Means and method for preparing viral vectors and uses of the same

Abstract

To provide methods of producing and purifying AAV viral vectors having low empty capsids, low host cell proteins, and / or low contaminating DNA, while retaining high potency; and to provide a pharmaceutical composition containing the vector.SOLUTION: A method of manufacturing an AAV viral vector comprises: a. culturing adherent cells; b. transfecting the adherent cells with one or more plasmids to enable production of the AAV viral vector; c. lysing the adherent cells to isolate the AAV viral vector; d. acidifying and clarifying the cell lysate of c; e. purifying the product of d using cation exchange chromatography; f. filtering the product of e using tangential flow filtration; g. ultracentrifuging the product of f in a cesium chloride buffer; h. collecting the AAV viral vector from the product of g; and i. formulating and filling the AAV viral vector collected in h. to produce a final pharmaceutical product.SELECTED DRAWING: None

Description

[Technical field]

[0001] Sequence Listing This application contains a sequence listing which has been submitted in ASCII format via EFS-Web, This specification is hereby incorporated by reference in its entirety. The ASCII copy is named AVEX-003001WO_ST25.txt. It has a size of 14,639 bytes.

[0002] Related Applications This application is a joint venture of U.S. Provisional Patent Application No. 62 / 583,035, filed November 8, 2017. Priority is claimed to the specification, the contents of which are incorporated herein by reference in their entirety. is incorporated herein by reference.

[0003] The present disclosure relates to methods for preparing and purifying viral particles, as well as compositions and uses comprising same. . [Background technology]

[0004] Adeno-associated viruses (AAVs) are members of the Parvoviridae family. The AAV genome contains approximately 4.7 kilobases (kb) and is made up of nonstructural Rep members. Two major open reading frames encoding the replication and structural Cap proteins The AAV coding region is a linear single-stranded DNA molecule consisting of a coding frame. is a hairpin fragment of approximately 145 nucleotides that functions as a primer during the initiation of DNA replication. Two cis-acting proteins containing interrupted palindromic sequences that can be folded into structures It is flanked by inverted terminal repeat (ITR) sequences. In addition to its role in DNA replication, IT The R sequence is involved in viral integration, rescue from the host genome, and viral assembly into mature virions. It has been shown that it is required for encapsidation of the nucleic acid (Muzyczka, (1992) Curr.Top.Micro.Immunol.158:97-129).

[0005] Multiple AAV serotypes exist, providing diverse tissue tropism. Known serotypes include, for example: , AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 AAV9 is described in U.S. Pat. No. 7,633,663. , 198,951 and Gao et al., J. Virol., 78:638 1-6388 (2004), which are hereby incorporated by reference in their entireties. Advances in AAV6 and AAV8 delivery have allowed for simple systemic i.v. or It was possible to transduce skeletal and cardiac muscles with these serotypes after intraperitoneal injection. Pacak et al.,Circ.Res.,99(4):3-9(20 06) and Wang et al., Nature Biotech. 23(3):32 1-8 (2005). However, AAVs have been used to To target cell types, surgical intraparenchymal injection is required. t al.,“Safety and tolerability of gene t therapy with an adeno-associated virus(AA V) borne GAD gene for Parkinson's diseases e:an open label,phase I trial.”Lancet,36 9:2097-2105;Marks et al.,“Gene delivery of AAV2-neurturin for Parkinson's diseases e:a double-blind,randomized,controlled t rial.” Lancet Neurol 9:1164-1172; and Worgal l et al.,“Treatment of late infantile ne uronal ceroid lipofuscinosis by CNS admi nistration of a serotype 2 adeno-associa ted virus expressing CLN2 cDNA.”Hum Gene See Ther, 19(5):463-74.

[0006] The nucleotide sequence of the AAV serotype 2 (AAV2) genome was determined by Srivastava et al. et al., J Virol, 45:555-564 (1983), and Ruf fing et al., J Gen Virol, 75:3385-3392(199 4) As corrected by. Within the ITR, there are viral DNA replication (rep), Contains cis-acting sequences that direct encapsidation / packaging and host cell chromosomal integration. Three AAV promoters (p5, p19, and p40 based on their relative mapping positions) were identified. Two AAV internal open reads, called rep and cap genes, are Two rep promoters (p5 and p19) drive the expression of the rep , differential splicing of a single AAV intron (nucleotides 2107 and 2227 In addition to the rep proteins in the rep gene, four rep proteins (rep78, rep68 , rep52, and rep40). The rep proteins have multiple enzymatic properties. These genes are responsible for the replication of the viral genome. It is expressed from the promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splice sites and non-consensus translation start sites are involved in these three Responsible for the production of the associated capsid protein. Single consensus polyadenylation site is located at mapping position 95 of the AAV genome. AAV life cycle and genetics For more information, see Muzyczka, Current Topics in Microbiology. in Immunology and Immunology, 158:97-129 (1992). It is being reviewed.

[0007] AAV-derived vectors have the potential to (i) infect a wide variety of non-dividing and -transducing cells, including muscle fibers and neurons; (ii) are capable of infecting (transducing) dividing cell types; Therefore, natural host cell responses to viral infection, such as interferon-mediated (iii) wild-type virus has not been associated with any pathology in humans (iv) a wild-type AAV that is capable of integrating into the host cell genome. In contrast, replication-defective AAV vectors generally remain episomal and therefore do not express oncogenes. (v) in contrast to other vector systems, there is limited risk of insertional mutagenesis or activation; However, AAV vectors do not induce significant immune responses (see ii) and are therefore not suitable for therapeutic transfection. because it results in long-term expression of the gene (if the gene product is not rejected) It is particularly attractive for the delivery of genetic material.

[0008] Self-complementary adeno-associated vectors (scAAV) are naturally occurring adeno-associated viruses (AAV) are viral vectors engineered for use in gene therapy. AAVs are designed so that the coding region forms an intramolecular double-stranded DNA template. The typical AAV genome is a single-stranded DNA template, so it is called “self-complementary.” The rate-limiting step in the AAV genome life cycle involves second strand synthesis. This is not the case for the V genome, which upon infection does not wait for cell-mediated second strand synthesis. Rather, the two complementary halves of the scAAV associate to form a vector that is ready for replication and transcription. This results in the formation of one double-stranded DNA (dsDNA) unit. Summary of the Invention

[0009] For example, fewer empty capsids, fewer host cell proteins, and / or less contaminating DNA. Scalable manufacturing and purification of AAV drug products with low dosages but high potency There remains a need to develop manufacturing methods.

[0010] The present disclosure provides methods for preparing purified viral particle preparations, including AAV particle preparations.

[0011] In some embodiments, the present disclosure provides: (a) 1 to 8×10 13 AAV9 viral vector -genomes / mL (vg / mL), (b) less than 7% empty viral capsids, (c) 1× 10 13 (d) less than about 100 ng / mL of host cell protein per vg / mL; and ×10 13 Approximately 5 × 10 per vg / mL 6 Drugs containing less than pg / mL residual host cell DNA A pharmaceutical composition is provided, wherein 1 to 8×10 13 Low number of AAV9 viral vector genomes / mL At least about 80% is functional.

[0012] In one embodiment, the AAV9 viral vector is a survival motor neuron (SMN) targeting In one embodiment, the AAV9 virus comprises a polynucleotide encoding a virion protein. The vector contains a polypeptide encoding the methyl-CpG binding protein 2 (MECP2) protein. In one embodiment, the AAV9 viral vector comprises a superoxygenase (SEQ ID NO: 1). We co-administered short hairpin RNA (shRNA) targeting steroid dismutase 1 (SOD1). In one embodiment, the AAV9 viral vector comprises a polynucleotide encoding Modified AAV2 ITR, chicken β-actin (CB) promoter, cytomegalovirus CMV immediate early enhancer, modified SV40 late 16s intron, bovine growth hormone It contains the Mon (BGH) polyadenylation signal, and the unmodified AAV2 ITRs.

[0013] The present disclosure provides pharmaceutical formulations. In some embodiments, the aqueous pharmaceutical formulation comprises: (a) a bioavailable liquid; AAV9 vector containing a polynucleotide encoding the SMN protein (b) virus vector, (b) Tris buffer, (c) magnesium chloride, (d) sodium chloride and (e) a poloxamer (e.g., poloxamer 188), wherein the pharmaceutical composition comprises In one embodiment of the formulation, the AAV9 viral vector is a modified AA V2 ITR, chicken β-actin (CB) promoter, cytomegalovirus (C MV) immediate early enhancer, modified SV40 late 16s intron, bovine growth hormone (B G-H) Further comprising a polyadenylation signal, and unmodified AAV2 ITRs. In one embodiment, the Tris buffer concentration is about 10 to 30 nM, for example, about 20 mM. In this embodiment, the pH of the formulation is about 7.7 to about 8.3, for example about pH 8.0 (e.g., USP <791> (as measured by the method of claim 1, which is incorporated by reference in its entirety). In an embodiment, the magnesium chloride concentration is about 0.5 to 1.5 mM, for example about 1 mM. In one embodiment of the formulation, the sodium chloride concentration is about 100 to 300 mM, for example about 200 mM. In one embodiment, the formulation is about 0.005% w / v poloxamer 18 Includes 8.

[0014] Another aspect of the disclosure is an AAV9 comprising a polynucleotide encoding an SMN protein. Viral vectors (e.g., compositions or formulations as disclosed herein) are administered intrathecally or intravenously. In a patient in need thereof, the I is administered to the patient by intravenous route. The present invention relates to a method for treating spinal muscular atrophy (SMA), wherein the patient is (a) 9 months of age or younger, (b) weigh at least about 2.6 kg; and (c) have biallelic SMN1 null mutations. and (d) having at least one functional copy of SMN2. In embodiments, the AAV9 viral vector comprises modified AAV2 ITRs, chicken β-amino acid sequence. Cutin (CB) promoter, cytomegalovirus (CMV) immediate early enhancer, Modified SV40 late 16s intron, bovine growth hormone (BGH) polyadenylation signal and unmodified AAV2 ITRs. In one embodiment, the patient weighs about 8.5 kg In one embodiment, the patient has at least one copy of the SMN2 gene. In one embodiment, the treatment is administered to a patient at age 6 months. In certain embodiments, the treatment is administered to a patient prior to the onset of muscle hypotonia, motor skill retardation, or the like. One or more of the following SMA symptoms may occur: inability to maintain balance, hunched posture, and joint hypermobility. In one embodiment, the patient is administered ELISA binding immunoassays prior to administration. have an anti-AAV9 antibody titer of 1:100 or less as determined by assay.

[0015] Also provided herein is an adeno-associated virus (AAV) vector as disclosed herein. The present invention relates to a method for treating spinal cord injury, the method comprising administering to a patient a composition or formulation comprising a marker. Methods for treating pediatric patients with spinal muscular atrophy (SMA) Type I are also disclosed.

[0016] The present disclosure also provides a method for the preparation of a method for the preparation of a methyl-CpG binding protein 2 (MECP2) protein. The AAV9 viral vector containing the polynucleotide is administered intrathecally or intravenously to patients. and a method for treating Rett syndrome in a patient in need thereof, comprising administering to Regarding.

[0017] The present disclosure also provides a small molecule hair inhibitor that targets superoxide dismutase 1 (SOD1). AAV9 viral vector containing a polynucleotide encoding pinRNA (shRNA) administering the drug to a patient in need thereof, including by intrathecal or intravenous route The present invention also relates to a method for treating amyotrophic lateral sclerosis (ALS) in a patient.

[0018] Another aspect of the present disclosure relates to a method of treating a patient suffering from SMA type I, comprising: (a) determining the patient's weight; (b) administering an AAV9 viral vector pharmaceutical composition to the patient; (c) obtaining a kit containing the AAV9 virus from the vial. administering the vector to a patient, wherein the viral vector concentration in each vial is is approximately 2.0 x 10 13 vg / mL, and the AAV9 viral vector expresses the SMN protein. and wherein the kit comprises the following number of vials: nothing.

[0019] [Table 1]

[0020] In one embodiment, the kit comprises a mutated AAV2 ITR, chicken β-actin (CB) promoter, cytomegalovirus (CMV) immediate early enhancer, modified SV 40 late 16s intron, bovine growth hormone (BGH) polyadenylation signal, and In one embodiment, the AAV comprises an AAV9 viral vector containing the AAV2 ITRs. The viral vector is approximately 1.0 × 10 14 ~2.5×10 14 Injection at a dose of vg / kg Therefore, it is administered.

[0021] Another aspect of the present disclosure is a polynucleotide encoding a survival motor neuron (SMN) protein. (a) a composition comprising an AAV9 viral vector and a surviving motor neuron (SM N) an AAV9 viral vector comprising a polynucleotide encoding a protein; (b) Tris buffer, (c) magnesium chloride, (d) sodium chloride, and (e) poloxamer. - (e.g., poloxamer 188) The present invention relates to a kit for treating patients suffering from Synaptic Mammary Glandular Atrophy (SMA).

[0022] Another aspect of the present disclosure is a polynucleotide encoding a survival motor neuron (SMN) protein. 20 mM Tris, 1 mM Approximately 2 in MgCl2, 200 mM NaCl, 0.005% w / v poloxamer 188. 0×10 13 Approximately 5.5 mL or approximately 8.3 mL of AAV9 formulated at a concentration of vg / mL The present invention relates to a kit comprising a vial containing a viral vector.

[0023] Another aspect of the present disclosure is a volume of a composition comprising a survival motor neuron (SMN) protein. A composition comprising an AAV9 viral vector containing a polynucleotide encoding a protein or ) AAVs containing a polynucleotide encoding a survival motor neuron (SMN) protein 9 viral vector, (b) Tris buffer, (c) magnesium chloride, (d) sodium chloride and (e) intravenous infusion of a formulation containing poloxamer (e.g., poloxamer 188). and administering to a patient in need thereof by do.

[0024] Another aspect of the present disclosure relates to a method for producing an AAV viral vector. The method for producing an AAV viral vector includes the steps of: (a) culturing adherent cells; Adherent cells are transfected with one or more plasmids to express AAV viral vectors. (c) lysing the adherent cells to isolate the 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 a cesium chloride (CsCl) buffer; and (h) harvesting the AAV viral vector from the product of (g).

[0025] Another aspect of the present disclosure relates to a method for purifying an AAV viral vector from a cell culture lysate. The method includes the steps of: (a) acidifying and clarifying a cell lysate; (c) purifying the product of (a) using cation exchange chromatography (CEX); (d) filtering the product of (b) by tangential flow filtration; (e) and (d) a step of ultracentrifuging the mixture using a 2 to 4 M cesium chloride (CsCl) buffer solution. (f) collecting the AAV viral vector from the product of (e); In one embodiment, the method comprises filtering the mixture by industry standard differential flow filtration. In one embodiment, the method is carried out in a 5×10 scale per manufacturing batch. 15 vg Super or 8 x 10 15 vg or 1×10 16 It produces yields of over vg.

[0026] Another aspect of the present disclosure is a method for the preparation of a medicament for the treatment of a pulmonary artery disease, comprising: Administering an AAV9 viral vector containing a polynucleotide encoding the SMN protein The present invention relates to a method for treating a patient with SMA type 1 by administering

[0027] Another aspect of the present disclosure is a method for producing ME as prepared by any of the methods herein. Administering an AAV9 viral vector containing a polynucleotide encoding the CP2 protein The present invention relates to a method for treating a patient with Rett Syndrome by administering

[0028] Another aspect of the present disclosure is a method for producing a SO AAV9 viral vector containing a polynucleotide encoding shRNA targeting D1 The present invention relates to a method for treating a patient with ALS by administering a medicament to the patient.

[0029] Another aspect of the present disclosure is a method for producing an SMN tank prepared by any of the methods herein. The present invention relates to an AAV9 viral vector comprising a polynucleotide encoding a protein.

[0030] Another aspect of the present disclosure is a method for producing an SMN tank prepared by any of the methods herein. Pharmaceutical compositions comprising AAV9 viral vectors containing polynucleotides encoding proteins Regarding.

[0031] Another aspect of the disclosure is an AAV9 comprising a polynucleotide encoding an SMN protein. A viral vector, a Tris buffer, a magnesium chloride solution, and a sodium chloride solution. wherein the pharmaceutical composition does not contain a preservative, and wherein the composition , produced by any of the methods herein.

[0032] Another aspect of the disclosure is an MECP2 antibody produced by any of the methods herein. The present invention relates to an AAV9 viral vector comprising a polynucleotide encoding a protein.

[0033] Another aspect of the disclosure is an MECP2 antibody produced by any of the methods herein. Pharmaceutical composition comprising an AAV9 viral vector containing a polynucleotide encoding a protein Related to the composition.

[0034] Another aspect of the disclosure is a method for producing SOD1 by any of the methods herein. AAV9 viral vector containing a polynucleotide encoding the target shRNA Regarding.

[0035] Another aspect of the disclosure is a method for producing a recombinant AAV comprising the steps of: (a) a modified AAV2 ITR and a vector comprising a recombinant AAV vector comprising a modified AAV2 ITR and a vector comprising a modified chicken β-actin (CB) Promoter, cytomegalovirus (CMV) immediate early enhancer, and modified SV40 The late 16s intron, the bovine growth hormone (BGH) polyadenylation signal, and the unmodified (b) a self-complementary AAV9 viral vector containing the modified AAV2 ITRs; (c) 20 mM Tris, (d) 200 mM NaCl, and (e) By intravenously administering a pharmaceutical composition containing 0.005% poloxamer 188, A method of treating SMA type 1 in a patient in need thereof, wherein the patient weighs 2.6 kg g to 8.5 kg. In one embodiment, the composition does not contain a preservative.

[0036] In one embodiment, the patient is (a) 9 months of age or younger; and (b) at least about 2.6 kg, (c) have a biallelic SMN1 null mutation or deletion, and (d) Has at least one functional copy of SMN2.

[0037] Another aspect of the disclosure is a method for producing a recombinant AAV comprising the steps of: (a) a modified AAV2 ITR and a vector comprising a recombinant AAV vector comprising a modified AAV2 ITR and a vector comprising a modified chicken β-actin (CB) Promoter, cytomegalovirus (CMV) immediate early enhancer, and modified SV40 The late 16s intron, the bovine growth hormone (BGH) polyadenylation signal, and the unmodified (b) a self-complementary AAV9 viral vector containing the modified AAV2 ITRs; (c) 20 mM Tris, (d) 200 mM NaCl, and (e) Suitable for or intended for intravenous administration of a pharmaceutical composition comprising 0.005% poloxamer 188 It relates to the composition produced.

[0038] In some embodiments, a composition or formulation is disclosed herein, wherein the composition or formulation The formulation contains at least one of the following: (a) 1.0 × 10 13 Approximately 0.09 per vg (b) less than about 30 μg / g (ppm) of cesium; (c) less than about 2 0–80 ppm of poloxamer 188; (d) 1.0 × 10 13 Approximately 0.22 n per vg g BSA, (e) 1.0 × 10 13 Approximately 6.8 × 10 per vg 5 Residual protease activity below pg Plasmid DNA, (f) 1.0 × 10 13 Approximately 1.1 × 10 per vg 5 Residual h below pg cDNA, and (g) 1.0 × 10 13 Contains less than about 4ng rHCP per vg.

[0039] In some embodiments, the present disclosure provides methods for producing intermediates (e.g., (a) culturing cells to produce a frozen intermediate; Step (b) Transfect the cultured cells with plasmids (e.g., three plasmids). (c) recovering the expanded virus 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 (g) optionally freezing the resulting purified virus particle intermediate preparation. In some embodiments, the upstream process includes further processing steps, in particular ., combined with further purification and formulation steps to produce the final pharmaceutical product.

[0040] In one embodiment, the working cell bank comprises HEK293 cells. are available in the art and suitable for use in the methods disclosed herein. Any suitable alternative cell type or derivative may be used.

[0041] In one embodiment, the transfection step utilizes polyethyleneimine. In one embodiment, the transfection step comprises the step of: For example, a triple vector using pSMN, pAAV plasmid and pHELP plasmid Including transfection.

[0042] In one embodiment, this step comprises lysing the transfected cells with a lysis buffer and a detergent. The method further comprises dissolving the mixture.

[0043] In one embodiment, the recovery step comprises recovering the product at a concentration of, for example, 50-200 U / ml, e.g. For example, treat with an endonuclease such as benzonase at a concentration of 75-150 U / ml to remove the residue. This includes reducing residual host cell DNA.

[0044] In one embodiment, the purification step involves depth filtration followed by removal of large molecular contaminants and cellular debris. A filter to remove bleeds, e.g., a 0.45 micron filter, but without vector gels. Any suitable depth filter may be used. A filter may be used.

[0045] In one embodiment, tangential flow filtration ("TFF") is 5-15 times, e.g., 6-10 times, concentrated eluate and at least 4 diavolumes volume), e.g. 6 diavolume diafiltration or 10 diavolume diafiltration. Diafilter with volume, 12 diavolume, or 15 diavolume Any suitable TFF filter may be used. In one embodiment, the TFF membrane is a cellulose membrane. It has a toe off.

[0046] Second, the present disclosure provides a downstream process for processing intermediates (e.g., frozen intermediates) into filtered drug substance. Downstream processing steps include acidification and clarification steps (using filtration) followed by followed by cation exchange chromatography, tangential flow filtration, and CsCl ultracentrifugation. and a further tangential flow filtration step, which results in purified AAV A filtered drug substance is produced in which the particles are suspended in a pharma- ceutically acceptable carrier.

[0047] In one embodiment, the acidification and clarification step comprises depth filtration followed by removal of large molecule contaminants. Filtration with a filter, such as a 0.45 micron filter, to remove particles and cell debris. In some embodiments, the pH adjustment is controlled. In some embodiments, a detergent is added, such as Tween. , the rate of Tween addition and the concentration range of Tween are controlled.

[0048] In one embodiment, the cation exchange chromatography is carried out using a 0.2 micron pore size chromatograph. Includes membrane-based chromatography resins using synthetic sulfonyl resins.

[0049] In one embodiment, the cesium chloride (CsCl) ultracentrifugation step is performed using 2-4M CsCl. 1, for example about 3M CsCl is used.

[0050] In another embodiment, the CsCl ultracentrifugation step is performed at about 40-50 k RPM for about 20-24 h. In another embodiment, the CsCl ultracentrifugation step is carried out at about 45k RPM. It will last for approximately 22 hours.

[0051] In one embodiment, tangential flow filtration ("TFF") is 5-15 times, e.g., 6-10 times, concentrated and at least 4 diavolumes, e.g., 6 diavolume diafiltration or 10 diavolume or 12 diavolume Achieve 10 diavolume or 15 diavolume diafiltration. In some embodiments, the TFF membrane has a 300 kDa cutoff.

[0052] In one embodiment, the eluent is below detectable levels of cesium (Cs). In the present study, the detection level of Cs was less than 50 parts per million (ppm). In another embodiment, the detection level of Cs is about 50-70 ppm. In one embodiment, the detection level of Cs is about 70 to 90 parts per million (ppm). In an embodiment, the detection level of Cs is about 90-110 parts per million (ppm). In an embodiment, the detection level of Cs is about 110-130 parts per million (ppm). In an embodiment, the detection level of Cs is about 130 to 150 parts per million (ppm). In this embodiment, the detection level of Cs is less than 150 parts per million (ppm).

[0053] Using these purification methods, 1 × 10 13 Vector genomes ("vg") / ml 5×10 6 Residual host cell DNA (hcDNA) less than pg / ml, e.g., 1×10 13 v 1.2 x 10 per g / mL 6 High-yield virus preparations containing less than pg / mL hcDNA For example, an AAV preparation (e.g., AAV9-SMN) can be prepared. 7.5×10 15 For a 5 kg patient receiving vg, a maximum of 100 mg / 5 kg dose would be 1.2×10 6 pg / mL x 7.5 x 10 15 vg / (1×10 13 vg / mL) = 8.4×10 7 pg of hcDNA = 84,000ng of hcDNA taken up In one embodiment, the preparation contains 1.0×10 13 5.0 x 10 per vg 5 Residual host cell DNA, less than pg, 1.0 × 10 13 2.0 x 10 per vg 5 Less than pg of residual host cell DNA, 1.0 × 10 13 1.1 × 10 per vg 5 Less than pg residual host Cellular DNA, 1.0×10 13 1.0 × 10 per vg 5 Sub-pg residual host cell DNA , 1.0×10 13 0.9 × 10 per vg 5 Residual host cell DNA, less than pg, 1.0× 10 13 0.8 × 10 per vg 5 Sub-pg residual host cell DNA, or any value in between Includes concentration.

[0054] In one embodiment, the AAV is a replication-deficient AAV9 having ITRs from AAV2, e.g. For example, scAAV9. In another embodiment, the AAV vector is In one embodiment, the SMN-encoding DNA is identified as GenBank Accession No. Conservative nucleotide substitutions in SMN DNA are also contemplated. (e.g., as set forth in GenBank Accession No. NM_000344.2, guanine to adenine change at position 625).

[0055] Another aspect of the present disclosure is the administration of the drug by (a) intravenous ("IV") injection or (b) intrathecal ("IT") administration. The present invention relates to a pharmaceutical composition comprising AAV particles in any suitable formulation.

[0056] In another embodiment, the pharmaceutical composition contains less than 10% empty capsids, less than 8% empty Capsid, <7% empty capsid, <5% empty capsid, <3% empty capsid or less than 1% empty capsids. In some embodiments, the pharmaceutical composition has about 5 % empty capsids. In one embodiment, the number of empty capsids is below the limit of detection. In some embodiments, the empty capsids may cause adverse reactions (e.g., immunological reactions) without therapeutic benefit. In some cases, the pharmaceutical composition may produce adverse reactions (immune, inflammatory, hepatic, and / or cardiac reactions). It is advantageous to have a low amount of empty capsids.

[0057] In another embodiment, the residual host cell proteins ("rHCPs") in the pharmaceutical composition is 1 x 10 13 rHCPs below 100 ng / ml per vg / ml, e.g., 1×10 13 40ng / ml or less of rHCP per vg / ml or 1×10 13 Per vg / ml In one embodiment, the rHCP is 1 to 50 ng / ml. The composition is 1.0 x 10 13 Less than 10 ng rHCP per vg or 1.0 × 10 13 <5 ng rHCP per vg, 1.0 × 10 13 <4ng rHCP per vg; or 1.0 x 10 13 Contains less than 3ng rHCP per vg, or any concentration in between .

[0058] In another embodiment, the residual host cell DNA ("hcDNA") in the pharmaceutical composition is , 1×10 13 5 × 10 per vg / ml 6 pg / ml or less of hcDNA, 1 × 10 13 1.2 × 10 per vg / ml 6 pg / ml or less of rHDNA or 1×10 13 vg / 1 x 10 per ml 5 pg / ml rHDNA ~ 1 x 10 13 1.2x per vg / ml 10 6 In one embodiment, the residual host cell DNA in the pharmaceutical composition is 1 pg / ml. is 1.0 × 10 13 5.0 x 10 per vg 5 Less than pg, 1.0 × 10 13 per vg 2.0×10 5 Less than pg, 1.0 × 10 13 1.1 × 10 per vg 5 Less than pg, 1.0 ×10 13 1.0 × 10 per vg 5 Less than pg of hcDNA, 1.0 × 10 13 vg hit 0.9×105 Less than pg of hcDNA, 1.0 × 10 13 0.8 × 10 per vg 5 p g hcDNA or any concentration in between.

[0059] In one embodiment, the residual plasmid DNA in the pharmaceutical composition is at least 1×10 13 v 1.7 x 10 per g / ml 6 pg / ml or less, or 1×10 13 1x per vg / ml 10 5 pg / ml to 1×10 13 1.7 × 10 per vg / ml 6 pg / ml. In an embodiment, the residual plasmid DNA in the pharmaceutical composition is greater than or equal to 1.0×10 13 vg Winnings 10.0 x 10 5 Less than pg, 1.0 × 10 13 8.0 x 10 per vg 5 Less than pg , or 1.0×10 13 6.8 x 10 per vg 5 Less than pg.

[0060] In certain embodiments, the pharmaceutical compositions disclosed herein comprise a dose of 1.0×10 13 vg hit Less than 0.5 ng per 1.0 × 10 13 <0.3 ng per vg, 1.0 × 10 13 v Less than 0.22 ng per g or 1.0 x 10 13 Less than 0.2 ng / vg or between In one embodiment, the medicament comprises a bovine serum albumin (BSA) in any concentration. The benzonase in the composition is 1.0 x 10 13 <0.2 ng per vg, 1.0 × 10 13 <0.1 ng per vg, 1.0 × 10 13 Less than 0.09 ng per vg, 1.0 ×10 13 In one embodiment, the concentration is less than or equal to 0.08 ng per vg. The poloxamer 188 in the pharmaceutical composition is about 10 to 150 ppm, about 15 to 10 In one embodiment, the selenium content in the pharmaceutical composition is about 0 ppm, or about 20 to 80 ppm. Sium is less than 50μg / g (ppm), less than 30μg / g (ppm), or less than 20μg / g (ppm), or any concentration therebetween.

[0061] In certain embodiments, the pharmaceutical compositions disclosed herein are characterized by, for example, SDS-PAGE. When determined by the above, 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 of total impurities. In some embodiments, the overall purity is greater than 90%, greater than 92%, as determined, for example, by SDS-PAGE. More than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or any value in between In one embodiment of the pharmaceutical composition, the non-single unspecified related impurity is a percentage of is greater than 5%, greater than 4%, as measured, for example, by SDS-PAGE. , greater than 3% or greater than 2%, or any percentage in between. The pharmaceutical composition is %, 91.9%, 92%, 93%, or any percentage in between. The percentage of filled capsids relative to sids (e.g., by analytical ultracentrifugation) In one embodiment of the pharmaceutical composition, the analytical The percentage of filled capsids measured in peak 1 by ultracentrifugation was 20–40%. 80%, 25-75%, 30-75%, 35-75%, or 37.4-70.3% In one embodiment of the pharmaceutical composition, the content measured in peak 2 by analytical ultracentrifugation is The percentage of packed capsids was 20–80%, 20–70%, 22–65%, and 2 4 to 62%, or 24.9 to 60.1%.

[0062] In one embodiment, the pharmaceutical composition disclosed herein is administered in a dose of 1.0 to 5.0 × 10 13 vg / mL, 1.2-3.0×10 13 vg / mL or 1.7 to 2.3 × 10 13 vg / Contains genomic titer in mL.

[0063] In one embodiment, the pharmaceutical compositions disclosed herein have a cytotoxicity of less than 5 CFU / mL, less than 4C Less than 3 CFU / mL, less than 2 CFU / mL, or less than 1 CFU / mL, or any level of bioburden therebetween. 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.

[0064] In one embodiment, USP, e.g., USP <785> (Incorporated by reference in its entirety The osmolality of the pharmaceutical compositions disclosed herein is 350 to 450 mO sm / kg, 370-440mOsm / kg, or 390-430mOsm / kg. In one embodiment, the pharmaceutical composition contains less than 1200 particles larger than 25 μm per container. Particles, less than 1000 particles larger than 25μm per container, less than 600 particles per container Particles larger than 25 μm, less than 500 particles larger than 25 μm per container, or any In one embodiment, the pharmaceutical composition contains 1000 units per container. Less than 0 particles >10μm, Less than 8000 particles >10μm per container Contains less than 6,000 particles larger than 10 μm per container or container.

[0065] In one embodiment, the pharmaceutical composition is administered in an amount of 0.5 to 5.0 × 10 13 vg / mL, 1.0 ~4.0×10 13 vg / mL, 1.5-3.0×10 13 vg / mL or 1.7-2. 3×10 13 It has a genomic titer of 0.01 vg / mL.

[0066] In one embodiment, the pharmaceutical compositions disclosed herein comprise one or more of the following: .0×10 13 Less than about 0.09 ng of benzonase per vg, about 30 μg / g (ppm ) less than cesium, approximately 20-80 ppm of poloxamer 188, 1.0 × 10 13 vg hit Approximately 0.22 ng of BSA per 1.0 × 10 13 Approximately 6.8 × 10 per vg 5 pg Residual plasmid DNA of less than 1.0 × 10 13 Approximately 1.1 × 10 per vg 5 Less than pg of residue Retained hcDNA, 1.0×10 13 Approximately less than 4ng rHCP per vg, pH 7.7-8. 3. Approximately 390-430 mOsm / kg, less than about 600 particles of ≧25 μm size per container Particles, less than about 6000 particles of ≥ 10 μm size per container, about 1.7 × 10 13 ~2 .3×10 13 Genomic titer in vg / mL, 1.0 × 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 Infectious titer in IU is 1.0 x 10 13 Approximately 100-300μg per vg of total protein, approximately 7.5 × 10 13 Δ7SM by viral vector at a dose of vg / kg Median survival of A mice was ≥24 days, with approximately 70-day survival based on in vitro cell-based assays. 130% relative potency and / or contains less than about 5% empty capsids.

[0067] In various embodiments, any of the viral particles discussed herein (e.g., A The present specification contains AAV SMN, AAV MECP2, or AAV SOD1 viral particles. The pharmaceutical compositions disclosed herein may vary within ±20%, ±15%, ±10%, or ±20% of a reference standard. In some embodiments, the potency is maintained at or within ±5% of the preferred in vitro potency. The efficacy or % functionality of the compound is measured using in vivo cell assays or in vivo animal models. The AAV SMN viral particles can be used in animal models of SMA, such as SMAΔ7 mice, or in vivo models of SMA. Suitable cell lines, such as primary neuronal cells isolated from the cortex of SMAΔ7 mice, It may be determined using a quantitative cell-based assay using progenitor cells (NPCs). In embodiments, efficacy is evaluated according to the methodology described by Foust et al., Nat. Biotechnol. .,28(3),pp.271-274(2010) against the reference standard. Any suitable reference standard may be used. Potency or % Functional AAV M eCP2 can be assayed using suitable in vitro cellular assays or in vivo animal models, such as those described by Guy et al., “Reversal of neurological defects in a mouse model of Rett syndrome.”Scie Mecp2 knockout mice as described in nce,315(5815):1143-7 Potency or % functional AAV SOD1 may be assessed using suitable in vitro cell lines. Cellular assays or in vivo animal models, e.g., Gurney et al., "Moto r neuron degeneration in mice that expre ss a human Cu,Zn superoxide dismutase mu tation.” Science, 264(5166):1772-5 D1 mutant mice. In one embodiment, the pharmaceutical composition comprises 7. 5×10 13 The median survival time in SMAΔ7 mice given a 500 mg / kg dose was When determined, the compound has an in vivo efficacy of greater than 15 days, greater than 20 days, greater than 22 days, or greater than 24 days. In an embodiment, the pharmaceutical composition, when tested by a cell-based assay, and / or 50-150%, 60-140%, or 70-130% improvement over a suitable control It has relative efficacy in vivo.

[0068] In one embodiment, the intravenous ("IV") formulation has a pH of 7.5 to 8.5, about 1 to 8× 10 13 viral vector genomes / mL (vg / mL), or 2 × 10 13 vg / ml~ 6×10 13 vg / ml genomic titer, and optionally 384-448 mOsm / kg In one embodiment, the IV formulation is in Tris buffer at pH 8.0. Contains MgCl2, NaCl, and pluronic F68.

[0069] In one embodiment, IV administration includes an AAV-9 vector carrying the SMN transgene. The surgeon must be able to provide appropriate care in the appropriate setting (e.g., interventional suite, operating room, dedicated procedure room, etc.). Under sterile conditions in a laboratory, the catheter is inserted into a peripheral vein (arm or leg) through an intravenous catheter. The prescribed dose is given as a single dose and infused slowly over approximately 30 to 60 minutes.

[0070] In another embodiment, the disclosure herein provides a method for administering rAAV9 and non-ionic surfactant to a patient. The present invention relates to a method for administering a polynucleotide to a subject in need thereof, including intrathecal ("IT") delivery of a low osmolarity contrast agent. The present invention provides compositions and methods for delivering rAAV9 to the central nervous system of a patient suffering from The polynucleotides may be derived from, for example, the brain, spinal cord, or other organelles. The drug is delivered to the medulla, glial cells, astrocytes and / or lower motor neurons. Examples of the low osmolarity contrast agent include iobitridol, iohexol, iomeprol, Iopamidol, Iopentol, Iopromide, Ioversol or Ioxilan In some embodiments, the polynucleotide is a survival motor neuron (SMN) polynucleotide. In one embodiment, the imaging agent is iohexol, e.g., iohexol. Omnipaque 180 (sold as Omnipaque 180, with a potency of 180 mg / mL) (Contains 388 mg of iohexol, the organic iodine equivalent).

[0071] In one embodiment, IT administration includes the use of a scAAV9 vector carrying the SMN transgene. The drug was diluted in normal saline and injected intrathecally into the lumbar spine for radiographic monitoring. Appropriate hyperdensity contrast media (O) approved for pediatric use and labeled for use in children The A carrying the SMN transgene is premixed with 100 mM MgCl2 (e.g., mnipaque 180). The total volume of the aqueous composition containing the AV-9 vector + contrast agent and / or saline is 5 mL. scAAV-9 vector carrying the contrast agent and SMN transgene The drugs may be co-formulated, co-packaged, or packaged and delivered separately to the patient facility.

[0072] Patients were placed in a PICU room or other appropriate setting (e.g., interventional Immediately transfer the patient to emergency care under sterile conditions in a special room, operating room, or dedicated procedure room. The aim of this study was to administer an scAAV-9 vector carrying the SMN transgene to the spinal cord. The facility will administer the catheter to the intact endothelium, with the beveled tip parallel to the dural fibers. needles may be used; this significantly reduces damage to the dural membrane and results in lower back pain, including in children. It has been shown to reduce the risk of cerebrospinal fluid leakage after vertebral puncture (Ebinger et al. al.,“Headache and Backache After Lumbar Puncture in Children and Adolescents:A Prospective Study.”Pediatrics,113(6):158 8-1592;Kiechl-Kohlendorfer et al., “Cereb rospinal Fluid Leakage After Lumbar Punc ture in Neonates:Incidence and Sonograph ic Appearance.”American Journal of Roent genology,181(1):231-234).

[0073] Sedation / anesthesia is recommended for any patient undergoing IT injections. Methods and dosing should be determined at each institution. The Trendelenburg position may be used during and after the procedure, at the discretion of the anesthesiologist at the facility. Sedation or anxiolysis sufficient to ensure anesthesia and immobility when the animal is given Patients should tilt their head down 30° for 15 minutes after IT therapy administration. The patient is then placed in Trendelenburg position to promote distribution to the cervical and brain regions.

[0074] The patient is in the lateral position and a styletted catheter is inserted into the subarachnoid space via lumbar puncture. It is inserted into the L3-L4 or L4-L5 interspinous space. Subarachnoid cannulation is done by using a catheter This is confirmed by the flow of clear cerebrospinal fluid (CSF) from the cerebrospinal fluid. The CSF is removed and the facility gas station The SMN tracer in the premixed contrast solution will be discarded according to guidelines. The scAAV-9 vector carrying the transgene is injected directly into the subarachnoid space.

[0075] In one embodiment, the present disclosure provides for intravenous or intrathecal administration of the pharmaceutical compositions disclosed herein. The present invention provides a method for treating a neurological disorder in a patient in need thereof, comprising delivering The parvovirus contains the self-complementary rAAV9 genome and the engineered transgene is The MN polynucleotide is included and the disease is SMA.

[0076] In another embodiment, the present disclosure provides a pharmaceutical composition disclosed herein administered in combination with an imaging agent. and a method for treating a neurological disorder in a patient in need thereof, the method comprising intraluminally delivering The parvovirus comprises a self-complementary rAAV9 genome and an engineered transfectant. the disease is SMA, and the imaging agent is omni It's paque 180.

[0077] In another embodiment, the present disclosure provides a pharmaceutical composition disclosed herein administered in combination with an imaging agent. Intraluminal delivery of type II, type III, or type I in a patient in need thereof. The present invention provides a method for treating type V SMA, wherein the parvovirus comprises a self-complementary rAAV9 genome. the engineered transgene comprises an SMN polynucleotide, and the imaging agent comprises an omn It's an ipaque 180.

[0078] In another embodiment, the present disclosure includes intravenous delivery of the pharmaceutical compositions disclosed herein. The present invention provides a method for treating SMA type 1 in a patient in need thereof, The virus contains a self-complementary rAAV9 genome, and the engineered transgene is the SMN gene. In some embodiments, the patient is 0 to 9 months of age. In one embodiment, the patient is 0 to 6 months of age. In another embodiment, the pediatric patient is up to about In some embodiments, the pediatric patient weighs about 8.5 kg or less. In some embodiments, the pediatric patient weighs about 2.6 kg or more.

[0079] In another embodiment, the present disclosure provides a method for the preparation of a pharmaceutical composition disclosed herein in a vial. To provide a kit for the treatment of SMA type 1 in patients in need thereof, including intravenous administration. In some embodiments, the patient's weight is measured and the dose is calculated based on the patient's weight. can be.

[0080] Unless otherwise defined, all technical and scientific terms used herein are intended to be understood as meaning those of ordinary skill in the art to which this disclosure pertains. It has the same meaning as commonly understood by one of ordinary skill in the art.

[0081] As used herein, the singular forms of words also include the term "a," "an," or "the," unless the context clearly dictates otherwise. For example, the terms "a," "an," and "the" are used interchangeably. It is understood that there is a singular or plural, and the term "or" is understood to be inclusive. "An element" means one or more elements.

[0082] Throughout this specification the words "comprising" or "including Variations such as "comprises" refer to the specified element, integer or step, or group. but any other element, integer or step, or It will be understood that the absence of any exclusion of a group of elements, integers or steps is implied. Throughout this specification the phrases "consisting of" or " Variations such as "consists of" include the specified elements, integers, or sequences. A step or a group of elements, integers or steps, and includes any other elements, integers or steps. is understood to imply the exclusion of a step or a group of elements, integers or steps. It will be understood that throughout this specification the phrase "consisting essentially of" "consist essentially of" or "consist essentially of" Variations such as "essentially of" refer to the specified element, integer or sequence. or a group of elements, integers or steps, and the disclosure and / or claims thereof Any other element, integer or step that does not materially affect the basic and novel characteristics of the present invention. It is understood to imply the inclusion of a step, or a group of elements, integers or steps. It would be.

[0083] Approximately means 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or This may be understood to be within 1%, 0.5%, 0.1%, 0.05%, or 0.01%. When used in connection with a percentage value, "about" means within ±1% (e.g., "about 5% " can be understood as within 4% to 6%), or within ±0.5% (for example, "about 5 "%" can be understood as being within 4.5% to 5.5%. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about." It will be decorated.

[0084] In the practice or testing of the present disclosure, methods similar or equivalent to those described herein may be used. Methods and materials may be used, suitable methods and materials are described below. All cited publications, patent applications, patents, and other references are incorporated by reference in their entirety. The references cited herein are admittedly prior art to the claimed disclosure. In the event of any conflict, the present specification, including definitions, will control. In this regard, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present invention will become apparent from the following detailed description and claims. .

[0085] Various objects and advantages of the present disclosure and a more complete understanding thereof may be obtained by considering the following drawings in conjunction with the accompanying drawings. As will become apparent from the following detailed description and the appended claims, This is easily understood. [Brief description of the drawings]

[0086] [Figure 1-1]Figure 1 shows the plasmid maps of pSMN, pHELP and pAAV. Figure 1A shows the plasmid map of pSMN. pSMN is a plasmid encoding information for a recombinant self-complementary AAV DNA genome expressing human survival motor neuron (SMN) cDNA under the control of a chicken-β-actin hybrid promoter with an immediate early cytomegalovirus (CMV) enhancer element. The SMN cDNA encodes a full-length functional protein. The expression cassette contains a modified intron sequence derived from Simian Virus 40 (SV40) and a bovine growth hormone (BGH) polyadenylation signal. This expression cassette (CMV-CB-SV40-SMN-BGHpA) is flanked by inverted terminal repeats (ITRs) from AAV2. The left ITR is modified to preferentially package the self-complementary AAV genome. The region between and including the ITRs is packaged together into a recombinant AAV9 capsid during the manufacture of the find drugs product. Important pSMN components that are not intended for packaging into the recombinant AAV genome include an open reading frame encoding kanamycin resistance (KanR) and the origin of replication (ori) derived from pUC. The ori and KanR regions are useful for plasmid construction. [Figure 1-2] FIG. 1B shows the plasmid map of the pHELP plasmid. The pHELP plasmid contains the trans-acting adenovirus components necessary for recombinant adeno-associated virus production. The pHELP plasmid contains the regions of the adenovirus genome that provide important factors for AAV replication, namely, E2A, E4, and VA RNA. The adenovirus E1 function involved in rAVV replication is provided by transfection of host 293 cells. However, the pHELP plasmid does not contain other adenovirus replication or structural genes. The adenovirus sequences present in this plasmid represent only about 28% (9,280 / 35,938) of the adenovirus genome and do not contain cis elements critical for replication, such as inverted terminal repeats. Therefore, infectious adenovirus is not expected to arise from such a production system. [Figure 1-3] Figure 1C shows the plasmid map of the AAV plasmid. The wild-type AAV genome contains two non-coding structural elements called inverted terminal repeats that flank the rep and cap open reading frames. Rep and cap encode the viral replication and capsid proteins, respectively. In the production of recombinant adeno-associated viral vectors; these viral ITRs are the only elements used in cis, while the viral open reading frame is supplied in trans. The AAV production method using transient transfection of adherent HEK293 cells splits the different genetic elements into separate plasmids, thereby addressing the cis / trans roles of those elements. The pAAV2 / 9 plasmid contains the open reading frames of the AAV2 rep gene and the AAV9 cap gene. [Diagram 2] FIG. 1 shows a process flow chart of the selection of HEK293 cells for exceptional adhesion and pre-master cell bank (MCB) banking. [Diagram 3] A summary of the selection of HEK293 cells for exceptional adherence and cell processing details for pre-master cell bank (MCB) banking is shown. [Figure 4] The upstream process flow for pharmaceutical ingredients is shown below. [Diagram 5] A flow diagram of the downstream process of pharmaceutical ingredients is shown below. [Figure 6] Inactivation of XMuLV by Tween 20 added up to 120 min is shown. [Figure 7] Inactivation of PRV by Tween 20 added up to 120 min is shown. [Figure 8] FIG. 1 shows a HEK293 cell expansion process flow for cell seeding density experiments. [Figure 9-1]Growth and metabolite profiles are shown. HEK 293 cells were seeded in duplicate at 12,000 and 8,000 cells / cm2 in bioreactors (pH 7.23, 37.0 °C, 55% dissolved oxygen (DO)). Cells were transfected with DNA plasmids / PEI 4 days (12,000 cells / cm2) and 5 days (8,000 cells / cm2) after seeding. Bioreactors were harvested 8 days (12,000 cells / cm2) and 9 days (8,000 cells / cm2) after seeding. pH and metabolite readings were taken daily on a Nova BioFlex. [Figure 9-2] This is a continuation of Figure 9-1. [Figure 9-3] This is a continuation of Figure 9-1. [Figure 10] Viral genome production as a function of cell seeding density (8000 or 12000 cells / cm2) and four different transfection time lengths (20 min, 1 h or 2 h) is shown. [Figure 11] 1 shows intermediate viral titers sampled at different filtration steps throughout the manufacturing process. [Figure 12] 1 shows the recovery of viral vectors and removal of host cell proteins (HCPs) in the TFF1 step. [Figure 13] FIG. 1 shows a HEK293 cell expansion process flow for cell seeding density experiments. [Figure 14-1] HEK 293 cells were seeded in duplicate at 8,000, 9,350, 10,700, and 12,050 cells / cm2 in bioreactors (pH 7.23, 37.0 °C, 55% DO). Five days after seeding, cells were transfected with DNA plasmid / PEI (1:1 m / m). pH and metabolite analysis was performed using a NOVA BioProfile 400. [Figure 14-2] This is a continuation of Figure 14-1. [Figure 14-3] This is a continuation of Figure 14-1. [Figure 14-4] This is a continuation of Figure 14-1. [Figure 14-5] This is a continuation of Figure 14-1. [Figure 15-1] Figure 1 shows drug substance production from four starting seeding densities in bioreactors. Comparison of viral titers and vector genomes recovered per unit surface area. [Figure 15-2] This is a continuation of Figure 15-1. [Figure 16] The manufacturing processes for Phase 1 (Process A) and Phase 3 (Process B) are shown. [Figure 17-1] A table showing the comparability and manufacturing consistency results is provided - Process A (Phase 1) and Process B (Phase 3) products. The Process B product is shown to have additional benefits when compared to Process A. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18] A pairwise comparison of Process A (Phase 1 lot NCHAAV9SMN0613) and Process B (Phase 3 lot 600156) is used to demonstrate equivalence between Process A and Process B. The Process B product is shown to have additional benefits when compared to Process A. [Figure 19] 1 shows an assessment of manufacturing consistency through pairwise comparison of Process B (Phase 3) lots 600156 and 600307. [Figure 20] 1 shows the stability profile of NCH lot NCHAAV9SMN0613 stored at real time storage conditions ≦−60° C. for 12 months. [Figure 21] The sedimentation coefficient (sec × 10-1 3) of the first phase material (NCHAAV9SMN0613) is shown, with empty capsids (7%) having a sedimentation coefficient of approximately 60 × 10-13 sec and full capsids having a sedimentation coefficient range of approximately 80 to 150 × 10-13 sec. [Figure 22] The sedimentation coefficient (sec × 10-13) of the third phase material (600156) is shown, with empty capsids (2%) having a sedimentation coefficient of approximately 60 × 10-13 sec and full capsids having a sedimentation coefficient range of approximately 80 to 150 × 10-13 sec. [Figure 23] The sedimentation coefficient (sec × 10-13) of the third phase material (600307) is shown, with empty capsids (4%) having a sedimentation coefficient of approximately 60 × 10-13 sec and full capsids having a sedimentation coefficient range of approximately 80 to 150 × 10-13 sec. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087] Moving the development of AAV gene therapy beyond animal models into clinical trials and / or therapeutic use To advance this goal, we must develop a scalable process capable of producing viral material suitable for human use. We developed a process.

[0088] In some embodiments, a "vector" refers to a plasmid, a phage, a transposon, , cosmids, chromosomes, viruses, virions, etc., linked to appropriate regulatory elements. Any genetic element that has the ability to replicate when in culture and that can transfer genetic sequences between cells Thus, the term includes cloning and expression vehicles, as well as viral vectors. This includes the tar.

[0089] In some embodiments, an "AAV vector" refers to, without limitation, AAV-1, AAV -2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8 and Vectors derived from adeno-associated virus serotypes, including AAV-9, are meant. The vector may contain one or more of the AAV wild-type genes, such as the rep and / or cap genes. The offspring are deleted in whole or in part but retain functional flanking ITR sequences. Functional ITR sequences are required for rescue, replication, and packaging of AAV virions. Thus, AAV vectors, as used herein, are defined as vectors that regulate viral replication and packaging. The ITRs are defined as at least including the sequences provided in the reference (e.g., functional ITRs). It is not necessary for the sequence to be a wild-type nucleotide sequence, but rather that the sequence be capable of functional rescue, replication and replication. Insofar as it provides a packaging, it may be modified, for example, by the insertion, deletion or substitution of nucleotides. In one embodiment, the vector has ITRs from AAV-2. The AAV-9 vector is an AAV-transducing vector that delivers the vector to the nucleus of a target cell. - Also meant is the protein coat or capsid which provides an efficient vehicle for delivery of nucleic acids.

[0090] In some embodiments, "scAAV" refers to a self-complementary adeno-associated virus (scAAV). AAV) is meant to refer to naturally occurring adeno-associated scAAV is a viral vector engineered from the AAV virus. They are called "self-complementary" because they are designed to form an intramolecular double-stranded DNA template. do.

[0091] In some embodiments, the term "vector-associated impurities" refers to any impurity other than authentic recombinant AAV particles. Vector-associated impurities include empty AAV capsids ( "empty" or "empty particles") and other than the intended vector genome AAV particles containing the polynucleotide sequence of These include AAV-encapsidated DNA impurities.

[0092] In some embodiments, a "recombinant virus" refers to the incorporation of a heterologous nucleic acid construct into, e.g., a particle. It means a virus that has been genetically altered by addition or insertion. "Recombinant" is taken from the "r" and For example, rAAV may refer to recombinant AAV. As used herein, "recombinant AAV" or "rAAV" is intended to encompass .

[0093] In some embodiments, an "AAV virion" refers to a wild-type (wt) AAV viral particle. AAV capsid protein envelope containing a linear, single-stranded AAV nucleic acid genome associated with the AAV capsid protein envelope. In this regard, any one AAV virion may contain any number of virus particles, including any one of the virions. A single-stranded AAV nucleic acid molecule in a complementary orientation, e.g., the "sense" strand or the "antisense" strand. can be packaged and both strands are equally infectious.

[0094] In some embodiments, the terms "recombinant AAV virion," "rAAV virion," "AAV vector particle," "complete capsid," and "complete particle" are used herein to refer to both. AAV transcripts that encapsidate a heterologous nucleotide sequence of interest flanked by AAV ITRs. rAAV virions are defined as infectious, replication-defective viruses that contain a protein coat. The AV vector-specifying sequence, AAV helper functions and accessory functions are introduced therein. In this way, the host cell is capable of carrying out subsequent gene delivery. Integrating AAV vectors (recombinant nucleotide sequences of interest) into infectious recombinant virion particles The AAV polypeptides that result in packaging of the AAV vector containing the nucleotide sequence of ... It becomes like this.

[0095] In some embodiments, the terms "empty capsid" and "empty particle" refer to AAV proteins. The plasmid contains a protein coat but contains a heterologous nucleotide sequence of interest flanked on either side by AAV ITRs. It refers to an AAV virion that is lacking in whole or in part a polynucleotide construct.

[0096] The term "host cell" refers to a host cell that contains an AAV helper construct, an AAV vector plasmid, an accessory - Can be used as a functional vector or recipient for other transfer DNA can be or have been used as such, for example, microorganisms, yeast cells, insect cells, and mammalian "Transfected cells" refers to cells that are related to the host or host cell that is transfected with the recombinant gene. This term includes the progeny of the original cell that is transfected. Thus, a "host cell," as used herein, generally refers to a cell that is capable of transfecting an exogenous DNA sequence. refers to a cell that has been infected with a single parent due to natural, accidental, or deliberate mutation. The descendants of a cell will not necessarily be completely similar to the original parent in morphology or in genomic or total DNA complement. It is understood that they are not necessarily identical.

[0097] In another embodiment, the term "AAV helper functions" refers to functions that are expressed to regulate the function of AAV gene products. and the product functions in trans for productive AAV replication. AAV helper functions are coding sequences derived from AAV that contain the major AAV promoters. Contains both open reading frames (ORFs), rep and cap. Rep expression The products include, inter alia: recognition, binding and nicking of the AAV DNA replication origin; DNA helical cleavage; and regulation of transcription from AAV (or other heterologous) promoters. The Cap expression product provides essential packaging functions. AAV helper functions, as used herein, are functions that are missing from an AAV vector. Used to complement the lance.

[0098] In one embodiment, the term "AAV helper construct" generally refers to a nucleotide sequence of interest. The deletion from the AAV vector to be used to generate a transduction vector for delivery of the sequence AAV helper constructs refer to nucleic acid molecules that contain nucleotide sequences that provide AAV functions. The construct confers transient expression of the AAV rep and / or cap genes required for AAV replication. helper constructs are commonly used to complement missing essential AAV functions; however, The construct lacks the AAV ITRs and cannot replicate or package itself. V helper constructs can be plasmids, phages, transposons, cosmids, viruses, or The vector may be in the form of a virion. Generally, the vector encodes both the Rep and Cap expression products. Several AAV vectors, including the plasmids pAAV / Ad and plM29+45, have been used. Looper constructs have been described, e.g., by Samulski et al. (1989) J. Virol.63:3822-3828; and McCarty et al. (19 91) See J. Virol. 65:2936-2945. Rep and / or Ca Several other vectors have been described that encode p expression products. See US Pat. Nos. 6,376,237 and 6,139,941.

[0099] In another embodiment, the term "transfection" refers to the uptake of foreign DNA by a cell. The term "introducing" is used to refer to when a cell has had exogenous DNA introduced inside its cell membrane. A number of transfection techniques are known in the art. For example, Graham et al. (1973) Virolo gy,52:456, Sambrook et al. (1989)Molecular Cloning,a laboratory manual,Cold Spring Harbor Laboratories, New York, Davis et al. l.(1986)Basic Methods in Molecular Biolo Gy, Elsevier, and Chu et al. (1981) Gene 13:19 See, e.g., 7. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell. You can enter.

[0100] As used herein, the term "cell line" refers to a cell line that exhibits continuous or sustained growth in vitro. Furthermore, during storage or transportation of such clonal populations, nuclei may be lost. It is known in the art that natural or induced changes in the type can occur. Therefore, cells derived from the cell lines referred to may not be strictly identical to the ancestral cells or cultures. In some embodiments, the cell lines referred to include such mutants. The terms "HEK293 cells," "293 cells," or grammatical equivalents thereof are used interchangeably herein. Host / packaging cells for use in the methods disclosed herein Refers to stocks.

[0101] In some embodiments, the term "elution fluid" may be used to elute a substance, depending on the context. In some embodiments, the term "eluent" may be understood to refer to the buffer used in the Depending on the context, eluted material, e.g., from a preceding purification step, e.g., assay or may be understood to refer to a product or material that is desirable for further purification.

[0102] In some embodiments, the methods described herein include methods for the manufacture of pharmaceutical and quasi-drug products. Management and quality control regulations (good manufacturing practice: G GMP is a regulatory practice to ensure pharmaceutical quality. For example, the Federal Drug Agency (FDA) GMP regulations establish control over the manufacturing process. Examples of current GMP regulations are: In some embodiments, the methods described herein include: GMP procedures are used for the production of AAV viral vectors on an industrial scale. Industrial-scale production of AAV viral vectors for therapeutic use is challenging due to scalability issues. Thus, in some embodiments, the methods described herein involve the use of AAV viruses. The vectors can be produced, for example, in adherent cells on an industrial scale and at a level of purity sufficient for administration to humans. The term "industrial scale" refers to, for example, a process in which the yield is 1000 mg / kg / production batch. 5×10 15 More than vg or 8 x 10 15 vg or 1×10 16 Like vg super, Methods for producing viral vectors in cells at greater than bench scale, e.g., commercial scale Refers to...

[0103] Upstream process In some embodiments, upstream processes are used to produce intermediates derived from working cell banks. wherein the upstream process includes (a) culturing cells, e.g., adherent cells; (b) culturing (c) transfecting the three plasmids into the cells, e.g., adherent cells; After the incubation period, the expanded virus particles are harvested from the cells, for example 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 purified virus particle intermediate preparation. In some embodiments, the intermediate preparation may be frozen. In embodiments, the intermediate preparation does not have to be frozen prior to downstream processing. In accordance with the present disclosure, the AAV prepared by the upstream process can be used as described herein. The AAV encodes shRNA targeting SOD1 and a polypeptide encoding MECP2. AAV containing a polynucleotide encoding SMN or an AAV containing a polynucleotide encoding SMN. In some embodiments, the upstream process is carried out on an industrial scale under GMP.

[0104] 1. Cell Line Transfection and Culturing In one aspect, a rAAV genome is disclosed herein. The rAAV genome comprises: A polypeptide (including, but not limited to, an SMN polypeptide) is encoded by a gene encoding the polypeptide. siRNAs and shRNAs directed against mutant proteins or regulatory sequences of their genes , antisense, and / or one or more sequences flanking the polynucleotide encoding the miRNA The polynucleotide contains the AAV ITRs described above. NA, specifically promoter DNA, enhancer DNA and polyadenylation signal The gene cassette is also operably linked to the sequence DNA to form a gene cassette. Intron sequences to facilitate processing of RNA transcripts when expressed in mammalian cells It may also include.

[0105] In some embodiments, the rAAV (e.g., rAAV9) genome can be prepared by any method including, but not limited to, Neurological disorders, including spinal cord and brain trauma / injury, stroke, and brain cancer, as well as Alzheimer's and the like. Non-limiting examples of known nervous system growth factors include nerve growth factor (NGF) and GF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), Neurotrophin-4 / 5 (NT-4 / 5), neurotrophin-6 (NT-6), ciliary Neoplastic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), fibroblasts Growth factor family (e.g., FGF1-15), leukemia inhibitory factor (LIF), insulin Certain members of the insulin-like growth factor family (e.g., IGF-1), neurturin, -Cephin, bone morphogenetic protein (BMP), immunophilin, transforming growth factor (T GF) growth factor family, neuregulin, epidermal growth factor (EGF), platelet-derived growth factor Factors (PDGF), Vascular Endothelial Growth Factor Family (e.g. VEGF165), Forista In general, the trophic factors or protective factors contemplated herein include, for example, Zinc finger transcription factors that regulate each of the protective factors are also contemplated. Thus, methods for modulating neuroimmune function include, but are not limited to, controlling microglia and astrocytes. Inhibition of logitocyte activation, for example by NFkB inhibition, or by siRNA, shRNA, or anti- NFkB (NFkB) signaling for neuroprotection by sense or miRNA in various cell types In yet another embodiment, the present invention contemplates the use of a medicament for the treatment of cancer, including the dual action of FkB and related pathways. The rAAV (e.g., rAAV9) genome encodes apoptosis inhibitors (e.g., bcl2, bc lxL) encoding a trophic factor or spinal cord injury-modulating protein for the treatment of spinal cord injury. or inhibitors of axon growth inhibitors (e.g., inhibitors of Nogo [Oertle et al. al.,The Journal of Neuroscience,23(13):5 The use of rAAVs (e.g., rAAV9) encoding the rAAV 393-5406 (2003) has also been reported. It is planned.

[0106] For the treatment of neurodegenerative disorders such as Parkinson's disease, rAAV (e.g., rAAV9) genomes are being developed. In various embodiments, the enzyme is an aromatic acid dopa decarboxylase (AADC), a tyrosine kinase inhibitor, Synthyl hydroxylase, GTP cyclohydrolase 1 (GTPCH1), apoptosis inhibitor Damaging factors (e.g., bcl2, bclxL), glial cell line-derived neurotrophic factor (GDNF) , an enzyme involved in the synthesis of the inhibitory neurotransmitter GABA or dopamine In a further embodiment, the rAAV (e.g., rAAV9) genome encodes, e.g., For example, it may encode a modulator of parkin and / or synuclein.

[0107] For the treatment of neurodegenerative disorders such as Alzheimer's disease, in some embodiments, Methods for increasing choline acetylcholine production are contemplated. In some embodiments, Methods for increasing levels of acetylcholinesterase (ChAT) A method of inhibiting the activity of AchE is contemplated.

[0108] In some embodiments, the rAAV (e.g., rAAV9) genome can be used to treat diseases such as Huntington's disease. Reducing mutant huntingtin protein (htt) expression for the treatment of neurodegenerative disorders siRNA, shRNA, antisense, and / or miRNA for use in methods of reducing Code A.

[0109] The rAAV (e.g., rAAV9) genome, in various embodiments, is used to treat neurological disorders such as ALS. siRNA, shRNA, antisense, and / or miRNA for use in the treatment of degenerative disorders The treatment involves the administration of TNF-α, nitric oxide, peroxynitrite, and and / or resulting in the downregulation of molecular markers of disease, such as nitric oxide synthase (NOS) .

[0110] In some embodiments, the vector comprises a superoxide dismutase (SDS) inhibitor for ALS. GDN for ALS or Parkinson's disease Small molecule targets for mutant proteins, such as neurotrophins like F or IGF1. It encodes an apin RNA (shRNA).

[0111] In one embodiment, the methods and materials described herein are used to treat ALS. ALS is a neurodegenerative disorder that results in progressive loss of motor neurons in the brain and spinal cord. It is a disease with symptoms including loss of ability to speak, eat, move and eventually breathe. Patients typically die within 3-5 years of diagnosis. 90-95% of ALS cases are due to idiopathic causes. Although it is unclear, some ALS cases are caused by a genetic mutation in the superoxide dismutase 1 (SOD1) gene. It is caused by spontaneous mutations, where the mutation causes a dominant gain of virulence. In mouse studies, knockout of SOD1 was shown to abolish disease, and thus Therapies that knock down levels of mutant SOD1 are believed to alleviate disease symptoms. can be obtained.

[0112] In some embodiments, the AAV vector targets SOD1 for ALS. An exemplary AAV encoding an shRNA against SOD1, e.g. For example, scAAV9 constructs are described in WO 2015031392 and U.S. Pat. Publication No. 2016272976, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, shRNA against SOD1 is provided in The loading AAV constructs may be prepared using the methods disclosed herein. In embodiments, such AAV constructs may be used to treat ALS. In embodiments, the SOD1 AAV is less than 10%, e.g., 7%, 5%, 4%, 3% In some embodiments, the SOD1 A antibody exhibits less than 1%, 2%, or 1% empty capsids. AV can be used in low amounts, e.g., as discussed herein with respect to the preparation and purification of AAV vectors. Bell residual host cell proteins, host cell DNA, plasmid DNA, and / or endonuclease As used herein, "AVXS-301" refers to the scAAV9 vector. A non-limiting example of a vector is a polynucleotide encoding an anti-human SOD1 shRNA. pSOD1sh, modified AAV2 ITR, human H1 promoter, and The modified and unmodified AAV2 ITRs were used in combination with anti-human SOD1 shRN It may be in either orientation (ie, 5' or 3') relative to the A expression cassette.

[0113] As used herein, the "pSOD1sh" vector plasmid refers to a SuperOxygen A short hairpin RNA (shRNA) targeting the expression of the steroid dismutase 1 (SOD1) gene was a polynucleotide encoding an anti-SOD1 shRNA cassette, Here, the cassette contains, for example, the "left" and "right" are flanked by adeno-associated virus inverted terminal repeat (ITR) sequences. In an embodiment, the polynucleotide encoding pSOD1sh is a human SOD1 mRNA. In some embodiments, the nucleic acid is transcribed into a short hairpin RNA that specifically targets the NA. The ITR sequences surrounding the polynucleotide encoding pSOD1sh may be naturally occurring, mutant, or are modified AAV ITR sequences. In some embodiments, at least one ITR sequence The sequence is a native, mutant or modified AAV2 ITR sequence. The TR flanks the polynucleotide encoding pSOD1sh. wherein the two ITR sequences are both natural, mutant or modified AAV2 ITR sequences. In some embodiments, the "left" ITR allows for the production of a self-complementary genome. The modified AAV2 ITR sequence and the "right" ITR is the native AAV2 ITR sequence. In some embodiments, the "right" ITR is modified to allow for the production of a self-complementary genome. AAV2 ITR sequence, and the "left" ITR is the native AAV2 ITR sequence. In one embodiment, the pSOD1sh vector is, for example, as described by Myslinksi. The Myslin further comprises a segment of the human H1 RNA promoter as shown in kski et al. “An unusually compact externa l promoter for RNA polymerase III transc ription of the human H1RNA gene.”Nucleic Acids Research, 29(12):2502-2509. In order to increase the size of the expression cassette, the pSOD1sh vector was randomly It further contains a unique stuffer sequence made up of a segment of the plasmid backbone. In some embodiments, the pSOD1sh vector comprises an anti-human SOD1 shRNA, modified AAV2 ITR, human H1 promoter, and a gene encoding the unmodified AAV2 ITR Contains nucleotides.

[0114] In one embodiment, the methods and materials described herein are used to treat neurological disorders such as Rett Syndrome. It may be used to treat Rett Syndrome, a rare developmental disorder first recognized in infancy. It is a neurological disorder, and in 90-95% of cases, it is caused by a mutation in the MECP2 gene on the X chromosome. Ruthie et al., “Rett syndrome is a aused by mutations in X-linked MECP2,enc oding methyl-CpG-binding protein 2.”Natu re Genetics, 23:185-188. They have only one copy of the X chromosome. Boys typically die shortly after birth, but girls who have two copies of the X chromosome usually die. They usually have one functional copy of this gene. Symptoms begin to appear between 6 and 18 months of age and are usually treated by hand massage or It is accompanied by characteristic symptoms such as hand-wringing, hand-slapping, hand-rubbing, hand-washing, or putting hands in the mouth. The disease is progressive and includes autistic-like behavior, irregular breathing, feeding and swallowing difficulties, developmental delays and seizures. There are 200 known mutations in the MECP2 gene that cause the disease. The severity of the disease varies widely among patients depending on the level of X-chromosome inactivation and dosage correction. Mouse studies show that MECP2 mutations do not cause neuronal death. This suggests that this is not a neurodegenerative disorder. sal of Neurological Defects in a Mouse M odel of Rett Syndrome.”Science,315(5815) ”1143-1147.

[0115] For embodiments relating to Rett Syndrome, the rAAV (e.g., rAAV9) genome can be, e.g., For example, it may encode methylcytosine-binding protein 2 (MeCP2). Exemplary AAV constructs, such as scAAV9 constructs, that include a polynucleotide encoding the AAV polypeptide are described in U.S. Pat. No. 9,415,121, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, a polypeptide comprising a polynucleotide encoding MeCP2 is provided. The AV construct may be prepared using the methods disclosed herein. In some embodiments, such AAV constructs may be used to treat Rett syndrome. In terms of morphology, MeCP2 AAV is less than 10%, for example, 7%, 5%, 4%, 3%, In some embodiments, the MeCP2 A exhibits less than 2%, or 1% empty capsids. AV can be used in low amounts, e.g., as discussed herein with respect to the preparation and purification of AAV vectors. Bell residual host cell proteins, host cell DNA, plasmid DNA, and / or endonuclease It presents a toxin.

[0116] As used herein, "AVXS-201" refers to a non-limiting example of a scAAV9 vector. Examples include a polynucleotide comprising a MECP2 cDNA expression cassette (e.g., pMECP2), modified AAV2 ITR, mouse Mecp2 promoter, modified SV40 Intron, minimal polyadenylation signal, and unmodified AAV2 ITR The modified and unmodified ITRs are either The orientation may be either 5' or 3'.

[0117] As used herein, a "pMECP2" vector plasmid refers to a MECP2 transcription factor. A polynucleotide encoding the protein, modified AAV2 ITR, mouse Mecp2 promoter, A modified SV40 intron, a minimal polyadenylation signal, and In some embodiments, pMECP2 comprises a modified AAV2 ITR. A vector construct comprising a polynucleotide encoding the MECP2 cDNA. A expression cassette, where the cassette contains, for example, a gene encoding the MECP2 gene. The "left" and "right" adeno-associated virus inverted terminal repeats (ITRs) of the polynucleotide In some embodiments, the polynucleotide encoding MECP2 is , a human MECP2 sequence, e.g., a naturally occurring human MECP2 sequence or an isoform thereof. In some embodiments, the ITR sequences are In some embodiments, the AAV ITR sequences are natural, mutant, or modified AAV ITR sequences. One ITR sequence is a native, mutant or modified AAV2 ITR sequence. In one embodiment, the two ITR sequences are both natural, mutant or modified AAV2 ITR sequences. In some embodiments, the "left" ITR allows for the production of a self-complementary genome. and the "right" ITR is the native AAV2 ITR sequence. In some embodiments, the "right" ITR allows for the production of a self-complementary genome. and the "left" ITR is the native AAV2 ITR sequence. In some embodiments, the pMECP2 vector contains the sequence of the mouse Mecp2 promoter. In some embodiments, the pMECP2 vector further comprises, but not all, of the segments. further comprises a Simian Virus 40 (SV40) intron. The pMECP2 vector is described, for example, in Levitt et al., "Definition of pMECP2 n of an efficient synthetic poly(A) site .” Genes & Development, 3:1019-1025 It further contains a minimal polyadenylation signal, as described above.

[0118] In some embodiments, the rAAV genome disclosed herein is The AAV DNA in the rAAV genome (e.g., ITRs) is , 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 AA The recombinant virus may be derived from any AAV serotype, including V-11. The nucleotide sequences of the genomes of these AAV serotypes are known in the art. For example, the complete genome of AAV-1 is provided in GenBank under accession number NC_002077. The complete genome of AAV-2 is available under GenBank accession numbers NC 001401 and Sri vastava et al., Virol., 45:555-564{1983). The complete genome of AAV-3 is available under GenBank accession number NC_1829; The complete genome of AAV-4 is provided in GenBank accession number NC_001829; The V-5 genome is available under GenBank accession number AF085716; The genomes are available under GenBank accession number NC_001862; AAV-7 and AAV At least a portion of the -8 genome is identified in GenBank accession numbers AX753246 and AX753247, respectively. and AX753249; the AAV-9 genome was provided by Gao et al., J. Virol. Rol., 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); and AAV-1 1 genome is provided in Virology, 330(2):375-383 (2004). do.

[0119] As used herein, a "pSMN" vector plasmid contains the SMN protein. The polynucleotide encoding the SMN cDNA is an expression cassette, The cassette can include, for example, a "left" and a "right" sequence of a polynucleotide encoding the SMN gene. In some embodiments, the sequence is flanked by adeno-associated virus inverted terminal repeat (ITR) sequences. Thus, a polynucleotide encoding SMN can be chosen from a human SMN sequence, e.g., a naturally occurring SMN sequence. The human SMN sequence or an isoform, variant, or mutant thereof. In embodiments, the ITR sequences are native, mutant, or modified AAV ITR sequences. In some embodiments, at least one ITR sequence is selected from the group consisting of a native, mutant, or modified AAV In some embodiments, the two ITR sequences are both naturally occurring. , mutant, or modified AAV2 ITR sequences. TR is a modified AAV2 ITR sequence that allows for the production of a self-complementary genome, The ITR of is a native AAV2 ITR sequence. In some embodiments, the "right" ITR is a modified AAV2 ITR sequence that allows for the production of self-complementary genomes, and the "left" I TR is a native AAV2 ITR sequence. It further contains a CMV enhancer / chicken beta-actin ("CB") promoter. In some embodiments, the pSMN plasmid is a Simian Virus 40 (SV40) insert. In some embodiments, the pSMN plasmid further comprises bovine growth hormone (BGH). GH) further comprising a polyadenylation (polyA) termination signal. Exemplary sequences that may be used for one or more of the following are shown in Table 1 below. All sequences shown in Table 1 below are used. In some embodiments, "AVXS-1 "01" refers to a vector that uses all of the sequences in Table 1 and is included within the term pSMN. Non-limiting examples of constructs.

[0120] In some embodiments, the pSMN vector comprises an SMN cDNA expression cassette, modified AAV2 ITR, chicken β-actin (CB) promoter, cytomegalovirus (CMV) immediate early enhancer, modified SV40 late 16s intron, bovine growth hormone (BGH) polyadenylation signal, and unmodified AAV2 ITRs. The unmodified ITRs can be oriented in either direction (i.e., 5' or may be on the 3' side).

[0121] In some embodiments, for example, during the manufacturing processes described herein, the vector construct The sequence is encapsidated, for example, into the AAV9 virion. Cysidation results in the production of stable, functional transgenes, e.g., a fully functional human SMN transgene. Capable of delivering genes, MECP2 transgenes, or anti-SOD1 shRNA In some embodiments, the capsidation is in a non-replicative recombinant AAV9 capsid. The capsid contains two truncated versions of VP1, VP2 and VP3, and all share the same 60 viruses produced by alternative splicing that have the C-terminal sequence of The proteins (VP1, VP2, VP3) are, for example, in a ratio of 1:1:10. In this embodiment, the product of this manufacturing process, e.g., a drug product, is a stable, fully functional human SM. Deliver the N transgene, MECP2 transgene, or anti-SOD1 shRNA In some embodiments, the capsid may comprise a non-replicating recombinant AAV9 capsid. VP2 and VP3 are two truncated forms of VP1, all of which share a common C-terminal sequence 60 viral proteins produced by alternative splicing, such as It contains VP1, VP2, and VP3 in a ratio of 1:1:10.

[0122] In some embodiments, the amount of functional viral vector is determined by a suitable in vitro cell assay. Determined by % of functional vg / mL when measured using serine or in vivo animal models For example, the percentage of functional AAV SMNs is determined in animal models of SMA, e.g., SMAΔ7 by relative potency using mice or a suitable cell line, e.g., SMAΔ7 mouse cortex. Quantitative cell-based assay using primary neural progenitor cells (NPCs) isolated from The percentage of functional AAV MeCP2 may be assayed using a suitable in vitro cell assay. In vivo animal models, such as Mecp2 knockout mice, The percent of functional AAV SOD1 may be determined using a suitable in vitro cell assay or in vivo It may also be assayed using an animal model, for example, an SOD1 mutant mouse.

[0123] The DNA sequence of an exemplary vector construct, such as AVXS-101, is shown in Table 1.

[0124] [Table 2]

[0125] In another embodiment, the DNA sequence of the AVXS-101 vector construct is provided in SEQ ID NO:1. To be: [ka] [ka]

[0126] In some embodiments, the SMN protein encoded by the pSMN plasmid The amino acid sequence of comprises: [ka]

[0127] In some embodiments, the AAV capsid proteins VP1, VP2, and VP3 are co-transfected. These have alternative initiation sites but share a common carboxy terminus. Below, VP1-specific amino acid sequences are shown in black and bold. The amino acid sequences common to all three capsid proteins are underlined and italicized. The amino acids that are selected are in bold and italics. [ka]

[0128] In one embodiment, the AAV capsid protein has the amino acid sequence shown in SEQ ID NO:3. The sequence is derived from the transcript encoding the sequence.

[0129] In another aspect, disclosed herein is a DNA plasmid comprising the rAAV genome. This DNA plasmid is used to generate infectious viral particles carrying the AAV9 capsid protein. To assemble the rAAV genome, an AAV helper virus (e.g., adenovirus) is used. Virus, E1-deleted adenovirus, or herpesvirus) into permissive cells. Techniques for producing rAAV particles are standard in the art and include: The AAV genome to be packaged, the rep and cap genes, and the helper gene In some embodiments, the production of rAAV involves the provision of a single The following components are present in the cells (herein referred to as packaging cells): rAA V genome, AAV rep and cap genes separate from (i.e., not present in) the rAAV genome The production of pseudotyped rAAV is described in detail in the literature. See, for example, International Publication No. WO 01 / 83692, which is incorporated herein by reference in its entirety. In various embodiments, the AAV capsid protein is modified. Modification of the capsid protein can facilitate delivery of the recombinant vector. For example, U.S. Patent Application Publication No. 2005 / 0053 is a method for producing a fluororesin-based fluororesin. No. 922 and U.S. Patent Application Publication No. 2009 / 0202490 (these disclosures See, for example, US Pat. No. 6,393,363, which is incorporated herein by reference in its entirety.

[0130] For general principles of rAAV production, see, e.g., Carter, 1992, Currell, et al. nt Opinions in Biotechnology, 1533-539; and Muzyczka,1992,CUM Topics in Microbial.an The various methods are reviewed in (D Immunol., 158:97-129). Ratschin et al.,Mol.Cell.Biol.4:2072(198 4);Hennonat et al.,Proc.Natl.Acad.Sci.US A,81:6466(1984);Tratschin et al.,Mol.Cel l.Biol.5:3251(1985);McLaughlin et al.,J. Virol.,62:1963(1988); and Lebkowski et al., 1988 Mol.Cell.Biol.,7:349(1988).Samulski et al. (1989, J. Virol., 63:3822-3828); US patent No. 5,173,414; International Publication No. WO 95 / 13365 and corresponding No. 5,658,776; WO 95 / 13392 ;International Publication No. 96 / 17947; PCT / US98 / 18600 ;International Publication No. 97 / 09441 (PCT / US96 / 14423 specification ); International Publication No. 97 / 08298 Brochure (PCT / US96 / 13872 Details International Publication No. 97 / 21825 (PCT / US96 / 20777) Detailed description); International Publication No. 97 / 06243 Pamphlet (PCT / FR96 / 01064 WO 99 / 11764; Perrin et al. 1995)Vaccine 13:1244-1250;Paul et al.(19 93)Human Gene Therapy 4:609-615;Clark et al. al. (1996) Gene Therapy 3:1124-1132; U.S. Pat. No. 5,786,211; U.S. Pat. No. 5,871,982; and U.S. Pat. The aforementioned publications are particularly relevant to rAAV production. With emphasis on the sections of these documents which are incorporated herein by reference in their entirety. It is incorporated herein by reference.

[0131] An exemplary method for producing packaging cells is to stably express all components necessary for AAV particle production. For example, the goal is to create a cell line that expresses the AAV rep and cap genes. a rAAV genome containing the AAV rep and cap genes separate from the rAAV genome; A plasmid (or multiple plasmids) containing a selectable marker such as a neomycin resistance gene. The AAV genome is integrated into the cell's genome using the GC tail method (Samulski et al.,1982,Proc.Natl.Acad.S6.USA,79:20 77-2081), addition of a synthetic linker containing a restriction endonuclease cleavage site (Lau ghlin et al., 1983, Gene, 23:65-73) or directly Slip-end ligation (Senapathy & Carter, 1984, J. Biol. (I. Chem., 259:4661-4666) Next, the packaging cell line is infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. In another example of a preferred method, the vector is an adenovirus or baculovirus rather than a plasmid. The rAAV genome and / or rep and cap genes are packaged using the virus. is introduced into cells.

[0132] Thus, the disclosure herein provides, in various embodiments, a method for producing infectious rAAV. The packaging cells are non-adherent cells cultured in suspension. In one embodiment, the cells may be HeLa cells, HELa cells, or adherent cells. Any suitable package, such as K293 cells and PerC.6 cells (cognate 293 strain), may be used. In one embodiment, the cell line is HEK 293. It is a cell.

[0133] To increase the yield of viral vector production, adherent cells were cultured and attached to culture flasks. In some embodiments, the downstream bioreactor may be selected for improved adhesion. Improved transfection efficiency and cell number at the seeding step. Cells can be detached from the cell culture surface by methods known in the art. The cells can be isolated by scraping or by incubation in a solution containing a protease. In an exemplary embodiment, the HEK293 cells may be washed with PBS. The cells may be dissociated with trypsin for approximately 2 minutes at room temperature. Growth medium containing serum may be added. Dissociation can be stopped by adding water or by repeatedly pipetting the suspension. The cell clumps may be dissociated. The cell suspension may then be pelleted, and the isolated pellets may be The cells may then be resuspended in a suitable complete growth medium. The cells are then seeded into new cell culture chambers. Cells that do not adhere to the surface after a certain period of time are considered to be cells that have not fully grown in cell culture medium. The cells may be removed by gentle aspiration along with the cell culture medium before being replaced with new medium. In some embodiments, the time for allowing the cells to adhere is about 2 hours, about 3 hours, about 4 hours, or about 6 hours. Once the cells have expanded, this step may be repeated for about 1 hour, about 5 hours, about 6 hours, or about 7 hours. This may be repeated to increase the fraction of cells that are strongly adherent to the culture flask. In some embodiments, this step is performed at least twice, at least three times, at least four times, at least In an exemplary embodiment, the HEK29 3 cells is 75 cm 2 The cells were seeded in a flask and allowed to adhere for 4 hours in a 37°C incubator. Afterwards, weakly adherent cells are removed by aspiration and the cell culture medium is replaced. In this study, the process of selecting strongly adherent cells was repeated over three cell culture passages. will be done.

[0134] In other embodiments, the rAAV9 (i.e., infectious encapsidated rAAV9 particles) are The rAAV genome disclosed herein includes a self-complementary rAAV genome. It is a genome.

[0135] In another embodiment, a rAAV is provided, such as rAAV9, referred to as "rAAV SMN." In some embodiments, the rAAV SMN genome is sequentially ITR, chicken-β-actin promoter with cytomegalovirus enhancer; SV40 intron, polynucleotide encoding SMN, polynucleotide derived from bovine growth hormone In some embodiments, the AAV2 ITR comprises a recombinant AAV2 ITR, a recombinant AAV2 ITR, and a recombinant AAV2 ITR. In the present invention, a polynucleotide encoding SMN is, for example, set forth in GenBank Accession No. MN_ 000344.2, GenBank Accession No. NM_017411, or any other suitable The human SMN gene is represented in or derived from the human SMN isoform. Target SMN sequences include the following sequences: [ka]

[0136] Conservative nucleotide substitutions of SMN DNA are also contemplated (see, e.g., GenBank (guanine to adenine change at position 625 of accession no. NM_000344.2 ). In some embodiments, the genome lacks AAV rep and cap DNA, and There is no AAV rep or cap DNA between the ITRs of the genome. Peptides include, but are not limited to, NCBI protein database number NP_00 In an embodiment, the human SMN1 polypeptide is represented by S MN DNA may be prepared by synthesising human SMN polypeptide (e.g., Uniprot accession no. Q1663 7, human SMN protein identified by isoform 1 (Q16637-1) Also included are polynucleotides encoding the SMN1 modulator polypeptide plastin. -3 (PLS3) is also contemplated [Oprea et al., Science 320( 5875):524-527 (2008)]. Sequences encoding other polypeptides are It may be replaced with N DNA.

[0137] Prior to transfection, cells are cultured in flasks or suitable bioreactors, or both. In some embodiments, the cells are expanded in a suitable culture medium at 4° C. In one embodiment, the culture medium may be expanded in a bioreactor that provides a continuous circulation of the culture medium. , cells are 200m 2 , 333m 2 , or 500m 2 iCELLis Bioreactor The culture medium contains 5-10% FBS, 4.5 g / L glucose, DMEM containing 4 mM L-glutamine. In some embodiments, adherent cells are resuspended in DMEM. The medium in the circulating medium bag is added and circulated through the bioreactor. The cell culture medium or any other medium is continuously pumped into the bioreactor using a peristaltic pump. Cells are cultured and transfected in flasks or bioreactors. Cells can be seeded at a density appropriate for the transfection. The seeding density depends on the cell type and the time until transfection. In some embodiments, the cells are about 8,000-16,000 cells / cm. 2 In one embodiment, HEK293 cells are seeded at 8,000 to 12,000 cells. / cm 2 are sown in.

[0138] Suitable methods for transducing and retransducing transduced cells into a subject are known in the art. In one embodiment, the cells are grown in vitro, e.g., in a suitable medium, to express the rAAV. The cells are then subjected to Southern blot and / or PCR etc. to identify cells carrying the DNA of interest. Screening using any conventional technique or by the use of selectable markers The vector can be transduced by

[0139] In some embodiments, the packaging cell line contains three plasmids: the AAV vector. A plasmid encoding or containing the vector sequence to be packaged into (e.g., pSMN, pMECP2transgene, or pSOD1sh), pHELP and pAAV2 / 9 are transfected. The transfection is not limited to However, electroporation, e.g., lipofectamine, cationic polymers, and cationic lipids, This can be done using any technique known in the art, including lipofection, which Any suitable transfection medium may be used. In one embodiment of the process, adherent human embryonic kidney (HEK293) cells are transfected with the triplex DNA plasmid. Polyethylenimine (PEI) co-precipitates are transfected. , scAAV9.CB.SMN vector (containing the CB promoter and a polynucleotide encoding SMN) The self-complementary AAV9 vectors containing the nucleotides were then transfected into large-scale adherent cell bioreactors. Triple DNA Plasmid Transfection into Adherent HEK293 Cells Using PEI Co-Precipitation in In one embodiment, the DM used for cell expansion is The EM growth medium is replaced with a modified DMEM transfection medium. It is formulated without calcium and L-glutamine. The medium was FBS-free, calcium-free, L-glutamine-free, and 4.5 g / L glutamine. In some embodiments, serum-free (e.g., FBS-free) DMEM is used. The transfection medium of the present invention improves the efficiency of transfection. In this study, the transfection medium was OptiMEM (Invitrogen / Th In one embodiment, three plasmids (pSMN, pH ELP and pAAV2 / 9) were mixed with PEI in the transfection medium and reacted. In some embodiments, the three plasmids are mixed together in a molar ratio of about 1:1:1. In some embodiments, the plasmid and PEI are mixed in a 1:1 ratio by weight of DNA:PEI. In some embodiments, the plasmid and PEI are mixed in a ratio of less than 1:1. In one embodiment, pSMN, pHELP and pAA are mixed in a weight ratio of 0.1:1. V2 / 9 is mixed in OptiMEM medium at a molar ratio of 1:1:1. In the present invention, PEI is added so that the ratio of DNA to PEI is 1:1 by weight. In embodiments, the reaction occurs for 0 to 60 minutes, or for 10 to 45 minutes, or for 20 to 30 minutes. In one embodiment, the reaction is allowed to occur for 15 to 30 minutes.

[0140] In one embodiment, the present disclosure provides a method for producing an AAV-based viral vector. This method involves (i) culturing adherent HEK293 cells in an industrial-scale bioreactor; (2) transfecting the adherent cells with the plasmid for less than 60 minutes. to allow for the production of AAV vectors, and optionally further processing, purification, preparation Applying formulation and filling steps to produce the pharmaceutical product. In its morphology, the scAAV9.CB.SMN vector is polyethylenimine (PEI " ) are produced using triple DNA plasmid transfection with co-precipitation. In one embodiment, the three plasmids utilized in this transfection are p SMN, pAAV2 / 9, and pHELP.

[0141] Transfection involves contacting the packaging cell line with the DNA-PEI co-precipitate. In some embodiments, the transfection may be performed by adding DNA The A-PEI co-precipitate is loaded into a medium recirculation bag. The DNA-PEI coprecipitate in the reaction medium circulates through the bioreactor and is completely absorbed into the growth medium. In some embodiments, the DNA-PEI co-transfection medium The precipitate is contacted with adherent cells in a bioreactor. The DNA-PEI coprecipitate in the reaction medium was incubated in the bioreactor for up to 2 hours. In some embodiments, the transfection is carried out for 1-2 hours. In some embodiments, transfection is performed in less than 1 hour, e.g., 10 minutes. In some embodiments, the transfer is performed for 20 minutes, 30 minutes, 40 minutes, or 50 minutes. In some embodiments, the transfection is carried out for 1-2 hours. Recirculate complete growth medium through the bioreactor to ensure complete incorporation of the transfection medium. It is stopped by replacing it.

[0142] 2. Recovery of Expanded Viral Particles After a suitable cell expansion time following transfection, in some embodiments, the cells In some embodiments, the cells are lysed to recover the viral particles. In some embodiments, the cells are dissociated from the reactor prior to initiation. Optionally, the viral particles are recovered without lysis. In some embodiments, the endonuclease is added to a final target concentration, e.g., in a bioreactor. Endonucleases degrade both DNA and RNA. In one embodiment, the endonuclease is available under the trade name Benzonase. Serratia marcescens, sold under the trademark Serratia marcescens, by EMD Millipore A genetically engineered endonuclease from Serratia marcescens clease (Eaves, Gnet al. J. Bact. 1963, 85, 273-278;Nestle,M.et al.J.Biol.Chem.1969, 244, 5219-5225). This enzyme was synthesized in strain K containing the pNUC1 production plasmid. Produced and purified from E. coli strain W3110, a mutant of 12 ( U.S. Pat. No. 5,173,418, hereby incorporated by reference in its entirety. Structurally, the protein is approximately 30 kDa with two critical disulfide bonds. It is a dimer of identical 245 amino acids of the Da subunit. ase® is a biocompatible polymer that can be used to detect all forms of DNA and RNA (single-stranded, double-stranded, linear and circular). It decomposes nucleic acids into 2-5 base long 5'-monophosphates, which are effective under a wide variety of working conditions. Digest to terminal oligonucleotides. Benzonase® The current regulations for manufacturing and quality control of pharmaceuticals and quasi-drugs (current goo d manufacturing practice (cGMP) and Thus, it can be used in industrial-scale protein and / or virus particle purification processes. Other endonucleases produced under cGMP conditions may also be purified using the methods disclosed herein. In one embodiment, benzonase can be used in the method. U / ml, e.g. 75-150 U / ml, e.g. to a final concentration of about 100 U / ml. In some embodiments, the addition of benzonase results in High vg production in the bioreactor is achieved while significantly reducing host cell DNA.

[0143] In some embodiments, the endonuclease is added prior to adding the lysis buffer to the reactor. In some embodiments, the cell lysis solution is allowed to mix with the adherent cells for up to 1 hour. Allow to mix for up to 2 hours, up to 3 hours, up to 4 hours, or up to 5 hours. In the present invention, the lysis buffer contains magnesium chloride and / or Tween-20 in a suitable buffer. In an exemplary embodiment, the lysis buffer may include 500 mM HEPES, 10% Tween 20, 20 mM MgCl2, pH 8.0. In some cases, salt sucrose solution (SSS) may be added to quench the benzonase reaction. In this formulation, SSS is added to a collection bag containing rinse buffer and mixed for 15 minutes. In an embodiment, the bioreactor is rinsed with a bioreactor rinse buffer and then The rinse solution is then poured into the harvest collection bag along with the quenched cell lysis solution and lysed cell contents. are collected together, all together comprising the bulk harvest. Bioreactor rinse buffer contains Tris, MgCl2, NaCl, Tween-20 and sucrose. In an exemplary embodiment, the bioreactor rinse buffer may include 2 0 mM Tris, 1 mM MgCl2, 500 mM NaCl, 1% Tween-20 w / v and 1% sucrose w / v, pH 8.1.

[0144] 3. Purification of Viral Particles After harvest, the bulk harvest virus particles are typically concentrated and purified by filtration. In one embodiment, the viral particles are purified by depth filtration followed by removal of larger molecular contaminants. and a filter to remove cell debris, e.g., a 0.45 μm filter, but The target genome is filtered by filtration through a filter that allows it to pass through. Any suitable depth filter may be used.

[0145] As understood in the art, depth filtration refers to the process of removing large amounts of large particles (e.g., A solution containing intact cells or cell debris can rapidly become clogged under such conditions. Refers to the use of porous filtration media for clarification, as compared to membrane filtration. Depth filtration media are available from Millipore, Pall, General Electric, and Sartorious.

[0146] Depth filtration target flow rates may be reduced to keep filter inlet pressures within specifications. After all the bulk harvest has been filtered, the depth filter may, in certain embodiments, be The diafiltration step used in the first tangential flow filtration step ("TFF1") The depth filter pool may be chased with a treatment buffer. The liquid is mixed. The depth filter pool liquid is then filtered through a 0.45 μm filter. The bulk harvest material may then be further clarified through a 0.45 μm filter. 1 buffer.

[0147] 4. Tangential flow filtration In various embodiments, tangential flow filtration is performed, e.g., It is used to concentrate the bulk harvest using low pressure filtration and to remove salts and proteins. Transient flow filtration (TFF) (also called cross-flow filtration (CFF)) is a technique that is well known to those skilled in the art. The technique is widely known and includes, but is not limited to, the equipment and protocols for its implementation in a wide range of situations. However, Pall Corporation, Port Washington, NY and and Spectrum Labs, Rancho Dominguez, CA. Generally, TFF involves recirculation of the retentate across the surface of the membrane. This gentle cross-flow feed may, in certain embodiments, minimize membrane fouling. In one embodiment, the TF The F step may be accomplished in a flat sheet system, as exemplified herein. Flat sheet systems may be used in large scale production, where such systems , a means to prevent excessive shear forces from being applied to the virus particles (e.g., an open flow channel, Alternatively, the TFF step may comprise a hollow fiber system, as exemplified herein. In one embodiment, the molecular weight cutoff (MW CO) is 200-400 kDa, for example, about 300 kDa.

[0148] In one embodiment, the TFF1 step is carried out using a 300 kDa MW cutoff regenerated cellulosic acid extract. This is performed using a gas membrane cassette, which is flushed and then rinsed with a NaOH solution. The solution is sanitized and equilibrated with TFF1 buffer. In one embodiment, the solution is sanitized and equilibrated with TFF1 buffer. 20 mM Tris, 1 mM MgCl2, 500 mM NaCl, 1% sucrose, pH 8 Includes .1.

[0149] In some embodiments, the concentration phase of the TFF1 step comprises a clarified harvest volume of The volume is selected to reduce the retentate by approximately 10-fold. After the target retentate volume is reached, diafiltration is performed. Diafiltration of the retentate may be performed in some cases. In some embodiments, the reaction can be carried out in about 6 diavolumes of TFF1 buffer. The retentate is diafiltered for about 5 to 20, or 10 to 15, or 1 The flow rate is 6 diavolumes of permeate total flow. Once this is achieved, the retentate may be concentrated again and recovered. To achieve this, a rinse of the membrane may be performed, for example two successive rinses.

[0150] 5. Intermediate products In some embodiments, the intermediate drug substance is then frozen on dry ice or in a freezer. and then transferred to storage at ≦−60° C. In another embodiment, prior to downstream processing There is no need to freeze the intermediate product.

[0151] In some embodiments, the cellulose may be used for further processing (e.g., as described herein). (for downstream purification) and multiple intermediate product raw materials Multiple intermediate product lots may be pooled prior to freezing and storage. In another embodiment, the multiple intermediate product drug lots may include frozen and stored lots. may be pooled after thawing.

[0152] downstream process In some embodiments, a downstream process is used to filter the intermediate product (e.g., a pooled intermediate product). In some embodiments, downstream processing steps include: (a) acidifying (b) purification and clarification (e.g., by filtration); (c) cation exchange chromatography; ) Tangential Flow Filtration ("TFF2"); (d) CsCl ultracentrifugation; (e) Viral and (f) further tangential flow filtration ("TFF3"). The purified AAV particles are suspended in a pharma- ceutical acceptable carrier. In some embodiments, downstream processing includes the step of producing a TFF1 intermediate followed by the step of producing a finished drug substance. The following preparation steps were performed: thawing and pooling of TFF1 intermediates, acidification and clarification, cation Exchange chromatography (CEX), tangential flow filtration (TFF2), CsC l Separation of intact / empty capsids by ultracentrifugation, tangential flow filtration (T Concentration / buffer exchange with FF3), filtration of TFF3 pool material to generate API, dilution of API This includes producing the drug product by distillation and filtration, storing the drug product and filling the drug product into vials. nothing.

[0153] In some embodiments, the downstream processes disclosed herein can be used to shRNA targeting AAV SMN, AAV MECP2, or SOD1 as described An intermediate containing an AAV encoding the

[0154] 1. Acidification and clarification of the intermediate In embodiments where the intermediates are frozen, downstream processing begins with thawing the TFF1 intermediate material. Detergents such as Tween 20 can be used to improve bulk survival at acidic pH. This may promote flocculation of host cell proteins and DNA. The pH of the TFF1 intermediate containing the thiol may then be lowered. The aggregates and precipitates are used as depth filters and to remove large molecular contaminants and cell debris. A filter, e.g., a 0.45 μm filter, but one through which the vector genome passes. The aqueous solution may be filtered through a filter that allows the aqueous solution to be removed. A depth filter may be used.

[0155] In one embodiment, Tween 20 is added to a final concentration of 10-20% Tween 20. In some embodiments, the TFF1 intermediate solution is slowly added to the TFF1 intermediate solution so that the T After adding ween 20, the target composition was 20 mM Tris, 1 mM MgCl2, 500 The solution is 36% Tween 20 in 1 mM NaCl, 1% sucrose m / v, pH 8.1. In some embodiments, the Tween 20 is slowly added over a period of about 1 to 6 hours. In some embodiments, Tween 20 is added slowly over a period of 3-6 hours. In some embodiments, Tween 20 is added slowly over a period of 4 hours. In some embodiments, the Tween 20 / TFF1 intermediate solution is incubated at room temperature overnight. In some embodiments, the Tween 20 / TFF1 intermediate solution is The solution is allowed to incubate at room temperature for 8 to 20 hours. The n20 / TFF1 intermediate solution is allowed to incubate at room temperature for 12-20 hours.

[0156] After incubation, any suitable acid may be added to the solution, such as Tween 20. In some embodiments, the pH of the TFF1 intermediate may be lowered by adding 1 M glycine, p H2.5 is added to achieve a target pH of 3.5±0.1. The pH is pH 3.0 to 4.0, about pH 3.3 to 3.7, about pH 3.4 to 3.6, or about pH Once the pH is within acceptable limits, pass the solution through a filter of any size. In an exemplary embodiment, a depth filter (e.g., Clarisolve POD) in series with a 0.45 μm filter (e.g., Opticap XL10 D urapore filters) or 0.8 / 0.45 μm PES filters are used.

[0157] 2. Cation Exchange Chromatography In various embodiments, a cation exchange (CEX) capture chromatography step is For example, the viral capsid may be used to bind host cell proteins, host cell DNA, and host cell lipids. The product is separated from the terpene, Tween 20 and other process-related impurities. The principles of Fie are well known in the art, but briefly, the method involves the steps of: It relies on charge-charge interactions between the positively charged particles to be charged and the negatively charged resin used. Generally, several diavolumes of buffer are first run until the pH and conductivity stabilize. The column is then equilibrated with 100% ethanol. The sample is then loaded and the column is washed with loading buffer. Finally, the sample of interest is eluted from the column using an elution buffer, and the sample is The presence of the sample of interest is detected by measuring the absorbance of the eluate. It can be put out.

[0158] In one embodiment, the CEX step includes a CIMmultus S03-8000 A composite column (sulfonyl) (2 μm narrow diameter) chromatography column is used. In one embodiment, the elution peak is collected starting from a sharp increase in OD280. OD280 may begin to increase when conductivity is between 80 and 85 mS / cm. CEX The eluate may be collected conventionally and may be collected in two fractions. The first fraction begins with a sharp increase in OD280 and contains 1.5 collection volumes ( In another embodiment, the second fraction is started immediately after the first fraction. 1.0 CV is collected. These two fractions are pooled and then diluted to pH 8.0±0.3. In one embodiment, the neutralization buffer is 1.0 M Tris, pH 7.0 at 20° C. Includes 9.1±0.1.

[0159] 3. Tangential Flow Filtration 2 In some embodiments, the concentration, removal of protein impurities, and subsequent CsCl ultracentrifugation steps A tangential flow filtration step (TFF) was performed to exchange the buffer into a suitable buffer for the elution step. 2) is used. Any suitable TFF membrane may be used. In some embodiments, the TFF The second step utilizes a 300 kD MWCO regenerated cellulose membrane.

[0160] In some embodiments, the concentration phase of this step is such that the volume of the CEX eluate is reduced. In one embodiment, the retentate is diluted 2-fold with diafiltration buffer. and the retentate is concentrated to its initial volume. The buffer consisted of 20 mM Tris, 2 mM MgCl2, 150 mM NaCl, 0.2% phosphate. TFF2 Na containing Loxamer 188, 1% sucrose, pH 8.1 ± 0.1, 20°C In such an embodiment, this step is performed using a fresh Diafiltration with the new buffer may be repeated until completion. In this case, the retentate was diluted 2-fold with CsCl-containing diafiltration buffer to obtain the retentate. The fluid is concentrated to its initial volume. In one embodiment, the CsCl-containing diafiltration tray The reaction buffer was 20 mM Tris, 2 mM MgCl2, 3 M CsCl, 0.2% porphyrin. TFF2 CsCl dialysis solution containing oxamer 188 at pH 8.1 ± 0.1, 20°C In such an embodiment, this step is carried out with a fresh buffer. Diafiltration may be repeated until completion of the CsCl diafiltration. After the treatment is complete, the retentate is then pumped to a defined volume that depends on the hold-up volume of the system. In some embodiments, the product recovery from the TFF2 system may be concentrated to volume. To maximize the rate, a rinse of the membrane is performed, for example two successive rinses.

[0161] 4.CsCl ultracentrifugation In some embodiments where AAV is used for in vivo gene transduction, the rAAV The final product contains minimal impurities and empty particles. Two methods for purifying AAV vectors These are ultracentrifugations using either iodixanol or CsCl gradients. One study comparing the two methods found higher benefits with iodixanol compared to CsCl. Although the AAV vectors were of high vector purity, the amount of empty viral capsids was higher. Strobel et al. s of Cesium Chloride-and Iodixanol-Based Purification of Recombinant Adeno-Assoc iated Viral Vectors for Preclinical Appl ications.”Human Gene Therapy Methods,26( 4):147-157. The use of CsCl has been shown to reduce the amount of empty viral capsids. However, CsCl can be toxic to cells and multiple cycles are required to remove residual CsCl. A purification step may be required, so shorter purification times such as Iodixanol (approximately 1 day) are recommended. This can lead to longer process times (approximately 3.5 days) compared to the conventional method. The multiple steps required to remove sCl frequently result in dramatic loss of rAAV. This can lead to low yields and recovery rates, often negating other benefits of the method. Hermens et al. “Purification of Re combinant Adeno-Associated Virus by Iodi xanol Gradient Ultracentrifugation Allow s Rapid and Reproducible Preparation of Vector Stocks for Gene Transfer in the N ervous System.”Human Gene Therapy,10:188 Furthermore, these two methods have been successfully used in the laboratory to generate preclinical samples. However, they are not scalable and therefore not suitable for large-scale production of commercial products. For example, Tomono et al., “Ultracentrifugation- free chromatography-mediated large-scale purification of recombinant adeno-assoc iated virus serotype 1(rAAV1).”Molecular Therapy-Methods & Clinical Development, 3:15058 (see "Cesium chloride (CsCl) or iodixanol density superconductivity"). "Centrifugal purification methods are not suitable for large-scale production."

[0162] In some embodiments, ultracentrifugation is used, e.g., to separate empty capsids from full capsids. Unexpectedly, the CsCl ultracentrifugation method disclosed herein is scalable. It is scalable and suitable for large-scale production of purified AAV vectors. The method may be carried out by, and may involve the use of a gradient buffer. Examples of gradient buffers include Examples of suitable glycerol include, but are not limited to, CsCl, sucrose, iodixanol, and other glycerols known in the art. Centrifugation can be performed in any centrifuge capable of reaching the desired g-forces, e.g. , automatic Optima XPN 1 with type 50.2 Ti rotor or equivalent rotor Ultracentrifugation can be performed in a 0.00 ultracentrifuge system or equivalent system. After ultracentrifugation, empty caps The capsids and intact capsids were separated into different bands in the tube, and material was extracted from the specific band. In some embodiments, this can be extracted by filtration after purification of TFF2. The used material is 241,600 to 302,000 g (approximately 40, In some embodiments, the TFF2 is purified by centrifugation at 10,000 to 50,000 rpm. The filtered material is centrifuged overnight. In some embodiments, the filtered material after TFF2 purification is The material is centrifuged for 16-24 hours. In some embodiments, the filtered TFF2 purified The material is centrifuged for 20-24 hours. In some embodiments, the filtered TFF2 purified The material is centrifuged at 15-25° C. In one embodiment, the filtered material after TFF2 purification is The material was heated to 302,000g (50,000 rpm on a 50.2 Ti rotor) at 20°C. In some embodiments, the CsCl centrifugation buffer is (a) containing CsCl, (b) MgCl2, (c) poloxamer 188 and (d) one or more of the components further comprising one or more of Tris. In an embodiment, the buffer for CsCl contains all of (a), (b), (c) and (d). In some embodiments, the buffer for CsCl is pH 7.5-8.5, or pH In one embodiment, a suitable buffer for CsCl centrifugation is 20 mM M Tris, 2 mM MgCl2, 3 M CsCl, 0.2% Poloxamer 188, pH 8 .1 ± 0.10. After the centrifugation step is complete, the tube is removed from the ultracentrifuge. In some embodiments, the top band, band A, contains empty capsids. In some embodiments, the second to third bands, bands B, C and D, are complete. In some embodiments, the AAV viral vector comprises a psyllid doublet band. In one embodiment, bands B, C, and D are collected using a 30 mL syringe. Insert the 18G needle attached to the syringe just below band D into the center of the tube. In other embodiments, using techniques known in the art and / or as described herein. The strands may be assayed for the presence of intact or empty capsids, and intact capsids may be assayed for the presence of empty capsids. The band containing the correct capsid may be collected.

[0163] The ratio of empty to non-empty viral capsids was determined by standard laboratory techniques. In some embodiments, the measurement is performed by absorbance measurement. In some embodiments, the measurement is performed by UV absorbance measurement. In this study, the total amount of capsid protein and the total amount of DNA were determined by UV absorbance measurement. In some embodiments, the measurement is performed by optical refractometry. In an embodiment, the measurement is performed by analytical ultracentrifugation.

[0164] In one embodiment, the AAV viral vectors collected after ultracentrifugation are empty capsids. and less than 7%, less than 5%, less than 3%, or less than 1% empty capsids. In one embodiment, the AAV viral vector collected after ultracentrifugation contains 1 to 10 empty capsids. In one embodiment, the AAV viral vector collected after ultracentrifugation has an empty 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. can be.

[0165] 5. Tangential Flow Filtration 3 to Generate Filtered Drug Substance In some embodiments, the TA-100001 is used to remove CsCl and enrich for intact vector capsids. A tangential flow filtration step (TFF3) is used. Filtration may be performed using a suitable membrane. In one embodiment, a 300 kDa MWCO A regenerated cellulose membrane is used. The membrane can retain the vector capsid. TFF3 The concentration phase of the work was designed to reduce the concentration of residual CsCl and the volume of the ultracentrifuged pool. In some embodiments, after the target retentate volume is reached, diafiltration may be performed. Diafiltration of the retentate will begin with a maximum of 10 diavolumes. In one embodiment, the suitable TFF3 buffer is (a) Tris, (b) MgCl2, (c) NaCl, or (d) Poloxamer The composition may include one or more of the following ingredients: Summer 188. In one embodiment, the preferred TF The F3 buffer may contain all of (a), (b), (c) and (d). In one embodiment, The TFF3 buffer is pH 7.5 to 8.5, pH 7.7 to 8.3, or pH 8.0. In one embodiment, a suitable TFF3 buffer is 20 mM Tris, 1 mM MgCl2, 200mM NaCl, 0.001% Poloxamer 188, pH 8.0±0 In another embodiment, a suitable TFF3 buffer comprises 20 mM Tris , 1 mM MgCl2, 200 mM NaCl, 0.005% Poloxamer 188, pH In one embodiment, the concentrated retentate is filtered through a 0.2 μm Pall Supor® EKV Sterilizing Grade Filters (Mini Kleenpak ) filter to produce a filtered drug substance. , and in the methods described herein, 5 x 10 per production batch 15 More than vg or 8 x 10 15 vg or 1×10 16 rAAV of over vg can be obtained.

[0166] Pharmaceutical Compositions The viral (e.g., AAV) particles purified by the methods disclosed herein are intended to be specific for human In some embodiments, the compound can be produced in high yields and with sufficient purity to be administered to elephants. , viral vectors are approximately 1–8 × 10 13 Viral vector genomes / mL (vg / mL ), or about 1.7 to 2.3 × 10 13 In some embodiments, the bolus is formulated at a concentration of 0.1 mg / mL. In the present study, the viral vector was approximately 1.9 to 2.1 × 10 13 Formulated at a concentration of vg / mL In some embodiments, the viral vector is about 2.0×10 13 vg / mL It is formulated at a concentration of

[0167] In some embodiments, the process for producing a viral vector includes the step of forming a vacant space that does not contain any nucleic acid material. The pharmaceutical composition containing low amounts of empty viral capsids (p (pharmaceutical composition) is a drug used to treat patients with immature immune systems. Patients, e.g., infants, may unnecessarily expose themselves to antigenic material (empty capsids, host cell proteins) without any therapeutic benefit. In some embodiments, this may be advantageous to avoid exposure of the endonuclease to harmful substances (proteins, host cell DNA). In some embodiments, such pharmaceutical compositions may be used to treat a variety of conditions, including the possibility of an infusion reaction or a broad immune response. This may reduce the likelihood of infection and improve the efficacy of treatment. Compared to capsids, empty capsids have different densities, so these two species are distributed along a gradient The separation can be accomplished by centrifugation or other methods known in the art. In some embodiments, the empty capsids are separated by ultracentrifugation. In one embodiment, the empty capsids are separated by CsCl gradient ultracentrifugation. In some embodiments, the empty capsids are separated by iodixanol gradient ultracentrifugation. In this embodiment, empty capsids are isolated by sucrose gradient ultracentrifugation.

[0168] The ratio of empty to non-empty viral capsids was determined by standard laboratory techniques. In some embodiments, the ratio is measured by absorbance measurements. In some embodiments, the ratio is measured by UV absorbance measurement. In this study, the total amount of capsid protein and the total amount of DNA were determined by UV absorbance measurement. In some embodiments, the measurement is determined by optical refractive index measurements. In other embodiments, the measurements are determined by analytical ultracentrifugation.

[0169] High levels of empty capsids could pose a challenge to the efficacy of viral vector therapy. In an embodiment, the pharmaceutical composition contains less than 10% empty capsids, less than 8% empty capsids. In another embodiment, the capsids have less than 7%, less than about 5%, less than 3%, or less than 1% empty capsids. In another embodiment, the pharmaceutical composition has 1-10% empty capsids. In another embodiment, the pharmaceutical composition comprises 6% empty capsids. Below empty capsids, 5% empty capsids, 4% empty capsids, 3% empty capsids, 2% or less empty capsids. In one embodiment, the number of empty capsids In another embodiment, the percentage of empty capsids is below the detection limit. In some embodiments, the percentage of empty capsular bag that is filled with the empty capsular bag is determined using, for example, AUC. Such a low percentage of empty capsids may be counterproductive to, for example, The composition has improved efficacy of treatment and / or reduced adverse events after administration to a patient when compared to a composition having In some embodiments, the effects of the present invention are reduced. The preparation method of the viral vector to be used may be the same as the previous methods, e.g., the adherent cell and / or These improved vacancies were compared to levels not using the purification method described in This results in a capsid fraction of .

[0170] Adherent cells used in the production of viral vectors in the viral vector production process Residual proteins from cells (e.g. HEK293 cells) cannot be completely separated and removed. Residual host cell proteins may induce an immune response. The amount of residual host cells cannot distinguish between viral capsid proteins and residual host cell proteins. The residual amount of the serotonin can be measured by any standard laboratory technique. The amount of purified host cell protein was measured by size exclusion or ion exchange chromatography. In some embodiments, the measurement can be performed by wiping with a parent cell specific antibody. In one embodiment, residual host cell proteins can be detected by ELISA. The amount of can be measured by enzyme-linked immunosorbent assay (ELISA). In an embodiment, the amount of residual host cell protein is measured by a commercially available ELISA kit. In some embodiments, the amount of residual host cell protein can be determined by Cygn Measured by the US Technologies HEK293 HCP ELISA kit It can be determined.

[0171] In another embodiment, the residual host cell protein in the pharmaceutical composition is less than 1×10 13 5 × 10 per vg / ml 6 pg / ml or less, 1×10 13 1.2 x 1 per vg / mL 0 6 pg / ml or less or 1×10 13 1 × 10 per vg / ml 5 pg / ml to 1×10 13 1.2 × 10 per vg / ml 6 pg / ml or 1×10 13 40 per vg / ml In one embodiment, the pharmaceutical composition comprises a 1.0×10 13 vg hit Contains less than 5, 4, 3, 2, 1 ng or less of residual host cell protein. In an embodiment, the pharmaceutical composition comprises 1.0×10 13 Residual host cells below 4ng per vg Contains cytoplasmic proteins.

[0172] In the viral vector production process, residues from adherent cells (e.g. HEK293 cells) Residual host cell DNA or residual plasmid transfected for the production of viral vectors It is possible that the mid DNA cannot be completely removed. filtration, tangential flow filtration, etc.) to remove residual host cell or plasmid DNA in the bulk. In one embodiment, the amount of residual host cell or plasmid DNA is measured by: In another embodiment, the residual host cell or plasmid DNA is The amount of is measured by quantitative PCR ( In another embodiment, the remaining host cells or plasmid D Measurement of the amount of NA is carried out by digital droplet PCR (ddPCR). In one embodiment, the amount of plasmid DNA is determined by the kanamycin resistance gene region of the plasmid. In another embodiment, the gene is determined using a qPCR assay with primers specific for the region. The amount of residual host cell DNA can be determined using commercially available qPCR assay kits, e.g., Thermo Scientific. resDNASEQ® Human Residual DNA Quantification Kit by oFisher, B Residual DNA quantification by iorad as determined by supermix or any equivalent product Reducing the amount of residual host cell or plasmid DNA may improve therapeutic outcomes. The therapeutic outcomes of the present invention can be improved by the treatments disclosed herein. Such compositions may be purified and / or selected for use in.

[0173] In one embodiment, the residual host cell DNA in the pharmaceutical composition is less than 1×10 13 vg 1.7 × 10 per ml 6 pg / ml or less, 1×10 13 1 × 10 per vg / ml 5 pg / ml to 1×10 13 1.2 × 10 per vg / ml 6 pg / ml. In one embodiment, the residual host cell DNA in the pharmaceutical composition is less than 1.0×10 13 per vg 3×10 5 , 2×10 5 , 1.1×10 5 , 1×10 5 pg or less or In an embodiment, the residual host cell DNA in the pharmaceutical composition is less than 1.0×10 13 1.1 × 10 per vg 5 pg or less.

[0174] In another embodiment, the residual plasmid DNA in the pharmaceutical composition is less than 1×10 13 v 1.7 x 10 per g / ml 6 pg / ml or less, 1×10 13 1 × 10 per vg / ml 5 pg / ml to 1×10 13 1.7 × 10 per vg / ml 6 pg / ml. In some embodiments, the residual plasmid DNA in the pharmaceutical composition is 1.0×10 13 vg hit 6.8×10 5 pg or less.

[0175] In one embodiment, the residual host cell DNA in the pharmaceutical composition is 1.0×10 13 vg hit 1.1×10 5 pg or less of residual plasmid DNA in the pharmaceutical composition. 1.0×10 13 6.8 x 10 per vg 5 pg or less.

[0176] In one embodiment, the residual host cell DNA in the pharmaceutical composition is 1.0×10 13 vg hit 1.1×10 5 pg or less of residual plasmid DNA in the pharmaceutical composition, .0×10 13 6.8 x 10 per vg 5 pg or less, and the residual amount in the pharmaceutical composition is Host cell protein is 1.0 × 10 13 Less than 4ng per vg.

[0177] In some embodiments, the amount of endotoxin in the pharmaceutical composition is 1.0×10 13 v Less than about 1 EU / mL per g / mL, 1.0 × 10 13 Approximately 0.75 EU per vg / mL / mL, less than 1.0 × 10 13 Less than about 0.5 EU / mL per vg / mL, 1.0 × 10 13 Less than about 0.4 EU / mL per vg / mL, 1.0 × 10 13 Approximately 0 per vg / mL Less than .35EU / mL, 1.0×10 13 Less than about 0.3 EU / mL per vg / mL, 1 .0×10 13 Less than about 0.25 EU / mL per vg / mL, 1.0 × 10 13 vg / m Less than approximately 0.2 EU / mL per liter, 1.0 x 10 13 Approximately 0.15 EU / vg / mL Less than 1.0 x 10 mL 13 Less than about 0.1 EU / mL per vg / mL, 1.0 × 10 1 3 Less than about 0.05 EU / mL per vg / mL or 1.0 x 10 13 Per vg / mL The amount of endotoxin is less than about 0.02 EU / mL. Such assays are known in the art, such as the amebocyte lysate (LAL) assay. In this embodiment, endotoxin is defined as ) <85> (herein incorporated by reference in its entirety).

[0178] In one embodiment, the bovine serum albumin (BSA) in the pharmaceutical composition is 1.0×10 13 <0.5 ng per vg, 1.0 × 10 13 Less than 0.3 ng per vg, or 1. 0×10 13 In one embodiment, the pharmaceutical composition The benzonase in 13 <0.2 ng per vg, 1.0 × 10 13 v Less than 0.1 ng per g or 1.0 x 10 13 It is less than 0.09 ng per vg.

[0179] In one embodiment, the pharmaceutical compositions disclosed herein include one or more of the following: Mu: 1.0×10 13 Less than about 0.09 ng of benzonase per vg, about 30 μg / g ( ppm) for cesium, approximately 20-80 ppm for poloxamer 188, and 1.0×10 13 Approximately 0.22 ng BSA per vg, 1.0 × 10 13 Approximately 6.8 × 10 per vg 5 Residual plasmid DNA, less than pg, 1.0 × 10 13 Approximately 1.1 × 10 per vg 5 pg Residual hcDNA: 1.0 × 10 13 Approximately less than 4ng rHCP per vg, pH 7.7 ~8.3, approximately 390-430 mOsm / kg, less than approximately 600 ≧25μm particles per container less than about 6000 particles of ≥ 10 μm size per container, less than about 1.7 × 10 1 3 ~2.3×10 13 Genomic titer in vg / mL, 1.0 × 10 13 Approximately 3.9 times per vg 10 8~8.4×10 10 Infectious titer in IU is 1.0 x 10 13 Approximately 100 to 30 per vg 0 μg total protein, approximately 7.5 × 10 13 Δ by viral vector at a dose of vg / kg Median survival of 7SMA mice ≥ 24 days, based on in vitro cell-based assay A relative potency of 70-130% and / or less than about 5% empty capsids.

[0180] In one embodiment, the pharmaceutical compositions disclosed herein contain one or more of the following, e.g. For example, all pH levels: 7.7 to 8.3 (e.g., USP <791> (When measured by , approximately 390-430 mOsm / kg (e.g., USP <785> (measured by Less than about 600 particles of size ≧25 μm per container (e.g., USP <787> By When measured, there are less than about 6000 particles of size ≥ 10 μm per container (e.g., US P <787> When measured by the 13 ~2.3×10 13 vg / mL genome titer, 1.0 × 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 IU's Infectious titer, 1.0 × 10 13 Approximately 100-300 μg of total protein per vg, e.g. For example, in vivo functionality tests as described in the specification In the experiment, the 13 Δ7SMA myeloma with viral vector at a dose of 500 mg / kg Median survival of mice ≥ 24 days, approximately 70-13 days based on in vitro cell-based assay 0% relative potency and / or less than about 5% empty capsids. The disclosed pharmaceutical compositions have an overall purity of 95% or greater (as determined, for example, by SDS-PAGE). In embodiments, the pharmaceutical compositions disclosed herein contain a non-single, non-specific associated Contains a series of impurities at levels greater than 2% (e.g., as determined by SDS-PAGE). In one embodiment, the pharmaceutical compositions disclosed herein have an endotoxin concentration of 0.75 EU / mL or less. Includes toxin levels (e.g., USP <85> (When measured by the method described above) The pharmaceutical compositions disclosed herein may be prepared, for example, as described in USP <71> Sterile as determined by The sexual test results show no proliferation.

[0181] High levels of residual host cell proteins, host cell DNA, plasmid DNA, and / or Endotoxins can pose a challenge to the efficacy of viral vector therapy. In some embodiments, residual host cell proteins, host cell DNA, plasmid DNA, and / or Such low amounts of endotoxin allow for better improved efficacy of the treatment following administration to a patient when compared to compositions having a higher amount; and and / or adverse events (e.g., inflammatory responses, liver injury) are reduced. The methods for preparing viral vectors disclosed herein are similar to previous methods, e.g., adherent cell and / or or levels not using the purification methods described herein. In some embodiments, the methods herein also include: Low amounts of residual host cell proteins, host cell DNA, and plasmids A virus vector with a reduced percentage of empty capsids in addition to DNA and / or endotoxin. This also allows the preparation of vectors.

[0182] In some embodiments, the amount of residual cesium after TFF, e.g., after a second TFF, is about In some embodiments, after TFF, e.g., after a second TFF, The amount of residual cesium is below about 30 μg / g. For example, the amount of residual cesium after the second TFF is less than about 20 μg / g. In some embodiments, the residual cesium in the pharmaceutical composition is 30 μg / g (ppm) or less. In this state, the amount of residual CsCl was measured by mass spectrometry and inductively coupled plasma mass spectrometry (ICP- MS), and / or another suitable method. The amount of residual cesium after the second TFF was measured below the limit of quantification, e.g., using ICP-MS. Rotate.

[0183] In some embodiments, the concentration of AAV viral vectors collected after the second TFF is Degrees are approximately 5 x 10 12 vg / ml or more, approximately 1×10 13 vg / ml or greater, or approximately 3 × 10 13 vg / ml or more.

[0184] In one embodiment, the pharmaceutical composition has one or more of the following: 1.0×10 13 Benzonase less than 0.09ng per vg, cesium less than 30μg / g (ppm) , approximately 20-80 ppm of poloxamer 188, 1.0 × 10 13 0.22n per vg g BSA, 1.0 × 10 13 6.8 x 10 per vg 5 Sub-pg residual plasmid DNA, 1.0 × 10 13 1.1 × 10 per vg 5 Less than pg of residual hcDNA, and 1 .0×10 13 <4ng rHCP per vg.

[0185] In another embodiment, the pharmaceutical composition has a molecular weight of within ±20%, within ±15%, or within ±20% of the reference standard. In one embodiment, the potency is between Fo and 10%, or between ±5%. ust et al., Nat.Biotechnol.,28(3),pp.271- 274 (2010) against a reference standard. A suitable reference standard may be used. In one embodiment, the pharmaceutical composition is administered to SMAΔ7 mice. Thus, when tested, it has a certain in vivo potency. In one embodiment, 13 Mice receiving the vg / kg dose for more than 15 days, more than 20 days, more than 22 days, or more than 24 days In one embodiment, the pharmaceutical composition is administered in a cell-based assay. 50 to 150%, 60 to 1 40% or have an in vitro relative potency of 70-130%.

[0186] Purified virus particles (e.g., The viral particles are formulated by known methods to produce pharmaceutical preparations. The compositions of the present disclosure can be prepared in a manner known in the art. The compositions can be formulated for administration to mammalian subjects, e.g., humans, using techniques such as those described herein. The delivery system may be intramuscular, intradermal, mucosal, subcutaneous, intravenous, intrathecal, an injectable depot device, or topical. It may be formulated for administration.

[0187] When the delivery system is formulated as a solution or suspension, the delivery system may be in an acceptable carrier, e.g. In an aqueous carrier, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronan, A variety of aqueous carriers may be used, such as hyaluronic acid. These compositions can be sterilized using conventional, well known sterilization techniques. The resulting aqueous solution may be sterilized by conventional techniques or may be sterile filtered. , may be packaged for immediate use or may be freeze-dried, where freeze-dried The preparation is combined with a sterile solution prior to administration.

[0188] The compositions, e.g., pharmaceutical compositions, may contain, for example, sodium acetate, Sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate esters, triethanolamine oleate, pH adjusters and buffers, isotonicity agents, wetting agents In some embodiments, the pharmaceutical composition may contain pharma- ceutically acceptable auxiliary substances such as In some other embodiments, the pharmaceutical composition does not include a preservative.

[0189] Viral vectors, e.g., the viral vectors in the compositions and formulations disclosed herein. The genomic titer of the vector can be determined by a number of standard methods. PCR with specific primers can provide a relative measurement, but requires smaller samples and For absolute measurements, quantitative PCR (qPCR) can be used. Droplet Digital PC R(ddPCR) is a method for performing digital PCR based on water-oil emulsion droplet technology. In this method, a sample is divided into tens of thousands of droplets, and PCR amplification of template molecules occurs in each individual droplet. There is no need to prepare a standard curve or have primers with high amplification efficiency. Therefore, ddPCR typically does not require as much sample as traditional PCR-based techniques. In one embodiment, the genomic titer of the viral vector is determined using PCR. In another embodiment, the genomic titer of the viral vector is determined using qPCR. In another embodiment, the genomic titer of the viral vector is determined using ddPC. For the method of determining the viral genome titer using ddPCR, see, for example, Lockheed Martin et al. et al., “Absolute Determination of Single -Stranded and Self-Complementary Adeno-A ssociated Viral Vector Genome Titers by Droplet Digital PCR,”Human Gene Therapy Methods, 25(2):115-125.

[0190] In some embodiments, the PCR-based method specifically targets the SMN gene. Detecting encapsidated AAV9 viral genome using designed primers and probes In another embodiment, the PCR-based method comprises the step of: Capsid fragments were isolated using specifically designed primers and probes targeting the promoter. In another embodiment, the PCR-based method detects and quantifies the transfected AAV9 viral genome. The method uses specifically designed primers and probes that target the CMV enhancer. The probe is used to detect and quantify encapsidated AAV9 viral genomes. In this study, the PCR-based method uses specifically designed primers that target the ITR sequences. and probes are used to detect and quantify encapsidated AAV9 viral genomes. In an embodiment, the PCR-based method comprises cloning the bovine growth hormone polyadenylation signal. Using specifically designed primers and probes to target encapsidated AAV9 vectors The virus genome is detected and quantified.

[0191] In some embodiments, the pharmaceutical composition has a pH of about 7.7 to 8.3 and a pH of about 390 to 43 In some embodiments, the pH is measured using a pH meter. In some embodiments, the pH is measured using a pH meter according to the United States Pharmacopeia (USP), for example <791> (incorporated by reference in its entirety) In some embodiments, the concentration of iodine is measured potentiometrically using compensated microelectrodes. Smolality is determined according to USP, e.g. USP <785> (incorporated by reference in its entirety) In some embodiments, osmolality is measured using a freezing point depression method based on the In another embodiment, the osmolality is measured using a pressure drop osmometer. It is measured using a penetrometer.

[0192] In one embodiment, the intravenous formulation comprises a pH of 7.5 to 8.5, 2×10 13 vg / ml ~6×10 13 Genomic titer of vg / ml and osmolality of 384-448 mOsm / kg In another embodiment, the intravenous formulation has a pH of 7.5 to 8.5, a concentration of 1.5× 10 13 vg / ml ~ 3.5 × 10 13 vg / ml genomic titer, and 384–448 m In another embodiment, the intravenous formulation has an osmolality of 7.5 to 100 Osm / kg. pH 8.5, 1.8 x 10 13 vg / ml ~ 2.2 × 10 13Genomic titer in vg / ml and an osmolality of 384 to 448 mOsm / kg. The IV formulation comprises about 0.1-2.0 mM MgCl. In one embodiment, the IV formulation In one embodiment, the IV formulation contains about 0.0 0.01% to 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.

[0193] In one embodiment, the IV formulation contains 1 mM Contains MgCl2, 200 mM NaCl, and 0.005% w / v poloxamer 188. In embodiments, the IV formulation comprises about 1×10 13 ~3×10 13 vg / mL or 1.7 × 1 0 13 ~2.3×10 13 Includes genomic titer in vg / mL.

[0194] Use of the pharmaceutical composition In another embodiment, the present disclosure provides a rAA having a genome comprising a polynucleotide. A method for delivering a polynucleotide to the central nervous system of a patient is disclosed, comprising administering V9. In some embodiments, the delivery comprises a rAA having a genome comprising the polynucleotide. V9. In some embodiments, a non-ionic low osmolarity contrast agent is also administered to the patient. Ionic low osmolarity contrast agents increase transduction of target cells in the central nervous system of patients In some embodiments, the rAAV9 genome is a self-complementary genome. In this example, the rAAV9 genome is a single-stranded genome.

[0195] In some embodiments, the polynucleotide is delivered to a brain region. Brain sites include, but are not limited to, the motor cortex and the brain stem. In some embodiments, the polynucleotide is delivered to the spinal cord. The octides are delivered to lower motor neurons. The polynucleotide is delivered to neural and glial cells. In some embodiments, the polynucleotide is delivered to glial cells. The cells are microglial cells, oligodendrocytes or astrocytes. In one embodiment, rAAV9 is used to deliver polynucleotides to Schwann cells.

[0196] Uses include, for example, lower motor neuron diseases such as SMA and ALS, and Pompe disease, This includes the treatment of lysosomal storage disorders, glioblastoma multiforme and Parkinson's disease. Addition disorders include, but are not limited to, activator deficiency / GM2 ganglioside stimulatory syndrome. α-mannosidosis, aspartylglucosaminuria, cholesteryl ester accumulation disease, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease, Barr's disease, fucosidosis, galactosialidosis, Gaucher's disease (type I, type II, type II Type I), GM1 gangliosidosis (infantile, late infantile / juvenile, adult / chronic), I cell disease / mucolipidosis II, infantile free sialic acid storage disease / ISSD, juvenile hexosarcoma Amidase A deficiency, Krabbe disease (infantile and late-onset), metachromatic leukodystrophy, Mucopolysaccharidosis disorders (pseudo-Hurler polydystrophy / mucolipidosis IIIA, MP Hurler syndrome type SI, Scheie syndrome type I, Hurler-Scheie syndrome type I Hunter syndrome, MPS II, Sanfilippo syndrome type A / MPS III Type A, Sanfilippo syndrome type B / MPS III type B, Sanfilippo syndrome type C / MP S III C, Sanfilippo syndrome D / MPS III D, Morquio A / M PS WA, Morquio B / MPS IVB, MPS IX Hyaluronidase deficiency MPS VI Maroteaux-Lamy syndrome, MPS VII Sly syndrome, Mucolipidosis Mucolipidosis type I / sialidosis, mucolipidosis type IIIC, mucolipidosis type IV), various Lactase deficiency, Niemann-Pick disease (types A, B, and C), neuronal ceroid lipofus Zinn's syndrome (CLN6 disease (atypical late infantile, late variant, early juvenile), Batten-J. Spielmeier-Voigt / Juvenile NCL / CLN3 Disease, Finnish Varian Late infantile CLN5, Jansky-Bielschowsky disease / late infantile CLN2 / TP P1 disease, Koofs / adult-onset NCL / CLN4 disease, Northern epilepsy / late infantile barrier CLN8, Santavuori-Haltia / infant CLN1 / PPT disease, β-mannosidosis, Pompe disease / Glycogen storage disease type II, Pycnodysplasia Sandhoff disease / adult-onset / GM2 gangliosidosis, Sandhoff disease Sandhoff disease / GM2 gangliosidosis-infantile type, Sandhoff disease / GM2 gangliosidosis -Juvenile, Schindler, Salla disease / sialic acid storage disease, Tay-Sachs / GM2 gangliosides Mycobacterial disease and Wolman's disease.

[0197] In further embodiments, the methods and materials are used to treat Rett Syndrome, Alzheimer's Disease, Treatment of nervous system disorders such as Parkinson's disease, Huntington's disease, or spinal cord and brain trauma / injury In one embodiment, the method is adapted for the treatment of nervous system injuries, including cerebral infarction, stroke, and brain cancer. Use of the methods and materials is indicated for the treatment of spinal muscular atrophy (SMA).

[0198] There are four types of SMA, which are traditionally classified according to age at onset and highest motor function attained. Both forms of SMA are inherited in an autosomal recessive manner and affect survival motor neurons. It is caused by mutations in the SMN1 gene. In humans, it is also called SMN2. They also carry a second, nearly identical copy of the SMN gene, which is involved in the differentiation of schizophrenia. Lefebvre et al “Identification and characterization of a spinal muscular atrophy-determining g ene.”Cell,80(1):155-65. Monani et al. “Spi nal muscular atrophy: a deficiency in au biquitous protein;a motor-neuron specifi c disease.” Neuron, 48(6):885-896. SMN1 and SM Both N2 genes express the SMN protein, but SMN2 has a translationally exclusive exon 7. contains a silent mutation that prevents inclusion of exon 7 in the SMN2 transcript. Thus, SMN2 is a transcription factor that encodes both the full-length SMN protein and a transcript lacking exon 7. As a result, SMN is produced in both the cytoplasmic and truncated forms, with the truncated form being the predominant form. The amount of functional full-length protein produced by N2 is greater than that produced by SMN1 Lorson et al. “A si ngle nucleotide in the SMN gene regulates s splicing and is responsible for spinal muscular atrophy.”PNAS,96(11)6307-6311. Monani et al,“A single nucleotide differ ence that alters splicing patterns disti nguishes the SMA gene SMN1 from the copy gene SMN2.”Hum Mol Genet 8(7):1177-1183 Although SMN2 cannot fully compensate for loss of the SMN1 gene, it does have a role in milder forms of SMA. Patients with CKD generally have higher SMN2 copy numbers. Lefebvre et al., “Cognitive Neuropathy rrelation between severity and SMN prote in level in spinal muscular atrophy.”Nat Genet 16(3):265-269. Park et al., “Spinal muscular atrophy:new and emerging insig hts from model mice.”Curr Neurol Neurosc i Rep 10(2):108-117. Note that the SMN2 copy number is the only Specifically, the c. The 859G>C variant has been reported as a positive disease modifier. Patients with A. posterior have a less severe disease phenotype. itive modified of spinal muscular atroph y in the SMN2 gene.”Am J Hum Genet 85(3) :408-413.

[0199] Type I SMA (also called infantile-onset or Werdnig-Hoffmann disease) is a condition in which the or when SMA symptoms appear by age 6 months. In this form, infants typically They have low muscle tone (hypotonia), a weak cry, and respiratory distress. Developmental milestones in which the child has difficulty swallowing and sucking and is able to sit without support These infants often have hypotonia, delayed motor skills, poor stability, Demonstrates one or more of the following SMA symptoms: hunched posture and joint hypermobility. In fact, these infants have two copies of the SMN2 gene, one on each chromosome 5. More than half of all new SMA cases are SMA type I.

[0200] Type II or intermediate SMA begins between 7 and 18 months of age when a child is no longer able to stand or stand independently. This is the case for SMA before the child is able to walk. Children with type 2 SMA generally have Both types have three SMN2 genes. SMA, also known as adult-onset SMA and Kugelberg-Welander disease, It causes varying degrees of weakness. Type III SMA occurs after 18 months of age and is characterized by small Children can stand and walk independently, but may require assistance. Type IV SMA develops slowly. People with SMA Type III or IV are able to walk in adulthood or later. Generally, there are four to eight SMN2 genes, which produce significant amounts of the full-length SMN protein. Quality can be produced.

[0201] In one embodiment, the term "treatment" includes rAAV as disclosed herein. An effective dose, or effective doses, of the composition are administered intravenously to an animal (including a human) in need thereof. Before the onset of the disorder / disease. If the dose is administered after the onset of the disorder / disease, the administration is prophylactic. Administration is therapeutic when administered. In embodiments, an effective dose is administered to treat the disorder / disease being treated. Alleviate (eliminate or reduce) at least one symptom associated with a disease condition a dose that slows or prevents progression to a disorder / disease state; A dose that slows or prevents the progression of the disease, reduces the extent of the disease, reduces (partially or is the dose that produces complete remission and / or extends survival. Examples of disease conditions that are addressed are provided herein.

[0202] In one embodiment, a composition comprising an rAAV of the present disclosure is administered to treat SMA in a patient in need thereof. In another embodiment, the composition comprising the rAAV of the present disclosure is administered intravenously to a patient with type I. The drug is administered intrathecally to patients with SMA types II, III, or IV in need thereof. will be done.

[0203] The present invention relates to administering an AAV9 viral vector via an intrathecal or intravenous route. A method for treating SMA type I in a patient in need thereof by In some embodiments, the patient is 0 to 9 months of age. 0-6 months of age. In some studies, viral vectors are used to treat patients with SMA type I. In some embodiments, the patient's weight is determined. In some embodiments, the patient weighs more than 2.6 kg. In this condition, patients weigh between 2.6 and 8.5 kg.

[0204] In some embodiments, the patient has a mutation, e.g., a nuclease, in one copy of the SMN1 gene. have a mutation (including any mutation that renders the encoded SMN1 non-functional) In some embodiments, the patient has a mutation in two copies of the SMN1 gene, e.g. In some embodiments, the patient has all copies of the SMN1 gene. In some embodiments, the patient has a mutation, e.g., a null mutation, in SMN1. In some embodiments, the patient has a deletion in one copy of the SMN1 gene. In some embodiments, the patient has a biallelic SMN1 deletion. have a SMN1 mutation, i.e., either a deletion or substitution in SMN1 on both alleles of the 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 one functional copy 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 one copy of the SMN2 gene. In some embodiments, the SMN gene does not have a c.859G>C substitution in exon 7 of the SMN gene. The genetic sequence of the SMN1 or SMN2 gene may be determined by whole genome sequencing. In another embodiment, the gene sequence and copy number of the SMN1 or SMN2 gene are In some embodiments, the sequence may be determined by high throughput sequencing. The gene sequence and copy number of the SMN1 or SMN2 gene were determined by microarray analysis. In some embodiments, the genetic sequence of the SMN1 or SMN2 gene may be determined. and copy number may be determined by Sanger sequencing. In the present study, the copy number of the SMN1 or SMN2 gene was determined by fluorescence in situ hybridization. This may be determined by fluorescence in situ hybridization (FISH).

[0205] In some embodiments, the patient exhibits one or more SMA symptoms. SMA symptoms include muscle tension, Symptoms may include hypotonia, delayed motor skills, inability to maintain balance, hunched posture, and joint hypermobility. In one embodiment, the neck posture is evaluated by having the patient sit cross-legged while supporting the shoulders (front and back). Neck stability is determined by the patient's ability to maintain an upright position. In some embodiments, spontaneous movement is observed while the patient is in a supine position; Motor skills are assessed by the patient's ability to lift elbows, knees, hands, and feet off a surface. In one embodiment, the patient's grip strength is measured by placing the patient's fingers in the palm of the hand and lifting the patient so that their shoulder is raised above the surface. Hypotonia and grip strength are measured by how quickly / In some embodiments, the head position is measured by how long the patient can maintain the grip. The patient is then rotated to the maximum extent possible and the ability of the patient to return the head to the midline is measured. In some embodiments, shoulder posture is assessed by supporting the head and trunk. The patient is seated and attempts to reach for a stimuli at shoulder height, which is located an arm's length away. This may be assessed by observing whether the arm is extended with elbow or shoulder flexion. In this embodiment, shoulder posture is also assessed by having the patient lie on their side with their hands at arm's length apart. Whether the patient reaches out by flexing the elbow or shoulder to reach for a stimuli at shoulder height in the vicinity In some embodiments, motor skills may be assessed by observing the patient's foot. Patients flex their hips or knees when stroked, tickled, or pinched In some embodiments, the present invention is evaluated by observing whether the patient is at risk of developing a pulmonary embolism. For example, CHOP INTEND allows for shoulder flexion, elbow flexion, hip adduction, neck flexion, head Shoulder extension, neck extension, and / or internal flexion of the spine may be assessed. Other SMA symptoms may be assessed based on CHOP INTEND.

[0206] In some embodiments, the patient is determined to be 100% 15 ... A patient who is treated after exhibiting symptoms (e.g., one or more symptoms) of SMA Type I, as defined by In some embodiments, the patient is treated before exhibiting symptoms of SMA Type I. In an embodiment, the patient is diagnosed with SMA Type 1 based on genetic testing prior to becoming symptomatic. can be.

[0207] Combination therapy is also contemplated herein. Combination, as used herein, means simultaneously. Combinations of methods include treatments given in a specific standard or sequential manner. The addition of standard medical treatments (e.g., riluzole in ALS) as well as combinations with novel therapies For example, other SMA therapies include those that modify or bind to pre-mRNA to disrupt its splicing. These include antisense oligonucleotides (ASOs) that alter the signaling pattern. ngh.et al.,“A multi-exon-skipping detect ion assay reveals surprising diversity o f splice isoforms of spinal muscular atr ophy genes.” Plos One, 7(11):e49595. In the present invention, nusinersen (U.S. Pat. Nos. 8,361,977 and 8,980,8 53 (hereby incorporated by reference) may also be used. The results showed that the SMN2 pre-mRNA was expressed in a 10-fold increase in the 5'-terminal region of the SMN2 mRNA. It is a approved ASO that promotes more efficient production of full-length SMN protein. In some embodiments, the AAV9 viral vector regulates splicing of In some embodiments, the method of treatment is administered in combination with a muscle enhancer. Therapeutic methods involving the V9 viral vector are administered in combination with a neuroprotective agent. In one embodiment, the therapeutic method including the AAV9 viral vector is an anti-SMN targeting antibody. In some embodiments, the drug is administered in combination with a sense oligonucleotide-based drug. In one embodiment, the treatment method includes administering an AAV9 viral vector in combination with nusinersen. In some embodiments, the therapeutic methods comprising the AAV9 viral vector include a myostatin inhibitor. In some embodiments, the therapeutic agent comprising the AAV9 viral vector is administered in combination with a therapeutic agent. The therapy is administered in combination with stamulumasu.

[0208] Delivery to postnatal individuals in need thereof is contemplated, although in utero delivery to fetuses may also be used. It is planned.

[0209] Methods of treating SMA type I patients using pharmaceutical compositions containing viral vectors are contemplated. In some embodiments, the viral vector is about 1-8×10 13 AAV9 virus vector In some embodiments, the antibody is formulated at a concentration of 10 ... The virus vector is approximately 1.7 to 2.3 × 10 13 It is formulated at a concentration of 500 mg / mL. In one embodiment, the viral vector is about 1.9 to 2.1 × 10 13 At a concentration of vg / mL In some embodiments, the viral vector is formulated to be about 2.0×10 13 vg / It is formulated at a concentration of 1 mL.

[0210] In some embodiments, where a viral vector is used to treat a patient with SMA type I, AV viral vectors (e.g., AAV SMN) are approximately 1.0–2.5 × 10 14 vg / k The viral vector is used to treat SMA type I in patients. In some embodiments, the AAV viral vector is about 1.1 x 10 14 For vg / kg Some viral vectors are used to treat SMA type 1 in patients In an embodiment, the AAV viral vector is infused into the patient over a period of about 45 to 70 minutes. In some embodiments, where a viral vector is used to treat a patient with SMA type I, the AA The V viral vector is infused into the patient over a period of approximately 60 minutes. In some embodiments in which a viral vector is used, the AAV viral vector is The patient is infused using a pump, peristaltic pump, or any other device known in the art. In some embodiments, a viral vector is used to treat a patient with SMA type I. Therefore, the AAV viral vector is infused into the patient using a syringe pump.

[0211] The titer of the administered rAAV viral vector can be determined, for example, by the specific rAAV, the method of administration, It will depend on the therapeutic goal, the individual, and the cell type or types being targeted. The titer of rAAV may be determined by standard methods in the art. Approximately 1 x 10 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 ,about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 or higher DN Dosages may range from 100 μg / mL to 100 μg / mL of vector genome (vg). Genomic titers may be expressed in units of genomic DNA as described herein, Lock et al. The nucleic acid sequence may be determined using ddPCR, or any other method known in the art. can be done.

[0212] Dosages may also vary based on the timing of administration to humans. The dose is about 1 × 10 11 vg / kg, approximately 1×10 12 vg / kg, approximately 1×10 13 vg / kg, approximately 1×10 14 vg / kg, approximately 1×10 15 vg / kg, approximately 1×10 16 v The amount of vector genome per kilogram of body weight may range from 1000 to 1000 g / kg, or even more. For neonates, the rAAV dosage is approximately 1 × 10 11 vg / kg, approximately 1×10 12 v g / kg, approximately 3×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 3×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 3×10 14 vg / kg, approximately 1×10 15 vg / kg, approximately 3 x 10 15 vg / kg, approximately 1×10 16 vg / kg, approx. 3×10 16 vg / k g or more of vector genomes per kilogram of body weight.

[0213] Dosages may also vary based on the timing of administration to humans. The amount is about 1 × 10 11 vg / kg / week, approximately 1×10 12 vg / kg / week, approx. 1×1 0 13 vg / kg / week, approximately 1×10 14 vg / kg / week, approximately 1×10 15 vg / kg / week , about 1×10 16 vg / kg / week or more vector gain per kilogram of body weight For neonates, the dosage of rAAV is approximately 1 x 10 11 vg / kg / week, approximately 1×10 12 vg / kg / week, approximately 3×10 12 vg / kg / week, approximately 1×10 13 vg / kg / week, approximately 3×10 13vg / kg / week, approximately 1×10 14 vg / kg / week, Approximately 3×10 14 vg / kg / week, approximately 1×10 15 vg / kg / week, approximately 3×10 15 vg / kg / week, approximately 1×10 16 vg / kg / week, approximately 3×10 16 vg / kg / week or more The range for vector genomes per kilogram of body weight per week is 1 x 10 for adults. 11 vg / 1.5kg / week, approximately 1×10 12 vg / 1.5kg / week, approximately 1×10 13 vg / 1.5kg / week, approx. 1×10 14 vg / 1.5kg / week, approximately 1×10 15 vg / 1.5 kg / week, approximately 1×10 16 vg / 1.5kg / week or more per kilogram of body weight Dosage of rAAV containing vector genome. For neonates, the dosage of rAAV is approximately 1 ×10 11 vg / 1.5kg / week, approximately 1×10 12 vg / 1.5kg / week, approximately 3×10 1 2 vg / kg / week, approximately 1×10 13 vg / 1.5kg / week, approximately 3×10 13 vg / 1.5 kg / week, approximately 1×10 14 vg / 1.5kg / week, approximately 3×10 14 vg / 1.5kg / week , about 1×10 15 vg / 1.5kg / week, approximately 3×10 15 vg / 1.5kg / week, approx. 1× 10 16 vg / 1.5kg / week, approximately 3×10 16 vg / 1.5kg / week or more This may be in the range of 1.5 genomes of vector per kilogram of body weight per week.

[0214] In one embodiment, the dose is about 1.1 x 10 per kg of patient body weight. 14 Vector Game In one embodiment, for a 5 kg patient, the dose is 0.5×1 0 14 ~5.0×10 14 In one embodiment, the patient will receive a full dose of vector genome. In some embodiments, the viral vector is administered in Tris-buffered saline. In the above, the viral vector is administered at a dose of about 5 to 20 mL / kg, about 10 to 20 mL / kg, or It is administered in approximately 5.5-6.5 mL / kg of Tris-buffered saline.

[0215] Dosages can be determined by a number of standard methods. Primed PCR can provide relative measurements but is not suitable for smaller samples and absolute measurements. ddPCR is a deoxyribonucleic acid (dPCR) method based on the water-oil emulsion droplet technology. This is a method for performing digital PCR. Baker et al., "Digital PCR hits its stride.”Nature Methods,9(6):54 1-544. Sykes et al., “Quantitation of targ ets for PCR by use of limiting dilution. "Biotechniques, 13(3) 444-449. The sample is divided into tens of thousands of droplets. The droplets are then split and PCR amplification of the template molecules is carried out in each individual droplet. Because there is no need to have highly efficient primers, ddPCR is typically It does not require as many samples as traditional PCR-based techniques. Examples of suitable PCR products include, but are not limited to, BioRad QX100 ddPCR and RainDanc In one embodiment, the method includes the use of Raindrop Digital PCR. The amount is determined using PCR. In another embodiment, the dosage is determined using qPCR. In another embodiment, the doses are determined using digital droplet PCR (ddPCR). In some embodiments, the PCR-based method targets the SMN gene. The encapsidated AAV9 virus was identified using specifically designed primers and probes. In another embodiment, the PCR-based method detects and quantifies the chicken genome. Specifically designed primers and probes targeting the β-actin promoter were used. In another embodiment, the encapsidated AAV9 viral genome is detected and quantified. Therefore, the PCR-based method uses specifically designed primers that target the CMV enhancer. Chromatin markers and probes are used to detect and quantitate the encapsidated AAV9 viral genome. In another embodiment, the PCR-based method uses specifically designed PCR products that target the ITR sequences. Detect and quantify the encapsidated AAV9 viral genome using the designed primers and probes. In another embodiment, the PCR-based method quantifies bovine growth hormone polyadenylation. The encapsidation was performed using specifically designed primers and probes that target the encapsidation signal. Detect and quantitate the induced AAV9 viral genome.

[0216] In one embodiment, 2.0×10 13 Using vg / ml as the target concentration of the drug product, Doses will be administered according to the table below.

[0217] [Table 3]

[0218] In some embodiments, the pharmaceutical composition comprising the AAV viral vector is administered within about 20 to 70 minutes. In some embodiments, the AAV virus is injected into the patient over a period of, for example, about 45 to 70 minutes. The pharmaceutical composition containing the vector is infused into the patient over a period of about 60 minutes. The pharmaceutical composition containing the AAV viral vector is used in an infusion pump, a peristaltic pump or a pump of the present invention. The patient is injected using any other device known in the art. The pharmaceutical composition containing the AAV viral vector is injected into the patient using a syringe pump. will be done.

[0219] Pre-screening of patients suitable for treatment, as well as the Administration of treatment to patients identified by the criteria described above is also contemplated. As a result, AAV-based gene therapy can generate both inflammatory and humoral immune responses. A proportion of potential patients with AAV have pre-existing antibodies to AAV. t al.,“Pre-existing anti-Adeno-Associate d Virus antibodies as a challenge in AAV gene therapy.”Hum Gene Ther Methods,24( 2):59-67. Boutin et al., “Prevalence of se rum IgG and neutralizing factors against adeno-associated virus(AAV)types 1,2,5, 6,8,and 9 in the healthy population:impl ications for gene therapy using AAV vect ors.” Hum Gene Ther. 21:704-712. Prior anti-AAV antibodies have been used to treat AAV gene therapy because they can interfere with successful transduction even in mice. This poses a serious obstacle to universal application of the law. In some embodiments, the level of anti-AAV9 antibody titer in the patient is determined prior to administration of the antibody. The levels of anti-AAV9 antibody titers in patients were determined by ELISA binding immunoassay. In some embodiments, the patient is administered an ELISA binding immunoassay prior to administration of the treatment. In some embodiments, the antibody has an anti-AAV9 antibody titer of 1:100 or less as determined by In the study, patients were randomly assigned to receive 1 mg / kg / day of treatment or 2 mg / kg / day of treatment, as determined by ELISA binding immunoassay prior to administration of treatment. In some embodiments, the patient has an anti-AAV9 antibody titer of 0.50 or less after treatment. Anti-AAV9 antibodies greater than 1:100 as determined by ELISA binding immunoassay titers and are monitored for 1-8 weeks or until titers fall below 1:100 In some embodiments, the patient is assessed for seroconversion by ELISA binding immunoassay after treatment. have an anti-AAV9 antibody titer of greater than 1:100 as determined by immunohistochemistry for 1 to 8 weeks, or until the titer falls below 1:50.

[0220] One approach to resolve high anti-AAV antibody titers is the use of immunosuppressants. The monoclonal anti-CD20 antibody rituximab in combination with lysine A reduces anti-AAV titers Mingozzi et al., “Pharm acological modulation of humoral immunity y in a nonhuman primate model of AAV gen e transfer for hemophilia B.”Mol Ther,20 Another approach is to use a platelet-based antibody depletion kit to deplete neutralizing antibodies before vector administration. The use of smapheresis. Monteilhet et al., “A 10 p patient case report on the impact of plas mapheresis upon neutralizing factors aga inst adeno-associated virus (AAV) types 1, 2, 6, and 8.” Mol Ther, 19(11): 2084-2091. Plus In mapheresis, blood is drawn from the patient and the plasma and blood cells are separated using a centrifuge or hollow fiber filter. The blood cells are then separated by either filtration or in treated plasma or saline. The fluid is then returned to the patient along with one of the following replacement fluids: 5% human albumin. A common use of is the removal of unwanted immunoglobulins, in which case the plasma Elimination represents an attractive approach to deplete anti-AAV antibodies. In certain embodiments, patients are administered ELISA binding immunoassays before and after treatment. had anti-AAV9 antibody titers of greater than 1:100 when treated with plasmapheresis In some embodiments, the patient is administered an ELISA binding immunoassay before or after treatment. have an anti-AAV9 antibody titer of greater than 1:50 as determined by plasmapheresis Treated with cis.

[0221] Pre-existing maternal antibodies against AAV9 may be transmitted to infants through breast milk or transplacental transfer in utero. In some embodiments, the patient may be screened by ELISA binding assay before or after treatment. have an anti-AAV9 antibody titer of greater than 1:100 as determined by immunoassay, In some embodiments, the patient is switched to ELISA before or after treatment. had an anti-AAV9 antibody titer of greater than 1:50 as determined by A binding immunoassay , and switched to artificial nutrition.

[0222] The condition of the patient may be monitored before and after administration of the treatment. Some patients with thrombocytopenia have a condition characterized by low platelet counts. Thrombocytopenia is a condition where the complete blood count is increased using a diluted blood sample in a hemocytometer. Thrombocytopenia can also be detected by examining the patient's blood (thin smear or peripheral It can also be detected by observing slides prepared with blood smears under a microscope. Normal human platelet counts range from 150,000 cells / ml to approximately 450,000 cells / ml. It is.

[0223] In some embodiments, the patient has a platelet count of greater than about 67,000 cells / ml prior to administration. or has greater than about 100,000 cells / ml, or greater than about 150,000 cells / ml. In some embodiments, the patient has a platelet count of less than about 150,000 cells / ml prior to administration. or less than about 100,000 cells / ml, or less than about 67,000 cells / ml , for 1 to 8 weeks, or a platelet count of more than about 67,000 cells / ml or more than about 100, The cells are monitored until they increase to greater than about 10,000 cells / ml, or to greater than about 150,000 cells / ml. In some cases, platelet counts after administration of the viral vector are less than approximately 67,000 cells / ml. In some embodiments, the patient may be treated with a platelet transfusion. In some embodiments, the patient is free of thrombocytopenia prior to administration of the viral vector. Patients have thrombocytopenia after administration of the viral vector for approximately 1 to 8 weeks or In some embodiments, the patient is monitored until he or she is no longer suffering from thrombocytopenia. The patient had thrombocytopenia after administration of vector and was treated with platelet transfusions.

[0224] Patient status will also be monitored using platelet, serum protein electrophoresis, serum gamma glutamate, and Glycotransferase (GGT), aspartate transaminase (AST) and Alanine aminotransferase (ALT), total bilirubin, glucose, creatinine Enzyme (CK), creatinine, blood urea nitrogen (BUN), electrolytes, alkaline phosphatases This may also involve standard blood tests to measure troponin I levels. Cardiac function is a common measure of heart health, and elevated levels are indicative of heart damage or heart-related conditions. In some embodiments, Troponin-I levels reflect In some embodiments, the patient is monitored for about 0.3, 1.0, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, The patient may have a Troponin-I level of less than 0.2, 0.15, or 0.1 μg / ml. In some embodiments, the patient has a blood glucose level of less than about 0.176 μg / ml prior to administration of the viral vector. In some embodiments, the patient may have a troponin-level. In some embodiments, the subject may have a troponin level of greater than about 0.176 μg / ml after administration. In this study, patients had troponin-I levels of approximately 0.176 μg / m after administration of the viral vector. Cardiac monitoring will be performed until the blood pressure drops below 1.

[0225] Aspartate transaminase (AST) and alanine aminotransferase ALT and total bilirubin are common measures of liver function, while creatinine is a measure of renal 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 has a hepatic vasculitis of less than about 8-40 U / L prior to administration of the viral vector. In some embodiments, the patient has a transaminase level. In some embodiments, the patient has an AST or ALT level of less than about 8-40 U / L prior to administration of the In this study, patients had bilirubin levels of less than 3.0 mg / dL prior to administration of the viral vector. In some embodiments, the patient has a blood glucose level of 1.8 mg / dL prior to administration of the viral vector. In some embodiments, the patient has a creatinine level of less than 100 mg / kg. and a hemoglobin (Hgb) level of 8-18 g / dL prior to administration of In this study, patients had a 20,000 / mm 3 Less than 100% white blood cells (WBC ) number.

[0226] The effectiveness of the treatment method may be determined using various motor skill tests before and after treatment. In detail, the Children's Hospital of Philadelphia Breast Cancer Center (CHCCH) and the Children's Hospital of Philadelphia Breast Cancer Center (CHCCH) were used to assess the motor skills of patients with SMA type I. Pediatric Neuromuscular Disease Screening (Children's Hospital of Philadelphia phia Infant Test of Neuromuscular Disorder Glanzman et al., “ The Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders(C HOP INTEND):Test development and reliabi lity.”Neuromuscular Disorders,20(3):155- 161. The CHOP INTEND test was performed at a mean age of 11.5 months (range 1.4-37.9 months). Test of Infant Motor Skills for 26 infants with type I SMA TIMP (T Motor Performance) and the newly invented SMA The Children's Hospital of Philadelphia SMA Strength Test (CHSMS) is a dynamic assessment of the pital of Philadelphia Test of Strength i n SMA:CHOP TOSS) was developed based on the evaluation of efficacy of treatment. The tests include, but are not limited to, CHOP INTEND, TIMP, CHOP T OSS, Peabody Developmental Motor Scales r Scales, Brazelton Neonat al Behavior Assessment, Motor Milestone Development Survey ( Motor Milestone Development Survey, Inter Ability Captured Thro ugh Interactive Video Evaluation: ACTIVE) , Bayley Scale of Infant Development lopment and compound motor action potential (CMAP) measurements. Other motor skill tests known in the art may also be included.

[0227] In some embodiments, the pretreatment baseline test is assessed using the CHOP INTEND scale. In one embodiment, the efficacy of the treatment is assessed by measuring CH during a follow-up visit. In some embodiments, the CHOP IN INTEND scale is used to determine the TEND measures neck alignment, righting reflex, and trunk movement in supported sitting, supine, and prone positions. In some embodiments, CHOP INTEND includes a measure of assisted effort. Includes measures of antigravity movement during turning, prone suspension and supported standing.

[0228] Many gene therapy studies involving AAV vectors have focused on antigen-specific targeting of AAV vectors. A targeted T cell response has been observed, which can be expected within 2-4 weeks after gene transfer. One possible consequence of the allele-specific T cell response is clearance and transduction of the transduced cells. This is due to the lack of expression of the IL-1 gene, which weakens the host immune response to AAV-based therapy. The patient may be given immunosuppressive drugs. In some embodiments, the patient is administered a viral vector. A glucocorticoid may be given prior to administration of the agonist. The patient may be given corticosteroids prior to administration of the viral vector. In some cases, the patient may be given oral steroids prior to administration of the viral vector. Examples of oral steroids include, but are not limited to, prednisone, prednisolone, Methylprednisolone, triamcinolone, betamethasone e) dexamethasone and hydrocortisone. In some embodiments, The oral steroid is or comprises prednisolone. Patients will begin prophylactic steroids at least 24 hours prior to viral vector administration. In some embodiments, the patient is administered an oral steroid at least once daily after administration of the viral vector. In some embodiments, the oral steroid is administered once daily for 30 days. In some embodiments, the oral steroid is administered twice daily. In an embodiment, the oral steroid is about 0.1 to 10 mg / kg, for example about 1 mg / kg. In some embodiments, the oral steroid is administered at a dose of about 0.1 to 10 mg. g / kg / day, e.g., about 1 mg / kg / day. In this embodiment, AST and ALT levels are monitored after administration of the viral vector. In such cases, oral steroid therapy may be used to improve AST and ALT levels, e.g., to meet clinical standards and Two times the upper limit of normal as determined by methods known in the art, or about 120 IU In some embodiments, oral steroid therapy is administered when the A ST and ALT levels are determined, for example, by clinical standards and methods known in the art. As long as the blood levels remain above twice the upper limit of normal or approximately 120 IU / L for more than 30 days, During continuous treatment with corticosteroids, the adrenal glands spontaneously increase their production of cortisol. If corticosteroid treatment is suddenly stopped, the body becomes deficient in cortisol. In some embodiments, the oral steroid is given to the patient for at least 30 days. In some embodiments, the steroid dose is gradually tapered according to a schedule. Oral steroid doses are adjusted so that AST and ALT levels are consistent with, for example, clinical standards and those known in the art. is less than twice the upper limit of normal, or about 120 IU / L, as determined by known methods. In some embodiments, the taper is 0.5 mg / kg over two weeks. kg / day, followed by a stepwise taper to 0.25 mg / kg / day for an additional 2 weeks. In some other embodiments, the taper of the oral steroid is at the discretion of the physician. It can be done.

[0229] kit Disclosed herein is also a kit for treating SMA in a patient in need thereof. Also provided are kits, wherein the kits comprise a ubiquitous IgG antibody comprising the SMN polynucleotide disclosed herein. The present invention relates to a method for treating SMA comprising administering to a patient a therapeutically effective amount or dose of a pharmaceutical composition comprising the steps of: As further disclosed herein, the kit may include an imaging agent (e.g., omni paque 180), and instructions for use of pharmaceutical preparations or compositions and imaging agents It further includes a book.

[0230] In some embodiments, the kit comprises a vial of the viral vector pharmaceutical composition. In some embodiments, the viral vector pharmaceutical composition comprises about 1.7 to 2.3 x 10 13 In some embodiments, the viral vector pharmaceutical composition has a concentration of about 1 vg / mL. .9~2.1×10 13 In some embodiments, the concentration of the viral vector is The pharmaceutical composition is about 2.0×1013 In some embodiments, the concentration is in vg / mL. Thus, 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 this case, the kit does not contain a 5.9 mL vial or contains at least one 5.9 mL vial. vials, at least two 5.9 mL vials or at least three 5.9 mL vials In some embodiments, the kit does not include or contains at least an 8.7 mL vial. At least one 8.7 mL vial, at least two 8.7 mL vials, at least three 1 x 8.7mL vial, at least 4 x 8.7mL vials, at least 5 x 8. Contains 7 mL vials, at least six 8.7 mL vials.

[0231] In some embodiments in which the kit is used to treat a patient with SMA type 1, the patient's weight is determined. In some embodiments, where the kit is used to treat a patient with SMA type I, the patient The patient weighs at least about 2.6 kg. The kit is used to treat SMA type I in a patient. In some embodiments, the patient weighs about 8.5 kg or less. In some embodiments for use in treating SMA Type I, the patient weighs between about 2.6 and 8.5 kg / m In some embodiments, where the kit is used to treat a patient with SMA Type 1, the kit The AAV viral vector from the vial in the kit is administered to the patient. In some embodiments, the AAV from a vial in the kit is used to treat SMA. The viral vector is approximately 1.0–2.5 × 10 14 The drug is administered to patients at a dose of 1000 mg / kg. In some embodiments, the kit is used to treat a patient with SMA type 1. The AAV viral vector from the iAL was approximately 1.1 × 10 14 Administered to patients at a dose of 1000 mg / kg In some embodiments, where the kit is used to treat a patient with SMA type I, The AAV viral vector from the vial in the kit is infused into the patient over a period of approximately 45 to 70 minutes. In some embodiments, where the kit is used to treat a patient with SMA type I, The AAV viral vector from the vial is infused into the patient over a period of approximately 60 minutes. In some embodiments, the vial in the kit is used to treat a patient with SMA type I. The AAV viral vectors of the present invention can be used in infusion pumps, peristaltic pumps, or other pumps known in the art. The kit may be used to treat SMA Type 1 in a patient. In some embodiments, the AAV viral vector from a vial in the kit is It is infused into the patient using a syringe pump.

[0232] In one embodiment, the vector is administered intravenously or intrathecally. The vector is administered intravenously. In one embodiment, the vector is administered intravenously using omnipaque 1 80. In another embodiment, the vector is administered intravenously with omnipaque. 180 is given intrathecally.

[0233] In another embodiment, as provided herein, rAAV is administered to target cells of a patient, including but not limited to, Methods of transducing cells, including epithelial or glial cells, are contemplated.

[0234] Transducing a patient's cells with the rAAV disclosed herein results in the production of a virion that is encoded by the rAAV. Thus, the present disclosure provides a method for the production of rAAV( The present invention provides a method for administering / delivering a nucleic acid molecule (e.g., encoding an SMN protein) to an animal or human patient. Such methods include transducing neural and / or glial cells with one or more rAAVs. The transduction is carried out with a gene cassette containing tissue-specific regulatory elements. For example, specific expression in neurons or specific expression in astrocytes may be achieved. Examples include neuron-specific enolase and glial fibrillary acid Protein promoters include inducible promoters that are under the control of ingested drugs. may also be developed.

[0235] In some embodiments, the vectors of the present disclosure are combined with imaging agents as described herein. Characteristics of the vectors of the present disclosure when used in combination and when not used in combination with an imaging agent It is contemplated that the transduction of cells is increased compared to the introduction. When the disclosed vectors are used in combination with imaging agents as described herein, Transduction of cells compared to transduction of the vectors of the present disclosure when not used in combination with a contrast agent. The percentage of the ion exchange rate is at least about 1%, or at least about 5%, at least about 10%, or 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 10 0%, at least about 120%, at least about 150%, at least about 180%, at least 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 increase In further embodiments, the cells are transduced using the vectors of the present disclosure as described herein. When used in combination with a contrast agent as specified, About 10% to about 50%, or about 10% to about 100%, compared to the transduction of the vector of the present disclosure. %, or about 5% to about 10%, or about 5% to about 50%, or about 1% to about 500%, or about 1 0% to about 200%, or about 10% to about 300%, or about 10% to about 400%, or about 10 Increases by 0% to about 500%, or by 150% to about 300%, or by 200% to about 500% .

[0236] The present disclosure also provides methods for administering the vectors and imaging agents of the present disclosure to the central nervous system of a patient in need thereof. When administered intrathecally, the vectors of the present disclosure improved patient survival compared to those administered without a contrast agent. In various embodiments, the vectors and methods of the present disclosure provide an improved method for the treatment of cancer. The contrast agent and the endothelial tissue are administered intrathecally into the central nervous system of the patient in need of the agent. In an embodiment, the vector and imaging agent are co-formulated and administered to the central nervous system or an area in need thereof. In another embodiment, the vector and imaging agent are administered intrathecally to a patient receiving central nervous system therapy. The system is provided in the same package for intrathecal administration to a patient in need thereof. In certain embodiments, the vectors and imaging agents of the present disclosure are administered to the central nervous system of a patient in need thereof. When administered intravenously, the vectors of the present disclosure have improved patient survival compared to when administered without a contrast agent. , 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 An increase of about 100%, at least about 150%, at least about 200% or more.

[0237] In some embodiments, the vectors of the present disclosure, when used in combination with imaging agents, Patients were placed in the Trendelenberg position (head down) It is contemplated that when the nuclease is placed in a nuclease-resistant position, transduction of the cells is further increased. In the present study, for example, the patient is placed in a head-down position for approximately 1 hour during or after intrathecal vector injection. 30 degrees, 15 to 30 degrees, 30 to 60 degrees, 60 to 90 degrees, or 90 to In various embodiments, the cells are transduced by the method described herein. The vectors of the present disclosure are administered as described in the following manner using contrast agents and Trendelenburg position. When used in combination with the Lenberg position, contrast and trend Used in combination with Trendelenberg position at least about 1%, or at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 4 0%, at least about 50%, at least about 60%, at least about 70%, at least about 8 0%, 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 45 In a further embodiment, the cell morphology is increased by at least about 500% or more. Transduction can involve the incorporation of vectors of the present disclosure into imaging agents and trendelenbull as described herein. When used in combination with the Trendelenberg position , contrast medium and Trendelenburg position ) in combination with the vectors of the present disclosure, 0%, or about 10% to about 100%, or about 5% to about 10%, or about 5% to about 50%, or About 1% to about 500%, or about 10% to about 200%, or about 10% to about 300%, or about 0% to about 400%, or about 100% to about 500%, or about 150% to about 300%, or about Increases by 200% to approximately 500%.

[0238] The present disclosure also relates to the use of vectors and imaging agents of the present disclosure in the Trendelenburg position. to the central nervous system of a patient in need of it in the Lenberg position. When administered intrathecally, patient survival was improved by the incorporation of the vectors of the present disclosure into imaging agents and Trendelenburg complexes. Patient survival when administered without the Trendelenberg position In various embodiments, the vectors and The contrast agent was administered in the Trendelenburg position. When administered to the central nervous system of a patient in need thereof, the survival of the patient is improved by the vector of the present disclosure. - contrast medium and Trendelenburg position n) at least about 1%, at least about 5%, or a smaller proportion of patients with 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 increase.

[0239] As used in this disclosure and the appended claims, the singular forms "a," "an," and "an" are intended to be used interchangeably. "The" includes plural referents unless the context clearly dictates otherwise. or optionally refers to the occurrence or non-occurrence of the subsequently described event or circumstance. and the description may include cases where the event or circumstance occurs and cases where it does not occur. For example, the phrase "a composition may optionally include a combination" means The composition may or may not include a combination of different molecules. The description includes both combinations and non-combinations (i.e., individual combinations). Ranges are defined herein to include both about and including one particular value. When such a range is expressed, it may be expressed as from and / or to about another particular value. The aspects of include from one particular value and / or to the other particular value. When values ​​are expressed as approximations, by the use of about, it is to be understood that the particular value forms another embodiment. It will be understood that the endpoints of each range are significant relative to the other endpoint. It will be understood that both endpoints are significant independently of the other endpoint.

[0240] The present disclosure is further illustrated by the following examples, which should not be construed as limiting. All references, patents, and published patent applications cited throughout this application, as well as figures, are incorporated herein by reference in their entirety. , which is incorporated herein by reference in its entirety for all purposes. EXAMPLES

[0241] The following examples are illustrative and are not intended to limit the scope of the disclosure described above. It should be done.

[0242] Example 1 - Creation of a pre-GMP master cell bank method Thaw: Single cell vial (1 x 10 6 Thaw the cells in a 37°C water bath for about 1 minute and then freeze for 5 min. Dilute the contents into 1 L of pre-warmed complete growth medium. Plate the cells in a T-25cm 2 Transfer to flask Incubate the plates in a 37 °C incubator for 4 days, replacing the culture medium with pre-warmed complete growth medium every day. While growing.

[0243] Selection for increased adhesion: Culture the cells using the following technique to select for cells that adhere strongly. I chose 25cm. 2 Cells were passaged after reaching 95% confluency in the flask. The cells were washed with 5 mL of PBS and then dehydrated with 0.5–1 mL of HyQTase. Dissociation was stopped by adding 5 mL complete growth medium and repeated. Cell clumps were dissociated by repeated pipetting. The cell suspension was then centrifuged at 200 x g for 4 min. The supernatant was discarded and the cell pellet was resuspended in 10 mL of complete growth medium. 75cm 2 After 4 hours of incubation in a 37°C incubator, Then, weakly adherent cells were washed away by aspirating the cell culture medium to isolate weakly adherent cells and non-adherent cells. Adherent cells were removed. The culture medium was replaced with 10 mL of pre-warmed complete growth medium. The process reduced cell mass by up to 35% of cell number by visual inspection. After incubation for 1 day, the cells were subcultured again. After three rounds of selection, the selected cell population was expanded (Figures 2 and 3). In the step, cells were cultured in 2 x 175 cm plates for a final volume of 25 mL. 2 The flasks were seeded. It was noted that there was a reduction in cell loss after the last medium change 4 hours after seeding.

[0244] Cell expansion: The cells are then enlarged to 2 × 175 cm 2 After the cells became confluent in the flask, The cells were washed with 15 mL PBS and then dissociated with 3 mL HyQTase. Incubate at room temperature for approximately 2 minutes. Dissociate by adding 10 mL of complete growth medium. The cell suspension was then centrifuged to give two cell pellets after aspiration of the supernatant. Each pellet was resuspended in 8 mL of complete growth medium and 2 mL of this concentrated cell suspension was added to 8 × 175cm 2 These flasks were filled with 20 mL of complete growth medium for a total of 2 Prepare by adding 2 mL of cell suspension and a split ratio of 1:4. The same procedure was used for the enlargement step, with the following changes: 4 × 175 cm 2 The flask is Enlarged at a 1:2 ratio and 4×175cm 2 Flasks were expanded at a split ratio of 1:3. This gives a total of 20 x 175 cm 2 It became a flask.

[0245] Collection: 20×175cm 2 The cells were harvested from the flask. The cells were washed with 15 mL of PBS. The cells were washed and then dissociated with 3 mL HyQTase as described above. Cell dissociation was stopped by adding 4 × 175 cm 2 Cell suspension from flask 1 The samples were collected in a 50 mL tube in addition to the 50 mL x 50 mL tube. The tubes were centrifuged to produce a cell pellet, which was then removed from the supernatant. Aspirate the supernatant and resuspend the cells in 10 mL of complete growth medium, resulting in a 50 mL cell suspension. I did.

[0246] Add this volume to 2 x 50 mL tubes for a total of 25 mL of cell suspension per tube. This sample was diluted 1:2 and used to measure the hemocytometer and Toluidine. Viable cell count per tube using Toludine (Trypan) Blue The number of viable cells in tube 1 sample was calculated as 1.99 × 10 6 Cells / mL, 3.98 ×10 6 A cell concentration of 100 cells / mL was obtained, and the tube 2 sample had a viable cell count of 2.4× 10 6 cells / mL (total 1 × 10 8 cells), 4.8 × 10 6 The cell concentration in cells / mL was obtained. (total 1.2 × 10 8 cells), so a total of 2.2 × 10 8 The cells were harvested. Centrifuge one tube again (6 min, 200 × g) and transfer the pellet to 10 mL (tube Resuspend the cells in 12 mL (tube 1) and 12 mL (tube 2) of freezing medium to a cell concentration of 1 × 10 7 Thin These two cell suspensions were pooled and transferred to 22 sterile cryovials. Add 1 mL aliquots (1 × 10 each) to the 7 The cells were then filled with 100 μl of ...

[0247] [Table 4]

[0248] The filled vials were then transferred to a freezing chamber with fresh isopropanol overnight - The vials were frozen at a controlled rate in a freezer at 80°C. The frozen vials were then placed in a liquid nitrogen bath. The 10 vials that will be banked in the GMP facility were transferred to 100 mL of vapor-phase liquid nitrogen. Transfer onto dry ice.

[0249] HEK293 cells from ATCC were thawed and adapted for increased adherence over three passages. After this, the project expanded and successfully created a seed bank. The samples were tested for the presence of pathogens (mycoplasma, fungi and bacteria). It was shown that the seed bank is suitable for master cell banking in a GMP facility.

[0250] Example 2 - Upstream process Intermediate cell bank derived from working cell banks using upstream processes (e.g., see Figure 4) The upstream process includes (a) culturing adherent cells; (b) culturing the cultured cells; In one embodiment, three plasmids as shown in FIG. 1 (e.g., the AAV S vectors described herein) are A step to transfect cells (containing MN) to enable production of AAV viral vectors. (c) lysing the adherent cells to isolate the AAV viral vector; The virus particles are purified 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 purified virus particle intermediate preparation. In certain embodiments, the AAV prepared by the upstream processes disclosed herein is shRNA encoding SOD1 or MECP2 as disclosed herein .

[0251] (a) Culture of adherent cells Thaw HEK293 cells and incubate them in a disposable flask for 7 h in a CO2 incubator. The thawed cells were washed with cell expansion growth medium, centrifuged, and resuspended in fresh lysates. The resuspended cells were placed in a flask containing Cell Expansion Growth Medium. Plate and incubate.

[0252] When the cells became confluent, they were washed with DPBS and incubated with TrypLE S. The flask was then removed with the enzyme solution. Cell expansion growth medium was added to the flask to induce enzyme me) Neutralize the solution and split the suspended cells into new flasks containing cell expansion growth medium. This expansion process was repeated seven times. In the final round, the suspension cells were reseeded in flasks. The cells were not reseeded, instead the cell slurry was inoculated into a bioreactor for further expansion.

[0253] Prior to inoculation, iCELLis 500 / 200m 2 or iCELLis 5 00 / 333m 2 An adherent cell bioreactor was prepared. Reactor unpacking, physical inspection, leak testing, tubing assembly installation, and probe flattening Equilibration was included. The bioreactor was equilibrated with cell expansion growth medium. pH (p H6.9~7.5), temperature (35℃~39℃), and dissolved oxygen (40~125%) are specified. After confirming that the cells were within the range of 4800–7000 cells / cm, the bioreactor was 2 (2 00m 2 Reactor) or 5000-12000 cells / cm 2 (333m 2rear The medium in the recirculating medium bag was seeded at the target seeding density of 100 μg / ml (for the 100 μg / ml vector). The cell slurry from the flask was added and circulated through the bioreactor.

[0254] (b) Transfection of adherent cells Four, five or six days after bioreactor inoculation, adherent HEK293 cells were inoculated with triplex DNA. The plasmid PEI co-precipitate was transfected. The three plasmids used were pSMN, pAAV2 / 9, and pHELP. Remove the spent DMEM growth medium from the bioreactor and transfection medium In large-scale adherent cell bioreactors, polyethyleneimine (PEI) ) triple DNA plasmid co-precipitated into adherent human embryonic kidney (HEK293) cells Use transfection to generate the scAAV9.CB.SMN vector. The vector plasmid pSMN contains the cDNA for human survival motor neuron protein (SMN). The three plasmids used in this transfection were pSMN(222m g), pAAV2 / 9 (333 mg), and pHELP (444 mg). Plasmids may be transfected at a molar ratio of 1:1:1. The medium was incubated until the bioreactor temperature was >30°C before adding the PEI-plasmid co-precipitate. The PEI-plasmid coprecipitation step was carried out in a bioreactor until equilibration was complete. Add the plasmid to the transfection medium and filter it through 0.2μ into the reaction bag. PEI was added to the transfection medium, which was then added to the reaction bag. The PEI-plasmid ratio is approximately 1:1 by weight. PEI-plasmid reactions are manually The mixture was mixed at 0.5° C. to form a uniform suspension, and the reaction was carried out for 15 to 30 minutes. At the end of the reaction time, the PEI-plasmid co-precipitate was transferred from the reaction bag to the bioreactor. The PEI-plasmid co-precipitation was incubated in the bioreactor for 1-2 h (another continuous incubation). After mixing for a period of time (described in Example 7), agitation was resumed. The transfection medium was recirculated through the bioreactor for 18-24 h prior to transfection.

[0255] Bioreactor day 6, 18-24 hours after transfection, Drain the filter and recirculate the transfection medium bag to the post-transfection After transfection, the medium was recirculated in the bioreactor while The bioreactor was refilled. On day 7, 18-24 hours after the medium change on day 6, recirculation The post-transfection medium in the bag is replaced with a fresh post-transfection medium bag. During this step, the bioreactor was not drained. Recirculation of the medium was usually Continue until collection on the 9th day.

[0256] (c) Lysis of transfected adherent cells After 9 days in the bioreactor, a final pre-harvest sample was taken from the reactor and all aliquots were The cell lysis step was started. Add 100 U / mL of benzoyl peroxidase to the bioreactor. Benzonase was added to the reactor. The benzonase was mixed in the reactor and dissolved and diluted in water. The buffer was mixed in the reactor at 15-25°C for 2 hours, and then the bioreactor was The contents of the actor were transferred to a collection bag. A saline sucrose solution ( SSS) was added to the harvest bag and mixed for 15 minutes. The bioreactor was then Rinse with quench buffer for 15 minutes, then transfer the rinse fluid to a collection bag. After the rinse was added to the collection bag, the contents were allowed to sit for 15 min. Mix and take bulk harvest sample.

[0257] (d) Preparation of virus particles by filtration and tangential flow filtration The combined bulk harvest was filtered through a POD depth filter into a collection bag. After the bulk harvest was filtered, the depth filter was chased with TFF1 buffer. The depth filter pool liquid was mixed and sampled. The pool was filtered through a 0.45 μm filter to further clarify the bulk harvest material. The .45 μm filter was chased with TFF1 buffer.

[0258] For TFF1 step, 5.0m 2 300kDa MW cutoff regenerated cellulosic acid The membrane cassette was flushed, sanitized with NaOH solution and equilibrated with TFF1 buffer. The concentration phase of the operation was designed to reduce the volume of the clarified harvest by approximately 10-fold. After the target retentate volume is reached, the diafiltration process begins. The retentate was diafiltered with 10 volumes of TFF1 buffer. Diafiltration of the residual solution should be performed with more than 6 diavolumes of TFF1 buffer, e.g. For example, 10 diavolume, 12 diavolume, or 15 diavolume. After a total permeate flow rate of 6 diavolumes was achieved, the retentate was again The concentrate was then collected in a collection bag. Two successive membrane rinses were performed to remove the eluate from the TFF system. The product recovery was maximized and an intermediate drug substance was produced.

[0259] (e) Freezing of intermediates In an LFH hood, aliquot the TFF1 intermediate into 1 or 2 liters of sterile PETG Bottle, then freeze on dry ice or in a freezer and transfer to a -60°C storage location. did.

[0260] [Table 5]

[0261] Example 3 - Downstream Processing Downstream steps (see, e.g., Figure 5) are used to process the TFF1 intermediate into filtered drug substance. In some embodiments, the downstream processes disclosed herein can be used to AAV SMN, AAV MECP2, or SOD1 targeting sh The downstream process steps may include processing intermediates containing AAV encoding RNA. (b) acidifying and clarifying the interstitial medium (by filtration); (c) tangential flow filtration ("TFF2"); (d) filtering the contents by ultracentrifugation using CsCl buffer; (e) separating the filled and empty viral capsids; (d) collecting the AAV viral vector from a second tank. The method includes filtering the mixture through a thermal flow filtration ("TFF3") step.

[0262] (a) Acidification and Clarification The TFF1 intermediate material from the upstream process (if previously frozen, thaw to room temperature) is mixed with The pooled TFF1 intermediates were mixed and a sample was taken and analyzed. The pooled TFF1 intermediate was diluted with 11–14% Tween 20 and then diluted with 10% Tween 20. Tween 20 inhibits the growth of bulk host cells under acidic pH conditions. This mixture was used to promote the flocculation of cellular proteins and DNA. The mixture was incubated for 12 to 20 hours. Then, acidification buffer (1M glycine) was added. The pH was then lowered to 3.3-3.7 by adding 1.1 ml 2 Clari solve and 2.2m 2 Millistak+C0HC depth filter and 0.45 The precipitate formed after lowering the pH was removed by filtration through a 1 μm polishing filter. This step resulted in an acidified and clarified TFF intermediate.

[0263] (b) Purification by cation exchange chromatography A cation exchange (CEX) chromatography step was used to isolate viral capsids. Separates proteins, DNA, and other process impurities, e.g., host cell lipids, TWEEN 20 This step was performed using an automated process chromatography system. CIMmultus S03-8000 Advanced Composite Column (Sulfonyl) (0.2μm A thin-diameter chromatography column (8.0 L) was used. The following buffer solutions and solutions were used: The following table shows:

[0264] [Table 6]

[0265] The acidified and clarified TFF intermediate (i.e., CEX load) was purified and equilibrated with C The conditions were such that the viral vector bound to the monolith column. The unbound material was washed from the column with CEX A buffer. The product was eluted from the resin with a gradient of CEX B buffer in the eluent. Starting from the beginning of the gradient, the reaction mixture was cooled to room temperature and then cooled to room temperature for 10 column volumes (CV) at 2.3 to 2.7 °C. The chromatography column was discarded after each batch (i.e., The CEX product eluate (fraction 2) was then neutralized with neutralizing buffer. The solution was neutralized to a pH of 7.7 to 8.3.

[0266] (c) Filtration by tangential flow filtration (TFF2) The TFF2 step concentrates the viral vector, removes protein impurities, and buffers The solution was exchanged into the appropriate buffer for the CsCl ultracentrifugation step. The neutralized CEX eluate was diluted with 0. 3m 2 The TFF system was equipped with a 300 kDa MWCO regenerated cellulose membrane. I understood.

[0267] The volume of the neutralized CEX eluate was reduced to the target retentate volume. Afterwards, diafiltration was started in discontinuous TFF mode (batch mode). The filtrate was diluted 2-fold with TFF2 NaCl diafiltration buffer and the retentate was diluted 2-fold with TFF2 NaCl diafiltration buffer. The TFF2 was concentrated to the final volume using NaCl diafiltration buffer. This was repeated until filtration was complete. The retentate was then diluted with TFF2 CsCl Dilution was performed 2-fold with diafiltration buffer and the retentate was concentrated to its initial volume. FF2 Diafiltration with NaCl diafiltration buffer is complete. This was repeated until complete.

[0268] Neutralized CEX eluate physical titer, system hold-up volume, system flow Based on the brush volume, and retentate density, the retentate is further concentrated to a final weight to obtain the desired target vector. The TFF2 CsCl diafiltration tray was then placed in a collection bag. After one flush cycle of the system with the purification buffer, the product was blown down and The product recovery from the TFF system was maximized. A sample of the TFF2 retentate (which is the retentate The TFF2 membrane cassette (containing the filtrate and flush) was taken for physical titration. was discarded after each batch (i.e., the TFF membranes were not reused).

[0269] [Table 7]

[0270] (d) CsCl ultracentrifugation The purpose of the ultracentrifugation step is to obtain complete encapsulation by using cesium chloride gradient ultracentrifugation. The aim of this study was to remove empty capsids from the sids. Add the TFF2 retentate to the ultracentrifuge tube. The tube was sealed. Place the tubes in an ultracentrifuge, such as the Tima XPN 100 Ultracentrifuge System or equivalent. The filled tubes were centrifuged at 45,000 rpm at 20° C. for 22 hours.

[0271] (e) Collection of AAV viral vectors After the centrifugation step is complete, remove the tubes from the ultracentrifuge and store them in the biosafety cabinet. The tube containing the product was mounted on a ring stand above a waste container. The lamp was positioned directly underneath the tube to capture the empty capsid band (Band A, top band A), a doublet band of intact capsids (band B and band C, doublet band B) The upper and lower bands, and the bottom band below the doublet (band D) were visualized. The needle attached to the syringe is used to puncture the tube to vent it, and the needle is used to insert the band. B, C, and D were removed. The collected material was transferred to a collection bag. The centrifugation pool (UC pool) was diluted with TFF2 buffer to obtain a consistent The diluted UC pool is then processed through a TFF3 step, resulting in a starting CsCl concentration of 100 mM. The buffers for the CsCl ultracentrifugation step are listed in the table below.

[0272] [Table 8]

[0273] (f) Filtration by tangential flow filtration (TFF3) In the TFF3 step, CsCl is removed and the complete vector is purified using the final formulation buffer. was concentrated. 2 Tangent with a 300 kDa MWCO regenerated cellulose membrane A single flow filtration system was used, and the viral vector was retained by the membrane.

[0274] After dilution, the volume of the UC pool was reduced to the target retentate volume. After the target volume was reached, Continuous diafiltration of the retentate volume was started. A sample of the retentate from the diafiltration was analyzed by physical chromatography. The retentate was further concentrated by targeting the permeate weight, and this The weight of is: 1) the retentate volume in the TFF system at the end of diafiltration; 2) 3) physical titer of UC pool after dilution; 4) target drug substance (DS) concentration; and 5) system flush solution. and the total volume of the filter flush solution, and 5) the density of the TFF3 buffer. The TFF3 membrane cassettes were discarded after each batch (ie, the cassettes were not reused).

[0275] [Table 9]

[0276] Perform two successive 20 mL rinses of the TFF membrane with TFF3 buffer to confirm the integrity of the TFF system. The vector was harvested from 0.2 mm Pall Supor® EKV sterile The rinse solution was collected using a grade filter (Mini Kleenpak). 3. Rinse the filter with buffer to recover any vector remaining on the filter and filter The final volume of the TFF3 pool (i.e., drug substance DS) was then adjusted. The DS was then divided into aliquots. The mixture was placed into 125 or 250 mL PETG bottles and frozen at <-60°C.

[0277] Example 4 - Formulation and filling Drug Products (DP) are single-dose, preservative-free, sterile, clear to slightly opaque, and colorless to pale White, 2.0×10 13 Non-replicating self-complementary AAV9 vectors at a target concentration of vg / ml The intravenous infusion solution was 20 mM Tris, 1 mM MgCl2, 200 mM NaCl, and 0.005% w / v poloxamer 188. The pH range of this solution was 7. It was 7 to 8.3.

[0278] [Table 10]

[0279] Fill sterile pre-made 10ml Crystal Zenith (CZ) vials with DP. Fill the vials, stopper them with sterile pre-made chlorobutyl rubber stoppers, and sterilize. The vials were sealed with sterile 20 mm flip-off aluminum seals. The subjects filled either a nominal fill volume of 8.3 mL. The target overfill was 0.4 mL. The vials were filled to a volume of 5.9±0.1 mL or 8.7±0.1 mL.

[0280] Example 5 - Potency Assay The relative potency of drug products was determined using a quantitative in vivo assay. An established mouse model of SMA disease was used. A pair of SMAΔ7 mice (Jac Kson Laboratories, #005025) is phenotypically normal, but Approximately 25% of offspring of the children are homozygous for the targeted SMN gene mutation and have SMA These mice showed signs of muscle weakness by day 5 and became ambulatory within the following week. The abnormalities can lead to falls. Jackson Laboratories has The mean survival time of animals with the current form is reported to be approximately 15 ± 2 days. A median survival of 16.3 days was demonstrated for untreated animals with an SMA-like phenotype (geometric mean; n=3 trials; 10 mice per trial).

[0281] When biologically active drug products are administered by intravenous (IV) infusion, the dose (vg / k The increase in survival time as a function of g is obtained. Drug product potency was measured. The potency of drug products and standards (vector genomes / mL; vg / mL) was mL) was determined by droplet digital polymerase chain reaction (ddPCR). The vector was diluted in saline and administered to mice with an SMA-like phenotype. Each of the three designated dose levels was achieved.

[0282] The results of an assay are considered acceptable if the assay meets the requirements. Assay suitability consists of the following: 1. Acceptance limit for negative control samples (15 ± 2 days, median survival time) 2. Acceptance limit for positive control samples (>40 days, median survival time) 3. Acceptance limits for reference standard median survival dose-response curves

[0283] In this test, a 0.9% viable vector was obtained using a leading vector batch (hereinafter, leading batch). The drug was administered at five different dose levels, including a 0 (zero) dose (untreated group) using saline. Median survival time (days) of SMAΔ7 mice administered the product (hereinafter, sample batch) A linear correlation between was determined. Table 10 shows the calibration curve using the preceding batch and the reference batch.

[0284] [Table 11]

[0285] The data from this study showed that the linear range of the median survival dose-response curve was 0–7.4 vg / mL. The linear regression curve of the prior batch reference standard was compared with the drug product sample. Establish the relative potency of drug product sample batches by comparison with the batch linear regression curve This was done using the ratio of the y-intercepts and slopes of each linear regression line (i.e., reference standard and test material). The percent relative efficacy calculation is described in the following formula (1): %RP = [(y-intercept / slope of test material)÷(y-intercept / slope of reference standard)]×100(1 )

[0286] The prior batch used in Phase 1 clinical trials was used as the reference batch, demonstrating 100% efficacy. was assigned.

[0287] Based on the above data, a preliminary assay was performed using the linear portion of the median survival dose-response curve. Acceptance criteria were established to determine the relative potency of drug product clinical batches as follows: A dose-response curve for median survival (days) was constructed using five dose (vg / kg) levels. Accomplished Doses of 0, 1.2E+13, and 7.4E+13 were selected to run a linear dose-response model. . The compatibility limit for saline (untreated group) was a median survival time of 13 to 17 days. The suitability limits for the reference standard samples were established as follows: 〇Slope ≧ 2.0E-13 〇y-intercept≧16.00 (day) ○y-intercept / slope ratio≧8.5E+13 ○R2≧0.7

[0288] The Δ7 mouse model was used to demonstrate the efficacy of SMA therapies, including drug products. 15 With a median survival time of ±2 days, TFF3 buffer solution (vehicle)-treated control animals provided a reliable baseline To provide a baseline control from which to measure product efficacy as increased median survival In drug product development work, the administered dose (vg / kg) can be logarithmically transformed to obtain the therapeutic The median survival time (in days) of treated SMAΔ7 neonatal mice was plotted against the median survival time (in days) of treated SMAΔ7 neonatal mice. Droplet digital PCR (ddPCR) affects survival in mouse models with linear correlation Three doses (excluding the vehicle treatment dose) were identified, as determined by genomic titers using CR For low, medium, and high potency standards, the standard titers (vg / mL) in Table 11 were used. In addition, both the zero point of the standard curve and the negative control were dissolved in TFF buffer (vehicle). The dose that demonstrates a survival time of ≥ 40 days (doubling of median survival time) will be used. A higher dose) was also included as a positive control.

[0289] [Table 12]

[0290] Dose solution preparation (see Table 12 for an example dilution scheme. Negative control - TFF3 buffer solution (drug product final formulation buffer) is used as the negative control Positive control - test article lot was diluted to 1.10 x 10 using TFF3 buffer solution. 14 vg / k g. See Table 12 for an example dilution scheme. Reference Standard Solution - The reference standard lot was prepared using TFF3 buffer solution as described in Table 11. Prepare two concentrations.

[0291] [Table 13]

[0292] Test Article Preparation - Test articles were diluted using TFF3 buffer solution. Dilutions were made in 50 μl of in the final total volume to yield the test dose (vg / kg) per mouse as set forth in Table 11. The dilutions were performed on 12 mice at that time and the minimum life span of the treated mice was calculated. One as a positive control with the goal of increasing median survival (days) to ≥ 40 days An additional volume of 100 ml was included.

[0293] Acceptance limits for control samples Negative control (untreated mice) - The assay acceptance limit for the negative control group was determined by the SMAΔ7 mice The median survival time was 15 ± 2 days. Both types of mice will be excluded from the analysis.

[0294] Positive control (group treated with target clinical dose) - The assay acceptance limit for the positive control group is The life span of the treated mice was at least ≥ 40 days median survival. Any mice that die within 10 days will be excluded from the analysis.

[0295] Acceptance limits for reference standard dose-response curves Reference standard for plotting median survival time (days) against administered dose (vg / mL) Assay suitability criteria will be determined for the dose-response curve. Assay Suitability See Table 13 for criteria.

[0296] [Table 14]

[0297] y-intercept / slope ratio - raw data versus administered dose (vg / kg) for reference standard and test article Determine the linear regression curve of median survival (days). For each linear regression, calculate the y-intercept versus slope. Calculate the ratio.

[0298] Results report Qualitative reporting of relative potency results - positive after adjudication of assay suitability criteria for each assay Determine that the median survival time (days) reading at one point for control material is ≧40 days If the median survival time in the positive control group is ≥ 40 days, the failure criteria are met. The test material may then be disposed of.

[0299] Quantitative reporting of relative potency results - After assay suitability criteria are determined for each assay, The relative efficacy of the substances is quantitatively determined. The positive control reached ≥ 40 days and 7.5 x 10 13 A median survival time of 31 ± 3 days was achieved for the group of mice corresponding to the upper standard dose of 500 vg / kg. Once established, the relative potency of the test substance can be reported. The percent relative potency (%RP) of the test substance ) is calculated using the y-intercept and slope of the linear regression of median survival time (days) dose response as follows: would be calculated as: %RP = 100% x [(test substance y-intercept / slope) ÷ (reference standard y-intercept / slope)]

[0300] Example 6: Surfactant inactivation test To reduce the effects of low pH and tween, pH 7.6 and 4~8% Tween-20 A sample of the drug substance TFF1 intermediate having the formula: The intermediate Tween-20 concentration was calculated by adding Tween-20 to the TFF1 intermediate before acidification. This low Tween-20 concentration is approximately 2.5 times lower than the complete process. This was considered the worst case scenario for surfactant-induced inactivation in drug substance processing. For the viral inactivation step by surfactant treatment, the test substances were 4–8% Tween The TFF-1 intermediate contained 1,2-dihydro-20. To determine activation capacity, duplicate experiments were performed using XMuLV and PRV, respectively. Test materials were spiked with virus at 0.5 °C. Virus was assayed using a plaque-forming infectivity assay. was used for quantification.

[0301] The TFF1 manufacturing step results in a detergent concentration range of 4-8% Tween-20 The TFF1 intermediates were pooled and further purified by 1H chromatography at a working temperature of 16-20 °C. 2% Tween-20 was added over a period of 12–20 h with mixing. Virus removal at a concentration of 8% Tween-20 and a controlled temperature of 16.0°C ± 0.1°C The duration of this inactivation step was 16 hours, compared to the typical process time of 16 hours. was 120 minutes.

[0302] For the inactivation step, a volume of test material is measured, equilibrated to the target temperature, and then infused with the virus. The inactivated challenge was prepared by spiking the samples of the spiked inactivated challenge. Samples were taken at 6 time points: <1 min, 15 min, 30 min, 60 min, 90 min, and The kinetics of inactivation over 120 min were demonstrated. After collection, each sample was diluted in growth medium and incubated for 120 min. Virus inactivation was stopped and assayed for virus. Large volumes of these time point samples were also used to increase the sensitivity of the assay for virus For this step, due to the presence of Tween-20 in the test material, The titer of the inactivation load is calculated from the titer of the spiked virus and the volume of the spiked virus. I calculated it.

[0303] The efficacy of detergent treatment (Tween-20) in inactivating viruses was measured after 90 minutes. The efficacy was demonstrated by LRV values ​​of greater than 4 log10 for both viruses at this time point. The kinetics of inactivation by detergent treatment are shown in Figures 6 and 7. Graph of virus inactivation rate over a 120 minute period during EEN-20 detergent treatment. Shown as:

[0304] Example 7: High seeding density, fast transfection and recovery, and DNA / PEI mixing Effect of times on API production High seeding density, one day faster transfection and recovery, and DNA / PEI mixing times The effect of each condition on DS production was determined. 2 In the bioreactor The results were judged in duplicate.

[0305] Materials and Methods Cell Scale-up HEK 293 cells were thawed and resuspended in DMEM supplemented with 10% FBS. Centrifuge at 209×g for 5 minutes at room temperature, then remove the supernatant and replace with fresh DMEM+10% FB. S was added. Cells were counted for viable cell density and viability and cultured in a 2×T175cm 2 Hula The cultures were then incubated at 37.0°C for 3 days until the cultures reached approximately 90% confluency. The flasks were incubated at 5% CO2 for 1 h. After each cell passage, the spent medium was removed and the flasks were 0.08mL / cm 2 Wash with PBS (-CaCl2, -MgCl2) and then flask TrypLE Select (0.04 mL / cm 2 ) at 37.0℃, 5%C Incubate for 2-3 min in 0.5% CO. DMEM + 10% FBS (0.04 mL / cm 2 The trypsin was quenched with 0.05% ethanol. The cells were expanded and plated according to the diagram in FIG.

[0306] Cell inoculation and monitoring Place HEK 293 cells in a bioreactor in growth medium (high glucose DMEM + 10% Australian FBS + 1:100 Penicillin Streptomycin p) Medium 8,000 cells / cm 2 and 12,000 cells / cm 2 At the target density of The process parameters were pH 7.23, 37.0°C, 55% solution. The oxygen concentration was set at 1.25 to 2.0 mM, and the linear velocity was set at 2 cm / s. 24 hours after seeding, the DMEM growth medium was Ground (0.188mL / cm 2 ) recirculation and set the recirculation rate to 12.5 mL / min. Daily samples were taken for offline pH, metabolites, and nutrients. , and read using Nova BioFlex. Four days after seeding (12,000 cells / cm 2 ), 5th day (8,000 cells / cm 2 ) and on the 9th day, three fibers were removed. , dissolved in 1:1:1 v / v PBS, A100 and B100 solution (ChemoMetec). The cells were then lysed and counted for total nuclei (NucleoCounter NC-200). The growth of the cultures was monitored.

[0307] Transfection 4th 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 then transfected with plasmid DNA and polyethylene. The DNA and PEI were transfected at a ratio of 1:1 mg / mg. The plasmid DNA was mixed in the ratio of pSMN plasmid, pAAV2 / 9 plasmid, and and pHELP plasmids at a mass ratio of 1:1.5:2, and were transfected into DMEM- / - Medium; high glucose, - CaCl2, - L-glutamine plus 0.2 μM filtered, reverse PEI was added to DMEM- / - medium and mixed by inversion. Next, PEI was added to the DNA, mixed by inversion, and incubated at 8,000 cells. / cm 2 (control) and 12,000 cells / cm 2 Incubate for 20 min at room temperature. The other two were cultured at 8,000 cells / cm. 2 The conditions were DNA / PEI for 1 hour and 2 hours. The DNA / PEI complex mixture was used to perform the four conditions. Two bioreactors were transfected at once. Each bioreactor was transfected with DNA / PE The I complex was added and incubated for 2 hours under process parameters. After 2 hours the recirculation loop was turned back on.

[0308] Medium change after transfection 24 hours after transfection, all the culture media in the bioreactor and recirculation loop were Remove the medium and replace with OptiMEM + 1:100 Pen Strep (0.132 mL / cm 2 ) and recirculated for 24 hours at process parameters. After a period of time, remove all medium from the recirculation only and add OptiMEM + 1:100 PenSt rep (12 mL / min) and recirculated at process parameters.

[0309] collect 8th day after cell inoculation (12,000 cells / cm 2 ) and day 9 (8,000 cells / cm 2 ), benzonase (100 U / mL), and lysis buffer (50 mM HEPES, 1% Chase with Tween 20 and incubate for 2 hours under process parameters. The bioreactor was drained and sucrose salt solution (500 mM NaCl, 1% w (v / v sucrose) was added and mixed by inverting. Reactor rinse buffer (500 mM NaCl, 1% w / v sucrose, 20 mM Tris Base, 1% v / v Tween 20, 1 mM MgCl2.6H2O) The bioreactor was drained and the bioreactor rinse was performed at the process parameters for 10 minutes. The buffer was pooled with the crude bulk harvest and mixed by inversion and added to the ddPCR assay. Sampled for Sei.

[0310] Depth Filtration and Tangential Flow Filtration 8th day after cell inoculation (12,000 cells / cm 2 ) and day 9 (control 8,000 cells / c m 2 ), for each condition (n=2 bioreactors), the bioreactors were harvested and the samples were The crude lysates were pooled. The pooled lysates were then purified using the Millistak C0HC Pod, 270cm 2 Filter and Millipak 40, 0.45 μm , Durapore, 200cm 2 Polish filter (EMD Millipore) Samples were taken after C0HC+0.45 and frozen at -80.0°C. The clarified lysate was then subjected to tangential flow Pellicon® 2 ultrafiltration. Filtration module PLCMK C 0.1m 2 Filter (EMD Millipore) The final product was concentrated by diafiltration using at least 6 diavolumes. Samples were obtained after TFF1 filtration and frozen at -80.0°C. All were analyzed for AAV2 / 9 titer and host cell proteins.

[0311] The plasmid was used to produce DS in a bioreactor. Data were collected in duplicate. Each of the conditions in the experiment corresponds to the bioreactor number and conditions shown in Table 14.

[0312] [Table 15]

[0313] Cell growth: Count cells on the day of seeding (day 0) and 4 days after seeding (12,000 cells / Nuclei were counted on days 1 (8,000 cells / cm2), 2 (10,000 cells / cm2), 5 (8,000 cells / cm2), and 9. Data were collected. , indicating that cells in all reactors grew exponentially between days 0 and 5. The number of nuclei on the 9th day after transfection was calculated from the number of nuclei on the 5th to 9th day in Bioreactor 221. This suggests that the total number of nuclei increased by 2.0-fold on the day 1. 8) None of the cultures showed significant growth during the 5th to 9th days. The increased growth in the nuclei was due to the distribution of cells on individual fibers used for total nuclei counting. This may be an artifact due to heterogeneity. Based on the data, it is likely that the cells in all bioreactors grew similarly.

[0314] pH, nutrients and metabolites: All bioreactor cultures showed a trend towards glucose consumption. The results were the same, so despite the increase in the growth curve of bioreactor 221, the cells This suggests that glucose was consumed at a similar rate. During the first 3 days, 12,000 cells / cm 2 The pH of bioreactors seeded with 8,000 cells / c was an average of 7.06. m 2 The average pH was 7.18. The pH decreased slightly with increased nutrient metabolism, and the ammonia Concomitant with higher ion levels, lactate increased by day 9. Day 6 (bioreactor 222) and Day 7 (bioreactor 223) , 226, 228) and then plateaued toward the end of production. This suggests the utilization of lactate as an energy source during this stage.

[0315] Production titer Measure viral genomes from recovered materials by digital droplet (ddPCR) 12,000 cells / cm 2 The titer was approximately 1.5-fold higher in the bioreactor seeded with The mean titer was 6.37E+10vg / mL (n=2), whereas 8,000 cells / cm 2 The control bioreactor inoculated with +10vg / mL (n=2). Titer data showed that high seeding density and early transfer one day It is suggested that transfection and harvesting support higher DS production yields. Control incubated with NA / PEI for 20 min (4.33E10vg / mL n=2) In comparison, the titer yield for DNA / PEI incubated for 1 hour was 1.4 min. The mean titer measurement showed a decrease of 1 (3.17E10vg / mL n=2) after 2 hours of For incubation, the mean titer measurement was 1.6-fold lower (2.67E1 0 vg / mL n=2). The data show that longer incubation times lead to a decrease in titer. This suggests that DNA and PEI can efficiently transfect HEK293 cells. This may be due to the formation of large complexes that are not transfectable. The virus production per mL and the table are compared to the production in a known process as The area values ​​are shown in FIG.

[0316] The virus titers measured at each step of the clarification and concentration steps are shown in Figure 11. Residual host cell proteins at each step during the F1 step are shown in Figures 12A-12B. show.

[0317] Example 8: Effect of seeding density on drug substance production The effect of seeding density on DS production was determined. Four seeding densities were evaluated and Each seeding density was run in duplicate in the culture.

[0318] Cell Scale-up HEK 293 cells were thawed and resuspended in DMEM supplemented with 10% FBS. Centrifuge at 209×g for 5 minutes at room temperature, then remove the supernatant and replace with fresh DMEM+10% FB. S was added. Cells were counted for viable cell density and viability and cultured in a 2×T175cm 2 Hula The cultures were then incubated at 37.0°C for 4 days until the cultures reached approximately 90% confluency. The flasks were incubated at 5% CO2 for 1 h. After each cell passage, the spent medium was removed and the flasks were 0.08mL / cm 2 Wash with PBS (-CaCl2, -MgCl2) and then flask TrypLE Select (0.04 mL / cm 2 ) at 37.0℃, 5%C Incubate for 2-3 min in 0.5% CO. DMEM + 10% FBS (0.04 mL / cm 2 The trypsin was quenched with 0.05% ethanol. The cells were expanded and plated according to the diagram in FIG.

[0319] Cell inoculation and monitoring Place HEK 293 cells in a bioreactor with 700 ml growth medium (high glucose DME M+10% Australian FBS+1:100 Pen Strep) in 4 target densities :8,000 cells / cm 2 ,9,350 cells / cm 2 , 10,700 cells / cm 2 and 1 2,050 cells / cm 2 Each was inoculated in duplicate with stirring. The temperature was set at pH 7.23, 37.0°C, 55% dissolved oxygen (DO), and a linear velocity of 2 cm / s. 24 hours after seeding, DMEM growth medium (total volume was now 0.188 mL / cm 2) recirculation was turned on to achieve a recirculation rate of 12.5 mL / min. Offline pH Daily samples were collected for metabolic, metabolic, and nutrient profiles and analyzed using Nova BioProfil The fibers were read using a 400 Hz CT scanner. Three fibers were removed on the 5th and 9th days after seeding. and then diluted with 1:1:1 v / v PBS, A100 and B100 solution (ChemoMetec). Lyse and count for total nuclei (NucleoCounter NC-200). The growth of the cultures was monitored.

[0320] Transfection Five days after cell inoculation, recirculation was stopped and the (recirculation volume) in each bioreactor chamber was 600 ml DMEM- / - medium (high glucose, -CaCl2, - L-glutamine). Plasmid DNA and polyethyleneimine ( PEIpro) was transfected at a mass ratio of 1:1. Plasmid DNA was transfected at a mass ratio of 1:1. A mass ratio of 5:2 (pSMN-3.56 mg, pAAV2 / 9-5.34 mg, and pHE Mix with LP-7.1 mg, add to 300 mL DMEM- / - medium, and filter using 0.2 μM filtration. PEI (16 mL) was added to 300 mL DMEM- / - The PEI and DNA mixture was added to the medium and mixed by inversion. The mixture was mixed by adding 600 ml of PEI / D The NA complex mixture was used to transfect two bioreactors, each with a corresponding seeding The PEI / DNA complex was added to each bioreactor and the process flow was repeated. Two hours after transfection, the cells were incubated at 4 °C for 2 h at 4 °C for 2 h. The was turned back on (12.5 mL / min).

[0321] Medium change after transfection 24 hours after transfection, all the culture media in the bioreactor and recirculation loop were Remove the medium and add OptiMEM (0.132 mL / cm 2 ) to process parameters The cells were recirculated at 12.5 mL / min for 24 hours. After 48 hours of transfection, The medium in the circulation bottle was replaced with fresh OptiMEM and recirculated at process parameters. (12mL / min).

[0322] collect Nine days after cell inoculation, benzonase (100 U / mL) was added and lysis buffer (50 mM HEPES, 1% Tween 20) and process parameters The bioreactor was drained and sucrose salt solution (500 mM NaCl, 1% w / v sucrose) was added and mixed by inversion. - Bioreactor rinse buffer (500 mM NaCl, 1% w / v sucrose, 20 mM M Tris base, 1% v / v Tween 20, 1 mM MgCl2.6H2O) for 15 min. The bioreactor was drained and the bioreactor rinse was performed at the process parameters for 10 minutes. The buffer was pooled with the crude bulk harvest and mixed by inversion and added to the ddPCR assay. Sampled for Sei.

[0323] The drug substance was produced in a bioreactor using plasmids. The cells were seeded at 100 dpi at 100 dpi and transfected and harvested on the same schedule (5 dpi, respectively). Data are presented in duplicate for each seeding density as shown in Table 15 below. Represents the

[0324] [Table 16]

[0325] Cell growth: Count cells on the day of seeding (day 0) and nuclei at days 5 and 9 after seeding. The data show that cells in all reactors grew exponentially between days 0 and 5. Despite the difference in starting seeding density, the number of nuclei indicated that the number of There was no significant difference in cell number between the groups. Five of the mice (221, 224, 225, 226, and 228) were cultured at 100°C for 10 min. This suggests that the number of cells doubled in the two reactors (222 and 223). As shown in Figure 1, the total number of nuclei increased by 1.4 times. In Reactor 227, the number of nuclei increased from the 5th to the 9th day. There was a 3.8-fold increase in total nuclei in the individual fibers used for counting. This may be an artifact due to uneven cell distribution. Based on the metabolite data, it is likely that cells in all reactors grew similarly. be.

[0326] pH, nutrients and metabolites: Glucose consumption in all bioreactor cultures (Figure 14 B) The tendency of glucose uptake was the same, indicating that the cells were able to grow with glucose despite the difference in starting seeding density. During the first 3 days, all cultures consumed off-line p H (Figure 14C) remained consistent (pH 7.25) and decreased with increasing nutrient metabolism. , and increased concomitantly with higher ammonium ion levels after 8 days (Figure 14E). Acid salts (Figure 14D) increased until day 6 and then plateaued toward the end of production. This suggests the use of lactate as an energy source during this stage. No significant differences in metabolite profiles were observed between reactors seeded at different times. This may be due to the minimal difference in starting seeding densities, <1.2-fold difference. do.

[0327] Production titer Measure viral genomes from recovered materials by digital droplet (ddPCR) 8,000 cells / cm 2 and 10,070 cells / cm 2 The titer is between the starting seeding densities of The mean values ​​were 3.99E+10±2.1E+09vg / mL (n=2) and and 3.70E+10±7.4E+09vg / mL (n=2). The reason could not be identified. However, the reactor seeded with 9,350 cells / cm2 had a yield of 5.02E+08vg / mL (n = 2), which is approximately 2 times the average titer of reactors seeded at adjacent densities. This difference is due to a lack of productivity at this seeding density rather than a lack of productivity. This was likely the result of an unspecified operator error during infection or recovery. cells / cm 2 Replicate reactors seeded with 100% ethanol demonstrated a 2-fold difference between each other, whereas The vector was 3.2E+10 vg / ml, within the range observed for low seeding densities. In contrast, the second reactor produced a titer of only 1.4E+10 vg / ml. Virus production per mL and surface area values ​​are shown in Figure 15A.

[0328] The seeding density and DS production were determined as shown in Figure 15B. +03 cells / cm 2 HEK 293 cells seeded at a range of The results showed that the lactic acid bacteria produced the same amount of lactic acid, pH, glucose consumption, lactate and ammonia production. This suggests that even a slightly higher seeding density does not adversely affect production. On the other hand, 12×10 3 cells / cm 2 The reactors seeded at a high seeding density showed a higher seeding rate than the other conditions. The results showed higher variability among duplicates, including lower mean titers. These results suggest that the method used in this study may not be optimal for production. From the results, for the bioreactor experiment, cells were cultured at 8 × 10 3 ~1×10 4 cells / cm 2 It is recommended to seed at densities in the range of

[0329] Example 9: Comparability assessment AVXS-101 drug product used in Phase 1 clinical trials (Step A) and pivotal clinical trials ( The primary objective is to evaluate the equivalence between the drug product used in step B) and the secondary objective is to evaluate the drug By comparing product lots 600156 and 600307, The purpose of the study was to evaluate the consistency of the results. Figure 16 shows the results of the Phase 1 (Step A) and Phase 3 (Step B) studies. The manufacturing process flows and their differences are shown.

[0330] The equivalence assessment was conducted by Nationwide Children's Hospital Phase 1 clinical drug product lot NCHAAV manufactured at NCH's Hospital Using 9SMN0613 and drug product lot 600156 manufactured by AveXis carried out.

[0331] product The following material lots were evaluated, as summarized in Table 16. Process A was used in this evaluation. Phase 1 clinical drug product lot NCHAAV9SMN0613 and AVXS using Process B A direct comparison of quality attributes obtained from -101 drug product lot 600156 was included. The results of release testing of Lot 600156 regarding the reproducibility and consistency of the scale-up process were The results were evaluated comprehensively together with Lot 600307.

[0332] [Table 17]

[0333] Manufacturing process overview 17A-B provide a summary of the equivalence results.

[0334] Comparability and manufacturing consistency assessment Process A and Process B materials were evaluated to be equivalent, and Process B materials were consistent. It has been evaluated that the Process B material also has additional benefits, such as advantages for industrial scale production. It was also decided.

[0335] Test Method pH Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and A pH analysis was performed on lot 600307 (Process B). The results for both processes were 7. The pH ranged from 0.9 to 8.0. This ensured that the pH of the process A and process B materials was equivalent. and that the Process B materials were consistent.

[0336] exterior Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and The appearance of lot 600307 (process B) was examined by visual inspection. The apparent difference in appearance of the results was due to the different vector concentrations (genome titers). The process B lot had a lower vector concentration than the process NCH AAV9SMN0613 lot. As a result, Lot NCH AAV9SMN0613 was more dilute and had a clearer, colorless On the other hand, the observation result for the process B lot was a colorless to white, slightly opaque solution. , due to approximately four times the concentration of virus particles in solution per mL.

[0337] Considering the different concentrations, the appearance of the Process A and Process B materials was rated as equivalent. ,Process B materials were evaluated as being consistent.

[0338] Osmolality Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and Lot 600307 (Process B) was tested for osmolality by freezing point depression. The results of the second process were also in the range of 410 to 415 mOSm / kg. The osmolality of the Process B materials was equivalent and the Process B materials were consistent. was proven.

[0339] Subvisible Particles Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and Lot 600307 (Process B) was examined for sub-visible particles using the light obscuration method. The results of both processes were well below the recommended limits for injectable drug products in the USP monographs. This confirmed that the number of subvisible particles in the process A and process B materials was equivalent, and and Process B materials were demonstrated to be consistent.

[0340] Genomic titer Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and Lot 600307 (Step B) was tested for genomic titer by ddPCR. AVXS Genomic titers for the -101 batches were expected to vary based on the target concentration at the time of manufacture. Degree B(3.7×10 13 vg / mL and 4.0 × 10 13 vg / ml) The NOM titer was 1.1 x 10 13 The genome titer of the antibody was at least three times that of the antibody (vg / mL). Therefore, process B was the superior method for large-scale production of AVXS-101.

[0341] Infectious titer Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and TCID for Lot 600307 (Process B) 50 The infectious titer was determined by the following procedure. 1.3×10 10 IU / mL and 6.7×10 9 In step A (5.9 × 10 10 The results showed that the rA It may be advantageous for large-scale production of AV, such as AVXS-101.

[0342] Total Protein Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and Lot 600307 (Process B) was tested for total protein by micro BCA. .0×10 13 When normalized to vg / mL, results for both steps ranged from 167 to 17 The normalized total protein values ​​ranged from 0.01 to 0.9 μg / mL. It was demonstrated that the process B materials were equivalent and that the process B materials were consistent.

[0343] In vivo relative potency Lot NCHAAV9SMN0613 was used as the standard for the assay. Based on the line, the potency results obtained for Lot 600156 and Lot 600307 The results showed that the process A and process B materials It was demonstrated that the process B materials were comparable and consistent.

[0344] Identification by Western Blot Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and Lot 600307 (Process B) was identified by Western blot analysis. The blot profiles and apparent fractionation of the major bands (VP1, VP2, and VP3) were The molecular weight values ​​were evaluated to be equivalent for Process A and Process B materials, and consistent for Process B materials. It was also appreciated that

[0345] % Empty Capsids by AUC Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and Lot 600307 (Process B) was examined for % empty capsids by AUC. Lot N The result for CHAAV9SMN0613 (Step A) was 7%. The results for 600307 (Step B) were 2% and 4%, respectively. %), the empty capsids produced were approximately 2 times smaller than those produced in step A (7%) as measured by AUC. Thus, step B produces an improved composition containing low concentrations of empty capsids. It was possible.

[0346] Identity and purity by SDS-PAGE Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B) and and Lot 600307 (Step B) were identified and Purity was examined. The % total purity in all steps was ≥ 98%, and the three capsid proteins were The binding patterns and apparent molecular weights of each were highly consistent. It was demonstrated that the process A and process B materials were equivalent and that the process B materials were consistent. It was proven.

[0347] Residual host cell proteins Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and Lot 600307 (Step B) was tested for residual host cell proteins by ELISA. All lots tested had results below the LOQ of the assay (8 ng / mL). These results demonstrated that the process A and process B materials were equivalent, and that the process B materials were It was proven that there was

[0348] Residual bovine serum albumin (BSA) Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and Lot 600307 (Process B) was tested for residual BSA. Results for all lots tested were The results were below the LOQ of the assay (0.50 ng / mL). and demonstrates equivalence of Process B materials and consistency of Process B materials.

[0349] Residual Benzonase Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and Lot 600307 (Process B) was tested for residual benzonase by ELISA. Results for all lots tested were below the LOQ of the assay (0.20 ng / mL). These results demonstrate the equivalence of Process A and Process B materials and the consistency of the Process B materials. This demonstrates that

[0350] residual host cell DNA Lot NCHAAV9SMN0613 (Process A), Lot 600156 (Process B), and Lot 600307 (Step B) was tested for residual host cell DNA by qPCR. .0×10 13 When normalized to vg / mL, the result for Step A was 3.7 x 10 5 pg / mL, while the results for step B were 0.76 x 10 mL, respectively.5 pg / mL and 0.68 ×10 5 Thus, step B resulted in a significantly lower residual hcDNA viral load. This has been used to produce large-scale vectors, such as rAAV, e.g., AVXS-101. This can be advantageous in manufacturing.

[0351] statistical analysis A statistical analysis was performed on the quantitative quality attributes. Comparisons were made between process A and process B as listed below. (NCHAAV9SMN0613) and each process B lot (600156 and 60030 7) were used in a pairwise fashion. The results are shown in Figures 18 and 19.

[0352] These studies demonstrate that Process B is a superior method for producing viral vectors. Process B consistently produced higher amounts of viral vector (genomic titers and sensitivities) It was less contaminated (less residual hcDNA) and less contaminated (as measured by staining titer). , there were fewer empty capsids.

[0353] Next-generation sequencing Next generation sequencing (NGS) was also performed to identify the AVXS-101 drug from step B. Establish the identity of the product Phase 3 material (by determining and / or confirming the genome sequence) and We assessed whether sequence variants (subpopulations) were present in these. Alignment of the sequence dataset against a reference sequence (pscSMN) identified the mutations. Complete (100%) breadth and sufficient depth of coverage across the entire genome to enable variant detection A total of four minor mutation sites were found; however, These are not true mutants, but rather are likely to be variants of regions of AAV that are difficult to sequence (e.g., Inverted end repeat sequences are notoriously difficult to sequence due to their low GC content and palindromic sequences. This appears to correspond to a sequencing error within the ITR range. See Table 17 for the results.

[0354] [Table 18]

[0355] Phase 1 Lot NCHAAV9SMN0613 Stability Profile Lot NCHAAV9SMN0613 was stored at ≤-60°C for 12 months. The stability results to date are shown in Figure 20. show.

[0356] A comparability study was completed for AVXS-101, which was used in a Phase 1 clinical trial. Manufactured by Nationwide Children's Phase 1 clinical drug product lot NCHAAV9SMN0613 and AveXis manufactured VXS-101 drug product lot 600156 was used. In addition, Process B lot The consistency of the production was determined using 600156 and 600307. both for process B) and manufacturing consistency (process B lot 600156 vs. 600307). Therefore, this study will use new and improved processes and analytical methods to validate AVXS-101 clinical trials. Determine the identity, quality, purity, and potency of ingredients and ensure consistency of manufacture and This made it possible to make a reliable evaluation.

[0357] Statistical analysis was performed on quantitative quality attributes. Comparison was made with process A lot NCHAAV9S Pairwise analysis was performed between MN0613 and Process B lot 600156. Compared to Process A Therefore, process B was a better method for producing a higher amount of viral vector with higher purity. For example, the viral vectors produced by process B were more sensitive than those produced by process A. The staining titer was 48% higher, the genomic titer was 8% higher, and the 10 μm diameter 92% fewer sub-visible particles > 25μm in diameter and 50% fewer sub-visible particles > 25μm in diameter , 100% fewer empty capsids, and 11% less residual hcDNA. , and were consistent compared with the test limits for each quality attribute.

[0358] Additionally, to ensure consistency in manufacturing using Process B, lots 600156 and 6003 Pairwise comparisons were performed using 07. Process B lots 600156 and 600307 The results of this initial pairwise comparison between the , and were consistent compared with the test limits for each quality attribute.

[0359] Based on the results, Phase 1 clinical drug product lot NCHAAV9S using Process A was AVXS-101 drug product lot 600156 using MN0613 and process B Due to the improved quality characteristics, process B resulted in a higher amount of viral vector and improved purity. This may be advantageous for large-scale production of rAAV, for example. These two material lots (Lots 600156 and 600307) produced from B It has been found to be reproducible and exhibits manufacturing consistency.

[0360] Example 10: Analytical Ultracentrifugation (AUC) Analysis Phase 1 (Process A, Lot NCHAAV9SMN0613) and Phase 3 (Process B, Lot Materials from the NCHAAs (600156 and 600307) were analyzed using the AUC method. AUC profiles of V9SMN0613, 600156, and 600307 (duplicate The results of the analysis (analyzed in a 3D-cell gate) are shown in Figures 21, 22, and 23, respectively.

[0361] The AUC analysis of each material was performed for the third phase materials with empty capsid contents of 2% and 4%, respectively. (Process B, Lots 600156 and 600307) Phase 1 material (Process A, Lot NCHAAV9SMN0613) exhibiting a 50% dimethyl ether (DMBE) content of 7%. exhibit similar sedimentation coefficients for empty and full capsids. The CsCl gradient ultracentrifugation step in the manufacturing process is Empty capsids can be more effectively separated from full capsids compared to the gradient ultracentrifugation preparation step. This is due to the fact that it is possible

[0362] AVXS-101 production lots using clinical and commercial formats were analyzed by ultracentrifugation using CsC When subjected to gradient purification, Nationwide Children's Hospital Phase 1 clinical trial material (NCHAAV) produced at the Elders Hospital 9SMN0613) and each subsequent production lot by AveXis. Phase 1 clinical drug product lots using Process A show three visible bands of capsid. AVXS-101 Drug Product Lot 60 using NCHAAV9SMN0613 and Process B Based on the AUC profiles of 0156, these materials appear to be comparable. AU of two material lots (lots 600156 and 600307) produced from process B The C profile was assessed as consistent.

[0363] Example 11 - Upstream process An upstream process was used to produce intermediates derived from the working cell bank. The method includes the steps of (a) culturing cells, (b) injecting the cultured cells with three processes as shown in FIG. (c) transfecting the cells with the plasmid; and (b) growing the virus from the cells after a culture period. (d) purifying the virus particles by filtration to remove any interacting particles. (e) removing any remaining cells or cell debris from step (d); and (f) subjecting the resulting purified virus particle intermediate to progressive flow filtration. The method includes the step of freezing the product.

[0364] Prior to transfection, cells are cultured in flasks or suitable bioreactors, or both. The cells were expanded in suitable culture media. One culture medium contained 10% FBS, 4.5 g / L glucose. In one embodiment, the flask is first Adherent cells are grown in the iCELLis Bioreactor and then transferred to the iCELLis Bioreactor. The adherent cells are then further expanded in a culture tube.

[0365] After cell expansion, transfect the triplex DNA-plasmid PEI co-precipitate into adherent HEK293 cells The three plasmids used in this transfection were pSMN, pAAV2 / 9, and pHELP. The DMEM growth medium used for cell expansion was modified DMEM. The DMEM transfection medium was replaced with F Contains no BS, no calcium, no L-glutamine, and contains 4.5g / L glucose Adherent HEK29 cells were cultured using PEI co-precipitates in a large-scale adherent cell bioreactor. 3. Triple DNA Plasmid Transfection into Cells Using scAAV9.CB.S A MN vector was constructed. This vector plasmid pSMN contains the human SMN cDNA. The three plasmids used in this transfection were pSMN(222m g), pAAV2 / 9 (333 mg), and pHELP (444 mg). The infection medium was adjusted to the bioreactor temperature before adding the PEI-plasmid co-precipitate. The bioreactor was equilibrated until the temperature reached >30°C. To do this, the plasmid was added to the transfection medium and filtered through 0.2 μl of the reaction mixture. The reaction included adding PEI to the transfection medium and then placing the The PEI-plasmid reaction was mixed manually to form a uniform suspension. The reaction is carried out for a period of 15-30 minutes. At the end of this reaction period, the bioreactor is The PEI-plasmid coprecipitate was added to the vector. The reactor was allowed to mix for 1-2 hours, after which recirculation was resumed. Prior to this, DMEM growth medium was recirculated through the bioreactor for 18-24 h.

[0366] The rAAV SMN genome (nucleotides 980 to 3336 of SEQ ID NO:1) is, in order, AV2 ITR, chicken β-actin promoter with cytomegalovirus enhancer , SV40 intron, (GenBank accession number NM_000344.2) The SMN-encoding DNA, the polyadenylation signal sequence derived from bovine growth hormone, and Conservative nucleotide substitutions in SMN DNA have also been proposed. (For example, the 625th position of GenBank accession number NM_000344.2) The genome lacks AAV rep and cap DNA. i.e., there is no AAV rep or cap DNA between the ITRs of the genome. MN polypeptides include, but are not limited to, NCBI protein database number N An example of the human SMN1 polypeptide is shown in P_000335.1. For the SMN vector, see Foust et al., Nature Biotech Nol., 28(3):271-274 (2010), in which the vector gene The sequence of the NOM insert is shown as nucleotides 980 to 3336 of SEQ ID NO:1).

[0367] Bioreactor day 6, 18-24 hours after transfection, bioreactor Drain the tube and transfer the DMEM recirculation media bag to a fresh 200 L OptiMEM transformer. After the infection, the medium was replaced with new medium. The bioreactor was refilled with 64 liters and the bioreactor was Recirculation in the aquarium was resumed. On the 7th day, 18 to 24 hours after the medium change on the 6th day, Freshly replenish the OptiMEM post-transfection medium in the recirculation bag (approximately 135 liters). The bioreactor was drained during this step and replaced with a fresh OptiMEM medium bag. Recirculation of medium continued until harvest on day 9.

[0368] After 9 days in the bioreactor, a final pre-harvest sample was taken from the reactor and the cells The dissolution process was started. Benzodiazepine was added to the bioreactor to a final concentration of 100 U / mL. After the benzonase was mixed in the reactor, 7.1 liters of benzonase was added to the reactor. The lysis solution was mixed in the reactor for 2 h before the first harvest step. 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) was added to the bag and mixed for 15 minutes. The solution quenched the benzonase in the harvest medium. The bioreactor was then The bioreactor was rinsed with reactor rinse buffer. 64 liters of bioreactor rinse buffer was added to the rinsing solution and mixed for 15 minutes. The rinse fluid was transferred to a common collection bag. After the rinse fluid was added to the collection bag, the contents were Mix for 15 minutes and take a bulk harvest sample.

[0369] The combined bulk harvest was filtered through a depth filter into a collection bag. After the harvest was filtered, the depth filter was placed in a 50 liter TFF1 diafiltration tank. The depth filter pool was mixed and the samples were washed with elution buffer. The depth filter pool was then filtered through a 0.45 μm filter to prepare the bulk solution. The harvested material was further clarified by filtering through a 0.45 μm filter with 6 liters of TFF1 buffer. To chase.

[0370] For TFF1 step, 5.0m 2 300kDa MW cutoff regenerated cellulose Flush the membrane cassette, sanitize with NaOH solution, and equilibrate with TFF1 buffer. The concentration phase of this operation was designed to reduce the volume of the clarified harvest by approximately 10-fold. After the target retentate volume is reached, the diafiltration process begins. The retentate was diafiltered with 6 volumes of TFF1 buffer. After the total permeate flow rate of the volume was achieved, the retentate was concentrated again and collected in a collection bag. Two successive membrane rinses were performed to maximize product recovery from the TFF system and to reduce intermediate The TFF1 intermediate was aliquoted into 1 or 2 liters in the LFH hood. Place in sterile PETG bottles and then freeze on dry ice or in a freezer at -60°C. was moved to a storage location.

[0371] [Table 19]

[0372] Example 12 - Downstream Processing Downstream processing was used to process the intermediate into filtered drug substance. Downstream processing steps included acidification and and a clarification step (using filtration), followed by cation exchange chromatography, Tangential Flow Filtration ("TFF2"), CsCl Ultracentrifugation and Further Tangential Flow Filtration ("TFF2") The TFF3 step allows the purified AAV particles to be purified and purified into a drug The resulting drug substance was filtered and suspended in a biologically acceptable carrier. The process included the following manufacturing steps after production of the TFF1 intermediate: thawing of the TFF1 intermediate and pooling, acidification and clarification, cation exchange chromatography (CEX), Tangier Total flow filtration (TFF2), intact / empty capsids by CsCl ultracentrifugation Separation of chromatographic data, concentration / buffer exchange by tangential flow filtration (TFF3), and TFF3 purification. Filtration of the drug substance to produce the drug product; dilution and filtration of the drug substance to produce the drug product; Storage and filling of drug products into vials.

[0373] The TFF1 intermediate material was thawed and mixed gently. Tween 20 was used to make the acidic solution. Promotes flocculation of bulk host cell proteins and DNA under H. CE TFF1 intermediate pool containing 15% Tween 20 for X-chromatography The pH of the solution was then lowered (pH 3.5). The solution was then passed through a depth filter and a 0.45 μm filter. The precipitate that formed after lowering the pH was removed by filtration at 40°C.

[0374] Tween 20 (20 mM Tris, 1 mM MgCl, 500 mM NaCl, 1 % sucrose m / v, pH 8.1 in 36% Tween 20 solution) was added to the TFF1 intermediate solution for 4 h. Add slowly over a period of 10 min to achieve a final concentration of 20% Tween 20. per kg of TFF1 intermediate / Tween spike pool after overnight incubation at Adding approximately 4 g of 1 M glycine pH 2.5 to TFF1 containing Tween 20 The pH of the intermediate was lowered to achieve the target pH of 3.5 ± 0.1. Afterwards, the solution was filtered through a Clarisolve POD depth filter in series with a 0.45 μm Opticap XL10 Durapore filter or 0.8 / 0.45μ PE These filters were then washed with CEX buffer A to remove the POD Flush twice the hold-up volume + 1 hold-up volume of the polishing filter. I did.

[0375] A cation exchange (CEX) capture chromatography step was used to isolate viral capsids. Proteins, DNA and other process impurities were separated. This step included the use of an automated process cleaner. The CIMmultus S03-8000 target was operated using a chromatography system. End composite column (sulfonyl) (pore size 2 μm) Chromatography column (8.0 L) The buffers and solutions are listed in the table below.

[0376] [Table 20]

[0377] The protein content of the clarified and acidified TFF1 intermediate was used to determine the CEX column load. The protein load on the CEX column was set to 70% of the maximum column capacity.

[0378] The elution peaks were manually collected starting from the sharp increase in OD280. increased when the conductivity was 80-85 mS / cm. The approximate volume of the CEX eluate (product) was about The volume was 20 liters or 2.5 CV (column volume). The CEX eluate was collected in two fractions. The first fraction started with a sharp increase in OD280 that converged over 1.5 CV. The second fraction started immediately after the first and was collected over 1.0 CV. The two fractions were neutralized to pH 8.0±0.30 using pH 9.0 neutralization buffer.

[0379] In the TFF2 step, the buffer is concentrated and purified by ultracentrifugation to remove protein impurities. The buffer was exchanged into the appropriate buffer for the step. The tangential flow filtration system was fitted with a 0.4 ml 2 (2 CEX cycles) or 0.2m 2 (1 CEX cycle) 300k MWCO It was used in conjunction with a regenerated cellulose membrane.

[0380] The concentration phase of this run reduced the volume of the CEX eluate. After the target retentate volume was reached, Diafiltration was started in discontinuous TFF mode (batch mode). Dilute 2-fold and perform 8 diavolumes of TFF2 NaCl diafiltration in discontinuous TFF mode. filtration buffer, followed by 8 diavolumes of TFF2 CsCl diafiltration. The mixture was then diafiltered with CsCl diafiltration buffer. After the rectification is complete, the retentate is pumped to a defined volume that depends on the hold-up volume of the system. Two successive membrane rinses were performed to maximize product recovery from the TFF2 system. It has grown large.

[0381] The retentate feed rate was set at 20% conversion rate to permeate (per 500 mL of retentate feed rate). Flow rate of 100 ml of permeate: 5 L / m 2 / min (0.1m 2 500mL per cassette The permeate flow rate was set at 100 s / min. The permeate flow rate was controlled by a clamp on the permeate tube. The permeate flow rate was controlled to maintain 20% of the normal flow rate.

[0382] [Table 21]

[0383] By using cesium chloride gradient ultracentrifugation, the complete capsid was separated by ultracentrifugation. The CsCl ultracentrifugation step uses a type 50.2 Ti roller to remove empty capsids. Automated Optima XPN 100 ultracentrifuge system or equivalent with a 300-mm diameter rotor or equivalent The filtered material after TFF2 purification was placed in an ultracentrifuge tube, and the solution was bubbled. The solution was added slowly along the inside of the tube wall to avoid contamination. The mixture was sealed using a handheld heat sealer and weighed at 302,000 g (50.2 Ti rotor) at 20°C. After the centrifugation step was completed, the tubes were removed from the ultracentrifuge. The tube containing the product was discarded. The tube was mounted on a ring stand above the container. The lamp was positioned directly below the tube. The empty capsid band (band A is the top band), the complete capsid doublet The doublet bands (bands B and C, the bands above and below the doublet) and the bands below the doublet The bottom band was marked on the tube. 18G needle attached to a 30mL syringe By inserting the band just below band D to the center of the tube, bands B, C, and D were removed. The collected material was transferred to a sterile 1 L PETG bottle. All centrifugations were Pool the material from the tubes into a sterile 1 L PETG bottle and perform the ultracentrifuge (UC) pooling. The buffers for the CsCl ultracentrifugation step are listed in the table below.

[0384] [Table 22]

[0385] In the TFF3 step, CsCl is removed and the final formulation buffer is used to elucidate the loaded vector The tangential flow filtration system was fitted with two 50 cm 2 300k MW The concentration phase of the TFF3 run was used to determine the concentration of residual CsCl. The retentate volume was then reduced by 1.0%. After the target retentate volume was reached, the diaphragm was Filtration was started. Diavolumes of the retentate were added to the column with 10 diavolumes of TFF3 buffer. After diafiltration was completed, the concentrated retentate was Filter the liquid through a 0.2 μm Pall Supor® EKV sterilizing grade filter (M The mixture was then passed through a pre-filter (indigo Kleenpak) and transferred to a second conical tube.

[0386] Vector was recovered from the TFF3 system by performing successive rinses of the membrane. TFF3 buffer was added to the primary conical tube holding the retentate. This material was then transferred to the cellulosic After recirculation, the flush solution was filtered through a 0.2 μm Pall Sup or(R) EKV Sterilizing Grade Filter (Mini Kleenpak) The TFF3 concentrate and partial pool were mixed and transferred to a second conical tube through a filter. , ≧4.5×10 13 vg / mL of Drug Substance (pooled TFF3 retentate + 2 rinses) ) to achieve a final vector concentration of 100 mM KCl.

[0387] Continuous rinsing of the membrane was performed to maximize product recovery from the TFF3 system. Add TFF3 buffer to the primary conical tube that holds the TFF3 retentate and initial flush material. The material was recirculated through a cellulose membrane. The secondary flush fluid was added to the 0.2μm Pall Sup until the weight determined by the or(R) EKV Sterilizing Grade Filter (Mini Kleenpak) This final concentrated solution was called the drug substance (DS). do.

[0388] [Table 23]

[0389] DS was fitted with a Pall Supor® EKV sterilizing grade filter (Mini Filter with Kleenpak and use sterile disposable assembly to create a sterile 1L glass bottle Prior to filtration of the TFF3 pool, the TFF3 buffer was filtered using a peristaltic pump. The filter was flushed by passing it through a filter and discarding it in a waste flush bag. The drug substance (DS) was then filtered through the flushed filter using a peristaltic pump to produce 1 L of diluted DS. Bacteria were collected in glass bottles. 5 x 10 13 Based on the target DS concentration of vg / mL, Add TFF3 buffer to the second conical tube holding S, 3.5 x 10 13 vg / m A diluted drug product ("DP") was prepared by filtering to a target concentration of L.

[0390] TFF3 buffer used for filter flushing and DS dilution for preparation of DP The following formulation is included:

[0391] [Table 24]

[0392] Filling DP into 5mL sterile pre-made Crystal Zenith (CZ) vials The containers were then sealed with sterile pre-made stoppers and sealed with sterile pre-made seals.

[0393] Example 13 - Potency Assay The relative potency of drug products was determined using a quantitative in vivo assay. An established mouse model of SMA disease was used. A pair of SMAΔ7 mice (Jac Kson Laboratories, #005025) is phenotypically normal, but Approximately 25% of offspring of the children are homozygous for the targeted SMN gene mutation and have SMA These mice showed signs of muscle weakness by day 5 and became ambulatory within the following week. The abnormalities can lead to falls. Jackson Laboratories has The mean survival time of animals with the current form is reported to be approximately 15 ± 2 days. A median survival of 16.3 days was demonstrated for untreated animals with an SMA-like phenotype (geometric mean; n=3 trials; 10 mice per trial).

[0394] When biologically active drug products are administered by intravenous (IV) infusion, the dose (vg / k The increase in survival time as a function of g is obtained. Drug product potency was measured. The potency of drug products and standards (vector genomes / mL; vg / mL) was mL) was determined by droplet digital polymerase chain reaction (ddPCR). The vector was diluted in saline and administered to mice with an SMA-like phenotype. Each of the three designated dose levels was achieved.

[0395] The results of an assay are considered acceptable if the assay meets the requirements. Assay suitability consists of the following: 4. Acceptance limit for negative control samples (15 ± 2 days, median survival time) 5. Acceptance limit for positive control samples (>40 days median survival) 6. Acceptance limits of the reference standard median survival dose-response curve

[0396] In this test, a 0.9% viable vector was obtained using a leading vector batch (hereinafter, leading batch). The drug was administered at five different dose levels, including a 0 (zero) dose (untreated group) using saline. The linear correlation between median survival time (days) of SMAΔ7 mice treated with the Table 24 shows the calibration curve using the preceding batch reference batch.

[0397] [Table 25]

[0398] The data from this study showed that the linear range of the median survival dose-response curve was 0–7.4 vg / mL. The linear regression curve of the prior batch reference standard was compared with drug product batch 8. Relative potency of drug product batch 816836 by comparing with 16836 linear regression curve This was done by determining the y-intercept and slope of each linear regression line (i.e., reference standard and test material). The percent relative efficacy calculation is described in the following formula (1): Can be: %RP = [(y-intercept / slope of test material)÷(y-intercept / slope of reference standard)]×100(1 )

[0399] The NCH0613 batch used in the Phase 1 clinical trial was used as the reference standard batch. An efficacy of 0% was assigned.

[0400] Based on the above data, a preliminary assay was performed using the linear portion of the median survival dose-response curve. Acceptance criteria were established to determine the relative potency of drug product clinical batches as follows: A dose-response curve for median survival (days) was constructed using five dose (vg / kg) levels. Accomplished Doses of 0, 1.2E+13, and 7.4E+13 were selected to run a linear dose-response model. . The compatibility limit for saline (untreated group) was a median survival time of 13 to 17 days. The suitability limits for the reference standard samples were established as follows: 〇Slope ≧ 2.0E-13 〇y-intercept≧16.00 (day) ○y-intercept / slope ratio≧8.5E+13 ○R2≧0.7

[0401] The Δ7 mouse model was used to demonstrate the efficacy of SMA therapeutics, including drug products. 1 With a median survival time of 5 days ± 2 days, untreated or saline-treated control animals were To provide a baseline control from which to measure product efficacy as increased median survival In drug product development work, the administered dose (vg / kg) is logarithmically transformed to obtain the therapeutic When plotted against median survival time (in days) of SMAΔ7 neonatal mice Droplet digital PCR (ddPCR) affects survival in mouse models with linear correlation Three doses (excluding the vehicle treatment dose) were identified, as determined by genomic titers using the For low, medium, and high potency standards, refer to the standard potency (vg / mL) in Table 26. In addition, both the calibration curve zero point and the negative control were treated with TFF buffer (vehicle) solution. The dose that demonstrates a survival of ≥ 40 days (a doubling of median survival) is used. A higher amount) was also included as a positive control.

[0402] [Table 26]

[0403] Dose solution preparation (see Table 26 for an example dilution scheme. Negative control - 0.9% saline is used as a negative control Positive control - test article lot was diluted 1.5 x 10 with saline. 14 Formulated in vg / kg do. Reference Standard Solution - The reference standard lot is prepared in saline at three concentrations as described in Table 25. Prepare at 37 °C.

[0404] [Table 27]

[0405] Test Article Preparation - Saline was used to dilute the test articles. Dilutions were 50 / mouse. The test doses (vg / kg) were calculated to yield the doses described in Table 26 in a final total volume of 10 μl. Dilutions were performed on 10 mice at that time point to ensure a minimum life span of ≥4 years for treated mice. One additional patient was added as a positive control with the goal of increasing median survival to 0 days. The volume included.

[0406] Acceptance limits for control samples Negative control (untreated mice) - The assay acceptance limit for the negative control group was determined by the SMAΔ7 mice The median survival time was 15 ± 2 days. Any mice that do not meet the criteria will be excluded from the analysis. If more than eight mice are used in a group, Up to 2 mice may be excluded for death within 10 days.

[0407] Positive control (group treated with target clinical dose) - The assay acceptance limit for the positive control group is The life span of the treated mice was at least ≥ 40 days median survival. Any mice that die within 10 days will be excluded from the analysis. If mice are used, up to 2 mice may be excluded for death within 10 days.

[0408] Acceptance limits for reference standard dose-response curves Reference standard for plotting median survival time (days) against administered dose (vg / mL) Assay suitability criteria will be determined for the dose-response curve. Assay Suitability See Table 27 for criteria.

[0409] [Table 28]

[0410] Y-intercept / slope ratio - raw versus administered dose (vg / kg) for reference standard and test article Determine the linear regression curve of median survival (days). For each linear regression, calculate the y-intercept versus slope. Calculate the ratio.

[0411] Results report Qualitative reporting of relative potency results - positive after adjudication of assay suitability criteria for each assay Determine that the median survival time (days) reading at one point for control material is ≧40 days If the median survival time in the positive control group is ≥ 40 days, the failure criteria are met. The test material may then be disposed of.

[0412] Quantitative reporting of relative potency results - After assay suitability criteria are determined for each assay, The relative efficacy of the substances is quantitatively determined. The positive control reached ≥ 40 days and 7.5 x 10 13 A median survival time of 31 ± 3 days was achieved for the group of mice corresponding to the upper standard dose of 500 vg / kg. Once established, the relative efficacy of the test substance can be reported. The percent relative efficacy (% RP) is calculated using the y-intercept and slope of linear regression of median survival time (days) dose-response as follows: This is calculated as: %RP = 100% x [(test substance y-intercept / slope) ÷ (reference standard y-intercept / slope)]

[0413] Example 14 - Single-dose gene replacement therapy for spinal muscular atrophy: Dosage study Spinal muscular atrophy (SMA) is a disease caused by a mutation in the gene encoding survival motor neuron 1 (SMN1). It is a severe childhood monogenic disorder caused by the loss or malfunction of the gene The incidence is approximately 1 in 10,000 births, with a carrier frequency of 1 in 54. It is characterized by the degeneration and loss of peripheral motor neurons, which leads to muscle atrophy. The disease is divided into four subtypes (1-4) based on age of onset and milestone achievement. SMA type 1 (SMA1) is the most severe form and is the most common genetic cause of infant death. There are two forms of SMN in MA; SMN1 is responsible for the functional production of the SMN protein SMN2 is the major gene that preferentially excludes exon 7 during splicing. As a result, they produce only a small fraction of functional SMN protein compared to SMN1. Thus, SMN2 copy number modifies the disease phenotype, and the presence of two copies of SMN2 modifies the disease phenotype. Infants with biallelic deletions of SMN1 and two copies of SMN2 have SMMA1. The risk of A1 is 97%.

[0414] Recent studies of the natural history of SMA1 (historical cohorts) have shown that among infants with the disease, The median age at onset of symptoms was 1.2 months (range, 0-4 months), and the disease was characterized by muscle tension. Characterized by hypotonia, severe weakness from early infancy, and the inability to sit without support Infants with SMA1 who had two copies of SMN2 had at least Noninvasive ventilation (considered equivalent to continuous ventilation) for at least 16 hours per day for 14 consecutive days The median age at need for bronchoscopy was 10.5 months. At 13.6 months, only 25% were alive without continuous ventilatory support, and at 20 months, As of now, 8% of patients have survived without this support. National Institutes of Health Another prospective, multicenter, historical study (NeuroNEXT), supported by the National Center for Neurology, showed that 8-month open tracheal The median surgery-free survival was 1.1 years (95% confidence interval, 6 to 17 years) for all SMA1 patients. After birth, respiratory and swallowing functions decline rapidly, eventually leading to the inability to maintain adequate nutrition and aspiration. Mechanical feeding (from a nasogastric or gastrostomy tube) to reduce the respiratory risks associated with Nutritional support is required. For most SMA1 patients, symptoms appear by age 3 months. Most patients require nutritional support by 12 months of age.

[0415] SMA1 patients are also receiving CHOP INTEND (Children's Hospital of Philadelphia Infant Neuromuscular Endocrinology). Failure to achieve major motor milestones and decline in function as measured by the MRI scale (CHOP INTEND ranges from 0 to 64, where the higher the score, the better.) This refers to good motor function and sensitivity to slight changes in motor function, such as antigravity movements of the limbs. In a historical analysis of 34 SMA1 patients, all but one did not achieve a score of at least 40 after age 6 months. NeuroNEXT cohort In CHOP, the INTEND score dropped by an average of 10.7 points between 6 and 12 months of age. I did.

[0416] Therapeutic strategies to increase SMN protein levels in motor neurons may improve efficacy of SMN2 One approach is to target the exon 7 subunit of SMN2. Nusine, an antisense oligonucleotide developed to inhibit splicing Central nervous system delivery of Lusene (Ionis Pharmaceuticals / Biogen) The drug improved weakness in a mouse model of severe SMA and reduced the severity of symptoms in affected mice. It has been shown to increase the average lifespan of mice from 16 to 25 days. December 2016 Nusinersen was approved by the Food and Drug Administration for the treatment of SMA in The drug was approved by the National Institute of Intent Research (NIR) in 4 children under 2 months of age. The loading dose is administered using repeated intrathecal injections.

[0417] Another potential SMA1 treatment is gene therapy, which involves the delivery of self-complementary adeno-associated AAV serotype 9 (scAAV9) (the coding region of this recombinant virus is an intramolecular double-stranded DNA fragment) A single intravenous injection delivering a copy of SMN in a NA [or self-complementary] template This technique induces SMN expression in motor neurons and peripheral tissues. It has been shown to reduce the effects of SMA in mouse models and improve the overall survival of these models. From the 15th day, the low dose (6.7 × 10 13 vg) is 28.5 day and higher doses of vector (2.0 × 10 per kilogram 14and 3.3 x 10 1 4 vg) has extended it to over 250 days.

[0418] SMN protein is ubiquitously expressed and SMA1 is expressed in many cell types (e.g., heart, pancreas, and skeletal muscles), as well as multiple systems (e.g., the autonomic and enteric nervous systems, the cardiovascular system, Given that the disease develops in the brain and pancreas, it is possible that the disease crosses the blood-brain barrier and reaches any area of ​​the spinal cord. In addition to targeting certain central nervous system neurons, systemic administration of AAV9-mediated gene therapy It may be advantageous to administer a hybrid cytomegalovirus enhancer-chicken β-activator. The self-complementary nature of this vector in combination with the sigma promoter allows for rapid and sustained expression of SMN. In April 2014, the present inventors developed a chicken β-actin promoter. Single dose with delivery of human survival motor neuron gene (hSMN) under control of a cytotoxic T cell received scAAV9 (scAAV9.CB.hSMN) (AVXS-101). A gene replacement therapy trial involving SMA1 infants has begun.

[0419] method Patients and Study Procedures: For the purposes of this study, all patients had a confirmed SMA1 genetic diagnosis, were homozygous for SMA1, and The patient had a combined SMN1 exon 7 deletion and two copies of SMN2. Patients with the c.859G→C disease-modifying variant in the genomic DNA were excluded. By 6 months of age, clinical signs include delayed motor skills, inability to maintain balance, hunched posture, and joint hypermobility. Demonstrating disease manifestations characterized by hypotonia as determined by assessment Those with active viral infections (HIV or hepatitis B or C seropositivity) Patients with concurrent diseases that pose unnecessary risks for gene transfer were excluded from this study. Patients who require invasive ventilatory support (tracheotomy with positive pressure) or who attend screening visits Patients with pulse oximetry <95% saturation at time of infusion were also excluded.

[0420] Patients were enrolled in two cohorts based on the dose of gene therapy they received. Cohort 1 Patients were treated with a low dose (6.7 × 10 per kilogram) 13 vg) for 5 months. patients in cohort 2 were treated with the high dose (2.0 × 10 per kilogram) 14 vg ) and were enrolled for one year. On the 30th day after administration, patient 1 of cohort 1 T cell responses were detected by IFN-γ ELISpot assay using AAV9 capsid 10 for >50 6 Spot-forming cells per PBMC ( A sharp increase in SFC was observed (normal, 10 6 <50 SFC per PBMC. Zolone was started at 2 mg / kg and administered for 35 days, with T cell responses and serum transaminases decreasing. As a result, the study protocol was amended so that patients 2-15 were Oral prednisolone at a dose of 1 mg per kilogram was administered 2 days prior to gene vector administration. The treatment started 4 hours ago and lasted for about 30 days. The treatment consisted of AS with maintenance prednisolone. T and ALT enzymes were below 120 IU / L and T cell responses were below 10 6 PBMs Continue until SFC is below 100 per C, then use prednisolone based on clinical judgment It was decided to gradually decrease.

[0421] The vector is delivered in normal saline (approximately 10-20 ml per kilogram); This was infused intravenously over approximately 60 minutes. Some patients had a gastrostomy or nasogastric tube at the time of enrollment. Enteral nutrition using a tube was required, but the choice was based on the wishes of the parents or attending physician. After enrollment in the study, all patients who required nutritional support were provided with a gastrostomy tube and were provided with a tube for the duration of the study. The tube was not removed.

[0422] Outcomes: The primary outcome was any treatment-related adverse event of grade 3 or greater. Secondary outcomes were time to death or need for continuous ventilatory support. The latter was at least in the absence of acute reversible disease or perioperative conditions. This was defined as at least 16 hours of respiratory support per day for 14 consecutive days. The goal is to achieve motor milestones (especially sitting without support) and CHOP INTEND score included.

[0423] In the application of the CHOP INTEND scale in SMA, a score of >40 was Maintenance of unsupported sitting is considered clinically meaningful. Measured and graded: Bayley Scales of Infant Development Infant and Toddler Development Gross Motor Subtest Sitting without support for at least 5 seconds according to item 22 ("Sitting without support"); World According to the World Health Organization (WHO) criteria Sitting without support for at least 10 seconds according to the WHO criteria and item 26 of the Bayley Scales mentioned above. Sitting unsupported for at least 30 seconds ("functional sitting independently"). Major motor miles The tone is based on the Ability C Mini captured by interactive video judgment. aptured Through Interactive Video Evalua tion-mini:ACTIVE-mini) was used to assess patients' videos independently. Compound muscle action potentials (CMAP) were recorded at baseline and The pathological condition of the muscle was evaluated by electrical impedance myogography. Quantification was performed by EIM.

[0424] Statistical Analysis: Safety analyses were performed in all patients, who were also randomized to primary survival (PFS) and Analysis (as defined above and in the protocol) and the CHOP INTEND scale These analyses of changes from baseline to 1 and 3 months were a...

Claims

1. a. 1 to 8 x 10 １３ AAV9 viral vector genomes / mL (vg / mL); b. less than about 7% empty viral capsids; c. 1 x 10 １３ less than about 100 ng / mL host cell protein per vg / mL; d. 1 x 10 １３ Approximately 5 x 10 per vg / mL ６ Residual host cell DNA less than pg / mL 1. A pharmaceutical composition comprising: and the above 1 to 8×10 １３ At least about 80 AAV9 viral vector genomes / mL % is functional.

2. The AAV9 viral vector encodes a survival motor neuron (SMN) protein. The composition of claim 1, comprising a polynucleotide that binds to the

3. The AAV9 viral vector is a methyl-CpG binding protein 2 (MECP2) polypeptide. The composition of claim 1, comprising a polynucleotide encoding a protein.

4. The AAV9 viral vector targets superoxide dismutase 1 (SOD1). The present invention relates to a method for the preparation of a nucleic acid molecule comprising the steps of: The composition of claim 1.

5. The AAV9 viral vector comprises a modified AAV2 ITR, chicken β-actin (C B) Promoter, cytomegalovirus (CMV) immediate early enhancer, modified SV40 The late 16s intron, the bovine growth hormone (BGH) polyadenylation signal, and the unmodified The composition of claim 2, comprising a modified AAV2 ITR.

6. 6. The method of claim 2 or 5, wherein the polynucleotide encodes the SMN protein of SEQ ID NO:

2. The composition described in

7. 7. Any of claims 2, 5, or 6, wherein the AAV9 viral vector comprises SEQ ID NO:

1. The composition described in claim 1.

8. 1.7 to 2.3 x 10 １３ 8. The method according to claim 1, further comprising administering to said patient a medicament ... The composition described.

9. 1.9 to 2.1 x 10 １３ 9. The method according to claim 1, further comprising administering to said patient a medicament ... The composition described.

10. Approximately 2×10 １３ The composition according to any one of claims 1 to 9, comprising AAV9 vg / mL. thing.

11. The composition of any one of claims 1 to 10, comprising less than about 5% empty capsids.

12. The composition of any one of claims 1 to 11, comprising less than about 3% empty capsids.

13. The composition of any one of claims 1 to 12, comprising less than about 1% empty capsids.

14. 1 to 2 x 10 １４ 2. The method of claim 1, comprising or consisting of an AAV9 viral vector of 14. The composition according to any one of claims 1 to 13.

15. 1.1 x 10 １４ 2. The method of claim 1, comprising or consisting of an AAV9 viral vector of 15. The composition according to any one of claims 1 to 14.

16. 1.7 x 10 １４ 16. The method according to claim 1, comprising administering to said patient a virus vector comprising the AAV9 virus vector of the present invention. The composition according to any one of claims 1 to 4.

17. The percentage of functional viral vector genomes can be determined by in vitro cell assays or in vitro The composition of any one of claims 1 to 16, as measured using an in vivo animal model.

18. AAV containing a polynucleotide encoding a survival motor neuron (SMN) protein 9. Viral vector, Tris buffer, magnesium chloride, sodium chloride, and poloxamer An aqueous pharmaceutical formulation comprising a poloxamer (e.g., poloxamer 188), wherein the pharmaceutical composition is A preparation that does not contain a preservative.

19. The AAV9 viral vector comprises a modified AAV2 ITR, chicken β-actin (C B) Promoter, cytomegalovirus (CMV) immediate early enhancer, modified SV40 The late 16s intron, the bovine growth hormone (BGH) polyadenylation signal, and the unmodified 20. The formulation of claim 18, further comprising a modified AAV2 ITR.

20. 18 or 19, wherein the polynucleotide encodes the SMN protein of SEQ ID NO:

2.

20. The formulation described in 19.

21. 21. Any one of claims 18 to 20, wherein the AAV9 viral vector comprises SEQ ID NO:

1. The formulation described in

22. 18 to 2, wherein the Tris buffer concentration is about 10 to 30 nM, for example about 20 mM.

1. The formulation according to any one of claims 1 to 9.

23. The pH of the formulation is about 7.7 to about 8.3, for example about pH 8.0 (e.g., USP <791 The formulation according to any one of claims 18 to 22, wherein the .alpha.-tocopherol content is 0.01 to 0.01% by mass spectrometry.

24. 10. The method of claim 1, wherein the magnesium chloride concentration is about 0.5 to 1.5 mM, for example about 1 mM.

24. The formulation according to any one of claims 8 to 23.

25. The sodium chloride concentration is about 100-300 mM, for example about 200 mM.

25. The formulation according to any one of 18 to 24.

26. 26. Any one of claims 18 to 25 comprising about 0.005% w / v poloxamer 188. The formulation described in

27. Osmolality of 390-430 mOsm / kg (measured, for example, by USP <785>) The formulation according to any one of claims 18 to 26, having a molecular weight of 1,000 to 1,000.

28. The AAV9 viral vector is in a composition according to any one of claims 1 to 17. The formulation according to any one of claims 18 to 27.

29. A formulation according to any one of claims 18 to 28 or any one of claims 2 or 5 to 17 The method includes administering the composition described in paragraph 1 to a patient by intrathecal or intravenous route.

1. A method for treating spinal muscular atrophy (SMA) type I in a patient in need thereof, comprising administering to said patient but, a. is 9 months of age or younger; b. weighs at least about 2.6 kg; c. have biallelic SMN1 null mutations or deletions; and d. having at least one functional copy of SMN2.

30. The AAV9 viral vector comprises a modified AAV2 ITR, chicken β-actin (C B) Promoter, cytomegalovirus (CMV) immediate early enhancer, modified SV40 The late 16s intron, the bovine growth hormone (BGH) polyadenylation signal, and the unmodified 30. The method of claim 29, comprising a modified AAV2 ITR.

31. 29. The method of claim 28, wherein the polynucleotide encodes the SMN protein of SEQ ID NO:

2.

30. The method according to claim 30.

32. 32. Any one of claims 29 to 31, wherein the AAV9 viral vector comprises SEQ ID NO:

1. The method according to

33. The viral vector is about 1 to 2.5 × 10 １４ This includes administering the drug at a dose of 100 mg / kg. The method according to any one of claims 29 to 32.

34. The viral vector was administered in an amount of about 1.1 × 10 １４ vg / kg 34. The method according to any one of claims 29 to 33.

35. 35. The method of claim 33 or 34, wherein the amount of viral vector genome is measured using ddPCR. The method according to

36. 36. The method of claim 29, wherein the patient weighs about 8.5 kg or less. method.

37. The patient has c.859G>C in exon 7 of at least one copy of the SMN2 gene The method of any one of claims 29 to 36, which has no substitutions.

38. 38. Any one of claims 29 to 37, wherein the treatment is administered to the patient before 6 months of age. The method according to

39. The treatment includes treating hypotonia, motor skill retardation, poor neck balance, hunched posture, and joint hypermobility. Administered to the patient before the onset of one or more selected SMA symptoms.

9. The method according to any one of claims 8 to 9.

40. The patient had a 1:100 antibody titer as determined by ELISA binding immunoassay prior to dosing.

40. The method of any one of claims 29 to 39, having an anti-AAV9 antibody titer of:

41. The patient had a 1:50 or greater IgG antibody titer as determined by ELISA binding immunoassay prior to dosing.

41. The method of any one of claims 29 to 40, wherein the patient has an anti-AAV9 antibody titer below 100 mg / mL.

42. The patient had a 1:100 immune response as determined by ELISA binding immunoassay after dosing. and for about 1-8 weeks or when the titer is less than 1:100 The method of any one of claims 29 to 41, wherein the concentration is monitored until it decreases.

43. The patient had a 1:100 immune response as determined by ELISA binding immunoassay after dosing. and for about 1-8 weeks or until the titer drops to less than 1:50 The method of any one of claims 29 to 42, wherein the patient is monitored until the patient's condition improves.

44. When the patient has a pre- or post-administration determined by ELISA binding immunoassay have an anti-AAV9 titer of greater than 1:100, e.g., before or after administration, and are switched to artificial feeding The method according to any one of claims 29 to 39,

45. When the patient has a pre- or post-administration determined by ELISA binding immunoassay have an anti-AAV9 titer of greater than 1:50 and are switched to artificial feeding, e.g., before or after administration The method according to any one of claims 29 to 39,

46. The patient had a 1:100 immune response as determined by ELISA binding immunoassay after dosing. and is treated with plasmapheresis.

46. ​​The method according to any one of claims 1 to 45.

47. The patient had a 1:50 response rate as determined by ELISA binding immunoassay after dosing. 29 to 30, having an anti-AAV9 titer greater than or equal to 100 mg / kg and treated with plasmapheresis.

46. ​​The method of any one of claims 46 to 47.

48. said patient having greater than about 67,000 cells / ml, or greater than about 100,000 cells / ml prior to administration 2. The method of claim 1, wherein the patient has a platelet count of greater than about 150,000 cells / ml or greater than about 150,000 cells / ml.

9. The method according to any one of claims 9 to 47.

49. said patient having less than about 67,000 cells / ml or less than about 100,000 cells / ml after administration / ml or less than about 150,000 cells / ml, and have a platelet count of about 1 to 8 For weeks, or when the platelet count is about 67,000 cells / ml, or about 100,000 cells / ml 10. The method of claim 1, wherein the antibody is monitored until the antibody concentration exceeds about 150,000 cells / ml or exceeds about 150,000 cells / ml.

9. The method according to any one of claims 9 to 48.

50. the patient has a platelet count below about 67,000 cells / ml after administration and receives a platelet transfusion The method according to any one of claims 29 to 49, wherein the patient is treated with

51. The method according to any one of claims 29 to 50, wherein the patient does not have thrombocytopenia prior to administration. How to.

52. The patient has thrombocytopenia following administration and continues to have thrombocytopenia for about 1 to 8 weeks or 52. The method according to any one of claims 29 to 51, wherein the patient is monitored until he or she is no longer thrombocytopenic. method.

53. Claims 29 to 52, wherein the patient has thrombocytopenia after administration and is treated with platelet transfusion.

2. The method according to claim 1 .

54. The patient has a troponin level of less than about 0.176 ug / ml prior to administration of the viral vector. The method of any one of claims 29 to 53, having a -I level.

55. The patient's Troponin-I level is monitored following administration of the viral vector. The method according to any one of claims 29 to 54.

56. After administration, the patient's troponin-I level is less than about 0.176 ug / ml.

56. The method of claim 54 or 55, wherein monitoring is performed.

57. The method according to any one of claims 29 to 56, wherein the patient has normal liver function prior to administration. method.

58. The patient has a hepatic transaminase level of less than about 8-40 U / L prior to administration. The method according to claim 57.

59. The hepatic transaminase is alanine transaminase (AST), asparagine 58. The method of claim 57, wherein the agonist is selected from the group consisting of an acid transaminase (ALT), ... and combinations thereof. The method according to

60. The patient has a bilirubin level of less than 3.0 mg / dL and less than 1.8 mg / dL prior to administration. creatinine level of less than 0.05 mg / dL, Hgb level of 8-18 g / dL, and / or Hgb level of about 20,000 / mm ３ 60. The method of any one of claims 29 to 59, wherein the patient has a white blood cell count of less than 100 mg / kg.

61. 29 to 6, wherein the viral vector is administered in Tris-buffered saline.

10. The method according to any one of claims 0 to 9.

62. The viral vector is administered at a concentration of about 5 to 20 mL / kg, about 10 to 20 mL / kg, or about 5 . 5-6.5 mL / kg of Tris-buffered saline. The method according to any one of claims 1 to 5.

63. 63. Any of claims 29 to 62, wherein the viral vector is injected over about 45 to 75 minutes.

3. The method according to claim 1 .

64. 64. Any one of claims 29 to 63, wherein the viral vector is infused over about 60 minutes. The method according to

65. 65. The method of claim 63 or 64, wherein the infusion comprises a syringe pump.

66. The patient is taking oral steroids at least 24 hours prior to administration of the viral vector. The method of any one of claims 29 to 65, wherein the

67. The patient is receiving oral steroids for at least 30 days after administration of the viral vector. The method of any one of claims 29 to 66, wherein a drug is administered.

68. 68. The method of claim 67, wherein the oral steroid is administered once a day.

69. 68. The method of claim 67, wherein the oral steroid is administered twice daily.

70. The patient exhibits elevated ALT and / or AST levels following administration of the viral vector. and the oral steroid is monitored for AST and / or ALT levels after 30 days. is continued until the blood glucose level is less than twice the upper limit of normal or less than about 120 IU / L.

70. The method according to any one of claims 1 to 69.

71. The patient is taking oral steroids and has AST and / or ALT levels less than twice the upper limit of normal. or less than about 120 IU / L. Method of posting.

72. 71. The method of any one of claims 66 to 70, wherein the oral steroid is administered at a dose of about 1 mg / kg. The method according to any one of claims 1 to 5.

73. After AST and ALT are less than twice the upper limit of normal or less than about 120 IU / L, 72. The method according to any one of claims 66 to 71, further comprising tapering oral steroid administration. Method of posting.

74. The taper was to 0.5 mg / kg / day for 2 weeks, followed by 0.25 mg / kg / day for an additional 2 weeks.

74. The method of claim 73, comprising a stepwise increase to 100 g / kg / day.

75. The oral steroid was administered at a dose of approximately 1 mg / kg for 30 days, followed by 0.5 mg / kg for 2 weeks. mg / kg / day, followed by a further taper down to 0.25 mg / kg / day over a 2-week period.

74. The method of any one of claims 66 to 73, comprising:

76. 76. Any of claims 66 to 75, wherein the oral steroid is prednisolone or an equivalent drug.

3. The method according to claim 1 .

77. 77. The method of claim 29, further comprising administering to the patient a muscle enhancer or a neuroprotectant.

2. The method according to any one of claims 1 to 11.

78. administering to said patient an antisense oligonucleotide targeted to SMN. The method according to any one of claims 29 to 77.

79. The method according to any one of claims 29 to 78, comprising administering to the patient nusinersen. Method of posting.

80. 80. The method according to any one of claims 29 to 79, comprising administering to the patient stamulamab. Method of posting.

81. Any of claims 29 to 80, wherein the efficacy is determined using the CHOP-INTEND scale.

3. The method according to claim 1 .

82. A method for treating pediatric patients with spinal muscular atrophy (SMA) type I, with or without disease onset. and an adeno-associated virus (AAV) vector according to any one of claims 2 or 5 to 28. administering to the patient a composition or formulation comprising:

83. A method for treating Rett Syndrome in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a compound according to claim 3 or 8. Administering the composition according to any one of claims 1 to 17 to the patient via an intrathecal or intravenous route. The method of claim 1,

84. A method for treating amyotrophic lateral sclerosis (ALS) in a patient in need thereof. The composition according to any one of claims 4 or 8 to 17 is administered intrathecally or intravenously. administering to said patient.

85. 1. A method for treating a patient suffering from SMA Type I, comprising: a. determining the patient's weight; b. By obtaining a kit containing a vial of an AAV9 viral vector pharmaceutical composition. wherein the kit comprises the following number of vials: 【Table 1】 c. The viral vector concentration in each vial was approximately 2.0 x 10 １３ vg / mL; d. The AAV9 viral vector comprises a polynucleotide encoding the SMN protein. and e. administering the AAV9 viral vector from the vial to the patient. ,method.

86. The AAV9 viral vector comprises a mutated AAV2 ITR, a chicken β-actin (CB) promoter, cytomegalovirus (CMV) immediate early enhancer, modified SV 40 late 16s intron, bovine growth hormone (BGH) polyadenylation signal, and 86. The method of claim 85, comprising the AAV2 ITRs.

87. 85. The method of claim 85, wherein the polynucleotide encodes the SMN protein of SEQ ID NO:

2.

86. The method according to claim 86.

88. 88. Any one of claims 85 to 87, wherein the AAV9 viral vector comprises SEQ ID NO:

1. The method according to

89. The AAV viral vector is about 1.0 × 10 １４ ~2.5 x 10 １４ For vg / kg 89. The method of any one of claims 85 to 88, wherein the amount is administered by injection.

90. The AAV viral vector is about 1.1 × 10 １４ Administered by injection at a dose of 1000 mg / kg.

90. The method according to any one of claims 85 to 89, wherein

91. 91. The method of claim 89 or 90, wherein the viral vector is infused over a period of about 45 to 70 minutes. How to.

92. 92. Any one of claims 89 to 91, wherein the viral vector is infused over about 60 minutes. The method according to

93. 93. The method of any one of claims 89 to 92, wherein the infusion comprises a syringe pump.

94. Any of claims 85 to 93, wherein the amount of viral vector genome is measured using ddPCR. The method according to any one of claims 1 to 5.

95. 85 to 87, wherein the dosage titer of the AAV9 viral vector is measured by ddPCR.

94. The method of any one of claims 94 to 94.

96. 96. The method of any one of claims 85 to 95, comprising administering a dose volume of 。 【Table 2】

97. The composition according to any one of claims 2 or 5 to 17 or any one of claims 18 to 28. A method for treating spinal muscular atrophy (SMA) type I comprising administering to a patient suffering from spinal muscular atrophy (SMA) type I a drug comprising administering to the patient ... A kit for treating patients with

98. comprising a polynucleotide encoding a survival motor neuron (SMN) protein; and pH 7.7-8.3, e.g., 20 mM Tris, 1 mM MgCl at about 8.0 ２ , 200m Approximately 2.0 x 10 in M ​​NaCl, 0.005% w / v Poloxamer 188 １３ vg / mL Approximately 5.5 mL or approximately 8.3 mL of AAV9 viral vector formulated at a concentration of A kit containing a vial containing

99. The AAV9 viral vector comprises a mutated AAV2 ITR, a chicken β-actin (CB) promoter, cytomegalovirus (CMV) immediate early enhancer, modified SV 40 late 16s intron, bovine growth hormone (BGH) polyadenylation signal, and 99. The kit of claim 98, comprising AAV2 ITRs.

100. 98. The method of claim 97, wherein the polynucleotide encodes the SMN protein of SEQ ID NO:

2.

99. The kit according to claim 99.

101. Any one of claims 98 to 100, wherein the AAV9 viral vector comprises SEQ ID NO:

1. Item 3. The kit according to item 2.

102. The amount of the viral vector genome is measured by ddPCR according to claims 98 to 101. The kit according to any one of claims 1 to 4.

103. A volume of the composition according to any one of claims 2 or 5 to 17 or claims 18 to 28 Administering the formulation according to any one of claims 1 to a patient in need thereof by intravenous infusion. A method for treating SMA type I, comprising:

104. When the patient weighs between 2.6 and 3.0 kg, the volume is 16.5 mL. The method of claim 103.

105. If the patient weighs 3.1-3.5 kg, the volume is 19.3 mL. The method of claim 103.

106. When the patient weighs 3.6 to 4.0 kg, the volume is 22.0 mL. The method of claim 103.

107. If the patient weighs 4.1-4.5 kg, the volume is 24.8 mL. The method of claim 103.

108. When the patient weighs between 4.6 and 5.0 kg, the volume is 27.5 mL. The method of claim 103.

109. When the patient weighs 5.1 to 5.5 kg, the volume is 30.3 mL. The method of claim 103.

110. When the patient weighs between 5.6 and 6.0 kg, the volume is 33.0 mL. The method of claim 103.

111. If the patient weighs between 6.1 and 6.5 kg, the volume is 35.8 mL. The method of claim 103.

112. If the patient weighs between 6.6 and 7.0 kg, the volume is 38.5 mL. The method of claim 103.

113. If the patient weighs 7.1-7.5 kg, the volume is 41.3 mL. The method of claim 103.

114. When the patient weighs between 7.6 and 8.0 kg, the volume is 44.0 mL. The method of claim 103.

115. If the patient weighs 8.1-8.5 kg, the volume is 46.8 mL. The method of claim 103.

116. A method for producing an AAV viral vector, comprising: a. culturing adherent cells; b. Transfecting the adherent cells with one or more plasmids to produce the AAV virus. To enable the production of rus vectors; c. lysing the adherent cells to isolate the AAV viral vector; d. Acidifying and clarifying the cell lysate of (c); e. Purify the product of (d) above using cation exchange chromatography (CEX). thing; f. filtering the product of (e) using tangential flow filtration (TFF). ； g. ultracentrifuging the product of (f) in a cesium chloride (CsCl) buffer; and h. recovering the AAV viral vector from the product of (g). The method includes:

117. The method of claim 116, wherein the AAV is AAV9.

118. 118. The method of claim 116 or 117, wherein the AAV is self-complementary (scAAV). 。

119. The method according to any one of claims 116 to 118, wherein the adherent cells are HEK293 cells. How to.

120. 120. Any of claims 116 to 119, wherein the adherent cells are selected for adherence prior to culturing.

13. The method according to claim 1.

121. The selection may include selecting for adherence by multiple subculturing of the adherent cells. The method according to any one of claims 116 to 120, comprising:

122. 122. The method according to claim 116, wherein the adherent cells are seeded in a bioreactor for culture.

3. The method according to claim 1 .

123. The bioreactor is a large-scale bioreactor capable of providing continuous circulation of cell culture medium.

123. The method of claim 122, wherein the reactor is an ororeactor.

124. The bioreactor is 200 m ２ Or 333m ２ 10. A bioreactor according to claim 1 . 22 or 123.

125. The bioreactor is 500 m ２ 122 or 123, which is a bioreactor. The method according to

126. The adherent cells are added to the medium in a recirculating medium bag, for example using a peristaltic pump to move the medium through the bag. The method according to any one of claims 122 to 125, wherein the electroreactor is circulated.

127. The peristaltic pumping continues while the adherent cells are seeded into a culture bioreactor. The method of claim 126.

128. The seeding density is about 8,000 to 12,000 cells / cm ２ Claims 122 to 12 8. The method according to any one of claims 7 to 7.

129. The transfection step includes transfecting the recirculation medium bag. Adding medium and pumping the transfection medium, for example using a peristaltic pump, The method according to any one of claims 122 to 128, comprising circulating the bioreactor. Method of posting.

130. 130. The method of claim 126, wherein the circulation, e.g., peristaltic pumping, is performed at 15 to 25° C.

2. The method according to any one of claims 1 to 11.

131. The transfection step comprises the use of an adenovirus helper plasmid (pHEL P) contacting the adherent cells with the Method of posting.

132. The transfection step is carried out using a plasmid encoding the AAV rep gene. The method according to any one of claims 116 to 131, comprising contacting the adherent cells with method.

133. The transfection step comprises transfecting a plasmid encoding the AAV cap gene. The method according to any one of claims 116 to 132, comprising contacting the adherent cells with method.

134. The transfection step contains AAV re vector on the same plasmid (pAAV). A plasmid encoding the AAV p gene and the AAV cap gene is contacted with the adherent cells. The method of any one of claims 116 to 133, comprising:

135. 135. The method of claim 132 or 134, wherein the AAV rep gene is rep2.

136. The method of claim 133 or 134, wherein the AAV cap gene is cap9.

137. The transfection step is carried out using a transfection agent polyethylenimine ( 136. The method of claim 116, comprising contacting the adherent cells with a soluble form of PEG (PEI). The method according to

138. The ratio of PEI to at least one of said plasmids is less than 1:1 by weight. The method of claim 137.

139. the ratio of PEI to at least one of said plasmids is about 1:1 by weight; The method of claim 137.

140. The transfection step may further comprise using a serum-free transfection medium. The method according to any one of claims 116 to 139, comprising contacting the adherent cells. Law.

141. The transfection step is a calcium-free transfection. The method according to any one of claims 116 to 140, comprising contacting a medium with the adherent cells. Method of posting.

142. The transfection step may be a glutamine-free transfection. The method according to any one of claims 116 to 141, comprising contacting a medium with the adherent cells. Method of posting.

143. 116-14, wherein the transfection step is carried out for 10-60 minutes.

3. The method according to any one of claims 2 to 3.

144. 116-14, wherein the transfection step is carried out for 10-30 minutes.

4. The method according to any one of claims 3 to 4.

145. 116-14, wherein the transfection step is carried out for less than 30 minutes 5. The method according to any one of claims 4 to 4.

146. 116-14, wherein the transfection step is carried out for 20-30 minutes.

6. The method according to any one of claims 5 to 5.

147. 116-14, wherein the transfection step is carried out for 15-30 minutes.

7. The method according to any one of claims 6 to 6.

148. 148. The method according to any one of claims 116 to 147, wherein the lysing step comprises whole cell lysis. Method of posting.

149. The lysis step may include using a lysis buffer supplemented with an endonuclease. The method of any one of claims 116 to 148, comprising:

150. 150. The method of claim 149, wherein the endonuclease is benzonase.

151. The lysing step comprises using a lysis buffer supplemented with TWEEN. The method of any one of claims 116 to 150.

152. Any of claims 116 to 151, wherein the dissolving step is carried out at 15 to 25°C.

13. The method according to claim 1.

153. Freezing the cell lysate of step (c) prior to the acidification step of step (d). The method of any one of claims 116 to 152, further comprising:

154. 1. A method for purifying an AAV viral vector from a cell culture lysate, comprising: a. acidifying and clarifying the cell lysate; b. Purifying the product of (a) above using cation exchange chromatography (CEX). Steps: c. filtering the product of (b) by tangential flow filtration; d. The product of (c) is ultracentrifuged using 2 to 4 M cesium chloride (CsCl) buffer. Steps: e. harvesting the AAV viral vector from the product of (d); f. filtering the product of (e) by tangential flow filtration The method includes:

155. The acidification step acidifies the cell lysate to a pH of about 3.0 to 4.

0. The method of any one of claims 116 to 154, comprising:

156. The acidification step acidifies the cell lysate to a pH of about 3.3 to 3.

7. The method of any one of claims 116 to 155, comprising:

157. The acidification step acidifies the cell lysate to a pH of about 3.4 to 3.

6. The method of any one of claims 116 to 156, comprising:

158. the acidification step comprising acidifying the cell lysate to a pH of about 3.

5. The method according to any one of claims 116 to 157.

159. Claims 116 to 158, wherein the ultracentrifugation is carried out at 40,000 to 50,000 rpm.

2. The method according to claim 1 .

160. Claims 116-15, wherein the ultracentrifugation is carried out at about 43,000-46,000 rpm 10. The method according to any one of claims 9 to 10.

161. 161. The method according to any one of claims 116 to 160, wherein the ultracentrifugation is carried out at 15 to 25°C. How to.

162. 162. The method according to any one of claims 116 to 161, wherein the ultracentrifugation is carried out for 16 to 24 hours. How to.

163. 163. The method according to any one of claims 116 to 162, wherein the ultracentrifugation is carried out for 20 to 24 hours. How to.

164. The method according to any one of claims 116 to 168, wherein the CsCl is at a concentration of about 3M. Law.

165. The cell lysate is incubated with Tween prior to the acidification step. The method according to any one of claims 116 to 164,

166. The cell lysate is incubated with Tween for approximately 8-20 hours prior to the acidification step.

166. The method of any one of claims 116 to 165, wherein the cell is incubated.

167. the clarification step comprises filtering the cell lysate through a depth filter; 167. The method of any one of claims 116 to 166.

168. The clarification step comprises filtering the cell lysate through a 0.45 micron filter. The method of any one of claims 116 to 167, comprising:

169. The method according to any one of claims 116 to 168, wherein the CEX comprises a sulfonyl resin. Law.

170. At least one TFF step was performed using cellulosic acid with a molecular weight cutoff of 300 kDa MW. and using a cation exchange membrane to reduce the eluate volume of said cation exchange step to at least 6 minutes. The method of any one of claims 116 to 169, wherein the ratio of the number of stimuli to the number of stimuli is reduced to 1.

171. At least one TFF step is performed using a cell with a molecular weight cutoff of about 300 kDa MW.

171. The method of any one of claims 116 to 170, comprising using a loin membrane.

172. 172. Any one of claims 116 to 171, wherein the CsCl buffer comprises about 3M CsCl. The method according to

173. The CsCl buffer is Tris, MgCl ２ and poloxamer 188.

13. The method according to any one of claims 116 to 172.

174. Any one of claims 116 to 173, wherein the CsCl buffer comprises about 20 mM Tris. The method according to

175. The CsCl buffer is about 2mM MgCl ２ Any of claims 116 to 174, including 13. The method according to claim 1.

176. The CsCl buffer contains poloxamer 188, optionally about 0.2% w / v poloxamer The method of any one of claims 116 to 175, comprising:

177. Any of claims 116 to 176, wherein the CsCl buffer has a pH of about 7.5 to 8.

5.

13. The method according to claim 1.

178. Any of claims 116 to 177, wherein the CsCl buffer has a pH of about 7.9 to 8.

2.

13. The method according to claim 1.

179. The number of empty viral capsids was determined by extracting the AAV viral capsid from the ultracentrifuged cell lysate.

178. The vector of claim 116, which is less than 7% of the total viral capsid after harvesting.

2. The method according to any one of claims 1 to 11.

180. The number of empty viral capsids was determined by extracting the AAV viral capsid from the ultracentrifuged cell lysate.

179. The vector of claim 116, which is less than 5% of the total viral capsid after harvesting.

2. The method according to any one of claims 1 to 11.

181. The number of empty viral capsids was determined by extracting the AAV viral capsid from the ultracentrifuged cell lysate. The vector of any one of claims 116 to 200, which is less than 3% of the total viral capsid after harvesting.

2. The method according to any one of claims 1 to 11.

182. The number of empty viral capsids was determined by extracting the AAV viral capsid from the ultracentrifuged cell lysate. The vector of any one of claims 116 to 201, which is less than 1% of the total viral capsid after harvesting.

2. The method according to any one of claims 1 to 11.

183. Any of claims 179 to 182, wherein the number of empty viral capsids is measured by AUC. The method according to any one of claims 1 to 5.

184. The AAV viral vector is extracted from the ultracentrifuged cell lysate using a syringe. The method of any one of claims 116 to 183, wherein the sample is collected by

185. The AAV viral vector collected after the second TFF step is purified by Tris, MgCl ２ 12. The method of claim 11, wherein the soluble soluble soluble salt is stored in a solution comprising 1,2-dichlorophenyl sodium chloride, ...

6. The method of any one of claims 6 to 184.

186. 186. The method of claim 185, wherein the solution comprises about 20 mM Tris.

187. The solution contains about 1 mM MgCl ２ 187. The method of claim 185 or 186, comprising:

188. 188. The method according to any one of claims 185 to 187, wherein the solution comprises about 200 mM NaCl. Method of posting.

189. 189. The method of claim 185, wherein the solution comprises about 0.005% w / v poloxamer 188.

2. The method according to any one of claims 1 to 11.

190. 190. The method according to any one of claims 185 to 189, wherein the solution has a pH of about 7.5 to 8.

5. How to.

191. 191. The method according to any one of claims 185 to 190, wherein the solution has a pH of about 7.7 to 8.

3. How to.

192. The AAV viral vector collected after the second TFF is less than about 30 μg / g Any one of claims 116 to 191 containing less than or about 20 μg / g CsCl. The method according to

193. The concentration of the AAV viral vector collected after the second TFF is about 3×10 １３ v The method of any one of claims 116 to 192, wherein the solubility is 100 g / ml or more.

194. Host cell proteins and / or host cell DNA are deterged from the cell lysate.

193. The method according to claim 116, wherein the pores are removed by flocculation with a sorbent. The method according to claim 5.

195. The AAV viral vector encodes a survival motor neuron (SMN) protein. The method of any one of claims 116 to 194, comprising a polynucleotide comprising

196. The AAV virus vector comprises a mutated AAV2 ITR, a chicken β-actin ( CB) Promoter, cytomegalovirus (CMV) immediate early enhancer, modified SV4 0 late 16s intron, bovine growth hormone (BGH) polyadenylation signal, and A 196. The method of any one of claims 116 to 195, comprising an AV2 ITR.

197. 195. The method of claim 195, wherein the polynucleotide encodes the SMN protein of SEQ ID NO:

2.

196. The method according to claim 196.

198. The method of claim 195 or 196, wherein the AAV viral vector comprises SEQ ID NO:

1. Law.

199. The plasmid encoding the SMN protein, the plasmid encoding the pAAV The plasmid encoding the pHELP was transfected in a 1:1:1 ratio.

200. The method of any one of claims 195 to 198, wherein the vector is transfected with

200. The AAV viral vector comprises a polynucleotide encoding the MECP2 protein. The method of any one of claims 116 to 194, comprising:

201. The AAV viral vector comprises a polynucleotide encoding an shRNA targeting SOD1.

195. The method of any one of claims 116 to 194, comprising a nucleotide.

202. SMN-TA as prepared by the method according to any one of claims 116 to 199. administering an AAV9 viral vector comprising a polynucleotide encoding a protein; 23. A method for treating a patient with SMA type 1 by

203. As prepared by the method of any one of claims 116 to 194 or 200 AAV9 viral vector comprising a polynucleotide encoding the MECP2 protein of A method for treating a patient with Rett Syndrome by administering

204. As prepared by the method of any one of claims 116 to 194 or 201 AAV9 virus containing a polynucleotide encoding shRNA targeting SOD1 of A method of treating a patient with ALS by administering a spectrophotometer.

205. SMN protein produced by the method according to any one of claims 116 to 199. An AAV9 viral vector comprising a polynucleotide encoding a gene.

206. SMN protein produced by the method according to any one of claims 116 to 199. A pharmaceutical composition comprising an AAV9 viral vector comprising a polynucleotide encoding a gene.

207. an AAV9 viral vector comprising a polynucleotide encoding an SMN protein; Aqueous pharmaceutical composition comprising a Tris buffer, a magnesium chloride solution, and a sodium chloride solution 200, wherein the pharmaceutical composition does not contain a preservative and the composition is a composition according to any one of claims 116 to 199. A composition produced by the method according to any one of claims 1 to 4.

208. ME produced by the method according to any one of claims 116 to 194 or 200. An AAV9 viral vector comprising a polynucleotide encoding a CP2 protein.

209. ME produced by the method according to any one of claims 116 to 194 or 200. The present invention includes an AAV9 viral vector comprising a polynucleotide encoding the CP2 protein. Pharmaceutical compositions.

210. SO produced by the method according to any one of claims 116 to 194 or 201. AAV9 viral vector containing a polynucleotide encoding shRNA targeting D1 Tha.

211. SO produced by the method according to any one of claims 116 to 194 or 201. AAV9 viral vector containing a polynucleotide encoding shRNA targeting D1 A pharmaceutical composition comprising a ter.

212. a. A modified AAV2 ITR and a chicken β-actin (CB) promoter and site A modified SV40 late 16s intron and a CMV immediate early enhancer. , a bovine growth hormone (BGH) polyadenylation signal and unmodified AAV2 ITRs. a self-complementary AAV9 viral vector comprising: b. 20 mM Tris, pH 8.0; c. 1mM MgCl2; d. 200 mM NaCl; and e. 0.005% Poloxamer 188 In a patient in need thereof, a pharmaceutical composition comprising 1. A method for treating SMA comprising: The method, wherein the patient weighs between 2.6 kg and 8.5 kg.

213. 213. The method of claim 212, wherein the composition does not contain a preservative.

214. 20. The patient comprising: a. is 9 months of age or younger; b. weighs at least about 2.6 kg; c. have biallelic SMN1 null mutations or deletions; and d. having at least one functional copy of SMN2; The method of claim 212 or 213.

215. a. A modified AAV2 ITR and a chicken β-actin (CB) promoter and site A modified SV40 late 16s intron and a CMV immediate early enhancer. , a bovine growth hormone (BGH) polyadenylation signal and unmodified AAV2 ITRs. a self-complementary AAV9 viral vector comprising: b. 20 mM Tris, pH 8.0; c. 1mM MgCl2; d. 200 mM NaCl; and e. 0.005% Poloxamer 188 A composition suitable for or manufactured for intravenous administration of a pharmaceutical composition comprising:

216. 216. The composition of claim 215, which is free of preservatives.

217. The method according to any one of claims 116 to 201, carried out on an industrial scale.

218. The AAV yield produced is 5×10 per production batch. １５ More than vg or 8 x 10 １５ v g or 1 x 10 １６ Any one of claims 116 to 201 or 217, wherein the The method described.

219. At least one of the following: a. 1.0 x 10 １３ less than about 0.09 ng benzonase per vg; b. Cesium less than about 30 μg / g (ppm); c. about 20-80 ppm of Poloxamer 188; d. 1.0 x 10 １３ less than about 0.22 ng BSA per vg; e. 1.0 x 10 １３ Approximately 6.8 x 10 per vg ５ Sub-pg residual plasmid DNA, f. 1.0 x 10 １３ Approximately 1.1 x 10 per vg ５ Sub-pg residual hcDNA, g. 1.0 x 10 １３ less than about 4 ng rHCP per vg; h. Approximately pH 7.7-8.3, i. Approximately 390-430mOsm / kg, j. less than about 600 particles >= 25 μm size per container; k. less than about 6000 particles >= 10 μm size per container; l. Approximately 1.7×10 １３ ~2.3 x 10 １３ Genomic titer in vg / mL; m. 1.0 x 10 １３ Approximately 3.9 x 10 per vg ８ ~8.4 x 10 １０ Infectious titer of IU 、 n. 1.0 x 10 １３ Approximately 100-300 μg total protein per vg; o. A relative efficacy of about 70-130%, and p. Less than about 5% empty capsids Any one of claims 1 to 17, 18 to 28, 82 to 84, or 205 to 211, comprising The composition or formulation described in.

220. At least one of the following: a. Approximately pH 7.7-8.3, b. Approximately 390-430mOsm / kg, c. less than about 600 particles >= 25 μm size per container; d. less than about 6,000 particles >= 10 μm size per container; e. Approximately 1.7×10 １３ ~2.3 x 10 １３ Genomic titer in vg / mL; f. 1.0 x 10 １３ Approximately 3.9 x 10 per vg ８ ~8.4 x 10 １０ Infectious titer of IU 、 g. 1.0 x 10 １３ Approximately 100-300 μg total protein per vg; h. Pluronic F-68 content of about 20-80 ppm; i. a relative efficacy of about 70-130%; j. 7.5 x 10 １３ In the Δ7SMN mouse model, the effect was observed at a dose of 1000 mg / kg for 24 days or more. median survival time, k. less than about 5% empty capsids; l. and a total purity of about 95% or greater; and m. about 0.75 EU / mL or less endotoxin Any one of claims 1 to 17, 18 to 28, 82 to 84, or 205 to 211, comprising The composition or formulation described in.

221. At least one of the following: a. 1.0 x 10 １３ less than about 0.09 ng benzonase per vg; b. Cesium less than about 30 μg / g (ppm); c. about 20-80 ppm of Poloxamer 188; d. 1.0 x 10 １３ less than about 0.22 ng BSA per vg; e. 1.0 x 10 １３ Approximately 6.8 x 10 per vg ５ Sub-pg residual plasmid DNA, f. 1.0 x 10 １３ Approximately 1.1 x 10 per vg ５ Sub-pg residual hcDNA, and g. 1.0 x 10 １３ Less than about 4 ng rHCP per vg Any one of claims 1 to 17, 18 to 28, 82 to 84, or 205 to 211, comprising The composition or formulation described in.

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