Means and methods for preparing viral vectors and uses thereof

Abstract

Methods of making and purifying viral particles are provided, as well as compositions and uses comprising the viral particles.

Description

[0001] This application is a divisional application of application No. 201880085757.1, filed on November 1, 2018, with the invention name “Means and methods for preparing viral vectors and their uses”.

[0002] Sequence Listing

[0003] This application contains a sequence listing that has been submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on October 31, 2018, is named AVEX-003001WO_ST25.txt, and is 14,639 bytes in size.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to U.S. Provisional Patent Application No. 62 / 583,035, filed November 8, 2017, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0006] The present disclosure relates to methods of preparing and purifying viral particles, as well as compositions and uses comprising the viral particles. Background Art

[0007] Adeno-associated virus (AAV) is a member of the Parvoviridae family. The AAV genome consists of a single-stranded DNA molecule comprising approximately 4.7 kilobases (kb) and consisting of two major open reading frames (encoding nonstructural Rep (replication) and structural Cap (capsid) proteins). The AAV coding region is flanked by two cis-acting inverted terminal repeats (ITRs), approximately 145 nucleotides in length, with interrupted palindromic sequences (which can fold into a hairpin structure that acts 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 encapsulation of viral nucleic acids into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. [Microbial Immunology Frontier] 158: 97-129).

[0008] There are many serotypes of AAV, and they provide different tissue tropisms. Known serotypes include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. AAV9 is described in U.S. Patent No. 7,198,951 and Gao et al., J. Virol. [Journal of Virology], 78: 6381-6388 (2004), the entire contents of which are incorporated herein by reference. Advances in the delivery of AAV6 and AAV8 have made it possible for these serotypes to transduce skeletal and cardiac muscle after simple systemic intravenous or intraperitoneal injection. See Pacak et al., Circ. Res. [Circulation Research], 99 (4): 3-9 (2006) and Wang et al., Nature Biotech. [Nature Biotechnology] 23 (3): 321-8 (2005). However, the use of AAV to target cell types within the central nervous system requires surgical intraparenchymal injection. See Kaplitt et al., “Safety and tolerability of gene therapy with an adeno-associated virus (AAV)borne GAD gene for Parkinson's disease: an open label, phase I trial.” Lancet, 369:2097-2105; Marks et al., “Gene delivery of AAV2-neurturin for Parkinson's disease: a double-blind, randomized, controlled trial.” Lancet Neurol, 9:1164-1172; and Worgall et al., “Treatment of late infantile neuronal ceroid lipofuscinosis by CNS administration of a serotype 2adeno-associated virus expressing CLN2cDNA. [Treatment of late infantile neuronal ceroid lipofuscinosis by CNS administration of a serotype 2 adeno-associated virus expressing CLN2 cDNA].” Hum Gene Ther [Human Gene Ther], 19(5):463-74.

[0009] The nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol [Journal of Virology], 45:555-564 (1983), as revised by Ruffing et al., J Gen Virol [Journal of Genetic Virology], 75:3385-3392 (1994). Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs. Three AAV promoters (designated p5, p19, and p40 due to their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. These two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The Rep proteins have multiple enzymatic properties that are ultimately responsible for replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0010] 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 myofibers and neurons; (ii) they lack viral structural genes, thereby eliminating natural host cell responses to viral infection, such as interferon-mediated responses; (iii) wild-type viruses have 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 are typically present as episomes, thereby 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), thereby allowing long-term expression of therapeutic transgenes (provided their gene products are not rejected).

[0011] Self-complementary adeno-associated vector (scAAV) is a viral vector for gene therapy constructed from naturally occurring adeno-associated virus (AAV). ScAAV is called "self-complementary" because the coding region is designed to form an intramolecular double-stranded DNA template. The rate-limiting step of the standard AAV genome life cycle includes second-strand synthesis because the typical AAV genome is a single-stranded DNA template. However, this is not the case for the scAAV genome. Upon infection, instead of waiting for cell-mediated second-strand synthesis, two complementary half-scAAVs will associate to form a double-stranded DNA (dsDNA) that is easily replicated and transcribed immediately.

[0012] There remains a need to develop scalable methods to manufacture and purify AAV drug products with, for example, low empty capsids / low host cell proteins and / or low contaminating DNA while maintaining high potency. Summary of the Invention

[0013] The present disclosure provides methods for preparing purified viral particle preparations, including AAV particle preparations.

[0014] In some embodiments, the present disclosure provides a pharmaceutical composition comprising (a) 1-8 x 10 13 AAV9 viral vector genomes / mL (vg / mL), (b) less than about 7% empty viral capsids, and (c) less than about 100 ng / mL host cell protein / 1x10 13 vg / mL, and (d) less than about 5 x 10 6 pg / mL residual host cell DNA / 1x 10 13 vg / mL, and 1-8x10 13 At least about 80% of the AAV9 viral vector genomes / mL are functional.

[0015] In one embodiment, the AAV9 viral vector comprises a polynucleotide encoding a survival motor neuron (SMN) protein. In one embodiment, the AAV9 viral vector comprises a polynucleotide encoding a methyl-CpG binding protein 2 (MECP2) protein. In one embodiment, the AAV9 viral vector comprises a polynucleotide encoding a short hairpin RNA (shRNA) targeting superoxide dismutase 1 (SOD1). In one embodiment, the AAV9 viral vector comprises 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.

[0016] The present disclosure provides pharmaceutical formulations. In some embodiments, the aqueous pharmaceutical formulation comprises (a) an AAV9 viral vector comprising a polynucleotide encoding a survival motor neuron (SMN) protein, (b) Tris buffer, (c) magnesium chloride, (d) sodium chloride, and (e) poloxamer (e.g., poloxamer 188), wherein the pharmaceutical composition does not comprise a preservative. In one embodiment of the formulation, the AAV9 viral vector further comprises 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. In one embodiment of the formulation, the Tris buffer concentration is about 10-30 nM, for example, about 20 mM. In one embodiment, the pH of the formulation is about 7.7 to about 8.3, for example, about pH 8.0 (e.g., as determined by USP <791> (incorporated by reference in its entirety)). In one embodiment of the formulation, the magnesium chloride concentration is about 0.5-1.5 mM, for example, about 1 mM. In one embodiment of the formulation, the sodium chloride concentration is about 100-300 mM, for example, about 200 mM. In one embodiment, the formulation comprises about 0.005% w / v poloxamer 188.

[0017] Another aspect of the present invention relates to a method for treating type 1 spinal muscular atrophy (SMA) in a patient in need thereof, the method comprising administering to the patient an AAV9 viral vector (e.g., a composition or formulation disclosed herein) comprising a polynucleotide encoding an SMN protein via an intrathecal or intravenous route, wherein the patient is (a) nine months of age or younger, (b) has a body weight of at least about 2.6 kg, (c) has a biallelic SMN1 null mutation or deletion, and (d) has at least one functional copy of SMN2. In one embodiment, the AAV9 viral vector comprises 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. In one embodiment, the patient weighs no more than about 8.5 kg. In one embodiment, the patient does not have a c.859G>C substitution in exon 7 of at least one copy of the SMN2 gene. In one embodiment, treatment is administered to the patient before the age of six months. In one embodiment, the treatment is administered to the patient prior to the onset of one or more SMA symptoms selected from the group consisting of hypotonia, delayed motor skills, poor head control, rounded shoulder posture, and joint hypermobility. In one embodiment, the patient has an anti-AAV9 antibody titer of 1:100 or less as determined by ELISA binding immunoassay prior to administration.

[0018] Also disclosed herein are methods of treating a pediatric patient with spinal muscular atrophy (SMA) type 1, with or without disease onset, comprising administering to the patient a composition or formulation comprising an adeno-associated viral (AAV) vector as disclosed herein.

[0019] The present disclosure also relates to methods of treating Rett syndrome in a patient in need thereof, comprising administering to the patient an AAV9 viral vector comprising a polynucleotide encoding a methyl-CpG-binding protein 2 (MECP2) protein via an intrathecal or intravenous route.

[0020] The present disclosure also relates to a method of treating amyotrophic lateral sclerosis (ALS) in a patient in need thereof, the method comprising administering to the patient an AAV9 viral vector via an intrathecal or intravenous route, the AAV9 viral vector comprising a polynucleotide encoding a short hairpin RNA (shRNA) targeting superoxide dismutase 1 (SOD1).

[0021] Another aspect of the present invention relates to a method of treating a patient with type I SMA, the method comprising the steps of: (a) determining the patient's weight, (b) obtaining a kit comprising vials of an AAV9 viral vector pharmaceutical composition, and (c) administering the AAV9 viral vector from the vials to the patient, wherein the concentration of the viral vector in each vial is about 2.0 x 10 13 vg / mL, wherein the AAV9 viral vector comprises a polynucleotide encoding an SMN protein; and wherein the kit comprises the following number of vials:

[0022]

[0023] In one embodiment, the kit comprises an AAV9 viral vector comprising a mutated 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 AAV2 ITR. In one embodiment, the AAV viral vector is expressed at about 1.0 x 10 14 -2.5x 10 14 Doses of vg / kg were administered by infusion.

[0024] Another aspect of the present disclosure relates to a kit for treating a patient with spinal muscular atrophy (SMA) type I, the kit comprising a vial containing a composition AAV9 viral vector comprising a polynucleotide encoding a survival motor neuron (SMN) protein or a formulation comprising (a) an AAV9 viral vector comprising a polynucleotide encoding a survival motor neuron (SMN) protein, (b) Tris buffer, (c) magnesium chloride, (d) sodium chloride, and (e) a poloxamer (e.g., poloxamer 188).

[0025] Another aspect of the present disclosure relates to a kit comprising a vial containing about 5.5 mL or about 8.3 mL of an AAV9 viral vector comprising a polynucleotide encoding a survival motor neuron (SMN) protein and at about 2.0 x 10 13 The concentration of vg / mL is prepared in 20 mM Tris, 1 mM MgCl2, 200 mM NaCl, 0.005% w / v Poloxamer 188 (pH 7.7-8.3, such as about 8.0).

[0026] Another aspect of the present disclosure relates to a method of treating type I SMA, comprising administering to a patient in need thereof by intravenous infusion a volume of a composition comprising an AAV9 viral vector comprising a polynucleotide encoding a survival motor neuron (SMN) protein or a formulation comprising (a) an AAV9 viral vector comprising a polynucleotide encoding a survival motor neuron (SMN) protein, (b) Tris buffer, (c) magnesium chloride, (d) sodium chloride, and (e) a poloxamer (e.g., poloxamer 188).

[0027] Another aspect of the present disclosure relates to a method for manufacturing an AAV viral vector. In one embodiment, the method for manufacturing an AAV viral vector comprises the following steps: (a) culturing adherent cells, (b) transfecting adherent cells with one or more plasmids to produce AAV viral vectors, (c) lysing adherent cells to isolate AAV viral vectors, (d) acidifying and clarifying the cell lysate of (c), (e) purifying the product of (d) using cation exchange chromatography (CEX), (f) filtering the product of (e) using tangential flow filtration (TFF), (g) ultracentrifuging the product of (f) in cesium chloride (CsCl) buffer; and (h) collecting the AAV viral vector from the product of (g).

[0028] Another aspect of the present disclosure relates to a method for purifying an AAV viral vector from a cell culture lysate, the method comprising the steps of: (a) acidifying and clarifying the cell lysate, (b) purifying the product of (a) using cation exchange chromatography (CEX), (c) filtering the product of (b) by tangential flow filtration, (d) ultracentrifuging the product of (c) using a 2-4 M cesium chloride (CsCl) buffer, (e) collecting the AAV viral vector from the product of (d), and (f) filtering the product of (e) by tangential flow filtration. In one embodiment, the method is performed on an industrial scale. In one embodiment, the method produces greater than 5 x 10 15 vg, or larger than 8x 10 15 vg or greater than 1x 10 16 Yield of vg / manufacturing batch.

[0029] Another aspect of the present disclosure relates to a method of treating a patient suffering from type 1 SMA by administering an AAV9 viral vector prepared according to any of the methods disclosed herein, the AAV9 viral vector comprising a polynucleotide encoding an SMN protein.

[0030] Another aspect of the present disclosure relates to a method of treating a patient suffering from Rett syndrome by administering an AAV9 viral vector prepared according to any of the methods herein, the AAV9 viral vector comprising a polynucleotide encoding a MECP2 protein.

[0031] Another aspect of the present disclosure relates to a method of treating a patient suffering from ALS by administering an AAV9 viral vector prepared according to any of the methods herein, the AAV9 viral vector comprising a polynucleotide encoding a shRNA targeting SOD1.

[0032] Another aspect of the present disclosure relates to an AAV9 viral vector comprising a polynucleotide encoding an SMN protein, the viral vector being prepared according to any of the methods herein.

[0033] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an AAV9 viral vector comprising a polynucleotide encoding an SMN protein, the pharmaceutical composition being prepared according to any of the methods described herein.

[0034] Another aspect of the present disclosure relates to an aqueous pharmaceutical composition comprising an AAV9 viral vector comprising a polynucleotide encoding an SMN protein, a Tris buffer, a magnesium chloride solution, and a sodium chloride solution, wherein the pharmaceutical composition does not comprise a preservative, and wherein the composition is prepared according to any of the methods described herein.

[0035] Another aspect of the present disclosure relates to an AAV9 viral vector comprising a polynucleotide encoding a MECP2 protein, the viral vector being prepared according to any of the methods herein.

[0036] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an AAV9 viral vector comprising a polynucleotide encoding a MECP2 protein, the pharmaceutical composition being prepared according to any of the methods described herein.

[0037] Another aspect of the present disclosure relates to an AAV9 viral vector comprising a polynucleotide encoding a shRNA targeting SOD1, the viral vector being prepared according to any of the methods herein.

[0038] Another aspect of the present disclosure relates to a method of treating type I SMA in a patient in need thereof by intravenously administering a pharmaceutical composition comprising (a) a 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 bovine growth hormone (BGH) polyadenylation signal, and an unmodified AAV2 ITR, (b) 20 mM Tris, pH 8.0, (c) 1 mM MgCl2, (d) 200 mM NaCl, and (e) 0.005% poloxamer 188, wherein the patient weighs 2.6 kg to 8.5 kg. In one embodiment, the composition does not contain a preservative.

[0039] In one embodiment, the patient is (a) nine months of age or younger, (b) has a body weight of at least about 2.6 kg, (c) has a biallelic SMN1 null mutation or deletion, and (d) has at least one functional copy of SMN2.

[0040] Another aspect of the present disclosure relates to a composition suitable for or manufactured for intravenous administration of a pharmaceutical composition comprising (a) a 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 bovine growth hormone (BGH) polyadenylation signal, and an unmodified AAV2 ITR, (b) 20 mM Tris, pH 8.0, (c) 1 mM MgCl2, (d) 200 mM NaCl, and (e) 0.005% poloxamer 188.

[0041] In some embodiments, disclosed herein are compositions or formulations, wherein the compositions or formulations comprise at least one of the following: (a) less than about 0.09 ng benzonase / 1.0 x 1013 vg, (b) less than about 30 μg / g (ppm) cesium, (c) about 20-80 ppm of poloxamer 188, (d) less than about 0.22 ng BSA / 1.0 x 10 13 vg, (e) less than about 6.8x 10 5 pg residual plasmid DNA / 1.0x 10 13 vg, (f) is less than about 1.1x 10 5 pg residual hcDNA / 1.0x 10 13 vg, and (g) less than about 4ng rHCP / 1.0 x 10 13 vg,

[0042] In some embodiments, the present invention provides an upstream process for producing an intermediate (e.g., a frozen intermediate) derived from a working cell bank, wherein the upstream process comprises the following steps: (a) culturing cells, (b) transfecting the cultured cells with a plasmid (e.g., three plasmids), (c) harvesting amplified viral particles from the cells after the culture period, (d) purifying the viral particles by filtration to remove any intact cells or cell debris, (e) subjecting the eluate from step (d) to tangential flow filtration, and (g) optionally freezing the resulting intermediate preparation of purified viral particles. In some embodiments, the upstream process is combined with further processing steps (e.g., further purification and formulation steps) to produce a final drug product.

[0043] In one embodiment, the working cell bank comprises HEK293 cells. In other embodiments, another cell type or derivative available in the art and suitable for use in the methods disclosed herein is used.

[0044] In one embodiment, the transfection step utilizes polyethyleneimine.In one embodiment, the transfection step comprises a triple vector transfection using a transgenic plasmid (e.g., pSMN, pAAV plasmid, and pHELP plasmid).

[0045] In one embodiment, the method further comprises lysing the transfected cells with a lysis buffer and a surfactant.

[0046] In one embodiment, the harvesting step comprises treating the product with an endonuclease (eg, at a concentration between 50-200 U / ml, such as at a concentration between 75-150 U / ml), such as benzonase, to reduce residual host cell DNA.

[0047] In one embodiment, the purification step comprises depth filtration followed by filtration through a filter that removes macromolecular contaminants and cellular debris, such as a 0.45 micron filter, but allows the vector genome to pass through. Any suitable depth filter can be used.

[0048] In one embodiment, tangential flow filtration ("TFF") achieves 5-15X (e.g., 6-10X) concentration of the eluate of step (d) and diafiltration of at least 4 diafiltration volumes (e.g., 6 diafiltration volumes) or 10 diafiltration volumes or 12 diafiltration volumes or 15 diafiltration volumes. Any suitable TFF filter can be used. In one embodiment, the TFF membrane is a cellulose membrane. In one embodiment, the TFF membrane has a cut-off value of 300 kDa.

[0049] Second, the present disclosure provides a downstream process for processing an intermediate (e.g., a frozen intermediate) into a filtered drug substance. The downstream process steps include acidification and clarification steps (using filtration), followed by cation exchange chromatography, tangential flow filtration, CsCl ultracentrifugation, and further tangential flow filtration steps to produce a filtered drug substance in which purified AAV particles are suspended in a pharmaceutically acceptable carrier.

[0050] In one embodiment, the acidification and clarification steps include depth filtration and subsequent filtration through a filter that removes macromolecular contaminants and cell debris, such as a 0.45 micron filter. In certain embodiments, pH regulation is controlled. As part of the steps, in one embodiment, a detergent such as Tween is added. In certain embodiments, the addition rate of Tween and the concentration range of the Tween are controlled.

[0051] In one embodiment, the cation exchange chromatography comprises using a membrane based chromatography resin of a composite sulfonyl resin having a pore size of 0.2 microns.

[0052] In one embodiment, the cesium chloride (CsCl) ultracentrifugation step uses 2-4 M CsCl, such as about 3 M CsCl.

[0053] In another embodiment, the CsCl ultracentrifugation step is performed at about 40-50 kRPM for about 20-25 hours. In another embodiment, the CsCl ultracentrifugation step is performed at about 45 kRPM for about 22 hours.

[0054] In one embodiment, tangential flow filtration ("TFF") achieves 5-15X (e.g., 6-10X) concentration of the eluate of step (d) and diafiltration of at least 4 diafiltration volumes (e.g., 6 diafiltration volumes) or 10 diafiltration volumes or 12 diafiltration volumes or 15 diafiltration volumes. In one embodiment, the TFF membrane has a cut-off of 300 kDa.

[0055] In one embodiment, the eluent has a detection level below cesium (Cs). In another embodiment, the detection level of Cs is below 50 parts per million (ppm). In another embodiment, the detection level of Cs is between about 50 and 70 ppm. In another embodiment, the detection level of Cs is between about 70 and 90 ppm. In another embodiment, the detection level of Cs is between about 90 and 110 ppm. In another embodiment, the detection level of Cs is between about 110 and 130 ppm. In another embodiment, the detection level of Cs is between about 130 and 150 ppm. In another embodiment, the detection level of Cs is less than 150 ppm.

[0056] These purification methods can be used to prepare high-yield viral preparations, including AAV preparations (e.g., AAV9-SMN), containing less than 5×10 6 pg / ml residual host cell DNA (hcDNA) / 1X 10 13 Vector genomes ("vg") / ml, for example, less than 1.2X 10 6 pg / mL hcDNA / 1X 10 13 vg / mL. Therefore, accept 7.5X 10 15 vg of 5 kg patients to obtain no more than 1.2 x 10 6 pg / mL*7.5X 10 15 vg / (1X 10 13 vg / mL)=8.4X 10 7 pg hcDNA = 84,000ng hcDNA / 5kg dose. In one embodiment, the formulation contains less than 5.0 x 10 5 pg residual host cell DNA / 1.0x 10 13 vg, less than 2.0x10 5 pg residual host cell DNA / 1.0x10 13 vg, less than 1.1x 10 5 pg residual host cell DNA / 1.0x 10 13 vg, less than 1.0x 10 5 pg residual host cell DNA / 1.0x 10 13 vg, less than 0.9x 10 5 pg residual host cell DNA / 1.0x10 13 vg, less than 0.8x 10 5 pg residual host cell DNA / 1.0x 10 13vg, or any concentration in between.

[0057] In one embodiment, the AAV is a replication-defective AAV9 with AAV2-derived ITRs, such as scAAV9. In another embodiment, the AAV vector carries the SMN transgene. In one embodiment, the DNA encoding SMN is provided in GenBank Accession No. NM_000344.2. Conservative nucleotide substitutions in the SMN DNA are also contemplated (e.g., a guanine to adenine change at position 625, as described in GenBank Accession No. NM_000344.2).

[0058] Another aspect of the invention relates to pharmaceutical compositions comprising AAV particles in a formulation suitable for (a) intravenous ("IV") injection or (b) intrathecal ("IT") administration.

[0059] In another embodiment, the pharmaceutical composition has less than 10% empty capsids, less than 8% empty capsids, less than 7% empty capsids, less than 5% empty capsids, less than 3% empty capsids, or less than 1% empty capsids. In some embodiments, the pharmaceutical composition has less than about 5% empty capsids. In one embodiment, the number of empty capsids is below the detection limit. In some embodiments, it is advantageous for a pharmaceutical composition to have a low amount of empty capsids because those empty capsids can produce adverse responses (e.g., immune responses, inflammatory responses, liver responses, and / or cardiac responses) that have no therapeutic benefit.

[0060] In another embodiment, the residual host cell protein ("rHCP") in the pharmaceutical composition is less than or equal to 100 ng / ml rHCP / 1X 10 13 vg / ml, for example less than or equal to 40ng / ml rHCP / 1X 10 13 vg / ml or 1-50ng / ml rHCP / 1X 10 13 In one embodiment, the pharmaceutical compositions disclosed herein contain less than 10 ng rHCP / 1.0 x 10 13 vg, or less than 5ng rHCP / 1.0x 10 13 vg, less than 4ng rHCP / 1.0x 10 13 vg, or less than 3ng rHCP / 1.0x10 13 vg, or any concentration in between.

[0061] In another embodiment, the residual host cell DNA ("hcDNA") in the pharmaceutical composition is less than or equal to 5 x 10 6 pg / ml hcDNA / 1X 10 13vg / ml, less than or equal to 1.2X 10 6 pg / ml rHDNA / 1X 10 13 vg / ml, or 1X10 5 pg / ml rHDNA / 1X 10 13 vg / ml to 1.2X 10 6 pg / ml / 1X 10 13 In one embodiment, the residual host cell DNA in the pharmaceutical composition is less than 5.0 x 10 5 pg / 1.0x 10 13 vg, less than 2.0x 10 5 pg / 1.0x 10 13 vg, less than 1.1x 10 5 pg / 1.0x 10 13 vg, less than 1.0x 10 5 pg hcDNA / 1.0x 10 13 vg, less than 0.9x 10 5 pgh cDNA / 1.0x 10 13 vg, less than 0.8x 10 5 pg hcDNA / 1.0x 10 13 vg, or any concentration in between.

[0062] In one embodiment, the residual plasmid DNA in the pharmaceutical composition is less than or equal to 1.7×10 6 pg / ml / 1X 10 13 vg / ml, or 1X 10 5 pg / ml / 1X 10 13 vg / ml to 1.7X10 6 pg / ml / 1X 10 13 In one embodiment, the residual plasmid DNA in the pharmaceutical composition is less than 10.0 x 10 5 pg / 1.0x 10 13 vg, less than 8.0x 10 5 pg / 1.0x 10 13 vg, or less than 6.8x 10 5 pg / 1.0x 10 13 vg.

[0063] In one embodiment, the pharmaceutical compositions disclosed herein contain less than 0.5 ng / 1.0 x 10 13 vg, less than 0.3ng / 1.0x 10 13vg, less than 0.22ng / 1.0x 10 13 vg, or less than 0.2ng / 1.0x 10 13 vg, or any concentration of bovine serum albumin (BSA) therebetween. In one embodiment, the benzonase in the pharmaceutical composition is less than 0.2 ng / 1.0 x 10 13 vg, less than 0.1ng / 1.0x 10 13 vg, less than 0.09ng / 1.0x 10 13 vg, less than 0.08ng / 1.0x10 13 In one embodiment, the pharmaceutical composition comprises poloxamer 188 at about 10-150 ppm, about 15-100 ppm, or about 20-80 ppm. In one embodiment, the pharmaceutical composition comprises cesium at less than 50 μg / g (ppm), less than 30 μg / g (ppm), or less than 20 μg / g (ppm), or any concentration therebetween.

[0064] In one embodiment, the pharmaceutical compositions disclosed herein comprise less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or any percentage in between, as determined, for example, by SDS-PAGE. In one embodiment, the total purity, for example, as determined by SDS-PAGE, is greater than 90%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or any percentage in between. In one embodiment of the pharmaceutical composition, no single unnamed related impurity is greater than 5%, greater than 4%, greater than 3% or greater than 2%, or any percentage in between, as measured, for example, by SDS-PAGE. In one embodiment, the pharmaceutical composition comprises a percentage of filled capsid relative to total capsid (e.g., Peak 1 + Peak 2 as measured by analytical ultracentrifugation) greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 91.9%, greater than 92%, greater than 93%, or any percentage in between. In one embodiment of the pharmaceutical composition, the percentage of filled capsid measured in Peak 1 by analytical ultracentrifugation is 20%-80%, 25%-75%, 30%-75%, 35%-75%, or 37.4%-70.3%. In one embodiment of the pharmaceutical composition, the percentage of filled capsid measured in Peak 2 by analytical ultracentrifugation is 20%-80%, 20%-70%, 22%-65%, 24%-62%, or 24.9%-60.1%.

[0065] In one embodiment, the pharmaceutical composition disclosed herein comprises 1.0-5.0 x 10 13 vg / mL, 1.2-3.0x10 13 vg / mL or 1.7-2.3 x 10 13 The genomic titer was vg / mL.

[0066] In one embodiment, the pharmaceutical compositions disclosed herein exhibit a bioburden of less than 5 CFU / mL, less than 4 CFU / mL, less than 3 CFU / mL, less than 2 CFU / mL, or less than 1 CFU / mL, or any concentration therebetween. In one embodiment, according to USP, e.g., USP <85> (incorporated by reference in its entirety) is less than 1.0 EU / mL, less than 0.8 EU / mL, or less than 0.75 EU / mL.

[0067] In one embodiment, according to USP, e.g., USP <785> (Incorporated by reference in its entirety) The pharmaceutical compositions disclosed herein have an osmolality of 350-450 mOsm / kg, 370-440 mOsm / kg, or 390-430 mOsm / kg. In one embodiment, the pharmaceutical composition comprises less than 1200 particles larger than 25 μm per container, less than 1000 particles larger than 25 μm per container, less than 600 particles larger than 25 μm per container, less than 500 particles larger than 25 μm per container, or any value therebetween. In one embodiment, the pharmaceutical composition comprises less than 10,000 particles larger than 10 μm per container, less than 8,000 particles larger than 10 μm per container, or less than 6,000 particles larger than 10 μm per container.

[0068] In one embodiment, the pharmaceutical composition has a concentration of 0.5-5.0 x 10 13 vg / mL, 1.0-4.0x 10 13 vg / mL, 1.5-3.0x 10 13 vg / mL or 1.7-2.3 x 10 13 The genomic titer was vg / mL.

[0069] In one embodiment, the pharmaceutical compositions disclosed herein comprise one or more of the following: less than about 0.09 ng benzonase / 1.0 x 10 13 vg, less than about 30 μg / g (ppm) of cesium, about 20-80 ppm of poloxamer 188, less than about 0.22 ng BSA / 1.0 x 10 13 vg, less than about 6.8x 10 5 pg residual plasmid DNA / 1.0x 10 13vg, less than about 1.1x10 5 pg residual hcDNA / 1.0x 10 13 vg, less than about 4ng rHCP / 1.0x 10 13 vg, pH 7.7-8.3, approximately 390-430 mOsm / kg, fewer than approximately 600 particles ≥ 25 μm / container, fewer than approximately 6,000 particles ≥ 10 μm / container, approximately 1.7x10 13 -2.3x 10 13 The genome titer of vg / mL is about 3.9x 10 8 -8.4x 10 10 IU / 1.0x 10 13 The infectivity titer of vg is about 100-300 μg / 1.0x10 13 vg of total protein, at about 7.5 x 10 13 Median survival of Δ7SMA mice ≥24 days with a vg / kg dose of viral vector, approximately 70%-130% relative potency based on in vitro cell-based assays and / or less than about 5% empty capsids.

[0070] In various embodiments, the pharmaceutical compositions disclosed herein comprising any of the viral particles discussed herein (e.g., AAV SMN, AAVMECP2, or AAV SOD1 viral particles) retain potency between ±20%, between ±15%, between ±10%, or between ±5% of a reference standard. In some embodiments, potency is measured using a suitable in vitro cell assay or in vivo animal model. For example, potency or % functional AAV SMN viral particles can be determined using an animal model of SMA (e.g., SMAΔ7 mice) or a cell-based quantitative assay using a suitable cell line (e.g., primary neural progenitor cells (NPCs) isolated from the cortex of SMAΔ7 mice). In one embodiment, potency is assessed relative to a reference standard using the method of Foust et al., Nat. Biotechnol., 28(3), pp. 271-274 (2010). Any suitable reference standard can be used. The potency or % functional AAV MeCP2 can be determined using a suitable in vitro cell assay or in vivo animal model, such as Mecp2 knockout mice (as in Guy et al., "Reversal of neurological defects in a mouse model of Rett syndrome." Science, 315(5815): 1143-7). The potency or % functional AAV SOD1 can be determined using a suitable in vitro cell assay or in vivo animal model, such as SOD1 mutant mice (as in Gurney et al., "Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation." Science, 264(5166): 1772-5). In one embodiment, the pharmaceutical composition has an in vivo potency that is determined by administration of 7.5 x 10 13 The pharmaceutical composition is administered at a dose of 500 mg / kg of SMAΔ7, and the median survival of the mice is greater than 15 days, greater than 20 days, greater than 22 days, or greater than 24 days. In one embodiment, the pharmaceutical composition has an in vivo relative potency of 50%-150%, 60%-140%, or 70%-130% relative to a reference standard and / or a suitable control as tested by a cell-based assay.

[0071] In one embodiment, the intravenous ("IV") formulation has a pH between 7.5 and 8.5 and a genome titer of about 1-8 x 10 13Viral vector genomes / mL (vg / mL), or between 2X 10 13 vg / ml-6X 10 13 vg / ml, and optionally an osmolarity of 384-448 mOsm / kg. In one embodiment, the IV formulation comprises MgCl2, NaCl, Pluronic F68 in Tris buffer at pH 8.0.

[0072] In one embodiment, for IV administration, an AAV-9 vector carrying the SMN transgene is administered once at the indicated dose in an appropriate setting (e.g., interventional suite, operating room, dedicated procedure room) under sterile conditions through an intravenous catheter inserted into a peripheral limb vein (arm or leg) and infused slowly over approximately 30-60 minutes.

[0073] In another embodiment, the present disclosure provides compositions and methods for delivering polynucleotides to the central nervous system of a patient in need thereof, comprising delivering rAAV9 and a non-ionic hypotonic contrast agent intrathecally ("IT") to the patient, wherein the rAAV9 comprises a self-complementary genome comprising the polynucleotide. The polynucleotides are delivered to, for example, the brain, spinal cord, glial cells, astrocytes, and / or lower motor neurons. Non-ionic hypotonic contrast agents are, for example, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, or ioxilan. In some embodiments, the polynucleotide is a survival motor neuron (SMN) polynucleotide. In one embodiment, the contrast agent is iohexol, such as iohexol 180 (sold as Omnipaque 180, containing 388 mg of iohexol, equivalent to 180 mg of organic iodine / mL).

[0074] In one embodiment, for IT administration, the scAAV9 vector carrying the SMN transgene is diluted with normal saline and premixed with an appropriate hyperbaric contrast agent (e.g., Omnipaque 180) approved and labeled for pediatric use for radiographic monitoring of injection via lumbar intrathecal injection. The total volume of the aqueous composition containing the AAV-9 vector carrying the SMN transgene plus the contrast agent and / or saline does not exceed 5 mL. The contrast agent and the scAAV-9 vector carrying the SMN transgene can be co-formulated, co-packaged, or packaged separately and delivered to the patient center.

[0075] Patients received the scAAV-9 vector carrying the SMN transgene via intrathecal injection under sterile conditions in the PICU patient room or other appropriate setting with immediate access to acute critical care management (e.g., interventional suite, operating room, dedicated procedure room). The site can be accessed using an atraumatic needle with the bevel inserted parallel to the dural fibers; this has been shown to significantly reduce dural injury and thus the risk of CSF leakage after lumbar puncture (Ebinger et al., “Headache and Backache After Lumbar Puncture in Children and Adolescents: A Prospective Study.” Pediatrics, 113(6):1588-1592; Kiechl-Kohlendorfer et al., “Cerebrospinal Fluid Leakage After Lumbar Puncture in Neonates: Incidence and Sonographic Appearance.” American Journal of Roentgenology, 181(1):231-234), including in children.

[0076] Sedation / anesthesia is recommended for all patients receiving IT injections. The method and medications are at the discretion of the local anesthesiologist but should include a sufficient level of sedation or anxiolysis to ensure analgesia and inactivity during and after the Trendelenburg position. The patient will be placed in the Trendelenburg position with the head tilted 30° downward for 15 minutes before the IT therapy is administered to enhance distribution to the cervical and brain regions.

[0077] The patient is placed in the lateral decubitus position, and a stylet catheter is inserted into the L3-L4 or L4-L5 interspinal space via lumbar puncture to enter the subarachnoid space. Subarachnoid cannulation is confirmed by the presence of clear cerebrospinal fluid (CSF) flowing from the catheter. CSF is removed and disposed of according to institutional guidelines. ScAAV-9 vector carrying the SMN transgene is injected directly into the subarachnoid space in premixed contrast solution.

[0078] In one embodiment, the present disclosure provides a method of treating a neurological disease in a patient in need thereof, the method comprising intravenously or intrathecally delivering a pharmaceutical composition disclosed herein, wherein the parvovirus comprises a self-complementary rAAV9 genome, wherein the engineered transgene comprises an SMN polynucleotide, and wherein the disease is SMA.

[0079] In another embodiment, the present disclosure provides a method of treating a neurological disease in a patient in need thereof, the method comprising intrathecally delivering a pharmaceutical composition disclosed herein and a contrast agent, wherein the parvovirus comprises a self-complementary rAAV9 genome, wherein the engineered transgene comprises an SMN polynucleotide, wherein the disease is SMA, and wherein the contrast agent is Omnipaque 180.

[0080] In another embodiment, the present disclosure provides a method of treating type II, III, or IV SMA in a patient in need thereof, the method comprising intrathecally delivering a pharmaceutical composition disclosed herein and a contrast agent, wherein the parvovirus comprises a self-complementary rAAV9 genome, wherein the engineered transgene comprises an SMN polynucleotide, and wherein the contrast agent is Omnipaque 180.

[0081] In another embodiment, the present disclosure provides a method of treating type I SMA in a patient in need thereof, the method comprising intravenously delivering a pharmaceutical composition disclosed herein, wherein the parvovirus comprises a self-complementary rAAV9 genome, and wherein the engineered transgene comprises an SMN polynucleotide. In some instances, the patient is 0-9 months old. In some instances, the patient is 0-6 months old. In other embodiments, the pediatric patient weighs up to about 8 kg. In some embodiments, the pediatric patient is about 8.5 kg or less. In some embodiments, the pediatric patient is about 2.6 kg or greater.

[0082] In another embodiment, the present disclosure provides a kit for treating type I SMA in a patient in need thereof, the kit comprising intravenously administering a pharmaceutical composition disclosed herein contained in a vial. In some embodiments, the patient's weight is measured and the dosage is calculated based on the patient's weight.

[0083] 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.

[0084] As used herein, the singular form of a word also includes the plural form of the word, unless the context clearly indicates otherwise; for example, the terms "a," "an," and "the" are to be understood as singular or plural, and the term "or" is to be understood as inclusive. For example, "an element" means one or more elements.

[0085] Throughout the specification, the word "comprising" or variations such as "comprises" should be understood to imply the inclusion of the stated elements, wholes or steps or groups of elements, wholes or steps, but not the exclusion of any other elements, wholes or steps or groups of elements, wholes or steps. Throughout the specification, the word "consisting of" or variations such as "consists of" should be understood to imply the inclusion of the stated elements, wholes or steps or groups of elements, wholes or steps, and the exclusion of any other elements, wholes or steps or groups of elements, wholes or steps. Throughout the specification, the word "consisting essentially of" or variations such as "consists essentially of" should be understood to imply the inclusion of the stated elements, wholes or steps or groups of elements, wholes or steps, and any other elements, wholes or steps or groups of elements, wholes or steps that do not materially affect the basic and novel characteristics of the disclosure and / or claims.

[0086] 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. When used with reference to a percentage value, "about" can be understood as within ±1% (e.g., "about 5%" can be understood as within 4%-6%) or within ±0.5% (e.g., "about 5%" can be understood as within 4.5%-5.5%). Unless otherwise clear from the context, all numerical values provided herein are modified by the term "about."

[0087] Although methods and materials similar or equivalent to those described herein can be used for 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 admitted to be the disclosed prior art for protection. In the event of a conflict, this specification (including definitions) will be taken as the criterion. In addition, the materials, methods and examples are merely illustrative and are not intended to be restrictive. Other features and advantages of the present disclosure will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Various objects and advantages of the present disclosure, as well as a more complete understanding of the present disclosure, 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:

[0089] Figure 1. Plasmid maps of pSMN, pHELP, and pAAV.

[0090] Figure 1A The plasmid map of pSMN is shown. pSMN is a plasmid encoding recombinant self-complementary AAV DNA genomic information that expresses human survival motor neuron (SMN) cDNA under the control of a chicken-β-actin hybrid promoter together with an immediate / early cytomegalovirus (CMV) enhancer element. The SMN cDNA encodes a full-length functional protein. The expression cassette contains modified intronic sequences from simian virus 40 (SV40) and a bovine growth hormone (BGH) polyadenylation signal. The expression cassette (CMV-CB-SV40-SMN-BGHpA) is flanked by AAV2-derived inverted terminal repeats (ITRs). The left ITR is modified to preferentially package the self-complementary AAV genome. During the production of the desired drug product, the region between and including the ITRs is packaged together into a recombinant AAV9 capsid. Key pSMN components that are not intended to be packaged into the recombinant AAV genome include an open reading frame (KanR) encoding kanamycin resistance and an origin of replication (ori) derived from pUC. The ori and KanR regions can be used for plasmid production.

[0091] Figure 1B The plasmid map of pHELP plasmid is shown. pHELP plasmid contains trans-acting adenoviral components necessary for the production of recombinant adeno-associated virus. pHELP plasmid contains regions in the adenoviral genome that provide important factors for AAV replication, namely E2A, E4 and VA RNA. The adenoviral E1 function involved in rAVV replication is provided by the transfected host 293 cells. However, pHELP plasmid does not contain other adenoviral replication or structural genes. The adenoviral sequences present in the plasmid only account for about 28% (9,280 / 35,938) of the adenoviral genome and do not contain cis elements that are critical for replication, such as inverted terminal repeats. Therefore, such a production system is not expected to produce infectious adenovirus.

[0092] Figure 1C Plasmid maps showing AAV plasmids. The wild-type AAV genome contains two noncoding structural elements, called inverted terminal repeats, flanking the rep and cap open reading frames. Rep and cap encode the 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 1 open reading frame is provided in trans. Methods for preparing AAV using transient transfection of adherent HEK293 cells resolve the cis / trans role of different genetic elements by separating them on different plasmids. The pAAV2 / 9 plasmid contains the open reading frames of the AAV2 rep gene and the AAV9 cap gene.

[0093] Figure 2A flow chart showing the process of selecting HEK293 cells for superior adherence and primary cell bank (MCB) construction.

[0094] Figure 3 Shown is an outline of the cell processing details for selecting HEK293 cells for superior adherence and primary cell bank (MCB) development.

[0095] Figure 4 Describe the upstream process flow chart of API.

[0096] Figure 5 Describe the downstream process flow chart of the API.

[0097] Figure 6 Shown is the inactivation of XMuLV by the addition of Tween 20 up to 120 minutes.

[0098] Figure 7 Inactivation of PRV by addition of Tween 20 up to 120 minutes is shown.

[0099] Figure 8 Describes the HEK 293 cell expansion process during cell seeding density experiments.

[0100] Figure 9A -E shows growth and metabolite profiles. HEK 293 cells were cultured at 12,000 and 8,000 cells / cm 2 Two replicates were inoculated into 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 ) cells were transfected with DNA plasmid / PEI. Eight days after inoculation (12,000 cells / cm 2 ) and nine days (8,000 cells / cm 2 ) The bioreactor was harvested. pH and metabolite readings were taken daily on the Nova BioFlex.

[0101] Figure 10 The cell seeding density (8000 or 12000 cells / cm 2 ) and four different lengths of transfection time (20 min, 1 h, or 2 h).

[0102] Figure 11 Virus titers are shown for intermediates sampled at different filtration steps throughout the manufacturing process.

[0103] Figure 12A -B shows viral vector recovery and host cell protein (HCP) clearance during the TFF1 step.

[0104] Figure 13 Describes the HEK 293 cell expansion process during cell seeding density experiments.

[0105] Figure 14A -E shows HEK 293 cells at 8,000 cells / cm 2 , 9,350 cells / cm 2 , 10,700 cells / cm 2 , 12,050 cells / cm 2 Two replicates were inoculated into bioreactors (pH 7.23, 37.0°C, 55% DO). Five days after inoculation, cells were transfected with DNA plasmid / PEI (1:1 m / m). pH and metabolite analysis was performed using a NOVA BioProfile 400.

[0106] Figure 15A -B shows a comparison of viral titer per unit surface area and harvested vector genomes from four starting inoculum densities for drug substance production in a bioreactor.

[0107] Figure 16 The Phase 1 (Process A) and Phase 3 pilot (Process B) manufacturing processes are shown.

[0108] Figure 17A - B provides a table illustrating comparability and manufacturing consistency results - Process A (Phase 1) and Process B (Phase 3) products. Compared to Process A, the Process B product shows additional benefits.

[0109] Figure 18 Comparability between Process A and Process B was shown using pairwise comparisons of Process A (Phase 1 Batch NCHAAV9SMN0613) and Process B (Phase 3 Batch 600156). The product of Process B showed additional benefits compared to Process A.

[0110] Figure 19 Manufacturing consistency assessment by pairwise comparison of Process B (Phase 3) batches 600156 and 600307 is shown.

[0111] Figure 20 Shown is the stability profile of NCH batch NCHAAV9SMN0613 stored at real-time storage conditions of ≤-60°C for 12 months.

[0112] Figure 21 The sedimentation coefficient (seconds x 10) for the Stage 1 material (NCHAAV9SMN0613) is shown. -13 ), showing a sedimentation coefficient of approximately 60 x 10 -13seconds of empty capsids (7%), and sedimentation coefficients ranging from approximately 80-150 x 10 -13 Seconds of full capsid.

[0113] Figure 22 Shows the sedimentation coefficient (seconds x 10) for the Stage 3 material (600156) -13 ), showing a sedimentation coefficient of approximately 60x10 -13 seconds of empty capsids (2%), and sedimentation coefficients ranging from approximately 80-150 x 10 -13 Seconds of full capsid.

[0114] Figure 23 Shows the sedimentation coefficient (seconds x 10) for the Stage 3 material (600307) -13 ), showing a sedimentation coefficient of approximately 60x10 -13 seconds of empty capsids (4%), and sedimentation coefficients ranging from approximately 80-150 x 10 -13 Seconds of full capsid. DETAILED DESCRIPTION

[0115] To advance the development of AAV gene therapies beyond animal models and into clinical research and / or therapeutic use, a scalable method for generating viral material suitable for human use is required.

[0116] In some embodiments, "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with appropriate control elements and which is capable of transferring gene sequences between cells. Thus, the term includes cloning and expression vectors, as well as viral vectors.

[0117] In some embodiments, "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. The AAV vector may have one or more AAV wild-type genes that are completely or partially deleted, such as rep and / or cap genes, but retains functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of AAV virions. Therefore, AAV vectors are defined herein to include at least those sequences that provide viral replication and packaging (e.g., functional ITRs) in cis. ITRs do not have to be wild-type nucleotide sequences 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 one embodiment, the vector is an AAV-9 vector having ITRs derived from AAV-2. "AAV vector" also refers to a protein shell or capsid that provides an effective vehicle for delivering vector nucleic acid to the nucleus of a target cell.

[0118] In some embodiments, "scAAV" refers to a self-complementary adeno-associated virus (scAAV), which is a viral vector for gene therapy engineered from a naturally occurring adeno-associated virus (AAV). scAAV is called "self-complementary" because the coding region has been engineered to form an intramolecular double-stranded DNA template.

[0119] In some embodiments, the term "vector-associated impurities" refers to all types of AAV particles other than genuine recombinant AAV particles. Vector-associated impurities include empty AAV capsids (also referred to as "empty" or "empty particles"), as well as AAV particles containing polynucleotide sequences other than the intended vector genome (also referred to as "AAV-encapsulated nucleic acid impurities" or "AAV-encapsulated DNA impurities").

[0120] In some embodiments, a "recombinant virus" refers to a virus that has been genetically altered, for example, by the addition or insertion of a heterologous nucleic acid construct into the particle. "Recombinant" may be abbreviated as "r," for example, rAAV may refer to recombinant AAV. As used herein, the term "AAV" is intended to include "recombinant AAV" or "rAAV."

[0121] In some embodiments, "AAV virions" refers to intact viral particles, such as wild-type (wt) AAV viral particles (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 having a complementary sense (e.g., "positive") strand or "antisense" strand can be packaged into any one AAV viral particle, and both strands are equally infective.

[0122] In some embodiments, the terms "recombinant AAV virions," "rAAV virions," "AAV vector particles," "whole capsids," and "whole particles" are defined herein as infectious, replication-defective viruses comprising an AAV protein shell that encapsulates a heterologous nucleotide sequence of interest flanked by AAV ITRs. rAAV virions are produced in suitable host cells that have sequences specifying the AAV vector, AAV helper functions, and accessory functions introduced therein. In this manner, the host cells are rendered capable of encoding AAV polypeptides that provide for packaging of the AAV vector (comprising the recombinant nucleotide sequence of interest) into infectious, recombinant virion particles for subsequent gene delivery.

[0123] In some embodiments, the terms "empty capsid" and "empty particle" refer to an AAV virion that includes an AAV protein capsid but lacks all or part of a polynucleotide construct comprising a heterologous nucleotide sequence of interest flanked by AAV ITRs.

[0124] The term "host cell" refers to, for example, a microorganism, yeast cell, insect cell, and mammalian cell that can or has been used as a recipient of an AAV helper construct, an AAV vector plasmid, an accessory function vector, or other transfer DNA. The term includes the progeny of the original cell that has been transfected. Thus, "host cell" as used herein generally refers to a cell that has been transfected with an exogenous DNA sequence. It will be understood that the progeny of a single parent cell may not necessarily be identical to the original parent in morphology or in terms of genome or total DNA complement sequence due to natural, accidental, or deliberate mutations.

[0125] In another embodiment, the term "AAV helper functions" 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 two major AAV open reading frames (ORFs), rep and cap. The Rep expression product has been shown to have many functions, including: recognition, binding and cleavage of the AAV DNA replication origin; DNA helicase activity; and regulation of transcription from an AAV (or other heterologous) promoter. 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.

[0126] In one embodiment, the term "AAV helper construct" generally refers to a nucleic acid molecule comprising a nucleotide sequence that provides an AAV function deleted from an AAV vector, which will be used to produce a transduction vector to deliver the target nucleotide sequence. AAV helper constructs are typically used to provide transient expression of the AAV rep and / or cap genes to supplement the missing AAV functions necessary for AAV replication; however, the helper construct lacks the AAV ITR and can neither replicate nor package itself. The AAV helper construct can be in the form of a plasmid, phage, transposon, cosmid, virus or virion. Many AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode Rep and Cap expression products. For example, see 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, for example, U.S. Patent Nos. 5,139,941 and 6,376,237.

[0127] In another embodiment, the term "transfection" is used to refer to the uptake of exogenous DNA by a cell, and when the exogenous DNA has been introduced into the interior of the cell membrane, the cell has been "transfected". Many transfection techniques are well known in the art. For example, see Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratory, 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 exogenous DNA moieties into a suitable host cell.

[0128] As used herein, the term "cell line" refers to a cell population capable of continuous or prolonged growth and division in vitro. It is also known in the art that during storage or transfer of such clonal populations, karyotype changes may occur spontaneously or induced. Therefore, cells derived from a cell line may not be identical to the ancestral cell or culture, and cell lines 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.

[0129] In some embodiments, the term "eluent" is understood in this context to refer to a buffer used to elute a substance. In some embodiments, the term "eluent" is understood in this context to refer to an eluted substance, such as a desired product or substance from a previous purification step, such as for assay or further purification.

[0130] In some embodiments, the methods described herein use Good Manufacturing Practices (GMP) and are performed on an industrial scale. GMP is a regulatory practice, such as those enforced by the Federal Drug Administration (FDA), to ensure the quality of drugs. GMP regulations establish controls over the manufacturing process. The FDA publishes examples of current GMP regulations. 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 producing AAV viral vectors on an industrial scale and at a purity level sufficient for administration to humans, for example, in adherent cells. The term "industrial scale" refers to production on a scale greater than a laboratory scale, such as a commercial scale (e.g., where the yield per production batch is greater than 5x 1015 vg, or larger than 8x 10 15 vg or greater than 1x 10 16 vg) Methods for producing viral vectors in cells.

[0131] Upstream Process

[0132] 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) harvesting amplified viral particles from the cells after the culture period, such as by total cell lysis, (d) purifying the viral particles by 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 the downstream process. In some embodiments, the AAV prepared using the upstream process disclosed herein is an AAV encoding an shRNA targeting SOD1, an AAV comprising a polynucleotide encoding MECP2, or an AAV comprising a polynucleotide encoding SMN, as described herein. In some embodiments, the upstream process is performed under GMP and on an industrial scale.

[0133] 1. Cell Line Transfection and Culture

[0134] In one aspect, disclosed herein is a rAAV genome. The rAAV genome comprises one or more AAV ITRs flanked by polynucleotides encoding polypeptides (including but not limited to SMN polypeptides) or encoding siRNA, shRNA, antisense and / or miRNA for control sequences of mutant proteins or their genes. The polynucleotides are operably linked to transcriptional control DNA, particularly promoter DNA, enhancer DNA, and polyadenylation signal sequence DNA, which function in target cells to form gene cassettes. The gene cassettes may also include intron sequences to facilitate processing of RNA transcripts when expressed in mammalian cells.

[0135] In some embodiments, the rAAV (e.g., rAAV9) genome encodes a trophic or protective factor for the treatment of neurodegenerative disorders including, but not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, and nervous system injuries including spinal cord and brain trauma / injury, stroke, and brain cancer. Non-limiting examples of known nervous system growth factors include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 / 5 (NT-4 / 5), neurotrophin-6 (NT-6), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor family (e.g., FGF 1-15), leukemia inhibitory factor (LIF), certain members of the insulin-like growth factor family (e.g., IGF-1), neurotrophin, persephin, bone morphogenetic protein (BMP), immunophilins, transforming growth factor (TGF) family of growth factors, neuregulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor family (e.g., VEGF 165), follistatin, Hif1, and others. Also generally contemplated are zinc finger transcription factors that regulate each of the nutritional or protective factors contemplated herein. In further embodiments, methods for modulating neuroimmune function are contemplated, including but not limited to inhibiting microglial and astrocyte activation by, for example, NFkB inhibition or NFkB effects on neuroprotection (dual effects of NFkB and related pathways in different cell types) via siRNA, shRNA, antisense or miRNA. In additional embodiments, the rAAV (e.g., rAAV9) genome encodes an inhibitor of apoptosis (e.g., bcl2, bclxL). rAAV (e.g., rAAV9) encoding a trophic factor or a repressor of a spinal cord injury regulatory protein or axonal growth inhibitor (e.g., a repressor of Nogo [Oertle et al., The Journal of Neuroscience, 23(13):5393-5406 (2003)] are also contemplated for use in treating spinal cord injury.

[0136] For the treatment of neurodegenerative disorders such as Parkinson's disease, the rAAV (e.g., rAAV9) genome encodes, in various embodiments, aromatic acid dopa decarboxylase (AADC), tyrosine hydroxylase, GTP-cyclohydrolase 1 (gtpch1), apoptosis inhibitors (e.g., bcl2, bclxL), glial cell line-derived neurotrophic factor (GDNF), inhibitory neurotransmitter aminobutyric acid (GABA), or enzymes involved in dopamine biosynthesis. In further embodiments, the rAAV (e.g., rAAV9) genome may encode, for example, modifiers of Parkin and / or synuclein.

[0137] To treat neurodegenerative disorders such as Alzheimer's disease, in some embodiments, methods of increasing acetylcholine production are contemplated. In some embodiments, methods of increasing choline acetyltransferase (ChAT) levels or inhibiting acetylcholinesterase (AchE) activity are contemplated.

[0138] In some embodiments, the rAAV (e.g., rAAV9) genome encodes siRNA, shRNA, antisense and / or miRNA for use in methods for reducing expression of mutant huntingtin protein (htt) to treat a neurodegenerative disorder such as Huntington's disease.

[0139] In various embodiments, the rAAV (e.g., rAAV9) genome encodes siRNA, shRNA, antisense and / or miRNA for the treatment of neurodegenerative disorders such as ALS. Treatment results in a decrease in the expression of disease markers such as TNF-α, nitric oxide, peroxynitrite and / or nitric oxide synthase (NOS).

[0140] In some embodiments, the vector encodes a short hairpin RNA (shRNA) directed against a mutated protein, such as superoxide dismutase (SOD, such as SOD-1) for ALS, or a neurotrophic factor for ALS or Parkinson's disease, such as GDNF or IGF1.

[0141] In one embodiment, the methods and materials described herein can be used to treat ALS. ALS is a neurodegenerative disease that causes the progressive loss of motor neurons in the brain and spinal cord, with symptoms including loss of the ability to speak, eat, move, and eventually breathe. The disease usually leads to death within 3-5 years of diagnosis. Although the cause of 90%-95% of ALS is unknown, some ALS is caused by mutations in the superoxide dismutase 1 (SOD1) gene, in which the mutations result in toxic dominant gain of function. Studies in mice have shown that knocking out the SOD1 gene does not cause disease, and therefore, therapies that knock down mutant SOD1 levels are thought to alleviate disease symptoms.

[0142] In some embodiments, the AAV vector encodes an shRNA targeting SOD1 for ALS. Exemplary AAVs encoding shRNAs for SOD1, such as scAAV9 constructs, are provided in WO 2015031392 and US2016272976 (the contents of which are incorporated herein in their entirety). In some embodiments, AAV constructs encoding shRNAs for SOD1 can be prepared using the methods disclosed herein. In some embodiments, these AAV constructs can be used to treat ALS. In some embodiments, the SOD1 AAV exhibits less than 10%, such as less than 7%, 5%, 4%, 3%, 2% or 1% empty capsids. In some embodiments, the SOD1 AAV exhibits low amounts of residual host cell proteins, host cell DNA, plasmid DNA and / or endotoxins, such as the levels discussed herein for preparing and purifying AAV vectors. As used herein, "AVXS-301" is a non-limiting example of an scAAV9 vector, i.e., comprising a polynucleotide encoding an anti-human SOD1 shRNA (e.g., pSOD1sh), a modified AAV2 ITR, a human H1 promoter, and an unmodified AAV2 ITR. The modified and unmodified ITRs can appear in either orientation (i.e., 5' or 3') relative to the anti-human SOD1 shRNA expression cassette.

[0143] As used herein, a "pSOD1sh" vector plasmid comprises a polynucleotide encoding a short hairpin RNA (shRNA) targeting superoxide dismutase 1 (SOD1) gene expression, i.e., an anti-SOD1 shRNA cassette, wherein the cassette is flanked by adeno-associated viral inverted terminal repeats (ITRs), such as the "left" and "right" polynucleotides encoding pSOD1sh. In some embodiments, the polynucleotide encoding pSOD1sh is transcribed into a short hairpin RNA that specifically targets human SOD1 mRNA. In some embodiments, the ITR sequences surrounding the polynucleotide encoding pSOD1sh are natural, variant, or modified AAV ITR sequences. In some embodiments, at least one ITR sequence is a natural, variant, or modified AAV2 ITR sequence. In some embodiments, the ITRs flank the polynucleotide encoding pSOD1sh. 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 for the generation of a self-complementary genome, while the "right" ITR is a natural AAV2 ITR sequence. In some embodiments, the "right" ITR is a modified AAV2 ITR sequence that allows for the generation of a self-complementary genome, while the "left" ITR is a native AAV2 ITR sequence. In some embodiments, the pSOD1sh vector further comprises a fragment of the human H1 RNA promoter, for example, as described by Myslinkski et al. "Anunusually compact external promoter for RNA polymerase III transcription of the human H1 RNA gene." Nucleic Acids Research, 29(12):2502-2509. In some embodiments, the pSOD1sh vector further comprises a unique stuffer sequence made from fragments of a random plasmid backbone to increase the size of the expression cassette. In some experiments, the pSOD1sh vector comprises a polynucleotide encoding an anti-human SOD1 shRNA, a modified AAV2 ITR, a human H1 promoter, and an unmodified AAV2 ITR.

[0144] In one embodiment, the methods and materials described herein can be used to treat neurodevelopmental disorders, such as Rett syndrome. Rett syndrome is a rare neurological disorder first recognized in infancy, with 90%-95% of cases caused by mutations in the MECP2 gene on the X chromosome. Ruthie et al., "Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2," Nature Genetics, 23:185-188. Boys with only one copy of the X chromosome typically die shortly after birth, while girls with two copies of the X chromosome typically have one functional copy of the gene. Symptoms begin between 6 and 18 months of age, with hallmark symptoms such as hand wringing or squeezing, hand clapping, rubbing, washing hands, or hand-to-mouth movements. The disorder is progressive and associated with severe disabilities, which can include autistic-like behavior, irregular breathing, difficulty eating and swallowing, growth retardation, and seizures. There are 200 known mutations in the MECP2 gene, and the severity of the disease varies from patient to patient, depending on the level of X-inactivation and dosage compensation. Studies in mice have shown that MECP2 mutations do not cause neuronal death, suggesting that it is not a neurodegenerative disorder. Guy et al., “Reversal of Neurological Defects in a Mouse Model of Rett Syndrome.” Science, 315(5815), 1143-1147.

[0145] For embodiments related to Rett syndrome, the rAAV (e.g., rAAV9) genome may encode, for example, methylcytosine binding protein 2 (MeCP2). Exemplary AAVs, such as scAAV9 (constructs comprising polynucleotides encoding MeCP2), are provided in U.S. Patent No. 9,415,121 (the contents of which are incorporated herein in their entirety). In some embodiments, AAV constructs comprising polynucleotides encoding MeCP2 can be prepared using the methods disclosed herein. In some embodiments, these AAV constructs can be used to treat Rett syndrome. In some embodiments, the MeCP2 AAV exhibits less than 10%, such as less than 7%, 5%, 4%, 3%, 2% or 1% empty capsids. In some embodiments, the MeCP2 AAV exhibits low amounts of residual host cell proteins, host cell DNA, plasmid DNA and / or endotoxins, such as the levels discussed herein for preparing and purifying AAV vectors.

[0146] As used herein, "AVXS-201" is a non-limiting example of a scAAV9 vector, i.e., comprising a polynucleotide (e.g., pMECP2) comprising a MECP2 cDNA expression cassette, a modified AAV2 ITR, a murine Mecp2 promoter, a modified SV40 intron, a minimal polyadenylation signal, and unmodified AAV2 ITRs. The modified and unmodified ITRs can appear in either orientation (i.e., 5' or 3') relative to the MECP2 cDNA expression cassette.

[0147] As used herein, the "pMECP2" vector plasmid comprises a polynucleotide encoding a MECP2 protein, a modified AAV2 ITR, a mouse Mecp2 promoter, a modified SV40 intron, a minimal polyadenylation signal, and an unmodified AAV2 ITR. In some embodiments, pMECP2 is a vector construct comprising a polynucleotide encoding a MECP2 protein (i.e., a MECP2 cDNA expression cassette, wherein the cassette is flanked by adeno-associated virus inverted terminal repeats (ITRs), such as the "left" and "right" sequences of the polynucleotide encoding the MECP2 gene). In some embodiments, the polynucleotide encoding MECP2 is a human MECP2 sequence, such as a naturally occurring human MECP2 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 for the generation of a self-complementary genome, while the "right" ITR is a native AAV2 ITR sequence. In some embodiments, the "right" ITR is a modified AAV2 ITR sequence that allows for the generation of a self-complementary genome, while the "left" ITR is a native AAV2 ITR sequence. In some embodiments, the pMECP2 vector further comprises a fragment of the mouse Mecp2 promoter, but not all of it. In some embodiments, the pMECP2 vector further comprises a simian virus 40 (SV40) intron. In some embodiments, the pMECP2 vector further comprises a minimal polyadenylation signal, e.g., as defined in Levitt et al., "Definition of an efficient synthetic poly(A) site." Genes & Development, 3:1019-1025.

[0148] In some embodiments, the rAAV genomes disclosed herein lack 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 the AAV serotypes are known. 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).

[0149] As used herein, a "pSMN" vector plasmid comprises a polynucleotide encoding an SMN protein, i.e., an SMN cDNA expression cassette, wherein the cassette is flanked by adeno-associated viral inverted terminal repeats (ITRs), e.g., the "left" and "right" polynucleotides encoding the SMN gene. In some embodiments, the polynucleotide encoding SMN is a human SMN sequence, e.g., a naturally occurring human SMN sequence, or an isoform, variant, or mutant thereof. In some embodiments, the ITR sequence is a native, variant, or modified AAV ITR sequence. In some embodiments, at least one ITR sequence is a native, variant, or modified AAV2 ITR sequence. In some embodiments, both ITR sequences are native, variant, or modified AAV2 ITR sequences. In some embodiments, the "left" ITR is a modified AAV2 ITR sequence that allows for the generation of a self-complementary genome, while the "right" ITR is a native AAV2 ITR sequence. In some embodiments, the "right" ITR is a modified AAV2 ITR sequence that allows for the generation of a self-complementary genome, while the "left" ITR is a native 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 (poly A) termination signal. Exemplary sequences that can be used for one or more of the above components 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.

[0150] 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 present in either orientation (i.e., 5' or 3') relative to the SMN cDNA expression cassette.

[0151] In some embodiments, for example, in the manufacturing processes described herein, the vector construct sequence is encapsulated into, for example, AAV9 viral particles. In these embodiments, the encapsulation is in a non-replicating recombinant AAV9 capsid that is capable of delivering a stable, functional transgene, such as a fully functional human SMN transgene, a MECP2 transgene, or an anti-SOD1 shRNA. In some embodiments, the capsid is composed of 60 viral proteins (VP1, VP2, VP3) in a 1:1:10 ratio, for example, produced by alternative splicing, whereby VP2 and VP3 are two truncated forms of VP1, both having a common C-terminal sequence. In some embodiments, the product of the manufacturing process, such as a pharmaceutical product, may comprise a non-replicating recombinant AAV9 capsid to deliver a stable, fully functional human SMN transgene, a MECP2 transgene, or an anti-SOD1 shRNA. In some embodiments, the capsid is composed of 60 viral proteins (VP1, VP2, VP3) in a 1:1:10 ratio generated by alternative splicing, wherein the alternative splicing makes VP2 and VP3 two truncated forms of VP1, both having a common C-terminal sequence.

[0152] In some embodiments, the amount of functional viral vector is determined by measuring the % of functional vg / mL using a suitable in vitro cell assay or in vivo animal model. For example, the % of functional AAV SMN can be determined by relative potency using an animal model of SMA (e.g., SMAΔ7 mice) or a cell-based quantitative assay using a suitable cell line (e.g., primary neural progenitor cells (NPCs) isolated from the cortex of SMAΔ7 mice). The % of functional AAV MeCP2 can be determined using a suitable in vitro cell assay or in vivo animal model (e.g., Mecp2 knockout mice). The % of functional AAV SOD1 can be determined using a suitable in vitro cell assay or in vivo animal model (e.g., SOD1 mutant mice).

[0153] The DNA sequences of exemplary vector constructs, such as AVXS-101, are described in Table 1.

[0154] Table 1: Summary components of AVXS-101 vector construct DNA sequence (all nt start and end positions are relative to SEQ ID NO: 1).

[0155]

[0156]

[0157] In another aspect, the DNA sequence of the AVXS-101 vector construct is provided below: SEQ ID NO: 1:

[0158]

[0159]

[0160] In some embodiments, the amino acid sequence of the SMN protein encoded by the pSMN plasmid comprises:

[0161] MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNGDICETSGPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASIDFKRETCVVVYTGYGNREEQNLSDLLSPICEVA NNIEQNAQENENESQVSTDESENSRSPGNKSDNIKPKSAPWNSFLPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSCWLPPFPSGPPIIPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRCSHSLN(SEQ ID NO:2).

[0162] In some embodiments, the AAV capsid proteins VP1, VP2, and VP3 are derived from the same transcript. They have alternative start sites but share a carboxyl terminus. Below, the VP1-specific amino acid sequence is shown in black and bold. The amino acid sequence shared by VP1 and VP2 is underlined and italicized. Amino acids shared by all three capsid proteins are shown in bold and italicized.

[0163]

[0164]

[0165] In one embodiment, the AAV capsid protein is derived from a transcript encoding the amino acid sequence shown in SEQ ID NO:3.

[0166] On the other hand, disclosed herein is a DNA plasmid comprising an rAAV genome. The DNA plasmid is transferred to cells that are infected with a helper virus (e.g., adenovirus, adenovirus or herpes virus deleted for E1) of AAV, so that the rAAV genome is assembled into infectious viral 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. In certain embodiments, the production of rAAV involves the following components present in a single cell (referred to herein as packaging cells): 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 functions. 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. Modification of the capsid protein is well 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 entireties.

[0167] The general principles of rAAV production are reviewed in, e.g., 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 and Cellular 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 and Cellular Biology] 5:3251 (1985); McLaughlin et al., J. Virol. [Journal of Virology], 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol. [Molecular and Cellular Biology], 7:349 (1988). Samulski et al. (1989, J. Virol., 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 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 foregoing documents are hereby incorporated by reference in their entirety, with particular emphasis on those portions of the documents relating to rAAV production.

[0168] An exemplary method for generating packaging cells is to generate a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or plasmids) comprising the following items is integrated into the genome of the cell: an rAAV genome lacking AAV rep and cap genes, an AAV rep and cap genes separated from the rAAV genome, and a selective marker (e.g., neomycin resistance gene). The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA [Proceedings of the National Academy of Sciences of the United States S6], 79: 2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene [gene], 23: 65-73) or by direct blunt end ligation (Senapathy and Carter, 1984, J. Biol. Chem. [Journal of Biological Chemistry], 259: 4661-4666). The packaging cells are then infected with a helper virus (such as an adenovirus). The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0169] Thus, the disclosure herein provides, in various embodiments, packaging cells for producing infectious rAAV. The packaging cells can be non-adherent cells cultured in suspension or adherent cells. In one embodiment, any suitable packaging cell line can be used, such as HeLa cells, HEK 293 cells, and PerC.6 cells (a homologous 293 cell line). In one embodiment, the cell line is HEK 293 cells.

[0170] To increase viral vector production yield, adherent cells can be cultured and selected to improve adhesion to the culture flask. In some embodiments, transfection efficiency and cell count are improved during the subsequent bioreactor inoculation step. During subculture, cells can be separated from the cell culture surface using methods known in the art. For example, cells can be floated by scraping or by incubating in a solution containing a protease. In one exemplary embodiment, HEK293 cells can be washed with PBS at room temperature and dissociated with trypsin for approximately 2 minutes. Dissociation can be terminated by adding growth medium containing serum, and cell clumps can be dissociated by repeated pipetting of the suspension. The cell suspension can then be pelleted, and the separated pellet can be resuspended in appropriate complete growth medium. The cells can then be seeded into a new cell culture chamber and allowed to adhere. Before completely replacing the cell culture medium with growth medium, cells that have not adhered to the surface after a period of time can be gently aspirated with the cell culture medium to remove them. In some embodiments, the period of time allowed for cell adhesion can be approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, or approximately 7 hours. After cell expansion, this process can be repeated to increase the proportion of cells that strongly adhere 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 a 75 cm 2 The flasks were allowed to adhere for 4 hours in a 37° C. incubator before 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.

[0171] In other embodiments, the rAAV9 (ie, infectious, encapsulated rAAV9 particles) comprises a rAAV genome disclosed herein. In one aspect, the rAAV genome is a self-complementary genome.

[0172] In another aspect, rAAV is provided, for example, rAAV9 designated "rAAV SMN." In some embodiments, the rAAV SMN genome comprises a first AAV2 ITR, a chicken β-actin promoter with a cytomegalovirus enhancer, an SV40 intron, a polynucleotide encoding SMN, a polyadenylation signal sequence from bovine growth hormone, and a second AAV2 ITR in the sequence. In some embodiments, the polynucleotide encoding SMN is a human SMN gene, such as described below or derived from GenBank Accession No. MN_000344.2, GenBank Accession No. NM_017411, or any other suitable human SMN isoform. Exemplary SMN sequences include the following:

[0173]

[0174] Conservative nucleotide substitutions in 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 lacks both 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 listed in NCBI Protein Database No. NP_000335.1. In some embodiments, SMN DNA includes a polynucleotide encoding a human SMN polypeptide (e.g., human SMN protein, isoform 1 (Q16637-1), identified by Uniprot Accession No. Q16637). Also contemplated is the SMN1-modifier polypeptide plectin-3 (PLS3) [Oprea et al., Science 320(5875):524-527 (2008)]. Sequences encoding other polypeptides may be substituted for SMN DNA.

[0175] Prior to transfection, cells are expanded in suitable culture medium in flasks or suitable bioreactors (or both). In some embodiments, cells can be expanded in a bioreactor that provides continuous circulation of cell culture medium. In one embodiment, cells are grown at 200 m 2 、333m 2 or 500m 2 iCELLis are expanded in bioreactors. One culture medium is DMEM with 5%-10% FBS, 4.5 g / L glucose, 4 mM L-glutamine. In some embodiments, adherent cells are added to culture medium in a recirculating 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 at an appropriate density in flasks or bioreactors for culture and transfection. The seeding density can depend on the cell type and the amount of time prior to transfection. In some embodiments, approximately 8,000-16,000 cells / cm 2 In one embodiment, cells are seeded at a density of 8,000 to 12,000 cells / cm 2 Seed HEK293 cells.

[0176] 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 the cells (e.g., in an appropriate culture medium) and selecting for those cells carrying the DNA of interest using conventional techniques such as Southern blotting and / or PCR or by using a selectable marker.

[0177] In some embodiments, the packaging cell line is transfected with three plasmids: a plasmid encoding or comprising 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 using liposome amines, cationic polymers, and cationic lipids. Any suitable transfection medium can be used. In one embodiment of the transfection method, adherent human embryonic kidney (HEK293) cells are transfected with a triple DNA plasmid polyethyleneimine (PEI) coprecipitation. In one embodiment, the triple DNA plasmid is transfected into adherent HEK293 cells using PEI coprecipitation in a large-scale adherent cell bioreactor to produce a scAAV9.CB.SMN vector (a self-complementary AAV9 vector comprising a CB promoter and a polynucleotide encoding SMN). In one embodiment, a modified DMEM transfection medium is used instead of the DMEM growth medium used for cell expansion. The medium does not contain calcium and L-glutamine. In one embodiment, the transfection medium is DMEM containing no FBS, no calcium, no L-glutamine, and 4.5 g / L glucose. In certain embodiments, transfection medium containing no serum (e.g., no FBS) improves transfection efficiency. In one embodiment, the transfection medium is OptiMEM (Invitrogen / Thermo Fisher). In one embodiment, three plasmids (pSMN, pHELP, and pAAV2 / 9) are mixed with PEI in a transfection medium and reacted. In certain embodiments, the three plasmids are mixed together in a molar ratio of about 1:1:1. In certain embodiments, the plasmids and PEI are mixed in a 1:1 ratio by weight of DNA:PEI. In certain embodiments, the plasmids and PEI are mixed in a ratio of less than 1:1 by weight of DNA:PEI. In one embodiment, pSMN, pHELP, and pAAV2 / 9 are mixed in a molar ratio of 1:1:1 in OptiMEM medium. In such an embodiment, PEI is added so that the weight ratio of DNA:PEI is 1:1. In some embodiments, the reaction is allowed to occur for 0-60 minutes, or 10-45 minutes, or 20-30 minutes. In one embodiment, the reaction is allowed to occur for 15-30 minutes.

[0178] 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 a plasmid for less than 60 minutes to enable production of the AAV vector; and optionally applying further processing, purification, formulation, and filling steps to produce a drug product. In one embodiment of the method, a scAAV9.CB.SMN vector is produced using a triple DNA plasmid transfection using polyethyleneimine ("PEI") co-precipitation. In one embodiment, the three plasmids used for the transfection are pSMN, pAAV2 / 9, and pHELP.

[0179] 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 culture medium recirculation bag. In certain embodiments, the DNA-PEI coprecipitate in the transfection medium circulates into a bioreactor and replaces the growth medium completely. In certain embodiments, the DNA-PEI coprecipitate in the transfection medium is allowed to contact the adherent cells in the bioreactor. In certain embodiments, the DNA-PEI coprecipitate in the transfection medium is allowed to contact the adherent cells in the bioreactor for 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, by recirculating complete growth medium through the bioreactor and replacing the transfection medium completely, transfection is stopped.

[0180] 2. Harvesting Amplified Viral Particles

[0181] 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 initiating the cell lysis process. In some embodiments, the cells are lysed in situ. Optionally, the viral particles are harvested without lysis. In some embodiments, an endonuclease is added, for example, circulated into the bioreactor to a final target concentration. The endonuclease is an endonuclease that can degrade 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. [Biochemistry] 1969, 244, 5219-5225), which is named Commercially available (EMD Millipore). 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 incorporated herein 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. 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 into 5'-monophosphate-terminated oligonucleotides 2-5 bases in length. It is produced according to current good manufacturing practices (cGMP) and can therefore be used for industrial-scale methods of protein and / or viral particle purification. Other endonucleases produced under cGMP conditions can also be used in the purification methods disclosed herein. In one embodiment, benzonase is added to a bioreactor to a final concentration of 50-200 U / ml, for example 75-150 U / ml, for example about 100 U / mL. In certain embodiments, adding benzonase significantly reduces host cell DNA while allowing high vg to be produced in the bioreactor.

[0182] In certain embodiments, before lysis buffer is added to the reactor, endonuclease is allowed to mix. In certain embodiments, cell lysis solution is allowed to mix with 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 certain embodiments, lysis buffer may include magnesium chloride and / or Tween-20 in a suitable buffer. In an exemplary embodiment, lysis buffer is 500mM HEPES, 10% Tween-20, 20mM MgCl , pH 8.0. A salt sucrose solution (SSS) that quenches the Benzonase reaction can be added to terminate the lysis reaction. In certain embodiments, SSS is added to a harvest bag comprising rinse buffer and mixed for 15 minutes. In certain embodiments, the bioreactor is rinsed with bioreactor rinse buffer, and then the rinse fluid is collected in a harvest collection bag together with the quenched cell lysis solution and the cracked cell contents (all of which together constitute batch harvests). In certain embodiments, the bioreactor rinse buffer may include Tris, MgCl , NaCl, Tween-20 and sucrose. In one exemplary embodiment, the bioreactor wash buffer comprises 20 mM Tris, 1 mM MgCl2, 500 mM NaCl, 1% Tween-20 w / v, and 1% sucrose w / v (pH 8.1).

[0183] 3. Purification of Viral Particles

[0184] After harvesting, the bulk harvested viral particles can be concentrated and purified (typically by filtration). In one embodiment, the viral particles are filtered by depth filtration followed by filtration through a filter that removes macromolecular contaminants and cellular debris, such as a 0.45 μm filter, but allows the vector genome to pass through. Any suitable depth filter can be used.

[0185] As understood in the art, depth filtration refers to the use of a porous filter medium to clarify a solution containing a large number of large particles (e.g., intact cells or cell debris), in contrast to membrane filtration, which can quickly become clogged under such conditions. A variety of depth filtration media with different pore sizes are commercially available from various manufacturers such as Millipore, Pall, General Electric, and Sartorious.

[0186] The target flow rate for depth filtration can be reduced to maintain the filter inlet pressure within a specified range. In certain embodiments, once all of the bulk harvest has been filtered, the depth filter can be chased with diafiltration buffer in the subsequent first tangential flow filtration step ("TFF1"). The depth filter pool is mixed. The depth filter pool can then be filtered through a 0.45 μm filter to further clarify the bulk harvest material. The 0.45 μm filter is then chased with TFF1 buffer.

[0187] 4. Tangential flow filtration

[0188] In various embodiments, tangential flow filtration is used to concentrate batch harvests, and remove salts and proteins, such as 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 for implementing its equipment and scheme in a wide range of situations can be commercially available from multiple manufacturers, including but not limited to the Pall Corporation (Pall Corporation) of Port Washington, New York and the Spectrum Labs (Spectrum Labs) of Rancho Dominguez, California. Typically, TFF can relate to the recirculation of the retentate across the membrane surface. In certain embodiments, this gentle cross-flow feed can minimize membrane fouling, maintain high filtration rate, and provide high product recovery. In one embodiment, the TFF step can be realized with a flat plate system, as illustrated herein. The flat plate system can be used in large-scale production, wherein the system has a device (e.g., open flow channel) to prevent excessive shearing forces on viral particles. Alternatively, the TFF step can be realized with a hollow fiber system, as illustrated herein. In one embodiment, the molecular weight cut-off (MWCO) of the TFF system is between 200-400 kDa, such as about 300 kDa.

[0189] 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 NaOH solution and equilibrated with TFF1 buffer. In one embodiment, the TFF1 buffer comprises 20 mM Tris, 1 mM MgCl2, 500 mM NaCl, 1% sucrose, pH 8.1.

[0190] In certain embodiments, the concentration stage of the TFF1 step is selected as the volume reduction of the clarification harvest by about 10x. Once the target retentate volume is reached, diafiltration operation can be started. In certain embodiments, the retentate described in the TFF1 buffer diafiltration of about 6 diafiltration volumes can be used. In certain embodiments, the retentate described in the TFF1 buffer diafiltration of about 5-20 or 10-15 or 12 diafiltration volumes is used. Once the infiltration total flow rate of 6 diafiltration volumes has been reached, retentate can be concentrated again and collected. Rinsing can be performed, for example twice continuous rinsing of membrane, to increase the product recovery rate of intermediate bulk drug.

[0191] 5. Intermediate products

[0192] In some embodiments, the intermediate drug substance can then be frozen on dry ice or in a freezer and then transferred to storage at ≤ -60° C. In other embodiments, the intermediate product does not need to be frozen prior to downstream processing.

[0193] In some embodiments, multiple batches of intermediate material are pooled together for further processing (e.g., for purification by a downstream process, e.g., as described herein). The multiple batches of intermediate material can be pooled before freezing and storage. In other embodiments, the multiple batches of intermediate material can be pooled after thawing the frozen and stored batches.

[0194] Downstream Process

[0195] In some embodiments, the intermediate (e.g., pooled intermediate) is processed into a filtered drug substance using a downstream process. 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 in which the purified AAV particles are suspended in a pharmaceutically acceptable carrier. In some embodiments, the downstream process comprises the following manufacturing steps after 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 full / empty capsid separation, tangential flow filtration (TFF3) for concentration / buffer exchange, filtration of the TFF3 pool material to produce the drug substance, drug substance dilution and filtration to produce the drug product, drug product storage, and filling of the drug product into vials.

[0196] In some embodiments, the downstream processes disclosed herein can be used to process intermediates comprising AAV SMN, AAV MECP2, or AAV encoding a SOD1-targeting shRNA as described herein.

[0197] 1. Acidification and clarification of intermediates

[0198] In the embodiment of frozen intermediates, the downstream process starts by thawing the TFF1 intermediate material. Detergents, such as Tween 20, can be used to promote flocculation of batch host cell proteins and DNA at acidic pH. The pH value of the TFF1 intermediate containing detergent can then be reduced. The flocculants and precipitates formed when the pH is reduced can then be removed by filtering the solution through a depth filter and a filter (which removes macromolecular contaminants and cell debris, such as a 0.45 μm filter, but allows the vector genome to pass through). Any suitable depth filter can be used.

[0199] In one embodiment, Tween 20 is slowly added to the TFF1 intermediate solution to reach a final concentration of Tween 20 between 10% and 20%. In some embodiments, the target composition after adding Tween 20 is a solution of 36% Tween 20 in 20mM Tris, 1mM MgCl2, 500mM NaCl, 1% sucrose m / v (pH 8.1). In some embodiments, Tween 20 is slowly added over about 1-6 hours. In some embodiments, Tween 20 is slowly added over 3-6 hours. In some embodiments, Tween 20 is slowly added over 4 hours. In some embodiments, the Tween 20 / TFF1 intermediate solution is allowed to incubate 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.

[0200] In some embodiments, the solution of the present invention can be prepared by adding a tween 20 having a TFF1 intermediate. After incubation, the pH value 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 value is within an acceptable range, the solution can pass through a filter of any size. In an exemplary embodiment, a depth filter (for example, Clarisolve POD) consistent with a 0.45 μm filter (for example, Opticap XL10Durapore filter) or a 0.8 / 0.45 μm PES filter is used.

[0201] 2. Cation Exchange Chromatography

[0202] 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 brief, the method relies on the charge-charge interaction between the positively charged particles to be separated and the negatively charged resin used. Typically, the column is first balanced by running a small amount of diafiltration volume 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 sample containing the fraction is collected. The presence of the target sample can be detected by optical absorbance measurement of the eluent.

[0203] In one embodiment, the CEX step uses a CIMmultus S03-8000 advanced composite column (sulfonyl) (2 μm pore) chromatographic column. In one embodiment, the elution peak is collected starting from a sharp rise in OD280. When the conductivity is between 80-85 mS / cm, OD280 begins to rise. The CEX eluate can be collected according to conventional procedures and can be divided into two fractions for collection. In one embodiment, the first fraction starts at a sharp rise in OD280 and collects 1.5 column volumes (CV). In another embodiment, the second fraction starts immediately after the first fraction and collects 1.0 CV. The two fractions are pooled and then neutralized to pH 8.0 ± 0.30. In one embodiment, the neutralization buffer comprises 1.0 M Tris, pH 9.1 ± 0.1 at 20° C.

[0204] 3. Tangential flow filtration 2

[0205] In certain embodiments, a tangential flow filtration step (TFF2) is used to concentrate, remove protein impurities, and exchange the buffer with a suitable buffer for a subsequent CsCl ultracentrifugation step. Any suitable TFF membrane can be used. In one embodiment, the TFF2 step utilizes a 300kD MWCO regenerated cellulose membrane.

[0206] 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 diafiltration buffer and concentrated to its original volume. In one embodiment, the diafiltration buffer is TFF2 NaCl diafiltration buffer, which contains 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 diafiltration with the new buffer is complete. In one embodiment, the retentate is diluted 2-fold with diafiltration buffer containing CsCl and concentrated to its original volume. In one embodiment, the diafiltration buffer containing CsCl is TFF2 CsCl diafiltration buffer, which contains 20 mM Tris, 2 mM MgCl2, 3 M CsCl, 0.2% Poloxamer 188, pH 8.1±0.1 at 20°C. In such an embodiment, the process can be repeated until the diafiltration with the new buffer is complete. Once the CsCl diafiltration is complete, the retentate can then be concentrated to a specified volume, depending on the system hold-up volume. In certain embodiments, rinsing, such as two consecutive rinses of the membrane, is performed to maximize product recovery from the TFF2 system.

[0207] 4. CsCl ultracentrifugation

[0208] 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 the two methods showed that iodixanol-derived AAV vectors had higher vector purity but more empty viral capsids than 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 result in a dramatic loss of rAAV, resulting in low yields and recoveries, often offsetting the other advantages 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 methods work well in the laboratory for producing preclinical samples, they are not scalable and therefore unsuitable 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”).

[0209] In some embodiments, an ultracentrifugation step is used, for example, to separate empty capsids from full capsids. Surprisingly, 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 gradient buffers known in the art. Centrifugation can be performed in any centrifuge capable of achieving the desired g-force, such as an automatic Optima XPN 100 ultracentrifuge system or an equivalent system equipped with a 50.2Ti rotor or an equivalent rotor. After ultracentrifugation, empty capsids and full capsids are separated into different bands in a test tube, and material can be extracted from a specific band. In some embodiments, the filtered material purified by TFF2 is centrifuged at 241,600-302,000 g (about 40,000-50,000 rpm in a 50.2Ti rotor). In some embodiments, the filtered material purified by TFF2 is centrifuged overnight. In certain embodiments, the filtered material purified by TFF2 is centrifuged for 16-24 hours. In certain embodiments, the filtered material purified by TFF2 is centrifuged for 20-24 hours. In certain 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) at 20°C for 17 hours. In certain embodiments, the buffer for CsCl centrifugation may have one or more of the following ingredients: including (a) CsCl, also including one or more of (b) MgCl2, (c) poloxamer 188 and (d) Tris. In certain embodiments, the buffer for CsCl may include all of (a), (b), (c) and (d). In certain embodiments, the buffer for CsCl has pH 7.5-8.5, or pH 7.9-8.2. In one embodiment, a suitable buffer for CsCl centrifugation is 20mM Tris, 2mM MgCl2, 3M CsCl, 0.2% poloxamer 188, pH 8.1±0.10. After the centrifugation step is complete, the tube can be removed from the ultracentrifuge. In some embodiments, the highest band, band A, contains empty capsids. In some embodiments, the second highest band, bands B, C, and D contain double bands of full capsids. In some embodiments, a syringe is used to collect the AAV viral vector. In one embodiment, bands B, C, and D are removed by an 18G needle connected to a 30mL syringe, and the syringe is inserted just below band D to the middle of the tube. In other embodiments, techniques known in the art and / or the techniques described herein can be used to analyze the presence of full capsids or empty capsids in the bands, and the bands containing full capsids are collected.

[0210] The ratio of empty viral capsids to non-empty viral capsids 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 determined by 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.

[0211] In one embodiment, the AAV viral vector collected after ultracentrifugation has less than 8% empty capsids, less than 7% empty capsids, less than 5% empty capsids, less than 3% empty capsids, or less than 1% empty capsids. 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 determined as a percentage of total capsids.

[0212] 5. Tangential flow filtration 3 to produce filtered API

[0213] In certain embodiments, a tangential flow filtration step (TFF3) is used to remove CsCl and concentrate the full carrier capsid. Tangential flow filtration can be performed using a suitable membrane. In one embodiment, a regenerated cellulose membrane with a 300kDa MWCO 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 certain embodiments, once the target retentate volume is reached, diafiltration begins. 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, the component including (a) Tris, (b) MgCl , (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, the 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 MgCl2, 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 MgCl2, 200 mM NaCl, 0.005% poloxamer 188, pH 8.0 ± 0.1 at 20°C. In one embodiment, 0.2 μm Pall The concentrated retentate is filtered through an EKV sterile grade filter (MiniKleenpak) to produce filtered drug substance. In some embodiments, the methods described herein produce greater than 5 x 10 15 vg, or greater than 8x 10 15 vg or greater than 1x 10 16 vg's rAAV / manufacturing batch / .

[0214] Pharmaceutical composition

[0215] Virus (e.g., AAV) particles purified according to the methods disclosed herein can be produced in high yields with sufficient purity to allow them to be administered to human subjects. In some embodiments, the concentration of the purified virus (e.g., AAV) particles is about 1-8 x 10 13 Viral vector genomes / mL (vg / mL) or approximately 1.7-2.3 x 10 13 The viral vector is prepared at a concentration of about 1.9-2.1 x 10 13 In some embodiments, 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.

[0216] In certain embodiments, in the production process of viral vectors, empty viral capsids that do not contain nucleic acid material can be generated. Pharmaceutical compositions comprising low amounts of empty viral capsids may be advantageous because they avoid exposing patients (e.g., infants) with immature immune systems to unnecessary exposure to antigenic material (empty capsids, host cell proteins, host cell DNA) without therapeutic benefit. In certain embodiments, such pharmaceutical compositions can reduce potential infusion reactions or more extensive immune responses, and can improve therapeutic efficacy. Compared with full viral capsids with genomic material, empty capsids have different densities, thereby allowing separation of the two species 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.

[0217] The ratio of empty viral capsids to non-empty viral capsids can be measured by standard laboratory techniques. In some embodiments, the ratio is measured by optical 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 determined by UV absorbance measurement. In some embodiments, the measured value is determined by optical refractive index measurement. In some other embodiments, the measured value is determined by analytical ultracentrifugation.

[0218] 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% empty capsids, less than about 5% empty capsids, less than 3% empty capsids, 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 determined as the percentage of total capsids (e.g., using AUC). In some embodiments, for example, compared with compositions with higher percentage empty capsids, these low percentage 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 existing methods (e.g., methods that do not use adherent cells and / or the purification methods described herein).

[0219] In the production process of viral vectors, residual proteins from adherent cells (e.g., HEK293 cells) used to produce viral vectors may not be completely separated. Residual host cell proteins are likely to trigger an immune response. The amount of residual host cells can be measured by any standard laboratory technique, and these techniques can distinguish between viral capsid proteins and 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, measurement can be completed by Western blotting of 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 kit.

[0220] In another embodiment, the residual host cell protein in the pharmaceutical composition is less than or equal to 5×10 6 pg / ml / 1X 10 13 vg / ml, less than or equal to 1.2X 10 6 pg / ml / 1X 10 13 vg / mL or 1X 10 5 pg / ml / 1X10 13 vg / ml to 1.2X 106 pg / ml / 1X 10 13 vg / ml or less than or equal to 40ng / ml / 1X 10 13 In one embodiment, the pharmaceutical composition comprises less than or equal to 5 ng, 4 ng, 3 ng, 2 ng, 1 ng or less of residual host cell protein / 1.0 x 10 13 In one embodiment, the pharmaceutical composition comprises less than or equal to 4 ng of residual host cell protein / 1.0 x 10 13 vg.

[0221] During the production process of viral vectors, residual host cell DNA from adherent cells (e.g., HEK293 cells) or residual plasmid DNA transfected to produce viral vectors may not be completely removed. Purification processes (such as acidification, clarification, tangential flow filtration, etc.) remove batches of residual host cells or plasmid DNA. In one embodiment, the measurement of the amount of residual host cells or plasmid DNA is performed by PCR. In another embodiment, the measurement of the amount of residual host cells 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 cells or plasmid DNA is performed by digital droplet PCR (ddPCR). In one embodiment, the amount of plasmid DNA is determined using qPCR assays using primers specific to the kanamycin resistance gene region of the plasmid. In another embodiment, the amount of residual host cell DNA is determined by a commercial qPCR assay kit, such as the qPCR kit from ThermoFisher. Human Residual DNA Quantitation Kit, Biorad's Residual DNA Quantitation 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 treatments disclosed herein.

[0222] In one embodiment, the residual host cell DNA in the pharmaceutical composition is less than or equal to 1.7×10 6 pg / ml / 1X 10 13 vg / ml, 1X 10 5 pg / ml / 1X 10 13 vg / ml to 1.2X10 6 pg / ml / 1X 10 13 In one embodiment, the residual host cell DNA in the pharmaceutical composition is less than or equal to 3 x 10 5 , 2x 10 5 , 1.1x 10 5 , 1x 10 5pg or less / 1.0 x 10 13 In an embodiment, the residual host cell DNA in the pharmaceutical composition is less than or equal to 1.1x10 5 pg / 1.0x 10 13 vg.

[0223] In another embodiment, the residual plasmid DNA in the pharmaceutical composition is less than or equal to 1.7×10 6 pg / ml / 1X 10 13 vg / ml, 1X 10 5 pg / ml / 1X 10 13 vg / ml to 1.7X10 6 pg / ml / 1X 10 13 In another embodiment, the residual plasmid DNA in the pharmaceutical composition is less than or equal to 6.8 x 10 5 pg / 1.0x 10 13 vg.

[0224] In one embodiment, the residual host cell DNA in the pharmaceutical composition is less than or equal to 1.1 x 10 5 pg / 1.0x10 13 vg, and the residual plasmid DNA in the pharmaceutical composition is less than or equal to 6.8 x 10 5 pg / 1.0x 10 13 vg.

[0225] In one embodiment, the residual host cell DNA in the pharmaceutical composition is less than or equal to 1.1 x 10 5 pg / 1.0x 10 13 vg, and the residual plasmid DNA in the pharmaceutical composition is less than or equal to 6.8 x 10 5 pg / 1.0x 10 13 vg, and the residual host cell protein in the pharmaceutical composition is less than or equal to 4ng / 1.0x 10 13 vg.

[0226] In some embodiments, the amount of endotoxin in the pharmaceutical composition is less than about 1 EU / mL / 1.0 x 10 13 vg / mL, less than about 0.75EU / mL / 1.0x 10 13 vg / mL, less than about 0.5EU / mL / 1.0x 10 13 vg / mL, less than about 0.4EU / mL / 1.0x 10 13 vg / mL, less than about 0.35EU / mL / 1.0x 1013 vg / mL, less than about 0.3EU / mL / 1.0x 10 13 vg / mL, less than about 0.25EU / mL / 1.0x 10 13 vg / mL, less than about 0.2EU / mL / 1.0x 10 13 vg / mL, less than about 0.15EU / mL / 1.0x 10 13 vg / mL, less than about 0.1EU / mL / 1.0x 10 13 vg / mL, less than about 0.05EU / mL / 1.0x 10 13 vg / mL or less than about 0.02EU / mL / 1.0x 10 13 vg / 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 Pharmacopoeia ("USP"). <85> (incorporated herein by reference in its entirety) for endotoxin determination.

[0227] In one embodiment, the bovine serum albumin (BSA) in the pharmaceutical composition is less than 0.5 ng / 1.0 x 10 13 vg, less than 0.3ng / 1.0x 10 13 vg, or less than 0.22ng / 1.0x 10 13 In one embodiment, the benzonase in the pharmaceutical composition is less than 0.2 ng / 1.0 x 10 13 vg, less than 0.1ng / 1.0x 10 13 vg, or less than 0.09ng / 1.0x10 13 vg.

[0228] In one embodiment, the pharmaceutical compositions disclosed herein comprise one or more of the following: less than about 0.09 ng benzonase / 1.0 x 10 13 vg, less than about 30 μg / g (ppm) of cesium, about 20-80 ppm of poloxamer 188, less than about 0.22 ng BSA / 1.0 x 10 13 vg, less than about 6.8x 10 5 pg residual plasmid DNA / 1.0x 10 13 vg, less than about 1.1x10 5 pg residual hcDNA / 1.0x 10 13 vg, less than about 4ng rHCP / 1.0x 10 13vg, pH 7.7-8.3, approximately 390-430 mOsm / kg, fewer than approximately 600 particles ≥ 25 μm / container, fewer than approximately 6,000 particles ≥ 10 μm / container, approximately 1.7x10 13 -2.3x 10 13 The genome titer of vg / mL is about 3.9x 10 8 -8.4x 10 10 IU / 1.0x 10 13 The infectivity titer of vg is about 100-300 μg / 1.0x10 13 vg of total protein, at about 7.5 x 10 13 Median survival of Δ7SMA mice ≥24 days with a vg / kg dose of viral vector, approximately 70%-130% relative potency based on in vitro cell-based assays and / or less than about 5% empty capsids.

[0229] In one embodiment, the pharmaceutical compositions disclosed herein comprise one or more, e.g., all, of the following: pH 7.7-8.3 (e.g., as determined by USP <791> ), about 390-430 mOsm / kg (e.g., as measured by USP <785> less than about 600 particles ≥ 25 μm in size per container (e.g., as measured by USP <787> less than about 6000 particles ≥ 10 μm in size per container (e.g., as measured by USP <787> measured), approximately 1.7 x 10 13 -2.3x 10 13 The genome titer of vg / mL is about 3.9x 10 8 -8.4x 10 10 IU / 1.0x 10 13 The infectivity titer of vg is about 100-300 μg / 1.0 x 10 13 vg of total protein, at about 7.5 x 10 13vg / kg dose of viral vector, a median survival of Δ7SMA mice of ≥24 days (e.g., in an in vivo functional assay, such as described herein), about 70%-130% relative potency according to an in vitro cell-based assay and / or less than about 5% empty capsids. In embodiments, the pharmaceutical compositions disclosed herein comprise an overall purity greater than or equal to 95% (e.g., as determined by SDS-PAGE). In embodiments, the pharmaceutical compositions disclosed herein do not comprise a single unnamed related impurity at a level greater than 2% (e.g., as determined by SDS-PAGE). In embodiments, the pharmaceutical compositions disclosed herein comprise an endotoxin level less than or equal to 0.75 EU / mL (e.g., as determined by USP <85> In the examples, the pharmaceutical compositions disclosed herein are tested for no growth in a sterility test (e.g., by USP <71> Measurement).

[0230] High levels of residual host cell proteins, host cell DNA, plasmid DNA and / or endotoxins may pose a challenge to the efficacy of viral vector therapy. In certain embodiments, these relatively low amounts of residual host cell proteins, host cell DNA, plasmid DNA and / or endotoxins improve therapeutic efficacy and / or reduce adverse events (such as inflammatory response, liver damage) after being administered to the patient, for example, compared with compositions of these substances with a higher amount. In certain embodiments, compared with the levels in existing methods (such as methods without using adherent cells and / or purification methods described herein), the method for preparing viral vectors disclosed herein provides these improved levels. In certain embodiments, the method herein also allows for the preparation of viral vectors with a reduced empty capsid percentage except for a small amount of residual host cell proteins, host cell DNA, plasmid DNA and / or endotoxins.

[0231] 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 the quantitative limit (e.g., using ICP-MS).

[0232] 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 1x 10 13 vg / ml, or greater than or equal to about 3 x 1013 vg / ml.

[0233] In one embodiment, the pharmaceutical composition has one or more of the following: less than 0.09 ng benzonase / 1.0 x 10 13 vg, less than 30 μg / g (ppm) of cesium, about 20-80 ppm of poloxamer 188, less than 0.22 ng BSA / 1.0x10 13 vg, less than 6.8x 10 5 pg residual plasmid DNA / 1.0x 10 13 vg, less than 1.1x 10 5 pg residual hcDNA / 1.0x10 13 vg, and less than 4ng rHCP / 1.0x 10 13 vg.

[0234] In another embodiment, the pharmaceutical composition maintains potency of ±20%, ±15%, ±10% or ±5% of a reference standard. In one embodiment, potency is assessed relative to a reference standard using the method of Foust et al., Nat. Biotechnol., 28(3), pp. 271-274 (2010). Any suitable reference standard may be used. In one embodiment, the pharmaceutical composition has in vivo potency as tested in SMAΔ7 mice. In one embodiment, administration of 7.5 x 10 13 The test mice at a vg / kg dose had a median survival of greater than 15 days, greater than 20 days, greater than 22 days, or greater than 24 days. In one embodiment, the pharmaceutical composition has an in vitro relative potency of 50%-150%, 60%-140%, or 70%-130% relative to a reference standard and / or a suitable control as tested by a cell-based assay.

[0235] Viral particles (e.g., viral particles) purified according to the present disclosure can be formulated according to known methods to prepare 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. Specifically, the delivery system can be formulated for intramuscular, intradermal, mucosal, subcutaneous, intravenous, intrathecal, injectable storage devices, or topical administration.

[0236] 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, for example, 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 can be combined with a sterile solution before administration.

[0237] Compositions, such as pharmaceutical compositions, may contain pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusters and buffers, tonicity adjusters, wetting agents, and the like, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like. In some embodiments, the pharmaceutical composition contains a preservative. In some other embodiments, the pharmaceutical composition does not contain a preservative.

[0238] The genomic titer of viral vectors, such as the genomic titer of viral vectors in the compositions and formulations disclosed herein, can be determined using a variety of standard methods. PCR with viral vector-specific primers can provide relative measurements, but quantitative PCR (qPCR) can be used for smaller samples and absolute measurements. Droplet digital PCR (ddPCR) is a digital PCR method based on water-oil emulsion droplet technology. The sample is separated into tens of thousands of droplets, and PCR amplification of the template molecule occurs in each individual droplet. There is no need to make a standard curve or to use primers with high amplification efficiency, so ddPCR does not typically use large amounts of samples like traditional PCR-based techniques. In one embodiment, PCR is used to determine the genomic titer of the viral vector. In another embodiment, qPCR is used to determine the genomic titer of the viral vector. In another embodiment, ddPCR is used to determine the genomic titer of the viral vector. Methods for determining viral genome titers using ddPCR are 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.

[0239] In some embodiments, the PCR-based method uses primers and probes specifically designed to target the SMN gene to detect and quantify the encapsulated 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 encapsulated 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 encapsulated 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 encapsulated 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 encapsulated AAV9 viral genome.

[0240] In some embodiments, the pharmaceutical composition is approximately pH 7.7-8.3 and has an osmotic pressure molarity of 390-430 mOsm / kg. In some embodiments, pH is measured using a pH meter. In some embodiments, a microelectrode with temperature compensation is used according to standards set by the United States Pharmacopoeia (USP), e.g., <791> (incorporated by reference in its entirety), pH was measured by potentiometric titration. In some embodiments, pH was measured according to USP, e.g., USP <785> (incorporated by reference in its entirety), freezing point depression is used to measure osmolality. In some embodiments, vapor pressure depression osmometers are used to measure osmolality. In other embodiments, membrane osmometers are used to measure osmolality.

[0241] In one embodiment, the pH of the intravenous formulation is between 7.5 and 8.5 and the genome titer is 2×10 13 vg / ml-6X 10 13 vg / ml, and an osmotic pressure molar concentration of 384-448mOsm / kg. In another embodiment, the pH value of the intravenous injection formulation is between 7.5 and 8.5, and the genome titer is 1.5X 10 13 vg / ml-3.5X 10 13 vg / ml, and an osmotic pressure molar concentration of 384-448mOsm / kg. In another embodiment, the pH value of the intravenous injection formulation is between 7.5 and 8.5, and the genome titer is 1.8X 10 13 vg / ml-2.2X 10 13vg / ml, with an osmolarity of 384-448 mOsm / kg. In one embodiment, the IV formulation comprises about 0.1-2.0 mM MgCl2. 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, for example, at a pH of 7.5-8.5.

[0242] In one embodiment, the IV formulation comprises 1 mM MgCl2, 200 mM NaCl, 0.005% w / v Poloxamer 188 in 20 mM Tris buffer (pH 8.0). In an embodiment, the IV formulation comprises about 1 x 10 13 Up to 3x 10 13 vg / mL or 1.7 x 10 13 to 2.3x 10 13 The genomic titer was vg / mL.

[0243] Uses of the pharmaceutical composition

[0244] In other embodiments, disclosed herein are methods for delivering a polynucleotide to the central nervous system of a patient, the methods comprising administering an rAAV9 having a genome comprising the polynucleotide. In some embodiments, the delivery is intrathecal delivery of the polynucleotide to the central nervous system of the patient, the methods comprising administering an rAAV9 having a genome comprising the polynucleotide. In some embodiments, a non-ionic hypotonic contrast agent is also administered to the patient. The non-ionic hypotonic contrast agent increases 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.

[0245] In some embodiments, the polynucleotide is delivered to a brain region. Brain regions contemplated for delivery include, but are not limited to, the motor cortex and the brainstem. In some embodiments, the polynucleotide is delivered to the spinal cord. In some embodiments, the polynucleotide is delivered to lower motor neurons. Embodiments of the present 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.

[0246] Uses include, for example, treatment of lower motor neuron diseases such as SMA and ALS as well as Pompe disease, lysosomal storage disorders, glioblastoma multiforme, and Parkinson's disease. Lysosomal storage disorders 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 (types I, II, and III), GM1 gangliosidosis (infantile, late infantile / juvenile, adult / chronic), I-cell disease / mucolipidosis II, infantile free sialic acid storage disease / ISSD, juvenile hexosaminidase A deficiency, Krabbe disease (infantile onset, late onset), metachromatic leukodystrophy, mucopolysaccharidoses (Pseudo-Hurler polydystrophy / mucolipidosis IIIA, MPSI Hurler syndrome), and leukodystrophy. Syndrome), MPS I Scheie Syndrome, MPS I Hurler-Scheie Syndrome, MPS II Hunter syndrome, Sanfilippo syndrome type A / MPS III A, Sanfilippo syndrome type B / MPS III B, Sanfilippo syndrome type C / MPS IIIC, Sanfilippo syndrome type D / MPS IIID, Morquio type A / MPS WA, Morquio type B / MPS IVB, MPS IX Hyaluronidase deficiency, MPS VI Maroteaux-Lamy syndrome, MPS VII Sly syndrome, mucolipidosis I / sialidosis, mucolipidosis IIIC, mucolipidosis IV), multiple sulfatase deficiency, Niemann-Pick disease (types A, B, and C), neuronal ceroid lipofuscinosis (CLN6 disease (atypical late infantile, late-onset variant, early juvenile), Batten-Spielmeyer-Vogt syndrome / juvenile NCL / CLN3 disease, Finnish variant late infantile CLN5, Jansky-Bielschowsky disease / late infantile CLN2 / TPP1 disease, Kufs / adult-onset NCL / CLN4 disease, Northern epilepsyEpilepsy / Variant Late Infantile CLN8, Santavuori-Haltia / Infantile CLN1 / PPT Disease, Beta-Mannosidosis, Pompe Disease / Glycogen Storage Disease Type II, Pycnodystrophy, Sandhoff Disease / Adult-Onset GM2 Gangliosidosis, Sandhoff Disease / GM2 Gangliosidosis - Infantile, Sandhoff Disease / GM2 Gangliosidosis - Juvenile, Schindler Disease, Salla Disease / Sialic Acid Storage Disease, Tay-Sachs Disease / GM2 Gangliosidosis, Wolman Disease.

[0247] In further embodiments, the methods and materials are indicated for use in treating neurological diseases, such as Rett syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease, or for treating neurological injuries, including spinal cord and brain trauma / injury, stroke, and brain cancer. In one embodiment, the methods and materials are indicated for use in treating spinal muscular atrophy (SMA).

[0248] There are four types of SMA, which are generally classified by age at onset and peak motor function achieved. All forms of SMA are inherited in an autosomal recessive manner and are caused by mutations in the survival motor neuron 1 (SMN1) gene. Humans also carry a second, almost 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, but SMN2 contains a translationally silent mutation in exon 7, which results in the ineffective inclusion of exon 7 in the SMN2 transcript. Thus, SMN2 produces both the full-length SMN protein and a truncated version of SMN lacking exon 7, with the truncated version being the predominant form. As a result, SMN2 produces much less functional, full-length protein than does SMN1 (70%-90% less). 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 patterns distinguishes the SMA gene SMN1 from the copy gene SMN2." Hum Mol Genet, 8(7): 1177-1183. Although SMN2 cannot fully compensate for the loss of the SMN1 gene, patients with a weakened form of SMA generally have a higher number of SMN2 copies.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 specific mutation have a milder disease phenotype. Prior et al., “A positive modification of spinal muscular atrophy in the SMN2 gene.” Am J Hum Genet 85(3):408-413.

[0249] Type I SMA (also known as infantile-onset or Werdnig-Hoffmann disease) refers to SMA symptoms that appear at birth or by 6 months of age. In this type, infants typically have low muscle tone (hypotonia), a weak cry, and difficulty breathing. They often have trouble swallowing and sucking, and they don't reach the developmental milestone of being able to sit up unassisted. They often show one or more of the symptoms of SMA chosen from hypotonia, delayed motor skills, poor head control, rounded shoulder posture, and joint hypermobility. Typically, these infants have two copies of the SMN2 gene, one on each chromosome 5. More than half of new cases of SMA are SMA Type I.

[0250] Type II, or intermediate, SMA is when SMA develops between the ages of 7 months and 18 months and before children are able to stand or walk independently. Children with type 2 SMA generally have at least three SMN2 genes. Late-onset SMA (also called types III and IV SMA, mild SMA, adult-onset SMA, and Kugelberg-Welander disease) causes varying degrees of weakness. Type III SMA develops after 18 months, and children can stand and walk independently, although they may need assistance. Type IV SMA develops in adulthood, and people with it are able to walk by adulthood. People with type III or IV SMA typically have four to eight SMN2 genes, from which considerable amounts of full-length SMN protein are produced.

[0251] 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 disorder / disease develops, the administration is preventative. If the dose is administered after the disorder / disease develops, the administration is therapeutic. In an embodiment, an effective dose is a dose that alleviates (eliminates or alleviates) at least one symptom associated with the disorder / disease state being treated, slows or prevents progression to the disorder / disease state, slows or prevents the progression of the disorder / disease state, reduces the extent of the disease, results in (partial or complete) remission of the disease and / or prolongs survival. Examples of disease states contemplated for treatment are listed herein.

[0252] In one embodiment, a composition comprising a rAAV of the present disclosure is administered intravenously to a patient in need thereof having SMA Type I. In another embodiment, a composition comprising a rAAV of the present disclosure is administered intrathecally to a patient in need thereof having SMA Type II, Type III, or Type IV.

[0253] Disclosed herein is a method for treating 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 viral vector is used to treat type I SMA in a patient, and the patient's weight is determined. In some embodiments, the patient's weight is less than 8.5 kg. In some embodiments, the patient's weight is greater than 2.6 kg. In some embodiments, the patient's weight is 2.6-8.5 kg.

[0254] In some embodiments, the patient has a mutation, such as a null mutation (including any mutation that renders the encoded SMN1 nonfunctional), in one copy of the SMN1 gene. In some embodiments, the patient has a mutation, such as a null mutation, in both copies of the SMN1 gene. In some embodiments, the patient has a mutation, such as a null mutation, in all copies of the SMN1 gene. In some embodiments, the patient has a deletion in one copy of the SMN1 gene. In some embodiments, the patient has a deletion in both 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 the c.859G>C substitution in exon 7 of at least one copy of the SMN2 gene. In some embodiments, the gene sequence of the SMN1 or SMN2 gene can be determined by whole genome sequencing. In other embodiments, the gene sequence and copy number of the SMN1 or SMN2 gene can be determined by high-throughput sequencing. In some embodiments, the gene sequence and copy number of the SMN1 or SMN2 gene can be determined by microarray analysis. In some embodiments, the gene 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).

[0255] In some embodiments, the patient exhibits 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 determined by placing the patient in a sitting position with assistance at the shoulders (front and back). Head control is assessed by the patient's ability to keep their head upright. In some embodiments, spontaneous movements are observed when the patient is in a supine position, and motor skills are assessed by the patient's ability to lift their elbows, knees, hands, and feet off the surface. In some embodiments, the patient's grip strength is measured by placing their fingers in the patient's palm and lifting the patient until their shoulders are off the surface. Hypotonia and grip strength are measured by how long the patient maintains a grip. In some embodiments, head control is assessed by placing the patient's head in the maximum available rotation and measuring the patient's ability to turn the head back toward the midline. In some embodiments, shoulder posture can be assessed by sitting the patient with head and trunk support and observing whether the patient flexes the elbow or shoulder to reach a stimulus placed at shoulder level at arm's length. In some embodiments, shoulder posture can also be assessed by placing the patient in a side-lying position and observing whether the patient flexes their elbow or shoulder to reach a stimulus placed at arm's length at shoulder level. In some embodiments, motor skills are assessed by observing whether the patient flexes their hip or knee when their 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 can be assessed by known clinical measurements (e.g., CHOP INTEND). Other SMA symptoms can be assessed according to known clinical measurements, such as CHOP INTEND.

[0256] In some embodiments, treatment is performed after a patient exhibits symptoms (e.g., one or more symptoms) of SMA Type 1 as determined using one of the tests described herein. In some embodiments, a patient is treated before they exhibit symptoms of SMA Type 1. In some embodiments, a patient is diagnosed with SMA Type 1 based on a genetic test before they develop symptoms.

[0257] Combination therapies are also contemplated herein. Combinations as used herein include simultaneous treatment or sequential treatment. Combinations of methods may include the addition of certain standard medical treatments (e.g., riluzole in ALS), as well as combinations with novel therapies. For example, other treatments for SMA include antisense oligonucleotides (ASOs), which alter binding to pre-mRNAs and alter their splicing patterns. Singh et al., "A multi-exon-skipping detection assay reveals surprising diversity of splice isoforms of spinal muscular atrophy genes." Plos One, 7(11): e49595. In one embodiment, nusinersen (U.S. Pat. Nos. 8,361,977 and 8,980,853, incorporated herein by reference) may be used. Nusindrone is an approved ASO that targets intron 6, exon 7, or intron 7 of the SMN2 pre-mRNA, regulating the splicing of SMN2 to more efficiently produce the full-length SMN protein. In some embodiments, the treatment method comprising the AAV9 viral vector is administered in combination with a muscle-enhancing agent. In some embodiments, the treatment method comprising the AAV9 viral vector is administered in combination with a neuroprotective agent. In some embodiments, the treatment method comprising the AAV9 viral vector is administered in combination with an antisense oligonucleotide-based drug targeting SMN. In some embodiments, the treatment method comprising the AAV9 viral vector is administered in combination with Nusindrone. In some embodiments, the treatment method comprising the AAV9 viral vector is administered in combination with a myostatin inhibitory drug. In some embodiments, the treatment method comprising the AAV9 viral vector is administered in combination with sidarucizumab.

[0258] While delivery after birth to an individual requiring labor is contemplated, intrauterine delivery to a fetus is also contemplated.

[0259] Methods of treating patients with type I SMA using pharmaceutical compositions comprising viral vectors are contemplated. In some embodiments, the dosage of 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 In some embodiments, the viral vector is prepared at a concentration of about 1.9-2.1 x 10 13 In some embodiments, 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.

[0260] In some embodiments, wherein the viral vector is used to treat type I SMA in a patient, the AAV viral vector (e.g., AAVSMN) is expressed at a concentration of about 1.0-2.5 x 10 14 In some embodiments, wherein the viral vector is used to treat type I SMA in a patient, the AAV viral vector is administered at a dose of about 1.1 x 10 14 vg / kg dose is administered to the patient. In some embodiments, the viral vector is used to treat type I SMA in a patient, and the AAV viral vector is infused into the patient over about 45-70 minutes. In some embodiments, the viral vector is used to treat type I SMA in a patient, and the AAV viral vector is infused into the patient over about 60 minutes. In some embodiments, the viral vector is used to treat type I SMA in a patient, and 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 viral vector is used to treat type I SMA in a patient, and the AAV viral vector is infused into the patient using a syringe pump.

[0261] The titer of the rAAV viral vector to be administered will vary depending on, for example, the specific rAAV, the mode of administration, the therapeutic goal, the individual, and the cell type or cells being targeted, and can be determined by standard methods in the art. The titer of rAAV can be about 1×10 6 , about 1X 10 7 , about 1X 10 8 , about 1X 10 9 , about 1X 10 10 , about 1X 10 11 , about 1X 10 12 , about 1X10 13 , about 1X 10 14 or more DNA enzyme resistant particles (DRP) / ml. Dosage can also be expressed in units of vector genomes (vg). Genomic titer can be determined using ddPCR (Lock et al.) described in this application or any other method known in the art.

[0262] The dosage may also vary depending on the time of administration to humans. These dosage ranges for rAAV in adults may be approximately 1×10 11 vg / kg, about 1X 10 12 vg / kg, about 1X 10 13 vg / kg, about 1X 10 14 vg / kg, about 1X 10 15 vg / kg, about 1X10 16vg / kg, or more vector genomes / kg body weight. For neonates, rAAV doses can range from about 1 x 10 11 vg / kg, about 1X10 12 vg / kg, about 3X 10 12 vg / kg, about 1X 10 13 vg / kg, about 3X 10 13 vg / kg, about 1X 10 14 vg / kg, about 3X 10 14 vg / kg, about 1X 10 15 vg / kg, about 3X 10 15 vg / kg, about 1X 10 16 vg / kg, about 3X 10 16 vg / kg or more vector genomes / kg body weight.

[0263] The dosage may also vary depending on the time of administration to humans. These dosage ranges for rAAV in adults may be approximately 1×10 11 vg / kg / week, about 1X 10 12 vg / kg / week, about 1X 10 13 vg / kg / week, about 1X 10 14 vg / kg / week, about 1X 10 15 vg / kg / week, about 1X 10 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 1X 10 12 vg / kg / week, about 3X 10 12 vg / kg / week, about 1X 10 13 vg / kg / week, about 3X10 13 vg / kg / week, about 1X 10 14 vg / kg / week, about 3X 10 14 vg / kg / week, about 1X 10 15 vg / kg / week, about 3X 10 15 vg / kg / week, about 1X 10 16 vg / kg / week, about 3X 10 16 vg / kg / week, or more vector genomes / kg body weight / week. The rAAV dose in adults is 1X10 11 vg / 1.5kg / week, about 1X 10 12 vg / 1.5kg / week, about 1X 10 13 vg / 1.5kg / week, about 1X1014 vg / 1.5kg / week, about 1X 10 15 vg / 1.5kg / week, about 1X 10 16 vg / 1.5kg / week or more vector genomes / kg body weight. For neonates, the dose range of rAAV can be about 1×10 11 vg / 1.5kg / week, about 1X 10 12 vg / 1.5kg / week, about 3X 10 12 vg / kg / week, about 1X10 13 vg / 1.5kg / week, about 3X 10 13 vg / 1.5kg / week, about 1X 10 14 vg / 1.5kg / week, about 3X 10 14 vg / 1.5kg / week, about 1X 10 15 vg / 1.5kg / week, about 3X 10 15 vg / 1.5kg / week, about 1X 10 16 vg / 1.5kg / week, about 3X 10 16 vg / 1.5kg / week, or more vector genomes / 1.5kg body weight / week.

[0264] In one embodiment, the dose is about 1.1 x 10 14 Vector genomes / kg (vg / kg) of patient weight. In one embodiment, a 5 kg patient would receive 0.5 x 10 14 Up to 5.0X 10 14 In 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.

[0265] Dosage can be determined by a number of standard methods. PCR with viral vector-specific primers can provide relative measurements, but qPCR can be used for smaller samples and absolute measurements. ddPCR is a digital PCR method based on water-oil emulsion droplet technology. Baker et al., "Digital PCR hits its stride." Nature Methods, 9(6): 541-544. Sykes et al., "Quantitation of targets for PCR by use of limiting dilution." Biotechniques, 13(3): 444-449. The sample is separated into tens of thousands of droplets, and PCR amplification of the template molecule occurs in each individual droplet. There is no need to prepare a standard curve or to use primers with high amplification efficiency, so ddPCR does not typically use as many samples as traditional PCR-based techniques. Examples of commercially available ddPCR machines include, but are not limited to, the BioRad QX100 ddPCR and the RainDance Raindrop Digital PCR. In one embodiment, the dose is determined using PCR. In another embodiment, the dose is determined using qPCR. In another embodiment, the dose 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 encapsulated 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 encapsulated 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 encapsulated 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 encapsulated 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 encapsulated AAV9 viral genome.

[0266] In one aspect, 2.0X 10 13 vg / ml as the target concentration of the drug product, and the doses were administered according to the table below.

[0267] Table 2: Dosage

[0268] Patient weight range (kg) <![CDATA[Dose volume a (mL)]]> 2.6-3.0 16.5 3.1-3.5 19.3 3.6-4.0 22.0 4.1-4.5 24.8 4.6-5.0 27.5 5.1-5.5 30.3 5.6-6.0 33.0 6.1-6.5 35.8 6.6-7.0 38.5 7.1-7.5 41.3 7.6-8.0 44.0 8.1-8.5 46.8

[0269] a NOTE: Use the upper limit of the patient's weight range to calculate the dose volume.

[0270] In some embodiments, the pharmaceutical composition comprising an 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 an AAV viral vector is infused into the patient over about 60 minutes. In some embodiments, the pharmaceutical composition comprising an 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 an AAV viral vector is infused into the patient using a syringe pump.

[0271] It is also contemplated that patients may be pre-screened for treatment and that treatment be administered to patients identified according to the criteria disclosed herein. AAVs can elicit both cellular and humoral immune responses. Therefore, a small proportion of patients who may receive AAV gene therapy have pre-existing anti-AAV antibodies. 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 can prevent successful transduction, pre-anti-AAV antibodies pose a serious obstacle to the widespread application of AAV gene therapy. In some embodiments, the anti-AAV9 antibody titer level in the patient is determined prior to administration of the AAV viral vector. In some embodiments, the anti-AAV9 antibody titer level in the patient is determined by ELISA combined with immunoassay. In some embodiments, the patient's anti-AAV9 antibody titer is 1:100 or less, as determined by ELISA combined with immunoassay prior to administration of the treatment. In some embodiments, the patient has an anti-AAV9 antibody titer of 1:50 or less as determined by ELISA binding immunoassay prior to administration of treatment. In some embodiments, the patient has an anti-AAV9 antibody titer of greater than 1:100 as determined by ELISA binding immunoassay after treatment and 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 binding immunoassay after treatment and is monitored for 1-8 weeks or until the titer decreases to less than 1:50.

[0272] One approach to overcoming high anti-AAV antibody titers is to use immunosuppressant drugs. The monoclonal anti-CD20 antibody rituximab combined with cyclosporine 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, 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 the plasma and blood cells are separated by centrifugation or hollow fiber filtration. The blood cells are then returned to the patient along with treated plasma or a replacement solution (such as 4.5% human albumin in saline). A common use of therapeutic apheresis is to remove unwanted immunoglobulins, but in this case, plasmapheresis is an attractive method to deplete anti-AAV antibodies. In some embodiments, the patient has an anti-AAV9 antibody titer greater than 1:100 before or after treatment (as determined by ELISA combined immunoassay) and is treated with plasmapheresis. In some embodiments, the patient has an anti-AAV9 antibody titer greater than 1:50 before or after treatment (as determined by ELISA binding immunoassay) and is treated with plasmapheresis.

[0273] Pre-existing maternal antibodies to AAV9 may be transferred to the infant patient via breast milk or intrauterine placental transfer. In some embodiments, the patient has an anti-AAV9 antibody titer greater than 1:100 (as determined by ELISA binding immunoassay) before or after treatment and is switched to formula feeding. In some embodiments, the patient has an anti-AAV9 antibody titer greater than 1:50 (as determined by ELISA binding immunoassay) before or after treatment and is switched to formula feeding.

[0274] The patient's condition can be monitored before and after treatment is administered. Some patients who have received AAV-based therapies have developed thrombocytopenia, a condition characterized by a low platelet count. Thrombocytopenia can be detected by performing a complete blood count on a hematology counter using a diluted blood sample. Thrombocytopenia can also be detected by viewing a slide prepared from the patient's blood (a thin blood film or a peripheral blood smear) under a microscope. A normal human platelet count ranges from 150,000 cells / ml to approximately 450,000 cells / ml.

[0275] In certain embodiments, the patient's platelet count is 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 certain embodiments, the patient's platelet count is 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 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, the patient can be treated with platelet transfusion. In certain embodiments, the patient does not have thrombocytopenia before administration of the viral vector. In certain embodiments, the patient develops thrombocytopenia after administration of the viral vector and is monitored for about 1-8 weeks or until the patient does not have thrombocytopenia. In some embodiments, the patient develops thrombocytopenia following administration of the viral vector and is treated with platelet transfusions.

[0276] Monitoring a patient's condition may also involve standard blood tests that measure 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 levels. Troponin I levels are a general measure of heart health, with elevated levels reflecting heart damage or heart-related conditions. In some embodiments, troponin I levels are monitored after administration of the viral vector. In some embodiments, prior to 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, prior to 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 greater 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.

[0277] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and total bilirubin are general measures of liver function, while creatinine tracks kidney function. Elevated levels of AST, ALT, or total bilirubin may indicate liver dysfunction. In some embodiments, the patient has normal liver function prior to administration of the viral vector. In some embodiments, the patient's liver transaminase levels are less than about 8-40 U / L prior to administration of the viral vector. In some embodiments, the patient's AST or ALT levels are less than about 8-40 U / L prior to administration of the viral vector. In some embodiments, the patient's bilirubin levels are less than 3.0 mg / dL prior to administration of the viral vector. In some embodiments, the patient's creatinine levels are less than 1.8 mg / dL prior to administration of the viral vector. In some embodiments, the patient's hemoglobin (Hgb) levels are between 8-18 g / dL prior to administration of the viral vector. In some embodiments, the patient's white blood cell (WBC) count is less than 20,000 cells / mm2 prior to administration of the viral vector. 3 .

[0278] The efficacy of treatment methods can be determined using various motor skill tests before and after treatment. In particular, the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) was developed to assess motor skills in 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 children with type I SMA with a mean age of 11.5 months (range, 1.4-37.9 months) using the Test of Infant Motor Performance (TIMP) and the Children's Hospital of Philadelphia Test of SMA Strength (CHOP TOSS), a newly designed SMA motor assessment. Testing of treatment efficacy is not limited to the CHOPINTEND test, but can also include other motor skill tests known in the art, including but not limited to TIMP, CHOP TOSS, Peabody Development Motor Scales, Brazelton Neonatal Behavior Assessment tests, Motor Milestone Development Survey, Abilities Captured by Interactive Video Assessment (ACTIVE), Bayley Scale of Infant Development, and measurement of the Compound Motor Action Potential (CMAP).

[0279] In some embodiments, a pre-treatment baseline test is performed using the CHOP INTEND scale. In one embodiment, the CHOP INTEND scale is used during follow-up to determine the efficacy of treatment. In some embodiments, CHOP INTEND includes measurements of head control, righting reflexes, and trunk movements in supported sitting, supine, and prone positions. In some embodiments, CHOP INTEND includes measurements of assisted turning, supine hanging, and anti-gravity movements in supported standing.

[0280] In many gene therapy studies involving AAV vectors, antigen-specific T cell responses to AAV vectors have been observed and may occur between 2 and 4 weeks after gene transfer. One possible outcome of such antigen-specific T cell responses is clearance of transduced cells and loss of transgene expression. In order to suppress the host's immune response to AAV-based therapies, immunosuppressants may be administered to the patient. In some embodiments, glucocorticoids may be administered to the patient before the viral vector is administered. In some embodiments, corticosteroids may be administered to the patient before the viral vector is administered. In some embodiments, oral steroids may be administered to the patient before the viral vector is administered. Examples of oral steroids include, but are not limited to, prednisone, prednisolone, methylprednisolone, triamcinolone acetonide, betamethasone, dexamethasone, and hydrocortisone. In some embodiments, oral steroids are or comprise prednisolone. In some embodiments, the patient begins using prophylactic steroids at least 24 hours before the viral vector is administered. In some embodiments, oral steroids are administered to the patient for at least 30 days after the viral vector is administered. In some embodiments, oral steroids are administered once daily. In some embodiments, oral steroids are administered twice daily. In certain embodiments, oral steroids are administered at a dosage of about 0.1-10 mg / kg, for example, about 1 mg / kg. In certain embodiments, oral steroids are administered at a dosage of about 0.1-10 mg / kg / day, for example, about 1 mg / kg / day. In certain embodiments, the levels of AST and ALT are monitored after administration of the viral vector. In such embodiments, oral steroid treatment is administered when AST and ALT levels exceed twice or about 120 IU / L of the upper limit of normal (for example, determined by clinical standards and methods known in the art). In certain embodiments, oral steroid treatment is administered orally for more than 30 days as long as AST and ALT levels exceed twice or more than about 120 IU / L of the upper limit of normal (for example, determined by clinical standards and methods known in the art). During the continuous treatment period of corticosteroids, the adrenal glands naturally reduce the production of cortisol. If corticosteroid treatment is stopped suddenly, the body may experience a lack of cortisol. In some embodiments of administering oral steroids to patients for at least 30 days, the steroid dosage is slowly tapered on a timetable. In some embodiments, the oral steroid dose is tapered when AST and ALT levels fall below two times the upper limit of normal (e.g., as determined by clinical criteria and methods known in the art) or about 120 IU / L. In some embodiments, tapering comprises a stepwise decrease 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 oral steroid dose is tapered at the physician's discretion.

[0281] Reagent test kit

[0282] The present invention also provides a kit for treating SMA in a patient in need thereof, wherein the kit comprises one or more doses of a pharmaceutical composition comprising an effective amount or dose of a viral vector comprising an SMN polynucleotide disclosed herein and, depending on the type of SMA (as further disclosed herein), a contrast agent (e.g., Omnipaque 180), and instructions for how to use the pharmaceutical formulation or composition and the contrast agent.

[0283] In some embodiments, the kit comprises a vial of a viral vector pharmaceutical composition. In some embodiments, the concentration of the viral vector pharmaceutical composition is about 1.7-2.3 x 10 13 In some embodiments, the concentration of the viral vector pharmaceutical composition is about 1.9-2.1 x 10 13 In some embodiments, the concentration of the viral vector pharmaceutical composition is about 2.0x10 13 vg / mL. In some embodiments, the vial contains about 5.9 mL of the viral vector pharmaceutical composition. In some embodiments, the vial contains about 8.7 mL of the viral vector pharmaceutical composition. In some embodiments, the kit does not contain a 5.9 mL vial, contains at least one 5.9 mL vial, contains at least two 5.9 mL vials, or contains at least three 5.9 mL vials. In some embodiments, the kit does not contain an 8.7 mL vial, contains at least one 8.7 mL vial, contains at least two 8.7 mL vials, contains at least three 8.7 mL vials, contains at least four 8.7 mL vials, contains at least five 8.7 mL vials, or contains at least six 8.7 mL vials.

[0284] In some embodiments of the kit for treating SMA Type I in a patient, the patient's weight is determined. In some embodiments of the kit for treating SMA Type I in a patient, the patient's weight is at least about 2.6 kg. In some embodiments of the kit for treating SMA Type I in a patient, the patient's weight is no more than about 8.5 kg. In some embodiments of the kit for treating SMA Type I in a patient, the patient's weight is between about 2.6 and 8.5 kg. In some embodiments of the kit for treating SMA Type I in a patient, the AAV viral vector in the vial of the kit is administered to the patient. In some embodiments of the kit for treating SMA Type I in a patient, the AAV viral vector in the vial of the kit is administered to the patient at a rate of about 1.0 to 2.5 x 10 14 vg / kg is administered to a patient. In some embodiments where the kit is used to treat type I SMA in a patient, the AAV viral vector in the vial of the kit is administered at a dose of about 1.1 x 10 14In some embodiments of the kit for treating type I SMA in a patient, the AAV viral vector in the vial of the kit is infused into the patient over about 45-70 minutes. In some embodiments of the kit for treating type I SMA in a patient, the AAV viral vector in the vial of the kit is infused into the patient over about 60 minutes. In some embodiments of the kit for treating type I SMA in a patient, the AAV viral vector in the vial of the kit is infused into the patient using an infusion pump, a peristaltic pump, or any other device known in the art. In some embodiments of the kit for treating type I SMA in a patient, the AAV viral vector in the vial of the kit is infused into the patient using a syringe pump.

[0285] In one embodiment, the vector is administered intravenously or intrathecally. In one embodiment, the vector is administered intravenously. In one embodiment, the vector is administered intravenously with Omnipaque 180. In another embodiment, the vector is administered intrathecally with Omnipaque 180.

[0286] In another aspect, methods of transducing target cells in a patient, including but not limited to neural cells or glial cells, with rAAV are contemplated herein.

[0287] The rAAV transduction of patient cells disclosed herein can result in sustained expression of polypeptides or RNA encoded by rAAV. Therefore, the present disclosure provides methods for administering / delivering rAAV (e.g., encoding SMN protein) to animals or human patients. These methods include transducing neural cells and / or glial cells with one or more rAAVs. Transduction can be performed with a gene cassette comprising tissue-specific control elements. For example, a promoter that allows specific expression in neurons or specific expression in astrocytes. Examples include neuron-specific enolase and glial fibrillary acid protein promoters. Inducible promoters under the control of drug intake can also be developed.

[0288] In some aspects, it is contemplated that the transduction of cells is increased when the vectors of the present disclosure are used in combination with a contrast agent as described herein, relative to the transduction when the vectors of the present disclosure are not used in combination with the contrast agent. In various embodiments, the transduction of cells is increased by at least about 1%, or at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, at least about 150%, at least about 180%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, or more, relative to the transduction when the vectors of the present disclosure are not used in combination with the contrast agent. In other embodiments, transduction of cells is increased by about 10% to about 50%, or about 10% to about 100%, or about 5% to about 10%, or about 5% to about 50%, or about 1% to about 50%, or about 10% to about 200%, or about 10% to about 300%, or about 10% to about 400%, or about 100% to about 500%, or about 150% to about 300%, or about 200% to about 500% when the vectors of the present disclosure are used in combination with a contrast agent described herein, relative to transduction when the vectors of the present disclosure are not used in combination with a contrast agent.

[0289] The present invention also provides aspects in which intrathecal administration of a vector and a contrast agent of the present invention to the central nervous system of a patient in need thereof results in increased survival of the patient relative to the survival of the patient when the vector of the present invention is administered in the absence of a contrast agent. In various embodiments, the vector and contrast agent of the present invention are administered separately intrathecally to the central nervous system of a patient in need thereof. In other embodiments, the vector and contrast agent are co-formulated and administered intrathecally to the central nervous system or a patient in need thereof. In other embodiments, the vector and contrast agent are provided in the same packaging for intrathecal administration to the central nervous system or a patient in need thereof. In various embodiments, administration of the vectors of the present disclosure and a contrast agent to the central nervous system of a patient in need thereof results in an increase in the patient's survival by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or more relative to the patient's survival when the vectors of the present disclosure are administered in the absence of the contrast agent.

[0290] In some aspects, it is contemplated that when the vectors of the present disclosure are used in combination with a contrast agent and when the patient is placed in the Trendelenburg position (head-down position), transduction of cells is further increased. In some embodiments, for example, during or after intrathecal vector infusion, the patient is tilted in the head-down position from about 1 degree to about 30 degrees, from about 15 to about 30 degrees, from about 30 to about 60 degrees, from about 60 to about 90 degrees, or from about 90 to about 180 degrees. In various embodiments, transduction of cells is increased by at least about 1%, or at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, at least about 150%, at least about 180%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, or more when a vector of the present disclosure is used in combination with a contrast agent and Trendelenburg as described herein, relative to transduction when the vector of the present disclosure is not used in combination with a contrast agent and Trendelenburg. In other embodiments, transduction of cells is increased by about 10% to about 50%, or about 10% to about 100%, or about 5% to about 10%, or about 5% to about 50%, or about 1% to about 50%, or about 10% to about 200%, or about 10% to about 300%, or about 10% to about 400%, or about 100% to about 500%, or about 150% to about 300%, or about 200% to about 500% when a vector of the present disclosure is used in combination with a contrast agent and Trendelenburg as described herein, relative to transduction when the vector of the present disclosure is not used in combination with a contrast agent and Trendelenburg.

[0291] The present invention also provides aspects wherein intrathecal administration of a vector of the present invention and a contrast agent to the central nervous system of a patient in need thereof in the Trendelenburg position results in a further increase in the patient's survival relative to the patient's survival when the vector of the present invention is administered in the absence of a contrast agent and in the Trendelenburg position. In various embodiments, administration of a vector of the present invention and a contrast agent to the central nervous system of a patient in need thereof in the Trendelenburg position results in an increase in the patient's survival relative to the patient's survival when the vector of the present invention is administered in the absence of a contrast agent and in the Trendelenburg position by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or more.

[0292] Unless the context clearly dictates otherwise, as used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents. "Optional" or "optionally" means that the event or circumstance described subsequently may or may not occur, and that the description includes examples in which the event, circumstance, or component occurs as well as examples in which the event or circumstance does not occur. For example, the phrase "optional composition may comprise a combination" means that the composition may comprise a combination of different molecules or may not comprise a combination, such that the description includes both the combination and the exclusion of the combination (i.e., the individual members of the combination). Ranges can be expressed herein as from "about" one particular value, and / or to "about" other particular values. When such ranges are expressed, another aspect includes from a particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each range are important relative to the other endpoints and are independent of the other endpoints.

[0293] The present disclosure is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents, and published patent applications cited in this application, as well as the figures, are hereby incorporated by reference in their entirety for all purposes.

[0294] Examples

[0295] The following examples should be considered illustrative, rather than limiting, of the scope of the above disclosure.

[0296] Example 1 - Generation of Pre-GMP Master Cell Bank

[0297] method

[0298] Thaw: Single cell vial (1x 10 6 Thaw 100 cells in a 37°C water bath for approximately 1 minute and dilute the contents in 5 mL of pre-warmed complete growth medium. Transfer the cells to a T-25cm 2 flasks and grown in a 37°C incubator for 4 days, replacing the medium with pre-warmed complete growth medium every day.

[0299] Selection for increased adherence: The cells were cultured using the following technique to select for strongly adherent cells. 2When the flask reaches 95% confluence, the cells are subcultured. The cells are washed with 5 mL of PBS and then dissociated with 0.5-1 mL of HyQTase for approximately 2 minutes at room temperature. Dissociation is terminated by adding 5 mL of complete growth medium and repeatedly pipetting to dissociate cell clumps. The cell suspension is then centrifuged at 200 x g for 4 minutes. The supernatant is discarded and the cell pellet is resuspended in 10 mL of complete growth medium. The cells are transferred to a 75 cm 2 After incubation for 4 hours in a 37°C incubator, weakly adherent and non-adherent cells were removed by aspirating the cell culture medium. The culture medium was replaced with 10 mL of pre-warmed complete growth medium. By visual inspection, this process reduced the cell mass by up to 35%. The cells were incubated for another 2 days before being subcultured again. This selection process (consisting of changing the culture medium 4 hours after inoculation) was performed three times ( Figure 2 and Figure 3 In the final selection step, cells were plated onto 2 x 175 cm 2 The final volume of the flask was 25 mL. Note the reduction in cell loss after the media change in the last 4 hours post-inoculation.

[0300] Cell expansion: Once cells are grown in 2 x 175 cm 2 The cells were then expanded after confluence in the flask. The cells were washed with 15 mL of PBS and then dissociated with 3 mL of HyQTase and incubated at room temperature for about 2 minutes. Dissociation was terminated by adding 10 mL of complete growth medium. Once the supernatant was aspirated, the cell suspension was centrifuged to produce 2 cell pellets. Each pellet was resuspended in 8 mL of complete growth medium and 2 mL of this concentrated cell suspension was added to 8 x 175 cm 2 Flasks were prepared by adding 20 mL of complete growth medium to give a total of 22 mL of cell suspension and a 1:4 split ratio. The next expansion step used the same procedure with the following changes: 4 x 175 cm 2 Flasks were expanded with a 1:2 split ratio and 4 x 175 cm 2 The flasks were expanded at a split ratio of 1:3. This resulted in a total of 20 x 175 cm 2 flask.

[0301] Harvest: from 20 x 175cm 2 Cells were collected from the flasks. The cells were washed with 15 mL of PBS and then dissociated with 3 mL of HyQTase as described above. Cell dissociation was terminated by adding 10 mL of complete growth medium and collected into 50 mL tubes containing 4 x 175 cm 2Add the cell suspension from the flask to one 50 mL tube. This results in five 50 mL tubes, each containing 40 mL of cell suspension. Centrifuge the tubes to produce a cell pellet, aspirate the supernatant, and resuspend the cells in 10 mL of complete growth medium to yield a 50 mL cell suspension.

[0302] The volume was divided into 2 x 50 mL tubes, for a total of 25 mL of cell suspension in each tube. The 1:2 diluted sample was used to calculate the viable cell count of each tube of cells using a hemocytometer and toludine (Trypan) blue. The viable cell count of tube 1 sample was 1.99 x 10 6 cells / mL, yielding 3.98 x 10 6 The cell concentration was 10 cells / mL, and the viable cell count in tube 2 was 2.4 x 10 6 cells / mL (total 1x 10 8 cells), yielding 4.8 x 10 6 cells / mL (total 1.2 x 10 8 Thus, a total of 2.2 x 10 8 Both tubes were centrifuged again (6 min, 200 x g) and the pellets were resuspended in 10 mL (tube 1) and 12 mL (tube 2) of freezing medium, respectively, to adjust the cell concentration to 1 x 10 7 cells / mL. Pool the two cell suspensions and aliquot 1 mL (each containing 1 x 10 7 cells) were filled into 22 sterile cryovials (Table 3).

[0303] Table 3: Calculation of total harvested cells

[0304]

[0305] The filled vials were then transferred to a freezer with fresh isopropyl alcohol and placed in a -80°C freezer overnight for controlled rate freezing. The frozen vials were then transferred to a liquid nitrogen tank in the vapor phase. Ten vials were transferred to a GMP facility on dry ice for storage.

[0306] HEK293 cells from ATCC were thawed and successfully cultured for three passages to allow for increased adherence before expansion and seed bank establishment. The seed bank was tested for growth and the presence of adventitious agents (mycoplasma, fungi, and bacteria). Testing demonstrated that the seed bank was suitable for use as a master cell bank in a GMP facility.

[0307] Example 2 - Upstream Process

[0308] Upstream processes (see e.g. Figure 4) for producing an intermediate derived from a working cell bank, wherein the upstream process comprises the following steps: (a) culturing adherent cells, (b) transfecting the cultured cells with three plasmids as shown in FIG1 (e.g., comprising an AAV SMN as described herein) to enable production of the AAV viral vector, (c) lysing the adherent cells to isolate the AAV viral vector, (d) purifying the viral particles by filtration to remove any intact cells or cell debris, and (e) subjecting the purified product of (d) to tangential flow filtration, and (f) freezing the resulting intermediate preparation of purified viral particles. In an alternative embodiment, the AAV prepared using the upstream process disclosed herein encodes an shRNA targeting SOD1 or MECP2 disclosed herein.

[0309] (a) Cultivation of adherent cells

[0310] HEK293 cells were thawed and expanded for seven passages in disposable flasks in a CO2 incubator. The thawed cells were washed with cell expansion growth medium, centrifuged, and resuspended in fresh cell expansion growth medium. The resuspended cells were seeded into flasks containing cell expansion growth medium and incubated.

[0311] When the cells reached confluence, they were washed with DPBS and removed from the flask with TrypLE Select enzyme solution. Cell expansion growth medium was added to neutralize the enzyme solution, and the suspended cells were separated and re-seeded into new flasks containing cell expansion growth medium. This expansion process was repeated seven times. In the final iteration, the suspended cells were not re-seeded into the flask, and the cell slurry was inoculated into the bioreactor for further expansion.

[0312] Preparation of iCELLis 500 / 200m 2 or iCELLis 500 / 333m 2 The adherent cell bioreactor is used for inoculation prior to inoculation. Preparation activities include unpacking the disposable bioreactor, physical inspection, leak testing, connecting the tubing components, and balancing the probes. The bioreactor is then filled with cell expansion growth medium to equilibrate the bioreactor. Once pH (pH 6.9-7.5), temperature (35°C-39°C), and dissolved oxygen (40%-125%) are confirmed to be within the specified range, the bioreactor is plated at 4800-7000 cells / cm 2 (For 200m 2 reactor) or 5000-12000 cells / cm 2 (For 333m 2 The cell slurry from the previous step was added to the culture medium in the recirculating medium bag and circulated through the bioreactor.

[0313] (b) Transfection of adherent cells

[0314] On the 4th, 5th or 6th day after bioreactor inoculation, adherent HEK293 cells were transfected with triple DNA plasmid PEI coprecipitation. The 3 plasmids used for this transfection were pSMN, pAAV2 / 9 and pHELP. The DMEM growth medium used for cell expansion was removed from the bioreactor and replaced with transfection medium. Polyethylenimine ("PEI") coprecipitation was used in a large-scale adherent cell bioreactor and triple DNA plasmids were transfected into adherent human embryonic kidney (HEK293) cells to prepare scAAV9.CB.SMN vectors. The vector plasmid pSMN contains the cDNA of human motor neuron survival protein (SMN). The 3 plasmids used for this transfection were pSMN (222 mg), pAAV2 / 9 (333 mg) and pHELP (444 mg). Plasmids can be transfected in a 1:1:1 molar ratio. Before adding the PEI-plasmid coprecipitation, the transfection medium was balanced in the bioreactor until the bioreactor temperature>30°C. The PEI-plasmid coprecipitation process includes adding the plasmid to the transfection medium, and carrying out 0.2 μ filtration into the reaction bag. PEI is added to the transfection medium and then added to the reaction bag. The weight ratio of the PEI-plasmid is about 1: 1. Artificial mixing of the PEI-plasmid reaction forms a homogeneous suspension, and the reaction is through 15-30 minutes. At the end of the reaction time, the PEI-plasmid coprecipitation is transferred to the bioreactor from the reaction bag. Before restarting stirring, the PEI-plasmid coprecipitation is mixed in the bioreactor for 1-2 hours (alternative duration has been described in Example 7). The transfection medium is recirculated in the bioreactor for 18-24 hours, and then the next culture medium replacement is carried out.

[0315] On bioreactor day 6, 18-24 hours after transfection, the bioreactor is drained and the transfection medium recirculation bag is replaced with post-transfection medium. The bioreactor is refilled with post-transfection medium and recirculated within the bioreactor. On day 7, 18-24 hours after the media change on day 6, the post-transfection medium in the recirculation bag is replaced with a fresh bag of post-transfection medium. The bioreactor is not drained during this step. Recirculation of the medium continues until harvest, typically on day 9.

[0316] (c) Lysis of transfected adherent cells

[0317] After 9 days in the bioreactor, the final pre-harvest sample was extracted from the reactor and the whole cell lysis process was started. Benzonase was added to the bioreactor to a final concentration of 100 U / mL. Benzonase was mixed in the reactor and lysis buffer was added to the reactor. The lysis buffer was mixed in the reactor at 15°C-25°C for 2 hours, and then the contents of the bioreactor were transferred to a harvest bag. A salt sucrose solution (SSS) to quench the Benzonase reaction was added to the harvest bag and mixed for 15 minutes. The bioreactor was then rinsed with bioreactor rinse buffer for 15 minutes, and the rinse was then collected into a harvest collection bag together with the quenched cell lysate. Once the rinse was added to the collection bag, the contents were mixed for 15 minutes and a batch harvest sample was taken.

[0318] (d) Preparation of viral particles by filtration and tangential flow filtration

[0319] The combined bulk harvest is filtered through a POD depth filter into a collection bag. Once all the bulk harvest has been filtered, the depth filter is chased with TFF1 buffer. The depth filter pool is mixed and sampled. The depth filter pool is then filtered through a 0.45 μm filter to further clarify the bulk harvest material. The 0.45 μm filter is then chased with TFF1 buffer.

[0320] For the TFF1 step, 5.0m 2 The regenerated cellulose membrane box of 300kDa MW cut-off value is rinsed, sterilized with NaOH solution and balanced with TFF1 buffer.The concentration stage of this operation is designed to reduce the volume of the clarified harvest by about 10x.Once the target retentate volume is reached, diafiltration operation is started.With the TFF1 buffer diafiltration retentate of 6 diafiltration volumes.Alternatively, the retentate described in diafiltration can be used, for example, 10 diafiltration volumes, 12 diafiltration volumes or 15 diafiltration volumes, with the TFF1 buffer that surpasses 6 diafiltration volumes.Once the infiltration total flow that reaches 6 diafiltration volumes, concentrate the retentate again and gather in the crops in the collecting bag.Membrane is carried out to twice continuous rinsing, so that the product recovery from the TFF system is maximized, thereby produces intermediate bulk drug.

[0321] (e) Frozen intermediates

[0322] The TFF1 intermediate was aliquoted into 1 or 2 L sterile PETG bottles in the LFH hood, then frozen on dry ice or in a freezer and transferred to -60 °C for storage.

[0323] Table 4: Buffers used in upstream processes

[0324]

[0325]

[0326] Example 3 - Downstream Process

[0327] Use downstream processes (see e.g. Figure 5 ) Processing the TFF1 intermediate into a filtered drug substance. In some embodiments, the downstream processes disclosed herein can be used to process intermediates comprising AAV SMN, AAVMECP2, or an AAV shRNA encoding a target of SOD1 as described herein. The downstream process steps include: (a) acidification and clarification of the intermediate (using filtration), (b) purification using cation exchange chromatography, (c) filtration using tangential flow filtration ("TFF2"), (d) ultracentrifugation using CsCl buffer to separate filled and empty viral capsids, (e) collection of the AAV viral vector, and (e) filtration of the collected AAV viral vector using a second tangential flow filtration ("TFF3") step.

[0328] (a) Acidification and clarification

[0329] The TFF1 intermediate material from the upstream process (if previously frozen, thawed to room temperature) is pumped into the bag with a mixer. The pooled TFF1 intermediate is mixed and a sample is taken to determine the titer. The pooled TFF1 intermediate is immediately treated by adding 11%-14% Tween 20. Tween 20 is used to promote batch flocculation of host cell proteins and DNA under acidic pH conditions. The mixture is incubated for 12-20 hours. The pH is then reduced to pH 3.3-3.7 by adding acidifying buffer (1M glycine). The solution is then filtered through 1.1m 2 Clarisolve and 2.2m 2 A Millistak+COHC depth filter and a 0.45 μm polishing filter were used to remove the precipitate formed after the pH was lowered. This process yielded an acidified and clarified TFF intermediate.

[0330] (b) Cation exchange chromatography purification

[0331] A cation exchange (CEX) chromatography step is used to separate viral capsids from proteins, DNA, and other process impurities (e.g., host cell lipids, Tween 20). This step utilizes a CIMmultus S03-8000 Advanced Composite Column (Sulfonyl) (0.2 μm pore) chromatography column (8.0 L) operated using an automated process chromatography system. Buffers and solutions are shown in the following table:

[0332] Table 5: Buffers and solutions for one CEX cycle

[0333]

[0334] The acidified and clarified TFF intermediate (i.e., CEX load) is loaded onto a clean and equilibrated CEX column. The conditions allow the viral vector to bind to the monolithic column. Unbound material is washed from the column with CEX A buffer. The product is eluted from the resin using a gradient of CEX B buffer in CEX A buffer. Fraction 1 is collected starting at the beginning of the elution gradient, collecting 10 column volumes (CV) with a specified volume of 2.3-2.7 CV. The chromatographic column is discarded after each batch (i.e., the chromatographic column is not reused). The CEX product eluate (fraction 2) is then neutralized to a pH of 7.7-8.3 using neutralization buffer.

[0335] (c) Tangential flow filtration (TFF2)

[0336] The TFF2 step concentrates the viral vector, removes protein impurities, and exchanges the buffer to the appropriate buffer for the CsCl ultracentrifugation step. The neutralized CEX eluate is washed with a 0.3 ml 2 TFF system processing with a 300 kDa MWCO regenerated cellulose membrane.

[0337] Reduce the volume of the neutralized CEX eluate to the target retentate volume. Once the target retentate volume is reached, begin diafiltration in discontinuous TFF mode (batch mode). Dilute the retentate 2-fold with TFF2 NaCl diafiltration buffer and concentrate it to its initial volume. Repeat this process until diafiltration is complete with TFF2 NaCl diafiltration buffer. Then, dilute the retentate 2-fold with TFF2 CsCl diafiltration buffer and concentrate it to its initial volume. Repeat this process until diafiltration is complete with TFF2 NaCl diafiltration buffer.

[0338] Based on the physical titer of the neutralized CEX eluate, the system holdup volume, the system flush volume, and the retentate density, the retentate is further concentrated to a final mass to achieve the desired target vector concentration and recovered into a collection bag. A flush cycle of the system using TFF2 CsCl diafiltration buffer is followed by a product drain to maximize product recovery from the TFF system. A sample of the TFF2 retentate (which includes the retentate and flush) is taken for physical titer measurement. The TFF2 membrane cassette is discarded after each batch (i.e., the TFF membrane is not reused).

[0339] Table 6: Buffers for TFF2

[0340]

[0341] (d) CsCl ultracentrifugation

[0342] The purpose of the ultracentrifugation step is to remove empty capsids from full capsids using cesium chloride gradient ultracentrifugation. Add the TFF2 retentate to an ultracentrifuge tube and seal the tube. Place the tube in an ultracentrifuge, such as an automated Optima XPN 100 ultracentrifuge system or an equivalent system equipped with a 50.2Ti rotor or equivalent. Centrifuge the filled tube at 45,000 rpm for 22 hours at 20°C.

[0343] (e) Collection of AAV viral vectors

[0344] After centrifugation step is completed, test tube is taken out from ultracentrifuge and is placed in biological safety cabinet.The pipe containing product is installed on the ring support above waste container.Place lamp just below test tube so that the following are visualized: empty capsid band (band A, the highest band), full capsid double band (band B and band C, the upper band and the lower band of double band) and the lowest band (band D) below double band.Pierce pipe with the needle attached on syringe so that pipe is ventilated, remove band B, C and D with needle.The material collected is transferred in collecting bag.Dilution ultracentrifugation pool (UC pool) collected with TFF2 buffer, to reach consistent initial CsCl concentration in TFF2 load material.The UC pool of dilution is handled in TFF3 step.The buffer of CsCl ultracentrifugation step is listed in the following table:

[0345] Table 7: Buffers for CsCl ultracentrifugation

[0346]

[0347] (f) Tangential flow filtration (TFF3)

[0348] The TFF3 step removes CsCl and concentrates the whole support using the final formulation buffer. 2 The viral vectors were retained by the membrane.

[0349] The volume of the diluted UC pool was reduced to the target retentate volume. Once the target volume was reached, continuous diafiltration was initiated at a constant retentate volume. The retentate was diafiltered with TFF3 buffer. A sample of the diafiltered retentate was taken for physical potency determination. The retentate was further concentrated by targeting the permeate weight, which was calculated from: 1) the volume of the retentate in the TFF system at the end of diafiltration, 2) the physical potency of the diluted UC pool, 3) the target drug substance (DS) concentration, 4) the combined volume of the system flush and filter flush, and 5) the density of the TFF3 buffer. The TFF3 membrane cassette was discarded after each batch (i.e., the cassette was not reused).

[0350] Table 8: Buffers for TFF3

[0351]

[0352] The TFF membrane was rinsed twice with TFF3 buffer in succession at 20 mL to recover the support from the TFF system. The rinses were passed through a 0.2 mm Pall Recover the drug substance through an EKV sterilizing grade filter (Mini Kleenpak). Rinse the filter with TFF3 buffer to recover any remaining vector in the filter and adjust the final volume of the filtered TFF3 pool (i.e., drug substance DS). Aliquot the DS into 125 mL or 250 mL PETG bottles and freeze at <-60°C.

[0353] Example 4 - Formulation and Filling

[0354] The drug product (DP) is a single-dose, preservative-free, sterile, clear to slightly opaque, colorless to off-white, and is supplied at a target concentration of 2.0 x 10 13 vg / ml of non-replicating, self-complementary AAV9 vector was infused intravenously. DP contained 20 mM Tris, 1 mM MgCl2, 200 mM NaCl, and 0.005% w / v Poloxamer 188. The pH of the solution ranged from 7.7 to 8.3.

[0355] Table 9: Drug Product Plant Operations - Buffer Composition

[0356]

[0357] DP was filled into sterile, ready-to-use, 10 ml Crystal Zenith (CZ) vials, stoppered with sterile, ready-to-use, chlorobutyl elastomer stoppers, and sealed with sterile, 20 mm flip-top aluminum seals. Vials were filled to a nominal fill volume of 5.5 mL or 8.3 mL. The target overfill was 0.4 mL, with the vials filled to 5.9 ± 0.1 mL or 8.7 ± 0.1 mL.

[0358] Example 5 - Potency Determination

[0359] The relative efficacy of the drug product is measured using a quantitative in vivo assay. The assay uses an established mouse model of SMA disease. Breeding pairs of the SMAΔ7 mouse strain (Jackson Laboratories, #005025) are phenotypically normal, but approximately 25% of their offspring are homozygous for the targeted SMN gene mutation and display an SMA-like phenotype. By day 5, they develop signs of muscle weakness, and over the next week, they develop an abnormal gait and a tendency to fall. Jackson Laboratories reports that the average survival time of animals with an SMA-like phenotype is approximately 15±2 days. Pilot studies have shown that the median survival time of untreated animals with an SMA-like phenotype is 16.3 days (geometric mean; n=3 studies; 10 mice per study).

[0360] The bioactive drug product, administered by intravenous (IV) infusion, produces an increase in survival time as a function of dose (vg / kg). Drug product potency is measured relative to a reference material (previous batch of vector). The potency of the drug product and reference material (vector genomes / mL; vg / mL) is determined by droplet digital polymerase chain reaction (ddPCR). The vector is diluted in saline to achieve each of three specific dose levels, which will be administered to mice with an SMA-like phenotype.

[0361] If the assay passes suitability, the assay result is considered acceptable. Assay suitability consists of the following:

[0362] 1. Acceptance limit for negative control samples (15 ± 2 days, median survival)

[0363] 2. Acceptance limit for positive control samples (>40 days, median survival)

[0364] 3. Acceptance limits for the reference standard median survival dose-response curve

[0365] In this study, a previous batch of vehicle (hereinafter referred to as the previous batch) was used to determine the linear correlation between the median survival (days) of SMAΔ7 mice when the drug product (hereinafter referred to as the sample batch) was administered at five different dose levels, including a dose of 0 (zero) using a 0.9% saline solution (untreated group). Table 10 shows the calibration curve using the previous batch reference batch.

[0366] Table 10: Calibration curves over time for five dose levels of AVXS-101 using a previous batch of standard reference

[0367]

[0368] *Not Determined (ND) - Median survival (days) not reached as of August 24, 2017

[0369] The data from this study showed that the linear range of the median survival dose-response curve was 0-7.4 vg / kg. The relative potency of the drug product sample batches was established by comparing the linear regression curve of the previous batch of the reference standard with the linear regression curve of the drug product sample batch. This was done by using the ratio of the y-intercept and slope of each linear regression line (i.e., the reference standard and the test article). The percent relative potency calculation is described by the following equation (1):

[0370] %RP = [(y-intercept of test product / slope) ÷ (y-intercept of reference standard / slope)] x 100 (1)

[0371] A previous batch used in the Phase 1 clinical trial was used as the reference standard batch and was assigned a potency of 100%.

[0372] The above data established preliminary assay acceptance criteria using the linear portion of the median survival dose-response curve to determine the relative potency of the drug product clinical batches as follows:

[0373] Median survival (days) dose-response curves were generated using 5 dose (vg / kg) levels

[0374] Doses 0, 1.2E+13, and 7.4E+13 were selected to run a linear dose-response model.

[0375] • The limit of suitability for saline (untreated group) was a median survival of 13 to 17 days.

[0376] The applicability limits of the reference standard samples are:

[0377] οSlope ≥ 2.0E-13

[0378] οy intercept ≥ 16.00 (days)

[0379] οy intercept / slope ratio ≥ 8.5E+13

[0380] οR2≥0.7

[0381] The efficacy of SMA therapies (including drug products) was demonstrated using a Δ7 mouse model. The median survival was 15 days ± 2 days, and control animals treated with TFF3 buffer solution (vehicle) provided a reliable baseline control from which product efficacy could be measured as an increase in median survival. The development work for the drug product identified three (3) doses (excluding the vehicle-treated dose) that were determined by genomic titers using droplet digital PCR (ddPCR) and that affected the survival of the mouse model in a linear manner when the administered dose (vg / kg) was logarithmically transformed and plotted against the median survival (days) of treated SMAΔ7 newborn mice. The low, medium, and high titer standards are shown in Table 11 for the standard titers (vg / mL). In addition, TFF buffer (vehicle) solution was used for the zero (0) calibration curve point and negative control. Doses that showed a survival of ≥ 40 days (doses that showed greater than a doubling of the median survival) were also included as positive controls.

[0382] Table 11: Target doses

[0383]

[0384] Dose solution formulation (See Table 12 for an example of a dilution scheme.

[0385] Negative control - TFF3 buffer solution (final formulation buffer for drug product) is used as a negative control

[0386] Positive control: TFF3 buffer solution was used at 1.10 x 10 14 Prepare test article batches at 500 μg / kg. See Table 12 for an example of a dilution scheme.

[0387] Reference Standard Solutions - Reference standard batches were prepared using TFF3 buffer solution at the three concentrations described in Table 11.

[0388] Table 12: Dilution scheme for reference standards and test samples (example)

[0389]

[0390] Test Article Preparation - Test article was diluted using TFF3 buffer. Dilutions were calculated to produce the test dose (vg / kg) shown in Table 11 for each mouse in a final total volume of 50 μl. 12 mice were diluted simultaneously, with one additional volume used as a positive control, with the goal of increasing the minimum lifespan of treated mice to a median survival (days) of ≥40 days.

[0391] Acceptance limit for control samples

[0392] Negative control group (untreated mice) - The acceptance limit for the negative control group was that SMAΔ7 mice achieved a median survival of 15 ± 2 days. In addition, any mice that died within ≤ 10 days were excluded from the analysis.

[0393] Positive control (group treated at the target clinical dose) - The acceptance limit for the positive control group was a minimum lifespan of treated mice with a median survival of ≥40 days. In addition, any mice that expired within ≤10 days were excluded from the analysis.

[0394] Acceptance limits of the reference standard dose-response curve

[0395] Assay suitability criteria will be determined with reference to a standard linear dose response curve that plots median survival (days) versus administered dose (vg / mL). Assay suitability criteria are shown in Table 13.

[0396] Table 13: Assay Suitability Criteria (Reference Standard Linear Dose-Response Curve)

[0397]

[0398] Y-Intercept / Slope Ratio - Linear regression curves measuring median survival (days) for the reference standard and the test article versus the administered dose (vg / kg) were calculated. The ratio of the y-intercept to the slope of each linear regression was calculated.

[0399] Report results

[0400] Qualitative Reporting of Relative Potency Results—Evaluate the assay suitability criteria for each assay before determining a single-point median survival (days) reading of the positive control material ≥ 40 days. If the median survival of the positive control is ≥ 40 days, the test article may be disposed of if the following criteria are met.

[0401] Quantitative Reporting of Relative Potency Results—Evaluate the assay suitability criteria for each assay before quantitative determination of the relative potency of the test article. Once the positive control has reached ≥40 days and has reached the representative upper standard dose of 7.5 x 10 13 The relative efficacy of the test article can be reported based on a median survival of 31 ± 3 days for the group of mice receiving vg / kg. The relative efficacy percentage (%RP) of the test article will be calculated using the y-intercept and slope of the linear regression of the median survival (days) dose-response as follows:

[0402] %RP = 100% * [(test product y-intercept / slope) ÷ (reference standard y-intercept / slope)]

[0403] Example 6: Surfactant inactivation study

[0404] To isolate the effects of low pH and Tween, surfactant inactivation studies were performed using a sample of the drug substance TFF1 intermediate with a pH of 7.6 and 4%-8% Tween-20. The Tween-20 concentration in the TFF1 intermediate was approximately 2.5-fold lower than when Tween-20 was added to the TFF1 intermediate prior to acidification throughout the process. This lower Tween-20 concentration is considered a worst-case scenario for surfactant-driven inactivation in the drug substance process. The test article for the viral inactivation step by surfactant treatment was a TFF-1 intermediate containing 4%-8% Tween-20. To evaluate the ability of the surfactant treatment step to inactivate viruses, the test article was spiked with viruses using both XMuLV and PRV in replicate experiments. Viruses were quantified using a plaque-forming infectivity assay.

[0405] The TFF1 manufacturing step produces a surfactant concentration range of 4%-8% Tween-20. The TFF1 intermediate is pooled, to which an additional 12% Tween-20 is added at an operating temperature of 16°C-20°C, with a mixing time of 12-20 hours. The viral clearance process is performed at a Tween-20 concentration of 4%-8% and a controlled temperature of 16.0°C ± 0.1°C. The inactivation process lasts 120 minutes, compared to a typical process time of 16 hours.

[0406] For the inactivation process, the inactivated load was prepared by measuring the volume of the test article, balancing to the target temperature, and then adding the virus. Samples of the added inactivated load were removed at six (6) time points to demonstrate the kinetics of inactivation over time: <1 minute, 15 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes. After collection, each sample was diluted in growth medium to stop viral inactivation and subjected to viral assay. In order to improve the detection sensitivity at 90 minutes and 120 minutes, a large number of samples at these time points were also subjected to viral assay. Due to the presence of Tween-20 in the test article for this step, the titer of the inactivated load was calculated by the titer of the added virus and the volume of the added virus.

[0407] The viral inactivation effect by surfactant treatment (Tween-20) was shown to be effective as evidenced by LRV values greater than 4 log10 for both viruses at the 90 minute time point. Figure 6 and Figure 7 Shown is a graph of the virus inactivation rate during Tween-20 surfactant treatment over 120 minutes.

[0408] Example 7: Effects of Higher Seeding Density, Earlier Transfection and Harvest Times, and DNA / PEI Mixing Time on API Production

[0409] The effects of higher seeding density, transfection and harvesting 1 day earlier, and DNA / PEI mixing time on DS production were evaluated. 2 Each condition was evaluated in duplicate in the bioreactors.

[0410] Materials and Methods

[0411] Cell scale-up

[0412] HEK 293 cells were thawed and resuspended in DMEM supplemented with 10% FBS. The cells were centrifuged at 209 x g for 5 min at room temperature, the supernatant was removed, and fresh DMEM + 10% FBS was added. The cells were counted for viable cell density and viability and plated on 2 x T175 cm 2 flasks and incubate at 37.0°C, 5% CO2 for three days until the culture reaches approximately 90% confluence. For each cell passage, the spent medium is removed and the cells are washed with 0.08 mL / cm 2 The flask was washed with PBS (-CaCl2, -MgCl2) and then eluted with TrypLE Select (0.04 mL / cm 2 ) and incubate the flask at 37.0°C, 5% CO2 for 2-3 minutes. Trypsin was added to the flask with DMEM + 10% FBS (0.04 mL / cm 2 ) quenched. Figure 8 Amplification and inoculation were performed according to the diagram in the figure.

[0413] Cell seeding and monitoring

[0414] HEK 293 cells were plated at 8,000 cells / cm 2 and 12,000 cells / cm 2 The target density of the cells was inoculated into the bioreactor in growth medium (high glucose DMEM + 10% Australian FBS + 1:100 penicillin-streptomycin (Pen Strep)) under stirring. The process parameters were set to pH 7.23, 37.0°C, dissolved oxygen (DO) 55%, and a linear velocity of 2 cm / s. 24 hours after inoculation, the cells were recirculated with DMEM growth medium (0.188 mL / cm 2 ) was turned on to a recirculation rate of 12.5 mL / min. Offline pH, metabolites, and nutrients were read using Nova BioFlex for daily samples. On the fourth day after inoculation (12,000 cells / cm 2 ), Day 5 (8,000 cells / cm 2) and on the ninth day, three fibers were removed and lysed with a 1:1:1 v / v solution of PBS, A100, and B100 (ChemoMetec), and total cell nuclei were counted (NucleoCounter NC-200) to monitor culture growth.

[0415] Transfection

[0416] On the fourth day after cell inoculation (12,000 cells / cm 2 ) and day 5 (8,000 cells / cm 2 ), recirculation was stopped, and the cells in each bioreactor were transfected with plasmid DNA and polyethyleneimine (PEI). DNA and PEI were mixed at a ratio of 1:1 mg / mg. Plasmid DNA was transfected with pSMN plasmid at a mass ratio of 1:1.5:2, and pAAV2 / 9 plasmid and pHELP plasmid were added to DMEM- / - medium; high glucose, -CaCl2, -L-glutamine and 0.2 μM filtered and mixed by inversion. PEI was added to DMEM- / - medium and mixed by inversion. PEI was then added to the DNA, mixed by inversion, and incubated at room temperature for 20 minutes (for 8,000 cells / cm 2 (control) and 12,000 cells / cm 2 For the other two 8,000 cells / cm 2 Conditions were defined, with DNA / PEI incubations for 1 and 2 hours. For each of the four conditions, the DNA / PEI complex mixture was used to transfect two bioreactors. The DNA / PEI complex was added to each bioreactor and incubated for two hours at the process parameters. The recirculation loop was reopened two hours after transfection.

[0417] Change medium after transfection

[0418] 24 hours after transfection, all medium in the bioreactor and recirculation loop was removed and replaced with OptiMEM+1:100 Pen Strep (0.132 mL / cm 2 ) was replaced and recirculated for 24 hours at process parameters. 48 hours after transfection, all medium was removed from the recirculation and replaced with OptiMEM+1:100 Pen Strep (12 mL / min) and recirculated at process parameters.

[0419] Harvest

[0420] Eight days after cell seeding (12,000 cells / cm 2 ) and day 9 (8,000 cells / cm 2), Benzonase (100 U / mL) was added, washed with lysis buffer (50 mM HEPES, 1% Tween 20), and incubated for two hours under process parameters. The bioreactor was drained, sucrose saline (500 mM NaCl, 1% w / v sucrose) was added, and mixed by inversion. Under process parameters, the bioreactor was washed with bioreactor rinse buffer (500 mM NaCl, 1% w / v sucrose, 20 mM Tris base, 1% v / v Tween 20, 1 mM MgCl2.6H2O) for about 15 minutes. The bioreactor was drained, and the bioreactor rinse buffer was pooled with the crude batch harvest, mixed by inversion, and sampled for ddPCR determination.

[0421] Depth filtration and tangential flow filtration

[0422] On the eighth day (12,000 cells / cm 2 ) and day 9 (control 8,000 cells / cm 2 ), harvest cells after inoculation of bioreactors, sample and pool the crude lysate for each condition (n=2 bioreactors). Pooled lysate was then passed through a Millistak COHC Pod, 270 cm 2 Filter and Millipak40, 0.45μm, double pore, 200cm 2 The mixture was clarified with a polishing filter (EMD Millipore). Samples were taken after COHC + 0.45 and frozen at -80.0°C. The mixture was then filtered through a tangential flow 2 ultrafiltration modules PLCMKC 0.1m 2 The clarified lysate was concentrated using a filter (EMD Millipore). The final product was diafiltered using at least 6 diafiltration volumes. TFF1 filtered samples were obtained and frozen at -80.0°C. All samples were submitted for AAV2 / 9 titer and host cell protein.

[0423] DS production in bioreactors using plasmids. Data represent duplicate runs for each condition, and the corresponding bioreactor numbers and conditions are shown in Table 14.

[0424] Table 14: Bioreactor numbers and corresponding conditions.

[0425]

[0426] Cell growth: Cells were counted on the day of seeding (day 0) and on day 4 after seeding (12,000 cells / cm 2 ), day 5 (8,000 cells / cm 2) and counted nuclei on day 9. The data indicate that cells in all reactors grew exponentially between day 0 and day 5. Following transfection, nuclei counts on day 9 indicated that bioreactor 221 had a 2.0-fold increase in total nuclei from day 5 to day 9. All other reactors (222 to 228) did not show significant growth between day 5 and day 9. The increase in growth in bioreactor 221 is likely an artifact of the uneven distribution of cells on the individual fibers used for total nuclei counts. Figure 9A -E The metabolite data shown indicate that cells in all bioreactors likely grew similarly.

[0427] pH, nutrients, and metabolites: The glucose consumption trend was the same in all bioreactor cultures, indicating that despite the increased growth curve in bioreactor 221, the cells consumed glucose at a similar rate. 2 The average pH of the bioreactor inoculated with 8,000 cells / cm 2 The pH of the bioreactors inoculated at 220 nmol / L was an average of 7.18. The pH decreased slightly with increasing nutrient metabolism and then increased by day 9 with increasing ammonium ion levels. Lactate increased until day 5 (bioreactors 221, 223, 224, 225, 227) and day 6 (bioreactors 222, 226, 228) before stabilizing near the end of production, indicating the use of lactate as an energy source during this period.

[0428] Production titer

[0429] Viral genomes from harvested material were measured using digital droplet PCR (ddPCR). 2 The bioreactor was inoculated at a titer of 8,000 cells / cm 2 The inoculated control bioreactors (average titer of 4.33E+10 vg / mL (n=2)) were about 1.5 times higher, with an average titer of 6.37E+10 vg / mL (n=2). The titer data showed that inoculation at a higher density, transfection one day earlier, and harvesting supported higher DS production. Compared to the control (4.33E10 vg / mL, n=2) in which DNA / PEI was incubated for 20 minutes, the titer production of DNA / PEI incubated for one hour showed a 1.4-fold decrease in the measured average titer (3.17E10 vg / mL, n=2), while the average titer of the measurement of incubation for two hours showed a 1.6-fold decrease (2.67E10 vg / mL, n=2). The data showed that longer incubation times resulted in a decrease in titer. This may be due to the formation of large complexes between DNA and PEI, which were unable to efficiently transfect HEK293 cells. Figure 10Virus yield / mL and surface area values are given and compared to the yield in a known process as a positive control.

[0430] The viral titer was measured at each step of the clarification and concentration steps as Figure 11 The residual host cell proteins at each step during TFF1 are shown in Figure 12A -B.

[0431] Example 8: Effect of inoculation density on API production

[0432] The effect of inoculation density on DS production was investigated. Four inoculation densities were evaluated, each in duplicate in bioreactors.

[0433] Cell scale-up

[0434] HEK 293 cells were thawed and resuspended in DMEM supplemented with 10% FBS. The cells were centrifuged at 209 x g for 5 min at room temperature, the supernatant was removed, and fresh DMEM + 10% FBS was added. The cells were counted for viable cell density and viability and plated on 2 x T175 cm 2 flasks and incubated at 37.0°C, 5% CO2 for four days until the culture reached approximately 90% confluence. For each cell passage, the spent medium was removed and the cells were washed with 0.08 mL / cm 2 The flask was washed with PBS (-CaCl2, -MgCl2) and then eluted with TrypLE Select (0.04 mL / cm 2 ) and incubate the flask at 37.0°C, 5% CO2 for 2-3 minutes. Trypsin was added to the flask with DMEM + 10% FBS (0.04 mL / cm 2 ) quenched. Figure 13 Amplification and inoculation were performed according to the diagram in the figure.

[0435] Cell seeding and monitoring

[0436] Bioreactors were inoculated with HEK 293 cells at four target densities, each in duplicate: 8,000 cells / cm 2 , 9,350 cells / cm 2 , 10,700 cells / cm 2 and 12,050 cells / cm 2The cells were grown in 700 ml of growth medium (high glucose DMEM + 10% Australian FBS + 1:100 Pen Strep) with stirring. The process parameters were set at pH 7.23, 37.0°C, dissolved oxygen (DO) 55%, and a linear velocity of 2 cm / s. 24 hours after inoculation, recirculation with DMEM growth medium (total volume now 0.188 mL / cm2) was turned on to a recirculation rate of 12.5 mL / min. Offline pH, metabolites, and nutrients were read using a NovaBioProfile 400 for daily samples. On the fifth and ninth days after inoculation, three fibers were removed and lysed with a 1:1:1 v / v solution of PBS, A100, and B100 (ChemoMetec), and total nuclei were counted (NucleoCounter NC-200) to monitor culture growth.

[0437] Transfection

[0438] On the fifth day after cell inoculation, recirculation was stopped and the culture medium in each bioreactor chamber (not a recirculation bottle) was replaced with 600ml DMEM- / - culture medium (high glucose, -CaCl2, -L-glutamine). Each reactor was transfected with plasmid DNA and polyethyleneimine (PEIpro) in a mass ratio of 1:1. Plasmid DNA was mixed with a mass ratio of 1:1.5:2 (pSMN-3.56mg, pAAV2 / 9-5.34mg, and pHELP-7.1mg), added to 300ml DMEM- / - culture medium, filtered at 0.2 μM, and mixed by inversion. PEI (16ml) was added to 300ml DMEM- / - culture medium and mixed by inversion. PEI and DNA mixture were combined, mixed by inversion and incubated at room temperature for 20 minutes. Each 600ml PEI / DNA complex mixture was used to transfect two bioreactors, repeated for each corresponding inoculation density. PEI / DNA complex was added to each bioreactor and incubated for two hours under process parameters. The recirculation loop was returned two hours after transfection (12.5 mL / min).

[0439] Change medium after transfection

[0440] 24 hours after transfection, all medium in the bioreactor and recirculation loop was removed and replaced with OptiMEM (0.132 mL / cm 2 ) and recirculated at process parameters (12.5 mL / min) for 24 hours. After 48 hours of transfection, the medium in the recirculation bottle was replaced with fresh OptiMEM and recirculated at process parameters (12 mL / min).

[0441] Harvest

[0442] On the ninth day after cell inoculation, Benzonase (100 U / mL) was added, washed with lysis buffer (50 mM HEPES, 1% Tween 20), and incubated for two hours under process parameters. The bioreactor was drained and sucrose saline (500 mM NaCl, 1% w / v sucrose) was added and mixed by inversion. Under process parameters, the bioreactor was washed with bioreactor rinse buffer (500 mM NaCl, 1% w / v sucrose, 20 mM Tris base, 1% v / v Tween 20, 1 mM MgCl2.6H2O) for 15 minutes. The bioreactor was drained, and the bioreactor rinse buffer was pooled with crude batch harvest, mixed by inversion, and sampled for ddPCR determination.

[0443] Plasmids were used to produce the drug substance in bioreactors. The bioreactors were inoculated at different densities and transfected and harvested on the same schedule (5 and 9 days after inoculation, respectively). As shown in Table 15 below, the data represent duplicates for each inoculation density:

[0444] Table 15: Starting inoculum density Bioreactor number

[0445] <![CDATA[8,000 cells / cm 2 > 221,222 <![CDATA[9,350 cells / cm 2 > 223,224 <![CDATA[10,700 cells / cm 2 > 225,226 <![CDATA[12,050 cells / cm 2 > 227,228

[0446] Cell Growth: Cells were counted on the day of inoculation (day 0), and nuclei were counted on days 5 and 9 after inoculation. The data showed that cells in all reactors grew exponentially between days 0 and 5. Despite the different starting inoculation densities, nuclei counts did not indicate major differences in cell numbers between groups on day 5. After transfection, nuclei counts on day 9 showed that the total number of nuclei in five reactors (221, 224, 225, 226, 228) doubled from day 5 to day 9. The total number of nuclei in two reactors (222, 223) increased by 1.4 times, as shown in Figure 5. Figure 14A As shown in Figure 2, the total number of nuclei in reactor 227 increased 3.8-fold from day 5 to day 9. This discrepancy is likely an artifact of uneven cell distribution between the individual fibers used for counting. Figure 14B - For the metabolite data shown in E, cells in all reactors likely grew similarly.

[0447] pH, nutrients and metabolites: glucose consumption ( Figure 14B ) The trend was the same in all bioreactor cultures, indicating that despite the different starting inoculum densities, the cultures consumed glucose at similar rates. Offline pH ( Figure 14C ) remained consistent (pH 7.25) in all cultures for the first three days, decreased with increasing nutrient metabolism, and increased after day 8 with increasing ammonium ion levels ( Figure 14E Lactate ( Figure 14D ) increased until day 6 and then stabilized near the end of production, indicating that lactate was utilized as an energy source during this phase. No significant differences in metabolite profiles were observed between reactors inoculated at different starting densities. This may be because the differences in starting inoculation density were small, <1.2-fold.

[0448] Production titer

[0449] Viral genomes were measured from harvested material using digital droplet PCR (ddPCR). The starting inoculation density was 8,000 cells / cm 2 and 10,070 cells / cm 2 The titers of the reactors seeded at 9,350 cells / cm2 were comparable, with an average of 3.99E+10±2.1E+09 vg / mL (n=2) and 3.70E+10±7.4E+09 vg / mL (n=2), respectively. For reasons that cannot be determined, the reactors seeded at 9,350 cells / cm2 showed an average titer measurement of 5.02E+08 vg / mL (n=2), which is approximately 2 logs lower than the average titer of the reactors seeded at the flanking density. This difference is most likely the result of unidentified operator errors during transfection or harvesting rather than a lack of productivity at this seeding density. 2 The inoculated replicate reactors showed a two-fold difference from each other, with one reactor yielding a titer of 3.2E+10 vg / ml, within the range observed for lower inoculation densities, while the second reactor produced a titer of only 1.4E+10 vg / ml. Virus yield / mL and surface area values are shown in Table 1. Figure 15A shown.

[0450] like Figure 15B As shown, the seeding density and DS yield were evaluated at 8E+03 to 10E+03 cells / cm 2 HEK 293 cells seeded within the range showed consistent growth profiles, pH, glucose consumption, lactate, and ammonia production. In addition, comparable titers were produced, indicating that slightly higher seeding densities did not negatively impact yield. In contrast, at 12 x 10 3 cells / cm 2 The reactors inoculated at a higher density of 10 showed more variability between replicates, including lower mean titers compared to the other conditions, suggesting that the method used in this experiment may not be the optimal production method. These results support the use of a 10-μm slurry at 8x 10 3 and 1x 10 4 cells / cm 2 Bioreactor experiments were performed by inoculating cells within a range of densities.

[0451] Example 9: Comparability Assessment

[0452] The primary objective was to assess comparability between the AVXS-101 drug product used in the Phase 1 clinical study (Process A) and the drug product used in the pivotal clinical study (Process B). A secondary objective was to assess manufacturing consistency using Process B by comparing drug product batches 600156 and 600307. Figure 16 Represents the manufacturing process flows for Phase 1 (Process A) and Phase 3 trials (Process B) and the differences between them.

[0453] Comparability assessments were performed using Phase 1 clinical drug product batch NCHAAV9SMN0613 manufactured by Children's National Hospital (NCH) and drug product batch 600156 manufactured by AveXis.

[0454] product

[0455] The following material batches were evaluated, as summarized in Table 16. The evaluation included a direct comparison of the quality attributes of results from Phase 1 clinical drug product batch NCHAAV9SMN0613 using Process A and AVXS-101 drug product batch 600156 using Process B. In addition, an overall evaluation of the release test results from batches 600156 and 600307 was performed to determine the reproducibility and consistency of the scaled-up process.

[0456] Table 16: Comparability of AVXS-101 Drug Product to be Assessed Between Process A (Phase 1) and Process B (Phase 3) and Manufacturing Consistency of Process B

[0457]

[0458] Manufacturing Process Overview

[0459] Figure 17A -B provides a summary of the comparable results.

[0460] Comparability and manufacturing consistency assessment

[0461] The Process A and Process B materials were assessed to be comparable, and the Process B material was assessed to be consistent. The Process B material was also determined to have additional benefits, such as for industrial scale production.

[0462] Test methods

[0463] pH

[0464] pH analysis was performed on batches NCHAAV9SMN0613 (Process A), 600156 (Process B), and 600307 (Process B). The results for both processes ranged from 7.9 to 8.0. This indicates that the pH values of the Process A and Process B materials are comparable, and that the Process B material is consistent.

[0465] Appearance

[0466] A visual inspection of the appearance of batches NCH AAV9SMN0613 (Process A), 600156 (Process B), and 600307 (Process B) was performed. The apparent difference in appearance results between Process A and Process B is due to the different vector concentrations (genomic titers). Batch NCH AAV9SMN0613 has a lower vector concentration than the Process B batch. As a result, batch NCH AAV9SMN0613 is more diluted, resulting in a more transparent and colorless solution, while the colorless to white and slightly opaque observation of the Process B batch is due to approximately 4 times the concentration of viral particles per mL in the solution.

[0467] Taking into account the concentration differences, the appearance of the Process A and Process B materials were assessed to be comparable, and the Process B material was assessed to be consistent.

[0468] Osmolality

[0469] Osmolality was measured by freezing point depression for batches NCHAAV9SMN0613 (Process A), 600156 (Process B), and 600307 (Process B). The results for both processes ranged from 410 to 415 mOSm / kg. This indicates that the osmolality of the Process A and Process B materials is comparable, and that the Process B material is consistent.

[0470] Subvisible particles

[0471] Subvisible particle counts were measured by light obscuration for batches NCHAAV9SMN0613 (Process A), 600156 (Process B), and 600307 (Process B). Results from both methods were well below the recommended limits in the USP monograph for parenteral drug products. This demonstrates that the subvisible particle counts for the Process A and Process B materials are comparable and that the Process B material is consistent.

[0472] Genomic titer

[0473] Genomic titers were measured by ddPCR for batches NCHAAV9SMN0613 (Process A), batch 600156 (Process B), and batch 600307 (Process B). The genomic titers of AVXS-101 batches are expected to fluctuate based on the target concentration in production. The genomic titers produced by Process B (3.7 x 10 13 vg / mL and 4.0x 10 13 vg / ml) than the genome titer of process A (1.1x 10 13 vg / mL) was at least 3-fold higher, thus process B is a better approach for large-scale manufacturing of AVXS-101.

[0474] Infectious titer

[0475] Infectious titers were measured by TCID50 for batches NCHAAV9SMN0613 (Process A), batch 600156 (Process B), and batch 600307 (Process B). Process B (1.3 x 10 10 IU / mL and 6.7x 10 9 IU / ml) than process A (5.9x 10 10 IU / mL) produced an average of 66% higher infectious titers, which may be advantageous, for example, for large-scale manufacturing of rAAVs such as AVXS-101.

[0476] Total protein

[0477] Total protein was measured by micro-BCA for batch NCHAAV9SMN0613 (process A), batch 600156 (process B), and batch 600307 (process B). Normalized to 1.0 x 10 13 vg / mL, with the results for both methods ranging from 167-179 μg / mL. The normalized total protein values indicate that the Process A and Process B materials are comparable, and that the Process B material is consistent.

[0478] Relative potency in vivo

[0479] Batch NCHAAV9SMN0613 was used as the reference material for the assay. Based on the reference standard curve, potency results obtained for batches 600156 and 600307 were 97% and 96%, respectively. These results demonstrate that process A and process B materials are comparable and that process B material is consistent.

[0480] Identity determined by Western blotting

[0481] Batch NCHAAV9SMN0613 (Process A), Batch 600156 (Process B), and Batch 600307 (Process B) were identified by Western blotting. The blot patterns and apparent molecular weight values of the major bands (VP1, VP2, and VP3) were assessed to be comparable for Process A and Process B materials, and the Process B material was also assessed to be consistent.

[0482] AUC empty capsid %

[0483] Empty capsid % measurements were performed by AUC on batch NCHAAV9SMN0613 (Process A), batch 600156 (Process B), and batch 600307 (Process B). The result for batch NCHAAV9SMN0613 (Process A) was 7%. The results for batches 600156 and 600307 (Process B) were 2% and 4%, respectively. By AUC measurement, Process B (2% and 4%) produced approximately two times less empty capsids than Process A (7%). Thus, Method B is capable of producing an improved composition containing a lower concentration of empty capsids.

[0484] Identity and purity determined by SDS-PAGE

[0485] Identity and purity determinations (Process A) were performed by SDS-PAGE on batch NCHAAV9SMN0613, batch 600156 (Process B), and batch 600307 (Process B). The total purity % for both processes was ≥98%, and the band patterns and apparent molecular weights for each of the three capsid proteins were highly consistent. These results indicate that the Process A and Process B materials are comparable, and that the Process B materials are consistent.

[0486] Residual host cell proteins

[0487] Residual host cell protein measurements were performed by ELISA on batch NCHAAV9SMN0613 (Process A), batch 600156 (Process B), and batch 600307 (Process B). For the assay, the results for all batches were < LOQ (8 ng / mL). These results indicate that the Process A and Process B materials are comparable, and that the Process B materials are consistent.

[0488] Residual bovine serum albumin (BSA)

[0489] Residual BSA measurements were performed on batch NCHAAV9SMN0613 (Process A), batch 600156 (Process B), and batch 600307 (Process B). For the assay, the results for all batches were < LOQ (0.50 ng / mL). These results indicate that the Process A and Process B materials are comparable, and that the Process B materials are consistent.

[0490] Residual Benzonase

[0491] Residual Benzonase measurements were performed by ELISA on batch NCHAAV9SMN0613 (Process A), batch 600156 (Process B), and batch 600307 (Process B). For the assay, the results for all batches were < LOQ (0.20 ng / mL). These results indicate that the Process A and Process B materials are comparable, and that the Process B materials are consistent.

[0492] Residual host cell DNA

[0493] Residual host cell DNA was measured by qPCR for batch NCHAAV9SMN0613 (process A), batch 600156 (process B), and batch 600307 (process B). Normalized to 1.0 x 10 13 vg / mL, and the result of process A was 3.7x 10 5 pg / mL, and the results of process B were 0.76x 10 5 pg / mL and 0.68 x 10 5 Thus, process B produces viral vectors with significantly lower residual hcDNA, which may be advantageous, for example, for large-scale manufacturing of rAAVs such as AVXS-101.

[0494] Statistical analysis

[0495] Statistical analysis was performed on the quantitative quality attributes. Pairwise comparisons were performed between the Process A batch (NCHAAV9SMN0613) and each of the Process B batches (600156 and 600307), as listed below. These results are shown in the table below. Figure 18 and 19 shown.

[0496] These studies demonstrate that process B is a superior method for producing viral vectors. Process B consistently produces higher quantities of viral vector (measured by genomic titer and infectious titer), with lower impurities (lower residual hcDNA), and fewer empty capsids.

[0497] Next-generation sequencing

[0498] Next generation sequencing (NGS) was also performed to confirm (determine and / or validate the genomic sequence) identity and to assess the presence of sequence variation (subpopulations) in the AVXS-101 drug product Phase 3 material from Process B. Alignment of the sequence dataset to the sponsor-provided reference sequence (pscSMN) revealed full (100%) breadth and sufficient depth of coverage of the full length of the genome to enable detection of variation. A total of four minor variant sites were noted, however these appear to represent sequencing errors within difficult-to-sequence regions (e.g., the inverted terminal repeats (ITRs) of AAV, which are notoriously difficult to sequence due to their high GC content and palindromic sequences) rather than true variation. See Table 17 for sequencing results.

[0499] Table 17: DNA Sequencing Results for AVXS-101 Phase 3 Batch 600156 from Process B

[0500]

[0501] Phase 1 Batch NCHAAV9SMN0613 Stability Curve

[0502] Batch NCHAAV9SMN0613 was stored at ≤-60°C for 12 months. The batch was analyzed at each time point. No adverse trends were noted. Figure 20 Stability results to date are shown.

[0503] A comparability study of AVXS-101 for Phase 1 clinical studies was completed. The evaluation was performed using Phase 1 clinical drug product batch NCHAAV9SMN0613 manufactured at Children's National Hospital and AVXS-101 drug product batch 600156 manufactured by AveXis. In addition, manufacturing consistency was evaluated using Process B batches 600156 and 600307. For the comparability evaluation (Process A compared to Process B) and manufacturing consistency (Process B batch 600156 compared to 600307), the study evaluated the identity, quality, purity and potency of the AVXS-101 clinical trial material using new and improved processes and analytical methods to enable a robust assessment of comparability and manufacturing consistency.

[0504] Statistical analysis of quantitative quality attributes was performed. Pairwise comparisons were performed between Process A, lot NCHAAV9SMN0613, and Process B, lot 600156. Process B was the superior method, producing higher quantities of purer viral vectors compared to Process A. For example, compared to Process A, Process B produced viral vectors with 48% higher infectious titers, 8% higher genomic titers, 92% fewer subvisible particles larger than 10 μm, 50% fewer subvisible particles larger than 25 μm, 100% fewer empty capsids, and 11% less residual hcDNA. All results were consistent relative to the test limits for each quality attribute.

[0505] Additionally, to establish manufacturing consistency using Process B, a pairwise comparison was performed using Lots 600156 and 600307. The results from this initial pairwise comparison between Process B Lots 600156 and 600307 demonstrated consistency in manufacturing. All results were also consistent relative to the test limits for each quality attribute.

[0506] Based on the evaluation of the results, the resulting quality attributes of Phase 1 clinical drug product batch NCHAAV9SMN0613 using process A and AVXS-101 drug product batch 600156 using process B demonstrated that process B produced higher quantities of viral vector and improved purity, which could be advantageous, for example, for large-scale manufacturing of rAAV. In addition, two batches of material produced from process B (batches 600156 and 600307) were found to be reproducible, further demonstrating manufacturing consistency.

[0507] Example 10: Analytical Ultracentrifugation (AUC) Analysis

[0508] Material from Phase 1 (Process A, batch NCHAAV9SMN0613) and Phase 3 (Process B, batches 600156 and 600307) were analyzed using the AUC method. Figure 21 、 Figure 22 and Figure 23 AUC profiles are shown for NCHAAV9SMN0613, 600156, and 600307, respectively (analyzed in duplicate).

[0509] AUC analysis of each material showed similar sedimentation coefficients for empty and full capsids, with the Stage 1 material (Process A, Batch NCHAAV9SMN0613) exhibiting an elevated empty capsid content (7%) compared to the Stage 3 materials (Process B, Batch 600156 and 600307) which had empty capsid contents of 2% and 4%, respectively. This was attributed to the CsCl gradient ultracentrifugation manufacturing step in Process B being able to more effectively separate empty from full capsids compared to the iodixanol gradient ultracentrifugation manufacturing step employed in Process A.

[0510] In the Phase 1 clinical trial material (NCHAAV9SMN0613) produced at Children's National Hospital and each subsequent production batch at AveXis, AVXS-101 production batches used in clinical and commercial demonstrations consistently showed three visible capsid bands when subjected to a CsCl gradient purification process using ultracentrifugation. Based on the AUC profiles of Phase 1 clinical drug product batch NCHAAV9SMN0613 using Process A and AVXS-101 drug product batch 600156 using Process B, these materials were considered comparable. In addition, the AUC profiles of the two batches of material produced by Process B (Batch 600156 and 600307) were assessed to be consistent.

[0511] Example 11 - Upstream Process

[0512] An intermediate derived from a working cell bank is produced using an upstream process, wherein the upstream process comprises the following steps: (a) culturing cells, (b) transfecting the cultured cells with the three plasmids shown in Figure 1, (c) harvesting amplified viral particles from the cells after a culture period, (d) purifying the viral particles by filtration to remove any intact cells or cell debris, (e) subjecting the eluate from step (d) to tangential flow filtration, and (f) freezing the resulting intermediate preparation of purified viral particles.

[0513] Prior to transfection, cells are expanded in appropriate culture media in flasks or suitable bioreactors (or both). One culture medium is DMEM with 10% FBS, 4.5 g / L glucose, and 4 mM L-glutamine. In one embodiment, adherent cells are initially grown in flasks and then transferred to an iCELLis bioreactor for further adherent cell expansion within the bioreactor.

[0514] After cell expansion, adherent HEK293 cells were transfected with a triple DNA plasmid PEI co-precipitation. The three plasmids used for this transfection were pSMN, pAAV2 / 9, and pHELP. Modified DMEM transfection medium was used instead of the DMEM growth medium used for cell expansion. DMEM transfection medium does not contain FBS, calcium, L-glutamine, and 4.5 g / L glucose. In a large-scale adherent cell bioreactor, the triple DNA plasmid was transfected into adherent HEK293 cells using the PEI co-precipitation method to prepare the scAAV9.CB.SMN vector. The vector plasmid pSMN contains the cDNA of human SMN. The three plasmids used for this transfection were pSMN (222 mg), pAAV2 / 9 (333 mg), and pHELP (444 mg). Before adding the PEI-plasmid co-precipitation, the transfection medium was equilibrated in the bioreactor until the bioreactor temperature was >30°C. The PEI-plasmid coprecipitation process involves adding the plasmid to the transfection medium and filtering it through a 0.2μ filter into a reaction bag. PEI is added to the transfection medium and then added to the reaction bag. The PEI-plasmid reaction is manually mixed to form a homogeneous suspension, and the reaction is allowed to proceed for 15-30 minutes. At the end of the reaction time, the PEI-plasmid coprecipitate is added to the bioreactor. The PEI-plasmid coprecipitate is allowed to mix in the bioreactor for 1-2 hours before recirculation is restarted. The DMEM growth medium is recirculated in the bioreactor for 18-24 hours before the next medium change.

[0515] The rAAV SMN genome (nucleotides 980-3336 of SEQ ID NO: 1) comprises the AAV2 ITR, the chicken β-actin promoter with a cytomegalovirus enhancer, an SV40 intron, the DNA encoding SMN described in GenBank Accession No. NM_000344.2, a polyadenylation signal sequence from bovine growth hormone, and another AAV2 ITR. Conservative nucleotide substitutions in the SMN DNA are also contemplated (e.g., a guanine to adenine change at position 625 of GenBank Accession No. NM_000344.2). The genomes lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the genome. Contemplated SMN polypeptides include, but are not limited to, the human SMN1 polypeptide listed in NCBI Protein Database No. NP_000335.1. The rAAV9 SMN vector is described in Foust et al., Nature Biotechnology 28(3):271-274 (2010), where the sequence of the vector genome insert is shown as nucleotides 980-3336 of SEQ ID NO: 1).

[0516] On the 6th day of bioreactor, 18-24 hours after transfection, bioreactor was emptied and the DMEM recirculation culture medium bag was replaced with 200 liters of fresh OptiMEM transfection culture medium. Bioreactor was refilled with 64 liters and the recirculation in the bioreactor was restarted. On the 7th day, 18-24 hours after the 6th day of changing culture medium, the OptiMEM transfection culture medium (about 135 liters) in the recirculation bag was replaced with a bag of fresh OptiMEM culture medium. In this step, bioreactor was not emptied. The recirculation of culture medium continued until the 9th day of the results.

[0517] After 9 days in the bioreactor, the final pre-harvest sample was extracted from the reactor and the cell lysis process was started. Benzonase was added to the bioreactor to a final concentration of 100 U / mL. After Benzonase was mixed in the reactor, 7.1 liters of lysis solution were added to the reactor. Before the first harvest step, the lysis solution was mixed in the reactor for 2 hours. At the end of the 2-hour lysis, the contents of the bioreactor were transferred to a harvest bag. 8.9 liters of salt sucrose solution (SSS) were added to the harvest bag and mixed for 15 minutes. The SSS solution quenched the Benzonase in the harvest culture medium. The bioreactor was then rinsed with bioreactor rinse buffer. For bioreactor rinsing, 64 liters of bioreactor rinse buffer were added to the bioreactor and mixed for 15 minutes. The rinse solution was then transferred to a common harvest collection bag. Once the rinse solution was added to the collection bag, the contents were mixed for 15 minutes and a batch harvest sample was taken.

[0518] The combined bulk harvest is filtered through a depth filter into a collection bag. Once all the bulk harvest has been filtered, the depth filter is chased with 50 liters of TFF1 diafiltration buffer. The depth filter pool is mixed and sampled. The depth filter pool is then filtered through a 0.45 μm filter to further clarify the bulk harvest material. The 0.45 μm filter is then chased with 6 liters of TFF1 buffer.

[0519] For the TFF1 step, 5.0m 2 A regenerated cellulose membrane cassette with a 300 kDa MW cutoff is rinsed, disinfected with NaOH solution, and equilibrated with TFF1 buffer. The concentration phase of this operation is designed to reduce the volume of the clarified harvest by approximately 10x. Once the target retentate volume is reached, the diafiltration operation begins. The retentate is diafiltered with 6 diafiltration volumes of TFF1 buffer. Once a total permeate flux of 6 diafiltration volumes is reached, the retentate is concentrated again and harvested into a collection bag. The membrane is rinsed twice in succession to maximize product recovery from the TFF system, thereby producing the intermediate drug substance. The TFF1 intermediate is aliquoted into 1 or 2 L sterile PETG bottles in an LFH hood, then frozen on dry ice or in a freezer and transferred to -60°C for storage.

[0520] Table 18: Buffers used in upstream processes

[0521]

[0522]

[0523] Example 12 - Downstream Process

[0524] The intermediate is processed into a filtered drug substance using a downstream process. The downstream process steps include an acidification and clarification step (using filtration), followed by cation exchange chromatography, tangential flow filtration ("TFF2"), CsCl ultracentrifugation and a further tangential flow filtration step ("TFF3") to produce a filtered drug substance in which the purified AAV particles are suspended in a pharmaceutically acceptable carrier. Specifically, the downstream process comprises the following manufacturing steps after 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 full / empty capsid separation, tangential flow filtration (TFF3) for concentration / buffer exchange, filtration of the TFF3 pool material to produce the drug substance, drug substance dilution and filtration to produce the drug product, drug product storage and filling of the drug product into vials.

[0525] Thaw the TFF1 intermediate material and mix gently. At acidic pH, Tween 20 is used to promote bulk flocculation of host cell proteins and DNA. The pH of the TFF1 intermediate pool containing 15% Tween 20 is lowered for CEX chromatography (pH 3.5). The precipitate formed after the pH reduction is removed by filtering the solution through a depth and 0.45 μm filter.

[0526] Tween 20 (36% Tween 20 solution in 20 mM Tris, 1 mM MgCl2, 500 mM NaCl, 1% sucrose m / v, pH 8.1) was slowly added to the TFF1 intermediate solution over 4 hours to achieve a final concentration of 20% Tween 20. After incubation overnight at room temperature (RT), the pH of the Tween 20 containing TFF1 intermediate was lowered to a target pH of 3.5 ± 0.1 by adding approximately 4 g of 1 M glycine pH 2.5 per kg of TFF1 intermediate / Tween spike pool. Once the pH was within the acceptable range, the solution was passed through a Clarisolve POD depth filter in series with a 0.45 μm Opticap XL10 dual-pore filter or a 0.8 / 0.45 μm PES filter, and the filter was then rinsed with CEX buffer A for two times the POD filter retention volume plus one polishing filter retention volume.

[0527] A cation exchange (CEX) capture chromatography step was used to separate the viral capsid from proteins, DNA, and other process impurities. This step utilized a CIMmultus S03-8000 Advanced Composite Column (Sulfonyl) (2 μm pore) column (8.0 L) operated using an automated process chromatography system. The buffers and solutions are shown in the table below:

[0528] Table 19: Buffers and solutions for one CEX cycle

[0529]

[0530] The CEX column loading was determined by the protein content of the clarified, acidified TFF1 intermediate. The protein loading of the CEX column was set to 70% of the maximum column capacity.

[0531] Manually collect the elution peak starting at the sharp rise in OD280. The OD280 rises when the conductivity is between 80-85 mS / cm. An appropriate volume of CEX eluate (product) is approximately 20 liters or 2.5 CV (column volumes). Collect the CEX eluate in two fractions. The first fraction begins at the sharp rise in OD280 and is collected for 1.5 CV. The second fraction begins immediately after the first and is collected for 1.0 CV. Neutralize both fractions to pH 8.0 ± 0.30 with pH 9.0 neutralization buffer.

[0532] The TFF2 step concentrates, removes protein impurities, and exchanges the buffer for the appropriate buffer for the CsCl ultracentrifugation step. 2 (2 CEX cycles) or 0.2m 2 (1 CEX cycle) 300k MWCO regenerated cellulose membrane was used in combination.

[0533] The concentration phase of this operation reduces the volume of the CEX eluate. Once the target retentate volume is reached, diafiltration is initiated in discontinuous TFF mode (batch mode). The retentate is diluted 2X and diafiltered with 8 diafiltration volumes of TFF2 NaCl diafiltration buffer, followed by diafiltration in discontinuous TFF mode with 8 diafiltration volumes of TFF2 CsCl diafiltration buffer. Once the CsCl diafiltration is complete, the retentate is concentrated to a specified volume, depending on the system hold-up volume. Two consecutive rinses of the membrane are performed to maximize product recovery from the TFF2 system.

[0534] The retentate feed rate was set to 5 L / m 2 / min(500mL / min / 0.1m 2 The permeate flow rate was controlled by a clamp on the permeate tube to maintain the permeate flow rate at 20% of the retentate feed rate.

[0535] Table 20: Buffers for TFF2

[0536]

[0537] Ultracentrifugation can be used to remove empty capsids from full capsids using cesium chloride gradient ultracentrifugation. The CsCl ultracentrifugation step uses an automated Optima XPN 100 ultracentrifugation system or an equivalent system equipped with a 50.2Ti rotor or equivalent rotor. The filtered material purified by TFF2 is slowly added to the ultracentrifuge tube along the inside of the tube wall without introducing bubbles into the solution. The filled tube is sealed with a handheld heat sealer and centrifuged at 302,000g (50,000 rpm in a 50.2Ti rotor) at 20°C for 17 hours. After the centrifugation step is complete, the test tube is removed from the ultracentrifuge and placed in a biosafety cabinet. The tube containing the product is mounted on a ring stand above the waste container. A light is placed under the test tube and the empty capsid band (band A is the highest band), the full capsid double band (band B and band C, the upper and lower bands of the double band), and the lowest band below the double band are marked on the test tube. Bands B, C, and D were removed by an 18G needle connected to a 30 mL syringe, which was inserted just below band D to the middle of the tube. The collected material was transferred to a sterile 1 L PETG bottle. The material from all centrifuge tubes was pooled into a sterile 1 L PETG bottle to create an ultracentrifugation (UC) pool. The buffers for the CsCl ultracentrifugation step are listed in the table below:

[0538] Table 21: Buffers for CsCl ultracentrifugation

[0539]

[0540] The TFF3 step removes CsCl and concentrates the whole support using the final formulation buffer. 2 The TFF3 process is designed to reduce the concentration of residual CsCl and the volume of the UC pool. Once the target retentate volume is reached, diafiltration is initiated. The retentate is diafiltered with 10 diafiltration volumes of TFF3 buffer. Once diafiltration is complete, the concentrated retentate is passed through a 0.2 μm Pall The tubes were transferred through an EKV sterile grade filter (MiniKleenpak) into a secondary conical tube.

[0541] The membrane is rinsed continuously to recover the vector from the TFF3 system. TFF3 buffer is added to the primary conical tube that previously contained the TFF3 retentate. This material is recirculated through the cellulose membrane. After recirculation, the rinse is passed through a 0.2 μm Pall Transfer to a secondary conical tube using an EKV sterile grade filter (Mini Kleenpak). Combine the TFF3 concentrate and the fraction pool to obtain a final vector concentration ≥ 4.5 × 10 13 vg / mL of drug substance (pooled TFF3 retentate + two rinses).

[0542] Perform continuous rinsing of the membrane to maximize product recovery from the TFF3 system. Add TFF3 buffer to the primary conical tube that previously contained the TFF3 retentate and initial rinse material. This material is recirculated through the cellulose membrane. The secondary rinse is passed through a 0.2 μm Pall The solution was transferred to a secondary conical tube using an EKV sterilizing grade filter (Mini Kleenpak) until a defined weight was reached in the secondary conical tube. The final concentrated solution was designated as the drug substance (DS).

[0543] Table 22: Buffers for TFF3

[0544]

[0545] Using Pall Filter the DS through an EKV sterilizing grade filter (Mini Kleenpak) into a sterile 1 L glass bottle (using sterile disposable components). Before filtering the TFF3 pool, rinse the filter by passing TFF3 buffer through the filter using a peristaltic pump and discarding it into a waste rinse bag. The drug substance (DS) is then filtered through the rinsed filter using a peristaltic pump and collected into a 1 L sterile glass bottle. Based on 5 x 10 13 vg / mL of DS, TFF3 buffer was added to the secondary conical tube containing DS and passed through the filter to prepare a diluted drug product ("DP") to 3.5 x 10 13 Target concentration in vg / mL.

[0546] TFF3 buffer used for filter washing and DS dilution to prepare DP was composed of the following formulation.

[0547] Table 23: Drug Product Plant Operations - Buffer Composition

[0548]

[0549] DP was filled into 5 mL sterile, ready-to-use Crystal Zenith (CZ) vials, stoppered with sterile, ready-to-use stoppers, and sealed with sterile, ready-to-use seals.

[0550] Example 13 - Potency Determination

[0551] The relative efficacy of the drug product is measured using a quantitative in vivo assay. The assay uses an established mouse model of SMA disease. Breeding pairs of the SMAΔ7 mouse strain (Jackson Laboratories, #005025) are phenotypically normal, but approximately 25% of their offspring are homozygous for the targeted SMN gene mutation and display an SMA-like phenotype. By day 5, they develop signs of muscle weakness, and over the next week, they develop an abnormal gait and a tendency to fall. Jackson Laboratories reports that the average survival time of animals with an SMA-like phenotype is approximately 15±2 days. Pilot studies have shown that the median survival time of untreated animals with an SMA-like phenotype is 16.3 days (geometric mean; n=3 studies; 10 mice per study).

[0552] The bioactive drug product, administered by intravenous (IV) infusion, produces an increase in survival time as a function of dose (vg / kg). Drug product potency is measured relative to a reference material (previous batch of vector). The potency of the drug product and reference material (vector genomes / mL; vg / mL) is determined by droplet digital polymerase chain reaction (ddPCR). The vector is diluted in saline to achieve each of three specific dose levels, which will be administered to mice with an SMA-like phenotype.

[0553] If the assay passes suitability, the assay result is considered acceptable. Assay suitability consists of the following:

[0554] 4. Acceptance limit for negative control samples (15 ± 2 days, median survival)

[0555] 5. Acceptance limit for positive control samples (>40 days, median survival)

[0556] 6. Acceptance Limits for Reference Standard Median Survival Dose-Response Curves

[0557] In this study, a previous batch of vehicle (hereinafter referred to as the previous batch) was used to determine the linear correlation between the median survival (days) of SMAΔ7 mice when the drug product was administered at five different dose levels, including a dose of 0 (zero) using 0.9% saline solution (untreated group). Table 24 shows the calibration curve using the previous batch reference batch.

[0558] Table 24: Calibration curves using five dose levels of AVXS-101

[0559]

[0560] * Not Determined (ND) - Median survival (days) not reached as of August 24, 2017

[0561] The data from this study showed that the linear range of the median survival dose-response curve was 0-7.4 vg / kg. The relative potency of drug product batch 816836 was established by comparing the linear regression curve of the previous batch of reference standard with the linear regression curve of drug product batch 816836. This was done by using the ratio of the y-intercept and slope of each linear regression line (i.e., reference standard and test article). The percent relative potency calculation is described by the following equation (1):

[0562] %RP = [(y-intercept of test product / slope) ÷ (y-intercept of reference standard / slope)] x 100 (1)

[0563] The NCH0613 batch used in the Phase 1 clinical trial was used as the reference standard batch and was assigned a potency of 100%.

[0564] The above data established preliminary assay acceptance criteria using the linear portion of the median survival dose-response curve to determine the relative potency of the drug product clinical batches as follows:

[0565] Median survival (days) dose-response curves were generated using 5 dose (vg / kg) levels

[0566] Doses 0, 1.2E+13, and 7.4E+13 were selected to run a linear dose-response model.

[0567] • The limit of suitability for saline (untreated group) was a median survival of 13 to 17 days.

[0568] The applicability limits of the reference standard samples are:

[0569] οSlope ≥ 2.0E-13

[0570] οy intercept ≥ 16.00 (days)

[0571] οy intercept / slope ratio ≥ 8.5E+13

[0572] οR2≥0.7

[0573] The efficacy of SMA therapies (including drug products) was demonstrated using a Δ7 mouse model. The median survival was 15 days ± 2 days, and untreated or saline-treated control animals provided a reliable baseline control from which product efficacy could be measured as an increase in median survival. The development work for the drug product identified three (3) doses (excluding the vehicle-treated dose) that were determined by genomic titer using droplet digital PCR (ddPCR) and that affected the survival of the mouse model in a linear manner when the administered dose (vg / kg) was log-transformed and plotted against the median survival (days) of treated SMAΔ7 newborn mice. The low, medium, and high titer standards are shown in Table 26 for the standard titers (vg / mL). In addition, TFF buffer (vehicle) solution was used for the zero (0) calibration curve point and negative control. Doses that showed a survival of ≥ 40 days (doses that showed greater than a doubling of the median survival) were also included as positive controls.

[0574] Table 25: Target doses

[0575]

[0576]

[0577] Dosage Solution Formulation (See Table 26 for an example of a dilution regimen.

[0578] Negative control - Use 0.9% saline as a negative control

[0579] Positive control - saline at 1.5 x 10 14 vg / kg to prepare the test batches.

[0580] Reference Standard Solutions - Reference standard batches were prepared using saline solution at the three concentrations described in Table 25.

[0581] Table 26: Dilution scheme for reference standards and test samples (example)

[0582]

[0583] Test Article Preparation - Dilute the test article using saline solution. Calculate the dilution to produce the test dose (vg / kg) shown in Table 26 for each mouse in a final total volume of 50 μl. Perform the dilution on 10 mice simultaneously, using an additional volume as a positive control, with the goal of increasing the minimum lifespan of treated mice to a median survival of ≥40 days (days).

[0584] Acceptance limit for control samples

[0585] Negative control group (untreated mice) - The acceptance limit for the negative control group was that the SMAΔ7 mice reached a median survival of 15 ± 2 days. In addition, any mice that expired within ≤ 10 days were excluded from the analysis. If more than 7 mice were used in a group, up to 2 mice could be excluded due to expiration within ≤ 10 days.

[0586] Positive Control (Group Treated at the Target Clinical Dose) - The acceptance limit for the positive control group was a minimum lifespan of treated mice with a median survival of ≥40 days. In addition, any mice that expired within ≤10 days were excluded from analysis. If more than 7 mice were used in a group, up to 2 mice could be excluded due to expiration within ≤10 days.

[0587] Acceptance limits of the reference standard dose-response curve

[0588] Assay suitability criteria will be determined with reference to a standard linear dose response curve that plots median survival (days) versus administered dose (vg / mL). Assay suitability criteria are shown in Table 27.

[0589] Table 27: Assay Suitability Criteria (Reference Standard Linear Dose-Response Curve)

[0590]

[0591] Y-Intercept / Slope Ratio - Linear regression curves measuring median survival (days) for the reference standard and the test article versus the administered dose (vg / kg) were calculated. The ratio of the y-intercept to the slope of each linear regression was calculated.

[0592] Report results

[0593] Qualitative Reporting of Relative Potency Results—Evaluate the assay suitability criteria for each assay before determining a single-point median survival (days) reading of the positive control material ≥ 40 days. If the median survival of the positive control is ≥ 40 days, the test article may be disposed of if the following criteria are met.

[0594] Quantitative Reporting of Relative Potency Results—Evaluate the assay suitability criteria for each assay before quantitative determination of the relative potency of the test article. Once the positive control has reached ≥40 days and has reached the representative upper standard dose of 7.5 x 10 13 The relative efficacy of the test article can be reported based on a median survival of 31 ± 3 days for the group of mice receiving vg / kg. The relative efficacy percentage (%RP) of the test article will be calculated using the y-intercept and slope of the linear regression of the median survival (days) dose-response as follows:

[0595] %RP = 100% * [(test product y-intercept / slope) ÷ (reference standard y-intercept / slope)]

[0596] Example 14 - Single-Dose Gene Replacement Therapy Treatment of Spinal Muscular Atrophy: Dosing Study

[0597] Spinal muscular atrophy (SMA) is a severe monogenic childhood disorder caused by the absence or dysfunction of the gene encoding survival motor neuron 1 (SMN1). The disease affects approximately 1 in 10,000 live births, with a carrier frequency of 1 in 54. SMA is characterized by degeneration and loss of lower motor neurons, leading to muscle atrophy. The disease is divided into four subtypes (1 to 4) based on age of onset and milestone attainment. SMA type 1 (SMA1) is the most severe form and the most common genetic cause of infant mortality. There are two forms of SMN; SMN1 is the primary gene responsible for producing functional SMN protein. SMN2 preferentially excludes exon 7 during splicing, resulting in only a smaller fraction of functional SMN protein compared to SMN1. Therefore, SMN2 copy number modifies the disease phenotype, and the presence of two copies of SMN2 is associated with SMA1.

[0598] Infants with biallelic deletion of SMN1 and two copies of SMN2 have a 97% risk of developing SMA1.

[0599] Recent studies of the natural history of SMA1 (historical cohorts) have shown that the median age of symptom onset in infants with the disease is 1.2 months (range, 0 to 4 months), and the disease is characterized by hypotonia, severe weakness beginning in early infancy, and the inability to sit unsupported. Among infants with SMA1 who have two copies of SMN2, the median age at death or the need for noninvasive ventilation for at least 16 hours per day for at least 14 consecutive days (considered equivalent to permanent ventilation) is 10.5 months. In one cohort of affected children, only 25% survived without permanent ventilatory support at 13.6 months, and 8% survived without such support by 20 months. Another prospective, multicenter historical study involving patients with two copies of SMN2, sponsored by the National Institutes of Health (NeuroNEXT), showed a median tracheostomy-free survival of 8 months (95% confidence interval, 6 to 17 months). All patients with SMA1 have a steep postnatal decline in respiratory and swallowing function and eventually require instrumental nutritional support (via a nasogastric or gastrostomy tube) to maintain adequate nutrition and reduce the risk of respiratory problems associated with aspiration. For patients with SMA1 who develop symptoms at 3 months of age, most require feeding support by 12 months of age.

[0600] SMA1 patients also fail to achieve major milestones in motor function and have functional decline, as measured on the CHOPINTEND (Children's Hospital of Philadelphia Infant Neuromuscular Disability Test) scale, which ranges from 0 to 64, with higher scores indicating better motor function. This is a tool that is sensitive to small changes in motor function, such as anti-gravity movement of a limb. In a historical analysis of 34 SMA1 patients, all but one did not achieve a score of at least 40 after 6 months of age. In the NeuroNEXT cohort, the average CHOPINTEND score decreased by 10.7 points from 6 to 12 months of age.

[0601] Therapeutic strategies to increase SMN protein levels in motor neurons have focused on enhancing the effectiveness of SMN2. One approach involves central nervous system delivery of nusindromic acid (Ionis Pharmaceuticals / Biogen), an antisense oligonucleotide developed to inhibit exon 7 splicing of SMN2. This drug has been shown to improve weakness in a severe mouse model of SMA and increase the median lifespan of affected mice from 16 to 25 days. In December 2016, nusindromic acid received Food and Drug Administration approval for the treatment of SMA. The drug is administered by repeated intrathecal injections after four loading doses within the first two months of life.

[0602] A potential alternative treatment for SMA1 is a gene therapy given as a one-time intravenous administration that delivers a single copy of SMN in a self-complementary adeno-associated virus serotype 9 (scAAV9). (The coding region of this recombinant virus forms an intramolecular double-stranded DNA [or self-complementary] template.) This approach induced SMN expression in motor neurons and peripheral tissues, which counteracted the effects of SMA in mouse models and was effective at low doses (6.7 x 10 13 vg / kg body weight) extended the mean survival in this model from 15 days to 28.5 days, and at higher doses (2.0 x 10 14 and 3.3x 10 14 vg / kg) carrier to more than 250 days.

[0603] In addition to crossing the blood-brain barrier and targeting central nervous system neurons in all regions of the spinal cord, systemic administration of AAV9-mediated gene therapy may be advantageous because SMN protein is ubiquitously expressed and SMA1 affects multiple systems (e.g., the autonomic and enteric nervous systems, the cardiovascular system, and the pancreas) and many cell types (e.g., the heart, pancreas, and skeletal muscle). The self-complementary nature of the vector, combined with the hybrid cytomegalovirus enhancer-chicken β-actin promoter, enables rapid and sustained expression of SMN. In April 2014, we initiated a gene replacement therapy study involving infants with SMA1 who received a one-time dose of scAAV9 (scAAV9.CB.hSMN) (AVXS-101), which delivered the human survival motor neuron gene (hSMN) under the control of the chicken β-actin promoter.

[0604] method

[0605] Patients and study procedures : For the purposes of the study, all patients had a genetically confirmed diagnosis of SMA1, homozygous SMN1 exon 7 deletion, and two copies of SMN2. Patients carrying the c.859G→C disease modifier in SMN2 exon 7 were excluded. Selected patients had onset of disease from birth to 6 months of age, characterized by clinically assessed hypotonia with delayed motor skills, poor head control, rounded shoulder posture, and joint hypermobility. Patients with active viral infection (including positive serology for HIV or hepatitis B or C) or concomitant diseases that posed unnecessary risk for gene transfer were excluded from the study. Patients requiring invasive ventilatory support (positive pressure tracheostomy) or with a pulse oximetry saturation <95% at the screening visit were also excluded.

[0606] Patients were enrolled in two cohorts based on the dose of gene therapy administered. Patients in cohort 1 received a low dose (6.7 x 10 13 vg / kg) and were enrolled over a 5-month period; those in cohort 2 received a high dose (2.0 x 10 14 vg / kg) and were enrolled over the course of one year. On day 30 after dosing, an IFN-γ ELISpot assay performed on patient 1 in cohort 1 detected a T cell response and showed that every 10 6 A sudden spike in >50 spot-forming cells (SFC) directed against AAV9 capsids in peripheral blood mononuclear cells (PBMCs) (normal is 10 6<50 SFCs in PBMCs). Prednisolone was started at 2 mg / kg and maintained for 35 days until T cell responses and serum transaminases decreased. As a result, the experimental protocol was modified, and patients 2 to 15 received oral prednisolone at a dose of 1 mg / kg / day for approximately 30 days, starting 24 hours before administration of the gene vector. Treatment with prednisolone was continued until AST and ALT enzymes decreased to levels below 120 IU / L and T cell responses decreased to 100 SFCs / 10 6 If the number of PBMCs is less than 50, prednisolone can be gradually reduced based on clinical judgment.

[0607] The vector was delivered in normal saline (approximately 10 to 20 ml / kg) which was infused intravenously over a period of approximately 60 minutes. At the time of enrollment, some patients required enteral feeding via a gastrostomy tube or nasogastric tube, the choice of which was based on the preference of the parents or attending physician. Once enrolled in the study, all patients requiring nutritional support received a gastrostomy tube, and these tubes were not removed during the study.

[0608] ending The primary outcome was safety, defined as any grade 3 or higher treatment-related adverse event. Secondary outcomes were time to death or the need for permanent ventilatory assistance. The latter was defined as at least 16 hours of ventilatory assistance per day for at least 14 days in the absence of acute, reversible illness or perioperative conditions. Exploratory outcomes included motor milestone achievement (specifically, unassisted sitting) and the CHOP INTEND score.

[0609] Maintaining a score of 40 or above using the CHOP INTEND scale has been considered clinically meaningful in SMA. Unassisted sitting was assessed and classified according to the following criteria: unassisted sitting for at least 5 seconds according to item 22 of the Bayley Scales of Infant Development's gross motor subtest ("unassisted sitting"); unassisted sitting for at least 10 seconds according to World Health Organization (WHO) criteria ("unassisted sitting according to WHO criteria"); and unassisted sitting for at least 30 seconds according to item 26 of the aforementioned Bayley scale ("independent functional sitting"). Major motor milestones were confirmed by reviewing the patient's video recordings using the Ability Captured Through Interactive Video Evaluation-mini (ACTIVE-mini) by an independent reviewer. Compound muscle action potentials (CMAPs) were recorded from surface electrodes at baseline and every 6 months after infusion. Muscle pathology was quantified by electrical impedance myography (EIM).

[0610] Statistical analysisSafety analyses were performed on all patients included in the primary analysis of survival (as defined above and in the protocol) and in the analysis of change from baseline to 1 and 3 months on the CHOP INTEND scale. This change from baseline to each study visit was analyzed using a mixed-effects model for repeated measures. The mixed model included fixed effects for cohort and visit, as well as a covariate for baseline score. Milestone achievement and nutritional and ventilatory support were analyzed in Cohort 2. Statistical analyses were performed using SAS software, version 9.4. All comparisons with the historical cohort are descriptive only.

[0611] result

[0612] patient Of the 16 patients screened, one was excluded due to a persistently elevated anti-AAV9 antibody titer (>1:50). Of the 15 patients enrolled in the study, 3 were enrolled in the low-dose cohort 1 and 12 were enrolled in the high-dose cohort 2. The mean age at treatment was 6.3 months (range, 5.9 to 7.2) in cohort 1 and 3.4 months (range, 0.9 to 7.9) in cohort 2 (Table 28).

[0613] Table 28: Demographic and clinical characteristics of 15 patients

[0614]

[0615] Survival and permanent ventilation By the end of the study, all patients were at least 20 months old and no longer required permanent mechanical ventilation; the median age at the last pulmonary assessment was 30.8 months in Cohort 1 and 25.7 months in Cohort 2. In contrast, only 8% of patients in the historical cohort did not require permanent mechanical ventilation. At 29 months of age, one patient in Cohort 1 required permanent ventilation for hypersalivation. After salivary gland ligation, the need for noninvasive ventilation decreased by 25% to 15 hours per day.

[0616] Motor function assessment All patients in Cohorts 1 and 2 achieved increases from baseline in their scores on the CHOP INTEND scale, and these changes were maintained during the study. Patients in Cohort 2 achieved a mean increase of 9.8 points at 1 month and 15.4 points at 3 months (P < .001 for both comparisons); 11 patients achieved and maintained a score of 40 or higher. At the study cutoff of August 7, 2017, patients in Cohort 1 had achieved a mean increase of 7.7 points from a mean baseline of 16.3, and patients in Cohort 2 had achieved a mean increase of 24.6 points from a mean baseline of 28.2.

[0617] Movement milestones in Cohort 2Of the 12 patients in Cohort 2, 11 were able to sit unassisted for at least 5 seconds, 10 for at least 10 seconds, and 9 for at least 30 seconds (Table 31). Eleven patients achieved head control, nine were able to roll over, and two were able to crawl, pull to a stand, stand independently, and walk independently. Eleven patients achieved speech. No patient in the historical cohort achieved any of these motor milestones, and speech was rarely achieved.

[0618] Table 29: Event-free survival and motor and other milestones for the 12 patients in Cohort 2.

[0619]

[0620] Pulmonary and nutritional status of cohort 2 : Of the 12 patients in cohort 2, 10 did not require noninvasive ventilation at baseline, and 7 did not require ventilatory assistance at the last follow-up visit (Table 29). At baseline, 7 patients did not require enteral feeding, including 1 who subsequently required a gastrostomy tube placement after gene replacement therapy, which may be related to scoliosis surgery. Of the 5 patients who were receiving enteral feeding before gene replacement therapy, 11 of the 12 patients achieved or maintained independent swallowing ability at the last follow-up, and 4 were able to be fed orally.

[0621] Security By the end of the study, a total of 56 serious adverse events were observed in 13 patients across the two cohorts. Of these, two events were determined by the investigators to be treatment-related Grade 4 events based on laboratory values according to the Common Terminology Criteria for Adverse Events (Table 30). Patient 1 in cohort 1 had elevated serum aminotransferase levels (31 times the upper limit of the normal range for alanine aminotransferase (ALT) and 14 times the upper limit of the normal range for aspartate aminotransferase (AST), without other liver function abnormalities (i.e., total and indirect bilirubin and alkaline phosphatase), and without clinical manifestations. As described above, these elevations were attenuated by prednisolone treatment, which was subsequently administered to the remaining patients. One patient in cohort 2 required additional prednisolone to attenuate elevated serum ALT and AST levels (ALT was 35 times the upper limit of the normal range and AST was 37 times the upper limit of the normal range). Of the 241 nonserious adverse events, 3 were considered to be treatment-related, including asymptomatic elevations in serum aminotransferase levels (ALT and AST, both less than 10 times the upper limit of the normal range) in 2 patients, which resolved without additional prednisolone treatment (Table 0). There were no other abnormalities in liver function tests. Of the 15 patients, 14 had respiratory disorders, which frequently result in death or require tracheostomy in children with SMA1.

[0622] Table 30: Adverse events.

[0623]

[0624]

[0625] A single intravenous infusion of an adeno-associated viral vector containing DNA encoding SMN in patients with SMA1 resulted in longer survival than in historical cohorts of the disease. All 15 patients exceeded the previously reported median survival age of 10.5 months free of permanent ventilation for SMA1 patients with two copies of SMN2. All patients also exceeded the 20-month benchmark, at which point only 8% of patients with this disease survive without permanent ventilation. All but one of the 12 patients in Cohort 2 achieved motor milestones not reported in the historical cohort. The gains in motor function were clinically meaningful, as reflected by improvements in feeding (fingers), sitting, and speaking. The majority of patients who did not require supportive care at enrollment were free of nutritional support (6 of 7 patients) and ventilatory support (7 of 10 patients) at their last follow-up visit. In both cohorts, patients' scores on the CHOP INTEND scale increased from baseline. Within the first month of Cohort 2, the average increase was 9.8 points, while in the historical cohort of the NeuroNEXT study, the average decrease was more than 10 points between months 6 and 12.

[0626] Preclinical studies of SMN gene replacement therapy in the SMNΔ7 mouse model have shown that early treatment, presumably while motor neurons are still intact, improves survival and motor function in mice. The clinical findings in our early treatment studies reflect the trends in preclinical studies. Two patients were able to crawl, stand, and walk unsupported after early treatment. Both patients had a family history of SMA, which may have facilitated early diagnosis. Although motor function continued to improve in all patients in both cohorts of our study, preclinical and clinical data suggest that early treatment and newborn screening for SMA are beneficial.

[0627] Serious adverse events due to AAV gene replacement therapy were limited to elevations in serum aminotransferase levels approximately 3 weeks after treatment in two patients without other liver enzyme abnormalities; the elevations in two other patients did not reach the cutoff for the definition of a serious adverse event (ie, greater than 10 times the normal range). Treatment with prednisolone attenuated the elevations in liver enzymes. One patient was not screened due to the presence of anti-AAV9 antibodies, which is consistent with population-based studies showing low anti-AAV9 seropositivity in children and young adults and increased anti-AAV9 seropositivity in individuals older than 40 years. However, the presence of viral antibodies may be a limitation of AAV gene replacement therapy.

[0628] This study used a single-group design with a historical cohort as a control, which was one of the limited options when the natural history of the disease was well characterized and fatal. To enroll a homogenous sample similar to published historical studies, we restricted the enrollment to include only symptomatic SMA1 patients with biallelic SMN1 mutations and two copies of SMN2 and excluded patients with the c.859G→C genetic modifier in exon 7 of SMN2, as this genetic modifier predicts a milder disease phenotype. However, such gene replacement therapy need not be limited to symptomatic patients or those with a specific genomic subtype.

[0629] In conclusion, a single intravenous infusion of a high-dose adeno-associated viral vector containing DNA encoding SMN in patients with SMA1 resulted in prolonged survival, improved motor function, and increased scores on the CHOP INTEND scale to levels not previously observed in this disease. These improvements resulted in a lower percentage of patients requiring supportive care than in historical studies. No reduction in motor function or clinical decline was reported during up to 2 years of follow-up. Several patients had transient and asymptomatic elevations in aminotransferase levels. Further studies are needed to assess the long-term safety and durability of gene replacement therapy in patients with SMA1.

[0630] Example 15 - Pharmacokinetics of scAAV9.CB.hSMN

[0631] Conventional clinical pharmacokinetic studies are not applicable to gene replacement therapy products. However, scAAV9.CB.hSMN vector shedding studies, which assess the amount of vector eliminated from the body through body fluids and excretion, are a measurement method that can be used instead of conventional pharmacokinetic studies for gene replacement therapies.

[0632] During the clinical study, vector shedding after scAAV9.CB.hSMN infusion was investigated at multiple time points. Saliva, urine, and stool samples were collected weekly until day 30, then monthly until month 12, and every 3 months thereafter. Samples from five patients were analyzed for scAAV9.CB.hSMN vector shedding by droplet digital polymerase chain reaction until the month 18 visit. 14 A therapeutic dose of 500 vg / kg was administered to 5 patients for scAAV9.CB.hSMN vector shedding analysis.

[0633] scAAV9.CB.hSMN was detected in shedding samples after infusion. Concentrations of scAAV9.CB.hSMN in urine and saliva were 0.1% to 0.01% of the initial in vivo concentration on day 1 after infusion, after which concentrations dropped below the limit of quantification. In stool, levels of 10% to 30% of the initial in vivo concentration were detectable on day 1 after infusion. One patient showed a peak concentration in stool on day 14 after infusion, which was 280% of the initial in vivo concentration. In contrast, three patients with available data had in vivo concentrations <1% of the initial concentration on day 14 after infusion, and the concentrations decreased by approximately 4 logs (10,000-fold) over 30 days after infusion. Overall, scAAV9.CB.hSMN was primarily cleared from the body in the stool, and by day 60 after infusion, it was below the limit of quantification in the stool.

[0634] Example 16 - Non-clinical toxicology testing

[0635] Animal pharmacology: After infusion of the scAAV9.CB.hSMN vector in the Δ7 SMA disease mouse model (SMNΔ7 mice), body weight increased, righting behavior improved, survival was significantly prolonged in a dose-dependent manner, and SMA-related heart defects were restored to normal compared with untreated SMNΔ7 mice.

[0636] Animal Toxicology: Following intravenous infusion in mice, the vector and transgene were widely distributed, with highest expression in the heart and liver, and also substantial expression in the brain and spinal cord. In the pivotal 3-month Good Laboratory Practice (GLP)-compliant mouse toxicology study, the primary target organs of toxicity were the heart and liver. scAAV9.CB.hSMN vector-related findings in the ventricles included dose-related inflammation / edema and fibrosis, and in the atria included inflammation and thrombosis. Liver findings included hepatocellular hypertrophy, Kupffer cell activation, and scattered hepatocellular necrosis. In mice, no adverse effect level (NoAEL) was established for scAAV9.CB.hSMN vector-related cardiac and liver findings, and the maximum tolerated dose was defined as 1.5 x 10 14 vg / kg, which...

Claims

1. A pharmaceutical composition comprising a.1-8×10 13 AAV9 viral vector genomes / mL (vg / mL); b. Less than about 7% empty viral capsids; c. Less than about 100 ng / mL host cell protein / 1×10 13 vg / mL; d. Less than about 5×10 6 pg / mL residual host cell DNA / 1×10 13 vg / mL; And wherein said 1-8×10 13 At least about 80% of the AAV9 viral vector genomes / mL are functional.

2. The composition of claim 1, wherein the AAV9 viral vector comprises a polynucleotide encoding a survival motor neuron (SMN) protein, a polynucleotide encoding MeCpG 2, or a polynucleotide encoding a SOD1 shRNA.

3. The composition of claim 1, wherein the AAV9 viral vector comprises 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.

4. The composition of claim 2, wherein the polynucleotide encodes the SMN protein of SEQ ID NO:

2.

5. The composition of claim 1, wherein the AAV9 viral vector comprises SEQ ID NO:

1.

6. The composition of claim 1, comprising 1.7–2.3×10 13 AAV9 vg / mL.

7. The composition of claim 1, wherein the composition is an aqueous pharmaceutical product.

8. The composition of claim 7, wherein the aqueous pharmaceutical formulation comprises Tris buffer, MgCl2, NaCl, and poloxamer 188.

9. The composition of claim 7, wherein the aqueous pharmaceutical formulation does not contain a preservative.

10. The composition of claim 8, wherein (i) the concentration of Tris buffer is about 10-30 nM; (ii) the pH of the formulation is from about 7.7 to about 8.3; (iii) MgCl2 concentration is about 0.5-1.5 mM; (iv) NaCl concentration is about 100-300 mM; (v) the formulation comprises about 0.005% w / v poloxamer 188; or (vi) The aqueous pharmaceutical formulation has an osmolarity of 390-430 mOsm / kg.

11. A method of treating spinal muscular atrophy (SMA) type 1 in a patient in need thereof, comprising administering to the patient the composition of claim 2, wherein the patient: a. Nine months old or younger; b. Weight of at least 2.6 kg; c. have biallelic SMN1 null mutations or deletions; and d. Have at least one functional copy of SMN2.

12. The method of claim 11, wherein the viral vector is present at a concentration of about 1-2.5×10 14 vg / kg dose was administered.

13. The method of claim 11, wherein the patient has a body weight of no more than about 8.5 kg.

14. The method of claim 11, wherein the patient does not have a c.859G>C substitution in exon 7 of the SMN2 gene in at least one copy.

15. The method of claim 11, wherein the composition is administered to the patient at less than 6 months of age, or before the onset of one or more SMA symptoms selected from the group consisting of hypotonia, delayed motor skills, poor head control, rounded shoulder posture, and joint hypermobility.

16. The method of claim 11, wherein the viral vector is administered in Tris-buffered saline at about 5-20 mL / kg, about 10-20 mL / kg, or about 5.5-6.5 mL / kg.

17. The method of claim 11, wherein efficacy is determined using the CHOP INTEND scale.

18. The method of claim 11, comprising administering to the patient a dose volume selected from the group consisting of: a 16.5 mL dose for a patient weighing 2.6-3.0 kg, a 19.3 mL dose for a patient weighing 3.1-3.5 kg, a 22.0 mL dose for a patient weighing 3.6-4.0 kg, a 24.8 mL dose for a patient weighing 4.1-4.5 kg, a 27.5 mL dose for a patient weighing 4.6-5.0 kg, a 5.1-5.5 kg, a dose of 30.3 mL for patients weighing 5.6-6.0 kg, a dose of 33.0 mL for patients weighing 5.6-6.5 kg, a dose of 35.8 mL for patients weighing 6.6-7.0 kg, a dose of 38.5 mL for patients weighing 6.6-7.0 kg, a dose of 41.3 mL for patients weighing 7.1-7.5 kg, a dose of 44.0 mL for patients weighing 7.6-8.0 kg, and a dose of 46.8 mL for patients weighing 8.1-8.5 kg.

19. The method of claim 11, wherein the composition is administered to a patient in need thereof by intravenous or intrathecal infusion.

20. The method of claim 19, wherein the viral vector is infused over about 45-75 minutes.

21. A kit comprising a vial containing about 5.5 mL or about 8.3 mL of an AAV9 viral vector comprising a polynucleotide encoding a survival motor neuron (SMN) protein and containing about 2.0×10 13 The concentration of vg / mL was prepared in 20 mM Tris, 1 mM MgCl2, 200 mM NaCl, 0.005% w / v Poloxamer 188, pH 7.7-8.3.

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