are homozygous for a survival motor neuron gene (SMN1)- / - mutation. In some aspects, the SMN1- / - mutation comprises a deletion of SMN1 exon 7 (Δ7). In some aspects, the incubation step c) is followed by fixation and permeabilization of the first plurality of terminally differentiated NPCs.

[0016] In another aspect, the present disclosure provides a method comprising the following items: (g) providing a second plurality of terminally differentiated NPCs; (h) transducing the second plurality of terminally differentiated NPCs with a reference standard comprising the viral vector; (i) incubating the transduced second plurality of terminally differentiated NPCs under conditions sufficient to express the protein of interest; (j) contacting the second plurality of terminally differentiated NPCs from (i) with a molecule specific for the protein of interest; (k) imaging the second plurality of terminally differentiated NPCs to obtain an integrated fluorescence intensity / cell (IFI-C) assay readout; and (l) comparing the IFI-C of the first plurality of terminally differentiated NPCs with the IFI-C of the second plurality of terminally differentiated NPCs; thereby determining the relative potency of the viral vector of the test sample relative to the reference standard.

[0017] In some aspects of the methods of the disclosure, the second plurality of terminally differentiated NPCs are homozygous for an SMN1- / - mutation. In some aspects, the SMN1- / - mutation comprises a deletion of SMN1 exon 7 (Δ7).

[0018] In some aspects, the incubating step (i) is followed by fixation and permeabilization of the second plurality of terminally differentiated NPCs.

[0019] In some aspects, the first plurality of terminally differentiated NPCs and the second plurality of terminally differentiated NPCs are produced by terminally differentiating neural progenitor cells isolated from the cortex of SMN1- / - mouse embryos. In some aspects, the neural progenitor cells (NPCs) are terminally differentiated by: (a) culturing the NPCs in a serum-free medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to form neurospheres; (b) dissociating the neurospheres to produce dissociated NPCs; and (c) culturing the dissociated NPCs in a serum-rich medium without growth factors to produce terminally differentiated NPCs.

[0020] In some aspects of the methods of the present disclosure, the first plurality of cells and the second plurality of cells are transduced at at least two different multiplicities of infection (MOI) of the viral vector by the test sample and the reference standard. In some aspects, the first plurality of cells and the second plurality of cells are transduced at 5 different MOIs of the viral vector in the test sample and the reference standard. In some aspects, the 5 MOIs include 300,000, 150,000, 75,000, 37,500, 18,750 viral particles / cell.

[0021] In some aspects of the methods of the present disclosure, the comparing step (1) comprises plotting a standard curve of MOI versus IFI-C for each of the test sample and the reference standard. In some aspects, the comparing step (1) comprises calculating a linear regression of log MOI versus IFI-C for each of the test sample and the reference standard, thereby obtaining a test sample slope and a reference standard slope.

[0022] In some aspects of the methods of the present disclosure, determining the relative efficacy of the viral vector is performed by parallel line analysis (PLA), and wherein the PLA includes measuring the slope ratio of the test sample slope relative to the reference standard slope. In some aspects, the reference standard slope is greater than or equal to 1.02E+05. In some aspects, the slope ratio is between 0.69-1.45. In some aspects, the slope ratio is between 0.75 and 1.33.

[0023] In some aspects of the methods of the present disclosure, the methods include calculating the coefficient of variation of the linear regression of the sample. In some aspects, the coefficient of variation is between 15.6% and 29.5%. In some aspects, the coefficient of variation is less than or equal to 40%, less than or equal to 30%, or less than or equal to 20%.

[0024] In some aspects of the methods of the present disclosure, the methods include calculating the R of the linear regression of the test sample and the reference standard. 2 In some aspects, the R values ​​of the test sample and the reference standard are 2 Value greater than or equal to 0.95.

[0025] In some aspects of the methods of the present disclosure, the methods include calculating an assay dynamic window for the reference standard. In some aspects, the assay dynamic window is greater than or equal to 2.69.

[0026] In some aspects of the methods of the present disclosure, the protein of interest is a survival motor neuron (SMN1) protein. In some aspects, the SMN1 protein comprises the amino acid sequence of SEQ ID NO:3.

[0027] In some aspects of the methods of the present disclosure, the viral vector is adeno-associated virus serotype 9 (AAV9). In some aspects, the viral vector comprises a sequence encoding a cytomegalovirus (CMV) enhancer / chicken-β-actin hybrid promoter (CB) operably linked to a sequence encoding the SMN1 protein. In some aspects, the viral vector comprises an AAV inverted terminal repeat (ITR) from AAV serotype 2 (AAV2) DNA. In some aspects, the viral vector comprises a sequence of SEQ ID NO: 1.

[0028] In some aspects of the methods of the disclosure, the cells are passaged 8 to 15 times prior to transduction with the viral vector.

[0029] In some aspects of the methods of the disclosure, the step of incubating the terminally differentiated NPCs after transduction is performed for about 69-75 hours (h).

[0030] In some aspects of the methods of the present disclosure, the molecule specific for the target protein includes an antibody, an antibody fragment or an aptamer. In some aspects, the antibody includes an antibody specific for the target protein. In some aspects, the anti-target protein antibody is provided at a concentration of about 4 μg / mL. In some aspects, the anti-target protein antibody is provided at a concentration of about 2 μg / mL. In some aspects, the molecule includes a detectable label.

[0031] In some aspects of the methods of the present disclosure, the methods further include contacting the terminally differentiated NPCs with a second molecule that specifically recognizes the molecule specific for the protein of interest. In some aspects, the second molecule includes a detectable label. In some aspects, the second molecule includes an antibody, an antibody fragment, or an aptamer.

[0032] In some aspects of the methods of the disclosure, following the fixation and permeabilization steps, the terminally differentiated NPCs are contacted with an anti-nuclear detectable marker.

[0033] In some aspects of the methods of the disclosure, the terminally differentiated NPCs are on a solid surface. In some aspects, the solid surface is coated with poly-D-lysine. In some aspects, the terminally differentiated NPCs are seeded at a density of 20,000 cells / well.

[0034] In another aspect, the method of measuring or quantifying viral infectious titer in a plurality of cells further comprises optimizing the multiplicity of infection (MOI) of the plurality of cells.

[0035] In another related aspect, the plurality of cells are transduced with the viral vector prior to step a). In another aspect, the incubating step b) is followed by fixation and permeabilization of the plurality of cells.

[0036] In another related aspect, the step of determining the relative potency of the viral vector test sample is performed by parallel line analysis (PLA) relative to a standard curve of a reference standard after linear regression data fitting.

[0037] In another aspect, the viral vector is an adeno-associated virus serotype 9 (AAV9), which contains a cDNA expressing SMN1 protein under the control of a cytomegalovirus (CMV) enhancer / chicken-β-actin hybrid promoter (CB), and AAV inverted terminal repeats (ITRs) from AAV serotype 2 (AAV2) DNA.

[0038] In another related aspect, the cells transduced with the viral vector are terminally differentiated non-dividing cells.

[0039] In another aspect, the cell is derived from a neural progenitor cell in an SMN1- / - genetic background (mTD-NPC-Δ7).

[0040] In another aspect, the IFI-C readout reflects a measure of protein expression.

[0041] In another aspect, the molecule specific for the protein of interest comprises an antibody, an antibody fragment or an aptamer. In another aspect, the antibody comprises an antibody specific for the protein of interest.

[0042] In another aspect, the molecule comprises a detectable label.

[0043] In another aspect, the method further comprises washing the cells to remove the molecule specific for the protein of interest.

[0044] In another aspect, the method further comprises contacting the cells with a second molecule that specifically recognizes the molecule specific for the protein of interest. In another aspect, the second molecule comprises a detectable label. In another aspect, the second molecule comprises an antibody, an antibody fragment, or an aptamer. In another aspect, after the fixation and permeabilization steps, the cells are contacted with an anti-nuclear detectable label.

[0045] In another aspect, the method allows for quantitative measurement of dose-dependent increases in the levels of the protein of interest.

[0046] In another aspect, the protein of interest is survival motor neuron (SMN1) protein.

[0047] The present disclosure provides kits comprising: (a) a plurality of cells capable of being transduced with a viral vector; (b) a viral vector encoding a protein of interest; (c) a first molecule capable of binding to the protein of interest; (d) a second molecule capable of binding to the first molecule, wherein the second molecule comprises a detectable label; and, (e) instructions for use in an imaging assay.

[0048] The present disclosure provides a method for producing a pharmaceutical composition comprising a viral vector containing a transgene, the method comprising: (a) producing the viral vector containing the transgene; (b) measuring the viral vector according to the method for measuring the transgene of the present disclosure; and (c) formulating the viral vector containing the transgene in a pharmaceutical composition.

[0049] The present disclosure provides a method of treating a patient in need thereof with a therapy comprising a viral vector containing a transgene, the method comprising: (a) assaying the viral vector containing the transgene according to the method of measuring transgene expression of the present disclosure; and (b) administering the viral vector containing the transgene to the patient.

[0050] In some aspects of the methods of the present disclosure, 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% relative to a reference standard. In some aspects, the relative potency of the viral vector is at least 90% relative to the reference standard.

[0051] In some aspects of the methods of the disclosure, the potency of the viral vector in the pharmaceutical formulation is within 5% of the potency of the reference standard, within 10% of the potency of the reference standard, or within 20% of the potency of the reference standard.

[0052] Any of the above aspects may be combined with any other aspect.

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0054] As used herein, unless the context clearly indicates otherwise; otherwise, the singular form of a word also includes the plural form of the word; as an example, the terms "a, an" and "the" are understood to be singular or plural, and the term "or" is understood to be inclusive. For example, "an element" means one or more elements.

[0055] Throughout this specification, the word "comprising" or variations such as "comprises" shall be understood to imply the inclusion of the stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps. Throughout this specification, the word "consisting of" or variations such as "consists of" shall be understood to imply the inclusion of the stated elements, integers or steps, or groups of elements, integers or steps, and the exclusion of any other elements, integers or steps, or groups of elements, integers or steps. Throughout this specification, the word "consisting essentially of" or variations such as "consists essentially of" shall be understood to imply the inclusion of the stated elements, integers or steps, or groups of elements, integers or steps, and any other elements, integers or steps, or groups of elements, integers or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0056] About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term "about".

[0057] Although methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not considered to be prior art for the claimed disclosure. When a conflict occurs, the present specification including the definitions shall prevail. In addition, materials, methods, and examples are illustrative only and are not intended to be restrictive. Other features and advantages of the present disclosure will be clear from the following specific embodiments and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0059] Any of the above aspects and embodiments may be combined with any other aspect or embodiment disclosed herein in the Summary and / or Detailed Description sections.

[0060] Various objects and advantages of the present invention, as well as a more complete understanding of the present invention, will become apparent and more readily appreciated by referring to the following detailed description and appended claims taken in conjunction with the accompanying drawings, in which:

[0061] Figure 1 Demonstrates the master / working library system.

[0062] Figure 2 Demonstrates a hierarchical master / work library system.

[0063] Figures 3A to 3C Terminal differentiation of NPCs derived from the germinal cortex of SMNΔ7 mice is shown. Figure 3A )NPCs are derived from the mouse SMN- / - germinal cortex at approximately e14.5 (middle image) and ( Figure 3B ) were grown as proliferative neurospheres in the presence of mitogens (growth factors EGF and FGF). Figure 3C )When removed from growth factors and placed in serum-based medium, neurospheres can be dissociated and ultimately differentiated into CNS cells including GFAP+ astrocytes.

[0064] Figure 4 Images of mTD-NPC-Δ7 transduced with AAV9-eGFP at the indicated MOIs are shown.

[0065] Figures 5A to 5B Cell density at 20,000 cells / well (nuclear staining) is shown ( Figure 5A ) and at a cell density of 10,000 cells / well (nuclear staining) ( Figure 5B ) Cropped image covering about 40% of the holes.

[0066] Figure 6 Shown are images of SMN1 (2B1) and cell nucleus (Hoechst 33342) staining at 72 hours after transduction with AAV9 vector (lot number NCHAAV9SMN0613).

[0067] Figure 7Images of GFP and SMN1 staining of mTD-NPC-Δ7 transduced with AAAV9 vector batch NCHAAV9SMN0613 are shown (top) (bottom image is control group).

[0068] Figures 8A to 8B Nuclear staining of mTD-NPC-Δ7 plated on uncoated plates is shown (montage of images covering 40% of the well) ( Fig. 8A ), and nuclear staining of mTD-NPC-Δ7 seeded on poly-D-lysine coated plates (montage of images covering 40% of the well) ( Figure 8B ).

[0069] Figures 9A to 9B Images of AAV9-eGFP transduced mTD-NPC-Δ7 at 72 hours post-transduction are shown ( Fig.9A ) and images of AAV9-eGFP transduced mTD-NPC-Δ7 at 48 hours after transduction ( Fig. 9B ).

[0070] Fig.10 Images of SMN1 (green) and nucleus (blue) staining of mTD-NPC-Δ7 transduced with SMN1-encoding AAV9 vector (72 hours post-transduction) are shown.

[0071] Fig.11 Shown are images of SMN1 (green) and nucleus (blue) staining for mTD-NPC-Δ7 transduced with NCH0613 at various multiplicities of infection (MOI) (72 hours post-transduction).

[0072] Fig.12 Curve fits of the 12-point dose data of integrated fluorescence intensity / cell (IFI-C) are shown (left: untransformed, right: log-transformed on the x-axis).

[0073] Fig.13 Shown is a fitted line plot of log MOI versus IFI-C.

[0074] Fig.14 A scatter plot of IFI-C versus log MOI is presented, showing that the lines for all five replicates are very close. All five replicates passed the pairwise concurrency test (all p values>0.25).

[0075] Figures 15A to 15C The application of in vitro relative potency determination of AAV9 vectors in a quantitative infectivity assay using an mNPC-based assay platform is demonstrated.

[0076] Figures 16A to 16B Demonstrated proof-of-concept studies to establish infection titers against EC50 AAV9 vector infectivity assay.

[0077] Fig.17 is a graph showing equivalent dose-dependent increases in SMN using three different anti-SMN antibody concentrations.

[0078] Fig.18 An example image of a cell nucleus acquired using the CellInsight High-Content Screening System.

[0079] Fig.19 is a graph showing the linearity between measured relative potency and expected relative potency. Squares indicate geometric means and circles indicate individual values. The x-axis shows expected relative potency (%) and the y-axis shows measured relative potency (%).

[0080] Figures 20A to 20B is a pair of graphs showing the specificity exhibited by staining cells transduced with SMN1-encoding AAV9 vectors or MECP2-encoding AAV9 vectors using SMN1 antibodies. Fig. 20A Cells stained with anti-SMN1 antibodies are shown. Open circles indicate control cells transduced with SMN1 encoding AAV9 vectors, and solid circles indicate cells transduced with MECP2 encoding AAV9 vectors. The x-axis shows log2 MOI (multiplicity of infection), and the y-axis shows integrated fluorescence intensity / cell (IFI-C). Fig. 20B Cells transduced with MECP2 encoding AAV9 vectors and stained with anti-MECP2 antibodies are shown. The x-axis shows log2 MOI, and the y-axis shows IFI-C. Specificity is demonstrated by the following: although AAV9-MECP2 showed successful transduction of cells when stained with anti-MECP2 antibodies, when cells transduced with AAV9-SMN1 vectors, but not AAV9-MECP2 vectors, were stained with anti-SMN1 antibodies, the IFI-C signal indicating exogenous SMN1 protein expression showed a dose-dependent increase with increasing MOI doses, as indicated by the dose-dependent increase of IFI-C.

[0081] Fig.21 The plate layout used in the uniformity study is shown.

[0082] Fig. 22 is a graph showing a dose-dependent increase in IFI-C. mTD-NPC-Δ7 cells were transduced with AAV9-SMN1 or AAV9-MECP2 vectors, stained with anti-SMN1 or anti-MeCP2, and IFI-C was measured.

[0083] Fig.23Is an image montage of an in vitro relative potency assay plate. Columns 1-7 are stained with anti-SMN1 antibody. Columns 8-11 are stained with anti-MeCP2 antibody.

[0084] Fig.24 is a graph showing a dose-dependent increase in IFI-C in response to AAV9-MECP2 transduction.

[0085] Fig.25 is a graph showing a summary of the slope ratio estimates for the samples to the reference standard.

[0086] Fig.26A The plasmid map of pSMN is shown. pSMN is a plasmid encoding the information of the recombinant self-complementary AAV DNA genome, which expresses human motor neuron survival (SMN) cDNA under the control of a chicken-β-actin hybrid promoter with an immediate / early cytomegalovirus (CMV) enhancer element. SMN cDNA encodes a full-length functional protein. The expression cassette contains a modified intron sequence derived from Simian Virus 40 (SV40) and a bovine growth hormone (BGH) polyadenylation signal. The expression cassette (CMV-CB-SV40-SMN-BGHpA) is flanked by inverted terminal repeats (ITRs) from AAV2 sources. The left ITR is modified to preferentially package the self-complementary AAV genome. The region between and including the ITR is packaged together into the recombinant AAV9 capsid during the manufacture of the final drug product. The key pSMN components that are not intended to be packaged into the recombinant AAV genome include an open reading frame encoding resistance to kanamycin (KanR) and a replication origin (ori) derived from pUC. The ori and KanR regions can be used for plasmid production.

[0087] Fig.26B The plasmid map of pHELP plasmid is shown. The pHELP plasmid contains trans-acting adenoviral components necessary for the production of recombinant adeno-associated viruses. The pHELP plasmid contains regions in the adenoviral genome that provide factors important for AAV replication, namely E2A, E4 and VA RNA. The adenoviral E1 function involved in rAAV replication is provided by transfecting host 293 cells. However, the pHELP plasmid does not contain other adenoviral replication or structural genes. The adenoviral sequences present in this plasmid represent only about 28% (9,280 / 35,938) of the adenoviral genome and do not contain cis elements that are essential for replication, such as inverted terminal repeats. Therefore, it is not expected that infectious adenovirus will be produced from this production system.

[0088] Fig.26CThe plasmid map of the AAV plasmid is shown. The wild-type AAV genome contains two non-coding structural elements (called inverted terminal repeats) that flank the rep and cap open reading frames. Rep and cap encode viral replication and capsid proteins, respectively. In the production of recombinant adeno-associated viral vectors, the viral ITRs are the only elements used in cis, while the viral open reading frames are provided in trans. The method of preparing AAV using transient transfection of adherent HEK293 cells solves the cis / trans effects of different genetic elements by dividing them into separate plasmids. The pAAV2 / 9 plasmid contains the open reading frames of the AAV2 rep gene and the AAV9 cap gene.

[0089] Fig. 27 A process flow diagram for selecting HEK293 cells for exceptional adherence and pre-master cell bank (MCB) construction is shown.

[0090] Fig.28 A summary of the cell processing details used to select HEK293 cells for exceptional adherence and pre-master cell bank (MCB) construction is shown.

[0091] Fig.29 The drug substance upstream process flow diagram is described.

[0092] Fig.30 A drug substance downstream process flow diagram is described.

[0093] Fig.31 Inactivation of XMuLV by Tween 20 added up to 120 min is shown.

[0094] Fig.32 Inactivation of PRV by Tween 20 added up to 120 min is shown.

[0095] Fig.33 The HEK 293 cell expansion process during a cell seeding density experiment is described.

[0096] Figures 34A to 34E Growth and metabolite profiles are shown. HEK 293 cells were cultured at 12,000 and 8,000 cells / cm 2 Duplicate inoculations were performed in bioreactors (pH 7.23, 37.0°C, 55% dissolved oxygen (DO)). Four days after inoculation (12,000 cells / cm 2 ) and five days (8,000 cells / cm 2 ) were transfected with DNA plasmid / PEI. Eight days after seeding (12,000 cells / cm 2 ) and nine days (8,000 cells / cm 2) The bioreactor was harvested. pH and metabolite readouts were taken daily on the Nova BioFlex.

[0097] Fig.35 The viral genome production is shown as a function of cell seeding density (8000 or 12000 cells / cm 2 ) and four different transfection time lengths (20 min, 1 h or 2 h).

[0098] Fig.36 Shown are viral titers from intermediates sampled at different filtration steps throughout the manufacturing process.

[0099] Fig.37A and 37B Viral vector recovery and host cell protein (HCP) clearance at the TFF1 step are shown.

[0100] Fig.38 The HEK 293 cell expansion process during a cell seeding density experiment is described.

[0101] Figures 39A to 39E HEK 293 cells were cultured at 8,000 cells / cm 2 , 9,350 cells / cm 2 , 10,700 cells / cm 2 , 12,050 cells / cm 2 Bioreactors (pH 7.23, 37.0°C, 55% DO) were inoculated in duplicate. Cells were transfected with DNA plasmid / PEI (1:1 m / m) five days after inoculation. pH and metabolite analysis were performed using NOVA BioProfile 400.

[0102] Figures 40A to 40B Drug substance production from four starting inoculation densities in the bioreactor is shown. A comparison of virus titer and vector genomes harvested per unit surface area is shown.

[0103] Fig.41 Phase 1 (Process A) and Phase 3 trial (Process B) manufacturing processes are shown.

[0104] Figures 42A to 42B Tables showing comparability and manufacturing consistency results are provided - Process A (Phase 1) and Process B (Phase 3) products. Process B products are shown to have additional benefits compared to Process A. 1 NCH ​​Phase 1 batch AAV9SMN0613 was manufactured prior to the current “proposed testing limit” for genomic titer measured by ddPCR. The genomic titer value for this batch was reestablished in August 2017 using a modified SOP-137 (v3).2 The difference in genomic titer results between process A and process B batches is due to different manufacturing target concentrations. Batch NCH AAV9SMN0613 was initially assayed at 1.1x10 13 vg / mL, while AAV9-SMN1 lots 600156 and 600307 were formulated at a lower target titer concentration of 4.0x10 vg / mL when measured by the same method. 13 vg / mL target titer concentration. 3 Every 1.0x10 13 vg / mL adjustment results in a range from 2.0x10 13 vg / mL to 6.0x10 13 vg / mL. Actual values ​​have been multiplied by the following factors to provide the appropriate specifications for each 1.0x10 13 Values ​​of vg / mL: 1 / 1.06 (Batch NCH AAV9SMN0613), 1 / 3.7 (Batch 600156) and 1 / 4.0 (Batch 600307). 4 The difference in appearance results between Process A and Process B is due to different vector concentrations (genomic titers. Batch NCH AAV9SMN0613 has a significantly lower vector concentration than the Process B batch. Therefore, Batch NCH AAV9SMN0613 is more dilute, resulting in a clearer and colorless solution, while the colorless to white and slightly opaque observations for the Process B batch result from a nearly 4-fold higher concentration of viral particles per mL of solution. 5 Actual results are not for 1.0x10 13 vg / mL were adjusted because the results were below the LOQ of the respective methods. 6 Lot NCHAAV9SMN0613 was designated as the initial potency reference standard for SOP-285 and was assigned a potency value of 100%. All results were generated using SOP-285v5.

[0105] Fig.43 Pairwise comparisons using Process A (Phase 1 Batch NCHAAV9SMN0613) and Process B (Phase 3 Batch 600156) show comparability between Process A and Process B. The Process B product was shown to have additional benefits compared to Process A.

[0106] Fig.44 Manufacturing consistency assessment performed by pairwise comparison of Process B (Phase 3) Lots 600156 and 600307 is shown.

[0107] Fig.45Shown is the stability profile of NCH batch NCHAAV9SMN0613 stored over 12 months at real-time storage conditions of ≤ -60°C.

[0108] Fig.46 The sedimentation coefficient (s x10 -13 ), showing that the empty capsid (7%) has a size of approximately 60x10 -13 s, and the complete capsid has a sedimentation coefficient of about 80-150x10 -13 s sedimentation coefficient range.

[0109] Fig.47 The sedimentation coefficient (s x10 -13 ), showing that the empty capsid (2%) has a size of approximately 60x10 -13 s, and the complete capsid has a sedimentation coefficient of about 80-150x10 -13 s sedimentation coefficient range.

[0110] Fig.48 The sedimentation coefficient (s x10 -13 ), showing that the empty capsid (4%) has a size of approximately 60 x10 -13 s, and the complete capsid has a sedimentation coefficient of about 80-150x10 -13 s sedimentation coefficient range. DETAILED DESCRIPTION

[0111] The present disclosure provides a cell-based quantitative in vitro potency assay that uses cells that allow AAV9 vector transduction to evaluate potency, intended for batch processing of AAV9 drug products expressing target proteins. In another embodiment, the viral vector drug product is an AAV9 vector expressing the SMN1 protein. The assay utilizes terminally differentiated non-dividing cells derived from neural progenitor cells in an SMN1- / - genetic background (terminally differentiated cells derived from NPCs, hereinafter referred to as mTD-NPC-Δ7), which have the ability to be effectively transduced by non-replicating AAV9 vectors. Using mTD-NPC-Δ7 as an in vitro cell model system, a cell-based 5-day quantitative assay was developed to measure the dose-dependent increase in SMN1 protein levels after transduction of SMN1-encoding AAV9 vectors at increasing multiplicity of infection (MOI) using a commercially available monoclonal antibody specific for SMN protein by a high-content imaging system. In some embodiments, a cell-based in vitro assay can measure the potency of a vector sample relative to a reference standard.

[0112] Cell-based assays

[0113] The present disclosure provides a cell-based in vitro assay for measuring the efficacy of an AAV vector encoding a protein of interest. In some embodiments, the protein of interest is SMN1, and SMN1 expression from the vector in terminally differentiated non-dividing cells lacking SMN1 is measured using the methods described herein.

[0114] In one embodiment, the disclosure provides methods for measuring transgene expression, the methods comprising the steps of: (a) culturing a plurality of cells, wherein the cells comprise a viral vector, wherein the viral vector comprises a transgene, wherein the culturing is under conditions sufficient to express a protein of interest from the transgene; (b) incubating the plurality of cells to allow transgenic expression of the protein of interest to ensu; (c) contacting the plurality of cells with a molecule specific for the protein of interest; (d) imaging the cells to obtain an integrated fluorescence intensity / cell (IFI-C) assay readout; and, (e) assaying expression of the transgene based on the IFI-C readout.

[0115] In another embodiment, a method for measuring or quantifying viral infection titers in a plurality of cells is provided, the method comprising the steps of: (a) culturing a plurality of cells, wherein the cells comprise a viral vector, wherein the viral vector comprises a transgene, wherein the culturing is under conditions sufficient to express a protein of interest from the transgene; (b) incubating the plurality of cells to allow transgenic expression of the protein of interest to ensue; (c) contacting the plurality of cells with a molecule specific for the protein of interest; (d) imaging the cells to obtain an integrated fluorescence intensity / cell (IFI-C) assay readout; and, (e) assaying expression of the transgene based on the IFI-C readout.

[0116] In another embodiment, the methods of measuring or quantifying viral infection titers in a plurality of cells further comprise optimizing the multiplicity of infection (MOI) of the plurality of cells. In another embodiment, an infectivity assay for measuring or quantifying viral infection titers in a plurality of cells is provided.

[0117] In another embodiment, the plurality of cells are transduced with the viral vector prior to step (a). In another embodiment, the incubation step (b) is followed by fixation and permeabilization of the plurality of cells.

[0118] In another embodiment, the step of determining the relative potency of the viral vector is performed by performing parallel line analysis (PLA) relative to the standard curve of the reference standard after linear regression data fitting. Parallel line determination is a method for calculating relative potency. In some embodiments, relative potency is calculated for dilution determination.

[0119] In some embodiments, the methods include (a) providing a first plurality of terminally differentiated neural progenitor cells (NPCs); (b) transducing the first plurality of terminally differentiated NPCs with a test sample comprising a viral vector comprising a sequence encoding a protein of interest; (c) incubating the transduced first plurality of terminally differentiated NPCs under conditions sufficient to express the protein of interest; (d) contacting the first plurality of terminally differentiated NPCs from (c) with a molecule specific for the protein of interest; (e) imaging the first plurality of terminally differentiated NPCs to obtain an integrated fluorescence intensity / cell (IFI-C) assay readout; and (f) assaying expression of the protein of interest based on the IFI-C readout.

[0120] As used herein, a "test sample" refers to a sample comprising an AAV viral vector comprising a sequence encoding a protein of interest whose titer and / or potency is unknown and which is to be determined using the methods described herein.

[0121] In some embodiments, the methods include (g) providing a second plurality of terminally differentiated NPCs; (h) transducing the second plurality of terminally differentiated NPCs with a reference standard comprising the viral vector; (i) incubating the transduced second plurality of terminally differentiated NPCs under conditions sufficient to express the protein of interest; (j) contacting the second plurality of terminally differentiated NPCs from (i) with a molecule specific for the protein of interest; (k) imaging the second plurality of terminally differentiated NPCs to obtain an integrated fluorescence intensity / cell (IFI-C) assay readout; and (l) comparing the IFI-C of the first plurality of terminally differentiated NPCs with the IFI-C of the second plurality of terminally differentiated NPCs; thereby determining the relative potency of the viral vector of the test sample relative to the reference standard.

[0122] In some embodiments, the methods include providing a third plurality of terminally differentiated NPCs, transducing the third plurality of terminally differentiated NPCs with an assay control comprising the viral vector, incubating the transduced third plurality of terminally differentiated NPCs under conditions sufficient to express the protein of interest; contacting the third plurality of terminally differentiated NPCs with a molecule specific for the protein of interest; imaging the third plurality of terminally differentiated NPCs to obtain an integrated fluorescence intensity / cell (IFI-C) assay readout; and comparing the IFI-C of the third plurality of terminally differentiated NPCs to the IFI-C of the first plurality of terminally differentiated NPCs and / or the second plurality of terminally differentiated NPCs; thereby determining the effectiveness of the in vitro potency assay. In some embodiments, the assay control is a positive control.

[0123] In some embodiments, multiple terminally differentiated NPCs are cultured in parallel, transduced with test samples, reference samples, and optional assay controls, incubated, stained, and imaged. For example, multiple terminally differentiated NPCs can be cultured in the same 96-well plate, transduced with test samples, reference samples, and optional assay controls, and subjected to further downstream processing.

[0124] In some embodiments, the first plurality of cells and the second plurality of cells are transduced with viral vectors from the test sample and the reference standard at at least two different multiplicities of infection (MOI). In some embodiments, the at least two different MOIs include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 MOIs. In some embodiments, the at least two different MOIs include 5 different MOIs. In some embodiments, the 5 MOIs include 300,000, 150,000, 75,000, 37,500, 18,750 viral particles / cell.

[0125] In some embodiments, the methods further comprise transducing a third plurality of

[0126] In another embodiment, the viral vector or a pharmaceutical composition comprising the viral vector retains a potency of between ±20%, between ±15%, between ±10%, preferably ±5% of a reference standard. In one embodiment, the potency is assessed relative to a reference standard using the methods disclosed herein. Any suitable reference standard may be used.

[0127] As used herein, "reference standard" refers to a composition comprising an AAV vector whose concentration and / or potency is known, the AAV vector encoding a protein of interest. Exemplary reference standards include AAV-SMN1 vectors stored at less than or equal to -60°C until use, thawed once, and stored at 2°C-8°C for less than one week.

[0128] 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% relative to a reference standard. In some embodiments, the relative potency of the viral vector is at least 90% relative to a reference standard.

[0129] The methods of the present invention measure the potency of proteins expressed by transgenes present in viral vectors disclosed herein, wherein the transgenes are intended for delivery to the brain. Brain regions envisioned for delivery include, but are not limited to, the motor cortex and brainstem. In some embodiments, the transgenes are delivered to the spinal cord. In some embodiments, the transgenes are delivered to lower motor neurons. Embodiments of the present invention employ rAAV9 to deliver transgenes to nerves and glial cells. In some embodiments, the glial cells are microglia, oligodendrocytes, or astrocytes. In some embodiments, rAAV9 is used to deliver transgenes to Schwann cells.

[0130] Uses of the viral vectors disclosed herein are indicated, for example, for the treatment of lower motor neuron diseases, such as SMA and ALS, as well as Pompe disease, lysosomal storage diseases, glioblastoma multiforme, and Parkinson's disease. Lysosomal storage diseases include, but are not limited to, activator deficiency / GM2 gangliosidosis, alpha-mannosidosis, aspartylglucosaminuria, cholesterol ester storage disease, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, galactosialidosis, Gaucher Disease (Type I, Type II, Type III), GM1 gangliosidosis (infantile, late infantile / juvenile, adult / chronic), I-cell disease / mucolipidosis type II, infantile free sialic acid storage disease / IS SD, juvenile hexosaminidase A deficiency, Krabbe disease (infantile onset, late onset), metachromatic leukodystrophy, mucopolysaccharidosis (Pseudo-Hurler polydystrophy, polydystrophy / mucolipidosis type IIIA, MPS I Harlan syndrome, MPS I Scheie syndrome, MPS I Harlan-Schaie syndrome, MPS II Hunter syndrome, Sanfilippo syndrome type A / MPS III A, Sanfilippo syndrome type B / MPS III B, Sanfilippo syndrome type C / MPS III C, Sanfilippo syndrome type D / MPS III D, Morquio type A / MPS WA, Morquio type B / MPS IVB, MPS IX hyaluronidase deficiency, MPS VI Maroteaux-Lamy, MPS VII Sly syndrome, Mucolipidosis type 1 / sialidosis, Mucolipidosis type IIIC, Mucolipidosis type IV), multiple sulfatase deficiency, Niemann-Pick diseaseDisease) (type A, B, C), neuronal ceroid lipofuscinosis (CLN6 disease (atypical late infantile, late onset, early juvenile), Batten-Spielmeyer-Vogt / juvenile NCL / CLN3 disease, Finnish variant late infantile CLN5, Jansky-Bielschowsky / late infantile CLN2 / TPP1 disease, Kufs / adult-onset NCL / CLN4 disease, Northern epilepsy / variant late infantile CLN8, Santavuori Haltia / infantile CLN1 / PPT disease, beta-mannosidosis, Pompe disease / glycogen storage disease type II, pycnodystrophy, Sandhoff disease ( Disease / Adult-Onset / GM2 Gangliosidosis, Sandhoff Disease / GM2 Gangliosidosis-Infantile, Sandhoff Disease / GM2 Gangliosidosis-Juvenile, Schindler Disease, Salla Disease / Sialidosis, Tay-Sachs Disease / GM2 Gangliosidosis, Wolman Disease.

[0131] Use of the viral vectors disclosed herein, for example, for the treatment of SMA is indicated.

[0132] In further embodiments, use of these methods and materials for treating neurological diseases such as Rett syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease, or for treating nervous system injuries, including spinal cord and brain trauma / injury, stroke, and brain cancer is indicated.

[0133] "Treatment" includes the step of administering an effective dose or effective multiple doses of a composition comprising the rAAV of the present invention intravenously or via the intrathecal route to an animal (including a human) in need thereof. If the dose is administered before the development of the disorder / disease, the administration is preventive. If the dose is administered after the development of the disease, the administration is therapeutic. In an embodiment of the present invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, slows or prevents progression to a disorder / disease state, slows or prevents progression of a disorder / disease state, reduces the extent of the disease, causes disease (partial or complete) relief, and / or prolongs survival. Examples of disease states envisioned to be treated by the methods of the present invention are listed above.

[0134] Terminally differentiated neural progenitor cells (NPCs)

[0135] Provided herein are protocols for generating terminally differentiated NPCs and using terminally differentiated NPCs in cell-based in vitro potency assays to determine the relative potency of AAV vectors encoding a protein of interest. In some embodiments, the potency of the AAV vector is determined relative to a reference standard. The protocols provided herein can be used to determine the potency of AAV-SMN1 vectors in pharmaceutical compositions (e.g., drug substances and drug product compositions), and to assess vector stability.

[0136] Materials for carrying out the protocol will be known to those of ordinary skill in the art. Exemplary materials include tissue culture treated flasks (T75, T150 and T175), polypropylene centrifuge tubes 15 and 50 mL with caps, pipettes (single channel P1000, P200 and P20 and 8 or 12 channel P1000 and P300), Corning BioCoat poly-D-lysine 96-well plates (Corning 354640), optically clear plate seals (Fisher brand 8408240), reagent storage containers, PIPET-AID, Cellometer slides (Nexcelom, CHT4-SD100-002), 96-well DeepWell TM Polypropylene microplates, low-binding 1.5 mL microcentrifuge tubes, 70% (v / v) isopropyl alcohol (IPA), and dry ice. However, equivalent materials and reagents may be used.

[0137] As provided in the examples herein, NPCs are collected from the cortex of embryos of SMAΔ7 (SMN1- / -) mouse strains at embryonic stage about 14.5 (e14.5). These cells can then be dissociated into single cells. During culture, the cells form neurospheres of 3-dimensional colonies of undifferentiated cells. After about 3-5 days, the neurospheres can then be passaged by dissociating into single cells and forming secondary balls. In order to terminally differentiate NPCs, the neurospheres can then be dissociated and inoculated in 0.5 mL (24-well plates, Falcon) at 1E+06 cells / well, or inoculated in 100 μL (96-well plates, Corning) serum-rich medium without growth factors at 2E+05 cells / well. About 24 hours after differentiation, the cells become terminally differentiated into the glial lineage.

[0138] As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. It is further known in the art that spontaneous or induced changes in karyotype may occur during storage or transfer of such clonal populations. Thus, cells derived from a reference to a cell line may not be identical to the ancestral cell or culture, and reference to a cell line includes such variants.

[0139] In one embodiment, the terminally differentiated non-dividing cells disclosed herein for use in the disclosed cell-based assays are derived from neural progenitor cells in an SMN1- / - genetic background (terminally differentiated cells derived from NPCs, referred to herein as "mTD-NPC-Δ7"). These cells have the ability to be efficiently transduced by non-replicating AAV9 vectors.

[0140] In some embodiments, NPC-Δ7 cells isolated from mouse blastoderm and cultured as described herein are frozen (eg, at less than or equal to -60°C) and then used in cell-based in vitro potency assays.

[0141] In some embodiments, NPC-Δ7 cells are thawed using the following protocol. Before use, the complete growth medium is preheated in a 37°C water bath or equivalent for at least 30 minutes. Frozen cryovials of mNPC cells are removed from a liquid nitrogen reservoir. The vial is kept on dry ice until it is ready to thaw, then thawed quickly in a 37°C water bath, vortexing occasionally to ensure thawing. The surface of the vial is wiped with 70% (v / v) isopropanol (IPA), and the contents are then transferred to a 50mL centrifuge tube using a sterile pipette in a BSC. After thawing the cells, the cryoprotectant is slowly diluted to prevent osmotic shock. About 10-20mL is usually sufficient to overcome toxic effects. 10-20mL of warm complete growth medium is added dropwise, while gently mixing by vortexing, followed by centrifugation at 300xg for 5 minutes at 20°C. The supernatant is aspirated, and the tube is then gently agitated to break up the cell pellet. Add an appropriate volume (eg, 1.0-2.0 mL) of warm complete growth medium to the cells and mix.

[0142] In some embodiments, viable cell count and viability are obtained. In some embodiments, the viable cell count is > 60.0% viable in order to proceed.

[0143] In some embodiments, cells are transferred to a tissue culture flask, 10.0 mL of complete growth medium is added, and the flask is gently shaken to ensure even distribution. The flask is then incubated at 37°C and 5% CO. 2 The cells were incubated for at least 72 hours at 4 °C and then tested for growth and viability.

[0144] NPC cell culture

[0145] In some embodiments, the NPC cells are homozygous for a mutation in SMN1 (SMN1- / -). In some embodiments, the mutation in SMN1- / - is a null mutation. In some embodiments, SMN1- / - is a deletion of exon 7 (Δ7), and the cells are referred to as NPC-Δ7 cells. In some embodiments, the NPCs are isolated or derived from a mouse blastoderm.

[0146] In some embodiments, NPC-Δ7 cells are cultured through one or more rounds of passaging prior to use in a cell-based in vitro potency assay described herein.

[0147] In some embodiments, NPC-Δ7 cells are used in the assay starting from the second passage after thawing.

[0148] In some embodiments, NPC-Δ7 cells are used in the cell-based in vitro potency assays described herein at passages 8-15. Thawing is not considered a passage. In some embodiments, cells are used to passage 15. For example, if the working cell bank is frozen at passage 6 (P6) or later, when the cells are thawed, the passage number they retain is P6. After the appropriate number of days of cell proliferation, the cells are passaged by dissociation with Accumax and become P7. At P7, cells cannot be used for assays.

[0149] In some embodiments, NPC-Δ7 cells are passaged (P8) by dissociation with Accumax and can then be used for in vitro potency assays.

[0150] As the cells proliferate in suspension, they form 3-dimensional colonies called neurospheres. In some embodiments, cells are passaged every 4±1 days, for example to prevent the neurospheres from growing too large and becoming necrotic in the center.

[0151] Exemplary cell passage scheme is described as follows.Before use, basal medium and complete growth medium are preheated 30 minutes in 37 ℃ of water baths or equivalent.Exemplary basal medium comprises DMEM / F12, GlutaMAX supplement, 2%B27 supplement (50x) and 1% antibiotic-antimycotic.Exemplary complete growth medium comprises basal medium, 0.1% heparin (5mg / mL), 0.02%bFGF (basic fibroblast growth factor) recombinant human protein (100g / mL) and 0.005%EGF (epidermal growth factor) recombinant human protein solution (1mg / mL).

[0152] In some embodiments, to passage the cells, the flask containing the cells is removed from the incubator and the surface of the flask is rinsed with the medium containing the cells. The cells are transferred from the flask to a 50 mL conical tube and centrifuged at 300 x g for 5 minutes. The supernatant is aspirated without disturbing the cell pellet and 200.0 μL Accumax is added. The cell pellet is gently ground and then incubated at room temperature for 30 ± 10 minutes.

[0153] At the end of the Accumax incubation, Accumax was neutralized by pre-warmed basal medium. In some embodiments, 400.0 μL of pre-warmed basal medium was added and the cells were gently triturated to completely dissociate into single cells.

[0154] In some embodiments, an additional 400.0 μL of pre-warmed basal medium was added to bring the total volume to 1.0 mL.

[0155] In some embodiments, the cells are diluted to an acceptable cell density range. Exemplary cell density ranges include a range of 5.00E+05 cells / mL to 1.00E+07 for cell counting. However, one of ordinary skill will be able to adjust the cell density range to an appropriate cell counting method.

[0156] In some embodiments, sales from multiple flasks with the same cell reference / batch at the same passage number are pooled prior to cell counting.

[0157] In some embodiments, the cells are mixed and then a sample of the cells is removed to determine viable cell count and viability.

[0158] In some embodiments, mTD NPC-Δ7 (terminally differentiated NPCΔ7) plates are prepared when the viability of each cell count is ≥60.0%, ≥70.0%, ≥80.0% or ≥90.0%. In some embodiments, mTD NPC-Δ7 (terminally differentiated NPCΔ7) plates are prepared when the viability of each cell count is ≥80.0%.

[0159] In some embodiments, the in vitro potency assay is continued only if the viability of each cell count is ≥ 70.0% viable.

[0160] Terminally differentiated NPC

[0161] The present disclosure provides methods of terminally differentiating NPCs to generate terminally differentiated NPCs for use in the cell-based in vitro potency assays described herein. In some embodiments, the terminally differentiated NPCs comprise a homozygous deletion of exon 7 of SMN1 (mTD NPC-Δ7 cells).

[0162] In some embodiments, a plating medium is used to terminally differentiate NPCs. An exemplary plating medium comprises DMEM / F12, GlutaMAX supplement, 2% B27 supplement (50x), 1% antibiotic-antimycotic, and 10% FBS.

[0163] In some embodiments, cells are diluted to 20,000 cells / well / 100 μL (or 2.00×10 5In some embodiments, the cells are diluted to a density of 5,000 cells / well / 100 μL, 10,000 cells / well / 100 μL, 15,000 cells / well / 100 μL, 20,000 cells / well / 100 μL, 25,000 cells / well / 100 μL, or 30,000 cells / well / 100 μL.

[0164] In some embodiments, 100 μL of cells were gently mixed and added to the wells of a 96-well poly-D-lysine coated plate.

[0165] In some embodiments, one or more plates are allowed to rest at ambient temperature for 25 ± 5 minutes, after which the plated cells are placed in a 37°C, 5% CO 2 incubator.

[0166] In some embodiments, the plated cells were incubated at 37°C, 5% CO 2 Place in the incubator for 24 hours ± 2 hours before transduction.

[0167] Transduction

[0168] In one embodiment, the term "transfection" is used interchangeably with the term "transduction" herein, and the term or its grammatical equivalent is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" or "transduced" when the foreign DNA has been introduced inside the cell membrane. A variety of transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier; and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more foreign DNA moieties into suitable host cells.

[0169] Suitable methods for transducing cells are known in the art. In one embodiment, cells are transduced in vitro by combining a viral vector with cells. In one embodiment, cells are transduced in vitro by combining an AAV9 vector with cells. In one embodiment, cells are transduced in vitro by combining an SMN1 encoding AAV9 vector with cells. In one embodiment, AAV9 is combined with cells about 24 hours after differentiation. In another embodiment, it is combined with cells about 12-24 hours after differentiation. In another embodiment, it is combined with cells about 24-32 hours after differentiation.

[0170] In one embodiment, any suitable transfection medium can be used. In one embodiment, the DMEM serum-free growth medium used for cell expansion is replaced with a modified DMEM transfection medium. In one embodiment, the transfection medium is DMEM without FBS, without calcium, without L-glutamine but with 4.5 g / l glucose.

[0171] In a specific embodiment, the step of incubating the cells after transduction is performed for about 69-75 hours. In some embodiments, the step of incubating the cells after transduction is performed for about 24-48 hours, 48-69 hours, or about 75-90 hours.

[0172] In one embodiment, transduction of a patient's cells with the rAAV of the invention results in sustained expression of the polypeptide or RNA encoded by the rAAV.

[0173] In another embodiment, the cell transduced with the viral vector is a terminally differentiated non-dividing cell. In another embodiment, the cell transduced with the viral vector is a terminally differentiated non-dividing primary cell, such as mTD NPC-Δ7 cells.

[0174] In some embodiments, mTD NPC-Δ7 cells are transduced with a test sample and a reference standard comprising an AAV vector containing a transgene encoding an SMN1 protein. In some embodiments, mTD NPC-Δ7 cells are transduced with a test sample, a reference standard, and an assay control (e.g., a positive control, sometimes referred to as a control) comprising an AAV vector containing a transgene encoding an SMN1 protein.

[0175] In some embodiments, AAV9-SMN1 vector reference standard (RS), control (Crtl), and test samples are prepared as follows. Aliquots of reference standard (RS), control, and test samples are thawed at ambient temperature. Before use, the formulation buffer and plate culture medium are preheated in a 37°C water bath or equivalent for at least 30 minutes. Samples are pre-diluted to 1.00E+12 vg / mL in a 1.5mL microcentrifuge tube using appropriate preheated formulation buffer (the protocol can be modified for other concentrations).

[0176] In some embodiments, serial dilutions are performed to prepare different MOIs to generate a plot of MOI versus IFI-C. For example, a starting concentration of 300K MOI can be serially diluted to generate RS, test, and assay / positive control samples at 150K MOI, 75K MOI, 37.5K MOI, and 18.75K MOI.

[0177] In some embodiments, samples prepared at the prepared MOI are gently mixed and immediately dispensed at an angle to the wall of the corresponding well of a 96-well plate. The plate is transferred to an incubator (37°C ± 1°C, 5 ± 1% CO2). The plate is incubated for 72 ± 2 hours.

[0178] Cell staining

[0179] Provided herein are methods for staining cells for a protein of interest, e.g., an SMN protein, such as SMN1 or SMN2, e.g., SMN1. These methods for staining cells can be used in the cell-based in vitro potency assays described herein.

[0180] In one embodiment, the molecule specific for the protein of interest comprises an antibody, an antibody fragment, or an aptamer. In another embodiment, the antibody comprises an antibody specific for the protein of interest.

[0181] Antibodies exist as complete immunoglobulins or as many well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide bonds in the hinge region to produce F(ab')2, i.e., Fab dimers, which are themselves light chains connected to VH-CH1 by disulfide bonds. F(ab')2 can be reduced under mild conditions to break the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimers into Fab' monomers. Fab' monomers are essentially Fabs with a portion of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, edited by WE Paul, Raven Press, New York (1999)). Although various antibody fragments are defined according to the digestion of intact antibodies, it will be appreciated by those skilled in the art that such Fab' fragments, etc., can be synthesized de novo by chemical methods or using recombinant DNA methods. Therefore, the term antibody as used herein also includes antibody fragments produced by modifying the entire antibody or synthesized de novo using recombinant DNA methods. Antibodies include single chain antibodies, including single chain Fv (sFv or scFv) antibodies, in which the variable heavy chain and variable light chain are joined together (directly or through a peptide linker) to form a contiguous polypeptide.

[0182] As explained above (see previous definitions), antibodies used herein optionally include F(ab)2, F(ab')2, Fab, Fab', scFv, etc., depending on the specific requirements of the embodiment. Some embodiments utilize alternative immunoglobulins, such as IgM, IgA, IgD, and IgE. In addition, all possible isotypes of various immunoglobulins are also covered in the current embodiments. Therefore, IgG1, IgG2, IgG3, etc. are all possible molecules for use in the present invention.

[0183] In one embodiment, the anti-target protein antibody is provided at a concentration of about 4 μg / mL. In another embodiment, the anti-target protein antibody is provided at a concentration of about 1-4 μg / mL, 4-8 μg / mL, 8-12 μg / mL, or 12-16 μg / mL. In one embodiment, the anti-target protein antibody is provided at a concentration of about 2 μg / mL.

[0184] In another embodiment, the molecule includes a detectable label to achieve detection. Labels include, for example, chelated lanthanide metals (such as europium), platinum group metals (such as ruthenium), fluorescent dyes (especially including xanthine derivatives, such as fluorescein and rhodamine or any derivatives of both), fluorescent proteins (such as green fluorescent protein (GFP) and its derivatives yellow fluorescent protein (YFP) and red fluorescent protein (RFP)), radioactive labels (such as iodine 125 and actinium-225) and other similar detectable labels known in the art. In some embodiments, detectable labels that do not require a washing step are used.

[0185] Fluorescent marker groups are usually distinguished from each other based on one or more of their excitation spectra, emission spectra or fluorescence lifetimes. By guiding the excitation light of different wavelengths at the cell, the fluorescence level produced by any one or more different detectable labels can then be determined. Alternatively, detectable labels are selected to have distinguishable fluorescence emission maxima, for example, they emit light or fluoresce with significantly different wavelengths. In operation, a single light source is guided at the cell. Then, the fluorescence emission from the cell is passed through filters that separate different fluorescent emitters, and then these different fluorescent emitters are quantified. When selecting distinguishable excitation or emission maxima, it is usually preferred that the excitation or emission spectrum of a mark such as a reference mark does not overlap significantly with the excitation or emission spectrum of another mark. Specifically, although there is usually a maximum excitation or emission wavelength for different marks, there is typically a wider wavelength range with some excitation or emission. Typically, select a mark so that there is substantially no overlap between the excitation or emission spectra of two marks, for example, when detecting a mark, less than 10% of the fluorescence is due to the overlap with another mark.

[0186] In one embodiment, the measurement of fluorescence intensity provides a measure of the expression of the target protein that has occurred inside the cell. In another embodiment, the integrated fluorescence intensity / cell (IFI-C) value is calculated for each dose of the viral vector provided in the cell-based assay disclosed herein and repeated. In another embodiment, the IFI-C readout reflects a measure of the expression of the target protein.

[0187] In one embodiment, the method provided herein further comprises washing the cells to remove molecules specific to the target protein. In another embodiment, after washing, the method further comprises contacting the cells with a second molecule that specifically recognizes a molecule specific to the target protein. In another embodiment, the second molecule comprises a detectable label. In another embodiment, the second molecule comprises an antibody, an antibody fragment, or an aptamer.

[0188] In another embodiment of the methods provided herein, after the fixation and permeabilization steps, the cells are contacted with an anti-nuclear detectable marker. Detectable markers for detecting nuclei may include, but are not limited to, DAPI, propidium iodide (PI), Hoechst, 470、RedDot TM 2 Far-red nuclear stain or dye NucFix TM In another embodiment, use Live 488 and Live 650 Nuclear Stain. Live Nuclear Stain specifically stains nuclei in live or fixed cells without the need for washing.

[0189] In one embodiment, after staining with at least one detectable label disclosed herein, the method further comprises the step of collecting one or more cell images, wherein the image shows the expression of the protein of interest in the cell as determined by detecting a detectable signal provided by at least one detectable label upon excitation with light. In addition, the step of obtaining the image is followed by performing image analysis.

[0190] In one embodiment, after separation and / or before performing a cell-based assay disclosed herein, the viability of the cells disclosed herein is assessed. Methods for assessing cell viability are well known in the art and include, for example, trypan blue staining (or an equivalent cell viability reagent according to the cell counter used), microscopic observation, etc.

[0191] In one embodiment, a plurality of cells are seeded on a solid surface. In another embodiment, the solid surface comprises any type of plate known in the art, including 24 or 96-well plates. In another embodiment, the plate used in the assay is suitable for imaging the cells. In another embodiment, the solid surface is coated with poly-D-lysine.

[0192] In another embodiment, the cells are seeded on a solid surface at a density of 20,000 cells. In another embodiment, the cells are seeded on a solid surface at a density of 10,000 cells. In another embodiment, the cells are seeded on a solid surface at a density of 10,000-20,000 cells. In another embodiment, the cells are seeded at a density of 20,000 cells / well. In another embodiment, the cells are seeded at a density of 10,000 cells / well. In another embodiment, the cells are seeded at a density of 10,000-20,000 cells / well.

[0193] In one embodiment, the transgene comprises a polynucleotide encoding a survival motor neuron (SMN1) protein. In another embodiment, the protein of interest expressed by cells transduced with a viral vector disclosed herein is a survival motor neuron (SMN1) protein.

[0194] In some embodiments, the transduction plate containing the terminally differentiated and transduced cells described herein is removed from the incubator at 72 hours ± 2 hours for cell staining.

[0195] In some embodiments, cells are fixed according to the protocol described below. 50.0 μL of 4% paraformaldehyde or an appropriate volume and concentration is gently added to the wells. The plate is then incubated at ambient room temperature, for example, for 5 to 7 minutes. After incubation, the 4% paraformaldehyde is aspirated from each well and the wells are washed with 250.0 μL DPBS (Dublin's phosphate buffered saline).

[0196] In some embodiments, the plates can be stored at 2°C-4°C. In some embodiments, the plates can be stored for up to 3 days. When storing the plates, remove the DPBS and add 250.0 μL of fresh DPBS before storing the plates.

[0197] In some embodiments, the cells are permeabilized with Triton X-100. In some embodiments, DPBS is gently aspirated from each well and 50.0 μL of 0.1% Triton X-100 is gently added. The plate can then be incubated at ambient room temperature, for example, for 5 to 7 minutes, 0.1% Triton X-100 is aspirated from each well, and the wells are washed with DPBS.

[0198] Primary antibody incubation

[0199] Exemplary primary antibodies include mouse monoclonal anti-SMN antibody (clone 2B1), Santa Cruz sc-32313XS lot number C2818 at a 1:500 dilution, Santa Cruz sc-32313XS lot number F2118 at a 1:1000 dilution, and EMD Millipore lot number 3054700 at a 1:500 dilution. For example, to prepare 4.0 mL of staining solution at a 1:500 dilution of antibody, 8.0 μL of anti-SMN antibody was added to 4.0 mL of 1% BSA in DPBS.

[0200] In some embodiments, incubation with the primary antibody is at ambient room temperature for 120 to 150 minutes.

[0201] In some embodiments, after primary antibody incubation, cells are washed with DPBS.

[0202] Secondary antibody incubation

[0203] Exemplary secondary antibodies include final 2 μg / mL goat anti-mouse IgG (H+L) AlexaFluor Plus 488 (at 1:1000 dilution) and 2 μg / mL nuclear dye Hoechst 33342 (1:5000 dilution) in 1% BSA in DPBS. Hoechst 33342 nuclear dye can be pre-diluted by adding 10.0 μL Hoechst 33342 nuclear dye to 40.0 μL distilled water.

[0204] In some embodiments, cells are incubated in the secondary antibody for 60 to 80 minutes at ambient room temperature, protected from light (eg, covered with foil).

[0205] Aspirate the secondary antibody solution from each well and wash the cells with DPBS.

[0206] In some embodiments, the plate is then sealed with a clear optical plate seal and imaged.

[0207] Calculate relative potency

[0208] The present disclosure provides methods for determining the effectiveness of a vector in a test sample. In some embodiments, the effectiveness of a vector in a test sample is determined relative to a reference standard (RS). In some embodiments, the reference standard comprises the same vector as the test sample, but the characteristics of the reference standard (vector concentration, effectiveness, etc.) are known.

[0209] In some embodiments, the relative potency calculation of the vector in the test sample compared to the vector in the reference standard is performed using parallel lines analysis (PLA). PLA is a method used to compare dose response curves, for example, IFI-C as a result of MOI or log MOI.

[0210] In some embodiments, PLA includes fitting a separate linear model to the Log 2 MOI is relative to IFI-C. In some embodiments, the IFI-C of an assay control (eg, a positive control) is also measured simultaneously, and the MOI or Log relative to IFI-C is also calculated for the assay control. 2 MOI. In some embodiments, the IFI-C for each of the test sample, reference standard, and optional assay control is averaged from multiple assay readouts (eg, 2, 3, 4, 5, or more replicates) at each MOI.

[0211] In some embodiments, the R-squared value (R) of the linear regression is calculated using the least squares method for the test samples, reference standards, and assay controls. 2 ), intercept, and slope estimates.

[0212] In some embodiments, the ratio of the slope estimate of the test sample relative to the reference standard is used. The parallelism between the test sample and the reference standard is assessed by quantifying the slope of the sample against the reference standard. The sample slope is parallel to the slope of the reference standard if the slope ratio is within the empirical range established between the assay control sample and the reference standard, where:

[0213] y=α+βlog 2 (MOI)+e(M2.1)

[0214] where y is the integrated fluorescence intensity / cell from a given MOI level; α and β are the intercept and slope of the linear regression line, respectively, and e is the residual.

[0215] In some embodiments, a common slope model is used in PLA. In some embodiments, for each test sample and reference standard and optional assay control, a linear regression model (M2.2) with a separate intercept and a common slope is fitted to: The assay readout of the integrated fluorescence intensity on the plate / cell (IFI-C) relative log 2 The expected MOI value of the transformation, where:

[0216] y=α+βlog 2 (MOI)+e(M2.1)

[0217] And where y i is the integrated fluorescence intensity / cell at a given MOI level from sample i (i∈{sample, reference standard}); α i is the individual intercept for sample i (i∈{sample, reference standard}); β is the common slope and e is the residual.

[0218] In some embodiments, the IFI-C for each of the test and reference samples and optional assay controls is averaged from multiple assay readouts (eg, 2, 3, or more replicates) at each MOI.

[0219] In some embodiments, the relative potency is calculated as follows: The relative potency of the test sample is calculated as follows based on the intercept or slope estimate from the model (M2.2):

[0220]

[0221] In some embodiments, if the coefficient of variation percentage (CV%), linear regression fit R 2 The assay plate is considered valid if the assay value, the assay kinetic window of the reference standard (RS), the slope ratio of the assay control (e.g., positive control) relative to the RS, the relative potency of the assay control, and the slope ratio of the test sample relative to the RS meet certain criteria.

[0222] In some embodiments, the CV% of the IFI-C of the test sample is less than or equal to 40%, less than or equal to 30%, less than or equal to 20%, less than or equal to 10%, or less than or equal to 10%. In some embodiments, the CV% of the IFI-C of the test sample is less than or equal to 20%.

[0223] In some embodiments, the R 2 ≥0.99, ≥0.95, ≥0.90, ≥0.89, or ≥0.85.

[0224] In some embodiments, the R 2 ≥0.95.

[0225] In some embodiments, the reference standard (RS) has an assay dynamic window (maximum signal / background signal cells only) of ≥2.0, ≥2.1, ≥2.2, ≥2.3, ≥2.4, ≥2.5, ≥2.6, ≥2.69, ≥2.8, ≥2.9, or ≥3.0. In some embodiments, the reference standard (RS) has an assay dynamic window (maximum signal / background signal cells only) of ≥2.69.

[0226] In some embodiments, the reference standard (RS) has a slope > 1.02E+05.

[0227] In some embodiments, the slope ratio of the assay control (e.g., positive control) relative to the reference standard is within 0.60-1.5. In some embodiments, the slope ratio of the assay control (e.g., positive control) relative to the reference standard is within 0.70-1.4. In some embodiments, the slope ratio of the assay control (e.g., positive control) relative to the reference standard is within 0.75-1.33.

[0228] In some embodiments, for each test sample, the mean is estimated for the natural log-transformed relative potency results and standard deviation and associated 95% confidence limits; and calculated the geometric mean relative potency and percentage coefficient of variation

[0229] In certain embodiments, log(y ij )=μ i +∈ ij (M2.1), where y ij is the relative potency from the recovery sample i; μ i is the mean log-transformed relative potency from recovered sample i, and is the random within-measurement residual.

[0230] In some embodiments, the overall intermediate precision is calculated between the test samples. In some embodiments, log(y ijk )=μ i +θ j +∈ ijk (M2.2) to calculate the intermediate log(y ijk )=μ i +θ j +∈ ijk Precision; where y ijk is the relative potency from recovered sample i; μ i is the average log-transformed relative potency of recovered sample i; is the random between-assay effect from assay run j; and is the random within-measurement residual.

[0231] In some embodiments, overall repeatability is calculated between test samples. In some embodiments, log(y iljk )=μ i +α l θ j +∈ ijk (M2.3) Calculate the overall repeatability; where y ijkis the relative potency from recovered sample i; μ i is the average log-transformed relative potency of recovered sample i; is the random between-laboratory effect from laboratory l; is the random between-assay effect from assay run j nested in laboratory l; and is the random within-measurement residual.

[0232] In some embodiments, the parallelism of the slope ratio is assessed. The parallelism of the in vitro relative potency assay can be determined by comparing the slope ratio of the test sample to the reference standard, i.e., the slope 样品 / Slope 标准品 The slope was estimated from the linear regression model between IFI-c and log2-transformed MOI level.

[0233] In some embodiments, the slope ratios are log-transformed for analysis. In some embodiments, the mean is estimated for the natural log-transformed slope ratios of each test sample and the assay control (e.g., positive control) sample. and standard deviation And then calculate the geometric mean relative potency and percentage coefficient of variation

[0234] The methods for performing the statistical analysis described herein are known to those of ordinary skill in the art. For example, statistical analysis can be performed using statistical software JMP Pro 13.2.1, R, Matlab, etc.

[0235] Vector stability

[0236] Provided herein are methods for assessing the stability of a vector using the cell-based in vitro potency assay described herein.

[0237] In some embodiments, the methods include maintaining a vector stability sample at a specific temperature (e.g., 20° C.-25° C.) for a certain length of time, and then comparing the relative potency of the vector stability sample to a reference standard that has not been maintained at the specific temperature. For example, the vector stability sample can be maintained at 20° C.-25° C. for 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 4 months, and the potency of the vector stability sample can be compared to a reference standard maintained at -60° C. or below for the same length of time using the methods provided herein.

[0238] Carrier

[0239] The present disclosure provides viral vectors as gene therapy biological products, which are intended to be developed for treating pediatric patients diagnosed with diseases. The effectiveness of the vectors provided herein and the pharmaceutical compositions comprising these vectors can be determined using the cell-based in vitro efficacy assays described herein. In one embodiment, the disease is SMA, such as SMA type 1, SMA type 2, SMA type 3, SMA type 4, or a combination thereof. In one embodiment, the disease is SMA type 1, which is a severe neuromuscular disease characterized by the loss of motor neurons due to genetic defects in the SMN1 gene that is important for the survival of motor neurons. In some embodiments, the viral vector is composed of a non-replicating, non-integrating, recombinant self-complementary adeno-associated virus serotype 9 (AAV9), which is contained under the control of a cytomegalovirus (CMV) enhancer / chicken-β-actin hybrid promoter (CB) to express the cDNA of the SMN1 protein and two AAV inverted terminal repeats (ITRs) from AAV serotype 2 (AAV2) DNA.

[0240] In one embodiment, the term "vector" refers to any genetic element that is capable of replication and can transfer gene sequences between cells when associated with appropriate control elements, such as plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, etc. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0241] In one embodiment, the term "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8 and AAV-9, preferably AAV-9. In the AAV vector, one or more of the AAV wild-type genes (preferably rep and / or cap genes) can be deleted in whole or in part, but retains functional flanking ITR sequences. Functional ITR sequences are essential for the rescue, replication and packaging of AAV virions. Therefore, the AAV vector is defined herein as at least including those sequences (e.g., functional ITRs) required for viral replication and packaging in cis. ITR does not have to be a wild-type nucleotide sequence, and can be changed, for example, by insertion, deletion or substitution of nucleotides, as long as the sequence provides functional rescue, replication and packaging. In a preferred embodiment, the vector is an AAV-9 vector with ITRs from AAV-2 sources. "AAV vector" also means a protein shell or capsid that provides an efficient vehicle for delivering vector nucleic acid to the nucleus of a target cell.

[0242] In one embodiment, the term "scAAV" refers to a self-complementary adeno-associated virus (scAAV), which is a viral vector engineered from a naturally occurring adeno-associated virus (AAV) for use in gene therapy. scAAV is called "self-complementary" because the coding region has been designed to form an intramolecular double-stranded DNA template.

[0243] In one embodiment, rAAV genome is provided herein. The rAAV genome comprises one or more AAV ITRs, and the one or more AAV ITRs are flanked by coding polypeptides (including but not limited to SMN polypeptides) or polynucleotides encoding siRNA, shRNA, antisense and / or miRNA for the control sequence of a mutant protein or its gene. The polynucleotides are operably connected to transcription control DNA, particularly promoter DNA and polyadenylation signal sequence DNA, that work in target cells to form a gene cassette. The gene cassette can also include intron sequences to promote the processing of RNA transcripts when expressed in mammalian cells.

[0244] In some embodiments, the rAAV9 genome encodes siRNA, shRNA, antisense and / or miRNA for use in methods of reducing mutant huntingtin protein (htt) expression to treat neurodegenerative disorders such as Huntington's disease.

[0245] In various embodiments, the rAAV9 genome encodes siRNA, shRNA, antisense and / or miRNA for use in the treatment of neurodegenerative disorders such as ALS. Treatment results in decreased expression of molecular markers of the disease such as TNFα, nitric oxide, peroxynitrite and / or nitric oxide synthase (NOS).

[0246] In some embodiments, the vector encodes a short hairpin RNA against a mutant protein of ALS (such as superoxide dismutase) or a neurotrophic factor of ALS or Parkinson's disease (such as GDNF or IGF1).

[0247] In some embodiments, the use of the viral vectors of the invention for treating neurodevelopmental disorders such as Rett syndrome is indicated. For embodiments related to Rett syndrome, the rAAV9 genome may encode, for example, methylcytosine binding protein 2 (MeCP2).

[0248] The rAAV genome disclosed herein may lack AAV rep and cap DNA. The AAV DNA (e.g., ITR) in the rAAV genome may be from any AAV serotype from which a recombinant virus may be derived, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, and AAV-11. The nucleotide sequences of the genomes of AAV serotypes are known in the art. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC 001401 and Srivastava et al. Virol. [Virology], 45:555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the complete genome of AAV-5 is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least a portion of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004).

[0249] In another embodiment, a DNA plasmid comprising the rAAV genome of the present invention is provided. The DNA plasmid is transferred to a cell that can be infected with a helper virus (e.g., adenovirus, adenovirus or herpes virus deleted by EI) of AAV so that the rAAV genome is assembled into infectious virus particles with AAV9 capsid protein. The technology for producing rAAV particles (wherein the AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to cells) is standard in the art. The production of rAAV requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from the rAAV genome (i.e., not in the rAAV genome), and the helper virus function components. The production of pseudotype rAAV is disclosed in, for example, WO01 / 83692, which is incorporated herein by reference in its entirety.

[0250] In various embodiments, the AAV capsid protein can be modified to enhance the delivery of the recombinant vector. Modification of the capsid protein is generally known in the art. See, for example, US 2005 / 0053922 and US 2009 / 0202490, the disclosures of which are incorporated herein by reference in their entirety.

[0251] The general principles of rAAV production are reviewed, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, CUM Topics in Microbial. And Immunol., 158:97-129). Various methods are described in the following literature: Ratschin et al., Mol. Cell. Biol. [Molecular Cell Biology] 4:2072 (1984); Hennonat et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. [Molecular Cell Biology] 5:3251 (1985); McLaughlin et al., J. Virol. [Journal of Virology], 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol. [Molecular Cell Biology], 7:349 (1988). Samulski et al. (1989, J. Virol. [Journal of Virology], 63:3822-3828); U.S. Patent No. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent No. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy [Gene Therapy] 3: 1124-1132; U.S. Pat. No. 5,786,211; U.S. Pat. No. 5,871,982; and U.S. Pat. No. 6,258,595. The above documents are hereby incorporated by reference in their entirety, with particular emphasis on those parts of the documents related to rAAV production. Therefore, the present invention provides packaging cells that produce infectious rAAV. In one embodiment, any suitable packaging cell line can be used, such as HeLa cells, HEK-293 cells and PerC.6 cells (homologous 293 line), preferably HEK293 cells.In other embodiments, the invention provides rAAV9 (ie, infectious encapsidated rAAV9 particles) comprising the rAAV genome of the invention. In one aspect of the invention, the rAAV genome is a self-complementary genome.

[0252] In another embodiment, the method allows for quantitative measurement of dose-dependent increases in the levels of a protein of interest.

[0253] In other embodiments, the invention provides rAAV9 (ie, infectious encapsidated rAAV9 particles) comprising the rAAV genome of the invention. In one aspect of the invention, the rAAV genome is a self-complementary genome.

[0254] In another embodiment, rAAV is provided, such as rAAV9 named "rAAV9-SMN" or "AAV9-SMN1". The rAAV SMN genome has, in order, AAV2 ITRs, a chicken β-actin promoter with a cytomegalovirus enhancer, an SV 40 intron, SMN encoding DNA listed in (GenBank Accession No. NM_000344.2), a polyadenylation signal sequence from bovine growth hormone, and another AAV2 ITR. Exemplary SMN coding sequences include the following:

[0255] 1 CCACAAATGT GGGAGGGCGA TAACCACTCG TAGAAAGCGGT GAGAAGTTAC TACAAGCGGT61 CCTCCCGGCC ACCGTACTGT TCCGCTCCCA GAAGCCCCGG GCGGCGGAAG TCGTCACTCT 121TAAGAAGGGA CGGGGCCCCA CGCTGCGCAC CCGCGGGTTT GCTATGGCGA TGAGCAGCGG 181CGGCAGTGGT GGCGGCGTC CGGAGCAGGA GGATTCCGTG CTGTTCCGGC GCGGCACAGG 241CCAGAGCGAT GATTCTGACA TTTGGGATGA TACAGCACTG ATAAAAGCAT ATGATAAAGC 301TGTGGCTTCA TTTAAGCATG CTCTAAAGAA TGGTGACATT TGTGAAACTT CGGGTAAACC 361AAAAACCACA CCTAAAAGAA AACCTGCTAA GAAGAATAAA AGCCAAAAGA AGAATACTGC 421AGCTTCCTTA CACAGTGGA AAGTTGGGGA CAAATGTTCT GCCATTTGGT CAGAAGACGG 481TTGCATTTAC CCAGCTACCA TTGCTTCAAT TGATTTTAAG AGAGAAACCT GTGTTGTGGT 541TTACACTGGA TATGGAAATA GAGAGGAGCA AAATCTGTCC GATCTACTTT CCCCAATCTG 601TGAAGTAGCT AATAATATAG AACAGAATGC TCAAGAGAAT GAAAATGAAA GCCAAGTTTC 661AACAGATGAA AGTGAGAACT CCAGGTTCCC TGGAAATAAA TCAGATAACA TCAAGCCCAA 721ATCTGCTCCA TGGAACTCTT TTCTCCCTCC ACCACCCCCCC ATGCCAGGGC CAAGACTGGG 781ACCAGGAAAG CCAGGTCTAA AATTCAATGG CCCACCACCG CCACCGCCAC CACCACCACC 841CCACTTACTA TCATGCTGGC TGCCTCCATTTCCTTCTGGA CCACCAATAA TTCCCCCACC 901ACCTCCCATA TGTCCAGATT CTCTTGATGA TGCTGATGCT TTGGGAAGTA TGTTAATTTC 961ATGGTACATG AGTGGCTATC ATACTGGCTA TTATATGGGT TTCAGACAAA ATCAAAAAGA 1021AGGAAGGTGC TCACATTCCT TAAATTAAGG AGAAATGCTG GCATAGAGCA GCACTAAATG 1081ACACCACTAA AGAAACGATC AGACAGATCT GGAATGTGAA GCGTTATAGA AGATAACTGG 1141CCTCATTTC TCAAAATATC AAGTGTGG AAAGAAAAAAA GGAAGTGGAA TGGGTAACTC 1201TTCTTGATTA AAAGTTATGT AATAACCAAA TGCAATGTGA AATATTTTAC TGGACTCTTT 1261TGAAAAACCA TCTGTAAAAG ACTGGGTGGG GGGTGGGAGG CCAGCACGGT GGTGAGGCAG 1321TTGAGAAAAT TTGAATGTGG ATTAGATTTT GAATGATATT GGATAATTAT TGGTAATTTT 1381ATGGCCTGTG AGAAGGGTGT TGTAGTTTAT AAAAGACTGT CTTAATTTGC ATACTTAAGC 1441ATTTAGGAAT GAAGTGTTAG AGTGTCTTAA AATGTTTCAA ATGGTTTAAC AAAATGTATG 1501TGAGGCGTAT GTGGCAAAAT GTTACAGAAT CTAACTGGTG GACATGGCTG TTCATTGTAC 1561TGTTTTTTTC TATCTTCTAT ATGTTTAAA GTATATAATA AAAATATTTA ATTTTTTTTT 1621 A(SEQID NO:2)。

[0256] Conservative nucleotide substitutions of SMN DNA are also contemplated (e.g., a guanine to adenine change at position 625 of GenBank Accession No. NM_000344.2). In some embodiments, the genome of rAAV lacks AAV rep and cap DNA, i.e., no AAV rep or cap DNA is present between the ITRs of the genome. Contemplated SMN polypeptides include, but are not limited to, the human SMN1 polypeptide and its isoforms listed in NCBI Protein Database No. NP_000335.1. Contemplated SMN polypeptides also include, but are not limited to, the human SMN2 polypeptide and any isoform of the SMN polypeptide. It is also contemplated that sequences encoding other polypeptides such as the SMN1-modifier polypeptide plectin-3 (PLS3) [Oprea et al., Science 320(5875):524-527 (2008)] may replace SMN DNA. rAAV9 SMN vectors are described in Foust et al., Nature Biotechnology 28(3):271-274 (2010).

[0257] In one embodiment, the viral vector is an adeno-associated virus serotype 9 (AAV9) comprising a cDNA expressing the SMN1 protein under the control of a cytomegalovirus (CMV) enhancer / chicken-β-actin hybrid promoter (CB), and an AAV inverted terminal repeat (ITR) from an AAV serotype 2 (AAV2) DNA. In another embodiment, the AAV is a replication-deficient AAV9 with AAV2-derived ITRs, preferably scAAV9. In one embodiment, the AAV vector carries an SMN transgene. In a preferred embodiment, the SMN encoding DNA is listed in GenBank Accession No. NM_000344.2. Conservative nucleotide substitutions of the SMN DNA are also envisioned (e.g., a guanine to adenine change at position 625 as shown in GenBank Accession No. NM_000344.2).

[0258] In some embodiments, the term "vector-related impurities" refers to all types of AAV particles other than true recombinant AAV particles. Vector-related impurities include empty AAV capsids (also referred to as "empty bodies" or "empty particles"), and AAV particles containing polynucleotide sequences other than the expected vector genome (also referred to as "AAV encapsidated nucleic acid impurities" or "AAV encapsidated DNA impurities").

[0259] In some embodiments, "recombinant virus" means a virus that has been genetically altered, for example, by adding or inserting a heterologous nucleic acid construct into a particle. "Recombinant" may be abbreviated as "r," for example, rAAV may refer to recombinant AAV. The term "AAV" as used herein is intended to encompass "recombinant AAV" or "rAAV."

[0260] In some embodiments, "AAV virion" means a complete virus particle, such as a wild-type (wt) AAV virus particle (comprising a linear single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat). In this regard, a single-stranded AAV nucleic acid molecule with complementary sense (e.g., "sense" or "antisense" strand) can be packaged into any one AAV virion, and both strands have equal infectivity.

[0261] In some embodiments, the terms "recombinant AAV virions", "rAAV virions", "AAV vector particles", "complete capsids" and "complete particles" are defined herein as infectious replication-defective viruses comprising an AAV protein coat that encapsidates a heterologous nucleotide sequence of interest flanked on both sides by AAV ITRs. rAAV virions are produced in suitable host cells into which sequences specifying the AAV vector, AAV helper functions and auxiliary functions have been introduced. In this way, the host cells are enabled to encode AAV polypeptides that provide for packaging the AAV vector (containing the recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.

[0262] In some embodiments, the terms "empty capsid" and "empty particle" refer to an AAV virion that includes an AAV protein coat but lacks in whole or in part a polynucleotide construct comprising a heterologous nucleotide sequence of interest flanked on both sides by AAV ITRs.

[0263] The term "host cell" means, for example, microorganisms, yeast cells, insect cells and mammalian cells, which can or have been used as receptors for AAV auxiliary constructs, AAV vector plasmids, auxiliary function vectors or other transfer DNA. The term includes the progeny of the original cells that have been transfected. Therefore, as used herein, "host cell" generally refers to a cell that has been transfected with an exogenous DNA sequence. It should be understood that the progeny of a single parent cell is not necessarily identical to the original parent in morphology or genome or total DNA complement due to natural, accidental or deliberate mutations.

[0264] In another embodiment, the term "AAV helper function" refers to AAV-derived coding sequences that can be expressed to provide AAV gene products that in turn act in trans for productive AAV replication. Thus: AAV helper functions include both the major AAV open reading frames (ORFs) rep and cap. The Rep expression product has been shown to have many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The Cap expression product provides the necessary packaging function. AAV helper functions are used herein to complement missing AAV functions in AAV vectors in trans.

[0265] In one embodiment, the term "AAV auxiliary construct" generally refers to a nucleic acid molecule that includes a nucleotide sequence that provides an AAV function that is missing in an AAV vector for producing a transduction vector for delivering a nucleotide sequence of interest. AAV auxiliary constructs are generally used to provide transient expression of AAV rep and / or cap genes to supplement the missing AAV functions necessary for AAV replication; however, the auxiliary construct lacks AAV ITRs and can neither replicate nor package itself. AAV auxiliary constructs can be in the form of plasmids, phages, transposons, cosmids, viruses, or virions. Many AAV auxiliary constructs have been described, such as the commonly used plasmids pAAV / Ad and plM29+45 that encode both Rep and Cap expression products. See, for example, Samulski et al. (1989) J. Virol. [Journal of Virology] 63: 3822-3828; and McCarty et al. (1991) J. Virol. [Journal of Virology] 65: 2936-2945. Many other vectors encoding Rep and / or Cap expression products have been described. See, e.g., U.S. Patent Nos. 5,139,941 and 6,376,237.

[0266] In another embodiment, the term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when the foreign DNA has been introduced inside the cell membrane. A variety of transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456; Sambrook et al. (1989) Molecular Cloning, alaboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier; and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more foreign DNA moieties into a suitable host cell.

[0267] As used herein, the term "cell line" refers to a cell population capable of continuous or prolonged growth and division in vitro. It is further known in the art that spontaneous or inductive changes may occur in the karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell lines mentioned may not be identical to the ancestral cells or cultures, and the cell lines mentioned include such variants. In some embodiments, the terms "HEK293 cells", "293 cells" or their grammatical equivalents are used interchangeably herein and refer to the host / packaging cell line used in the methods disclosed herein.

[0268] In some embodiments, the term "eluent" can be understood in context to refer to a buffer for eluting a substance. In some embodiments, the term "eluent" can be understood in context to refer to an eluted substance, e.g., a desired product or substance from a previous purification step (e.g., for assay or further purification).

[0269] In some embodiments, the methods described herein are performed using good manufacturing practices (GMP) and at an industrial scale. GMP is a regulatory specification, such as those enforced by the Federal Drug Administration (FDA) to ensure the quality of drugs. GMP regulations establish controls on manufacturing processes. Examples of current GMP regulations are issued by the FDA. In some embodiments, the methods described herein use GMP procedures to produce AAV viral vectors on an industrial scale. To date, industrial-scale production of AAV viral vectors for gene therapy has been challenging due to scalability issues. Therefore, in some embodiments, the methods described herein provide advantages by, for example, producing AAV viral vectors in adherent cells at an industrial scale and at a purity level sufficient for administration to humans. The term "industrial scale" refers to a method for producing viral vectors in cells at a scale greater than a laboratory scale (e.g., a commercial scale), such as a yield of more than 5x10 in each manufacturing batch. 15 vg, or more than 8x10 15 vg or more than 1x10 16 In the case of vg.

[0270] Imaging

[0271] As part of the cell-based assays disclosed herein, image analysis can be performed using a high-content imaging platform (CellInsight CX5). This platform enables quantitative measurement of intracellular protein expression on a per-cell basis (integrated fluorescence intensity / cell). In addition, CellInsight CX5 allows for appropriate throughput for batch processing and stability studies.

[0272] In high-content screening, cells are first incubated with the substance, and after a period of time, the structural and molecular components of the cells are analyzed. The most common analysis involves labeling proteins with fluorescent tags, and finally using automated image analysis to measure changes in cell phenotype. By using fluorescent tags with absorption and emission maxima, several different cellular components can be measured in parallel. In addition, imaging enables the detection of changes at the subcellular level (e.g., cytoplasm versus nucleus versus other organelles). Therefore, a large number of data points can be collected for each cell (see Proll G, Steinle L, F, Kumpf M, Moehrle B, Mehlmann M, Gauglitz G (August 2007). "Potential of label-free detection in high-content-screening applications". J Chromatogr A. 1161(1-2):2-8). High-content screening automates the extraction of multicolor fluorescence information from specific fluorescence-based reagents incorporated into cells (Giuliano and Taylor (1995), Curr. Op. Cell Biol. 7:4; Giuliano et al. (1995) Ann. Rev. Biophys. Biomol. Struct. 24:405). Cells are analyzed using optical systems that can measure spatial dynamics as well as temporal dynamics. (Farkas et al. (1993) Ann. Rev. Physiol. 55:785; Giuliano et al. (1990), Optical Microscopy for Biology. B. Herman and K. Jacobson (eds.), pp. 543-557. Wiley-Liss, New York; Hahn et al. (1992) Nature 359:736; Waggoner et al. (1996) Hum. Pathol. 27:494). The concept is to treat each cell as a "well" with spatiotemporal information about the activity of the labeled components.

[0273] In one embodiment, a cell screening system is provided, the cell screening system comprising: a high power fluorescence optical system having a microscope objective, an XY stage suitable for holding a plate having an array of positions for holding cells and having means for moving the plate to align the positions with the microscope objective, and means for moving the plate in a direction to achieve focus; a digital camera; a light source having optical means for directing excitation light to cells in the array of positions and means for directing fluorescence emitted from the cells to the digital camera; and a computer device for receiving and processing digital data from the digital camera, wherein the computer device comprises: a digital frame grabber for receiving images from the camera, a display for user interaction and displaying assay results, a digital storage medium for data storage and archiving, and means for controlling, acquiring, processing, and displaying results. Methods for using such a system are disclosed in U.S. Pat. No. 6,756,207, which is incorporated herein in its entirety.

[0274] After the plate scan is complete, the system's image viewing, data viewing, and summary viewing tools can be used to view images and data. All images, data, and settings from the scan can be archived in the system's database for later viewing or docking with a network information management system. Data can also be exported to other third-party statistical packages to tabulate results and generate other reports. As the final stage of a complete scan, a report can be generated for one or more statistical information of the measured features. The user can use an interactive report generator to generate a graphical report based on the data summarized hole by hole for the scan area of ​​the plate. This report can include a summary of the statistical information of the holes in a tabular and graphical format and identification information about the sample.

[0275] Methods for imaging plates are known to those of ordinary skill in the art. Exemplary imaging platforms include the CellInsight high content screening (HCS) platform, which uses HSC studio software and standardized protocols. However, equivalent imaging and / or software platforms may also be used to practice the methods of the present disclosure.

[0276] An exemplary CellInsight setup for data acquisition includes the following settings. Assay settings: Imaging mode: Fluorescence 1 (F1) % Fluorescence 2 (F2); Collect bright field images; F1 image cell type: Mouse ES cells; Description: mNPC fluorophore AOVC-535-403; Fluorescence empirical value: 700.0ms; F2 image cell type: Mouse ES cells; Description: mNPC fluorophore P1 VC-660-503; Fluorescence empirical value: 5000.0ms; Set dilution factor for assay: 2.000; Display F1, F2 percentage: F1 / (F1+F2)*100%. Cell type settings: Mouse ES cells; Cell diameter: minimum 9.0 microns, maximum 30.0 microns; Roundness: 0.10; Contrast enhancement: 0.40; Declustering edge factor: 0.5; Declustering Th factor: 1.0; Background adjustment: 1.0. Trypan blue viability parameters: Dead cell diameter: minimum 8.0 μm, maximum 30 μm; Sensitivity: 1.0; Uniformity: 150; Contrast enhancement of very dark dead cells: 0.60. Protocol settings: Objective: 20x; Field size: 455.4x455.4 μm; Camera: X1; Camera acquisition mode: 1104x1104 (2x2 pixel binning); Autofocus with software; Software focus channel: 1; Autofocus interval: 1. Channel 1 settings: Composite, Channels included in camera gain: 2; Light intensity (in %): 100; Imaging mode: Wide field; Dye: 386-23_BGRFRN_BGRFRN; Depth of field: 6.563; Fixed exposure time; Target %: 25; Exposure time (seconds): 0.08. Channel 2 settings: Channels included in composite, Camera gain: 2; Light intensity (in %): 100; Imaging mode: Wide field; Dye: 485-20_BGRFRN_BGRFRN; Depth of field: 6.563; Fixed exposure time; Target %: 25; Exposure time (sec): 0.08.

[0277] Methods for producing AAV vectors

[0278] Provided herein are methods for producing AAV vectors, and pharmaceutical compositions comprising these AAV vectors, and methods for determining the efficacy of these AAV vectors using the cell-based efficacy assays described herein. In some embodiments, the AAV vector comprises a sequence encoding SMN1. In some embodiments, the relative efficacy 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 99.9%, at least 100%, at least 110%, at least 120%, at least 130%, or at least 140% relative to a reference standard.

[0279] In some embodiments, the relative potency of the viral vector is at least 90% compared to a reference standard.

[0280] The cell-based relative potency assay as described herein can be used to determine the relative potency of AAV-SMN1 vectors and is intended for use in batch handling and stability testing of AAV-SMN1 vector drug substances and drug products.

[0281] Upstream Process

[0282] In some embodiments, an upstream process is used to produce an intermediate derived from a working cell bank, wherein the upstream process comprises the following steps: (a) culturing cells, such as adherent cells, (b) transfecting the cultured cells, such as adherent cells, with three plasmids, (c) after the culture period, harvesting amplified viral particles from the cells, such as by whole cell lysis, (d) purifying the viral particles via filtration to remove any intact cells or cell debris, (e) subjecting the eluate from step (d) to tangential flow filtration, and (f) optionally freezing the resulting intermediate preparation of the purified viral particles. In some embodiments, the intermediate preparation can be frozen. In other embodiments, the intermediate preparation does not need to be frozen before downstream processing. In some embodiments, the AAV prepared using the upstream process disclosed herein is an AAV comprising a polynucleotide encoding SMN1 as described herein. In some embodiments, the upstream process is performed under GMP and on an industrial scale.

[0283] 1. Cell Line Transfection and Culture

[0284] In one aspect, the present invention discloses rAAV genome. The rAAV genome comprises one or more AAV ITRs, and the one or more AAV ITRs are flanked by coding polypeptides (including but not limited to SMN polypeptides) or polynucleotides encoding siRNA, shRNA, antisense and / or miRNA for the control sequence of a mutant protein or its gene. The polynucleotides are operably connected to transcription control DNA, particularly promoter DNA, enhancer DNA and polyadenylation signal sequence DNA, that work in target cells to form a gene cassette. The gene cassette can also include intron sequences to promote the processing of RNA transcripts when expressed in mammalian cells.

[0285] In some embodiments, the rAAV genome disclosed herein lacks AAV rep and cap DNA. The AAV DNA (e.g., ITR) in the rAAV genome can be from any AAV serotype from which recombinant viruses can be derived, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, and AAV-11. The nucleotide sequences of the genomes of AAV serotypes are known in the art. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC 001401 and Srivastava et al. Virol. [Virology], 45:555-564 {1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the complete genome of AAV-5 is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least a portion of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004).

[0286] As used herein, the "pSMN" vector plasmid comprises a polynucleotide encoding an SMN protein, i.e., an SMN cDNA expression cassette, wherein the cassette is flanked, for example, by adeno-associated virus inverted terminal repeat (ITR) sequences on the "left" and "right" sides of the polynucleotide encoding the SMN gene. In some embodiments, the polynucleotide encoding SMN is a human SMN sequence, such as a naturally occurring human SMN sequence or an isoform, variant, or mutant thereof. In some embodiments, the ITR sequence is a natural, variant, or modified AAV ITR sequence. In some embodiments, at least one ITR sequence is a natural, variant, or modified AAV2 ITR sequence. In some embodiments, both ITR sequences are natural, variant, or modified AAV2 ITR sequences. In some embodiments, the "left" ITR is a modified AAV2 ITR sequence that allows the production of a self-complementary genome, and the "right" ITR is a natural AAV2 ITR sequence. In some embodiments, the "right" ITR is a modified AAV2 ITR sequence that allows the production of a self-complementary genome, and the "left" ITR is a natural AAV2 ITR sequence. In some embodiments, the pSMN plasmid further comprises a CMV enhancer / chicken beta-actin ("CB") promoter. In some embodiments, the pSMN plasmid further comprises a simian virus 40 (SV40) intron. In some embodiments, the pSMN plasmid further comprises a bovine growth hormone (BGH) polyadenylation (polyA) termination signal. Exemplary sequences that can be used for one or more of the components discussed above are shown in Table 1 below. In some embodiments, all of the sequences shown in Table 1 below are used. In some embodiments, "AVXS-101" is a non-limiting example of a vector construct that uses all of the sequences in Table 1 and falls within the scope of the term pSMN.

[0287] In some embodiments, the pSMN vector may comprise an SMN cDNA expression cassette, a modified AAV2 ITR, a chicken β-actin (CB) promoter, a cytomegalovirus (CMV) immediate / early enhancer, a modified SV40 late 16s intron, a bovine growth hormone (BGH) polyadenylation signal, and an unmodified AAV2 ITR. The modified and unmodified ITRs may be entered in either orientation (i.e., 5' or 3') relative to the SMN cDNA expression cassette.

[0288] In some embodiments, for example, during the manufacturing process described herein, the vector construct sequence is encapsidated into, for example, an AAV9 virion. In these embodiments, encapsidation is in a non-replicating recombinant AAV9 capsid capable of delivering a stable functional transgene (e.g., a fully functional human SMN transgene). In some embodiments, the capsid is composed of 60 viral proteins (VP1, VP2, VP3) produced by alternative splicing, for example, at a ratio of 1:1:10, and the alternative splicing makes VP2 and VP3 two truncated forms of VP1, all with a common C-terminal sequence. In some embodiments, the product of the manufacturing process (e.g., a drug product) may include a non-replicating recombinant AAV9 capsid to deliver a stable fully functional human SMN transgene. In some embodiments, the capsid is composed of 60 viral proteins (VP1, VP2, VP3) produced by alternative splicing at a ratio of 1:1:10, and the alternative splicing makes VP2 and VP3 two truncated forms of VP1, all with a common C-terminal sequence.

[0289] The DNA sequences of exemplary vector constructs, such as AVXS-101 (AAV9-SMN1), are described in Table 1.

[0290] Table 1. Summary of DNA sequence components of AVXS-101 vector construct (all nucleotide start and end positions are related to SEQ ID NO: 1)

[0291]

[0292]

[0293] In another aspect, the DNA sequence of the AVXS-101 vector construct is provided in SEQ ID NO: 1:

[0294]

[0295]

[0296] In some embodiments, the amino acid sequence of the SMN protein encoded by the pSMN plasmid comprises:

[0297] MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNGDICETSGPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASIDFKRETCVVVYTGYGNREEQNLSDLLSPICEVA NNIEQNAQENENESQVSTDESENSRSPGNKSDNIKPKSAPWNSFLPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSCWLPPFPSGPPIIPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRCSHSLN(SEQ ID NO:3).

[0298] In some embodiments, the modified AAV2 ITR comprises a sequence of nucleotides 1-106 of SEQ ID NO: 1. In some embodiments, the cytomegalovirus (CMV) enhancer / chicken-β-actin hybrid promoter (CB) comprises a sequence of nucleotides 153-432 of SEQ ID NO: 1. In some embodiments, the sequence of the cDNA expressing the SMN1 protein comprises a sequence of nucleotides 1003-1887 of SEQ ID NO: 1. In some embodiments, the sequence of the SV40 intron comprises a sequence of nucleotides 774-870 of SEQ ID NO: 1. In some embodiments, the sequence of the bovine growth hormone (BGH) polyadenylation signal comprises a sequence of nucleotides 1973-2204 of SEQ ID NO: 1. In some embodiments, the unmodified AAV2 ITR comprises a sequence of nucleotides 2217-2359 of SEQ ID NO: 1.

[0299] In some embodiments, the AAV capsid proteins VP1, VP2, and VP3 are derived from the same transcript. These have alternative start sites but share a carboxyl terminus. Below, the VP1 specific amino acid sequence is shown in bold and bold. The amino acid sequence shared by VP1 and VP2 is underlined and italicized. The amino acids shared by these three capsid proteins are bold and italicized.

[0300]

[0301] In one embodiment, the AAV capsid protein is derived from a transcript encoding the amino acid sequence shown in SEQ ID NO:4.

[0302] In another aspect, the present invention discloses a DNA plasmid comprising a rAAV genome. The DNA plasmid is transferred to a cell that can be infected with a helper virus (e.g., adenovirus, adenovirus or herpes virus deleted by E1) of AAV so that the rAAV genome is assembled into an infectious virus particle with AAV9 capsid protein. The technology for producing rAAV particles (wherein the AAV genome to be packaged, rep and cap genes and helper virus functions are provided to cells) is standard in the art. In certain embodiments, the production of rAAV involves the following components present in a single cell (represented herein as packaging cells): rAAV genome, AAV rep and cap genes separated from the rAAV genome (i.e., not in the rAAV genome) and helper virus functional components. The production of pseudotyped rAAV is disclosed in, for example, WO 01 / 83692, which is incorporated herein by reference in its entirety. In various embodiments, the AAV capsid protein can be modified to enhance the delivery of the recombinant vector. The modification of the capsid protein is generally known in the art. See, for example, US 2005 / 0053922 and US 2009 / 0202490, the disclosures of which are incorporated herein by reference in their entirety.

[0303] The general principles of rAAV production are reviewed, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, CUM Topics in Microbial. and Immunol., 158:97-129). Various methods are described in the following literature: Ratschin et al., Mol. Cell. Biol. [Molecular Cell Biology] 4:2072 (1984); Hennonat et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. [Molecular Cell Biology] 5:3251 (1985); McLaughlin et al., J. Virol. [Journal of Virology], 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol. [Molecular Cell Biology], 7:349 (1988). Samulski et al. (1989, J. Virol. [Journal of Virology], 63:3822-3828); U.S. Patent No. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent No. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy [Gene Therapy] 3: 1124-1132; U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595. The above documents are hereby incorporated by reference in their entirety, with particular emphasis on those parts of the documents related to rAAV production.

[0304] An exemplary method for producing packaging cells is to produce a cell line that stably expresses all necessary components for AAV particle production. For example, a plasmid (or plasmids) comprising an rAAV genome lacking AAV rep and cap genes, an AAV rep and cap gene separated from the rAAV genome, and a selective marker (such as a neomycin resistance gene) is integrated into the cell genome. The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. [Proceedings of the National Academy of Sciences of the United States], 79: 2077-2081), adding a synthetic linker containing a restriction endonuclease cleavage site (Laug hlin et al., 1983, Gene [Gene], 23: 65-73), or by direct blunt end connection (Se napathy and Carter, 1984, J. Biol. Chem. [Journal of Biological Chemistry], 259: 4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. This approach has the advantage that the cells are selectable and suitable for large-scale production of rAAV.Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0305] Thus, in various embodiments, the disclosure herein provides packaging cells that produce infectious rAAV. The packaging cells can be non-adherent cells or adherent cells cultured in suspension. In one embodiment, any suitable packaging cell line can be used, such as HeLa cells, HEK293 cells, and PerC.6 cells (homologous 293 line). In one embodiment, the cell line is HEK 293 cells.

[0306] In order to increase the viral vector production, adherent cells can be cultivated and selected for improved adhesion to culture bottles. In certain embodiments, the transfection efficiency and cell count during the subsequent bioreactor inoculation step are improved. During the subculture, cells can be separated from the cell culture surface by methods known in the art. For example, cells can be lifted by scraping or by incubating in a solution comprising protease. In an exemplary embodiment, HEK293 cells can be washed with PBS and dissociated with trypsin for about 2 minutes at room temperature. Dissociation can be stopped by adding a growth medium containing serum, and cell clumps can be dissociated by repeatedly pipetting the suspension. Then the cell suspension can be precipitated, and the separated precipitate is resuspended in a suitable complete growth medium. Then the cells can be seeded in a new cell culture chamber, and it can be adhered. After a period of time, cells that do not adhere to the surface can be removed by gently aspirating with cell culture medium, and then the cell culture medium is completely replaced with growth medium. In certain embodiments, the time period allowing cells to adhere can be about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours or about 7 hours. After the cells have expanded, the process can be repeated to increase the fraction of cells that are strongly adhered to the culture flask. In some embodiments, the process is repeated at least 2 times, at least 3 times, at least 4 times, at least 5 times, or any suitable number of times. In an exemplary embodiment, HEK293 cells are seeded in 75 cm 2 The cells were placed in a flask and allowed to adhere for 4 hours in a 37° C. incubator, after which weakly adherent cells were removed by aspiration and the cell culture medium was replaced. In an exemplary embodiment, the process of selecting strongly adherent cells was repeated for three cell culture passages.

[0307] In other embodiments, the rAAV9 (ie, infectious encapsidated rAAV9 particles) comprises a rAAV genome disclosed herein. In one aspect, the rAAV genome is a self-complementary genome.

[0308] Prior to transfection, the cells are expanded in a suitable culture medium, in a flask or in a suitable bioreactor, or both. In some embodiments, the cells can be expanded in a bioreactor that provides continuous circulation of the cell culture medium. In one embodiment, the cells are expanded at 200 m 2 、333m 2 , or 500m 2iCELLis are expanded in bioreactors. One culture medium is DMEM with 5%-10% FBS, 4.5g / L glucose, 4mM L-glutamine. In some embodiments, adherent cells are added to the culture medium in a recirculating culture medium bag and circulated through the bioreactor. In some embodiments, a peristaltic pump is used to continuously recirculate the cell culture medium or any other culture medium through the bioreactor. Cells can be seeded in flasks or bioreactors at a suitable density for culture and transfection. The seeding density can depend on the cell type and the amount of time until transfection. In some embodiments, about 8000-16000 cells / cm 2 In one embodiment, cells are seeded at 8000-12,000 cells / cm 2 Seeding of HEK293 cells.

[0309] Suitable methods for transduction and reintroduction of transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with cells, e.g., in an appropriate culture medium, and selecting those cells that carry the DNA of interest using conventional techniques such as Southern blot and / or PCR, or by using a selectable marker.

[0310] In some embodiments, the packaging cell line is transfected with three plasmids: a plasmid encoding or containing a vector sequence to be packaged in an AAV vector (e.g., pSMN, pMECP2 transgene, or pSOD1sh), pHELP, and pAAV2 / 9. Transfection can be performed using any technique known in the art, including but not limited to electroporation, lipofection, such as with lipofectamine, cationic polymers, and cationic lipids. Any suitable transfection medium can be used. In one embodiment of the transfection process, adherent human embryonic kidney (HEK293) cells are transfected by triple DNA plasmid polyethyleneimine (PEI) coprecipitation. In one embodiment, the scAAV9.CB.SMN vector (a self-complementary AAV9 vector comprising a CB promoter and a polynucleotide encoding SMN) is produced in a large-scale adherent cell bioreactor using triple DNA plasmid transfection (coprecipitation with PEI) to adherent HEK293 cells. In one embodiment, the DMEM growth medium used for cell expansion is replaced with a modified DMEM transfection medium. This medium is formulated without calcium and L-glutamine. In one embodiment, the transfection medium is DMEM without FBS, without calcium, without L-glutamine, but with 4.5 g / L glucose.

[0311] In some embodiments, transfection medium without serum (e.g., without FBS) improves transfection efficacy. In one embodiment, transfection medium is OptiMEM (Invitrogen / ThermoFisher). In one embodiment, three plasmids (pSMN, pHELP, and pAAV2 / 9) are mixed with PEI in transfection medium and reacted. In some embodiments, three plasmids are mixed in about 1:1:1 molar ratio. In some embodiments, plasmids and PEI are mixed in 1:1 amount ratio. In some embodiments, plasmids and PEI are mixed with a DNA:PEI weight ratio less than 1:1. In one embodiment, pSMN, pHELP, and pAAV2 / 9 are mixed in OptiMEM medium in a 1:1:1 molar ratio. In this embodiment, PEI is added so that DNA:PEI is 1:1 by weight. In some embodiments, the reaction is allowed to occur for 0-60 minutes, 10-45 minutes, or 20-30 minutes. In one embodiment, the reaction is allowed to occur for 15-30 minutes.

[0312] In one embodiment, the present disclosure provides a method for manufacturing an AAV-based viral vector, the method comprising the steps of: (i) culturing adherent HEK293 cells in an industrial scale bioreactor, (2) transfecting the adherent cells with plasmids for less than 60 minutes to enable production of AAV vectors, and optionally applying further processing, purification, formulation and filling steps to produce a pharmaceutical product. In one embodiment of this method, a scAAV9.CB.SMN vector is produced using a triple DNA plasmid transfection co-precipitated with polyethyleneimine ("PEI"). In one embodiment, the three plasmids used for this transfection are pSMN, pAAV2 / 9 and pHELP.

[0313] Transfection can be carried out by contacting the packaging cell line with the DNA-PEI coprecipitate. In certain embodiments, the DNA-PEI coprecipitate in the transfection medium is filled into a medium recirculation bag. In certain embodiments, the DNA-PEI coprecipitate in the transfection medium is circulated in a bioreactor and completely replaces the growth medium. In certain embodiments, the DNA-PEI coprecipitate in the transfection medium is contacted with the adherent cells in the bioreactor. In certain embodiments, the DNA-PEI coprecipitate in the transfection medium is contacted with the adherent cells in the bioreactor for up to two hours. In certain embodiments, transfection occurs for one to two hours. In certain embodiments, transfection occurs for less than one hour, for example, 10 minutes, 20 minutes, 30 minutes, 40 minutes or 50 minutes. In certain embodiments, transfection occurs for one to two hours. In certain embodiments, transfection is stopped by recirculating the complete growth medium through the bioreactor and completely replacing the transfection medium.

[0314] 2. Harvesting the Amplified Virus Particles

[0315] After a suitable cell expansion period after transfection, in some embodiments, the cells are lysed and the viral particles are harvested. In some embodiments, the cells are dissociated from the reactor before the cell lysis process is initiated. In some embodiments, the cells are lysed in situ. Optionally, viral particles are harvested without lysis. In some embodiments, an endonuclease is added, for example, circulated to the bioreactor to the final target concentration. The endonuclease can be an endonuclease that degrades both DNA and RNA. In one embodiment, the endonuclease is a genetically engineered endonuclease from Serratia marcescens (Eaves, GN et al. J. Bact. [Journal of Bacteriology] 1963, 85, 273-278; Nestle, M. et al. J. Biol. Chem. [Journal of Biological Chemistry] 1969, 244, 5219-5225), known as the universal nuclease (EMD Millipore Corporation). The enzyme is produced and purified from E. coli strain W3110 (a mutant of strain K12) containing the pNUC1 production plasmid (U.S. Patent No. 5,173,418, which is hereby incorporated by reference in its entirety). Structurally, the protein is a dimer of identical subunits of approximately 30 kDa of 245 amino acids (with two important disulfide bonds). Universal nuclease Degrades all forms of DNA and RNA (single-stranded, double-stranded, linear and circular) and is effective under a wide range of operating conditions, digesting nucleic acids to 5'-monophosphate-terminated oligonucleotides 2-5 bases in length. It is produced according to current good manufacturing practices (cGMP), and therefore can be used in industrial-scale processes for purifying proteins and / or viral particles. Other endonucleases produced under cGMP conditions can also be used in the purification methods disclosed in the present application. In one embodiment, the full-strength nuclease is added to a bioreactor to a final concentration of between 50-200U / ml, such as between 75-150U / ml, such as about 100U / mL. In certain embodiments, the addition of the full-strength nuclease significantly reduces host cell DNA while allowing high vg production in a bioreactor.

[0316] In some embodiments, the endonuclease is mixed before the lysis buffer is added to the reactor. In some embodiments, the cell lysis solution is mixed with the adherent cells for up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, or up to 5 hours. In some embodiments, the lysis buffer may include magnesium chloride and / or Tween-20 in a suitable buffer. In an exemplary embodiment, the lysis buffer is 500 mM HEPES, 10% Tween 20, 20 mM MgCl 2 , pH 8.0. A salt sucrose solution (SSS) that quenches the omnipotent nuclease reaction can be added to stop the cleavage reaction. In some embodiments, SSS is added to a harvest bag containing a wash buffer and mixed for 15 minutes. In some embodiments, the bioreactor is rinsed with a bioreactor wash buffer, and the wash solution is then collected in a harvest collection bag together with the quenched cell lysis solution and the lysed cell contents, all of which together constitute a batch harvest. In some embodiments, the bioreactor wash buffer may comprise Tris, MgCl2, NaCl, Tween-20, and sucrose. In an exemplary embodiment, the bioreactor wash buffer comprises 20 mM Tris, 1 mM MgCl2 at pH 8.1, and 1 mM MgCl2 at pH 8.1. 2 , 500 mM NaCl, 1% Tween-20 w / v, and 1% sucrose w / v.

[0317] 3. Purification of Viral Particles

[0318] After harvesting, the bulk viral particle harvest can be concentrated and purified typically via filtration. In one embodiment, the viral particles are filtered by depth filtration followed by filtration through a filter (e.g., a 0.45 μm filter) that removes macromolecular contaminants and cell debris but allows the vector genome to pass therethrough. Any suitable depth filter can be used.

[0319] As understood in the art, depth filtration refers to the use of porous filter media to clarify solutions containing large quantities of large particles (e.g., intact cells or cell debris), whereas membrane filtration would quickly become clogged under such conditions. Depth filtration media of a variety of different pore sizes are commercially available from multiple manufacturers such as Millipore, Pall, General Electric, and Sartorious.

[0320] The target flow rate for the depth filtration may be reduced to keep the filter inlet pressure within specification. In certain embodiments, once all of the bulk harvest has been filtered, the depth filter may be driven out with the diafiltration buffer used in the subsequent first tangential flow filtration step ("TFF1"). The depth filter pools are combined. The depth filter pools may then be filtered through a 0.45 μm filter to further clarify the bulk harvest material. The 0.45 μm filter is then driven out with TFF1 buffer.

[0321] 4. Tangential flow filtration

[0322] In various embodiments, tangential flow filtration is used to concentrate batch harvests, and salts and proteins are removed, for example, using tangential flow filtration. Tangential flow filtration (TFF) (also referred to as cross-flow filtration CFF) is well known to those skilled in the art, and the equipment and schemes for its implementation in a wide range of situations are commercially available from multiple manufacturers, including but not limited to Pall Corporation in Port Washington, New York and Spectrum Labs in Rancho Dominguez, California. Typically, TFF may involve the recirculation of retentate across the membrane surface. In certain embodiments, this gentle cross-flow feed can minimize membrane fouling, maintain a high filtration rate and provide a high product recovery. In one embodiment, the TFF step can be implemented with a flat sheet system, as illustrated herein. Flat sheet systems can be used for large-scale production, wherein such systems are provided with devices (e.g., open flow channels) that prevent excessive shear forces on viral particles. Alternatively, the TFF step can be implemented with a hollow fiber system, as illustrated herein. In one embodiment, the molecular weight cutoff (MWCO) of the TFF system is between 200-400 kDa, such as about 300 kDa.

[0323] In one embodiment, the TFF1 step is performed using a regenerated cellulose membrane cassette with a 300 kDa MW cutoff. The cassette is rinsed and sterilized with a NaOH solution and equilibrated with a TFF1 buffer. In one embodiment, the TFF1 buffer comprises 20 mM Tris, 1 mM MgCl2, 500 mM NaCl, 1% sucrose, pH 8.1.

[0324] In certain embodiments, the concentration stage of the TFF1 step is selected to reduce the volume of the harvest of clarification by about 10 times. Once the target retentate volume is reached, diafiltration operation can be started. In certain embodiments, the retentate can be diafiltered with the TFF1 buffer of about 6 diafiltration volumes. In certain embodiments, the retentate can be diafiltered with the TFF1 buffer of about 5-20 or 10-15 or 12 diafiltration volumes. Once the total permeate flow of 6 diafiltration volumes has been achieved, the retentate can be reconcentrated and harvested. The flushing of membrane (for example, flushing twice in a row) can be performed to increase the product recovery rate of intermediate drug substance.

[0325] 5. Intermediate products

[0326] In some embodiments, the intermediate drug substance can then be frozen on dry ice or in a freezer and then transferred to storage < -60° C. In other embodiments, the intermediate product does not need to be frozen prior to downstream processing.

[0327] In some embodiments, multiple batches of intermediate product materials are pooled together for further processing (e.g., purification by downstream processes, e.g., as described herein). The multiple batches of intermediate product materials can be pooled before freezing and storage. In other embodiments, the multiple batches of intermediate product materials can be pooled after thawing the frozen and stored batches.

[0328] Downstream Process

[0329] In some embodiments, a downstream process is used to process an intermediate (e.g., a pooled intermediate) into a filtered drug substance. In some embodiments, the downstream process steps include: (a) acidification and clarification (e.g., using filtration), (b) cation exchange chromatography, (c) tangential flow filtration ("TFF2"), (d) CsCl ultracentrifugation, (e) collection of viral vectors, and (f) further tangential flow filtration ("TFF3") to produce a filtered drug substance, wherein the purified AAV particles are suspended in a pharmaceutically acceptable carrier. In some embodiments, the downstream process includes the following manufacturing steps subsequent to the production of the TFF1 intermediate: thawing and pooling of the TFF1 intermediate, acidification and clarification, cation exchange chromatography (CEX), tangential flow filtration (TFF2), CsCl ultracentrifugation for complete / empty capsid separation, tangential flow filtration (TFF3) for concentration / buffer exchange, TFF 3 pooled material filtration to produce drug substance, dilution and filtration of the drug substance to produce drug product, storage of the drug product, and filling of the drug product into vials.

[0330] In some embodiments, the downstream processes disclosed herein can be used to process intermediates comprising AAV SMN as described herein.

[0331] 1. Acidification and clarification of intermediates

[0332] In the embodiment where the intermediate is frozen, the downstream process starts with thawing the TFF1 intermediate material. Detergents (e.g., Tween 20) can be used to promote flocculation of most host cell proteins and DNA at acidic pH. The pH of the detergent containing the TFF1 intermediate can then be reduced. The floccules and precipitates formed when the pH is reduced can be removed by filtering the solution through a depth filter and removing macromolecular contaminants and cell debris but allowing the vector genome to pass through a filter (e.g., 0.45 μm filter). Any suitable depth filter can be used.

[0333] In one embodiment, Tween 20 is slowly added to the TFF1 intermediate solution to achieve a final concentration between 10%-20% Tween 20. In some embodiments, the target composition after adding Tween 20 is 20 mM Tris, 1 mM MgCl 2 , 36% Tween 20 solution in 500mM NaCl, 1% sucrose m / v, pH 8.1. In some embodiments, Tween 20 is added slowly over a period of about 1-6 hours. In some embodiments, Tween 20 is added slowly over 3-6 hours. In some embodiments, Tween 20 is added slowly over 4 hours. In some embodiments, the Tween 20 / TFF1 intermediate solution is incubated overnight at room temperature. In some embodiments, the Tween 20 / TFF1 intermediate solution is incubated at room temperature for 8-20 hours. In an exemplary embodiment, the Tween 20 / TFF1 intermediate solution is incubated at room temperature for 12-20 hours.

[0334] After incubation, the pH of the Tween 20 containing the TFF1 intermediate can be reduced by adding any suitable acid. In certain embodiments, 1M glycine (pH 2.5) is added to achieve a target pH of 3.5 ± 0.1. In certain embodiments, the target pH is pH 3.0-4.0, about pH 3.3-3.7, about pH 3.4-3.6, or about pH 3.5. Once the pH is within an acceptable range, the solution can be passed through a filter of any size. In an exemplary embodiment, a deep filter (e.g., Clarisolve POD) is used in conjunction with a 0.45 μm filter (e.g., Opticap XL10Durapore filter) or a 0.8 / 0.45 μm PES filter.

[0335] 2. Cation Exchange Chromatography

[0336] In various embodiments, a cation exchange (CEX) capture chromatography step is used, for example, to separate viral capsids from host cell proteins, host cell DNA, host cell lipids, Tween 20 and other process-related impurities. The principle of cation exchange chromatography is well known in the art, but in short, this method relies on the charge-charge interaction between the positively charged particles to be separated and the negatively charged resin used. Generally speaking, the column is first balanced by flowing through several diafiltration volumes of buffer until pH and conductivity are stable. The sample is then loaded and the column is washed with loading buffer. Finally, the target sample is eluted from the column using elution buffer, and the fractions containing the sample are collected. The presence of the target sample can be detected by optical absorbance measurement of the eluent.

[0337] In one embodiment, the CEX step utilizes a CIMmultus S03-8000 Advanced Composite Column (Sulfonyl) (2 μm pore) chromatographic column. In one embodiment, the elution peak is collected when the OD280 rises sharply. When the conductivity is between 80-85 mS / cm, the OD280 will begin to rise. The CEX eluate can be collected according to conventional procedures and can be collected in two fractions. In one embodiment, the first fraction starts when the OD280 rises sharply, and 1.5 collection volumes (CV) are collected. In another embodiment, the second fraction starts immediately after the first fraction and 1.0 CV is collected. The two fractions are pooled and then neutralized to pH 8.0 ± 0.30. In one embodiment, the neutralization buffer contains 1.0 M Tris, pH 9.1 ± 0.1 at 20 ° C.

[0338] 3. Tangential flow filtration 2

[0339] In some embodiments, a tangential flow filtration step (TFF2) is used to concentrate, remove protein impurities, and exchange the buffer into an appropriate buffer for a subsequent CsCl ultracentrifugation step. Any suitable TFF membrane may be used. In one embodiment, the TFF2 step utilizes a 300 kD MWCO regenerated cellulose membrane.

[0340] In some embodiments, the concentration stage of this step is designed to reduce the volume of the CEX eluate. In one embodiment, the retentate is diluted 2-fold with a diafiltration buffer and the retentate is concentrated to its original volume. In one embodiment, the diafiltration buffer is a TFF2 NaCl diafiltration buffer containing: 20 mM Tris, 2 mM MgCl2, 150 mM NaCl, 0.2% Poloxamer 188, 1% sucrose, pH 8.1±0.1 at 20°C. In such embodiments, this process can be repeated until the diafiltration with the new buffer is complete. In one embodiment, the retentate is diluted 2-fold with a diafiltration buffer containing CsCl and the retentate is concentrated to its original volume. In one embodiment, the diafiltration buffer containing CsCl is a TFF2 CsCl diafiltration buffer containing: 20 mM Tris, 2 mM MgCl 2 , 3M CsCl, 0.2% poloxamer 188, pH 8.1 ± 0.1 at 20°C. In such embodiments, this process can be repeated until the diafiltration with new buffer is completed. Once the CsCl diafiltration is completed, the retentate can then be concentrated to a specified volume, which depends on the system hold-up volume. In some embodiments, flushing of the membrane (e.g., two consecutive flushes) is performed to maximize the product recovery from the TFF2 system.

[0341] 4. CsCl ultracentrifugation

[0342] In some embodiments where AAV is used for in vivo gene transduction, the final product of rAAV can contain minimal impurities and empty particles. Two methods for purifying AAV vectors are ultracentrifugation using either iodixanol gradients or CsCl gradients. A study comparing these two methods demonstrated that iodixanol produced AAV vectors with higher vector purity but with more empty viral capsids compared to CsCl. Strobel et al. "Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications." Human Gene Therapy Methods, 26(4): 147-157. Although the use of CsCl results in a smaller amount of empty viral capsids, CsCl can be toxic to cells and multiple purification steps may be required to remove residual CsCl, resulting in a longer processing time (about 3.5 days) compared to shorter methods such as iodixanol (about 1 day). A different study showed that the many steps to remove residual CsCl often resulted in significant rAAV losses, resulting in low yields and recoveries, which often offset the other benefits of the method. Hermens et al. "Purification of Recombinant Adeno-Associated Virus by Iodixanol Gradient Ultracentrifugation Allows Rapid and Reproducible Preparation of Vector Stocks for Gene Transfer in the Nervous System." Human Gene Therapy, 10: 1885-1891. Furthermore, while both of these approaches work well in the laboratory for generating preclinical samples, they are not scalable and therefore not suitable for large-scale production of commercial products.See, e.g., Tomono et al., “Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1).” Molecular Therapy-Methods & Clinical Development, 3:15058 (“Purification methods using cesium chloride (CsCl) or iodixanol density ultracentrifugation are not suitable for large-scale production”).

[0343] In some embodiments, an ultracentrifugation step is used, for example, to separate empty capsids from complete capsids. Unexpectedly, the CsCl ultracentrifugation method disclosed herein is scalable and suitable for large-scale production of purified AAV vectors. Ultracentrifugation can be performed by analytical ultracentrifugation, and may involve the use of gradient buffers. Examples of gradient buffers include, but are not limited to, CsCl, sucrose, iodixanol, and other buffers known in the art. Centrifugation can be performed in any centrifuge capable of reaching the required g-force, such as an automated Optima XPN100 ultracentrifuge system or an equivalent system equipped with a 50.2Ti rotor or an equivalent rotor. After ultracentrifugation, empty capsids and complete capsids are separated into different zones in the tube, and can be extracted by extracting material from a specific zone. In some embodiments, the filtered material purified by TFF2 is centrifuged at 241,600-302,000g (about 40,000-50,000rpm in a 50.2Ti rotor). In some embodiments, the filtered material purified by TFF2 is centrifuged overnight. In some embodiments, the filtered material purified by TFF2 is centrifuged for 16-24 hours. In some embodiments, the filtered material purified by TFF2 is centrifuged for 20-24 hours. In some embodiments, the filtered material purified by TFF2 is centrifuged at 15°C-25°C. In one embodiment, the filtered material purified by TFF2 is centrifuged at 302,000g (50,000rpm in a 50.2Ti rotor) for 17 hours at 20°C. In some embodiments, the buffer for CsCl centrifugation may have one or more of the following components, including (a) CsCl, further comprising (b) MgCl 2, (c) poloxamer 188 and (d) one or more of Tris. In some embodiments, the buffer for CsCl can include all of (a), (b), (c) and (d). In some embodiments, the buffer for CsCl has a pH of 7.5-8.5 or pH 7.9-8.2. In one embodiment, a suitable buffer for CsCl centrifugation is 20 mM Tris, 2 mM MgCl 2 , 3M CsCl, 0.2% poloxamer 188, pH 8.1±0.10. After the centrifugation step is completed, the tube can be removed from the ultracentrifuge. In some embodiments, the highest zone, i.e., zone A, contains empty capsids. In some embodiments, the second highest zone, i.e., zones B, C, and D contain complete capsid double zones. In some embodiments, a syringe is used to collect the AAV viral vector. In one embodiment, zones B, C, and D are removed by an 18G needle attached to a 30mL syringe, and the needle is inserted into the middle of the tube just below zone D. In other embodiments, techniques known in the art and / or as described herein can be used to determine the presence of complete or empty capsids in the zone, and to collect the zone containing the complete capsid.

[0344] The ratio of empty viral capsid to non-empty viral capsid can be measured by standard laboratory techniques. In some embodiments, the measurement is performed by optical absorbance measurement. In some embodiments, the measurement is performed by UV absorbance measurement. In some embodiments, the total amount of capsid protein and the total amount of DNA can be measured according to UV absorbance measurement. In some embodiments, the measurement is performed by optical refractive index measurement. In some other embodiments, the measurement is performed by analytical ultracentrifugation.

[0345] In one embodiment, the AAV viral vector collected after ultracentrifugation has less than 8% empty capsids, less than 7% empty capsids, less than 5%, less than 3% or less than 1%. In one embodiment, the AAV viral vector collected after ultracentrifugation has 1%-10% empty capsids. In one embodiment, the AAV viral vector collected after ultracentrifugation has 2%-8% empty capsids. In one embodiment, the number of empty capsids is below the detection limit. In another embodiment, the percentage of empty capsids is measured as a percentage of total capsids.

[0346] 5. Tangential flow filtration 3 to produce filtered drug substance

[0347] In some embodiments, a tangential flow filtration step (TFF3) is used to remove CsCl and concentrate the complete carrier capsid. Suitable membranes can be used for tangential flow filtration. In one embodiment, a 300kDa MWCO regenerated cellulose membrane is used. The carrier capsid can be retained by the membrane. The concentration stage of the TFF3 operation can be designed to reduce the concentration of residual CsCl and the volume of the ultracentrifugation pool. In some embodiments, once the target retentate volume is reached, diafiltration is started. The retentate is diafiltered with a suitable TFF3 buffer of up to 10 diafiltration volumes. In one embodiment, a suitable TFF3 buffer may include one or more of the following components, which components include (a) s, (b) MgCl 2 , (c) NaCl or (d) poloxamer 188. In one embodiment, a suitable TFF3 buffer may include all of (a), (b), (c) and (d). In one embodiment, a TFF3 buffer has a pH of 7.5-8.5, pH 7.7-8.3 or pH 8.0. In one embodiment, a suitable TFF3 buffer comprises 20 mM Tris, 1 mM MgCl 2 , 200 mM NaCl, 0.001% Poloxamer 188, pH 8.0 ± 0.1 at 20°C. In another embodiment, a suitable TFF3 buffer comprises 20 mM Tris, 1 mM MgCl 2 , 200 mM NaCl, 0.005% Poloxamer 188, pH 8.0 ± 0.1 at 20°C. In one embodiment, the concentrated retentate is concentrated using a 0.2 μm Pall EKV sterilizing grade filter (Mini Kleenpak) filter to produce filtered drug substance. In some embodiments, the methods described herein produce more than 5x10 15 vg, or more than 8x10 15 vg or more than 1x10 16 vg rAAV.

[0348] Pharmaceutical composition

[0349] Virus (e.g., AAV) particles purified according to the methods disclosed herein can be produced in high yields with sufficient purity to be administered to human subjects. In some embodiments, the efficacy of a pharmaceutical composition comprising the AAV particles described herein is determined using a cell-based in vitro potency assay as described herein. In some embodiments, the AAV particles are purified in a concentration of about 1-8 x 10 13 Viral vector genomes / mL (vg / mL) or about 1.7-2.3x10 13The viral vector is formulated at a concentration between about 1.9-2.1 x 10 13 The viral vector is prepared at a concentration of about 2.0 x 10 13 The viral vector was prepared at a concentration of vg / mL.

[0350] In certain embodiments, during the production process of viral vector, empty viral capsids not containing nucleic acid material may be produced. Pharmaceutical compositions comprising low amounts of empty viral capsids may be advantageous because they avoid unnecessary exposure of patients (e.g., infants) with immature immune systems to antigenic materials (empty capsids, host cell proteins, host cell DNA) without therapeutic benefit. In certain embodiments, such pharmaceutical compositions can reduce potential transfusion reactions or more extensive immune responses, and can improve therapeutic efficacy. Compared with complete viral capsids with genomic material, empty capsids have different densities, thereby these two materials can be separated by gradient centrifugation or other methods known in the art. In certain embodiments, empty capsids are separated by ultracentrifugation. In certain embodiments, empty capsids are separated by CsCl gradient ultracentrifugation. In other embodiments, empty capsids are separated by iodixanol gradient ultracentrifugation. In certain embodiments, empty capsids are separated by sucrose gradient ultracentrifugation.

[0351] The ratio of empty viral capsid to non-empty viral capsid can be measured by standard laboratory techniques. In some embodiments, the ratio is measured by absorbance measurement. In some embodiments, the ratio is measured by UV absorbance measurement. In some embodiments, the total amount of capsid protein and the total amount of DNA can be measured by UV absorbance measurement. In some embodiments, the measurement is measured by optical refractive index measurement. In some other embodiments, the measurement is measured by analytical ultracentrifugation.

[0352] High levels of empty capsids may challenge the efficacy of viral vector therapy. In one embodiment, the pharmaceutical composition has less than 10% empty capsids, less than 8% empty capsids, less than 7%, less than about 5%, less than 3%, less than 1% empty capsids. In another embodiment, the pharmaceutical composition has 1%-10% empty capsids. In another embodiment, the pharmaceutical composition has 2%-8% empty capsids. In another embodiment, the pharmaceutical composition has less than or equal to 6% empty capsids, 5% empty capsids, 4% empty capsids, 3% empty capsids, 2% empty capsids or less. In one embodiment, the number of empty capsids is below the detection limit. In another embodiment, the percentage of empty capsids is measured, for example, using AUC as a percentage of total capsids. In some embodiments, for example, compared with compositions with higher percentages of empty capsids, these low percentages of empty capsids improve therapeutic efficacy and / or reduce adverse events (e.g., inflammatory response, liver damage) after being administered to patients. In some embodiments, the methods of making viral vectors disclosed herein provide these improved percentages of empty capsids compared to levels in prior methods (e.g., those that do not use adherent cells and / or the purification methods described herein).

[0353] During the production process of viral vectors, residual proteins from adherent cells (e.g., HEK293 cells) used to produce viral vectors may not be completely isolated. Residual host cell proteins have the potential to trigger an immune response. The amount of residual host cells can be measured by any standard laboratory technique that can distinguish viral capsid proteins from residual host cell proteins. In certain embodiments, the amount of residual host cell proteins can be measured by size exclusion or ion exchange chromatography. In certain embodiments, the measurement can be performed by Western blotting using parental cell-specific antibodies. In one embodiment, the amount of residual host cell proteins can be measured by enzyme-linked immunosorbent assay (ELISA). In certain embodiments, the amount of residual host cell proteins can be measured by commercial ELISA kits. In certain embodiments, the amount of residual host cell proteins can be measured by Cygnus Technologies HEK293 HCP ELISA kits.

[0354] In another embodiment, the residual host cell protein in the pharmaceutical composition is 1×10 13 vg / ml is less than or equal to 5X10 6 pg / ml, each 1X10 13 vg / mL is less than or equal to 1.2X10 6 pg / ml or 1X10 13 vg / ml 1X10 5 pg / ml to 1X10 13vg / ml 1.2X10 6 pg / ml or 1X10 13 vg / ml is less than or equal to 40ng / ml. In one embodiment, the pharmaceutical composition comprises 1.0x10 13 vg is less than or equal to 5, 4, 3, 2, 1 ng or less of residual host cell protein. In one embodiment, the pharmaceutical composition comprises per 1.0x10 13 vg less than or equal to 4ng of residual host cell protein.

[0355] During the production process of the viral vector, residual host cell DNA from adherent cells (e.g., HEK293 cells) or residual plasmid DNA transfected to produce the viral vector may not be completely removed. Purification processes (e.g., acidification, clarification, tangential flow filtration, etc.) remove most of the residual host cell or plasmid DNA. In one embodiment, the measurement of the amount of residual host cell or plasmid DNA is performed by PCR. In another embodiment, the measurement of the amount of residual host cell or plasmid DNA is performed by quantitative PCR (qPCR) using primers specific to the host cell or plasmid sequence. In another embodiment, the measurement of the amount of residual host cell or plasmid DNA is performed by digital droplet PCR (ddPCR). In one embodiment, the amount of plasmid DNA is measured using a qPCR assay using primers specific to the kanamycin resistance gene region of the plasmid. In another embodiment, the amount of residual host cell DNA is measured by a commercial qPCR assay kit, for example, Thermo Fisher Scientific's Human residual DNA quantification kit, Biorad's residual DNA quantification Supermix or any equivalent product. Reducing the amount of residual host cell or plasmid DNA can improve treatment outcomes, and such compositions can be purified and / or selected for use in the treatment disclosed herein.

[0356] In one embodiment, the residual host cell DNA in the pharmaceutical composition is 1×10 13 vg / ml is less than or equal to 1.7X10 6 pg / ml, each 1X10 13 vg / ml 1X10 5 pg / ml to 1X10 13 vg / ml 1.2X10 6 pg / ml. In one embodiment, the residual host cell DNA in the pharmaceutical composition is 1.0x10 13 vg is less than or equal to 3x10 5 , 2x10 5 , 1.1x10 5, 1x10 5 pg or less. In an embodiment, the residual host cell DNA in the pharmaceutical composition is 1.0x10 13 vg is less than or equal to 1.1x10 5 pg.

[0357] In another embodiment, the residual plasmid DNA in the pharmaceutical composition is 1×10 13 vg / m is less than or equal to 1.7X10 6 pg / ml, each 1X10 13 vg / ml 1X10 5 pg / ml to 1X10 13 vg / ml 1.7X10 6 In another embodiment, the residual plasmid DNA in the pharmaceutical composition is 1.0x10 13 vg is less than or equal to 6.8x10 5 pg.

[0358] In one embodiment, the residual host cell DNA in the pharmaceutical composition is 1.0×10 13 vg is less than or equal to 1.1x10 5 pg, and the residual plasmid DNA in the pharmaceutical composition is 1.0x10 13 vg is less than or equal to 6.8x10 5 pg.

[0359] In one embodiment, the residual host cell DNA in the pharmaceutical composition is 1.0×10 13 vg is less than or equal to 1.1x10 5 pg, and the residual plasmid DNA in the pharmaceutical composition is 1.0x10 13 vg is less than or equal to 6.8x10 5 pg, and the residual host cell protein in the pharmaceutical composition is 1.0x10 13 vg is less than or equal to 4ng.

[0360] In some embodiments, the amount of endotoxin in the pharmaceutical composition is 1.0 x 10 13 vg / mL is less than about 1EU / mL, and every 1.0x10 13 vg / mL is less than about 0.75EU / mL, per 1.0x10 13 vg / mL is less than about 0.5EU / mL, and every 1.0x10 13 vg / mL is less than about 0.4EU / mL, and every 1.0x10 13 vg / mL is less than about 0.35EU / mL, per 1.0x1013 vg / mL is less than about 0.3EU / mL, and every 1.0x10 13 vg / mL is less than about 0.25EU / mL, per 1.0x10 13 vg / mL is less than about 0.2EU / mL, and every 1.0x10 13 vg / mL is less than about 0.15EU / mL, per 1.0x10 13 vg / mL is less than about 0.1EU / mL, and every 1.0x10 13 vg / mL is less than about 0.05EU / mL or per 1.0x10 13 vg / mL is less than about 0.02EU / mL. Methods for determining the amount of endotoxin are known in the art, such as the Limulus amebocyte lysate (LAL) test. In the examples, the amount of endotoxin is determined according to the United States Pharmacopeia ("USP"). <85> (incorporated herein by reference in its entirety) to assay for endotoxin.

[0361] In one embodiment, the bovine serum albumin (BSA) in the pharmaceutical composition is 1.0 x 10 13 Vg is less than 0.5ng, per 1.0x10 13 vg is less than 0.3ng or per 1.0x10 13 In one embodiment, the universal nuclease in the pharmaceutical composition is 1.0x10 13 Vg is less than 0.2ng, per 1.0x10 13 vg is less than 0.1ng or per 1.0x10 13 vg is less than 0.09ng.

[0362] In one embodiment, the pharmaceutical composition disclosed herein comprises one or more of the following: 13 vg less than about 0.09ng of universal nuclease, less than about 30μg / g (ppm) of cesium, about 20-80ppm of poloxamer 188, and 1.0x10 13 vg is less than about 0.22ng BSA, per 1.0x10 13 vg is less than about 6.8x10 5 pg of residual plasmid DNA, each 1.0x10 13 vg is less than about 1.1x10 5 pg of residual hcDNA, each 1.0x10 13 vg less than about 4ng rHCP, pH 7.7-8.3, about 390-430mOsm / kg, less than about 600 particles ≥25μm per container, less than about 6000 particles ≥10μm per container, about 1.7x10 13 -2.3x1013 vg / mL genome titer, per 1.0x10 13 vg about 3.9x10 8 -8.4x10 10 IU infection titer, per 1.0x10 13 vg is about 100-300μg of total protein, at about 7.5x10 13 vg / kg dose of viral vector, a median survival of ≥24 days in Δ7SMA mice, about 70%-130% relative potency based on a cell-based in vitro assay, and / or less than about 5% empty capsids.

[0363] In one embodiment, the pharmaceutical composition disclosed herein comprises one or more, e.g., all, of the following: pH 7.7-8.3 (e.g., as described in USP <791> ), about 390-430 mOsm / kg (e.g., as measured according to USP <785> less than about 600 particles ≥ 25 μm in size per container (e.g., as measured in accordance with USP <787> less than about 6000 particles ≥ 10 μm in size per container (e.g., as measured in accordance with USP <787> Measured), about 1.7x10 13 -2.3x10 13 vg / mL genome titer, per 1.0x10 13 vg about 3.9x10 8 -8.4 x10 10 IU infection titer, per 1.0x10 13 vg is about 100-300μg of total protein, at about 7.5x10 13 vg / kg dose of viral vector, a median survival of ≥ 24 days in Δ7SMA mice, about 70%-130% relative potency according to a cell-based in vitro assay, and / or less than about 5% empty capsid. In an embodiment, the pharmaceutical composition disclosed herein comprises a total purity greater than or equal to 95% (e.g., as determined by SDS-PAGE). In an embodiment, the pharmaceutical composition disclosed herein does not contain a single unnamed related impurity (e.g., as determined by SDS-PAGE) at a level greater than 2%. In an embodiment, the pharmaceutical composition disclosed herein comprises an endotoxin level less than or equal to 0.75EU / mL. In an embodiment, the pharmaceutical composition disclosed herein is tested for no growth in a sterility test.

[0364] High levels of residual host cell proteins, host cell DNA, plasmid DNA and / or endotoxins may challenge the efficacy of viral vector treatment. In certain embodiments, for example, compared with compositions with a higher amount, these low amounts of residual host cell proteins, host cell DNA, plasmid DNA and / or endotoxins improve therapeutic efficacy and / or reduce adverse events (e.g., inflammatory response, liver injury) after being applied to the patient. In certain embodiments, compared with the levels in existing methods (e.g., not using those of adhesion-type cells and / or purification methods described herein), the method for preparing viral vectors disclosed herein provides these improved levels. In certain embodiments, in addition to the residual host cell proteins, host cell DNA, plasmid DNA and / or endotoxins of amount, the method herein also allows the preparation of viral vectors with reduced empty capsid percentages.

[0365] In some embodiments, the amount of residual cesium after TFF (e.g., the second TFF) is less than about 50 μg / g. In some embodiments, the amount of residual cesium after TFF (e.g., the second TFF) is less than about 30 μg / g. In some embodiments, the amount of residual cesium after TFF (e.g., the second TFF) is less than about 20 ug / g. In some embodiments, the residual cesium in the pharmaceutical composition is less than or equal to 30 ug / g (ppm). In some embodiments, the amount of residual CsCl can be measured by mass spectrometry, inductively coupled plasma mass spectrometry (ICP-MS) and / or another suitable method. In some embodiments, the amount of residual cesium after the second TFF is less than, for example, the quantitative limit using ICP-MS.

[0366] In some embodiments, the concentration of the AAV viral vector collected after the second TFF is greater than or equal to about 5×10 12 vg / ml, greater than or equal to about 1x10 13 vg / ml or greater than or equal to about 3x10 13 vg / ml.

[0367] In one embodiment, the pharmaceutical composition has one or more of the following: 13 vg less than 0.09ng universal nuclease, less than 30μg / g (ppm) cesium, about 20-80ppm poloxamer 188, per 1.0x10 13 vg less than 0.22ng BSA, per 1.0x10 13 vg less than 6.8x10 5 pg of residual plasmid DNA, each 1.0x10 13 vg less than 1.1x10 5 pg of residual hcDNA and 1.0x10 13 vg less than 4ng of rHCP.

[0368] In some embodiments, the potency of the pharmaceutical composition is measured using a cell-based in vitro potency assay as described herein. In some embodiments, the pharmaceutical composition comprises a viral vector, wherein 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% relative to a reference standard. In some embodiments, the pharmaceutical composition comprises a viral vector, wherein the relative potency of the viral vector is at least 90% relative to a reference standard.

[0369] Viral particles (e.g., viral particles) purified according to the present disclosure can be formulated according to methods known for preparing pharmaceutically useful compositions. The compositions of the present disclosure can be formulated for administration to mammalian subjects, such as humans, using techniques known in the art. In particular, the delivery system can be formulated for intramuscular, intradermal, mucosal, subcutaneous, intravenous, intrathecal, injectable reservoir devices, or topical administration.

[0370] When the delivery system is formulated as a solution or suspension, the delivery system is in an acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, such as water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid, etc. These compositions can be sterilized by conventional, well-known sterilization techniques, or can be aseptically filtered. The resulting aqueous solution can be packaged for use as is or lyophilized, and the lyophilized preparation is combined with a sterile solution before administration.

[0371] Compositions (e.g., pharmaceutical compositions) may contain pharmaceutically acceptable auxiliary substances close to physiological conditions, such as pH regulators and buffers, tension regulators, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. In some embodiments, the pharmaceutical composition comprises a preservative. In some other embodiments, the pharmaceutical composition does not comprise a preservative.

[0372] The genomic titer of viral vectors (such as those in the compositions and formulations disclosed herein) can be determined in a variety of standard ways. PCR using primers specific to viral vectors can provide relative measurements, but quantitative PCR (qPCR) can be used for fewer samples and absolute measurements. Droplet digital PCR (ddPCR) is a method for performing digital PCR based on water-oil emulsion droplet technology. The sample is divided into tens of thousands of droplets, and PCR amplification of the template molecule occurs in each individual droplet. There is no need to draw a standard curve or use primers with high amplification efficiency, so ddPCR typically does not use as many samples as traditional PCR-based techniques. In one embodiment, the genomic titer of the viral vector is determined using PCR. In another embodiment, the genomic titer of the viral vector is determined using qPCR. In another embodiment, the genomic titer of the viral vector is determined using ddPC. Methods for determining viral genome titers using ddPCR have been described, for example, in Lock et al., “Absolute Determination of Single-Stranded and Self-Complementary Adeno-Associated Viral Vector Genome Titers by Droplet Digital PCR,” Human Gene Therapy Methods, 25(2): 115-125.

[0373] In some embodiments, the PCR-based method uses primers and probes specifically designed to target the SMN gene to detect and quantify the encapsidated AAV9 viral genome. In other embodiments, the PCR-based method uses primers and probes specifically designed to target the chicken β-actin promoter to detect and quantify the encapsidated AAV9 viral genome. In other embodiments, the PCR-based method uses primers and probes specifically designed to target the CMV enhancer to detect and quantify the encapsidated AAV9 viral genome. In other embodiments, the PCR-based method uses primers and probes specifically designed to target the ITR sequence to detect and quantify the encapsidated AAV9 viral genome. In other embodiments, the PCR-based method uses primers and probes specifically designed to target the bovine growth hormone polyadenylation signal to detect and quantify the encapsidated AAV9 viral genome.

[0374] In some embodiments, the pharmaceutical composition is about pH 7.7-8.3 and has an osmotic pressure concentration of 390-430 mOsm / kg. In some embodiments, pH is measured using a pH meter. In some embodiments, pH is measured according to standards set by the United States Pharmacopeia (USP) (e.g., <791> ) (incorporated by reference in its entirety) is measured potentiometrically using a microelectrode with temperature compensation. In some embodiments, osmolarity is measured according to USP (e.g., USP <785> ) (incorporated by reference in its entirety) using freezing point depression. In some embodiments, osmolarity is measured using a vapor pressure reduction osmometer. In other embodiments, osmotic pressure is measured using a membrane osmometer.

[0375] In one embodiment, the intravenous formulation has a pH between 7.5 and 8.5, 2×10 13 vg / ml-6X10 13 vg / ml and a molar osmotic pressure concentration of 384-448mOsm / kg. In another embodiment, the intravenous formulation has a pH between 7.5 and 8.5, 1.5X10 13 vg / ml-3.5X10 13 vg / ml and a molar osmotic pressure concentration of 384-448mOsm / kg. In another embodiment, the intravenous formulation has a pH between 7.5 and 8.5, 1.8X10 13 vg / ml-2.2X10 13 vg / ml and a molar osmotic pressure concentration of 384-448mOsm / kg. In one embodiment, the IV formulation contains about 0.1-2.0mM MgCl 2 In one embodiment, the IV formulation comprises about 100-300 mM NaCl. In one embodiment, the IV formulation comprises about 0.001%-0.01% w / v Poloxamer 188. In one embodiment, the IV formulation is an aqueous formulation in 10-30 mM Tris buffer, e.g., at pH 7.5-8.5.

[0376] In one embodiment, the IV formulation comprises 1 mM MgCl in 20 mM Tris buffer at pH 8.0. 2 , 200 mM NaCl, 0.005% w / v Poloxamer 188. In an embodiment, the IV formulation comprises about 1 x 10 13 Up to 3x10 13 vg / mL or 1.7x10 13 Up to 2.3x10 13 The genome titer was vg / mL.

[0377] Uses of the pharmaceutical composition

[0378] The present disclosure provides a method of treating a patient in need thereof with a therapy comprising a viral vector containing a transgene, the method comprising: assaying the viral vector containing a transgene according to the cell-based in vitro potency assay described herein, and administering the viral vector containing a transgene to the patient. In some embodiments, the viral vector is formulated in a pharmaceutical composition.

[0379] In other embodiments, methods for delivering polynucleotides to the central nervous system of a patient are disclosed herein, comprising administering an rAAV9 having a genome that will include the polynucleotides. In some embodiments, delivery is intrathecal delivery of the polynucleotides to the central nervous system of the patient, comprising administering an rAAV9 having a genome that will include the polynucleotides. In some embodiments, a non-ionic low-osmotic contrast agent is also administered to the patient. The non-ionic low-osmotic contrast agent can increase the transduction of target cells in the central nervous system of the patient. In some embodiments, the rAAV9 genome is a self-complementary genome. In other embodiments, the rAAV9 genome is a single-stranded genome.

[0380] In some embodiments, nonionic low permeability contrast agents are also administered to patients. More specifically, the present invention provides methods for delivering vectors to the central nervous system of patients in need, including delivering rAAV9 and nonionic low permeability contrast agents intrathecally to patients, wherein rAAV9 comprises polynucleotides encoding target proteins. In some embodiments, the target protein is SMN1. Polynucleotides are delivered to, for example, the brain, spinal cord, glial cells, astrocytes and / or lower motor neurons. Nonionic low permeability contrast agents are, for example, iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol or ioxilan. In some embodiments, polynucleotides are survival of motor neurons (SMN) polynucleotides. Exemplary iohexol radiopaque agents include [Omnipaque.TM. (iohexol, N,N'-bis(2,3-dihydroxypropyl)-5-[N(2,3-dihydroxypropyl)-acetamido]-2,4,6-triiodo-isophthalamide), GE Healthcare, Waukesha, Wis.]. In some embodiments, the vector and contrast agent are administered intrathecally, and intrathecal diffusion is recorded using real-time continuous fluoroscopy.

[0381] In some embodiments, the polynucleotides are delivered to brain regions. Brain regions contemplated for delivery include, but are not limited to, the motor cortex and brainstem. In some embodiments, the polynucleotides are delivered to the spinal cord. In some embodiments, the polynucleotides are delivered to lower motor neurons. Embodiments of the disclosure use rAAV9 to deliver polynucleotides to nerves and glial cells. In some embodiments, the glial cells are microglia, oligodendrocytes, or astrocytes. In some embodiments, rAAV9 is used to deliver polynucleotides to Schwann cells.

[0382] In some embodiments, use of these methods and materials for treating spinal muscular atrophy (SMA) is indicated.

[0383] There are four types of SMA, which are conventionally classified by age of onset and the highest motor function achieved. All forms of SMA are autosomal recessive and are caused by mutations in the survival motor neuron 1 (SMN1) gene. Humans also carry a second, nearly identical copy of the SMN gene, called SMN2. Lefebvre et al. "Identification and characterization of a spinal muscular atrophy-determining gene." Cell, 80(1):155-65. Monani et al. "Spinal muscular atrophy: a deficiency in a ubiquitous protein; a motor-neuron specific disease." Neuron, 48(6):885-896. Both the SMN1 and SMN2 genes express the SMN protein, however SMN2 contains a translationally silent mutation in exon 7, which results in the inclusion of an ineffective exon 7 in the SMN2 transcript. Thus, SMN2 produces both the full-length SMN protein and a truncated form of SMN lacking exon 7, with the truncated form being the predominant form. Thus, the amount of functional full-length protein produced by SMN2 is much less (70%-90% less) than the amount of functional full-length protein produced by SMN1. Lorson et al. "A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy." PNAS, 96(11): 6307-6311. Monani et al., "A single nucleotide difference that alters splicing patternsdistinguishes the SMA gene SMN1 from the copygene SMN2." Hum Mol Genet [Human Molecular Genetics] 8(7): 1177-1183. Although SMN2 cannot fully compensate for the loss of the SMN1 gene, patients with milder forms of SMA often have more copies of SMN2.Lefebvre et al., "Correlation between severity and SMN protein level in spinal muscular atrophy." Nat Genet 16(3):265-269. Park et al., "Spinal muscular atrophy: new and emerging insights from model mice." Curr Neurol Neurosci Rep 10(2):108-117. It is important to note that SMN2 copy number is not the only phenotypic modifier. In particular, the c.859G>C variant in exon 7 of the SMN2 gene has been reported as a positive disease modifier. Patients with this particular mutation have a less severe disease phenotype. Prior et al., “A positive modification of spinal muscular atrophy in the SMN2 gene.” Am J Hum Genet 85(3):408-413.

[0384] When SMA symptoms appear at birth or before 6 months of age, it is Type I SMA (also called infantile-onset or Werdnig-Hoffmann disease). In this type, infants typically have low muscle tone (hypotonia), a weak cry, and difficulty breathing. They often have difficulty swallowing and sucking, and do not reach the developmental milestone of being able to sit up without assistance. They often show one or more of the following SMA symptoms: hypotonia, delayed motor skills, poor head control, rounded shoulder posture, and joint hypermobility. Typically, these infants have two copies of the SMN2 gene, one copy on each chromosome 5. More than half of all new SMA cases are SMA Type I.

[0385] When SMA develops between 7 and 18 months of age and before children can stand or walk independently, it is type II or intermediate SMA. Children with type 2 SMA usually have at least three SMN2 genes. Late-onset SMA (also known as type III and IV SMA, mild SMA, adult-onset SMA, and Kugelberg-Welander disease) causes different levels of weakness. Type III SMA develops after 18 months, and children can stand and walk independently, but they may need help. Type IV SMA develops in adulthood, and people are able to walk during their adulthood. People with type III or IV SMA usually have between four and eight SMN2 genes, which can produce quite a lot of full-length SMN protein.

[0386] In one embodiment, the term "treatment" includes the step of administering an effective dose or effective multiple doses of a composition comprising rAAV as disclosed herein intravenously or via an intrathecal route to an animal (including a human) in need thereof. If the dose is administered before the development of the disorder / disease, the administration is preventive. If the dose is administered after the development of the disease, the administration is therapeutic. In an embodiment, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, slows or prevents progression to a disorder / disease state, slows or prevents the progression of a disorder / disease state, reduces the extent of the disease, causes the disease to be alleviated (partially or completely), and / or prolongs survival. Examples of disease states envisioned for treatment are listed herein.

[0387] In one embodiment, a composition comprising a rAAV of the disclosure is administered intravenously to a patient with SMA (e.g., SMA Type I) in need thereof. In some embodiments, the patient with SMA is less than 2 years old. In some embodiments, the patient has a biallelic mutation in the SMN1 gene. In another embodiment, a composition comprising a rAAV of the disclosure is administered intrathecally to a patient with SMA Type II, III, or IV in need thereof. In some embodiments, a composition comprising a rAAV of the disclosure is used to treat a pediatric patient less than 2 years old who has SMA and has a biallelic mutation in the SMN1 gene.

[0388] Disclosed herein is a method for treating SMA (e.g., type I SMA) in a patient in need thereof by administering an AAV9 viral vector via an intrathecal or intravenous route. In some embodiments, the patient is 0-9 months old. In some other embodiments, the patient is 0-6 months old. In some embodiments, the patient is less than 2 years old. In some embodiments, the patient is less than 2 years old and has a biallelic mutation in the SMN1 gene. In some embodiments where a viral vector is used to treat a patient's SMA (e.g., type I SMA), the patient's weight is determined. In some embodiments, the patient has a weight of less than 13.5 kg. In some embodiments, the patient has a weight of less than 8.5 kg. In some embodiments, the patient has a weight of more than 2.6 kg. In some embodiments, the patient has a weight of 2.6-8.5 kg. In some embodiments, the patient has a weight of 2.6-13.5 kg.

[0389] In some embodiments, the patient has a mutation in one copy of the SMN1 gene, such as a null mutation (encompassing any mutation that renders the encoded SMN1 nonfunctional). In some embodiments, the patient has a mutation in two copies of the SMN1 gene, such as a null mutation. In some embodiments, the patient has a mutation in all copies of the SMN1 gene, such as a null mutation. In some embodiments, the patient has a deletion in one copy of the SMN1 gene. In some embodiments, the patient has a deletion in two copies of the SMN1 gene. In some embodiments, the patient has a biallelic SMN1 mutation, i.e., a deletion or substitution of SMN1 in both alleles of a chromosome. In some embodiments, the patient has at least one functional copy of the SMN2 gene. In some embodiments, the patient has at least two functional copies of the SMN2 gene. In some embodiments, the patient has at least two functional copies of the SMN2 gene. In some embodiments, the patient has at least three functional copies of the SMN2 gene. In some embodiments, the patient has at least four functional copies of the SMN2 gene. In some embodiments, the patient has at least five functional copies of the SMN2 gene. In some embodiments, the patient does not have a c.859G>C substitution in exon 7 of at least one copy of the SMN2 gene. In some embodiments, the genetic sequence of the SMN1 or SMN2 gene can be determined by whole genome sequencing. In other embodiments, the genetic sequence and copy number of the SMN1 or SMN2 gene can be determined by high-throughput sequencing. In some embodiments, the genetic sequence and copy number of the SMN1 or SMN2 gene can be determined by microarray analysis. In some embodiments, the genetic sequence and copy number of the SMN1 or SMN2 gene can be determined by Sanger sequencing. In some embodiments, the copy number of the SMN1 or SMN2 gene can be determined by fluorescence in situ hybridization (FISH).

[0390] In some embodiments, the patient shows one or more SMA symptoms.SMA symptoms may include hypotonia, delayed motor skills, poor head control, rounded shoulder posture and joint hypermobility.In some embodiments, poor head control is measured by placing the patient in a sitting position and assisting in the shoulder (front and back).Head control is assessed by the patient's ability to keep his head upright.In some embodiments, spontaneous movement is observed when the patient is in a supine position, and motor skills are assessed by the patient's ability to lift his elbows, knees, hands and feet off the surface.In certain embodiments, the patient's grip is measured by placing fingers in the patient's palm and lifting the patient until his shoulder leaves the surface.Hypotonia and grip are measured by how fast / how long the patient maintains grip.In some embodiments, head control is assessed by placing the patient's head in the maximum possible rotation and measuring the ability of the patient's head to turn back to the midline.In some embodiments, shoulder posture can be assessed by making the patient sit with the head and trunk supported, and observing whether the patient flexes the elbow or shoulder to reach the stimulus placed at the shoulder level at arm length. In some embodiments, shoulder posture may also be assessed by placing the patient in the lateral decubitus position and observing whether the patient flexes the elbow or shoulder to reach for a stimulus placed at shoulder level at arm's length. In some embodiments, motor skills are assessed by observing whether the patient flexes their hip or knee when the foot is stroked, tickled or pinched. In some embodiments, shoulder flexion, elbow flexion, hip adduction, neck flexion, head extension, neck extension and / or spinal flexion may be assessed by known clinical measures, such as CHOP INTEND. Other SMA symptoms may be evaluated according to known clinical measures, such as CHOP INTEND.

[0391] In some embodiments, the patient is treated after he or she exhibits symptoms (e.g., one or more symptoms) of SMA (e.g., SMA Type I) as determined using one of the tests described herein. In some embodiments, the patient is treated before he or she exhibits symptoms of SMA (e.g., SMA Type I). In some embodiments, the patient is diagnosed with SMA (e.g., SMA Type I) based on genetic testing before the onset of symptoms.

[0392] Combination therapy is also contemplated herein. Combinations as used herein include simultaneous treatment or sequential treatment. The combination of methods may include the addition of certain standard medical treatments (e.g., riluzole (ALS) in ALS), as well as combinations with novel therapies. For example, other therapies for SMA include antisense oligonucleotides (ASOs) that change the binding to precursor mRNA and change its splicing pattern. Singh et al., "A multi-exon-skipping detection assay reveals surprising diversity of splice isoforms of spinal muscular atrophy genes." Plos One [Public Library of Science Comprehensive], 7 (11): e49595. In one embodiment, nusinersen (U.S. Pat. No. 8,361,977 and US 8,980,853, which are incorporated herein by reference) may be used. Nusinersen is an approved ASO that targets intron 6, exon 7, or intron 7 of SMN2 pre-mRNA to regulate the splicing of SMN2 to more efficiently produce full-length SMN protein. In some embodiments, a treatment method comprising an AAV9 viral vector is administered in combination with a muscle enhancer. In some embodiments, a treatment method comprising an AAV9 viral vector is administered in combination with a neuroprotectant. In some embodiments, a treatment method comprising an AAV9 viral vector is administered in combination with an antisense oligonucleotide-based drug targeting SMN. In some embodiments, a treatment method comprising an AAV9 viral vector is administered in combination with nusinersen. In some embodiments, a treatment method comprising an AAV9 viral vector is administered in combination with a drug that inhibits myostatin. In some embodiments, a treatment method comprising an AAV9 viral vector is administered in combination with stamulumab.

[0393] While postnatal delivery to an individual in need is contemplated, intrauterine delivery to a fetus is also contemplated.

[0394] Methods of treating patients with SMA (e.g., type I SMA) using pharmaceutical compositions comprising viral vectors are contemplated. In some embodiments, about 1-8 x 10 13 The viral vector is formulated at a concentration of about 1.7-2.3 x 10 AAV9 viral vector genomes / mL (vg / mL). 13 The viral vector is formulated at a concentration of about 1.9-2.1 x 10 13The viral vector is prepared at a concentration of about 2.0 x 10 13 The viral vector was prepared at a concentration of vg / mL.

[0395] In some embodiments where a viral vector is used to treat SMA (e.g., type I SMA) in a patient, an AAV viral vector (e.g., AAV SMN) is administered at a concentration of about 1.0-2.5 x 10 14 vg / kg is administered to a patient. In some embodiments where a viral vector is used to treat SMA (e.g., type I SMA) in a patient, the AAV viral vector is administered at a dose of about 1.1 x 10 14 In some embodiments, the AAV viral vector is infused into the patient over about 45-70 min. In some embodiments, the AAV viral vector is infused into the patient over about 6 ...

[0396] The titer of the rAAV viral vector to be administered will vary depending on, for example, the particular rAAV, the mode of administration, the therapeutic goal, the individual, and the cell type or cells being targeted, and can be determined by methods standard in the art. The titer of rAAV can range from about 1×10 6 , about 1X10 7 , about 1X10 8 , about 1X10 9 , about 1X10 10 , about 1X10 11 , about 1X10 12 , about 1X10 13 , about 1X10 14 or more DNase-resistant particles (DRP). Dosages can also be expressed in units of vector genomes (vg). Genomic titers can be measured using ddPCR as described in the present application, Lock et al., or any other method known in the art.

[0397] The dosage may also vary depending on the schedule of administration to humans. In adults, these dosages of rAAV may range from about 1×10 11 vg / kg, about 1X10 12 vg / kg, about 1X10 13vg / kg, about 1X10 14 vg / kg, about 1X10 15 vg / kg, about 1X10 16 vg / kg or more vector genomes / kg body weight. For neonates, the rAAV dose range can be about 1×10 11 vg / kg, about 1X10 12 vg / kg, about 3X10 12 vg / kg, about 1X10 13 vg / kg, about 3X10 13 vg / kg, about 1X10 14 vg / kg, about 3X10 14 vg / kg, about 1X10 15 vg / kg, about 3X10 15 vg / kg, about 1X10 16 vg / kg, about 3X10 16 vg / kg or more vector genomes / kg body weight.

[0398] The dosage may also vary depending on the schedule of administration to humans. In adults, these dosages of rAAV may range from about 1×10 11 vg / kg / week, about 1X10 12 vg / kg / week, about 1X10 13 vg / kg / week, about 1X10 14 vg / kg / week, about 1X10 15 vg / kg / week, about 1X10 16 vg / kg / week or more vector genomes / kg body weight. For neonates, rAAV doses can range from about 1×10 11 vg / kg / week, about 1X10 12 vg / kg / week, about 3X10 12 vg / kg / week, about 1X10 13 vg / kg / week, about 3X10 13 vg / kg / week, about 1X10 14 vg / kg / week, about 3X10 14 vg / kg / week, about 1X10 15 vg / kg / week, about 3X10 15 vg / kg / week, about 1X10 16 vg / kg / week, about 3X10 16 vg / kg / week or more vector genomes / kg body weight / week. In adults, rAAV doses can range from 1X10 11 vg / 1.5kg / week, about 1X1012 vg / 1.5kg / week, about 1X10 13 vg / 1.5kg / week, about 1X10 14 vg / 1.5kg / week, about 1X10 15 vg / 1.5kg / week, about 1X10 16 vg / 1.5kg / week or more vector genomes / kg body weight. For neonates, the rAAV dose range can be about 1×10 11 vg / 1.5kg / week, about 1X10 12 vg / 1.5kg / week, about 3X10 12 vg / kg / week, about 1X10 13 vg / 1.5kg / week, about 3X10 13 vg / 1.5kg / week, about 1X10 14 vg / 1.5kg / week, about 3X10 14 vg / 1.5kg / week, about 1X10 15 vg / 1.5kg / week, about 3X10 15 vg / 1.5kg / week, about 1X10 16 vg / 1.5kg / week, about 3X10 16 vg / 1.5kg / week or more vector genomes / 1.5kg body weight / week.

[0399] In some embodiments, the dose retains 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% of the potency relative to a reference standard at the same dose. In some embodiments, the dose retains at least 90% of the potency relative to a reference standard at the same dose. In some embodiments, the relative potency is determined using a cell-based in vitro relative potency assay as described herein.

[0400] In one embodiment, the dose is about 1.1×10 14 Vector genome / kg patient body weight (vg / kg). In one embodiment, a 5 kg patient will receive 0.5×10 14 Up to 5.0X10 14In one embodiment, the viral vector is administered in Tris-buffered saline. In one embodiment, the viral vector is administered in about 5-20 mL / kg, about 10-20 mL / kg, or about 5.5-6.5 mL / kg of Tris-buffered saline.

[0401] The dose can be determined in a variety of standard ways. PCR using primers specific to viral vectors can provide relative measurements, but qPCR can be used for fewer samples and absolute measurements. ddPCR is a method for performing digital PCR based on water-oil emulsion droplet technology. Baker et al., "Digital PCR hits its stride [digital PCR has made great progress]." Nature Methods [Natural Methods], 9 (6): 541-544. Sykes et al., "Quantitation of targets for PCR by use of limiting dilution [quantitatively used for PCR targets by using limited dilution]." Biotechniques [Biotechniques], 13 (3) 444-449. The sample is divided into tens of thousands of droplets, and PCR amplification of the template molecule occurs in each individual droplet. There is no need to draw a standard curve or use primers with high amplification efficiency, so ddPCR typically does not use as many samples as traditional PCR-based technologies. Examples of commercially available ddPCR machines include, but are not limited to, BioRad QX100 ddPCR and RainDance Raindrop digital PCR. In one embodiment, the dosage is determined using PCR. In another embodiment, the dosage is determined using qPCR. In another embodiment, the dosage is determined using digital droplet PCR (ddPCR). In some embodiments, the PCR-based method uses primers and probes specifically designed to target the SMN gene to detect and quantify the encapsidated AAV9 viral genome. In other embodiments, the PCR-based method uses primers and probes specifically designed to target the chicken β-actin promoter to detect and quantify the encapsidated AAV9 viral genome. In other embodiments, the PCR-based method uses primers and probes specifically designed to target the CMV enhancer to detect and quantify the encapsidated AAV9 viral genome. In other embodiments, the PCR-based method uses primers and probes specifically designed to target the ITR sequence to detect and quantify the encapsidated AAV9 viral genome. In other embodiments, the PCR-based method uses primers and probes specifically designed to target the bovine growth hormone polyadenylation signal to detect and quantify the encapsidated AAV9 viral genome.

[0402] In one aspect, the dosage is administered according to the following table, using 2.0X1013 vg / ml as the target concentration of the drug product.

[0403] Table 2. Dosage

[0404]

[0405]

[0406] a NOTE: Dose volume is calculated using the upper limit of the patient's weight range.

[0407] In some embodiments, the pharmaceutical composition comprising the AAV viral vector is infused into the patient over about 20-70 minutes, for example, over about 45-70 minutes. In some embodiments, the pharmaceutical composition comprising the AAV viral vector is infused into the patient over about 60 min. In some embodiments, the pharmaceutical composition comprising the AAV viral vector is infused into the patient using an infusion pump, a peristaltic pump, or any other device known in the art. In some embodiments, the pharmaceutical composition comprising the AAV viral vector is infused into the patient using a syringe pump.

[0408] Prescreening of patients amenable to treatment is also contemplated, as well as administration of treatment to patients identified according to the criteria disclosed herein. AAV can elicit both cellular and humoral immune responses. Therefore, a portion of potential patients for AAV-based gene therapy have pre-existing antibodies against AAV. Jeune et al., "Pre-existing anti-Adeno-Associated Virus antibodies as a challenge in AAV gene therapy." Hum Gene Ther Methods, 24(2): 59-67. Boutin et al., "Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors." Hum Gene Ther, 21: 704-712. Because even very low levels of antibodies may prevent successful transduction, previous anti-AAV antibodies constitute a serious obstacle to the widespread application of AAV gene therapy. In some embodiments, the level of anti-AAV9 antibody titer in the patient is determined prior to administration of the AAV viral vector. In some embodiments, the level of anti-AAV9 antibody titer in the patient is determined by ELISA combined immunoassay. In some embodiments, the patient has an anti-AAV9 antibody titer equal to or less than 1:100 as determined by ELISA combined immunoassay prior to administration of the treatment. In some embodiments, the patient has an anti-AAV9 antibody titer of equal to or less than 1:50 as determined by ELISA combined immunoassay prior to administration of the treatment. In some embodiments, the patient has an anti-AAV9 antibody titer of greater than 1:100 as determined by ELISA combined immunoassay after treatment, and the patient is monitored for 1-8 weeks or until the titer decreases to less than 1:100. In some embodiments, the patient has an anti-AAV9 antibody titer of greater than 1:100 as determined by ELISA combined immunoassay after treatment, and the patient is monitored for 1-8 weeks or until the titer decreases to less than 1:50.

[0409] One way to overcome high anti-AAV antibody titers is to use immunosuppressant drugs. The combination of anti-CD20 monoclonal antibody rituximab and cyclosporin A has been shown to effectively reduce anti-AAV titers. Mingozzi et al., "Pharmacological modulation of humoral immunity in a nonhuman primate model of AAV gene transfer for hemophilia B." Mol Ther [Molecular Therapy], 20: 1410-1416. Another approach is to use plasma exchange to deplete neutralizing antibodies before vector administration. Monteilhet et al., "A 10 patient case report on the impact of plasmapheresis upon neutralizing factors against adeno-associated virus (AAV) types 1, 2, 6, and 8." Mol Ther, 19(11): 2084-2091. During plasmapheresis, blood is drawn from the patient, and plasma and blood cells are separated by centrifugation or hollow fiber filtration. The blood cells are then returned to the patient along with processed plasma or a replacement fluid such as 4.5% human albumin in saline. A common use of therapeutic apheresis is to remove unwanted immunoglobulins, but in this case, plasmapheresis represents an attractive method to deplete anti-AAV antibodies. In some embodiments, the patient has an anti-AAV9 antibody titer of greater than 1:100 as determined by ELISA combined immunoassay before or after treatment and is treated with plasmapheresis. In some embodiments, the patient has an anti-AAV9 antibody titer above 1:50 as determined by ELISA binding immunoassay before or after treatment and is treated with plasma exchange.

[0410] Pre-existing maternal antibodies to AAV9 can be transferred to the infant patient via breast milk or placental transfer in the uterus. In some embodiments, the patient has an anti-AAV9 antibody titer of greater than 1:100 as determined by ELISA combined immunoassay before or after treatment and is switched to formula feeding. In some embodiments, the patient has an anti-AAV9 antibody titer of greater than 1:50 as determined by ELISA combined immunoassay before or after treatment and is switched to formula feeding.

[0411] Before and after administration of the treatment, the patient's condition can be monitored. Some patients who have received AAV-based treatments have experienced thrombocytopenia, a condition characterized by low platelet counts. Thrombocytopenia can be detected by a complete blood count on a hemacytometer using a diluted blood sample. Thrombocytopenia can also be detected by observing a slide (blood film or peripheral blood smear) prepared with the patient's blood under a microscope. The normal human platelet count ranges from 150,000 cells / ml to about 450,000 cells / ml.

[0412] In some embodiments, the patient has a platelet count higher than about 67,000 cells / ml or higher than about 100,000 cells / ml or higher than about 150,000 cells / ml before administration. In some embodiments, the patient has a platelet count lower than about 150,000 cells / ml or lower than about 100,000 cells / ml or lower than about 67,000 cells / ml before administration, and the patient is monitored for 1-8 weeks or until the platelet count increases to higher than about 67,000 cells / ml, or higher than about 100,000 cells / ml, or higher than about 150,000 cells / ml. In some embodiments where the platelet count is lower than about 67,000 cells / ml after administration of the viral vector, platelet transfusions can be used to treat the patient. In some embodiments, the patient does not suffer from thrombocytopenia before administration of the viral vector. In some embodiments, the patient suffers from thrombocytopenia after administration of the viral vector, and the patient is monitored for about 1-8 weeks or until the patient is free of thrombocytopenia. In some embodiments, the patient suffers from thrombocytopenia after administration of the viral vector, and the patient is treated with platelet transfusions.

[0413] Monitoring the patient's condition may also involve standard blood tests measuring the following items: platelets, serum protein electrophoresis, serum gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), total bilirubin, glucose, creatine kinase (CK), creatinine, blood urea nitrogen (BUN), electrolytes, alkaline phosphatase and amylase. Troponin I levels are a general measure of heart health, and elevated levels reflect heart damage or heart-related conditions. In some embodiments, Troponin-I levels are monitored after administration of the viral vector. In some embodiments, before administration of the viral vector, the patient may have a Troponin-I level of less than about 0.3, 0.2, 0.15 or 0.1 μg / ml. In some embodiments, before administration of the viral vector, the patient may have a Troponin-I level of less than about 0.176 μg / ml. In some embodiments, after administration of the viral vector, the patient may have a Troponin-I level higher than about 0.176 μg / ml. In some embodiments, the patient receives cardiac monitoring after administration of the viral vector until Troponin-I levels are less than about 0.176 μg / ml.

[0414] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and total bilirubin are general measures of liver function, while creatinine tracks renal function. Increased levels of AST, ALT or total bilirubin may indicate liver dysfunction. In some embodiments, the patient has normal liver function before administering the viral vector. In some embodiments, the patient has a liver transaminase level of less than about 8-40U / L before administering the viral vector. In some embodiments, the patient has an AST or ALT level of less than about 8-40U / L before administering the viral vector. In some embodiments, the patient has a bilirubin level of less than 3.0mg / dL before administering the viral vector. In some embodiments, the patient has a creatinine level of less than 1.8mg / dL before administering the viral vector. In some embodiments, the patient has a hemoglobin (Hgb) level between 8-18g / dL before administering the viral vector. In some embodiments, the patient has a hemoglobin (Hgb) level of less than 20000 / mm before administering the viral vector. 3 White blood cell (WBC) count.

[0415] The efficacy of the treatment method can be determined using a variety of tests for motor skills before and after treatment. In particular, the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) was developed to evaluate the motor skills of patients with type I SMA. Glanzman et al., "The Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND): Test development and reliability." Neuromuscular Disorders, 20(3): 155-161. The CHOP INTEND test was developed after evaluating 26 infants with a mean age of 11.5 months (1.4 to 37.9 months) with SMA Type I using the Test of Infant Motor Performance (TIMP) and The Children's Hospital of Philadelphia Test of Strength in SMA (CHOP TOSS), both newly designed for motor assessment in SMA. Testing of treatment efficacy is not limited to the CHOP INTEND test, but may also include other motor skill tests known in the art, including but not limited to TIMP, CHOP TOSS, Peabody Development Motor Scale, Brazelton Neonatal Behavior Assessment test, Motor Milestone Development Survey, Ability Captured Through Interactive Video Evaluation (ACTIVE), Bayley Scale of Infant Development, and measurements of compound motor action potentials (CMAP).

[0416] In some embodiments, baseline testing prior to treatment is performed using the CHOP INTEND scale. In one embodiment, the efficacy of treatment is determined during a follow-up visit using the CHOP INTEND scale. In some embodiments, CHOP INTEND includes measures of head control, righting response, trunk movement in supported sitting, supine, and prone positions. In some embodiments, CHOP INTEND includes measures of anti-gravity movement in assisted rolling, abdominal sling, and supported standing.

[0417] In many gene therapy studies involving AAV vectors, antigen-specific T cell responses to AAV vectors have been observed, and can be expected to be between 2-4 weeks after gene transfer. A possible result of this antigen-specific T cell response is the removal of transduced cells and the loss of transgenic expression. In order to suppress the host's immune response to AAV-based therapy, patients can be given immunosuppressants. In some embodiments, glucocorticoids can be given to patients before the administration of viral vectors. In some embodiments, corticosteroids can be given to patients before the administration of viral vectors. In some embodiments, oral steroids can be given to patients before the administration of viral vectors. Examples of oral steroids include but are not limited to prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, dexamethasone and hydrocortisone. In some embodiments, oral steroids are or include prednisolone. In some embodiments, the patient starts using prophylactic steroids at least 24 hours before the administration of viral vectors. In some embodiments, after the administration of viral vectors, oral steroids are given to patients for at least 30 days. In some embodiments, oral steroids are administered once a day. In some embodiments, oral steroids are administered twice daily. In some embodiments, oral steroids are administered at a dosage of about 0.1-10 mg / kg, for example, about 1 mg / kg. In some embodiments, oral steroids are administered at a dosage of about 0.1-10 mg / kg / day, for example, about 1 mg / kg / day. In some embodiments, the levels of AST and ALT are monitored after administration of the viral vector. In such embodiments, when AST and ALT levels are, for example, measured by clinical standards and methods known in the art, more than twice the upper limit of normal, or about 120 IU / L, oral steroids are administered for treatment. In some embodiments, as long as AST and ALT levels are, for example, measured by clinical standards and methods known in the art, more than twice the upper limit of normal, or more than about 120 IU / L, oral steroids are administered for treatment for more than 30 days. During continuous treatment with corticosteroids, the adrenal glands naturally reduce the production of cortisol. If corticosteroid treatment is stopped suddenly, the body may experience cortisol deficiency. In some embodiments where oral steroids are administered to patients for at least 30 days, the steroid dosage is gradually reduced slowly on schedule. In some embodiments, the oral steroid dose is gradually reduced when AST and ALT levels drop to less than twice the upper limit of normal, or about 120 IU / L, for example, as determined by clinical criteria and methods known in the art. In some embodiments, the gradual reduction includes a stepwise reduction to 0.5 mg / kg / day for 2 weeks, followed by 0.25 mg / kg / day for another 2 weeks. In some other embodiments, the gradual reduction of oral steroids is performed at the discretion of a physician.

[0418] Reagent test kit

[0419] In one embodiment, the present invention provides a kit comprising:

[0420] a. Multiple cells capable of being transduced with viral vectors;

[0421] b. a viral vector encoding a target protein;

[0422] c. a first molecule capable of binding to the target protein;

[0423] d. a second molecule capable of binding to the first molecule, wherein the second molecule comprises a detectable label; and,

[0424] e. Instructions for use in imaging assays.

[0425] In some embodiments of the kits of the present disclosure, the plurality of cells comprises neural progenitor cells (mTD-NPC-?7) in an SMN1- / - genetic background.

[0426] In some embodiments, the viral vector drug product is an adeno-associated virus serotype 9 (AAV9) comprising a cDNA expressing an SMN1 protein under the control of a cytomegalovirus (CMV) enhancer / chicken-β-actin hybrid promoter (CB), and two AAV inverted terminal repeats (ITRs) from AAV serotype 2 (AAV2) DNA. In some embodiments, the vector comprises the sequence of SEQ ID NO: 2. In some embodiments, the vector comprises the sequence of SEQ ID NO: 1. In some embodiments, the vector comprises a sequence encoding an SMN1 protein comprising the amino acid sequence of SEQ ID NO: 3.

[0427] In some embodiments, the first molecule comprises an anti-SMN1 antibody.Exemplary antibodies include the mouse monoclonal antibody 2B1 antibody.

[0428] In some embodiments, the second molecule comprises an antibody specific for said first molecule. In some embodiments, the second molecule comprises a detectable label.

[0429] It must also be noted that, as used in the present disclosure and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. Optional or optional means that the event or situation described subsequently may occur, or may not occur, and the description includes the situation where the event or situation occurs and the situation where it does not occur. For example, the phrase that the composition may optionally include a combination means that the composition may include a combination of different molecules or may not include a combination, so that the description includes both the combination and the absence of the combination (i.e., the individual members of the combination). The range can be expressed herein as from about one specific value and / or to about another specific value. When such a range is expressed, another aspect includes from a specific value and / or to another specific value. Similarly, when a value is expressed as an approximation by using the leading word about, it should be understood that the specific value forms another aspect. It should be further understood that the end value of each range is meaningful with respect to another end value and independently of another end value. All references cited in the present disclosure are hereby incorporated herein by reference in their entirety.

[0430] The present invention will now be described by the following examples. It should be understood that the foregoing is for exemplary purposes only and is not intended to limit the scope of the present invention. It will be appreciated by those skilled in the art that modifications may be made without departing from the spirit or scope of the present invention as set forth in the claims.

[0431] Examples of Examples

[0432] The present invention may be understood with reference to the following embodiments:

[0433] 1. A method for measuring transgene expression, the method comprising:

[0434] a) providing a first plurality of terminally differentiated neural progenitor cells (NPCs);

[0435] b) transducing the first plurality of terminally differentiated NPCs with a test sample comprising a viral vector comprising a sequence encoding a protein of interest;

[0436] c) incubating the transduced first plurality of terminally differentiated NPCs under conditions sufficient to express the protein of interest;

[0437] d) contacting the first plurality of terminally differentiated NPCs from (c) with a molecule specific for the protein of interest;

[0438] e) imaging the first plurality of terminally differentiated NPCs to obtain an integrated fluorescence intensity / cell (IFI-C) assay readout; and

[0439] f) determining the expression of the protein of interest based on the IFI-C readout.

[0440] 2. The method of embodiment 1, wherein the first plurality of terminally differentiated NPCs are homozygous for a survival motor neuron gene (SMN1) - / - mutation.

[0441] 3. The method of embodiment 2, wherein the SMN1- / - mutation comprises a deletion of SMN1 exon 7 (Δ7).

[0442] 4. The method of any one of embodiments 1-3, wherein the incubation step c) is followed by fixation and permeabilization of the first plurality of terminally differentiated NPCs.

[0443] 5. The method of any one of embodiments 1-4, comprising:

[0444] g) providing a second plurality of terminally differentiated NPCs;

[0445] h) transducing the second plurality of terminally differentiated NPCs with a reference standard comprising the viral vector;

[0446] i) incubating the transduced second plurality of terminally differentiated NPCs under conditions sufficient to express the protein of interest;

[0447] j) contacting the second plurality of terminally differentiated NPCs from (i) with a molecule specific for the protein of interest;

[0448] k) imaging the second plurality of terminally differentiated NPCs to obtain an integrated fluorescence intensity / cell (IFI-C) assay readout; and

[0449] l) comparing the IFI-C of the first plurality of terminally differentiated NPCs with the IFI-C of the second plurality of terminally differentiated NPCs;

[0450] The relative potency of the viral vector in the test sample relative to the reference standard is thereby determined.

[0451] 6. The method of embodiment 5, wherein the second plurality of terminally differentiated NPCs are homozygous for the SMN1- / - mutation.

[0452] 7. The method of embodiment 6, wherein the SMN1- / - mutation comprises a deletion of SMN1 exon 7 (Δ7).

[0453] 8. The method of embodiment 4 or 5, wherein the incubation step (i) is followed by fixation and permeabilization of the second plurality of terminally differentiated NPCs.

[0454] 9. The method of any one of embodiments 1-8, wherein the first plurality of terminally differentiated NPCs and the second plurality of terminally differentiated NPCs are generated by terminally differentiating neural progenitor cells isolated from the cortex of an SMN1- / - mouse embryo.

[0455] 10. The method of embodiment 9, wherein the neural progenitor cells (NPCs) are terminally differentiated by:

[0456] a. These NPCs were cultured in serum-free medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to form neurospheres;

[0457] b. dissociating the neurospheres to produce dissociated NPCs; and

[0458] c. Culturing these dissociated NPCs in serum-rich medium without growth factors; thereby generating terminally differentiated NPCs.

[0459] 11. The method of any one of embodiments 1-10, wherein the first plurality of cells and the second plurality of cells are transduced with the test sample and the reference standard at at least two different multiplicity of infection (MOI) of the viral vector.

[0460] 12. The method of embodiment 11, wherein the first plurality of cells and the second plurality of cells are transduced at 5 different MOIs of the viral vector in the test sample and the reference standard.

[0461] 13. The method of embodiment 12, wherein the 5 MOIs include 300,000, 150,000, 75,000, 37,500, and 18,750 viral particles / cell.

[0462] 14. The method of any one of embodiments 5-13, wherein the comparing step (1) comprises plotting a standard curve of MOI versus IFI-C for each of the test sample and the reference standard.

[0463] 15. The method of any one of embodiments 5-14, wherein the comparing step (1) comprises calculating a linear regression of log MOI versus IFI-C for each of the test sample and the reference standard to yield a test sample slope and a reference standard slope.

[0464] 16. The method of any one of embodiments 5-15, wherein determining the relative potency of the viral vector is performed by parallel lines analysis (PLA), and wherein the PLA comprises measuring the slope ratio of the test sample slope relative to the reference standard slope.

[0465] 17. The method of embodiment 16, wherein the reference standard slope is greater than or equal to 1.02E+05.

[0466] 18. The method of embodiment 16 or 17, wherein the slope ratio is between 0.69-1.45.

[0467] 19. The method of embodiment 16 or 17, wherein the slope ratio is between 0.75 and 1.33.

[0468] 20. The method of any one of embodiments 16-19, comprising calculating the coefficient of variation of the linear regression of the sample.

[0469] 21. The method of embodiment 20, wherein the coefficient of variation is between 15.6% and 29.5%.

[0470] 22. The method of embodiment 20, wherein the coefficient of variation is less than or equal to 40%, less than or equal to 30%, or less than or equal to 20%.

[0471] 23. The method of any one of embodiments 16-22, comprising calculating the R2 value of the linear regression of the test sample and the reference standard.

[0472] 24. The method of embodiment 23, wherein the R2 value of the test sample and the reference standard is greater than or equal to 0.95.

[0473] 25. The method of any one of embodiments 16-24, comprising calculating an assay dynamic window for the reference standard.

[0474] 26. The method of embodiment 25, wherein the assay dynamic window is greater than or equal to 2.69.

[0475] 27. The method of any one of embodiments 1-26, wherein the protein of interest is survival motor neuron (SMN1) protein.

[0476] 28. The method of embodiment 27, wherein the SMN1 protein comprises the amino acid sequence of SEQ ID NO:3.

[0477] 29. The method of embodiment 27 or 28, wherein the viral vector is adeno-associated virus serotype 9 (AAV9).

[0478] 30. The method of any one of embodiments 27-29, wherein the viral vector comprises a sequence encoding a cytomegalovirus (CMV) enhancer / chicken-β-actin hybrid promoter (CB) operably linked to a sequence encoding the SMN1 protein.

[0479] 31. The method of any one of embodiments 27-30, wherein the viral vector comprises AAV inverted terminal repeats (ITRs) from AAV serotype 2 (AAV2) DNA.

[0480] 32. The method of any one of embodiments 27-31, wherein the viral vector comprises the sequence of SEQ ID NO: 1.

[0481] 33. The method of any one of embodiments 1-32, wherein the cells are passaged 8 to 15 times prior to transduction with the viral vector.

[0482] 34. The method of any one of embodiments 1-33, wherein the IFI-C readout reflects a measurement of protein expression.

[0483] 35. The method of any one of embodiments 1-34, wherein the step of incubating the terminally differentiated NPCs after transduction is performed for about 69-75 hours.

[0484] 36. The method of any one of embodiments 1-35, wherein the molecule specific for the protein of interest comprises an antibody, an antibody fragment, or an aptamer.

[0485] 37. A method as described in Example 36, wherein the antibody comprises an antibody specific for the target protein.

[0486] 38. The method of embodiment 37, wherein the anti-target protein antibody is provided at a concentration of about 4 μg / mL.

[0487] 39. The method of embodiment 37, wherein the anti-target protein antibody is provided at a concentration of about 2 μg / mL.

[0488] 40. The method of any one of embodiments 1-39, wherein the molecule comprises a detectable label.

[0489] 41. The method of any one of embodiments 1-40, further comprising washing the terminally differentiated NPCs to remove the molecule specific for the protein of interest.

[0490] 42. The method of any one of embodiments 1-38, further comprising contacting the terminally differentiated NPCs with a second molecule that specifically recognizes the molecule specific for the protein of interest.

[0491] 43. The method of embodiment 42, wherein the second molecule comprises a detectable label.

[0492] 44. The method of embodiment 42 or 43, wherein the second molecule comprises an antibody, an antibody fragment, or an aptamer.

[0493] 45. The method of any one of embodiments 1-44, wherein after the fixation and permeabilization steps, the terminally differentiated NPCs are contacted with an anti-nuclear detectable marker.

[0494] 46. ​​The method of any one of embodiments 1-45, wherein the terminally differentiated NPCs are on a solid surface.

[0495] 47. The method of embodiment 46, wherein the solid surface is coated with poly-D-lysine.

[0496] 48. The method of embodiment 46 or 47, wherein the terminally differentiated NPCs are seeded at a density of 20,000 cells / well.

[0497] 49. The method of any one of embodiments 1-48, wherein the method allows for quantitative measurement of a dose-dependent increase in the level of the protein of interest.

[0498] 50. A kit comprising:

[0499] a. Multiple cells capable of being transduced with viral vectors;

[0500] b. a viral vector encoding a target protein;

[0501] c. a first molecule capable of binding to the target protein;

[0502] d. a second molecule capable of binding to the first molecule, wherein the second molecule comprises a detectable label; and,

[0503] e. Instructions for use in imaging assays.

[0504] 51. The kit of embodiment 50, wherein the plurality of cells comprises neural progenitor cells (NPCs).

[0505] 52. The kit of embodiment 51, wherein the NPCs are homozygous for the SMN1- / - mutation.

[0506] 53. The kit of embodiment 51, wherein the SMN1- / - mutation is a deletion of exon 7 (Δ7).

[0507] 54. A kit as described in any of embodiments 50-53, wherein the viral vector is an adeno-associated virus serotype 9 (AAV9), which contains a cDNA expressing SMN1 protein under the control of a cytomegalovirus (CMV) enhancer / chicken-β-actin hybrid promoter (CB), and two AAV inverted terminal repeats (ITRs) from AAV serotype 2 (AAV2) DNA.

[0508] 55. The kit of any one of embodiments 50-54, wherein the first molecule comprises an anti-SMN1 antibody.

[0509] 56. The kit of any one of embodiments 50-55, wherein the second molecule comprises an antibody specific for the first molecule.

[0510] 57. The kit of embodiment 56, wherein the second molecule comprises a detectable label.

[0511] 58. The kit of any one of embodiments 50-57, wherein the protein of interest is survival motor neuron (SMN1) protein.

[0512] 59. A method of producing a pharmaceutical composition comprising a viral vector containing a transgene, the method comprising:

[0513] a. Producing a viral vector containing the transgene;

[0514] b. determining the viral vector according to the method for measuring the transgene as described in any one of Examples 1-49; and

[0515] c. formulating the viral vector containing the transgene into a pharmaceutical composition.

[0516] 60. The method of embodiment 59, wherein producing the viral vector comprises:

[0517] a. Cultivate adherent cells;

[0518] b. transfecting the adherent cells with one or more plasmids to enable production of the AAV viral vector;

[0519] c. lysing the adherent cells to isolate the AAV viral vector;

[0520] d. acidifying and clarifying the cell lysate of (c);

[0521] e. purifying the product of (d) using cation exchange chromatography (CEX);

[0522] f. filtering the product of (e) using tangential flow filtration (TFF);

[0523] g. The product of (f) is subjected to ultracentrifugation in cesium chloride (CsCl) buffer; and

[0524] h. Collect the AAV viral vectors from the product of (g).

[0525] 61. The method of embodiment 60, wherein the AAV is AAV9.

[0526] 62. The method of embodiment 60 or 61, wherein the AAV is self-complementary (scAAV).

[0527] 63. The method of any one of embodiments 60-61, wherein the adherent cells are HEK293 cells.

[0528] 64. The method of any one of embodiments 60-63, wherein the adherent cells are selected for adherence prior to culturing.

[0529] 65. The method of any one of embodiments 60-64, wherein the selection comprises subculturing the adherent cells multiple times to select for adherence.

[0530] 66. The method of any one of embodiments 60-65, wherein the adherent cells are inoculated in a bioreactor for culture.

[0531] 67. The method of embodiment 66, wherein the bioreactor is a large-scale bioreactor that can provide continuous circulation of cell culture medium.

[0532] 68. A method as described in Example 66 or 67, wherein the bioreactor is 200m 2 、333m 2 or 500m 2 Bioreactor.

[0533] 69. The method of any one of embodiments 66-68, wherein the adherent cells are added to the medium in a recirculating medium bag and circulated through the bioreactor.

[0534] 70. The method of embodiment 69, wherein the cells are circulated using a peristaltic pump.

[0535] 71. The method of embodiment 70, wherein the peristaltic pumping is continuous while the adherent cells are inoculated in the bioreactor for culture.

[0536] 72. The method of embodiment 71, wherein the seeding density is about 8,000-12,000 cells / cm 2 .

[0537] 73. The method of any one of embodiments 60-72, wherein the transfection step comprises adding transfection medium to the recirculating medium bag and circulating the transfection medium through the bioreactor.

[0538] 74. The method of embodiment 73, wherein the transfection medium is circulated using a peristaltic pump.

[0539] 75. The method of embodiment 73 or 74, wherein the cycling occurs between 15°C-25°C.

[0540] 76. The method of any one of embodiments 60-75, wherein the transfection step comprises contacting the adherent cells with an adenoviral helper plasmid (pHELP).

[0541] 77. The method of any one of embodiments 60-76, wherein the transfecting step comprises contacting the adherent cell with a plasmid encoding the AAV rep gene.

[0542] 78. The method of any one of embodiments 60-77, wherein the transfection step comprises contacting the adherent cell with a plasmid encoding an AAV cap gene.

[0543] 79. The method of any one of embodiments 60-78, wherein the transfection step comprises contacting the adherent cells with a plasmid encoding the AAV rep gene and the AAV cap gene on the same plasmid (pAAV).

[0544] 80. The method of embodiment 77 or embodiment 79, wherein the AAV rep gene is rep2.

[0545] 81. The method of embodiment 78 or 79, wherein the AAV cap gene is cap9.

[0546] 82. The method of any one of embodiments 60-81, wherein the transfection step comprises contacting the adherent cells with the transfection agent polyethyleneimine (PEI).

[0547] 83. The method of embodiment 82, wherein the ratio of PEI to at least one of the plasmids is less than 1:1 by weight.

[0548] 84. The method of embodiment 82, wherein the ratio of PEI to at least one of the plasmids is about 1:1 by weight.

[0549] 85. The method of any one of embodiments 73-84, wherein the transfection step comprises contacting the adherent cells with a serum-free transfection medium.

[0550] 86. The method of any one of embodiments 60-85, wherein the transfection step comprises contacting the adherent cells with a transfection medium that does not contain calcium.

[0551] 87. The method of any one of embodiments 60-86, wherein the transfection step comprises contacting the adherent cells with a transfection medium that does not contain glutamine.

[0552] 88. The method of any one of embodiments 60-87, wherein the transfection step is performed for 10-60 minutes, 10-30 minutes, 20-30 minutes, 15-30 minutes, or for less than 30 minutes.

[0553] 89. The method of any one of embodiments 60-88, wherein the lysis step comprises whole cell lysis.

[0554] 90. The method of any one of embodiments 60-89, wherein the cleavage step comprises using a cleavage buffer supplemented with an endonuclease.

[0555] 91. A method as described in Example 90, wherein the nuclease is a universal nuclease.

[0556] 92. The method of any one of embodiments 60-91, wherein the lysis step comprises using a lysis buffer supplemented with Tween.

[0557] 93. The method of any one of embodiments 60-92, wherein the cleavage step is performed between 15°C-25°C.

[0558] 94. The method of any one of embodiments 60-93, further comprising freezing the cell lysate of step (c) prior to the acidification step of (d).

[0559] 95. The method of any one of embodiments 60-94, wherein the CsCl buffer is 2-4 M CsCl buffer.

[0560] 96. The method of any one of embodiments 60-94, wherein the CsCl is at a concentration of about 3M.

[0561] 97. The method of any one of embodiments 60-96, further comprising (i) filtering the product of (g) by tangential flow filtration.

[0562] 98. The method of any one of embodiments 60-98, wherein the acidification step comprises acidifying the cell lysate to a pH of about 3.0-4.0, about 3.3-3.7, or about 3.4-3.6.

[0563] 99. The method of embodiment 98, wherein the acidification step comprises acidifying the cell lysate to a pH of about 3.5.

[0564] 100. The method of any one of embodiments 60-99, wherein the ultracentrifugation is performed between about 40,000-50,000 rpm or between about 43,000-46,000 rpm.

[0565] 101. The method of any one of embodiments 60-100, wherein the ultracentrifugation is performed between 15°C-25°C.

[0566] 102. The method of any one of embodiments 60-101, wherein the ultracentrifugation is performed for 16-24 hours or 20-24 hours.

[0567] 103. The method of any one of embodiments 60-102, wherein the cell lysate is incubated with Tween prior to the acidification step.

[0568] 104. The method of any one of embodiments 60-103, wherein the cell lysate is incubated with Tween for about 8-20 hours prior to the acidification step.

[0569] 105. The method of any one of embodiments 60-104, wherein the clarification step comprises filtering the cell lysate through a depth filter.

[0570] 106. The method of any one of embodiments 60-105, wherein the clarification step comprises filtering the cell lysate through a 0.45 micron filter.

[0571] 107. The method of any one of embodiments 60-106, wherein the CEX comprises a sulfonyl resin.

[0572] 108. The method of any one of embodiments 60-107, wherein at least one TFF step comprises using a cellulose membrane with a molecular weight cutoff of 300 kDa MW.

[0573] 109. The method of any one of embodiments 60-108, wherein the TFF step reduces the eluate volume of the cation exchange step by at least six times.

[0574] 110. The method of any one of embodiments 60-109, wherein the CsCl buffer comprises Tris, MgCl2, and poloxamer 188.

[0575] 111. The method of embodiment 110, wherein the CsCl buffer comprises about 20 mM Tris.

[0576] 112. The method of embodiment 110 or 111, wherein the CsCl buffer comprises approximately 2 mM MgCl2.

[0577] 113. The method of any one of embodiments 110-112, wherein the CsCl buffer comprises poloxamer 188, optionally about 0.2% w / v poloxamer 188.

[0578] 114. The method of any one of embodiments 60-113, wherein the CsCl buffer is between about pH 7.5-8.5 or between about pH 7.9-8.2.

[0579] 115. The method of any one of embodiments 60-114, wherein after collecting the AAV viral vectors from ultracentrifuged cell lysates, the number of empty viral capsids is less than 7%, less than 5%, less than 3% or less than 1% of the total viral capsids.

[0580] 116. The method of embodiment 115, wherein the number of empty viral capsids is measured by analytical ultracentrifugation (AUC).

[0581] 117. The method of any one of embodiments 60-116, wherein the AAV viral vectors are collected from the ultracentrifuged cell lysate using a syringe.

[0582] 118. The method of any one of embodiments 60-117, wherein the AAV viral vectors collected after the second TFF step are stored in a solution comprising Tris, MgCl2, NaCl, and Poloxamer 188.

[0583] 119. The method of embodiment 118, wherein the solution comprises about 20 mM Tris.

[0584] 120. The method of embodiment 118 or 119, wherein the solution comprises about 1 mM MgCl2.

[0585] 121. The method of any one of embodiments 118-120, wherein the solution comprises about 200 mM NaCl.

[0586] 122. The method of any one of embodiments 118-121, wherein the solution comprises about 0.005% w / v Poloxamer 188.

[0587] 123. The method of any one of embodiments 118-122, wherein the solution is between about pH 7.5-8.5 or between about pH 7.7-8.3.

[0588] 124. The method of any one of embodiments 60-123, wherein the AAV viral vectors collected after the second TFF contain less than about 30 μg / g or less than about 20 μg / g of CsCl.

[0589] 125. The method of any one of embodiments 60-124, wherein the concentration of the AAV viral vector collected after the second TFF is greater than or equal to about 3x10 13 vg / ml.

[0590] 126. The method of any one of embodiments 60-125, wherein flocculation with a detergent is used to remove host cell proteins and / or host cell DNA from the cell lysate.

[0591] 127. The method of any one of embodiments 60-120, wherein the AAV viral vector comprises a polynucleotide encoding a survival motor neuron (SMN) protein.

[0592] 128. The method of any one of embodiments 76-127, wherein the plasmid encoding the SMN protein, the plasmid encoding the pAAV, and the plasmid encoding the pHELP are transfected at a ratio of 1:1:1.

[0593] 129. The method of any one of embodiments 60-128, wherein the pharmaceutical composition comprises:

[0594] a.1-8x10 13 AAV9 viral vector genomes / mL (vg / mL);

[0595] b. Less than about 7% empty viral capsids;

[0596] c. Every 1x10 13 vg / mL less than about 100 ng / mL of host cell protein;

[0597] d. Every 1x10 13 vg / mL is less than about 5x10 6 pg / mL of residual host cell DNA; and

[0598] Among them, the 1-8x10 13 At least about 80% of the AAV9 viral vector genomes / mL are functional.

[0599] 130. The method of embodiment 129, wherein the pharmaceutical composition comprises 1.7-2.3 x 10 13 AAV9vg / mL or 1.9-2.1x10 13 AAV9 vg / mL between.

[0600] 131. The method of embodiment 129, wherein the pharmaceutical composition comprises about 2 x 10 13 AAV9 vg / mL.

[0601] 132. The method of any one of embodiments 129-131, wherein the pharmaceutical composition comprises less than about 5% empty capsids, less than about 3% empty capsids, or less than about 1% empty capsids.

[0602] 133. The method of any one of embodiments 129-132, wherein the pharmaceutical composition comprises 1-2 x 10 14 vg of the AAV9 viral vector or 1.1x10 14 vg, or consisting of the AAV9 viral vector.

[0603] 134. The method of any one of embodiments 129-132, wherein the pharmaceutical composition comprises 1.7 x 10 14 The AAV9 viral vector of vg.

[0604] 135. The method of any one of embodiments 129-134, wherein the pharmaceutical composition is an aqueous pharmaceutical formulation.

[0605] 136. The method of embodiment 135, wherein the formulation comprises Tris buffer, magnesium chloride, sodium chloride, and poloxamer, and wherein the pharmaceutical composition does not comprise a preservative.

[0606] 137. The method of embodiment 136, wherein the poloxamer comprises poloxamer 188.

[0607] 138. The method of any one of embodiments 135-137, wherein the pH of the formulation is about 7.7 to about 8.3.

[0608] 139. The composition of embodiment 138, wherein the pH is about pH 8.0.

[0609] 140. The method of any one of embodiments 136-139, wherein the magnesium chloride concentration is about 0.5-1.5 mM.

[0610] 141. The method of embodiment 140, wherein the magnesium chloride concentration is about 1 mM.

[0611] 142. The method of any one of embodiments 136-141, wherein the sodium chloride concentration is about 100-300 mM.

[0612] 143. The method of embodiment 142, wherein the sodium chloride concentration is about 200 mM.

[0613] 144. The method of any one of embodiments 136-143, wherein the formulation comprises about 0.005% w / v Poloxamer 188.

[0614] 145. The method of any one of embodiments 136-144, wherein the formulation has an osmolarity of 390-430 mOsm / kg.

[0615] 146. The method of any one of embodiments 60-145, wherein the pharmaceutical formulation comprises at least one of:

[0616] a. Every 1.0x10 13 vg is less than about 0.09ng of universal nuclease,

[0617] b. less than about 30 μg / g (ppm) of cesium,

[0618] c. about 20-80 ppm of Poloxamer 188,

[0619] d. Every 1.0x10 13 vg is less than about 0.22ng BSA,

[0620] e. Every 1.0x10 13 vg is less than about 6.8x10 5 Residual plasmid DNA of pg,

[0621] f. Every 1.0x10 13 vg is less than about 1.1x10 5 pg of residual hcDNA,

[0622] g. Every 1.0x10 13 vg is less than about 4ng of rHCP,

[0623] h. pH about 7.7-8.3,

[0624] i. About 390-430 mOsm / kg,

[0625] j. Fewer than about 600 particles ≥ 25 μm in size per container,

[0626] k. Less than about 6,000 particles ≥ 10 μm in size per container,

[0627] l. About 1.7x1013-2.3x10 13 vg / mL genome titer,

[0628] m. Every 1.0x10 13 vg about 3.9x10 8 -8.4x10 10 The infectious titer of IU,

[0629] n.per 1.0x10 13 vg about 100-300μg total protein,

[0630] o. Relative potency of approximately 70%-130%, and

[0631] p. Less than about 5% empty capsids.

[0632] 147. The method of any one of embodiments 60-145, wherein the pharmaceutical formulation comprises at least one of:

[0633] a. pH about 7.7-8.3,

[0634] b. About 390-430mOsm / kg,

[0635] c. Fewer than approximately 600 particles ≥ 25 μm in size per container,

[0636] d. Less than about 6,000 particles ≥ 10 μm in size per container,

[0637] e. Approximately 1.7x10 13 -2.3x10 13 vg / mL genome titer,

[0638] f. Every 1.0x10 13 vg about 3.9x10 8 -8.4x10 10 The infectious titer of IU,

[0639] g. Every 1.0x10 13 vg about 100-300μg total protein,

[0640] h. Pluronic F-68 content of about 20-80 ppm,

[0641] i. Relative potency of about 70%-130%,

[0642] j. in 7.5x10 13 At a dose of 100 vg / kg, the median survival in the Δ7SMN mouse model was greater than or equal to 24 days.

[0643] k. Less than about 5% empty capsids,

[0644] l. and an overall purity of greater than or equal to about 95%, and

[0645] m. Less than or equal to about 0.75 EU / mL of endotoxin.

[0646] 148. The method of any one of embodiments 60-145, wherein the pharmaceutical formulation comprises at least one of:

[0647] a. Every 1.0x10 13 vg is less than about 0.09ng of universal nuclease,

[0648] b. less than about 30 μg / g (ppm) of cesium,

[0649] c. about 20-80 ppm of Poloxamer 188,

[0650] d. Every 1.0x10 13 vg is less than about 0.22ng BSA,

[0651] e. Every 1.0x10 13 vg less than about 6.8x105pg of residual plasmid DNA,

[0652] f. Every 1.0x10 13 vg is less than about 1.1x10 5 pg of residual hcDNA, and

[0653] g. Every 1.0x10 13 vg is less than about 4ng of rHCP.

[0654] 149. The method of any of embodiments 59-148, wherein 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% relative to a reference standard.

[0655] 150. The method of any one of embodiments 59-148, wherein the relative potency of the viral vector is at least 90% relative to the reference standard.

[0656] 151. The method of any of embodiments 59-148, wherein the potency of the viral vector in the pharmaceutical formulation is within 5% of the potency of the reference standard, within 10% of the potency of the reference standard, or within 20% of the potency of the reference standard.

[0657] 152. A method of treating a patient in need thereof with a therapy comprising a viral vector containing a transgene, the method comprising:

[0658] a. determining the transgene-containing viral vector according to the method for measuring transgene expression as described in any one of Examples 1-49; and

[0659] b. administering the viral vector containing the transgene to the patient.

[0660] 153. The method of embodiment 152, wherein the viral vector is formulated in a pharmaceutical composition.

[0661] 154. The method of embodiment 146, wherein the pharmaceutical composition comprises:

[0662] a.1-8x10 13 AAV9 viral vector genomes / mL (vg / mL);

[0663] b. Less than about 7% empty viral capsids;

[0664] c. Every 1x10 13 vg / mL less than about 100 ng / mL of host cell protein; and

[0665] d. Every 1x10 13 vg / mL is less than about 5x10 6 pg / mL of residual host cell DNA;

[0666] Among them, the 1-8x10 13 At least about 80% of the AAV9 viral vector genomes / mL are functional.

[0667] 155. The method of any one of embodiments 152-154, wherein the patient in need thereof suffers from SMA, such as spinal muscular atrophy (SMA) type I.

[0668] 156. The method of embodiment 155, wherein the patient in need thereof:

[0669] a. Two years of age or younger, optionally nine months of age or younger;

[0670] b. having a body weight of at least about 2.6 kg;

[0671] c. have biallelic SMN1 null mutations or deletions; and

[0672] d. Having at least one functional copy of SMN2.

[0673] 157. The method of any one of embodiments 153-156, wherein the composition is administered to the patient via an intrathecal or intravenous route.

[0674] 158. The method of any one of embodiments 152-157, wherein the viral vector is present in an amount of about 1-2.5 x 10 14 vg / kg dose.

[0675] 159. The method of any one of embodiments 152-157, wherein the viral vector is at about 1.1 x 10 14 vg / kg dose.

[0676] 160. A method as described in embodiment 158 ​​or 159, wherein the amount of the viral vector genome is measured using ddPCR.

[0677] 161. The method of any one of embodiments 155-160, wherein the patient has a body weight of no more than about 13.5 kg, optionally no more than about 8.5 kg.

[0678] 162. The method of any one of embodiments 155-161, wherein the patient does not have a c.859G>C substitution in exon 7 of at least one copy of the SMN2 gene.

[0679] 163. The method of any one of embodiments 155-162, wherein the treatment is administered to the patient before 2 years of age, optionally before 6 months of age.

[0680] 164. The method of any one of embodiments 155-163, wherein the treatment is administered to the patient prior to the onset of one or more SMA symptoms selected from: hypotonia, delayed motor skills, poor head control, rounded shoulder posture, and joint hypermobility.

[0681] 165. The method of any one of embodiments 155-164, wherein the patient has an anti-AAV9 antibody titer equal to or less than 1:100 or 1:50 prior to administration as determined by an ELISA binding immunoassay.

[0682] 166. The method of any one of embodiments 155-164, wherein the patient has an anti-AAV9 titer above 1:100 after administration as determined by an ELISA binding immunoassay, and the patient is monitored for about 1-8 weeks or until the titer decreases to below 1:100.

[0683] 167. The method of any one of embodiments 155-164, wherein the patient has an anti-AAV9 titer above 1:100 after administration as determined by an ELISA binding immunoassay, and the patient is monitored for about 1-8 weeks or until the titer decreases to below 1:50.

[0684] 168. The method of any one of embodiments 155-164, wherein the patient has an anti-AAV9 titer of greater than 1:100 as determined by an ELISA binding immunoassay before or after administration and is switched to formula feeding.

[0685] 169. The method of embodiment 168, wherein the patient is switched to formula feeding prior to or after administration.

[0686] 170. The method of any one of embodiments 155-164, wherein the patient has an anti-AAV9 titer of greater than 1:50 as determined by ELISA binding immunoassay before or after administration and is switched to formula feeding.

[0687] 171. The method of embodiment 170, wherein the patient is switched to formula feeding prior to or after administration.

[0688] 172. The method of any one of embodiments 155-171, wherein the patient has an anti-AAV9 titer of greater than 1:100 or greater than 1:50 after administration as determined by an ELISA binding immunoassay and is treated with plasma exchange.

[0689] 173. The method of any one of embodiments 155-172, wherein the patient has a platelet count of greater than about 67,000 cells / ml, or greater than about 100,000 cells / ml, or greater than about 150,000 cells / ml prior to administration.

[0690] 174. The method of any one of embodiments 155-173, wherein the patient has a platelet count of less than about 67,000 cells / ml, or less than about 100,000 cells / ml, or less than about 150,000 cells / ml after administration, and the patient is monitored for about 1-8 weeks or until the platelet count increases to about 67,000 cells / ml, or greater than about 100,000 cells / ml, or greater than about 150,000 cells / ml.

[0691] 175. The method of any one of embodiments 155-174, wherein the patient has a platelet count of less than about 67,000 cells / ml after administration and is treated with platelet transfusions.

[0692] 176. The method of any one of embodiments 155-175, wherein the patient does not suffer from thrombocytopenia prior to administration.

[0693] 177. The method of any one of embodiments 155-175, wherein the patient suffers from thrombocytopenia after administration, and the patient is monitored for about 1-8 weeks or until the patient no longer suffers from thrombocytopenia.

[0694] 178. The method of any one of embodiments 155-175, wherein the patient suffers from thrombocytopenia after administration and is treated with platelet transfusion.

[0695] 179. The method of any one of embodiments 155-178, wherein prior to administration of the viral vector, the patient has a troponin-I level of less than about 0.176 ug / ml.

[0696] 180. The method of any one of embodiments 155-179, wherein troponin-I levels in the patient are monitored after administration of the viral vector.

[0697] 181. The method of embodiment 179 or embodiment 180, wherein monitoring is performed after administration until the troponin-I level in the patient is less than about 0.176 ug / ml.

[0698] 182. The method of any one of embodiments 155-181, wherein the patient has normal liver function prior to administration.

[0699] 183. The method of embodiment 182, wherein the patient has a liver transaminase level of less than about 8-40 U / L prior to administration.

[0700] 184. The method of embodiment 183, wherein the liver transaminase is selected from alanine aminotransferase (AST), aspartate aminotransferase (ALT), and a combination thereof.

[0701] 185. The method of any one of embodiments 155-184, wherein the patient has a bilirubin level of less than 3.0 mg / dL, a creatinine level of less than 1.8 mg / dL, an Hgb level between 8-18 g / dL, and / or a white blood cell count of less than about 20,000 / mm3 prior to administration.

[0702] 186. The method of any one of embodiments 155-185, wherein the viral vector is administered in Tris-buffered saline.

[0703] 187. The method of any one of embodiments 155-186, wherein the viral vector is administered in about 5-20 mL / kg, about 10-20 mL / kg, or about 5.5-6.5 mL / kg of Tris-buffered saline.

[0704] 188. The method of any one of embodiments 155-187, wherein the viral vector is infused over about 45-75 minutes.

[0705] 189. The method of any one of embodiments 155-188, wherein the viral vector is infused over about 60 minutes.

[0706] 190. The method of Embodiment 188 or Embodiment 189, wherein the infusion comprises a syringe pump.

[0707] 191. The method of any one of embodiments 155-189, wherein oral steroids are administered to the patient at least 24 hours prior to administration of the viral vector.

[0708] 192. The method of any one of embodiments 155-191, wherein oral steroids are administered to the patient for at least 30 days after administration of the viral vector.

[0709] 193. The method of embodiment 192, wherein the oral steroid is administered once daily.

[0710] 194. The method of embodiment 193, wherein the oral steroid is administered twice daily.

[0711] 195. The method of any one of embodiments 191-194, wherein the patient is monitored for elevated levels of ALT and / or AST after administration of the viral vector, and wherein the oral steroid is continued after 30 days until AST and / or ALT levels are less than twice the upper limit of normal or less than about 120 IU / L.

[0712] 196. The method of any one of embodiments 191-195, wherein oral steroids are administered to the patient until AST and / or ALT levels are less than two times the upper limit of normal or less than about 120 IU / L.

[0713] 197. The method of any one of embodiments 191-195, wherein the oral steroid is administered at a dose of about 1 mg / kg.

[0714] 198. The method of any one of embodiments 191-197, further comprising gradually reducing the oral steroid administration after AST and ALT are below two times the upper limit of normal or below about 120 IU / L.

[0715] 199. The method of embodiment 198, wherein the taper comprises a stepwise increase to 0.5 mg / kg / day for 2 weeks, followed by 0.25 mg / kg / day for an additional 2 weeks.

[0716] 200. The method of any one of embodiments 191-198, comprising administering the oral steroid at a dose of about 1 mg / kg for 30 days, and then tapering to 0.5 mg / kg / day for 2 weeks, followed by 0.25 mg / kg / day for another 2 weeks.

[0717] 201. The method of any one of embodiments 191-200, wherein the oral steroid is prednisolone or an equivalent.

[0718] 202. The method of any one of embodiments 155-201, comprising administering a muscle enhancing agent or a neuroprotective agent to the patient.

[0719] 203. The method of any one of embodiments 155-202, comprising administering to the patient an antisense oligonucleotide targeting SMN.

[0720] 204. The method of any one of embodiments 155-203, comprising administering nusinersen sodium to the patient.

[0721] 205. The method of any one of embodiments 155-204, comprising administering sidarucizumab to the patient.

[0722] 206. The method of any one of embodiments 155-205, wherein efficacy is determined using the CHOP-INTEND scale.

[0723] 207. The method of any one of 155-206, wherein the patient has or does not have disease onset.

[0724] 208. The method of any one of embodiments 155-207, comprising:

[0725] a. Determine the patient's weight;

[0726] b. A kit comprising vials of an AAV9 viral vector pharmaceutical composition, wherein the viral vector concentration in each vial is about 2.0 x 10 13 vg / mL; and

[0727] Wherein the number and volume of vials in the kit are selected from the group consisting of:

[0728] When the patient weighs 2.6 to 3 kg, 2 vials of 7.9-8.8 mL of the composition / vial; when the patient weighs between 3.1 and 3.5 kg, 2 vials of 5.1 to 5.9 mL of the composition / vial and 1 vial of 7.9-8.8 mL of the composition / vial; when the patient weighs between 3.6 and 4.0 kg, 1 vial of 5.1 to 5.9 mL of the composition / vial and 2 vials of 7.9-8.8 mL of the composition / vial; when the patient weighs between 4.1 and 4.5 kg, 3 vials of 7.9-8.8 mL of the composition / vial; when the patient weighs between 4.6 and 5. When the patient is between 5.1 and 5.5 kg, 1 vial of 5.1 to 5.9 mL of the composition / vial and 3 vials of 7.9-8.8 mL of the composition / vial; when the patient is between 5.6 and 6.0 kg, 4 vials of 7.9-8.8 mL of the composition / vial; when the patient is between 6.1 and 6.5 kg, 2 vials of 5.1 to 5.9 mL of the composition / vial and 3 vials of 7.9-8.8 mL of the composition / vial;

[0729] When the patient is between 6.6 and 7.0 kg, 1 vial of 5.1 to 5.9 mL of the composition / vial and 4 vials of 7.9-8.8 mL of the composition / vial; when the patient is between 7.1 and 7.5 kg, 5 vials of 7.9-8.8 mL of the composition / vial; when the patient is between 7.6 and 8.0 kg, 2 vials of 5.1 to 5.9 mL of the composition / vial and 4 vials of 7.9-8.8 mL of the composition / vial; when the patient is between 8.1 and 8.5 kg, 1 vial of 5.1 to 5.9 mL of the composition / vial When the patient is between 8.6 and 9.0 kg, 6 vials of 7.9-8.8 mL of the composition / vial; when the patient is between 9.1 and 9.5 kg, 2 vials of 5.1 to 5.9 mL of the composition / vial and 5 vials of 7.9-8.8 mL of the composition / vial; when the patient is between 9.6 and 10.0 kg, 1 vial of 5.1 to 5.9 mL of the composition / vial and 6 vials of 7.9-8.8 mL of the composition / vial; when the patient is between 10.1 When the patient is between 10.6 and 11.0 kg, 2 vials of 5.1 to 5.9 mL of the composition / vial and 6 vials of 7.9 to 8.8 mL of the composition / vial; when the patient is between 11.1 and 11.5 kg, 1 vial of 5.1 to 5.9 mL of the composition / vial and 7 vials of 7.9 to 8.8 mL of the composition / vial; when the patient is between 11.6 and 12.0 kg, 8 vials of 7.9 to 8.8 mL of the composition / vial vials; when the patient is between 12.1 and 12.5 kg, 2 vials of 5.1 to 5.9 mL of the composition / vial and 7 vials of 7.9-8.8 mL of the composition / vial; when the patient is between 12.6 and 13.0 kg, 1 vial of 5.1 to 5.9 mL of the composition / vial and 8 vials of 7.9-8.8 mL of the composition / vial; and when the patient is between 13.1 and 13.5 kg, 9 vials of 7.9-8.8 mL of the composition / vial; and c. administering the AAV9 viral vector from the vials to the patient.

[0730] 209. The method of embodiment 208, wherein the AAV viral vector is at about 1.0 x 10 14 -2.5x10 14 Doses of vg / kg were administered by infusion.

[0731] 210. The method of 208 or 209, wherein the AAV viral vector is concentrated to about 1.1 x 1014 Doses of vg / kg were administered by infusion.

[0732] 211. The method of embodiment 209 or 210, wherein the viral vector is infused over about 45-70 minutes.

[0733] 212. The method of any one of embodiments 209-211, wherein the viral vector is infused over about 60 minutes.

[0734] 213. The method of any one of embodiments 209-212, wherein the infusion comprises a syringe pump.

[0735] 214. The method of any one of embodiments 209-213, wherein the amount of the viral vector genome is measured using ddPCR.

[0736] 215. The method of any one of embodiments 209-214, wherein the dose titer of the AAV9 viral vector is measured by ddPCR.

[0737] 216. The method of any one of embodiments 155-215, comprising administering a dose volume of: 16.5 mL when the patient's body weight is 2.6-3.0 kg, 19.3 mL when the patient's body weight is 3.1-3.5 kg, 22.0 mL when the patient's body weight is 3.6-4.0 kg, 24.8 mL when the patient's body weight is 4.1-4.5 kg, 27.5 mL when the patient's body weight is 4.6-5.0 kg, 30.3 mL when the patient's body weight is 5.1-5.5 kg, 33.0 mL when the patient's body weight is 5.6-6.0 kg, 35.8 mL when the patient's body weight is 6.1-6.5 kg, 38.5 mL when the patient's body weight is 6.6-7.0 kg, 41.3 mL when the patient's body weight is 7.1-7.5 kg, and 7.6 mL when the patient's body weight is 7.7-8.0 kg. .6-8.0kg is 44.0, when the patient's weight is 8.1-8.5kg is 46.8mL, when the patient's weight is 8.6-9.0kg is 49.5, when the patient's weight is 9.1-9.5kg is 52.3mL, when the patient's weight is 9.6-10.0kg is 55.0, when the patient's weight is 10.1-10.5kg is 57.8mL, when the patient's weight is 10 .6-11.0kg, 60.5mL when the patient's weight is 11.1-11.5kg, 63.3mL when the patient's weight is 11.6-12.0kg, 66.0mL when the patient's weight is 12.1-12.5kg, 68.8mL when the patient's weight is 12.6-13.0kg, and 71.5mL when the patient's weight is 13.1-13.5kg.

[0738] 217. The method of any one of embodiments 155-216, wherein the pharmaceutical composition comprises:

[0739] a. A self-complementary AAV9 viral vector, the self-complementary AAV9 viral vector comprising a modified AAV2 ITR, a chicken β-actin (CB) promoter, a cytomegalovirus (CMV) immediate / early enhancer, a modified SV40 late 16s intron, a sequence encoding an SMN1 polypeptide, a bovine growth hormone (BGH) polyadenylation signal, and an unmodified AAV2 ITR;

[0740] b. 20 mM Tris at pH 8.0;

[0741] c.1mM MgCl2;

[0742] d. 200 mM NaCl; and

[0743] e.0.005% Poloxamer 188;

[0744] The patient was less than 2 years old.

[0745] 218. The method of embodiment 217, wherein the composition does not contain a preservative.

[0746] 219. A method as described in any of Examples 152-218, wherein 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% relative to a reference standard.

[0747] 220. The method of any one of embodiments 152-218, wherein the relative potency of the viral vector is at least 90% relative to the reference standard.

[0748] 221. The method of any one of embodiments 152-218, wherein the potency of the viral vector in the pharmaceutical formulation is within 5% of the potency of the reference standard, within 10% of the potency of the reference standard, or within 20% of the potency of the reference standard.

[0749] Examples

[0750] Example 1: Generation of murine Δ7 cells

[0751] Purpose

[0752] This procedure describes the generation of cell lines and banks created from mouse primary neural progenitor cells (mNPCs) for use in assay qualification, validation, release and stability testing of manufactured gene replacement drug materials.

[0753] scope

[0754] This is particularly applicable to the generation of mouse primary neural progenitor cell lines defective in expressing SMN1 protein derived from SMAΔ7 mice, which have exon 7 deleted from the SMN gene, resulting in a type 1 spinal muscular atrophy (SMA) disease that resembles human type 1 SMA disease.

[0755] program

[0756] Generation of primary neural progenitor cells (NPCs)

[0757] Pregnant Δ7 (+ / -) mice were given a lethal dose of isoflurane on embryological day 14 ± 2 (E14 ± 2). After wiping with ethanol, the abdomen was opened with scissors and the embryos were removed. The embryos were placed in a culture dish layered with cold Hanks balanced buffered salt solution (HBSS). One embryo was isolated at a time by cutting with scissors, placing in a clean culture dish, and removing from the amniotic sac with forceps.

[0758] Remove the tail and place it in an Eppendorf tube for genotyping. The cortex is separated from the brain and the meninges are removed as much as possible. The cortex is cut into small pieces and placed in a 15 mL conical tube with 14 mL cold HBSS. Use clean tools to repeat these steps (in this section) for each embryo to avoid contaminating DNA and cells.

[0759] Centrifuge the conical tube containing the dissected tissue at 300 g for 5 min.

[0760] Isolation of neural progenitor cells

[0761] Use a clean pipette to aspirate the buffer solution from each conical tube between each tube. Add 200 μL Accumax to each sample and incubate at room temperature for 30 minutes as part of the cell dissociation process. Then add 400 μL of complete medium to each sample and grind the tissue by pipetting up and down to dissociate the cells. Perform this procedure for each sample before moving to the next step.

[0762] Add another 400 μL of complete medium to bring the total volume to 1 mL. Filter the cells using a 70 μm cell strainer into a new 15 mL conical tube.

[0763] Cells are counted using an automated cell counter by adding a volume of cells to a volume of Trypan Blue stain (or equivalent cell viability reagent, depending on the cell counter used) and adding the appropriate volume to a slide inserted into the automated cell counter.

[0764] The total volume of cells (1 mL) was added to 9 mL of complete medium in individually labeled T-75 tissue culture flasks and placed at 37°C, 5% CO. 2 Incubator under . Mouse primary cells were isolated directly from the blastoderm of SMA△7 mice and designated as passage 0 (P0). In order to ensure that one or more cell lines from at least one dissected embryo were defective in expressing SMN1 protein due to carrying two alleles of the SMN1 gene lacking its exon 7 (△7), at least 4 embryos were collected and dissected. Cell lines containing homozygous SMN1 (△7) were confirmed by PCR genotyping according to SOP-269. As described in the following passage section, the cell lines were continued to be passaged in parallel until the SMN1△7 genotype and appropriate cell growth were confirmed for one cell line. The remaining cell lines were terminated, and the selected cell line (SMN1△7) was continued to be passaged to produce one or more master cell banks and one or more working cell banks (as appropriate).

[0765] Passaging and freezing cells

[0766] Recommended volume:

[0767] Flask <![CDATA[Surface area (cm 2 )]]> Total volume of culture medium (mL) T-75 75 10-15 T-150 150 20-25 T-175 175 25-40

[0768] Neurospheres

[0769] When the cells are ready to be passaged (i.e., before the cells reach close to the maximum recommended density), remove the flask from the incubator and mix thoroughly.

[0770] The cells were transferred from the flask to a 50 mL conical tube and centrifuged at 300 g for 5 minutes. If a larger volume of cells in culture medium is collected, the centrifugation time can be increased. For example, if 50 mL is collected, the cells can be centrifuged at 300 g for 8 minutes.

[0771] The supernatant was aspirated and 200 μL Accumax was added. The cell pellet was stirred and triturated one to three times and then incubated at room temperature for 30 minutes.

[0772] Accumax was then neutralized by adding 400 μL of complete growth medium, and the cells were triturated 10-15 times to dissociate into single cells.

[0773] Add another 400 μL of complete medium to bring the total volume to 1 mL. Additional volumes of complete medium may be added to dilute the cells for easier counting.

[0774] A small sample of the cells was removed to determine the total number of cells and viability as above (using trypan blue and an automated cell counter). The derived cells were designated passage 1 (P1).

[0775] The P1 cell suspension was diluted to 100,000 cells / mL and the appropriate volume was transferred (via pipette) to a new cell culture dish and incubated at 37° C., 5% CO 2 .

[0776] The above passaging steps were repeated, and for each passaging cycle, one passage number was added to the previous passage.

[0777] Freezing cells

[0778] Prior to freezing in cryogenic vials, cells were examined microscopically for cell morphology, viability, density (cell concentration), and microbial contamination.

[0779] Cells were frozen in freezing medium and stored in cryogenic vials in a cryogenic storage container at a -80 °C freezer for 24-72 hours.

[0780] After the initial freeze, the vials were transferred to a liquid nitrogen freezer.

[0781] Cell bank construction

[0782] Master Cell Bank (MCB)

[0783] A master cell bank (MCB) is created by expanding cells from an initial source and, after quality control (QC) testing, continued expansion from the initial source, which is used to prepare the master cell bank and expanded to establish a working cell bank (see Figure 1 to Figure 2 ).

[0784] The master cell bank and a small portion of the working cell bank are isolated and maintained away from all other materials. The cells can be frozen, and then thawed and passaged for use in the assays disclosed herein as needed.

[0785] Example 2: Method development for a robust cell-based quantitative relative potency assay

[0786] Materials and methods

[0787] Isolation and passaging of neural progenitor cells (NPCs)

[0788] NPCs were collected from the embryonic cortex of the SMAΔ7 mouse strain (Jackson Laboratories) at embryonic day approximately 14.5 (e14.5). These cells were dissociated into single cells by incubation in Accumax (STEMCELL Technologies) at room temperature for 30 minutes and 5% CO 2 The cells were cultured in a serum-free medium (DMEM / F12, Gibco) containing EGF (Corning) and bFGF (Corning) as growth factors in a non-adherent tissue culture flask. During the culture period, the cells formed neurospheres that were 3-dimensional colonies of undifferentiated cells. After about 3-5 days, the neurospheres were dissociated into single cells by incubation in Accumax (Stem Cell Technologies) at room temperature for 30 minutes and formed into secondary spheres, a process called "passaging".

[0789] Differentiation of NPCs

[0790] For terminal differentiation of NPCs, neurospheres were dissociated by incubation in Accumax (Stem Cell Technologies) at room temperature for 30 minutes and seeded at 1E+06 cells / well in 0.5 mL (24-well plates, Falcon) or 2E+05 cells / well in 100 μL (96-well plates, Corning) of serum-rich medium without growth factors. 24 hours after differentiation, cells were terminally differentiated mainly into the glial lineage.

[0791] Transduction of mTD-NPC-Δ7

[0792] At 24 hours after differentiation, the medium was removed from the cells and 100 μL of medium containing AAV9 vectors was added to the cells. The AAV9 vectors used in these studies were AAV9-eGFP lot SAB-138 and SMN1 encoding AAV9 (AAV9-SMN1) vector drug product lots NCHAAV9SMN0613 (reference standard), 816836, and 600156.

[0793] SMN1 staining

[0794] At the designated time points after transduction, cells were fixed by adding 50 μL of 4% paraformaldehyde (Alfa Aesar) in PBS to each well of a 96-well plate (Corning). Cells were incubated in 4% paraformaldehyde for 5 min at room temperature (RT) and then washed with 250 μL PBS / well. Cells were then permeabilized at room temperature for 5 min using 0.1% Triton X-100 (Thermo Scientific) in PBS and then washed with 250 μL PBS. For staining of SMN1, mouse monoclonal antibody 2B1 (Thermo Fisher) was used at 4 μg / mL. Cells were incubated with 50 μL of antibody diluted in 1% BSA (Fisher) in PBS for 2 hours at room temperature and then washed with 250 μL PBS. Finally, cells were incubated with secondary antibodies (goat anti-mouse IgG Alexa 488, Thermo Fisher) and nuclear stain (Hoechst 33342, Invitrogen) in 50 μL 1% BSA in PBS for 1 hour at room temperature. After washing the cells with 250 μL PBS, the plates were sealed with optically clear plate sealers (Thermo Fisher) and imaged as described below.

[0795] Image acquisition and analysis

[0796] CellInsight CX5 (Thermo Fisher Scientific, hereinafter designated as CX5) was used for image acquisition. CX5 is an automated high-content imaging instrument that allows the measurement of the biological activity of single cells in multi-well plates. CX5 was operated as follows: HCS Studio (Thermo Fisher Scientific) was used to configure parameters such as objective lens and exposure time. Images were acquired with a fixed exposure time using a 20x objective lens for fluorescence detection of a high dynamic range. The captured images were then analyzed using HCS Studio. HCS Studio uses each set of algorithms and filters to define each cell using nuclear staining, and then uses this nuclear staining as a basis to identify cells. HCS Studio was used to analyze the integrated fluorescence intensity in each cell after SMN1 staining. This software also calculates the measured mean value of each well over the entire cell population, which represents the biological changes in the well. For data analysis, integrated fluorescence intensity / cell (IFI-C) was used as a measure of SMN1 protein expression.

[0797] result

[0798] Transduction of mouse terminally differentiated cells (mTD-NPC-Δ7) derived from neural progenitor cells

[0799] To assess the permissiveness of mTD-NPC-Δ7 to the non-replicating self-complementary AAV9-SMN1 (AVXS-101), mTD-NPC-Δ7 was transduced using an AAV9-eGFP vector in a 24-well plate format. Transduction was monitored using green fluorescence signals from eGFP expressed in transduced cells, and images were acquired 24 hours after transduction. MOI-dependent eGFP expression demonstrated that mTD-NPC-Δ7 cells were permissive to AAV9 vectors without the need for chemical treatment or cell engineering. Figure 4 It is shown that the number of eGFP-positive cells increased with increasing multiplicity of infection (MOI).

[0800] Feasibility of mTD-NPC-Δ7-based assay using 96-well plate format

[0801] In order to have reasonable throughput for an assay that could be used as a batch-processed potency assay, the feasibility of using a 96-well plate format was evaluated.

[0802] Uniformity of cell distribution and determination of cell plating density on 96-well plates

[0803] To be able to accurately capture biological changes using imaging techniques, it is critical that each well has the appropriate cell density. We tested and compared 20,000 cells / well ( Figure 5A ) and 10,000 cells / well ( Figure 5B ). At a density of 20,000 cells / well, cells are close enough to each other to foster an appropriate biological environment without extensively contacting each other for optimal image analysis.

[0804] Establishment of SMN1 staining method for mTD-NPC-Δ7 transduced with AAV9 vector in 96-well plate format SMN1 staining in mTD-NPC-Δ7

[0805] Several mouse monoclonal antibodies from commercial sources were screened for detection of SMN1 protein using immunostaining (data not shown), and a mouse monoclonal antibody (2B1) was identified that showed low background and a consistently good dynamic range of detection.

[0806] Specificity of SMN1 monoclonal antibody (2B1)

[0807] To ensure that the protein detected by 2B1 was not an artifact from the AAV9 vector or from the transduction procedure, AAV9-eGFP transduced mTD-NPC-Δ7 was stained with 2B1. When stained by 2B1, cells transduced at 100,000 MOI did not show any detectable signal, but demonstrated successful AAV9 transduction, which was confirmed by GFP expression ( Figure 7 ).

[0808] Evaluation of tissue culture plates coated with poly-D-lysine

[0809] To ensure minimal cell loss during the staining procedure, we evaluated the need to use poly-D-lysine-coated 96-well plates. Fig. 8A and Figure 8B Uncoated and typical tissue culture treated 96-well plates are shown ( Fig. 8A ) and poly-D-lysine coated plates ( Figure 8B ). When cells were plated and stained on uncoated plates, cell loss was observed and many of the remaining cells fell off. However, after all staining procedures were completed, cells plated on poly-D-lysine did not show this phenomenon. Therefore, we decided to use poly-D-lysine coated plates for further assay development experiments.

[0810] Establishment of mTD-NPC-Δ7-based quantitative imaging assay to measure SMN1 protein levels

[0811] Determination of assay duration for detection of transgene expression.

[0812] The assay duration for detecting transgene expression was determined to be 72 hours after transduction. Since mTD-NPC-Δ7 is terminally differentiated and non-dividing, transgenic SMN1 expression accumulates after transduction, which reflects transgene expression in non-dividing motor neurons after transduction of SMN1-encoding AAV9 vectors in the SMAΔ7 disease model or SMA patients. For evaluation, we used AAV9-eGFP as a tool to monitor transgene expression at two time points, 48 ​​hours after transduction and 72 hours after transduction. At 72 hours after transduction, most cells (up to 80%) were GFP-positive, with high fluorescence signals per cell, which provided a good assay window. However, at 48 hours after transduction, there were far fewer GFP-positive cells, with much lower fluorescence signals per cell.

[0813] Detection and analysis of MOI-dependent increases in SMN1 protein expression for three AAV9 vector batches

[0814] Using established assay conditions, mTD-NPC-Δ7 was transduced with three SMN1-expressing AAV9 vector batches (NCHAAV9SMN0613, 816836, or 600156) at three different MOIs, and the cells were stained with anti-SMN1 2B1 monoclonal antibody. Images obtained after staining showed that SMN1 expression increased with increasing MOI. In order to determine whether the fluorescent signal from immunostaining can be used to quantitatively evaluate SMN1 expression, the integrated fluorescence intensity of each cell (integrated fluorescence intensity / cell, IFI-C) was analyzed. This experiment was performed at n=3. For each of the clinical batches tested, IFI-C increased with increasing MOI (Table 3). The data demonstrate that the SMN1 expression of the three clinical batches when transduced at three given MOIs is comparable (Table 3). Fig.10 Representative images of mTD-NPC-Δ7 at 72 hours post-transduction are shown.

[0815] Table 3. Fluorescence intensity / cell data for each batch

[0816]

[0817] Table 4. Comparison of fluorescence intensity / cell data between batches

[0818] NCH0613 816836 600156 CV 10k 7.86E+05 7.91E+05 7.13E+05 5.75 50k 1.33E+06 1.49E+06 1.27E+06 8.08 100k 1.65E+06 1.85E+06 1.54E+06 9.28

[0819] Proof-of-concept data demonstrating that the mTD-NPC-Δ7-based assay is a dose-dependent, quantitative assay in a 96-well format

[0820] To determine the quantitative characteristics of this assay, a 12-point MOI transduction of AAV9-SMN1 vector batch NCHAAV9SMN0613 (n=5) was performed in 2-fold dilutions of the vector using the developed mTD-NPC-Δ7-based assay. From background (MOI=1) to the highest MOI (MOI=200K), there was a 4.8-fold increase in IFI-C, which demonstrated an acceptable background signal ratio. As shown in Table 3, the relative standard deviation (RSD) (or CV%) of SMN1IFI-C from 5 parallel assays in 12 MOIs was less than 5% (from 0.5% to 4%). Preliminary proof-of-concept (POC) data showed that the assay was accurate. Fig.11 Visual confirmation of the MOI-dependent increase in SMN1 expression with increasing MOI (only 10 MOIs are shown). To further evaluate whether this assay was quantitative and therefore suitable as a cell-based potency assay, the 12-point data were analyzed using an appropriate curve fitting algorithm. Fig.12 As shown in , the assay showed good curve fit using the hyperbolic model (R 2=0.994). When the x-axis is logarithmically transformed, the MOI (from 200K to 12.5K) falls within the linear range.

[0821] The dose-dependent fitting data suggested the possibility of utilizing an image-based assay to determine the relative potency of AAV9-SMN1 vectors by parallel line analysis (PLA).

[0822] Furthermore, together with data comparing three AAV9-SMN1 vector clinical batches, these data support proof-of-concept that robust quantitative measurement of SMN1 can be achieved using a cell-based in vitro assay.

[0823] Table 5. 12-point MOI IFI-C data

[0824]

[0825] Statistical methods

[0826] To evaluate the suitability of the mTD-NPC-Δ7-based imaging assay as a potential batch-handling potency assay, we determined 5 MOIs that supported parallel line analysis (PLA). The following statistical analysis demonstrated that parallel line analysis (PLA) can be used to calculate the relative potency of AAV9-SMN1 vectors using a linear regression model.

[0827] Integrated fluorescence intensity / cell (IFI-C)

[0828] Integrated fluorescence intensity / cell (IFI-C) values ​​were calculated for each dose and each replicate. IFI-C was the assay readout for each dose.

[0829] Linear dose-response model

[0830] Five replicates were obtained for each MOI measured and a linear regression model was applied and showed a good fit with 98.9% R2 after logarithmic transformation of the x-axis (dose).

[0831] Proof-of-Concept Study Using PLA to Determine Relative Potency (RP)

[0832] In a paired analysis, five sets of SMN1 IFI-C were used to perform a "simulated" relative potency calculation on the logMOI data, with one set of data as the "simulated reference standard" relative to another set of data as the "simulated test article". Using this approach, there are ten possible pairwise combinations, as listed below (Table 6). The relative potencies (%) of the 10 simulated test articles ranged from 93.6% to 101.8%, with CV% less than 5%. Statistical analysis demonstrated that the newly developed assay is quantitative, with a 16-fold linear range covering 5 MOIs separated by 2-fold.

[0833] The assay's robustness, validation, and verification were determined, followed by a final evaluation of the assay's utility as a potency assay intended for batch handling of AAV9 drug product.

[0834] Table 6. Relative potency calculation data

[0835] Comparison between replicates Relative efficacy (%) 1 and 2 100.6 1 and 3 99.7 1 and 4 101.3 1 and 5 93.6 2 and 3 100.8 2 and 4 99.3 2 and 5 107.4 3 and 4 98.5 3 and 5 106.5 4 and 5 108.2 Mean 101.6 Standard Deviation 4.5

[0836] Evaluation of the optimal SMN antibody (2B1) concentration for mTD-NPC-Δ7 immunostaining

[0837] An ad hoc PLA tool was used to further evaluate the effect of different anti-SMN (2B1) antibody concentrations on the staining of mTD-NPC-Δ7 transduced with an AAV9-SMN1 vector. Compared to other immunoassays (such as ELISA), the acceptable concentrations of primary and secondary antibodies used in image-based assays are generally wider. This is due to the ability of image-based assays enabled by the instrument's highly sensitive camera to detect a wide range of signals by adjusting the exposure time to the light source. Therefore, the main goal of antibody concentration optimization is to ensure that the selected primary or secondary antibody concentration is not a limiting factor for quantitative determination of the target protein level (e.g., SMN1) as determined by a dose-dependent response.

[0838] For this purpose, three different concentrations of SMN (2B1) antibody (4 μg / mL, 2 μg / mL and 1 μg / mL) were tested to assess the linearity of the dose-dependent response determined by linear regression. Briefly, 12 MOI doses prepared from a batch of SMN1-encoding AAV9 vectors were added to the wells as shown in Table 7, and the cells were then immunostained by 2 μg / mL, 1 μg / mL and 4 μg / mL of SMN antibody according to the plate layout. As described above, five doses ranging from 200K MOI to 12.5K MOI were fitted to a linear regression and showed excellent linearity (R ) for the dose-dependent increase in SMN protein levels for all three conditions tested. 2 =0.98). In addition, the interim PLA analysis data was used to calculate the relative potency % by taking the 2 μg / mL condition as the reference standard and comparing the 4 μg / mL and 1 μg / mL conditions to the reference standard. In this analysis, the three conditions of SMN antibody concentration were demonstrated to be comparable, with relative potency values ​​of 99.8%, 100%, and 97.2% and slope ratios (slope of the test condition to the slope of the reference standard) of 0.991, 1.000, and 0.980 for 4 μg / mL, 2 μg / mL (reference standard), and 1 μg / mL, respectively. Fig.17and Table 8). Taken together, these data demonstrate that the SMN1 antibody is not a limiting factor for the quantification of SMN protein expressed in mTD-NPC-Δ7 cells.

[0839] Given equivalent data obtained from three different concentrations of SMN1 antibody, a midpoint of 2 μg / mL was chosen for establishing the relative in vitro cell-based potency of SMN1-encoding AAV9 vectors. This concentration was chosen to avoid excessive depletion of SMN (2B1) antibody, but to ensure that slight changes in primary antibody concentration would not impair assay performance.

[0840] The secondary antibody was used at a concentration of 2 μg / mL based on the supplier's recommendations to ensure that the secondary antibody was not a limiting factor in the detection of the primary antibody bound to the target protein plated in the microplate format. Fig.17 The data shown in Table 8 establish the quantification of the dose-dependent increase in SMN with excellent linearity (R 2 0.98) and had comparable slopes as shown by the slope ratios in Table 8, confirming the presence of sufficient amounts of secondary antibody for immunostaining.

[0841] Table 7. Plate layout comparing three different SMN antibody concentrations

[0842]

[0843] Table 8. Comparison of three SMN antibody concentrations by PLA

[0844] SMN antibody concentration RP% The slope ratio of RS to the sample 4ug / mL 99.8％ 0.991 2ug / mL (used as reference standard) 100％ NA 1ug / mL 97.2％ 0.980

[0845] in conclusion

[0846] The above studies describe the successful identification of a novel primary mouse cell model system that is permissive for AAV9; the development of a cell-based in vitro method for measuring transgene (SMN1) expression by using a high-content image-based quantification system; and POC data demonstrating the potential applicability of the developed mTD-NPC-Δ7-based assay as a batch-handling potency assay after assay validation was completed.

[0847] Furthermore, it has been shown that the newly established cell-based assay system has the following characteristics:

[0848] Rapidly replicating NPCs enable the generation of a pool of cells that have a progenitor phenotype preserved before they differentiate;

[0849] mTD-NPC-Δ7 is naturally permissive to AAV9 transduction and is MOA-reflective;

[0850] Measurement of SMN1 expression levels in mTD-NPC-Δ7 showed a good assay window (signal:background>4) due to the low background of TD-NPCs (derived from SMN- / - mice) and the fact that these cells are non-dividing (allowing for accumulation of transgene products);

[0851] The assay window allows for quantitative measurement of dose-dependent increases in SMN1 protein levels;

[0852] The assay to measure IFI-C is robust and provides throughput in a 96-well plate format;

[0853] Statistical analysis supports the use of PLA to calculate relative potency with accuracy and reproducibility.

[0854] Example 3: Development of a novel approach to target AAV9 infectivity in mNPCs

[0855] A robust quantitative mechanism of action (MOA)-reflective AAV9 infectivity assay using the SMN- / -Δ7 mouse neural progenitor cell line (mNPC) and the high-content imaging system CellInsight was established. Using the mNPC-based assay platform, the in vitro relative potency assay of AAV9 vectors was developed as a robust quantitative infectivity assay.

[0856] Proof-of-concept data were obtained showing that the assay is superior to the current TCID50 infectivity assay in aspects such as its quantitative nature and easier and less cumbersome assay process.

[0857] A proof-of-concept study was conducted to establish the EC 50 AAV9 vector infectivity assay

[0858] Table 9. EC as infection titer 50

[0859]

[0860] The entire dose range of multiplicities of infection (MOI) covering both the lower and upper plates was determined to have an excellent 4-p fit (0 to 8000K MOI).

[0861] The EC expressed as viral genomes / ml (vg / mL) 50 The value is used as the infectious titer of the AAV9 vector drug product, and this value replaces the cumbersome and variable TCID50 determination.

[0862] Advantages of this infectivity assay using SMN- / - mNPC cells compared to the current TCID50-based assay using HeLaRC32 cells

[0863] Table 10. Comparison of two methods for determining AAV9 vector infectious titers.

[0864]

[0865] Example 4: Determination of the relative potency of SMN1-encoding AAV9 vector drug substance and drug product in vitro

[0866] The relative potency of the AAV9-SMN1 vector drug substance and drug product was determined using the cell-based in vitro quantitative relative potency assay developed in Examples 1-3.

[0867] The cell-based relative potency assay is a robust quantitative in vitro assay for determining relative potency, which is intended for batch handling and stability testing of SMN1-encoded AAV9 vector drug substances and drug products. The cells used for this assay are derived from mouse primary neural progenitor cells (NPCs) isolated from the cortex of SMNΔ7 mice, as described in Examples 1 and 2. SMNΔ7 mice are an in vivo animal model of SMA disease that is homozygous for the SMN1 knockout allele and homozygous for the allele of SMN2 with a deletion of exon 7. The cell model system used in this assay is terminally differentiated NPCs (mTD-NPC-Δ7). In mTD-NPC-Δ7 transduced with increasing doses of SMN1-encoded AAV9 vectors, the increasing levels of SMN protein expression are measured by staining with a monoclonal antibody specific for SMN.

[0868] The relative potency of samples relative to the reference standard (RS) was calculated using parallel lines analysis (PLA) described by the following equation, where α (intercept) and β (slope) are estimates of the linear regression line from the common slope model:

[0869]

[0870] The high-content imaging platform (CellInsight CX5) used in this assay enables quantitative measurement of intracellular protein expression on a per-cell basis (integrated fluorescence intensity / cell). In addition, the CellInsight CX5 platform allows for appropriate throughput for batch processing and stability studies.

[0871] Cell-based relative potency assays were performed using the following equipment: a biological safety cabinet (BSC), a CellInsight CX5 (Thermo Fisher CX51110), a 37 ± 1 °C, 5% CO 2 Humidified CO 2 Incubator, water bath set at 37°C or equivalent, centrifuge with temperature control, Cellometer K2 Image Cytometer, and microscope.

[0872] The following materials were used for the cell-based relative potency assay: tissue culture treated flasks (T75, T150, and T175), polypropylene centrifuge tubes with caps 15 and 50 mL, pipettes (single channel P1000, P200, and P20 and 8 or 12 channel P1000 and P300), Corning BioCoat Poly-D-Lysine 96-well plates (Corning 354640), optically clear plate seals (Fisher Brand 8408240), reagent storage containers, PIPET-AID, Cellometer slides (Nylon, CHT4-SD100-002), 96-well DeepWell TM Polypropylene microplates, low-binding 1.5 mL microcentrifuge tubes, 70% (v / v) isopropyl alcohol (IPA), and dry ice.

[0873] Cell-based relative potency assays were performed using the reagents listed in Table 11 below. Unless otherwise noted or retested, reagents were kept to the manufacturer's recommended expiration date.

[0874] Table 11. Reagents

[0875]

[0876]

[0877]

[0878] Determination procedure

[0879] NPC-Δ7 cells were thawed using the protocol outlined below.

[0880] Pre-warm complete growth medium in a 37 °C water bath or equivalent for at least 30 min prior to use.

[0881] Remove the frozen cryovial of mNPC cells from liquid nitrogen storage. Keep the vial on dry ice until it is ready to thaw. Thaw the vial quickly in a 37°C water bath, vortexing occasionally to ensure thawing.

[0882] Wipe the surface of the vial with 70% (v / v) isopropyl alcohol (IPA) and transfer the contents to a 50 mL centrifuge tube using a sterile pipette in the BSC. After thawing the cells, dilute the cryoprotectant slowly to prevent osmotic shock. About 10-20 mL is usually sufficient to overcome toxic effects.

[0883] 10-20 mL of warm complete growth medium was added dropwise while mixing gently by vortexing, followed by centrifugation at 300 x g for 5 minutes at 20 °C.

[0884] Aspirate the supernatant and gently agitate the tube to break up the cell pellet.

[0885] Add an appropriate volume (1.0-2.0 mL is recommended) of warm complete growth medium to the cells and mix gently with a pipette.

[0886] Live cell counts and viability were obtained using the protocol "NPCd7 Count Viability (AO / PI)" in the Cellometer K2 software. Viability was expected to be ≥ 60.0% viable for each count.

[0887] Transfer cells to a T75 tissue culture flask and record the volume of cells collected. Add 10.0 mL of complete growth medium and gently shake the flask to ensure even distribution.

[0888] The flask was then placed in a chamber set at 37°C and 5% CO. 2 Incubate in a 37°C, 5% CO incubator. 2 Cultures were incubated at 4 °C for at least 72 h and then tested for growth and viability. Cells were passaged every 4 ± 1 days.

[0889] Cultivation of NPC-Δ7 cells

[0890] From the second generation after thawing, cells are used in the assay. Thawing is not considered a passage. Cells are used to the 15th generation. For example, if the working cell bank is frozen at or after the 6th generation (P6), when the cells are thawed, the number of passages they retain is P6. After the appropriate number of days of cell proliferation, the cells are passaged by dissociation with Accumax and become P7. At P7, cells cannot be used for assay. When the cells are passaged again (P8) by dissociation with Accumax, these cells can be used in the assay.

[0891] Table 12. Examples of passage designations when thawing and passaging cells 1

[0892]

[0893]

[0894] 1 References in this table to passages at which cells were frozen are examples only.

[0895] As the cells proliferate in suspension, they form 3-dimensional colonies called neurospheres. To prevent the neurospheres from growing too large and becoming necrotic in the center, cells are passaged every 4 ± 1 days.

[0896] Cell passaging procedures

[0897] Cells were passaged according to the procedure described below.

[0898] Pre-warm basal medium and complete growth medium in a 37 °C water bath or equivalent for 30 min prior to use.

[0899] To passage the cells, the flask containing the cells was removed from the incubator and the surface of the flask was rinsed with the culture medium containing the cells.

[0900] Transfer cells from the flask to a 50 mL conical tube and centrifuge at 300 x g for 5 min.

[0901] Aspirate the supernatant without disturbing the cell pellet and add 200.0 μL Accumax. Gently triturate the cell pellet by pipetting up / down several times until the pellet is dissociated, then incubate at room temperature for 30 ± 10 minutes.

[0902] At the end of the Accumax incubation, neutralize Accumax by adding 400.0 μL of pre-warmed basal medium. Gently triturate the cells (eg, 10-15 times) to completely dissociate into single cells.

[0903] The basal medium of 400.0 μ L preheating is added again so that cumulative volume is 1.0 mL. The basal medium of additional volume dilutes cells to 5.00E+05 cells / mL to 1.00E+07 for the acceptable cell density range of cell counting. After cell has been dissociated with Accumax and neutralized with basal medium respectively, optionally at this moment, under the same passage number, the cells from multiple flasks with the same cell reference product / batch are brought together. Every kind of cell suspension (cell in 200.0 μ L Accumax by 800.0 μ L basal medium) of 1.0 mL in total can be merged. The basal medium of additional volume is added to dilute cells to 5.00E+05 cells / mL to 1.00E+07 for the acceptable cell density range of cell counting (for example, 1.0 mL cells from flask 1 and 1.0 mL cells from flask 2 are mixed, and then 3.0 mL basal medium is added to further dilute cells).

[0904] The cells were mixed and then a small sample of the cells was removed to determine the live cell count and viability using the protocol "NPCd7 Count Viability (AO / PI)" in the Cellometer K2 software. When the viability of each cell count was ≥80.0%, mTD NPC-Δ7 (terminally differentiated NPCΔ7) plates were prepared. To continue culturing the cells, the viability of each cell count must be ≥70.0% viable.

[0905] If the cell suspension concentration is too high, add additional volume of basal medium and mix thoroughly and re-determine the viable cell count and viability.

[0906] Dilute the cell suspension to 100,000 cells / mL in pre-warmed complete growth medium. Pipette the appropriate volume into a new cell culture dish and return the cells to 37°C ± 1°C, 5% CO. 2 incubator at 4 °C for 4 ± 1 days.

[0907] Example calculation per flask:

[0908] Total volume: 20.0mL, Average viable cell density: 4.50x10 6 cells / mL

[0909]

[0910]

[0911] Table 13. Recommended volumes for tissue culture flasks

[0912] Flask <![CDATA[Surface area (cm 2 )]]> Total volume of culture medium (mL) T-75 75 10-15 T-150 150 20-25 T-175 175 25-40

[0913] The passaging scheme is optionally repeated, with each passaging cycle adding one passage number to the previous passage.

[0914] Preparation of mTD NPC-Δ7 (terminally differentiated NPCΔ7) plates

[0915] Cells were plated using a plating medium different from the medium used to passage the cells. Plating medium allows NPC cells to terminally differentiate. At least 7 mL of cells in plating medium were prepared per plate.

[0916] Record viable cell counts and determine the appropriate volume of cells to be used for plating.

[0917] Dilute the cells to 20,000 cells / well / 100 μL (or 2.00 x 10 5 cells / mL) density.

[0918] Example calculations per plate:

[0919] Total volume: 8.0mL, Average viable cell density: 4.50x10 6 cells / mL

[0920]

[0921] ...

Claims

1. An in vitro method for measuring transgene expression, the method comprising: include: a) providing a first plurality of terminally differentiated neural progenitor cells (NPCs), wherein the first plurality of terminally differentiated NPCs comprises a homozygous SMN1- / - mutation; b) transducing the first plurality of terminally differentiated NPCs with a test sample comprising an AAV9 viral vector comprising a sequence encoding an SMN1 protein; c) incubating the transduced first plurality of terminally differentiated NPCs for 69-75 hours or 75-90 hours under conditions sufficient to express SMN1 protein; d) fixing and permeabilizing said first plurality of terminally differentiated NPCs; e) contacting said first plurality of terminally differentiated NPCs from d) with an antibody specific for SMN1 protein; f) imaging said first plurality of terminally differentiated NPCs to obtain an integrated fluorescence intensity / cell readout; g) determining the expression of SMN1 protein based on the integrated fluorescence intensity / cell readout; h) providing a second plurality of terminally differentiated NPCs, wherein the second plurality of terminally differentiated NPCs comprises a homozygous SMN1- / - mutation; i) transducing said second plurality of terminally differentiated NPCs with a reference standard comprising said AAV9 viral vector; j) incubating the transduced second plurality of terminally differentiated NPCs for 69-75 hours or 75-90 hours under conditions sufficient to express SMN1 protein; k) fixing and permeabilizing said second plurality of terminally differentiated NPCs; l) contacting said second plurality of terminally differentiated NPCs from k) with an antibody specific for SMN1 protein; m) imaging the second plurality of terminally differentiated NPCs to obtain an integrated fluorescence intensity / cell readout; and n) comparing the integrated fluorescence intensity / cell of the first plurality of terminally differentiated NPCs with the integrated fluorescence intensity / cell of the second plurality of terminally differentiated NPCs; Thus determining the relative potency of the AAV9 viral vector of the test sample relative to the reference standard, wherein the first and second plurality of terminally differentiated NPCs are generated by terminally differentiating neural progenitor cells isolated from the cortex of an SMN1- / - mouse embryo, wherein the terminal differentiation comprises: a. Neural progenitor cells isolated from the cortex of SMN1- / - mouse embryos were cultured in a serum-free medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to form neurospheres; b. dissociating the neurospheres to produce dissociated NPCs; and c. culturing the dissociated NPCs in a serum-rich medium without growth factors; Thus, the terminally differentiated NPCs are generated. The SMN1- / - mutation is a deletion of SMN1 exon 7.

2. The method of claim 1, wherein the first plurality of terminally differentiated NPCs and the second plurality of terminally differentiated NPCs are transduced by the test sample and the reference standard at at least two different multiplicity of infection (MOI) of the AAV9 viral vector.

3. The method of claim 2, wherein the first plurality of terminally differentiated NPCs and the second plurality of terminally differentiated NPCs are transduced with the test sample and the reference standard at 5 MOIs, wherein the 5 MOIs are 300,000, 150,000, 75,000, 37,500, and 18,750 viral particles / cell.

4. The method of claim 1, wherein the comparing step n) comprises plotting a standard curve of MOI versus integrated fluorescence intensity / cell for each of the test sample and the reference standard.

5. The method of claim 1, wherein the comparing step n) comprises calculating a linear regression of log MOI versus integrated fluorescence intensity / cell for each of the test sample and the reference standard to yield a test sample slope and a reference standard slope.

6. The method of claim 1, wherein determining the relative potency of the AAV9 viral vector is performed by parallel lines analysis (PLA), and wherein the PLA comprises measuring the slope ratio of the test sample slope relative to the reference standard slope.

7. The method of claim 6, wherein the reference standard slope is greater than or equal to 1.02E+05, and wherein the slope ratio is between 0.69-1.

45.

8. The method of claim 6, comprising calculating an assay dynamic window for the reference standard, and wherein the assay dynamic window is greater than or equal to 2.

69.

9. The method of claim 1, wherein the AAV9 viral vector comprises AAV serotype 2 inverted terminal repeats (ITRs).

10. The method of claim 9, wherein the AAV9 viral vector comprises a sequence encoding a cytomegalovirus (CMV) enhancer / chicken-β-actin hybrid promoter (CB) operably linked to a sequence encoding an SMN1 protein.

11. The method of claim 9, wherein the AAV9 viral vector comprises the sequence of SEQ ID NO:

1.

12. The method of claim 1, wherein the integrated fluorescence intensity / cell readout reflects a measure of protein expression.

13. The method of claim 1, wherein the antibody is provided at a concentration of 4 μg / mL.

14. The method of claim 1, wherein the antibody comprises a detectable label.

15. The method of claim 1, further comprising contacting the terminally differentiated NPCs with a second molecule that specifically recognizes an antibody specific for SMN1 protein.

16. The method of claim 15, wherein the second molecule comprises a detectable label.

17. The method of claim 15, wherein the second molecule comprises an antibody, an antibody fragment, or an aptamer.

18. The method of claim 1, wherein following the fixation and permeabilization steps, the terminally differentiated NPCs are contacted with an anti-nuclear detectable marker.

19. The method of claim 1, wherein the terminally differentiated NPCs are on a solid surface.

20. A method for producing a pharmaceutical composition comprising an AAV9 viral vector containing a transgene encoding an SMN1 protein, the method comprising: include: a. Produce an AAV9 viral vector containing a transgene encoding SMN1 protein; b. determining the AAV9 viral vector according to the method of any one of claims 1 to 19; and c. When the AAV9 viral vector in the test sample has a relative potency of at least 60% compared to the AAV9 viral vector in the reference standard, the AAV9 viral vector containing a transgene encoding the SMN1 protein is formulated in a pharmaceutical composition.

21. The method of claim 20, wherein the pharmaceutical composition comprises: a.1-8x 10 13 AAV9 viral vector genomes / mL; b. Less than 7% empty viral capsids; c. Every 1x 10 13 Host cell proteins with a concentration of less than 100 ng / mL; or d. Every 1x 10 13 vg / mL less than 5x 10 6 pg / mL of residual host cell DNA.

22. The method of claim 20, wherein the pharmaceutical composition comprises: a. Every 1.0x10 13 vg less than 0.09ng of universal nuclease, b. Less than 30ppm cesium, c. 20-80 ppm of Poloxamer 188, d. Every 1.0x10 13 vg less than 0.22ng BSA, e. Every 1.0x10 13 vg less than 6.8x10 5 Residual plasmid DNA of pg, f. Every 1.0x10 13 vg less than 1.1x10 5 pg of residual hcDNA, g. Every 1.0x10 13 rHCP with vg less than 4ng, h.pH 7.7-8.3, i.390-430mOsm / kg, j. Less than 600 particles ≥ 25 μm in size per container, k. Less than 6000 particles ≥ 10 μm in size per container, l.1.7x 10 13 -2.3x 10 13 vg / mL genome titer, m. Every 1.0x 10 13 vg 3.9x 10 8 -8.4x 10 10 The infectious titer of IU, n. Every 1.0x 10 13 vg 100-300 μg total protein, and o. Less than 5% empty capsids.

23. The method of claim 20, wherein the pharmaceutical composition comprises: a. pH 7.7-8.3, b.390-430mOsm / kg, c. Less than 600 particles ≥ 25 μm in size per container, d. Less than 6,000 particles ≥ 10 μm in size per container, e.1.7x 10 13 -2.3x 10 13 vg / mL genome titer, f. Every 1.0x 10 13 vg 3.9x 10 8 -8.4x 10 10 The infectious titer of IU, g. Every 1.0x 10 13 vg 100-300μg total protein, h.20-80ppm Pluronic F-68 content, i.at 7.5x 10 13 At a dose of 100 vg / kg, the median survival in the Δ7SMN mouse model was greater than or equal to 24 days. j. Less than 5% empty capsids, k. and an overall purity greater than or equal to 95%, and l. Endotoxin less than or equal to 0.75EU / mL.

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