RU2818529C2 - Agents and method of producing viral vectors and versions of their use - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: group of inventions relates to methods of producing and purifying AAV virus particles and use thereof. Method of producing viral vector based on AAV involves culturing adhesive cells; their transfection with plasmid(s) to ensure production of viral vector based on AAV; lysis of adhesive cells with recovery of viral vector based on AAV; acidification and clarification of the obtained cell lysate; purification of the obtained product using cation-exchange chromatography (CEX); filtration using tangential flow filtration (TFF); ultracentrifugation in a buffer with cesium chloride CsCl and collection of viral vectors based on AAV. Method of treating type I spinal muscular atrophy comprises administering a composition comprising an AAV vector to a patient, where the AAV-based vector contains a polynucleotide coding a motor neuron survival protein (SMN).

EFFECT: group of inventions provides obtaining a pharmaceutical product based on AAV, with low content of empty capsids, low content of host cell protein and/or low content of contaminating DNA, while maintaining high activity.

40 cl, 36 dwg, 36 tbl, 22 ex

Description

LIST OF SEQUENCES

This application contains a sequence listing that was filed in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The specified ASCII copy, created on October 31, 2018, is named AVEX-003001WO_ST25.txt and is 14639 bytes in size.

RELATED APPLICATIONS

This application claims benefit from U.S. Provisional Patent Application No. 62/583,035, filed November 8, 2017, the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE PRESENT INVENTION

The present invention relates to methods for producing and purifying viral particles and compositions and uses thereof.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV) belongs to the Parvoviridae family. The AAV genome consists of a linear, single-stranded DNA molecule that contains approximately 4.7 kilobase pairs (kb) and consists of two major open reading frames encoding the nonstructural protein Rep (replication) and the structural protein Cap (capsid). The AAV coding regions are flanked by two inverted terminal repeat (ITR) sequences that act when placed in cis, approximately 145 nucleotides in length, with spacer-separated palindromic sequences that can fold into hairpin structures that function as primers during the initiation of DNA replication. In addition to their role in DNA replication, ITR sequences have been shown to be required for viral integration, release from the host genome, and encapsidation of viral nucleic acid into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97 -129).

There are many AAV serotypes that have different tissue tropisms. Known serotypes include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11. AAV9 is described in US Pat. US No. 7,198,951 and Gao et al., J. Virol., 78: 6381-6388 (2004), which are incorporated herein by reference in their entirety. Advances in the delivery of AAV6 and AAV8 have made it possible for these serotypes to be transduced into skeletal and cardiac muscle following simple systemic intravenous or intraperitoneal injections. See Pacak et al., Circ. Res., 99(4): 3-9 (2006) and Wang et al., Nature Biotech. 23(3): 321-8 (2005). However, the use of AAV to target cell types in the central nervous system required surgical intraparenchymal administration. See Kaplitt et al., "Safety and tolerance of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial." Lancet, 369:2097-2105; Marks et al., "Gene delivery of AAV2-neurturin for Parkinson's disease: a double-blind, randomized, controlled trial." Lancet Neurol 9:1164–1172; and Worgall et al., "Treatment of late infantile neuronal ceroid lipofuscinosis by CNS administration of a serotype 2 adeno-associated virus expressing CLN2 cDNA." Hum Gene Ther 19(5):463–74.

The nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J. Virol, 45: 555-564 (1983), as revised by Ruffing et al., J Gen Virol, 75: 3385-3392 (1994). Sequences acting in cis that control viral DNA replication (rep), encapsidation/packaging, and integration into the host cell chromosome are contained in the ITR. Three AAV promoters (named p5, p19, and p40 for their relative locations on the map) initiate the expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19) in combination with differential splicing of the single AAV intron (at nucleotides 2107 and 2227) result in the formation of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins have several enzymatic properties that are essentially responsible for viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2 and VP3. Alternative splicing and nonconsensus translation start sites are responsible for the production of three related capsid proteins. The only consensus polyadenylation site is located at position 95 on the AAV genome map. The life cycle and genetics of AAV are discussed in Muzyczka, Microbiology and Immunology, 158: 97-129 (1992).

AAV-derived vectors are particularly attractive for the delivery of genetic material because (i) they are capable of infecting (transducing) a wide range of nondividing and dividing cell types, including muscle fibers and neurons; (ii) they lack viral structural genes, thereby preventing natural host cell responses to viral infection, such as interferon-mediated responses; (iii) wild-type viruses have never been associated with any pathology in humans; (iv) unlike wild-type AAVs, which are able to integrate into the host cell genome, replication-defective AAV vectors are typically maintained as episomes, thereby limiting the risk of insertional mutagenesis or oncogene activation; and (v) unlike other vector systems, AAV vectors do not elicit a significant immune response (see ii), thus allowing long-term expression of therapeutic transgenes (provided their gene products are not rejected).

Self-complementary adeno-associated vectors (scAAV) are viral vectors constructed from naturally occurring adeno-associated virus (AAV) for use in gene therapy. scAAV is called “self-complementary” because the coding region is designed such that it forms an intramolecular double-stranded DNA template. The rate-limiting step in the life cycle of a standard AAV genome involves second-strand synthesis, since the typical AAV genome is a single-stranded DNA template. However, this is not the case for scAAV genomes. Following infection, without waiting for cell-mediated second-strand synthesis, the two complementary halves of scAAV associate to form a single unit of double-stranded DNA (dsDNA), which is ready for immediate replication and transcription.

There remains a need to develop a scalable method for the preparation and purification of an AAV-based pharmaceutical product, eg, low in empty capsids, low in host cell protein, and/or low in contaminating DNA, while maintaining high activity.

SHORT DESCRIPTION

The present invention provides a method for preparing purified viral particle preparations, including AAV particle preparations.

In some embodiments, the present invention provides a pharmaceutical composition comprising (a) 1-8×10 ¹³ AAV9-based viral vector genomes/ml (g.v./ml), (b) less than about 7% empty viral capsids, ( c) less than about 100 ng/ml host cell protein per 1 x 10 ¹³ g/ml, and (d) less than about 5 x 10 ⁶ pg/ml residual host cell DNA per 1 x 10 ¹³ g .v./ml, and at least about 80% of the 1-8×10 ¹³ AAV9-based viral vector genomes/ml are functional.

In one embodiment, the AAV9-based viral vector contains a polynucleotide encoding a survival motor neuron (SMN) protein. In one embodiment, the AAV9-based viral vector contains a polynucleotide encoding a methyl-CpG binding protein 2 (MECP2) protein. In one embodiment, the AAV9-based viral vector contains a polynucleotide encoding a short hairpin RNA (shRNA) targeting superoxide dismutase 1 (SOD1). In one embodiment, the AAV9-based viral vector contains a modified AAV2 ITR, a chicken beta-actin (CB) promoter, a cytomegalovirus (CMV) immediate-early enhancer, a modified SV40 late 16s intron, a bovine growth hormone (BGH) polyadenylation signal, and an unmodified ITR AAV2.

The present invention provides a pharmaceutical composition. In some embodiments, the aqueous pharmaceutical composition comprises (a) an AAV9-based viral vector containing a polynucleotide encoding a survival motor neuron (SMN) protein, (b) Tris buffer, (c) magnesium chloride, (d) sodium chloride, and (e) a poloxamer (eg, poloxamer 188), wherein the pharmaceutical composition does not contain a preservative. In one embodiment of the composition, the AAV9-based viral vector further comprises a modified AAV2 ITR, a chicken beta-actin (CB) promoter, a cytomegalovirus (CMV) immediate-early enhancer, a modified SV40 late 16s intron, a bovine growth hormone (BGH) polyadenylation signal and unmodified AAV2 ITR. In one embodiment of the formulation, the concentration of Tris buffer is approximately 10-30 nM, such as approximately 20 mM. In one embodiment, the pH of the formulation is from about 7.7 to about 8.3, for example, the pH is about 8.0 (eg, when measured according to USP <791> (incorporated by reference in its entirety)). In one embodiment of the formulation, the concentration of magnesium chloride is approximately 0.5-1.5 mM, such as approximately 1 mM. In one embodiment of the formulation, the concentration of sodium chloride is approximately 100-300 mM, for example, approximately 200 mM. In one embodiment, the composition contains approximately 0.005% w/v. poloxamer 188.

Another aspect of the present invention is directed to a method of treating spinal muscular atrophy (SMA) type I in a patient in need thereof, comprising administering an AAV9-based viral vector containing a polynucleotide encoding an SMN protein (e.g., a composition or composition described herein) to the patient by the intrathecal or intravenous route, where (a) the patient is nine months of age or less, (b) the patient's body weight is at least about 2.6 kg, (c) the patient has biallelic null mutations or deletions in SMN1 , and (d) the patient has at least one functional copy of SMN2 . In one embodiment, the AAV9-based viral vector contains a modified AAV2 ITR, a chicken beta-actin (CB) promoter, a cytomegalovirus (CMV) immediate-early enhancer, a modified SV40 late 16s intron, a bovine growth hormone (BGH) polyadenylation signal, and an unmodified ITR AAV2. In one embodiment, the patient's body weight is no more than about 8.5 kg. In one embodiment, the patient does not have the c.859G>C substitution in exon 7 of at least one copy of the SMN2 gene. In one embodiment, the treatment agent is administered to a patient under 6 months of age. In one embodiment, the treatment agent is administered to the patient before the onset of one or more SMA symptoms selected from hypotonia, delayed motor skill development, poor head support, slouched posture, and joint hypermobility. In one embodiment, prior to administration, the patient has anti-AAV9 antibody titers of 1:100 or lower, as determined by enzyme-linked immunosorbent assay ELISA.

Also disclosed herein is a method of treating a pediatric patient with spinal muscular atrophy (SMA) type I, with or without disease manifestation, comprising administering to the patient a composition or formulation containing an adeno-associated virus (AAV) vector described herein.

The present invention is also directed to a method of treating Rett syndrome in a patient in need thereof, comprising administering an AAV9-based viral vector containing a polynucleotide encoding methyl CpG binding protein 2 (MECP2) to the patient by the intrathecal or intravenous route.

The present invention is also directed to a method of treating amyotrophic lateral sclerosis (ALS) in a patient in need thereof, comprising administering an AAV9-based viral vector containing a polynucleotide encoding a short hairpin RNA (shRNA) targeting superoxide dismutase 1 (SOD1) to the patient intrathecally or by intravenous route.

Another aspect of the present invention is directed to a method of treating a patient suffering from SMA type I, comprising the steps of (a) determining the patient's weight, (b) providing a kit containing vials of an AAV9-based viral vector pharmaceutical composition, and (c) administering the viral an AAV9-based vector from vials to a patient, wherein the concentration of the viral vector in each vial is approximately 2.0×10 ¹³ g/mL, wherein the AAV9-based viral vector contains a polynucleotide encoding the SMN protein; and the set contains the following number of bottles.

Patient weight (kg) Bottle 5.5 ± 0.4 ml Bottle 8.3 ± 0.4 ml Total bottles per set 2.6-3.0 0 2 2 3.1-3.5 2 1 3 3.6-4.0 1 2 3 4.1-4.5 0 3 3 4.6-5.0 2 2 4 5.1-5.5 1 3 4 5.6-6.0 0 4 4 6.1-6.5 2 3 5 6.6-7.0 1 4 5 7.1-7.5 0 5 5 7.6-8.0 2 4 6 8.1-8.5 1 5 6

In one embodiment, the kit contains an AAV9-based viral vector containing a mutated AAV2 ITR, a chicken beta-actin (CB) promoter, a cytomegalovirus (CMV) immediate-early enhancer, a modified SV40 late 16s intron, a bovine growth hormone polyadenylation signal ( BGH) and ITR AAV2. In one embodiment, the AAV-based viral vector is administered by infusion at a dose of approximately 1.0 x 10 ¹⁴ -2.5 x 10 ¹⁴ g/kg.

Another aspect of the present invention is directed to a kit for the treatment of a patient suffering from spinal muscular atrophy (SMA) type I, containing vials containing an AAV9-based viral vector composition containing a polynucleotide encoding a survival motor neuron (SMN) protein, or a composition containing (a) an AAV9-based viral vector containing a polynucleotide encoding a survival motor neuron (SMN) protein, (b) Tris buffer, (c) magnesium chloride, (d) sodium chloride, and (e) a poloxamer (eg, poloxamer 188).

Another aspect of the present invention is directed to a kit containing vials containing approximately 5.5 ml or approximately 8.3 ml of an AAV9-based viral vector containing a polynucleotide encoding a survival motor neuron (SMN) protein and formulated at a concentration of approximately 2.0 x 10 ¹³ g/ml in 20 mM Tris, 1 mM MgCl ₂ , 200 mM NaCl, 0.005% w/v. poloxamer 188 at a pH of 7.7-8.3, for example approximately 8.0.

Another aspect of the present invention is directed to a method of treating SMA type I, comprising administering a specified volume of a composition, wherein the AAV9 viral vector composition comprises a polynucleotide encoding a survival motor neuron (SMN) protein, or a composition comprising (a) an AAV9 viral vector containing a polynucleotide encoding a survival motor neuron (SMN) protein, (b) Tris buffer, (c) magnesium chloride, (d) sodium chloride, and (e) a poloxamer (eg, poloxamer 188), by intravenous infusion to a patient in need thereof .

Another aspect of the present invention is directed to methods for producing an AAV-based viral vector. In one embodiment, a method of producing an AAV viral vector includes the steps of (a) culturing the adherent cells, (b) transfecting the adherent cells with plasmid(s) to produce the AAV viral vector, (c) lysing the adherent cells to release the viral vector based on AAV, (d) acidifying and clarifying the cell lysate from (c), (e) purifying the product from (d) using cation exchange chromatography (CEX), (f) filtering the product from (e) using tangential flow filtration ( TFF), (g) ultracentrifuging the product from (f) in cesium chloride (CsCl) buffer; and (h) collecting AAV-based viral vectors from the product of (g).

Another aspect of the present invention is directed to a method for purifying an AAV viral vector from a cell culture lysate, comprising the steps of (a) acidifying and clarifying the cell lysate, (b) purifying the product from (a) using cation exchange chromatography (CEX), (c) filtration product from (b) by tangential flow filtration, (d) ultracentrifugation of product from (c) using 2-4 M cesium chloride (CsCl) buffer, (e) collection of AAV-based viral vectors from product from (d), (f) filtering the product from (e) by tangential flow filtration. In one embodiment, the method is carried out on an industrial scale. In one embodiment, the method produces a yield of greater than 5×10 ¹⁵ g.v., or greater than 8×10 ¹⁵ g.v., or greater than 1×10 ¹⁶ g.v. per production batch.

Another aspect of the present invention is directed to a method of treating a patient who has SMA type 1 by administering an AAV9-based viral vector containing a polynucleotide encoding an SMN protein prepared in accordance with any of the methods disclosed herein.

Another aspect of the present invention is directed to a method of treating a patient who has Rett syndrome by administering an AAV9-based viral vector containing a polynucleotide encoding the MECP2 protein produced in accordance with any of the methods described herein.

Another aspect of the present invention is directed to a method of treating a patient who has ALS by administering an AAV9-based viral vector containing a polynucleotide encoding an shRNA targeting SOD1 prepared in accordance with any of the methods described herein.

Another aspect of the present invention is directed to an AAV9-based viral vector containing a polynucleotide encoding an SMN protein produced in accordance with any of the methods described herein.

Another aspect of the present invention is directed to a pharmaceutical composition comprising an AAV9-based viral vector containing a polynucleotide encoding an SMN protein, prepared in accordance with any of the methods described herein.

Another aspect of the present invention is directed to an aqueous pharmaceutical composition containing an AAV9-based viral vector containing a polynucleotide encoding an SMN protein, a Tris buffer, a magnesium chloride solution and a sodium chloride solution, wherein the pharmaceutical composition does not contain a preservative, and where the composition is prepared in accordance with by any of the methods described in this document.

Another aspect of the present invention is directed to an AAV9-based viral vector containing a polynucleotide encoding the MECP2 protein produced in accordance with any of the methods described herein.

Another aspect of the present invention is directed to a pharmaceutical composition comprising an AAV9-based viral vector containing a polynucleotide encoding the MECP2 protein, prepared in accordance with any of the methods described herein.

Another aspect of the present invention is directed to an AAV9-based viral vector containing a polynucleotide encoding an shRNA targeting SOD1 prepared in accordance with any of the methods described herein.

Another aspect of the present invention is directed to a method of treating SMA type I in a patient in need thereof by intravenously administering a pharmaceutical composition comprising (a) an AAV9-based self-complementary viral vector containing an ITR modified AAV2, chicken beta actin (CB) promoter, immediately -cytomegalovirus (CMV) early enhancer, modified SV40 late 16s intron, bovine growth hormone (BGH) polyadenylation signal, and unmodified AAV2 ITR, (b) 20 mM Tris at pH 8.0, (c) 1 mM MgCl ₂ , ( d) 200 mM NaCl and (e) 0.005% poloxamer 188, with the patient's body weight ranging from 2.6 kg to 8.5 kg. In one embodiment, the composition does not contain a preservative.

In one embodiment, the patient (a) is nine months of age or less, (b) has a body weight of at least about 2.6 kg, (c) has biallelic null mutations or deletions in SMN1 , and (d) has at least one functional copy of SMN2.

Another aspect of the present invention is directed to a composition suitable or prepared for intravenous administration, wherein the pharmaceutical composition contains (a) an AAV9-based self-complementary viral vector containing a modified AAV2 ITR, a chicken beta-actin (CB) promoter, a cytomegalovirus immediate-early enhancer ( CMV), modified SV40 late 16s intron, bovine growth hormone (BGH) polyadenylation signal, and unmodified AAV2 ITR, (b) 20 mM Tris at pH 8.0, (c) 1 mM MgCl ₂ , (d) 200 mM NaCl and (e) 0.005% poloxamer 188.

In some embodiments, a composition or formulation is disclosed herein, wherein the composition or formulation contains at least one of the following: (a) less than about 0.09 ng of benzonase per 1.0 x 10 ¹³ g.v., (b) less than about 30 μg/g (ppm) cesium, (c) about 20-80 ppm poloxamer 188, (d) less than about 0.22 ng BSA per 1.0 x 10 ¹³ g.v., (e) less than about 6, 8 x 10 ⁵ pg of residual plasmid DNA at 1.0 x 10 ¹³ g., (f) less than approximately 1.1 x 10 ⁵ pg of residual hcDNA at 1.0 x 10 ¹³ g. and (g) less than about 4 ng rHCP per 1.0 x 10 ¹³ g.

In some embodiments, the present invention provides an accumulation process for producing an intermediate (e.g., a frozen intermediate) obtained from a working cell bank, the accumulation process comprising the steps of (a) culturing the cells, (b) transfecting the cultured cells with plasmids (e.g., three plasmids ), (c) collecting increased amounts of viral particles from the cells after the culture period, (d) purifying the viral particles by filtration to remove any intact cells or cellular debris, (e) subjecting the eluent from step (d) to tangential flow filtration, and (g) optionally freezing the resulting intermediate preparation of purified viral particles. In some embodiments, the accumulation process is combined with additional processing steps, such as, for example, additional purification and formulation steps to obtain the final pharmaceutical product.

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

In one embodiment, polyethylenimine is used in the transfection step. In one embodiment, the transfection step includes transfection with three vectors using a transgenic plasmid, for example pSMN, pAAV plasmid and pHELP plasmid.

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

In one embodiment, the collection step includes treating the product with an endonuclease, such as benzonase, for example, at a concentration of 50 to 200 IU/ml, for example, at a concentration of 75 to 150 IU/ml, to reduce the content of residual host cell DNA.

In one embodiment, the purification step includes depth filtration and subsequent filtration through a filter that removes macromolecular contaminants and cellular debris, such as a 0.45 micron pore size filter, but which allows vector genomes to pass through it. Any suitable depth filter can be used.

In one embodiment, tangential flow filtration ("TFF") achieves 5 to 15X, such as 6 to 10X, the eluent concentration from step (d) and at least 4 diavolumes, such as 6 diafiltration diavolumes, or 10 diavolumes , or 12 diavolumes, or 15 diavolumes of diafiltration. Any suitable filter for TFF can be used. In one embodiment, the membrane for TFF is a cellulose membrane. In one embodiment, the TFF membrane has a 300 kDa cutoff.

Additionally, the present invention provides an isolation and purification process for treating an intermediate (eg, a frozen intermediate) to obtain a filtered drug substance. The steps in the isolation and purification process include an acidification and clarification step (using filtration), followed by cation exchange chromatography, tangential flow filtration, ultracentrifugation with CsCl, and an additional tangential flow filtration step to obtain the filtered drug substance, where the purified AAV particles are suspended in a pharmaceutically acceptable carrier.

In one embodiment, the acidification and clarification step includes depth filtration and subsequent filtration through a filter that removes macromolecular contaminants and cellular debris, such as a 0.45 micron pore size filter. In some embodiments, the pH control is controlled. As part of this step, in one embodiment, a detergent, such as Tween, is added. In some embodiments, the rate of Tween addition and the range of Tween concentrations are controlled.

In one embodiment, the cation exchange chromatography provides a membrane chromatography resin using a sulfonyl group composite resin with a pore size of 0.2 microns.

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

In another embodiment, the CsCl ultracentrifugation step is carried out at approximately 40-50 thousand rpm for approximately 20-25 hours. In another embodiment, the CsCl ultracentrifugation step is carried out at approximately 45 thousand rpm for approximately 22 hours.

In one embodiment, tangential flow filtration ("TFF") achieves 5 to 15X, such as 6 to 10X, the eluent concentration from step (d) and at least 4 diavolumes, such as 6 diafiltration diavolumes, or 10 diavolumes , or 12 diavolumes, or 15 diavolumes of diafiltration. In one embodiment, the TFF membrane has a 300 kDa cutoff.

In one embodiment, the eluent is below the cesium (Cs) detection level. In another embodiment, the Cs detection level is below 50 per million (ppm). In another embodiment, the Cs detection level is approximately 50-70 ppm. In another embodiment, the Cs detection level is approximately 70-90 parts per million (ppm). In another embodiment, the Cs detection level is approximately 90-110 parts per million (ppm). In another embodiment, the Cs detection level is approximately 110-130 parts per million (ppm). In another embodiment, the Cs detection level is approximately 130-150 parts per million (ppm). In another embodiment, the Cs detection level is less than 150 parts per million (ppm).

Such purification methods can be used to produce high-yield viral preparations, including AAV preparations (eg, AAV9-SMN) that contain less than 5 x 10 ⁶ pg/ml residual host cell DNA (hcDNA) per 1 x 10 ¹³ vector genomes ("g.v.")/ml, for example, less than 1.2×10 ⁶ pg/ml hcDNA per 1×10 ¹³ g.v./ml. Thus, a 5 kg patient receiving 7.5 x 10 ¹⁵ g i.v. would receive up to no more than 1.2 x 10 ⁶ pg/mL*7.5 x 10 ¹⁵ g i.v. /(1×10 ¹³ g/ml) = 8.4×10 ⁷ pg hcDNA=84000 ng hcDNA per 5 kg dose. In one embodiment, the preparation contains less than 5.0 x 10 ⁵ pg of residual host cell DNA per 1.0 x 10 ¹³ g., less than 2.0 x 10 ⁵ pg of residual host cell DNA per 1.0 x 10 ¹³ years of age, less than 1.1×10 ⁵ pg of residual host cell DNA per 1.0×10 ¹³ years of age, less than 1.0×10 ⁵ pg of residual host cell DNA per 1.0×10 ¹³ years of age, less than 0.9×10 ⁵ pg of residual host cell DNA per 1.0×10 ¹³ years of age, less than 0.8×10 ⁵ pg of residual host cell DNA per 1.0×10 ¹³ , or any concentration in the range limited by these values.

In one embodiment, the AAV is a replication-defective AAV9, such as scAAV9 with ITRs derived from AAV2. In another embodiment, the AAV-based vector carries the SMN transgene. In one embodiment, the DNA encoding SMN is submitted to GenBank under accession number NM_000344.2. Conservative nucleotide substitutions in SMN DNA are also provided (eg, guanine to adenine substitution at position 625, as listed in GenBank accession no. NM_000344.2).

Another aspect of the present invention is directed to a pharmaceutical composition containing AAV particles in a formulation suitable for either (a) intravenous ("IV") injection or (b) intrathecal ("IT") administration.

In another embodiment, the pharmaceutical composition contains less than 10% empty capsids, less than 8% empty capsids, less than 7% empty capsids, less than 5% empty capsids, less than 3% empty capsids, or less than 1% empty capsids. In some embodiments, the pharmaceutical composition contains less than about 5% empty capsids. In one embodiment, the number of empty capsids is below the detection limit. In some embodiments, it is preferable for the pharmaceutical composition to have low levels of empty capsids because these empty capsids may cause an adverse reaction (eg, immune reaction, inflammatory reaction, liver reaction, and/or cardiac reaction) without providing a therapeutic effect. .

In another embodiment, the content of residual host cell protein ("rHCP") in the specified pharmaceutical composition is 100 ng/ml rHCP per 1x10 ¹³ g.v./ml or less, for example, is 40 ng/ml rHCP per 1x 10 ¹³ g/mL or less or 1-50 ng/mL rHCP per 1x10 ¹³ g/mL. In one embodiment, the pharmaceutical composition disclosed herein contains less than 10 ng rHCP per 1.0 x 10 ¹³ g, or less than 5 ng rHCP per 1.0 x 10 ¹³ g, less than 4 ng rHCP per 1.0 x 10 ¹³ g.v., or less than 3 ng rHCP per 1.0 x 10 ¹³ g.v., or any concentration within the range limited by these values.

In another embodiment, the content of residual host cell DNA (“hcDNA”) in the pharmaceutical composition is 5×10 ⁶ pg/ml hcDNA at 1×10 ¹³ g.v./ml or less is 1.2×10 ⁶ pg /ml rHDNA at 1×10 ¹³ g.v./ml or less or from 1×10 ⁵ pg/ml rHDNA at 1×10 ¹³ g.v./ml to 1.2×10 ⁶ pg/ml at 1× 10 ¹³ g./ml. In one embodiment, the content of residual host cell DNA in the specified pharmaceutical composition is less than 5.0×10 ⁵ pg per 1.0×10 ¹³ g., less than 2.0×10 ⁵ pg per 1.0×10 ¹³ g.v., less than 1.1×10 ⁵ pg on 1.0×10 ¹³ g.v., less than 1.0×10 ⁵ pg hcDNA on 1.0×10 ¹³ g.v., less than 0.9 ×10 ⁵ pg hcDNA per 1.0 x 10 ¹³ year, less than 0.8 x 10 ⁵ pg hcDNA per 1.0 x 10 ¹³ year, or any concentration within the range limited by these values.

In one embodiment, the residual plasmid DNA content in said pharmaceutical composition is 1.7 x 10 ⁶ pg/ml per 1 x 10 ¹³ g/ml or less, or 1 x 10 ⁵ pg/ml per 1 x 10 ¹³ g .v./ml to 1.7×10 ⁶ pg/ml per 1×10 ¹³ g./ml. In one embodiment, the content of residual plasmid DNA in the specified pharmaceutical composition is less than 10.0 x 10 ⁵ pg per 1.0 x 10 ¹³ g., less than 8.0 x 10 ⁵ pg per 1.0 x 10 ¹³ g. V. or less than 6.8 × 10 ⁵ pg per 1.0 × 10 ¹³ g.

In one embodiment, the pharmaceutical composition disclosed herein contains less than 0.5 ng per 1.0 x 10 ¹³ g, less than 0.3 ng per 1.0 x 10 ¹³ g, less than 0. 22 ng at 1.0×10 ¹³ g. or less than 0.2 ng per 1.0×10 ¹³ g. or any concentration within the range limited by these values of bovine serum albumin (BSA). In one embodiment, the content of benzonase in the specified pharmaceutical composition is less than 0.2 ng per 1.0 x 10 ¹³ g., less than 0.1 ng per 1.0 x 10 ¹³ g., less than 0.09 ng at 1.0×10 ¹³ g.v., less than 0.08 ng at 1.0×10 ¹³ g.v. or any concentration within the range limited by these values. In one embodiment, the content of poloxamer 188 in the specified pharmaceutical composition is about 10-150 ppm, about 15-100 ppm, or about 20-80 ppm. In one embodiment, the cesium content of said pharmaceutical composition is less than 50 μg/g (ppm), less than 30 μg/g (ppm), or less than 20 μg/g (ppm), or any concentration within the range limited to these values.

In one embodiment, the total impurity content of the pharmaceutical composition disclosed herein is, for example, as determined by SDS-PAGE, less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less 4%, less than 3%, less than 2%, or any percentage within the range limited by these values. In one embodiment, the overall purity, for example, as determined by SDS-PAGE, is greater than 90%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or any percentage value within the range limited by these values. In one embodiment of the pharmaceutical composition, the content of none of the individual unnamed related impurities, for example, as determined by SDS-PAGE, is greater than 5%, greater than 4%, greater than 3%, or greater than 2%, or any percentage within a range limited these values. In one embodiment, the pharmaceutical composition provides a percentage of filled capsids relative to the total number of capsids (e.g., peak 1+peak 2 as measured by analytical ultracentrifugation) of greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89 %, more than 90%, more than 91%, more than 91.9%, more than 92%, more than 93%, or any percentage within the range limited by these values. In one embodiment of the pharmaceutical composition, the percentage of capsids filled, measured in peak 1 by analytical ultracentrifugation, is 20-80%, 25-75%, 30-75%, 35-75%, or 37.4-70.3%. In one embodiment of the pharmaceutical composition, the percentage of capsids filled, measured in peak 2 by analytical ultracentrifugation, is 20-80%, 20-70%, 22-65%, 24-62%, or 24.9-60.1%.

In one embodiment, the pharmaceutical composition disclosed herein contains a genomic titer of 1.0-5.0 x 10 ¹³ g/mL, 1.2-3.0 x 10 ¹³ g/mL or 1.7-2.3×10 ¹³ g/ml.

In one embodiment, a pharmaceutical composition disclosed herein has a demonstrated bioburden of less than 5 CFU/mL, less than 4 CFU/mL, less than 3 CFU/mL, less than 2 CFU/mL, or less than 1 CFU/mL, or any concentration in range limited by these values. In one embodiment, the amount of endotoxin in accordance with USP, such as USP clause <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.

In one embodiment, the osmolality of the pharmaceutical composition disclosed herein, in accordance with USP, e.g., USP Article <785> (incorporated by reference in its entirety), is 350-450 mOsm/kg, 370-440 mOsm/kg, or 390 -430 mOsm/kg. In one embodiment, the pharmaceutical composition contains less than 1200 particles greater than 25 microns per container, less than 1000 particles greater than 25 microns per container, less than 600 particles greater than 25 microns per container, less than 500 particles, whose size exceeds 25 microns, per container or any value within the range limited by these values. In one embodiment, the pharmaceutical composition contains less than 10,000 particles greater than 10 microns per container, less than 8,000 particles greater than 10 microns per container, or less than 6,000 particles greater than 10 microns per container.

In one embodiment, the pharmaceutical composition is characterized by a genomic titer of 0.5-5.0×10 ¹³ g.v./ml, 1.0-4.0×10 ¹³ g.v./ml, 1.5-3 .0×10 ¹³ g./ml or 1.7-2.3×10 ¹³ g./ml.

In one embodiment, the pharmaceutical composition disclosed herein comprises one or more of the following: less than about 0.09 ng of benzonase per 1.0 x 10 ¹³ g, less than about 30 μg/g (ppm) cesium, approximately 20-80 ppm poloxamer 188, less than approximately 0.22 ng BSA per 1.0 x 10 ¹³ year, less than approximately 6.8 x 10 ⁵ pg of residual plasmid DNA per 1.0 x 10 ¹³ year, less than approximately 1.1 x 10 ⁵ pg of residual hcDNA at 1.0 x 10 ¹³ yr, less than approximately 4 ng rHCP at 1.0 × 10 ¹³ yr, pH 7.7-8.3, approximately 390-430 mOsm/kg, less than approximately 600 particles ≥ 25 µm per container, less than approximately 6000 particles ≥ 10 µm per container, genomic titer of approximately 1.7 x 10 ¹³ -2.3 x 10 ¹³ g .v./ml, infectious titer of approximately 3.9 × 10 ⁸ -8.4 × 10 ¹⁰ IU per 1.0 × 10 ¹³ g., total protein content of approximately 100-300 μg per 1, 0 x 10 ¹³ g.i., median survival of ≥ 24 days in Δ7SMA mice at a viral vector dose of approximately 7.5 x 10 ¹³ g.i.v./kg, relative activity determined by in vitro cell-based assay , comprising 70-130%, and/or less than approximately 5% empty capsids.

In various embodiments, pharmaceutical compositions disclosed herein containing any of the viral particles discussed herein (e.g., AAV SMN, AAV MECP2, or AAV SOD1 viral particles) retain activity in the range of +20%, in the range of +15%, in the range of +10%, or in the range of +5% of that of the reference standard. In some embodiments, activity is measured using a suitable in vitro cell assay or in vivo animal model. For example, the activity or % of functional AAV SMN viral particles can be determined using an animal model of SMA, such as the SMAΔ7 mouse, or a cell-based quantitative assay using a suitable cell line, such as primary neural progenitor cells (NPCs) isolated from the cerebral cortex SMAΔ7 mice. In one embodiment, activity is assessed in comparison to a reference standard using the methods described in Foust et al., Nat. Biotechnol., 28(3), pp. 271-274 (2010). Any suitable reference standard may be used. The activity or % functional AAV MeCP2 can be analyzed using a suitable in vitro cell assay or an in vivo animal model, such as the Mecp2 gene knockout mouse, as described in Guy et al., "Reversal of neurological defects in a mouse model of Rett syndrome." Science, 315(5815):1143-7. The activity or % functional AAV SOD1 can be analyzed using a suitable in vitro cell assay or an in vivo animal model, such as the SOD1 mutant mouse, as described in Gurney et al., “Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation." Science, 264(5166):1772-5. In one embodiment, the pharmaceutical composition has in vivo activity as determined by the median survival value in an SMAΔ7 mouse dosed at 7.5 x 10 ¹³ g/kg for more than 15 days, more than 20 days, more than 22 days, or more than 24 days. In one embodiment, the pharmaceutical composition has a relative in vivo activity, when tested using a cell-based assay, of 50-150%, 60-140%, or 70-130% relative to the reference standard and/or appropriate control.

In one embodiment, the intravenous ("IV") formulation has a pH of 7.5 to 8.5, a genomic titer of about 1 to 8×10 ¹³ viral vector genomes/ml (g.v./ml), or 2 ×10 ¹³ g./ml to 6 × 10 ¹³ g./ml and optionally an osmolality of 384-448 mOsm/kg. In one embodiment, the IV formulation contains MgCl ₂ , NaCl, Pluronic F68 in Tris buffer at pH 8.0.

In one embodiment, for intravenous administration, an AAV-9-based vector carrying an SMN transgene is administered under sterile conditions in a suitable setting (eg, interventional suite, operating room, dedicated treatment room) in a single dose through a venous catheter inserted into a peripheral vein of the limb ( arms or legs) at the indicated dose and infuse slowly over approximately 30-60 minutes,

In another embodiment, the present invention provided herein provides compositions and methods for delivering a polynucleotide to the central nervous system of a patient in need thereof, comprising intrathecal (“IT”) delivery of rAAV9 and a nonionic low-osmolarity contrast agent to the patient, wherein rAAV9 contains a self-complementary genome containing a polynucleotide. The polynucleotide is delivered to, for example, the brain, spinal cord, glial cells, astrocyte and/or lower motor neuron. A non-ionic, low-osmolar contrast agent is, for example, iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol or ioxilan. In some embodiments, the polynucleotide is a survival motor neuron (SMN) protein. In one embodiment, the contrast agent is iohexol, such as iohexol 180 (sold as Omnipack 180 containing 388 mg iohexol, equivalent to 180 mg organic iodine per ml).

In one embodiment, for IT administration, the scAAV9-based vector carrying the SMN transgene is diluted in saline and premixed with a suitable hyperbaric contrast medium approved and labeled for pediatric use for radiographic injection monitoring by intrathecal lumbar injection (such as Omnipaque 180 ). The total volume of the aqueous composition containing the AAV-9-based vector carrying the SMN transgene, plus contrast medium and/or saline, will not exceed 5 ml. The contrast agent and the scAAV-9-based vector carrying the SMN transgene can be co-formulated, co-packaged, or packaged and delivered separately to the patient care center.

Patients receive the scAAV-9 vector carrying the SMN transgene via intrathecal injection under sterile conditions in the PICU or other appropriate setting (eg, intervention room, operating room, dedicated procedure room) with immediate access to acute critical care care . In situ, an atraumatic needle inserted beveled parallel to the dural fibers can be used as this has been shown to significantly reduce dural damage and therefore reduce the risk of CSF leakage after lumbar puncture (Ebinger et al., “Headache and Backache”). After Lumbar Puncture in Children and Adolescents: A Prospective Study." Pediatrics, 113(6):1588-1592; Kiechl-Kohlendorfer et al., "Cerebrospinal Fluid Leakage After Lumbar Puncture in Neonates: Incidence and Sonographic Appearance." American Journal of Roentgenology, 181(1):231-234), including in children.

Sedation/anesthesia is recommended for all patients receiving IT injections. The method and medications will be at the discretion of the local anesthesiologist, but should include a sufficient degree of sedation or anxiolysis to provide analgesia and immobilization for positioning in the Trendelenburg position during and after the procedure. Patients will be positioned in a 30° downward tilted Trendelenburg position for 15 minutes after IT therapy is administered to improve distribution to the cervical spine and brain.

Patients are placed in the lateral decubitus position and a catheter with a stylet is inserted by lumbar puncture into the interspinous space L3-L4 or L4-L5 into the subarachnoid space. Subarachnoid catheterization is confirmed by the flow of clear cerebrospinal fluid (CSF) from the catheter. CSF will be removed and disposed of in accordance with facility policies. The scAAV-9-based vector carrying the SMN transgene, in a pre-mixed contrast solution, is injected directly into the subarachnoid space.

In one embodiment, the present invention provides a method of treating a neurological disease in a patient in need thereof, comprising intravenous or intrathecal delivery of a pharmaceutical composition disclosed herein, wherein the parvovirus comprises a self-complementary rAAV9 genome, wherein the engineered transgene comprises an SMN polynucleotide, and wherein the disease is SMA.

In another embodiment, the present invention provides a method of treating a neurological disease in a patient in need thereof, comprising intrathecal delivery of a pharmaceutical composition disclosed herein with a contrast agent, wherein the parvovirus comprises a self-complementary rAAV9 genome, wherein the engineered transgene comprises an SMN polynucleotide, wherein the disease represents is SMA, and where the contrast agent is Omnipaque 180.

In another embodiment, the present invention provides a method of treating SMA type II, III or IV in a patient in need thereof, comprising intrathecal delivery of a pharmaceutical composition disclosed herein with a contrast agent, wherein the parvovirus comprises a self-complementary rAAV9 genome, wherein the engineered transgene comprises a polynucleotide SMN, where the disease is SMA, and where the contrast agent is Omnipaque 180.

In another embodiment, the present invention provides a method of treating SMA type I in a patient in need thereof, comprising intravenous delivery of a pharmaceutical composition disclosed herein, wherein the parvovirus comprises a self-complementary rAAV9 genome, and wherein the engineered transgene comprises an SMN polynucleotide. In some embodiments, the patient is 0-9 months old. In some embodiments, the patient is 0-6 months old. In other embodiments, the pediatric patient weighs no more than about 8 kg. In some embodiments, the pediatric patient weighs no more than about 8.5 kg or less. In some embodiments, the pediatric patient weighs approximately 2.6 kg or more.

In another embodiment, the present invention provides a kit for the treatment of SMA type I in a patient in need thereof, comprising intravenous administration of a pharmaceutical composition disclosed herein contained in vials. In some embodiments, the patient's weight is measured and the dose is calculated based on the patient's weight.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention relates.

As used herein, the singular form of a word also includes the plural form of a word unless the context clearly requires otherwise; By way of example, the singular and plural forms are to be understood as singular or plural, and the term “or” is to be understood as inclusive. By way of example, "element" means one element or more elements.

Throughout the specification, a word such as “comprising” or variations such as “comprises” will be understood to include said element, integer, stage or group of elements, integers or stages without excluding any other element, integer, stage or group elements, integers or stages. Throughout the specification, words such as “consisting of” or variations such as “consists of” will be understood to mean the inclusion of the specified element, integer, stage or group of elements, integers or stages, and the exclusion of any other element, integer, stages or groups of elements, integers or stages. Throughout the specification, words such as “essentially consisting of” or variations such as “essentially consisting of” will be understood to include said element, integer or stage, or group of elements, integers or stages and any other element, an integer or step, or group of elements, integers or steps that do not significantly affect the essential and novel features of the present invention and/or the claims.

"Approximately" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0. 05% or 0.01% of the declared value. When used in relation to a percentage, "about" can be understood as within ± 1% (for example, "about 5%" can be understood as within 4-6%) or ± 0.5% (for example, "about 5%" can be understood as within 4.5% - 5.5%). Unless the context otherwise requires, all numerical values given herein are modified by the term “approximately.”

Although methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. References cited herein are not considered to be prior art with respect to the claimed invention. In the event of a conflict, this description, including definitions, will control. Moreover, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

The various objects and advantages, as well as a more complete understanding of the present invention, will be apparent and better understood by reference to the following detailed description and the accompanying claims taken in conjunction with the accompanying drawings, which set forth as follows.

Fig. 1. Maps of plasmids pSMN, pHELP and pAAV.

In fig. Figure 1A shows a map of the pSMN plasmid. pSMN is a plasmid encoding information for a recombinant self-complementary AAV DNA genome that mediates cDNA expression for human motor neuron survival protein (SMN) under the control of a hybrid chicken beta-actin promoter with a cytomegalovirus (CMV) immediate-early 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. The expression cassette (CMV-CB-SV40-SMN-BGHpA) is flanked by inverted terminal repeats (ITRs) derived from AAV2. The left ITR is modified to preferentially package self-complementary AAV genomes. The regions between the ITRs, including the ITRs themselves, are co-packaged into recombinant AAV9 capsids during drug formulation of the present invention. Key components of pSMN that are not intended for packaging into recombinant AAV genomes include the open reading frame encoding kanamycin resistance (KanR) and origin of replication (ori) derived from pUC. The ori and KanR regions are useful for producing plasmids.

In fig. Figure 1B shows the plasmid map of the pHELP plasmid. The pHELP plasmid contains trans-acting adenovirus components necessary for the production of recombinant adeno-associated viruses. Plasmid pHELP contains regions of the adenovirus genome that provide factors that are important for AAV replication, namely E2A, E4 and VA RNA. The adenovirus E1 functions involved in rAVV replication are provided by transfection of line 293 host cells. However, the pHELP plasmid does not contain other replication-responsive or adenovirus structural genes. The adenovirus sequences present on this plasmid constitute only ∼28% (9280/35938) of the adenovirus genome and do not contain cis -elements critical for replication, such as inverted terminal repeats. Therefore, it is expected that no infectious adenovirus will be produced in such a production system.

In fig. 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 replication protein and capsid protein of the virus, respectively. In the production of recombinant adeno-associated viral vectors, viral ITRs are the only elements used in cis , while viral open reading frames are provided in trans . When using the transient transfection method of adherent HEK293 cells to create AAV, cis/trans roles for various genetic elements are ensured by distributing them into separate plasmids. Plasmid pAAV2/9 contains open reading frames for the AAV2 rep gene and the AAV9 cap gene.

In fig. Figure 2 shows a flow diagram for selecting HEK293 cells for exceptional adhesion and storing them as a preliminary master cell bank (MCB).

In fig. 3 shows a list of components of the cell processing process for selecting HEK293 cells for exceptional adhesion and storage as a preliminary master cell bank (MCB).

In fig. 4 describes the flow diagram of the accumulation process for a medicinal substance.

In fig. 5 describes a flow chart of the isolation and purification process for a drug substance.

In fig. Figure 6 shows inactivation of XMuLV by Tween 20 added for no more than 120 minutes.

In fig. 7 shows inactivation of PRV by Tween 20 added for no more than 120 minutes.

In fig. 8 describes a flow chart for ensuring the proliferation of HEK 293 cells during experiments to determine cell seeding density.

In fig. 9A-E show growth and metabolite formation profiles. 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 four days (12,000 cells/cm ² ) and five days (8,000 cells/cm ² ) after seeding. Biomass was collected from bioreactors eight days (12,000 cells/cm ² ) and nine days (8,000 cells/cm ² ) after sowing. pH and metabolite production data were read daily using Nova BioFlex.

In fig. 10 shows viral genome production as a function of cell seeding density (8,000 or 12,000 cells/cm ² ) and four different transfection time lengths (20 minutes, 1 hour, or 2 hours).

In fig. Figure 11 shows virus titers from samples of intermediates collected at various filtration stages throughout the production process.

In fig. 12 A-B show the levels of viral vector recovery and host cell protein (HCP) removal at the TFF1 stage.

In fig. 13 describes a flow diagram for ensuring the proliferation of HEK 293 cells during experiments to determine cell seeding density.

In fig. 14 AE shows that HEK 293 cells were seeded in duplicate at 8000 cells/ ^cm2 , 9350 cells/ ^cm2 , 10700 cells/ ^cm2 , 12050 cells/ ^cm2 in bioreactors (at pH 7.23, 37.0°C , 55% DO). Cells were transfected with DNA plasmids/PEI (1:1 w/w) five days after plating. pH and metabolite formation analyzes were performed using NOVA BioProfile 400.

In fig. 15 A-B shows the level of drug production at four initial inoculation densities in bioreactors. Comparison of viral titer and amount of vector genome collected per unit surface area.

In fig. 16 shows methods for preparing a phase 1 (method A) and phase 3 trial (method B).

In fig. 17 A-B presents a table that illustrates the results of determining the comparability and homogeneity of the production process - products of method A (phase 1) and method B (phase 3). Method B products have been shown to have additional benefits over Method A.

In fig. 18 shows the comparability of Method A and Method B using a pairwise comparison of Method A (Phase 1, Series NCHAAV9SMN0613) and Method B (Phase 3, Series 600156). Method B products have been shown to have additional benefits over Method A.

In fig. Figure 19 shows the process homogeneity assessment for a pairwise comparison of batches 600156 and 600307 of Method B (Phase 3).

In fig. Figure 20 shows the stability profile of the NCHAAV9SMN0613 NCH series stored under real-time storage conditions at ≤ -60°C for 12 months.

In fig. 21 shows sedimentation coefficients (s×10 ^-13 ) for phase 1 material (NCHAAV9SMN0613) indicating empty capsids (7%) with a sedimentation coefficient of approximately 60×10 ^-13 s, and full capsids with a range of sedimentation coefficients of approximately 80-150 ×10 ^-13 sec.

In fig. 22 shows sedimentation coefficients (s×10 ^-13 ) for phase 3 material (600156) indicating empty capsids (2%) with a sedimentation coefficient of approximately 60×10 ^-13 s, and full capsids with a range of sedimentation coefficients of approximately 80-150 ×10 ^-13 sec.

In fig. 23 shows sedimentation coefficients (s×10 ^-13 ) for phase 3 material (600307) indicating empty capsids (4%) with a sedimentation coefficient of approximately 60×10 ^-13 s, and full capsids with a range of sedimentation coefficients of approximately 80-150 ×10 ^-13 sec.

DETAILED DESCRIPTION

To advance the development of AAV-based gene therapy beyond animal models and into clinical trials and/or therapeutic applications, a scalable process has been developed to produce viral material suitable for use in humans.

In some embodiments, "vector" means any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., that is capable of replication when associated with appropriate control elements and that can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles as well as viral vectors.

In some embodiments, "AAV-based vector" means a vector derived from an adeno-associated virus serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 , AAV-8 and AAV-9. AAV-based vectors may have complete or partial deletion of one or more of the wild-type AAV genes, such as the rep and/or cap genes, but retain functional ITR flanking sequences. Functional ITR sequences are required for AAV virion release, replication, and packaging. Thus, an AAV-based vector is defined herein as including at least those sequences that, in cis, mediate replication and packaging (eg, functional ITRs) of the virus. The ITR need not be wild-type nucleotide sequences, but may be altered, for example by insertion, deletion or substitution of nucleotides, as long as the sequences allow for functional release, replication and packaging. In one embodiment, the vector is an AAV-9-based vector with ITRs derived from AAV-2. Also, by “AAV-based vector” is meant a protein shell or capsid that provides an effective vehicle for delivering the vector nucleic acid into the nucleus of target cells.

In some embodiments, "scAAV" refers to a self-complementary adeno-associated virus (scAAV), which is a viral vector constructed from a naturally occurring adeno-associated virus (AAV) for use in gene therapy. scAAV is called “self-complementary” because the coding region is designed such that it forms an intramolecular double-stranded DNA template.

In some embodiments, the term “vector-associated contaminants” refers to all types of AAV particles other than full-fledged recombinant AAV particles. Vector-associated contaminants include empty AAV capsids (also referred to as “empty units” or “empty particles”) and AAV particles containing polynucleotide sequences other than the desired vector genome (also referred to as “AAV capsid-enclosed nucleic acid contaminants” or “embedded nucleic acid contaminants”). AAV capsid with DNA contaminants").

In some embodiments, “recombinant virus” means a virus that has been genetically altered, for example, by adding or inserting a heterologous nucleic acid construct into the particle. "Recombinant" can be abbreviated as "r", for example, rAAV can refer to recombinant AAV. As used herein, the term "AAV" is intended to cover "recombinant AAV" or "rAAV."

In some embodiments, “AAV virion” means a complete viral particle, such as a wild-type (wt) AAV viral particle (containing the AAV genome as a linear single-stranded nucleic acid associated with an AAV capsid protein shell). In this case, single-stranded AAV nucleic acid molecules of any complementary strand, for example, “sense” or “antisense” strands, can be packaged into any AAV virion, and both strands are equally infectious.

In some embodiments, the terms “recombinant AAV virion,” “rAAV virion,” “AAV vector particle,” “complete capsids,” and “complete particles” are defined herein as an infectious replication-defective virus containing an AAV protein coat enclosing a heterologous nucleotide sequence of interest that is flanked on both sides of the AAV ITR. The rAAV virion is produced in a suitable host cell into which sequences defining an AAV vector, AAV helper functional elements and accessory functional elements have been introduced. Thus, the host cell becomes capable of encoding AAV polypeptides that enable packaging of an AAV-based vector (containing the recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.

In some embodiments, the terms “empty capsid” and “empty particle” refer to an AAV virion that contains an AAV protein coat but lacks all or part of a polynucleotide construct containing a heterologous nucleotide sequence of interest flanked on both sides of the AAV ITR.

The term “host cell” refers to, for example, microorganisms, yeast cells, insect cells and mammalian cells that can be or have been used as recipients of an AAV helper construct, an AAV vector plasmid, a functional element vector or other transferred DNA. The term includes the progeny of the original cell that has been transfected. Thus, as used herein, “host cell” generally refers to a cell that has been transfected with an exogenous DNA sequence. It is known that the offspring of one parent cell may not necessarily be completely identical in morphology or in genomic or complete DNA complement to the original parent due to natural, accidental or intentional mutation.

In another embodiment, the term “AAV helper functional elements” refers to AAV-derived coding sequences that can be expressed to produce AAV gene products, which in turn function in trans to allow productive AAV replication. Thus, AAV helper functional elements include both of the major AAV open reading frames (ORFs), rep and cap . Rep expression products have been shown to have multiple functions including, among others, recognition, binding, and nicking of the AAV DNA origin of replication; DNA helicase activity and modulation of transcription from AAV (or other heterologous) promoters. Cap expression products provide the necessary packaging functional elements. AAV helper functional elements are used herein to complement AAV functional elements in trans that are not present in AAV-based vectors.

In one embodiment, the term “AAV helper construct” refers generally to a nucleic acid molecule that contains nucleotide sequences providing functional AAV elements removed from an AAV-based vector to be used to prepare a transducing vector for delivering a nucleotide sequence of interest. AAV helper constructs are commonly used to provide transient expression of AAV rep and/or cap genes to complement missing AAV functional elements that are required for AAV replication; however, helper constructs lack AAV ITRs and cannot replicate or package themselves. AAV helper constructs can be in the form of a plasmid, phage, transposon, cosmid, virus or virion. A number of AAV helper constructs have been described, such as the widely used plasmids pAAV/Ad and plM29+45, which encode the expression products of Rep and Cap. See, for example, Samulski et al. (1989) J. Virol. 63:3822-3828 and McCarty et al. (1991) J. Virol. 65:2936–2945. A number of other vectors have been described that encode expression products of Rep and/or Cap. See, for example, US Patent Nos. 5,139,941 and 6,376,237.

In another embodiment, the term "transfection" is used to describe the uptake of foreign DNA into a cell, and the cell has been "transfected" if the exogenous DNA has been introduced into the cell through the membrane. A number of transfection techniques are generally known in the prior art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA fragments into suitable host cells.

As used herein, the term “cell line” refers to a population of cells capable of continuous or prolonged growth and division in vitro. It is further known in the art that spontaneous or induced changes may occur in the karyotype during storage or transfer of such clonal populations. Consequently, cells derived from said cell line may not be exactly identical to the ancestral cells or cultures, and said cell line includes such variants. In some embodiments, the terms “HEK293 cells,” “293 cell line,” or grammatical equivalents thereof are used interchangeably herein and refer to the host/packaging cell line used in the methods disclosed herein.

In some embodiments, the term “eluent” may be understood in context to refer to a buffer used to elute a substance. In some embodiments, the term "eluent" may be understood in the context to refer to the eluted substance, for example, the desired product or substance, from a previous purification step, for example, for analysis or further purification.

In some embodiments, the methods described herein are carried out using Good Manufacturing Practices (GMP) and on an industrial scale. GMP is a regulatory practice, such as that used by the Federal Drug Administration (FDA), to ensure the quality of pharmaceutical products. GMP regulations establish control over production processes. Examples of current GMP regulatory requirements are published by the FDA. In some embodiments, the methods described herein employ GMP procedures to produce AAV-based viral vectors on an industrial scale. To date, the production of AAV-based viral vectors for gene therapy on an industrial scale has been challenging due to scalability issues. Thus, in some embodiments, the methods described herein provide the benefit of producing AAV-based viral vectors, for example, in adherent cells, on an industrial scale and at levels of purity sufficient for administration to humans. The term "industrial scale" refers to methods for producing viral vectors in cells on a scale larger than laboratory scale, for example, commercial scale, for example, where the yield is greater than 5×10 ¹⁵ g.v., or greater than 8×10 ¹⁵ g.i. , or more than 1×10 ¹⁶ g. per production batch.

ACCUMULATION PROCESS

In some embodiments, an accumulation process is used to produce an intermediate obtained from a working cell bank, wherein the accumulation process includes the steps of (a) culturing the cells, such as adherent cells, (b) transfecting the cultured cells, such as adherent cells, with three plasmids, (c) collecting an increased number of viral particles from the cells after the culture period, for example, by completely lysing the cells, (d) purifying the viral particles by filtration to remove any intact cells or cellular debris, (e) subjecting the eluent from step (d) to tangential filtration flow and (f) optionally freezing the resulting intermediate preparation of purified viral particles. In some embodiments, the intermediate preparation can be frozen. In other embodiments, the intermediate does not need to be frozen prior to the isolation and purification process. In some embodiments, the AAV produced through the accumulation process disclosed herein is an AAV encoding a shRNA targeting SOD1, an AAV containing a polynucleotide encoding MECP2, or an AAV containing a polynucleotide encoding SMN, as described herein. In some embodiments, the accumulation process is carried out according to GMP and on an industrial scale.

1. Transfection and cell line cultivation

In one aspect, rAAV genomes are disclosed herein. rAAV genomes contain one or more AAV ITRs flanking a polynucleotide encoding a polypeptide (including, but not limited to, the SMN polypeptide) or encoding siRNA, shRNA, antisense and/or miRNA directed to the mutated proteins or their gene regulatory sequences. The polynucleotide is operably linked to DNA sequences that provide transcriptional control, in particular promoter DNA, enhancer DNA and a DNA polyadenylation signal sequence, which function in target cells to form a gene cassette. The gene cassette may also include intronic sequences to facilitate processing of the RNA transcript when expressed in mammalian cells.

In some embodiments, the rAAV genome (e.g., rAAV9) encodes a trophic or protective factor for the treatment of neurodegenerative disorders including, but not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, along with nervous system injury including spinal cord and brain injury/damage, stroke and types of brain cancer. Non-limiting examples of known nervous system growth factors include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotropin-3 (NT-3), neurotropin-4/5 (NT-4/5), neurotropin-6 (NT -6), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), fibroblast family growth factor (eg, FGF 1-15), leukemia inhibitory factor (LIF), some members of the insulin-like growth factor family ( e.g., IGF-1), neuroturins, persefin, bone morphogenetic proteins (BMPs), immunophilins, transforming growth factor (TGF) family of growth factors, neuregulins, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular growth factor family endothelium (for example, VEGF 165), follistatin, Hif1 and others. Also commonly discussed are zinc finger transcription factors that control each of the trophic or protective factors discussed herein. In additional embodiments, methods of modulating neuroimmune function are contemplated, including, but not limited to, inhibiting microglial and astroglial activation through, for example, inhibition of NFkB or NFkB for neuroprotection (dual action of NFkB and related pathways in different cell types) via siRNA, shRNA, antisense or miRNA. In still further embodiments, the rAAV genome (eg, rAAV9) encodes an inhibitor of apoptosis (eg, bcl2, bclxL). Use of rAAV (eg, rAAV9) encoding a trophic factor or spinal cord injury modulating protein, or an axonal growth inhibitor suppressor (eg, Nogo suppressor [Oertle et al., The Journal of Neuroscience, 23(13):5393-5406 (2003) )] is also being considered for the treatment of spinal cord injury.

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

For the treatment of neurodegenerative disorders such as Alzheimer's disease, in some embodiments, methods of increasing acetylcholine production are contemplated. In some embodiments, methods of increasing acetylcholine transferase (ChAT) levels or inhibiting acetylcholinesterase (AchE) activity are contemplated.

The rAAV genome (eg, rAAV9) encodes, in some embodiments, siRNA, shRNA, antisense and/or miRNA for use in methods of reducing mutant Huntington protein (htt) expression for treating a neurodegenerative disorder such as Huntington's disease.

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

In some embodiments, the vectors encode short hairpin RNAs (shRNAs) targeting mutated proteins, such as superoxide dismutase (SOD, e.g., SOD-1) in the case of ALS, or neurotrophic factors, such as GDNF or IGF1, in the case of ALS or disease Parkinson's.

In one embodiment, the methods and materials described herein can be used to treat ALS. ALS is a neurodegenerative disease that results in the progressive loss of motor neurons in the brain and spinal cord, with symptoms including loss of the ability to speak, eat, move and eventually breathe. The disease usually leads to death within 3-5 years of diagnosis. Although the cause of 90-95% of ALS cases is unknown, a subset of ALS is caused by genetic mutations in the superoxide dismutase 1 (SOD1) gene, where the mutation causes a toxic dominant gain of function. Studies in mice show that knocking out SOD1 does not lead to disease, and therefore therapies that reduce levels of mutant SOD1 are thought to provide relief from disease symptoms.

In some embodiments, the AAV-based vector encodes shRNA targeting SOD1 for ALS. An exemplary AAV-based construct, such as scAAV9 encoding shRNA for SOD1, is provided in WO2015031392 and US2016272976, the contents of which are incorporated herein in their entirety. In some embodiments, an AAV-based construct encoding shRNA for SOD1 can be produced using methods disclosed herein. In some embodiments, such AAV-based constructs can be used to treat ALS. In some embodiments, SOD1 AAV exhibits less than 10%, such as less than 7%, 5%, 4%, 3%, 2%, or 1% empty capsids. In some embodiments, AAV SOD1 exhibits low amounts of residual host cell protein, host cell DNA, plasmid DNA, and/or endotoxin, such as the levels contemplated herein for the production and purification of AAV vectors. As used herein, "AVXS-301" is a non-limiting example of a scAAV9-based vector, i.e. containing a polynucleotide (eg, pSOD1sh) encoding an shRNA targeting human SOD1, a modified AAV2 ITR, a human H1 promoter, and an unmodified AAV2 ITR. Modified and unmodified ITRs can be in any orientation (ie, 5' or 3') relative to the shRNA expression cassette targeting human SOD1.

The vector plasmid "pSOD1sh" as used herein contains a polynucleotide encoding a short hairpin RNA (shRNA) targeting the expression of the superoxide dismutase 1 (SOD1) gene, i.e. a shRNA cassette targeting SOD1, where the cassette is flanked by adeno-associated virus inverted terminal repeat (ITR) sequences, eg, to the left and right of the polynucleotide encoding pSOD1sh. In some embodiments, the polynucleotide encoding pSOD1sh is transcribed into a short hairpin RNA that specifically targets human SOD1 mRNA. In some embodiments, the ITR sequences surrounding the polynucleotide encoding pSOD1sh are native, variant, or modified AAV ITR sequences. In some embodiments, the at least one ITR sequence is a native, variant, or modified AAV2 ITR sequence. In some embodiments, the ITRs are flanked by a polynucleotide encoding pSOD1sh. In some embodiments, both of the two ITR sequences are native, variant, or modified AAV2 ITR sequences. In some embodiments, the left ITR is a modified AAV2 ITR sequence that allows for the production of self-complementary genomes, and the right ITR is a native AAV2 ITR sequence. In some embodiments, the right ITR is a modified AAV2 ITR sequence that allows for the production of self-complementary genomes, and the left ITR is a native AAV2 ITR sequence. In some embodiments, the pSOD1sh vector further comprises a human H1 RNA promoter segment, such as those described by Myslinksi. Myslinkski et al. "An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene." Nucleic Acids Research, 29(12):2502–2509. In some embodiments, the pSOD1sh vector further comprises a unique spacer sequence derived from random plasmid backbone segments to increase the size of the expression cassette. In some embodiments, the pSOD1sh vector contains a polynucleotide encoding a shRNA targeting human SOD1, a modified AAV2 ITR, a human H1 promoter, and an unmodified AAV2 ITR.

In one embodiment, the methods and materials described herein can be used to treat neurodevelopmental disorders such as Rett syndrome. Rett syndrome is a rare neurological disorder first identified in infancy, resulting from mutations in the MECP2 gene on the X chromosome in 90-95% of cases. Ruthie et al., "Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2." Nature Genetics 23:185–188. Boys who have only one copy of the X chromosome typically die soon after birth, while girls who have two copies of the X chromosome usually have one functional copy of the gene. They begin to develop symptoms between 6 and 18 months of age, with typical symptoms including twisting or squeezing, flapping, rubbing, washing, or hand-to-mouth movements. The disease is progressive with significant disability, which may include autism-like behavior, breathing difficulties, feeding and swallowing difficulties, growth retardation and seizures. There are 200 known MECP2 gene mutations, and depending on the level of X inactivation and dose compensation, disease severity varies widely from patient to patient. Studies in mice show that the MECP2 mutation does not cause neuronal death, suggesting that it is not a neurodegenerative disorder. Guy et al, "Reversal of Neurological Defects in a Mouse Model of Rett Syndrome." Science , 315(5815)"1143-1147.

For embodiments related to Rett syndrome, the rAAV genome (eg, rAAV9) may encode, for example, methylcytosine binding protein 2 (MeCP2). An exemplary AAV construct, such as scAAV9, containing a polynucleotide encoding MeCP2 is provided in US Pat. No. 9,415,121, the contents of which are incorporated herein in its entirety. In some embodiments, an AAV-based construct comprising a polynucleotide encoding MeCP2 can be produced using the methods disclosed herein. In some embodiments, such AAV-based constructs can be used to treat Rett syndrome. In some embodiments, MeCP2 AAV exhibits less than 10%, such as less than 7%, 5%, 4%, 3%, 2%, or 1% empty capsids. In some embodiments, MeCP2 AAV exhibits low amounts of residual host cell protein, host cell DNA, plasmid DNA, and/or endotoxin, such as the levels discussed herein for the production and purification of AAV vectors.

As used herein, "AVXS-201" is a non-limiting example of a scAAV9-based vector, i.e. containing a polynucleotide (eg, pMECP2) containing a MECP2 cDNA expression cassette, a modified AAV2 ITR, a mouse Mecp2 promoter, a modified SV40 intron, a minimal polyadenylation signal, and an unmodified AAV2 ITR. Modified and unmodified ITRs can be in any orientation (ie, 5' or 3') relative to the MECP2 cDNA expression cassette.

The vector plasmid "pMECP2" as used herein contains a polynucleotide encoding the MECP2 protein, a modified AAV2 ITR, a mouse Mecp2 promoter, a modified SV40 intron, a minimal polyadenylation signal, and an unmodified AAV2 ITR. In some embodiments, pMECP2 is a vector construct comprising a polynucleotide encoding a MECP2 protein, i.e. a MECP2 cDNA expression cassette, where the cassette is flanked by adeno-associated virus inverted terminal repeat (ITR) sequences, for example, to the “left” and “to the right” of the polynucleotide encoding the MECP2 gene. In some embodiments, the polynucleotide encoding MECP2 is a human MECP2 sequence, such as the naturally occurring human MECP2 sequence or isoforms, variants, or mutants thereof. In some embodiments, the ITR sequences are native, variant, or modified AAV ITR sequences. In some embodiments, the at least one ITR sequence is a native, variant, or modified AAV2 ITR sequence. In some embodiments, both of the two ITR sequences are native, variant, or modified AAV2 ITR sequences. In some embodiments, the left ITR is a modified AAV2 ITR sequence that allows for the production of self-complementary genomes, and the right ITR is a native AAV2 ITR sequence. In some embodiments, the right ITR is a modified AAV2 ITR sequence that allows for the production of self-complementary genomes, and the left ITR is a native AAV2 ITR sequence. In some embodiments, the pMECP2 vector further comprises a segment, but not the entire, mouse Mecp2 promoter. In some embodiments, the pMECP2 vector further comprises a simian virus 40 (SV40) intron. In some embodiments, the pMECP2 vector further comprises a minimal polyadenylation signal, for example, as defined by Levitt et al., "Definition of an efficient synthetic poly(A) site." Genes & Development 3:1019–1025.

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

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

In some embodiments, the pSMN vector may comprise an SMN cDNA expression cassette, a modified AAV2 ITR, a chicken beta-actin (CB) promoter, a cytomegalovirus (CMV) immediate-early enhancer, a modified SV40 late 16s intron, a bovine growth hormone (BGH) polyadenylation signal ) and unmodified AAV2 ITR. Modified and unmodified ITRs can be in any orientation (ie, 5' or 3') relative to the SMN cDNA expression cassette.

In some embodiments, for example, during the production processes described herein, the vector construct sequence is incorporated into a capsid, such as AAV9 virions. In such embodiments, the implementation encapsids a non-replicating recombinant AAV9 capable of delivering a stable functional transgene, for example, a fully functional human SMN transgene, a MECP2 transgene, or an shRNA targeting SOD1. In some embodiments, the capsid is composed of 60 viral proteins (VP1, VP2, VP3), for example, in a 1:1:10 ratio, resulting from alternative splicing such that VP2 and VP3 are two truncated forms of VP1, all sharing C-terminal sequences. In some embodiments, the product of the manufacturing process, such as a drug product, may comprise a non-replicating recombinant AAV9 capsid to deliver a stable, fully functional human SMN transgene, MECP2 transgene, or shRNA targeting SOD1. In some embodiments, the capsid is composed of 60 viral proteins (VP1, VP2, VP3) in a 1:1:10 ratio resulting from alternative splicing such that VP2 and VP3 are two truncated forms of VP1, all sharing the same C-terminal sequences.

In some embodiments, the amount of functional viral vectors is determined by % functional g.v./ml measured using a suitable in vitro cell assay or in vivo animal model. For example, % functional AAV SMN can be analyzed by relative activity using an animal model of SMA, such as the SMAΔ7 mouse, or a cell-based quantitative assay using a suitable cell line, such as primary neural progenitor cells (NPCs) isolated from the cerebral cortex of SMAΔ7 mice . % functional MeCP2 AAV can be analyzed using a suitable in vitro cell assay or in vivo animal model, eg Mecp2 knockout mouse. % functional AAV SOD1 can be analyzed using a suitable in vitro cell assay or in vivo animal model, such as the SOD1 mutant mouse.

The DNA sequence of an exemplary vector construct, such as AVXS-101, is described in Table 1.

Table 1. List of DNA sequence components of the AVXS-101 vector construct (all nucleotide positions of the beginning and end are indicated in relation to SEQ ID NO: 1).

Start position End position Size (nucleotides) Description Non-limiting description of potential benefits "Left" mutated AAV2 ITR 1 106 106 Modification of the left-handed ITR by deletion of the terminal resolution site to promote hairpin formation from the genome Without being limited by theory, a given mutated ITR may provide a second generation self-complementary vector to maximize vector efficiency while providing lower systemic doses CMV enhancer/CB promoter 153 432 280 Part of the CMV immediate-early enhancer Without being limited by theory, this may allow constitutive expression of SMN at a high level 439 704 266 CB core promoter Intron SV40 774 870 97 Intron from SV40 (to enhance accumulation of stable levels of mRNA for translation) Without being limited by theory, this may provide increased gene expression Human cDNA SMN 1003 1887 885 Modified from accession number NM_017411 in Genbank Without being limited by theory, this may allow expression of full-length SMN protein Termination signal Poly A BGH 1973 2204 232 Poly A BGH Signal Without being limited by theory, this may provide Poly A mRNA SMN (transcription termination signal) for efficient gene expression at a high level "Right" ITR AAV2 2217 2359 143 Unmodified ITR AAV2 Without being limited by theory, a given AAV2 ITR in cis can mediate both viral DNA replication and vector packaging based on the AAV genome.

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

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg 50

ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg 100 gagtggaatt cacgcgtgga tctgaattca attcacgcgt ggtacctctg 150 gtcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 200 cccccgccca tt gacgtcaa taatgacgta tgttcccata gtaacgccaa 250 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 300 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 350 cgtcaatgac ggtaaatggc ccgcctggca ttatg cccag tacatgacct 400 tatgggactt tcctacttgg cagtacatct actcgaggcc acgttctgct 450 tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt 500 attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggggcg 550 cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 600 gaggtgcggc ggcagccaat cagagcggcg cg ctccgaaa gtttcctttt 650 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg 700 ggcgggagcg ggatcagcca ccgcggtggc ggcctagagt cgacgaggaa 750 ctgaaaaacc agaaagttaa ctggtaagtt tt gtcttttatt 800 tcaggtcccg gatccggtgg tggtgcaaat caaagaactg ctcctcagtg 850 gatgttgcct ttacttctag gcctgtacgg aagtgttact tctgctctaa 900 aagctgcgga attgtacccg cggccgatcc accggtccgg aattcccggg 950 atatcgtcga cccacgcgtc cgggccccac gctgcgcacc cgcgggtttg 1000 ctatggcgat gagcagcggc ggcagtggtg gcggcgtccc ggagca ggag 1050 gattccgtgc tgttccggcg cggcacaggc cagagcgatg attctgacat 1100 ttgggatgat acagcactga taaaagcata tgataaagct gtggcttcat 1150 ttaagcatgc tctaaagaat ggtgacattt gtgaaacttc gggtaaacca 1200 ctaaaagaaa acctgctaag aagaataaaa gccaaaagaa 1250 gaatactgca gcttccttac aacagtggaa agttggggac aaatgttctg 1300 ccatttggtc agaagacggt tgcatttacc cagctaccat tgcttcaatt 1350 gattttaaga gagaaacctg tgttgtggtt tacactggat atggaaatag 1400 agaggagcaa aatctgtccg atctactttc cccaatctgt gaagtagcta 1450 ataatataga acagaatgct caagagaatg a aaatgaaag ccaagtttca 1500 acagatgaaa gtgagaactc caggtctcct ggaaataaat cagataacat 1550 caagcccaaa tctgctccat ggaactcttt tctccctcca ccacccccca 1600 tgccaggggcc aagactggga ccaggaaagc caggtctaaa attcaatggc 1650 accgc caccgccacc accaccaccc cacttactat catgctggct 1700 gcctccatt ccttctggac caccaataat tcccccacca cctcccatat 1750 gtccagattc tcttgatgat gctgatgctt tgggaagtat gttaatttca 1800 tggtacatga gtggctatca tactggctat tatatgggtt ttagacaaaa 1850 tcaaaaagaa ggaaggtgct cacattcctt aaattaagga gaaatgct gg 1900 catagagcag cactaaatga caccactaaa gaaacgatca gacagatcta 1950 gaaagcttat cgataccgtc gactagagct cgctgatcag cctcgactgt 2000 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 2050 tgacc ctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 2100 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 2150 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg 2200 ggggagagatc gatctgagga acccctagtg atggagttgg ccactccctc 2250 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 2300 gc ccgggctt tgcccggggcg gcctcagtga gcgagcgagc gcgcagagag 2350 ggagtggcc 2359 (SEQ ID NO: 1).

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

MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNGDICETSGKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASIDFKRETCVVVYTGYGNREEQNLSDLLSPICEVANNIEQNAQENENESQVSTDESENSRSPGNKSDNIKPKSAPWNSFL PPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSCWLPPFPSGPPIIPPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRCSHSLN (SEQ ID NO: 2).

In some embodiments, the AAV capsid proteins are: VP1, VP2, VP3, are obtained from the same transcript. They have alternative start sites but a common carboxyl end. Below, the specific amino acid sequences of VP1 are shown in black and in bold. Amino acid sequences common to VP1 and VP2 are underlined and highlighteditalics. Amino acids common to all three capsid proteins are shown in bold anditalics.

1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD

61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ

121 AKKRLLEPLG LVEEAAK TAP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE

181 SVPDPQPIGE PPAAPSGVGS LT MASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI

241 TTSTRTWALP TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR

301 LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY QLPYVLGSAH

361 EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENV

421 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT INGSGQNQQT LKFSVAGPSN MAVQGRNYIP

481 GPSYRQQRVS TTVTQNNNSE FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS

541 LIFGKQGTGR DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG

601 ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT

661 AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VEFAVNTEGV

721 YSEPRPIGTR YLTRNL (SEQ ID NO: 3).

In one embodiment, the AAV capsid proteins are derived from a transcript encoding the amino acid sequence set forth in SEQ ID NO: 3.

In another aspect, DNA plasmids containing rAAV genomes are disclosed herein. DNA plasmids are transferred into cells that can be infected with an AAV helper virus (eg, adenovirus, E1 deletion adenovirus, or herpesvirus) to assemble the rAAV genome with AAV9 capsid proteins to produce infectious virus particles. Techniques for producing rAAV particles in which the AAV genome to be packaged, rep and cap genes, and functional elements of the helper virus are delivered into a cell are standard in the art. In some embodiments, rAAV production involves the following components present in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and viral functional elements helper The production of pseudotyped rAAV is disclosed, for example, in WO 01/83692, which is incorporated herein by reference in its entirety. In various embodiments, AAV capsid proteins can be modified to facilitate delivery of the recombinant vector. Modifications of capsid proteins are well known in the art. See, for example, US 2005/0053922 and US 2009/0202490, the disclosures of which are incorporated herein by reference in their entirety.

General principles for the production of rAAV are discussed, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, CUM Topics in Microbial. and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hennonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. 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. 5173414; WO 95/13365 and related US Patent No. 5658776; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132; US Patent No. 5786211; US Patent No. 5,871,982 and US Patent No. 6,258,595. The foregoing documents are hereby incorporated herein by reference in their entirety, with particular reference to those portions of the documents that relate to the production of rAAV.

An exemplary method for producing a packaging cell is to create a cell line that stably expresses the necessary components for the production of AAV particles. For example, a plasmid (or multiple plasmids) containing the rAAV genome without the AAV rep and cap genes, the AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, is integrated into the genome of the cell. AAV genomes have been introduced into bacterial plasmids through procedures such as GC tail extension (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or by direct blunt end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as an adenovirus. The advantages of this method are that the cells are selectable and suitable for large-scale production of rAAV. Other examples of suitable methods for introducing rAAV genomes and/or rep and cap genes into packaging cells use adenovirus or baculovirus rather than plasmids.

The present invention presented herein thus provides, in various embodiments, packaging cells that produce infectious rAAV. Packaging cells can be non-adherent cells, suspension cultured cells or adherent cells. In one embodiment, any suitable packaging cell line may be used, such as HeLa cells, HEK 293 cells, and PerC.6 cells (293 related line). In one embodiment, the cell line is HEK 293 cells.

To increase the yield of viral vector production, adherent cells can be cultured and selected based on improved adhesiveness to culture mats. In some embodiments, during subsequent seeding steps in the bioreactor, transfection efficiency is improved and cell number is increased. During subculture, cells can be separated from the surface of the cell culture by methods known in the art. For example, cells can be lifted by scraping or by incubation in a solution containing proteases. In an illustrative embodiment, HEK293 cells can be washed with PBS and separated with trypsin for ~2 minutes at room temperature. Separation can be stopped by adding culture media containing serum, and cell clumps can be separated by repeated pipetting of the suspension. The cell suspension can then be pelleted and the recovered pellet can be resuspended in suitable complete culture media. The cells can then be seeded into new cell culture chambers and allowed to adhere. Cells that do not adhere to the surface after a period of time can be removed by gentle aspiration of the cell culture media before completely replacing the cell culture media with growth media. In some embodiments, the period of time during which cell adherence is achieved may be about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, or about 7 hours. Once cell proliferation has been ensured, the process can be repeated to increase the fraction of cells that adhere strongly to the culture mats. In some embodiments, the process is repeated at least 2 times, at least 3 times, at least 4 times, at least 5 times, or any suitable number of times. In an exemplary embodiment, HEK293 cells are seeded into 75 cm ² surface area mats, allowed to adhere for 4 hours in a 37°C incubator before removing low adherent cells by aspiration and replacing cell culture media. In an illustrative embodiment, the process of selecting highly adhesive cells is repeated over three subcultures of the cell culture.

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

In another aspect, an rAAV is provided, such as rAAV9 called "SMN rAAV". In some embodiments, the rAAV SMN genome comprises in sequence a first AAV2 ITR, a chicken β-actin promoter with a cytomegalovirus enhancer, an SV40 intron, a polynucleotide encoding SMN, a polyadenylation signal sequence from bovine growth hormone, and a second AAV2 ITR. In some embodiments, the polynucleotide encoding SMN is a human SMN gene, such as those listed in GenBank accession number MN_000344.2, listed in GenBank accession number NM_017411, or derived from them, or any other suitable isoform of human SMN. An exemplary SMN sequence comprises the sequence:

1 CCACAAATGT GGGAGGGCGA TAACCACTCG TAGAAAGCGT GAGAAGTTAC TACAAGCGGT

61 CCTCCCGGCC ACCGTACTGT TCCGCTCCCA GAAGCCCCGG GCGGCGGAAG TCGTCACTCT

121 TAAGAAGGGA CGGGGCCCCA CGCTGCGCAC CCGCGGGTTT GCTATGGCGA TGAGCAGCGG

181 CGGCAGTGGT GGCGGCGTCC CGGAGCAGGA GGATTCCGTG CTGTTCCGGC GCGGCACAGG

241 CCAGAGCGAT GATTCTGACA TTTGGGATGA TACAGCACTG ATAAAAGCAT ATGATAAAGC

301 TGTGGCTTCA TTTAAGCATG CTCTAAAGAA TGGTGACATT TGTGAAACTT CGGGTAAACC

361 AAAAACCACA CCTAAAAGAA AACCTGCTAA GAAGAATAAA AGCCAAAAGA AGAATACTGC

421 AGCTTCCTTA CAACAGTGGA AAGTTGGGGA CAAATGTTCT GCCATTTGGT CAGAAGACGG

481 TTGCATTTAC CCAGCTACCA TTGCTTCAAT TGATTTTAAG AGAGAAACCT GTGTTGTGGT

541 TTACACTGGA TATGGAAATA GAGAGGAGCA AAATCTGTCC GATCTACTTT CCCCAATCTG

601 TGAAGTAGCT AATAATATAG AACAGAATGC TCAAGAGAAT GAAAATGAAA GCCAAGTTTC

661 AACAGATGAA AGTGAGAACT CCAGGTCTCC TGGAAATAAA TCAGATAACA TCAAGCCCAA

721 ATCTGCTCCA TGGAACTCTT TTCTCCCTCC ACCACCCCCC ATGCCAGGGC CAAGACTGGG

781 ACCAGGAAAG CCAGGTCTAA AATTCAATGG CCCACCACCG CCACCGCCAC CACCACCACC

841 CCACTTACTA TCATGCTGGC TGCCTCCATT TCCTTCTGGA CCACCAATAA TTCCCCCACC

901 ACCTCCCATA TGTCCAGATT CTCTTGATGA TGCTGATGCT TTGGGAAGTA TGTTAATTTC

961 ATGGTACATG AGTGGCTATC ATACTGGCTA TTATATGGGT TTCAGACAAA ATCAAAAAGA

1021 AGGAAGGTGC TCACATTCCT TAAATTAAGG AGAAATGCTG GCATAGAGCA GCACTAAATG

1081 ACACCACTAA AGAAACGATC AGACAGATCT GGAATGTGAA GCGTTATAGA AGATAACTGG

1141 CCTCATTTCT TCAAAATATC AAGTGTTGGG AAAGAAAAAA GGAAGTGGAA TGGGTAACTC

1201 TTCTTGATTA AAAGTTATGT AATAACCAAA TGCAATGTGA AATATTTTAC TGGACTCTTT

1261 TGAAAAACCA TCTGTAAAAG ACTGGGGTGG GGGTGGGAGG CCAGCACGGT GGTGAGGCAG

1321 TTGAGAAAAT TTGAATGTGG ATTAGATTTT GAATGATATT GGATAATTAT TGGTAATTTT

1381 ATGGCCTGTG AGAAGGGTGT TGTAGTTTAT AAAAGACTGT CTTAATTTGC ATACTTAAGC

1441 ATTTAGGAAT GAAGTGTTAG AGTGTCTTAA AATGTTTCAA ATGGTTTAAC AAAATGTATG

1501 TGAGGCGTAT GTGGCAAAAT GTTACAGAAT CTAACTGGTG GACATGGCTG TTCATTGTAC

1561 TGTTTTTTTC TATCTTCTAT ATGTTTAAAA GTATATAATA AAAATATTTA ATTTTTTTTT

1621 A (SEQ ID NO: 4).

Conservative nucleotide substitutions in SMN DNA are also considered (for example, the change from guanine to adenine at position 625 listed in GenBank under accession number NM_000344.2). In some embodiments, the genome lacks rep and cap AAV DNA, thus, there is no rep or cap AAV DNA between the ITRs of the genome. Subject SMN polypeptides include, but are not limited to, the human SMN1 polypeptide listed in the NCBI Protein Database under NP_000335.1. In embodiments, the SMN DNA comprises a polynucleotide that encodes a human SMN polypeptide (e.g., human SMN protein identified in Uniprot under accession number Q16637, isoform 1 (Q16637-1)). Also discussed is plastin-3 polypeptide modifying SMN1 (PLS3) [Oprea et al., Science 320(5875): 524-527 (2008)]. Sequences encoding other polypeptides may be replaced with SMN DNA.

Before transfection, cells are expanded in suitable culture media, in mattresses or in a suitable bioreactor, or both. In some embodiments, cells can be propagated in bioreactors that provide continuous circulation of cell culture media. In one embodiment, cells are propagated in iCELLis bioreactors with a surface area of 200 m ² , 333 m ² or 500 m ² . One culture medium is DMEM with 5-10% FBS, 4.5 g/L glucose, 4 mM L-glutamine. In some embodiments, adherent cells are added to the media in a recirculating media bag and circulated through the bioreactor. In some embodiments, the cell culture medium or any other medium is continuously recirculated through the bioreactor using a peristaltic pump. Cells can be seeded at suitable densities into mats or bioreactors for culture and transfection. The seeding density may depend on the cell type and the amount of time before transfection. In some embodiments, cells are seeded at approximately 8,000-16,000 cells/cm ² . In one embodiment, HEK293 cells are seeded at 8000-12000 cells/cm ² .

Suitable methods for transducing and reintroducing transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with cells, for example, in suitable media and screening for those cells that carry DNA of interest using traditional techniques such as Southern blot and/or PCR variants , or using selectable markers.

In some embodiments, the packaging cell line is transfected with three plasmids: a plasmid encoding or containing a vector sequence to be packaged into an AAV-based vector (eg, pSMN, pMECP2 transgene, or pSOD1sh), pHELP, and pAAV2/9. Transfection can be accomplished using any of the techniques known in the art, including, but not limited to, electroporation, lipofection, for example, lipofectamine, cationic polymers and cationic lipids. Any suitable transfection media may be used. In one embodiment of the transfection method, adherent human embryonic kidney cells (HEK293) are transfected by co-precipitation of three DNA plasmids and polyethylenimine (PEI). In one embodiment, the scAAV9.CB.SMN vector (an AAV9-based self-complementary viral vector containing a CB promoter and a polynucleotide encoding SMN) is produced using three DNA plasmids to transfect HEK293 adherent cells by co-precipitation with PEI in a large-scale adherent cell bioreactor. In one embodiment, the DMEM growth medium used for cell propagation is replaced with a modified DMEM transfection medium. This medium is formulated without calcium and L-glutamine. In one embodiment, the transfection medium is DMEM without FBS, without calcium, without L-glutamine and 4.5 g/L glucose. In some embodiments, a transfection medium without serum (eg, without FBS) improves transfection efficiency. In one embodiment, the transfection medium is OptiMEM (Invitrogen/Thermo Fisher). In one embodiment, three plasmids (pSMN, pHELP and pAAV2/9) are mixed together with PEI in the transfection medium and reacted. In some embodiments, the three plasmids are mixed together in a molar ratio of approximately 1:1:1. In some embodiments, plasmids and PEI are mixed in a 1:1 ratio by weight of DNA:PEI. In some embodiments, the plasmids and PEI are mixed in a ratio of less than 1:1 by weight of DNA:PEI. In one embodiment, pSMN, pHELP and pAAV2/9 are mixed in a 1:1:1 molar ratio in OptiMEM medium. In such an embodiment, PEI is added such that the DNA:PEI ratio is 1:1 by weight. In some embodiments, the reaction is carried out for 0-60 minutes, or 10-45 minutes, or 20-30 minutes. In one embodiment, the reaction is carried out for 15-30 minutes.

In one embodiment, the present invention provides a method for producing an AAV viral vector, comprising the steps of (i) culturing adherent HEK293 cells in an industrial scale bioreactor, (2) transfecting the adherent cells with plasmids in less than 60 minutes to produce an AAV vector, and optionally performing further processing, purification, formulation and filling steps to obtain a pharmaceutical product. In one embodiment of this method, the scAAV9.CB.SMN vector is produced using transfection with three DNA plasmids using co-precipitation with polyethylenimine ("PEI"). In one embodiment, the 3 plasmids used for this transfection are pSMN, pAAV2/9 and pHELP.

Transfection can be accomplished by contacting the packaging cell line with a DNA-PEI co-pellet. In some embodiments, the DNA-PEI co-pellet in the transfection medium is filled into a recycle media bag. In some embodiments, the co-pellet of DNA-PEI in the transfection medium is circulated in the bioreactor and completely replaces the growth medium. In some embodiments, the DNA-PEI co-pellet in the transfection medium is brought into contact with adherent cells in the bioreactor. In some embodiments, the DNA-PEI co-pellet in the transfection medium is brought into contact with adherent cells in the bioreactor for no more than two hours. In some embodiments, transfection occurs within one to two hours. In some embodiments, transfection occurs in less than one hour, such as 10 minutes, 20 minutes, 30 minutes, 40 minutes, or 50 minutes. In some embodiments, transfection occurs within one to two hours. In some embodiments, the transfection is stopped by recirculating the complete culture medium through the bioreactor and completely replacing the transfection medium.

2. Collection of increased amounts of viral particles

After a suitable period of cell proliferation following transfection, in some embodiments the cells are lysed and the viral particles are collected. In some embodiments, the cells are separated from the reactor before the cell lysis process is initiated. In some embodiments, the cells are lysed in situ. Optionally, the viral particles are collected without lysis. In some embodiments, an endonuclease is added, eg, circulated into the bioreactor to a final target concentration. An endonuclease can be one that destroys both DNA and RNA. In one embodiment, the endonuclease is a genetically engineered endonuclease from Serratia marcescens (Eaves, G. N. et al. J. Bact. 1963, 85, 273-278; Nestle, M. et al. J. Biol. Chem. 1969, 244, 5219 -5225), which is sold under the name Benzonase® (EMD Millipore). The enzyme is obtained and purified from E. coli strain W3110, a mutant of strain K12 containing the pNUC1 production plasmid (US Pat. No. 5,173,418, which is incorporated herein by reference in its entirety). Structurally, the protein is a dimer of identical subunits, 245 amino acids long, weighing approximately 30 kDa, with two important disulfide bonds. Benzonase® degrades all forms of DNA and RNA (single-stranded, double-stranded, linear and circular) and is effective over a wide range of operating conditions, degrading nucleic acids into 5'-monophosphate-terminated oligonucleotides of 2-5 bases in length. Benzonase® is produced according to current Good Manufacturing Practices (cGMP) and thus can be used in industrial scale processes for the purification of proteins and/or viral particles. Other endonucleases that are produced under cGMP conditions can similarly be used in the purification methods disclosed herein. In one embodiment, benzonase is added to the bioreactor to a final concentration of 50 to 200 IU/ml, such as 75 to 150 IU/ml, such as approximately 100 IU/ml. In some embodiments, the addition of benzonase significantly reduces the level of host cell DNA while still allowing high production of g.v. in a bioreactor.

In some embodiments, the endonuclease is stirred before adding lysis buffer to the reactor. In some embodiments, the cell lysis solution is mixed with the adherent cells for no more than 1 hour, no more than 2 hours, no more than 3 hours, no more than 4 hours, or no more than 5 hours. In some embodiments, the lysis buffer may contain magnesium chloride and/or Tween-20 in a suitable buffer. In an illustrative embodiment, the lysis buffer is 500 mM HEPES, 10% Tween 20, 20 mM MgCl ₂ , pH 8.0. Sucrose saline (SSS), which quenches the reaction with benzonase, can be added to stop the lysis reaction. In some embodiments, SSS is added to a biomass collection bag containing a wash buffer and mixed for 15 minutes. In some embodiments, the bioreactor is rinsed with bioreactor wash buffer and the rinses are then collected in a collection bag for collected biomass along with the quenched cell lysis solution and lysed cell contents, which together constitute the total collected biomass. In some embodiments, the bioreactor wash buffer may contain Tris, MgCl2, NaCl, Tween-20, and sucrose. In an illustrative embodiment, the bioreactor wash buffer contains 20 mM Tris, 1 mM MgCl ₂ , 500 mM NaCl, 1% Tween-20 w/v. and 1% sucrose w/v. at pH 8.1.

3. Purification of virus particles

Once collected, the total collected biomass of viral particles can be concentrated and purified, typically through filtration. In one embodiment, the viral particles are filtered by depth filtration followed by filtration through a filter that removes macromolecular contaminants and cellular debris, such as a 0.45 μm filter, but which allows vector genomes to pass through it. Any suitable depth filter can be used.

As is known in the art, depth filtration refers to the use of a porous filtration medium to clarify solutions containing significant amounts of large particles (eg, intact cells or cellular debris), as compared to membrane filtration, which would clog quickly under such conditions. A variety of depth filtration media with different pore sizes are commercially available from various manufacturers such as Millipore, Pall, General Electric and Sartorious.

The target flow rate for depth filtration can be reduced to maintain filter inlet pressure within specification. After all of the total collected biomass has been filtered, a diafiltration buffer used for the subsequent first tangential flow filtration stage ("TFF1") can be passed through the depth filter in some embodiments. The pool obtained using a depth filter is mixed. The pool obtained from the depth filter can then be filtered through a 0.45 µm pore size filter to further purify the total biomass material collected. TFF1 buffer is then passed through a 0.45 μm filter.

4. Filtration in tangential flow

In various embodiments, tangential flow filtration is used to concentrate the total collected biomass and remove salts and proteins, for example, using tangential flow filtration. Tangential flow filtration (TFF) (also called cross flow filtration, CFF) is well known to those skilled in the art, and equipment and protocols for performing it in a wide range of situations are commercially available from a variety of manufacturers, including without limitation Pall Corporation, Port Washington , New York, USA, and Spectrum Labs, Rancho Dominguez, California, USA. In general, TFF may involve recirculation of the retentate across the membrane surface. This smooth cross-flow delivery can, in certain embodiments, minimize membrane fouling, maintain high filtration rates, and provide high product recovery. In one embodiment, the TFF step can be performed by a flat sheet system exemplified herein. Flat sheet systems can be used in large-scale production where such systems are provided with a means (eg, a free flow channel) to prevent the virus particles from being affected by excessive shear forces. Alternatively, the TFF step can be carried out using a hollow fiber system exemplified herein. In one embodiment, the molecular weight cutoff (MWCO) of the TFF system is 200-400 kDa, for example approximately 300 kDa.

In one embodiment, the TFF1 step is performed using a regenerated cellulose membrane cassette with a cutoff of MW 300 kDa. The cassette is washed and disinfected with NaOH solution and equilibrated with TFF1 buffer. In one embodiment, the TFF1 buffer contains 20 mM Tris, 1 mM MgCl ₂ , 500 mM NaCl, 1% sucrose, pH 8.1.

In some embodiments, the TFF1 stage concentration phase is selected to reduce the volume of clarified harvested biomass by approximately 10-fold. Once the target retentate volume has been reached, diafiltration processes can begin. The retentate, in some embodiments, can be diafiltered with approximately 6 dia volumes of TFF1 buffer. In some embodiments, the retentate is diafiltered with about 5-20, or 10-15, or 12 diavolumes of TFF1 buffer. After reaching 6 diavolumes of the total permeate flow, the retentate can be concentrated and collected again. Rinses, for example two sequential rinses of the membrane, can be performed to increase product recovery from the drug intermediate.

5. Intermediate product

In some embodiments, the drug intermediate can then be frozen on dry ice or in a freezer and then stored at < -60°C. In other embodiments, the intermediate does not need to be frozen prior to the isolation and purification process.

In some embodiments, multiple batches of intermediate material are combined together for further processing (eg, purification through an isolation and purification process, such as those described herein). Multiple batches of the intermediate product may be combined prior to freezing and storage. In other embodiments, multiple batches of intermediate material may be combined after thawing frozen and stored batches.

ISOLATION AND PURIFICATION PROCESS

In some embodiments, an isolation and purification process is used to process the intermediate (eg, a combined intermediate) to produce a filtered drug substance. In some embodiments, the isolation and purification process steps include (a) acidification and clarification (eg, using filtration), (b) performing cation exchange chromatography, (c) tangential flow filtration ("TFF2"), (d) ultracentrifugation using CsCl, (e) collection of the viral vector, and (f) additional tangential flow filtration ("TFF3") to obtain the filtered drug substance, wherein the purified AAV particles are suspended in a pharmaceutically acceptable carrier. In some embodiments, the isolation and purification process includes the following production steps subsequent to obtaining the TFF1 intermediate: thawing and combining the TFF1 intermediate, acidification and clarification, cation exchange chromatography (CEX), tangential flow filtration (TFF2), ultracentrifugation with CsCl to separate full/empty capsids, tangential flow filtration (TFF3) to concentrate/buffer exchange, filtration of pooled material resulting from TFF3 to form drug substance, dilution and filtration of drug substance to obtain drug product, storing the medicinal product and filling bottles with the medicinal product.

In some embodiments, the isolation and purification process disclosed herein can be used to process an intermediate containing AAV SMN, AAV MECP2, or AAV encoding shRNA targeting SOD1 described herein.

1. Acidification and clarification of the intermediate product

In embodiments where the intermediate is frozen, the isolation and purification process is initiated by thawing the intermediate material resulting from TFF1. A detergent, such as Tween 20, can be used to stimulate flocculation of host cell bulk proteins and DNA under acidic pH conditions. The pH of the detergent-containing TFF1 intermediate can then be lowered. The flocculant and sediment formed by lowering the pH can then be removed by filtering the solution through a depth filter and a filter that removes macromolecular contaminants and cellular debris, such as a 0.45 μm pore size filter, but which allows vector genomes to pass through it. Any suitable depth filter can be used.

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

After incubation, the pH of the Tween 20-containing solution of the TFF1 intermediate can be lowered by adding any suitable acid. In some embodiments, 1 M glycine pH 2.5 is added to achieve a target pH of 3.5 ± 0.1. In some embodiments, the target pH is pH 3.0-4.0, about pH 3.3-3.7, about pH 3.4-3.6, or about pH 3.5. Once the pH is within the acceptable range, the solution can be passed through a filter of any pore size. In an exemplary embodiment, a depth filter (eg, Clarisolve POD) is used along with a 0.45 μm pore size filter (eg, Opticap XL10 Durapore filter) or a 0.8/0.45 μm pore size PES filter.

2. Cation exchange chromatography

In various embodiments, a cation exchange (CEX) capture chromatography step is used, for example, to separate viral capsids from host cell proteins, host cell DNA, host cell lipids, Tween 20, and other process-related impurities. The principles of cation exchange chromatography are well known in the art, but in short, the method is based on charge-charge interactions between the positively charged species to be isolated and the negatively charged resin used. In general, the column is first equilibrated by passing several diavolumes of buffer until the pH and conductivity are stabilized. The sample is then loaded and the column is washed with loading buffer. Finally, an elution buffer is used to elute the sample of interest from the column, and fractions containing the sample are collected. The presence of a sample of interest can be detected by measuring the absorbance of the eluent.

In one embodiment, a CIMmultus S03-8000 Advanced Composite Column (sulfonyl group) chromatography column (2 μm pore size) was used in the CEX step. In one embodiment, the elution peak is collected starting with a sharp increase in OD280. OD280 begins to rise when conductivity is 80-85 mS/cm. The eluate resulting from CEX can be collected according to a standard procedure and can be collected in two fractions. In one embodiment, the first fraction begins with a sharp increase in OD280 and is collected to 1.5 collection volumes (CV). In another embodiment, the second fraction begins immediately after the first fraction and is collected to 1.0 CV. Fractions are pooled and then neutralized to pH 8.0 ± 0.30. In one embodiment, the neutralization buffer contains 1.0 M Tris with a pH of 9.1 ± 0.1 at 20°C.

3. Filtration in tangential flow 2

In some embodiments, a tangential flow filtration step (TFF2) is used to concentrate, remove protein impurities, and buffer exchange to a suitable buffer for the subsequent CsCl ultracentrifugation step. Any suitable TFF membrane can be used. In one embodiment, the TFF2 step uses regenerated cellulose membranes with a MWCO of 300 kDa.

In some embodiments, the concentration phase of this step is designed to reduce the volume of eluate resulting from CEX. In one embodiment, the retentate is diluted 2-fold with diafiltration buffer and the retentate is concentrated to its original volume. In one embodiment, the diafiltration buffer is a NaCl-based diafiltration buffer at TFF2 that contains 20 mM Tris, 2 mM MgCl2, 150 mM NaCl, 0.2% poloxamer 188, 1% sucrose, pH 8.1 ± 0. 1 at 20°C. In such embodiments, this process may be repeated until diafiltration with new buffer is completed. In one embodiment, the retentate is diluted 2-fold with a diafiltration buffer containing CsCl, and the retentate is concentrated to its original volume. In one embodiment, the CsCl-containing diafiltration buffer is a CsCl diafiltration buffer at TFF2 that contains 20 mM Tris, 2 mM MgCl ₂ , 3 M CsCl, 0.2% poloxamer 188, pH 8.1 ± 0, 1 at 20°C. In such embodiments, this process may be repeated until diafiltration with new buffer is completed. Once diafiltration with CsCl is complete, the retentate is then concentrated to a predetermined volume, which depends on the retention volume in the system. In some embodiments, rinses are performed, such as two sequential membrane rinses, to maximize product recovery from the TFF2 system.

4. Ultracentrifugation with CsCl

In some embodiments, where AAV is used for in vivo gene transduction, the final rAAV product may contain minimal amounts of impurities and void particles. Two methods for AAV vector purification are ultracentrifugation using either an iodixanol gradient or a CsCl gradient. One study comparing the two methods demonstrated that iodixanol yielded AAV viral vectors with higher vector purity but yielded a higher amount of empty viral capsids compared to that of CsCl. Strobel et al. "Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications." Human Gene Therapy Methods, 26(4):147–157. Although the use of CsCl results in fewer empty viral capsids, CsCl can be toxic to cells and multiple purification steps may be required to remove residual CsCl, resulting in a process that takes longer (~3.5 days) compared to faster methods, such as with iodixanol (~1 day). Another study showed that using a large number of purification steps to remove residual CsCl often results in significant loss of rAAV, resulting in low yields and recovery rates, which often negates other benefits of the process. Hermens et al. "Purification of Recombinant Adeno-Associated Virus by Iodixanol Gradient Ultracentrifugation Allows Rapid and Reproducible Preparation of Vector Stocks for Gene Transfer in the Nervous System." Human Gene Therapy 10:1885–1891. Additionally, while these two methods work well in the laboratory to obtain samples for preclinical testing, they are not scalable and thus are not suitable for large-scale production of commercial products. See for example Tomono et al., "Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1)." Molecular Therapy - Methods & Clinical Development, 3:15058 (“purification methods using cesium chloride (CsCl) or iodixanol density gradient ultracentrifugation are not suitable for large-scale production”).

In some embodiments, an ultracentrifugation step is used, for example, to separate empty capsids from full capsids. Surprisingly, the CsCl ultracentrifugation method disclosed herein is scalable and suitable for large-scale production of purified AAV vectors. Ultracentrifugation may be carried out by analytical ultracentrifugation and may involve the use of gradient buffers. Examples of gradient buffers include, but are not limited to, those with CsCl, sucrose, iodixanol, and others known in the art. Centrifugation can be carried out in any centrifuge capable of achieving the required dynamic load, such as the Optima XPN 100 automatic ultracentrifugation system or an equivalent system equipped with a 50.2 Ti rotor or equivalent rotor. After ultracentrifugation, empty capsids and full capsids are separated into individual bands within the tube and can be recovered by withdrawing material from a particular band. In some embodiments, the TFF2 purified filter material is centrifuged at 241600-302000 g (~40000-50000 rpm in a 50.2 Ti rotor). In some embodiments, the TFF2 purified filter material is centrifuged overnight. In some embodiments, the TFF2 purified filter material is centrifuged for 16-24 hours. In some embodiments, the TFF2 purified filter material is centrifuged for 20-24 hours. In some embodiments, the TFF2 purified filter material is centrifuged at 15-25°C. In one embodiment, the TFF2 purified filter material is centrifuged at 302,000 g (50,000 rpm in a 50.2 Ti rotor) for 17 hours at 20°C. In some embodiments, the CsCl centrifugation buffer may comprise one or more of the following ingredients, including (a) CsCl, further comprising one or more of (b) MgCl ₂ , (c) poloxamer 188, and (d) Tris. In some embodiments, the CsCl buffer may include all of (a), (b), (c) and (d). In some embodiments, the CsCl buffer has a pH of 7.5-8.5 or a pH of 7.9-8.2. In one embodiment, a suitable buffer for CsCl centrifugation is 20 mM Tris, 2 mM MgCl ₂ , 3 M CsCl, 0.2% poloxamer 188, pH 8.1 ± 0.10. Once the centrifugation step is complete, the tubes can be removed from the ultracentrifuge. In some embodiments, the topmost band, band A, contains empty capsids. In some embodiments, the next highest bands, bands B, C, and D, contain paired bands with complete capsids. In some embodiments, AAV-based viral vectors are collected using a syringe. In one embodiment, bands B, C, and D are removed with an 18G needle attached to a 30 mL syringe inserted just below band D to the middle of the tube. In other embodiments, the bands can be analyzed for the presence of full or empty capsids using techniques known in the art and/or as described herein, and bands containing full capsids can be collected.

The ratio of empty to non-empty viral capsids can be measured using standard laboratory techniques. In some embodiments, the measurement is performed through optical density measurements. In some embodiments, the measurement is performed through UV optical density measurements. In some embodiments, the total amount of capsid proteins and the total amount of DNA can be determined from UV absorbance measurements. In some embodiments, the measurement is performed by measuring the optical refractive index. In some other embodiments, the measurement is performed using analytical ultracentrifugation.

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

5. Tangential flow filtration 3 to obtain filtered drug substance

In some embodiments, a tangential flow filtration (TFF3) step is used to remove CsCl and concentrate the complete vector capsids. Tangential flow filtration can be carried out using a suitable membrane. In one embodiment, regenerated cellulose membranes with a MWCO of 300 kDa are used. Vector capsids can be retained by membranes. The concentration phase of the TFF3 process can be designed to reduce the concentration of residual CsCl and the pool volume resulting from ultracentrifugation. In some embodiments, once the target retentate volume is reached, diafiltration is started. The retentate is diafiltered with no more than 10 diavolumes of a suitable TFF3 buffer. In one embodiment, a suitable TFF3 buffer may contain one or more of the following components, including (a) Tris, (b) MgCl ₂ , (c) NaCl, or (d) poloxamer 188. In one embodiment, a suitable TFF3 buffer may contain all of the following: (a), (b), (c) and (d). In one embodiment, a suitable buffer for TFF3 is pH 7.5-8.5, pH 7.7-8.3, or pH 8.0. In one embodiment, a suitable TFF3 buffer contains 20 mM Tris, 1 mM MgCl ₂ , 200 mM NaCl, 0.001% poloxamer 188, has a pH of 8.0 ± 0.1 at 20°C. In another embodiment, a suitable buffer for TFF3 contains 20 mM Tris, 1 mM MgCl ₂ , 200 mM NaCl, 0.005% poloxamer 188, has a pH of 8.0 ± 0.1 at 20°C. In one embodiment, the concentrated retentate is filtered using a Pall Supor® EKV sterilizing filter (Mini Kleenpak) with a pore size of 0.2 μm to obtain the filtered drug substance. In some embodiments, the methods described herein provide a yield of greater than 5 x 10 ¹⁵ g, or greater than 8 x 10 ¹⁵ g, or greater than 1 x 10 ¹⁶ g. rAAV per production batch.

Pharmaceutical compositions

Viral (eg, AAV) particles purified in accordance with the methods disclosed herein can be produced in high yield with a purity sufficient to be administered to a human subject. In some embodiments, the viral vector is formulated at a concentration of about 1-8×10 ¹³ viral vector genomes/ml (gv/ml) or about 1.7-2.3×10 ¹³ gv/ml. In some embodiments, the viral vector is formulated at a concentration of approximately 1.9-2.1×10 ¹³ g/mL. In some embodiments, the viral vector is formulated at a concentration of approximately 2.0 x 10 ¹³ g/mL.

In some embodiments, the viral vector production process may produce empty viral capsids that do not contain nucleic acid material. Pharmaceutical compositions containing small amounts of empty viral capsids may be advantageous because they avoid exposing patients, such as infants with immature immune systems, to antigenic material (empty capsids, host cell protein, host cell DNA), optionally without providing a therapeutic effect . In some embodiments, such pharmaceutical compositions may reduce potential infusion reactions or multispecific immune responses and may improve therapeutic efficacy. Compared to complete viral capsids containing genome material, empty capsids have different densities, allowing the two species to be separated by gradient centrifugation or other methods known in the art. In some embodiments, empty capsids are separated by ultracentrifugation. In some embodiments, empty capsids are separated using CsCl gradient ultracentrifugation. In other embodiments, empty capsids are separated using iodixanol gradient ultracentrifugation. In some embodiments, empty capsids are separated using sucrose gradient ultracentrifugation.

The ratio of empty to non-empty viral capsids can be measured using standard laboratory techniques. In some embodiments, the ratio is measured through optical density measurements. In some embodiments, the ratio is measured through UV absorbance measurements. In some embodiments, the total amount of capsid proteins and the total amount of DNA can be determined using UV absorbance measurements. In some embodiments, the value is determined by measuring the optical refractive index. In some embodiments, the value is determined using analytical ultracentrifugation.

High levels of empty capsids may pose a threat to the effectiveness of viral vector-based therapies. In one embodiment, the pharmaceutical composition contains less than 10% empty capsids, less than 8% empty capsids, less than 7%, less than about 5%, less than 3%, less than 1% empty capsids. In another embodiment, the pharmaceutical composition contains 1-10% empty capsids. In another embodiment, the pharmaceutical composition contains 2-8% empty capsids. In another embodiment, the pharmaceutical composition contains 6% empty capsids or less, 5% empty capsids, 4% empty capsids, 3% empty capsids, 2% empty capsids or less. In one embodiment, the number of empty capsids is below the detection limit. In another embodiment, the percentage of empty capsids is determined as a percentage of the total number of capsids, for example, using AUC. In some embodiments, such a low percentage of empty capsids increases the effectiveness of treatment and/or reduces side effects (eg, inflammatory reactions, liver damage) after administration to a patient, for example, compared to compositions that have higher percentages of empty capsids. In some embodiments, the methods for producing viral vectors disclosed herein provide such improved percentages of empty capsids compared to levels in prior methods, for example, those that do not use adherent cells and/or purification methods described herein .

During the viral vector production process, residual protein from adherent cells (eg, HEK293 cells) used to produce viral vectors may not be completely separated. Residual host cell proteins have the potential to trigger an immune response. The amount of host cell debris can be measured using any standard laboratory techniques that can distinguish between viral capsid proteins and host cell debris. In some embodiments, the amount of residual host cell proteins can be measured by size exclusion or ion exchange chromatography. In some embodiments, the measurement can be performed using Western blotting using antibodies specific for the parent cell. In one embodiment, the amount of residual host cell protein can be measured using an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the amount of residual host cell protein can be measured using a commercial ELISA kit. In some embodiments, the amount of residual host cell protein can be measured using the HEK293 HCP ELISA kit from Cygnus Technologies.

In another embodiment, the amount of residual host cell protein in said pharmaceutical composition is 5×10 ⁶ pg/ml per 1×10 ¹³ g.v./ml or less, is 1.2×10 ⁶ pg/ml per 1×10 ¹³ g.v./ml or less, or from 1×10 ⁵ pg/ml per 1×10 ¹³ g.v./ml to 1.2×10 ⁶ pg/ml per 1×10 ¹³ g.v./ ml, or is 40 ng/ml per 1×10 ¹³ g/ml or less. In one embodiment, the pharmaceutical composition contains 5 ng or less, 4, 3, 2, 1 ng or less of residual host cell protein per 1.0 x 10 ¹³ g. In one embodiment, the pharmaceutical composition contains 4 ng or less of residual host cell protein per 1.0 x 10 ¹³ g.

During the viral vector production process, residual host cell DNA from adherent cells (eg, HEK293 cells) or residual plasmid DNA transfected to produce viral vectors may not be completely removed. The purification method (eg, acidification, clarification, tangential flow filtration, etc.) removes the bulk of residual host cell or plasmid DNA. In one embodiment, measurement of the amount of residual host cell or plasmid DNA is performed using PCR. In another embodiment, measurement of the amount of residual host cell or plasmid DNA is performed using quantitative PCR (qPCR) with primers specific for host cell or plasmid sequences. In another embodiment, measurement of the amount of residual host cell or plasmid DNA is performed using digital droplet PCR (ddPCR). In one embodiment, the amount of plasmid DNA is determined using a qPCR assay with primers specific to the kanamycin resistance gene region of the plasmid. In another embodiment, amounts of residual host cell DNA are determined using commercial qPCR assay kits, for example, resDNASEQ© Human Residual DNA Quantitation Kit from ThermoFisher, Residual DNA Quantification Supermix from Biorad, or any equivalent product. Reducing the amount of residual host cell or plasmid DNA may improve the therapeutic effect, and such a composition can be purified and/or selected for use in the treatments disclosed herein.

In one embodiment, the amount of residual host cell DNA in said pharmaceutical composition is 1.7 x 10 ⁶ pg/ml per 1 x 10 ¹³ g.v./ml or less, from 1 x 10 ⁵ pg/ml per 1 x 10 ¹³ g./ml to 1.2×10 ⁶ pg/ml per 1×10 ¹³ g./ml. In one embodiment ^, the amount of residual host cell DNA in the pharmaceutical composition is ^3x105 or less, ^2x105 , 1.1x105, ^1x105 pg or less per ^1.0x1013 g. V. In embodiments, the amount of residual host cell DNA in said pharmaceutical composition is 1.1 x 10 ⁵ pg per 1.0 x 10 ¹³ g. or less.

In another embodiment, the amount of residual plasmid DNA in the specified pharmaceutical composition is 1.7 x 10 ⁶ pg/ml per 1 x 10 ¹³ g.v./ml or less, from 1 x 10 ⁵ pg/ml per 1 x 10 ¹³ g .v./ml to 1.7×10 ⁶ pg/ml per 1×10 ¹³ g./ml. In another embodiment, the amount of residual plasmid DNA in said pharmaceutical composition is 6.8 x 10 ⁵ pg per 1.0 x 10 ¹³ g. or less.

In one embodiment, the amount of residual host cell DNA in the pharmaceutical composition is 1.1 x 10 ⁵ pg per 1.0 x 10 ¹³ g. or less, and the amount of residual plasmid DNA in the specified pharmaceutical composition is 6.8×10 ⁵ pg per 1.0×10 ¹³ g. or less.

In one embodiment, the amount of residual host cell DNA in the pharmaceutical composition is 1.1 x 10 ⁵ pg per 1.0 x 10 ¹³ g. or less, and the amount of residual plasmid DNA in the specified pharmaceutical composition is 6.8×10 ⁵ pg per 1.0×10 ¹³ g. or less, and the amount of residual host cell protein in said pharmaceutical composition is 4 ng per 1.0 x 10 ¹³ g. or less.

In some embodiments, the amount of endotoxin in the pharmaceutical composition is less than about 1 IU/mL per 1.0 x 10 ¹³ g/mL, less than about 0.75 IU/mL per 1.0 x 10 ¹³ g/mL ml, less than approximately 0.5 IU/ml per 1.0×10 ¹³ g.v./ml, less than approximately 0.4 IU/ml per 1.0×10 ¹³ g.v./ml, less than approximately 0. 35 IU/ml per 1.0 × 10 ¹³ g./ml, less than approximately 0.3 IU/ml per 1.0 × 10 ¹³ g./ml, less than approximately 0.25 IU/ml per 1 .0×10 ¹³ g/mL, less than approximately 0.2 IU/ml per 1.0×10 ¹³ g/mL, less than approximately 0.15 IU/ml per 1.0×10 ¹³ g .v./ml, less than approximately 0.1 IU/ml per 1.0×10 ¹³ g./ml, less than approximately 0.05 IU/ml per 1.0×10 ¹³ g./ml or less than approximately 0.02 IU/ml per 1.0×10 ¹³ g/ml. Methods for determining the amount of endotoxin are known in the art, for example, reaction with horseshoe crab amebocyte lysate (LAL). In embodiments, endotoxin is assayed according to United States Pharmacopoeia (“USP”) <85> (incorporated herein by reference in its entirety).

In one embodiment, the bovine serum albumin (BSA) content of the pharmaceutical composition is less than 0.5 ng per 1.0 x 10 ¹³ g.v., less than 0.3 ng per 1.0 x 10 ¹³ g.v. or less than 0.22 ng per 1.0 × 10 ¹³ g. In one embodiment, the content of benzonase in the specified pharmaceutical composition is less than 0.2 ng per 1.0 x 10 ¹³ g., less than 0.1 ng per 1.0 x 10 ¹³ g. or less than 0.09 ng per 1.0×10 ¹³ g.

In one embodiment, the pharmaceutical composition disclosed herein comprises one or more of the following: less than about 0.09 ng of benzonase per 1.0 x 10 ¹³ g, less than about 30 μg/g (ppm) cesium, approximately 20-80 ppm poloxamer 188, less than approximately 0.22 ng BSA per 1.0 x 10 ¹³ year, less than approximately 6.8 x 10 ⁵ pg of residual plasmid DNA per 1.0 x 10 ¹³ year, less than approximately 1.1 x 10 ⁵ pg of residual hcDNA at 1.0 x 10 ¹³ yr, less than approximately 4 ng rHCP at 1.0 × 10 ¹³ yr, pH 7.7-8.3, approximately 390-430 mOsm/kg, less than approximately 600 particles ≥ 25 µm per container, less than approximately 6000 particles ≥ 10 µm per container, genomic titer of approximately 1.7 x 10 ¹³ -2.3 x 10 ¹³ g. ./ml, infectious titer of approximately 3.9×10 ⁸ -8.4×10 ¹⁰ IU per 1.0×10 ¹³ g., total protein content of approximately 100-300 μg per 1.0× 10 ¹³ g.i., median survival of ≥ 24 days in Δ7SMA mice at a viral vector dose of approximately 7.5 x 10 ¹³ g.i.v./kg, relative activity determined by in vitro cell-based assay of 70-30%, and/or less than approximately 5% empty capsids.

In one embodiment, the pharmaceutical composition disclosed herein comprises one or more, such as all, of the following: pH 7.7-8.3 (e.g., as measured according to <791> USP), approximately 390-430 mOsm/kg (e.g., when measured according to <785>USP), less than approximately 600 particles ≥ 25 µm per container (e.g., when measured according to <787>USP), less than approximately 6000 particles ≥ 10 µm per container (e.g., when measured according to <787> USP), genomic titer of approximately 1.7 x 10 ¹³ -2.3 x 10 ¹³ g/mL, infectious titer of approximately 3.9 x 10 ⁸ -8.4 x 10 ¹⁰ IU at 1.0 x 10 ¹³ yr, total protein approximately 100-300 μg at 1.0 × 10 ¹³ yr, median survival ≥ 24 days in Δ7SMA mice at approximately 7.5 viral vector dose ×10 ¹³ , for example, in an in vivo functionality test, such as the one described herein, the relative activity, as determined by an in vitro cell-based assay, is about 70-130%, and/or less than about 5% of empty capsids. In embodiments, the pharmaceutical composition disclosed herein has an overall purity of 95% or greater (eg, as determined by SDS-PAGE). In embodiments, the pharmaceutical composition disclosed herein is characterized in that no individual unnamed related impurity is greater than 2% (eg, as determined by SDS-PAGE). In embodiments, the pharmaceutical composition disclosed herein provides endotoxin levels of 0.75 IU/ml or less (eg, when measured according to <85>USP). In embodiments, the pharmaceutical composition disclosed herein exhibits no growth in a sterility test, for example, when measured according to USP <71>.

High levels of residual host cell protein, host cell DNA, plasmid DNA, and/or endotoxin may pose a threat to the effectiveness of viral vector-based therapies. In some embodiments, such low amounts of residual host cell protein, host cell DNA, plasmid DNA, and/or endotoxin increase the effectiveness of treatment and/or reduce side effects (e.g., inflammatory reactions, liver damage) after administration to a patient, e.g. with compositions that have higher quantities. In some embodiments, the methods for producing viral vectors disclosed herein provide such improved levels over those of prior methods, for example, those that do not use the adherent cells and/or purification methods described herein. In some embodiments, the methods described herein also produce viral vectors with reduced percentages of empty capsids in addition to small amounts of residual host cell protein, host cell DNA, plasmid DNA, and/or endotoxin.

In some embodiments, the amount of residual cesium after the TFF, such as the second TFF, is less than about 50 μg/g. In some embodiments, the amount of residual cesium after the TFF, such as the second TFF, is less than about 30 μg/g. In some embodiments, the amount of residual cesium after the TFF, such as the second TFF, is less than about 20 μg/g. In some embodiments, the amount of residual cesium in the pharmaceutical composition is 30 μg/g (ppm) or less. In some embodiments, the amount of residual CsCl can be measured using mass spectrometry, inductively coupled plasma mass spectrometry (ICP-MS), and/or other suitable method. In some embodiments, the amount of residual cesium after the second TFF is less than the limit of quantitation, for example, when using ICP-MS.

In some embodiments, the concentration of AAV-based viral vectors collected after the second TFF is about 5 x 10 ¹² g/mL or greater, is 1 x 10 ¹³ g/mL or greater, or is about 3 x 10 ¹³ g/ml or more.

In one embodiment, the pharmaceutical composition is characterized by one or more of the following: less than 0.09 ng benzonase per 1.0 x 10 ¹³ g, less than 30 μg/g (ppm) cesium, approximately 20-80 ppm poloxamer 188, less 0.22 ng BSA at 1.0×10 ¹³ years of age, less than 6.8×10 ⁵ pg of residual plasmid DNA at 1.0×10 ¹³ years of age, less than 1.1×10 ⁵ pg of residual hcDNA by 1.0×10 ¹³ g. and less than 4 ng rHCP per 1.0×10 ¹³ g.

In another embodiment, the pharmaceutical composition retains activity in the +20% range, +15% range, +10% range, or +5% range of activity of the reference standard. In one embodiment, activity is assessed in comparison to a reference standard using the methods described in Foust et al., Nat. Biotechnol., 28(3), pp. 271-274 (2010). Any suitable reference standard may be used. In one embodiment, the pharmaceutical composition has in vivo activity when tested in SMAΔ7 mice. In one embodiment, a test mouse given a dose of 7.5 x 10 ¹³ g/kg has a median survival of greater than 15 days, greater than 20 days, greater than 22 days, or greater than 24 days. In one embodiment, the pharmaceutical composition has a relative in vitro activity when tested using a cell-based assay of 50-150%, 60-140%, or 70-130% relative to the reference standard and/or appropriate control.

Viral particles purified in accordance with the present invention (eg, viral particles) can be formulated according to known methods to obtain a pharmaceutically usable composition. The compositions of the present invention can be formulated for administration to a mammalian subject, such as a human, using techniques known in the art. In particular, delivery systems can be formulated for intramuscular, intradermal, mucosal, subcutaneous, intravenous, intrathecal, depot-type, or topical administration.

If the delivery system is formulated as a solution or suspension, the delivery system is in a suitable vehicle, such as an aqueous vehicle. Various aqueous vehicles may be used, such as water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions can be sterilized using traditional, well-known sterilization techniques or can be subjected to sterile filtration. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.

The compositions, for example pharmaceutical compositions, may contain pharmaceutically acceptable excipients to provide near physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example sodium acetate, sodium lactate , sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanol aminooleate, etc. In some embodiments, the pharmaceutical composition contains a preservative. In some other embodiments, the pharmaceutical composition does not contain a preservative.

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

In some embodiments, PCR-based methods detect and quantify the encapsidated AAV9 viral genome using specially designed primers and probes that target the SMN gene. In other embodiments, PCR-based methods detect and quantify the encapsidated AAV9 viral genome using specially designed primers and probes targeting the chicken beta-actin promoter. In other embodiments, PCR-based methods detect and quantify the encapsidated AAV9 viral genome using specially designed primers and probes that target the CMV enhancer. In other embodiments, PCR-based methods detect and quantify the encapsidated AAV9 viral genome using specially designed primers and probes that target ITR sequences. In other embodiments, PCR-based methods detect and quantify the encapsidated AAV9 viral genome using specially designed primers and probes targeting the bovine growth hormone polyadenylation signal.

In some embodiments, the pharmaceutical composition has a pH of approximately 7.7-8.3 and has an osmolality of 390-430 mOsm/kg. In some embodiments, pH is measured using a pH meter. In some embodiments, pH is measured potentiometrically using a temperature-compensated microelectrode in accordance with standards established by the United States Pharmacopeia (USP), such as <791> (incorporated by reference in its entirety). In some embodiments, osmolality is measured using USP freezing point depression, such as USP <785> (incorporated by reference in its entirety). In some embodiments, osmolality is measured using a vapor pressure reduction osmometer. In other embodiments, osmolality is measured using a membrane osmometer.

In one embodiment, the intravenous formulation has a pH of 7.5 to 8.5, a genomic titer of 2x10 ¹³ g/mL - 6x10 ¹³ g/mL, and an osmolality of 384-448 mOsm/kg . In another embodiment, the composition for intravenous administration has a pH of 7.5 to 8.5, a genomic titer of 1.5×10 ¹³ g.v./ml - 3.5×10 ¹³ g.v./ml and an osmolality of 384- 448 mOsm/kg. In another embodiment, the composition for intravenous administration has a pH of 7.5 to 8.5, a genomic titer of 1.8×10 ¹³ g.v./ml - 2.2×10 ¹³ g.v./ml and an osmolality of 384- 448 mOsm/kg. In one embodiment, the IV composition contains approximately 0.1-2.0 mM MgCl ₂ . In one embodiment, the IV composition contains approximately 100-300 mM NaCl. In one embodiment, the IV composition contains approximately 0.001%-0.01% w/v. poloxamer 188. In one embodiment, the IV formulation is an aqueous formulation in 10-30 mM Tris buffer, for example, at pH 7.5-8.5.

In one embodiment, the IV formulation contains 1 mM MgCl ₂ , 200 mM NaCl, 0.005% w/v. poloxamer 188 in 20 mM Tris buffer at pH 8.0. In embodiments, the IV formulation provides a genomic titer of approximately 1 x 10 ¹³ to 3 x 10 ¹³ g/mL or 1.7 x 10 ¹³ to 2.3 x 10 ¹³ g/mL.

Application options for pharmaceutical compositions

In other embodiments, disclosed herein are methods of delivering a polynucleotide to the central nervous system of a patient, comprising administering rAAV9 with a genome containing the polynucleotide. In some embodiments, the delivery is intrathecal delivery of the polynucleotide to the central nervous system of the patient, involving the introduction of rAAV9 with a genome containing the polynucleotide. In some embodiments, the patient is also administered a non-ionic low-osmolar contrast agent. A non-ionic, low-osmolar contrast agent increases the level of transduction of target cells in the patient's central nervous system. In some embodiments, the rAAV9 genome is a self-complementary genome. In other embodiments, the rAAV9 genome is a single-stranded genome.

In some embodiments, the polynucleotide is delivered to a region of the brain. Areas of the brain contemplated for delivery include, but are not limited to, the motor cortex and the brainstem. In some embodiments, the polynucleotide is delivered to the spinal cord. In some embodiments, the polynucleotide is delivered to a lower motor neuron. In embodiments of the present invention, rAAV9 is used to deliver polynucleotides to neural and glial cells. In some embodiments, the glial cell is a microgliocyte, oligodendrogliocyte, or astrocyte. In some embodiments, rAAV9 is used to deliver a polynucleotide to a Schwann cell.

Uses include, for example, the treatment of lower motor neuron diseases such as SMA and ALS, as well as Pompe disease, lysosomal storage diseases, glioblastoma multiforme and Parkinson's disease. Lysosomal storage diseases include, but are not limited to, activator deficiency/Gm2 gangliosidosis, alpha-mannisidosis, aspartyl glucosaminuria, cholesteryl ester storage disease, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, galactosialidosis, Gaucher disease (type I, type II, type III), GM1 gangliosidosis (early, late childhood/juvenile, adult/chronic), I-cell disease/mucolipidosis II, childhood free sialic acid storage disease/ISSD, childhood hexosaminidase A deficiency, Krabbe disease (with early onset, late onset), metachromatic leukodystrophy, disorders representing mucopolysaccharidoses (Hurler pseudopolydystrophy/mucolipidosis IIIA, MPSI representing Hurler syndrome, MPSI representing Scheie syndrome, MPS I representing Pfaundler-Hurler-Ellis, MPS II, representing Hunter's disease, Sanfilippo syndrome type A/MPS III A, Sanfilippo syndrome type B/MPS III B, Sanfilippo syndrome C/MPS III C, Sanfilippo syndrome type D/MPS III D, Morquio syndrome type A/MPS WA, Morquio syndrome type B/MPS IVB, MPS IX representing hyaluronidase deficiency, MPS VI representing Maroteaux-Lamy syndrome, MPS VII representing Sly syndrome, mucolipidosis I/sialidosis, mucolipidosis IIIC, mucolipidosis type IV), multiple sulfatase deficiency disease Niemann-Pick (type A, type B, type C), neuronal ceroid lipofuscinosis (CLN6 disease (atypical late childhood, late onset variant, early childhood), Spielmeier-Vogt-Batten disease/childhood NCL/CLN3 disease, Finnish late infantile CLN5 variant, Bielschowsky Jansky disease/late childhood CLN2/TPP1 disease, Kufs disease/acquired NCL/CLN4 disease, northern epilepsy/late childhood CLN8 variant, Santavuori-Haltia disease/childhood CLN1/PPT disease, beta mannosidosis, Pompe disease/glycogenosis type II, pycnodysostosis, Sandhoff disease/acquired/GM2-gangliosidosis, Sandhoff disease/GM2-gangliosidosis-early, Sandhoff disease/GM2-gangliosidosis-infantile, Schindler's disease, Salla's disease/sialic acid storage disease, disease Tay-Sachs/GM2 gangliosidosis, Wolman disease.

In additional embodiments, use of the methods and materials is indicated for the treatment of a nervous system disorder, such as Rett syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease, or for the treatment of nervous system injury, including spinal cord and brain injury/damage, stroke, and types of cancer. brain. In one embodiment, the methods and materials are indicated for the treatment of spinal muscular atrophy (SMA).

There are four types of SMA, which are generally classified by age of onset and the highest level of motor function that is achieved. All forms of SMA are characterized by autosomal recessive inheritance and are caused by

mutations in the survival motor neuron 1 (SMN1) gene. Humans also carry a second, nearly identical copy of the SMN gene, called SMN2. Lefebvreet al."Identification and characterization of a spinal muscular atrophy-determining gene."Cell,80(1):155-65. Monani et al. "Spinal muscular atrophy: a deficiency in a ubiquitous protein; a motor-neuron specific disease."Neuron, 48(6):885-896. Both genes, SMN1 and SMN2, express the SMN protein, however, SMN2 contains a translationally silent mutation in exon 7, resulting in inefficient inclusion of exon 7 in SMN2 transcripts. Thus, SMN2 produces both full-length SMN protein and a truncated version of SMN lacking exon 7, with the truncated version being the predominant form. As a result, the amount of functional full-length protein produced by SMN2 significantly less (70-90%) than that produced by SMN1. Lorson et al. "A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy."PNAS, 96(11) 6307-6311. Monani et al, "A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2."Hum Mol Genet 8(7):1177-1183. Although SMN2 cannot fully compensate for the loss of the SMN1 gene, patients with milder forms of SMA usually have higher SMN2 copy numbers. Lefebvre et al., "Correlation between severity and SMN protein level in spinal muscular atrophy."Nat Genet 16(3):265-269. Park et al., "Spinal muscular atrophy: new and emerging insights from model mice."Curr Neurol Neurosci Rep 10(2):108-117. The caveat is that SMN2 copy number is not the only phenotypic modifier. In particular, the c.859G>C variant in exon 7 of the SMN2 gene has been reported as a positive disease modifier. Patients with this particular mutation have less severe disease phenotypes. Prior et al., “A positive modified of spinal muscular atrophy in the SMN2 gene.”Am J Hum Genet 85(3):408-413.

SMA type I (also called infantile or Werdnig-Hoffman disease) is characterized by symptoms of SMA being present at birth or appearing before 6 months of age. With this type, children usually have low muscle tone (hypotonia), weak cry, and difficulty breathing. They often have difficulty swallowing and sucking, and do not reach the stage of development where they can sit up without assistance. They often exhibit one or more symptoms of SMA, selected from hypotonia, delayed motor skill development, poor head support, slouched posture, and joint hypermobility. Typically, these children have two copies of the SMN2 gene, one on each chromosome 5. More than half of all new cases of SMA are SMA type I.

SMA type II or intermediate SMA is characterized by the onset of SMA occurring between 7 and 18 months of age and before the child can stand or walk independently. Children with SMA type 2 usually have at least three SMN2 genes. Late-onset SMA (also known as type III and type IV SMA, mild SMA, adult-onset SMA, and Kugelberg-Welander disease) results in varying levels of weakness. The onset of SMA type III is after 18 months of age, and children can stand and walk independently, although they may require assistance. SMA type IV occurs in adulthood, and people can walk well into their adult years. People with SMA type III or IV usually have four to eight SMN2 genes, from which sufficient amounts of full-length SMN protein can be made.

In one embodiment, the term “treatment” includes the step of administering intravenously or intrathecally an effective dose or multiple effective doses of a composition containing rAAV as disclosed herein to an animal (including a human) in need thereof. If the dose is administered before the development of the disorder/disease, the administration is prophylactic. If the dose is administered after the disorder/disease has developed, the administration is therapeutic. In embodiments, an effective dose is a dose that alleviates (or eliminates or reduces) at least one symptom associated with the disorder/disease condition being treated, that slows or prevents progression of the disorder/disease condition, that retards or prevents progression disorder/disease condition that reduces the severity of the disease, which leads to remission (partial or complete) of the disease and/or prolongs survival. Examples of disease states considered for treatment are provided herein.

In one embodiment, compositions containing rAAV of the present invention are administered intravenously to a patient in need thereof having SMA type I. In another embodiment, compositions containing rAAV of the present invention are administered intrathecally to a patient in need thereof having SMA types II. III or IV.

Disclosed herein is a method of treating SMA type I in a patient in need thereof by administering an AAV9-based viral vector via the intrathecal or intravenous route. In some embodiments, the patient is 0-9 months old. In some other embodiments, the patient is 0-6 months old. In some embodiments, where the viral vector is used to treat SMA type I in a patient, the patient's weight is determined. In some embodiments, the patient's body weight is less than 8.5 kg. In some embodiments, the patient's body weight is greater than 2.6 kg. In some embodiments, the patient's body weight is between 2.6 and 8.5 kg.

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

In some embodiments, the patient exhibits one or more symptoms of SMA. Symptoms of SMA may include hypotonia, delayed motor skill development, poor head support, slouched posture, and joint hypermobility. In some embodiments, poor head support is determined by placing the patient in a seated position with the legs in a ring shape with support at the shoulders (front and back). Head retention was assessed by the patient's ability to hold his head upright. In some embodiments, spontaneous movement is observed while the patient is in a supine position, and motor skills are assessed by the patient's ability to lift his or her elbows, knees, arms, and legs from a surface. In some embodiments, a patient's grip strength is measured by placing a finger in the patient's palm and lifting the patient until the patient's shoulder clears the surface. Hypotonia and grip strength are measured by how quickly/long the patient maintains their grip. In some embodiments, head retention is assessed by placing the patient's head in the maximum possible rotated position and measuring the patient's ability to rotate the head back toward the midline. In some embodiments, slouched posture can be assessed by placing the patient in a sitting position with the head and torso supported and observing whether the patient flexes the elbows or shoulder to achieve a stimulus that is at arm's length at shoulder level. In some embodiments, slouched posture may also be assessed by placing the patient in a side decubitus position and observing whether the patient flexes the elbows or shoulder to achieve a stimulus that is at arm's length at shoulder level. In some embodiments, motor skills are assessed by observing whether patients flex their hips or knees when their leg is stroked, tickled, or squeezed. In some embodiments, shoulder flexion, elbow flexion, hip abduction, neck flexion, head abduction, neck extension, and/or spinal flexion may be assessed using known clinical measurement systems, such as CHOP INTEND. Other SMA symptoms can be assessed according to established clinical measurement systems, such as CHOP INTEND.

In some embodiments, patients are treated after they exhibit symptoms of SMA type I (eg, one or more symptoms) determined using one of the tests described herein. In some embodiments, patients are treated before they develop symptoms of SMA type I. In some embodiments, patients are diagnosed with SMA type I based on genetic testing before they become symptomatic.

Combination therapies are also discussed in this document. The combination used herein involves either simultaneous treatment or sequential use of treatment agents. Combinations of treatments may involve the addition of certain standard medical treatments (eg, riluzole for ALS) and are also combinations with new therapies. For example, other therapies for SMA include antisense oligonucleotides (ASOs), which alter binding to pre-mRNAs and alter their splicing patterns. Singh. et al., “A multi-exon-skipping detection assay reveals surprising diversity of splice isoforms of spinal muscular atrophy genes.” Plos One, 7(11):e49595. In one embodiment, nusinersen (US Pat. No. 8,361,977 and US Pat. No. 8,980,853, incorporated herein by reference) may be used. Nusinersen is an approved ASO that targets intron 6, exon 7, or intron 7 of SMN2 pre-mRNA, modulating SMN2 splicing to more efficiently produce full-length SMN protein. In some embodiments, the treatment method includes an AAV9-based viral vector that is administered in combination with a muscle enhancer. In some embodiments, the treatment method includes an AAV9-based viral vector that is administered in combination with a neuroprotectant. In some embodiments, the treatment method includes an AAV9-based viral vector that is administered in combination with an antisense oligonucleotide drug that targets SMN. In some embodiments, the treatment method includes an AAV9-based viral vector that is administered in combination with nusinersen. In some embodiments, the treatment method includes an AAV9-based viral vector that is administered in combination with a myostatin inhibitory drug. In some embodiments, the treatment method includes an AAV9-based viral vector that is administered in combination with stamulumab.

Although delivery to the individual in need thereof is contemplated after birth, intrauterine delivery to the fetus is also contemplated.

Methods for treating patients with SMA type I using pharmaceutical compositions containing a viral vector are being considered. In some embodiments, the viral vector is formulated at a concentration of approximately 1-8×10 ¹³ AAV9 viral vector genomes/ml (gv/ml). In some embodiments, the viral vector is formulated at a concentration of approximately 1.7-2.3×10 ¹³ g/ml. In some embodiments, the viral vector is formulated at a concentration of approximately 1.9-2.1×10 ¹³ g/mL. In some embodiments, the viral vector is formulated at a concentration of approximately 2.0 x 10 ¹³ g/mL.

In some embodiments, where a viral vector is used to treat type I SMA in a patient, the AAV-based viral vector (eg, AAV SMN) is administered to the patient at a dose of approximately 1.0-2.5 x 10 ¹⁴ g i.v. kg. In some embodiments, where a viral vector is used to treat SMA type I in a patient, the AAV-based viral vector is administered to the patient at a dose of approximately 1.1 x 10 ¹⁴ g/kg. In some embodiments, where the viral vector is used to treat SMA type I in a patient, the AAV-based viral vector is infused into the patient over a period of approximately 45-70 minutes. In some embodiments, where the viral vector is used to treat SMA type I in a patient, the AAV-based viral vector is infused into the patient over approximately 60 minutes. In some embodiments, where a viral vector is used to treat SMA type I in a patient, infusion of the AAV-based viral vector into the patient is performed using an infusion pump, peristaltic pump, or any other equipment known in the art. In some embodiments, where the viral vector is used to treat SMA type I in a patient, the AAV-based viral vector is infused into the patient using a syringe pump.

The titers of the rAAV viral vector to be administered will vary depending on, for example, the specific rAAV, the route of administration, the purpose of treatment, the individual and the target cell type(s), and can be determined by standard methods in the art. rAAV titers can range from about 1×10 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 1x10 ¹³ , approximately 1x10 ¹⁴ or more DNase resistant particles (DRP) per ml. Doses can also be expressed in vector genome units (vg). Genomic titer can be determined using ddPCR, as described herein by Lock et al., or by any other methods known in the art.

Doses may also vary depending on the time of administration to the individual. Such rAAV doses may range from about 1 x 10 ¹¹ g/kg, about 1 x 10 ¹² g/kg, about 1 x 10 ¹³ g/kg, about 1 x 10 ¹⁴ g .v./kg, approximately 1×10 ¹⁵ g.v./kg, approximately 1×10 ¹⁶ g.v./kg or more vector genomes per kilogram of body weight in adults. For a neonate, doses of rAAV may range from approximately 1 x 10 ¹¹ g/kg, approximately 1 x 10 ¹² g/kg, approximately 3 x 10 ¹² g/kg, approximately 1 x 10 ¹³ g.v./kg, approximately 3×10 ¹³ g.v./kg, approximately 1×10 ¹⁴ g.v./kg, approximately 3×10 ¹⁴ g.v./kg, approximately 1×10 ¹⁵ g. w/kg, approximately 3×10 ¹⁵ g w/kg, approximately 1×10 ¹⁶ g w/kg, approximately 3×10 ¹⁶ g w/kg or more vector genomes per kilogram of body weight.

Doses may also vary depending on the time of administration to the individual. Such rAAV doses may range from about 1 x 10 ¹¹ g/kg/week, about 1 x 10 ¹² g/kg/week, about 1 x 10 ¹³ g/kg/week, approximately 1 x 10 ¹⁴ g/kg/week, approximately 1 x 10 ¹⁵ g/kg/week, approximately 1 x 10 ¹⁶ g/kg/week or more vector genomes per kilogram of body weight in adults. For a neonate, rAAV doses may range from approximately 1 x 10 ¹¹ g/kg/week, approximately 1 x 10 ¹² g/kg/week, approximately 3 x 10 ¹² g/kg/week , approximately 1×10 ¹³ g./kg/week, approximately 3×10 ¹³ g./kg/week, approximately 1×10 ¹⁴ g./kg/week, approximately 3×10 ¹⁴ g. w/kg/week, approximately 1×10 ¹⁵ g/kg/week, approximately 3×10 ¹⁵ g/kg/week, approximately 1×10 ¹⁶ g/kg/week, approximately 3x10 ¹⁶ g/kg/week or more vector genomes per kilogram of body weight per week. Doses of rAAV 1x10 ¹¹ g/1.5 kg/week, approximately 1x10 ¹² g/1.5 kg/week, approximately 1x10 ¹³ g/1.5 kg/ week, approximately 1×10 ¹⁴ g./1.5 kg/week, approximately 1×10 ¹⁵ g./1.5 kg/week, approximately 1×10 ¹⁶ g./1.5 kg /week or more vector genomes per kilogram of body weight in adults. For a neonate, rAAV doses may range from approximately 1 x 10 ¹¹ g/1.5 kg/week, approximately 1 x 10 ¹² g/1.5 kg/week, to approximately 3 x 10 ¹² g. w./kg/week, approximately 1×10 ¹³ g./1.5 kg/week, approximately 3×10 ¹³ g./1.5 kg/week, approximately 1×10 ¹⁴ g. /1.5 kg/week, approximately 3×10 ¹⁴ g./1.5 kg/week, approximately 1×10 ¹⁵ g./1.5 kg/week, approximately 3×10 ¹⁵ g. ./1.5 kg/week, approximately 1×10 ¹⁶ g./1.5 kg/week, approximately 3×10 ¹⁶ g./1.5 kg/week or more vector genomes by 1.5 kilogram of body weight per week.

In one embodiment, the dose is approximately 1.1 x 10 ¹⁴ vector genomes per kg (g.v./kg) of the patient's body weight. In one embodiment, a 5 kg patient would receive a total dose of 0.5 x 10 ¹⁴ to 5.0 x 10 ¹⁴ vector genomes. In one embodiment, the viral vector is administered in Tris-buffered saline. In one embodiment, the viral vector is administered in about 5-20 ml/kg, about 10-20 ml/kg, or about 5.5-6.5 ml/kg Tris-buffered saline.

The dose can be determined in a variety of standard ways. PCR with viral vector-specific primers can provide relative measurements, but qPCR can be used for smaller samples and absolute measurements. ddPCR is a digital PCR technique that is based on water-oil emulsion droplet technology. Baker et al., "Digital PCR hits its stride." Nature Methods , 9(6):541-544. Sykes et al., “Quantitation of targets for PCR by use of limiting dilution.” Biotechniques , 13(3)444-449. The sample is divided into tens of thousands of droplets, and PCR amplification of the template molecules occurs in each individual droplet. There is no need to construct a standard curve or primers with high amplification efficiency, so ddPCR does not typically use as much sample material as traditional PCR-based techniques. Examples of commercially available ddPCR devices include, but are not limited to, BioRad QX100 ddPCR and RainDance Raindrop Digital PCR. In one embodiment, the dose is determined using PCR. In another embodiment, the dose is determined using qPCR. In another embodiment, the dose is determined using digital droplet PCR (ddPCR). In some embodiments, PCR-based methods detect and quantify the encapsidated AAV9 viral genome using specially designed primers and probes that target the SMN gene. In other embodiments, PCR-based methods detect and quantify the encapsidated AAV9 viral genome using specially designed primers and probes targeting the chicken beta-actin promoter. In other embodiments, PCR-based methods detect and quantify the encapsidated AAV9 viral genome using specially designed primers and probes that target the CMV enhancer. In other embodiments, PCR-based methods detect and quantify the encapsidated AAV9 viral genome using specially designed primers and probes that target ITR sequences. In other embodiments, PCR-based methods detect and quantify the encapsidated AAV9 viral genome using specially designed primers and probes targeting the bovine growth hormone polyadenylation signal.

In one aspect, the dose is administered according to the following table using 2.0 x 10 ¹³ g/mL as the target drug product concentration.

Table 2. Dose Patient weight range (kg) Dose volume ^a (ml) 2.6-3.0 16.5 3.1-3.5 19.3 3.6-4.0 22.0 4.1-4.5 24.8 4.6-5.0 27.5 5.1-5.5 30.3 5.6-6.0 33.0 6.1-6.5 35.8 6.6-7.0 38.5 7.1-7.5 41.3 7.6-8.0 44.0 8.1-8.5 46.8 ^a NOTE: Dose volume is based on the upper limit of the patient's weight range.

In some embodiments, a pharmaceutical composition comprising an AAV-based viral vector is administered by infusion to a patient over about 20-70 minutes, such as over about 45-70 minutes. In some embodiments, a pharmaceutical composition comprising an AAV-based viral vector is administered by infusion to a patient over approximately 60 minutes. In some embodiments, a pharmaceutical composition comprising an AAV-based viral vector is administered by infusion to a patient using an infusion pump, peristaltic pump, or any other equipment known in the art. In some embodiments, a pharmaceutical composition comprising an AAV-based viral vector is administered by infusion to a patient using a syringe pump.

Preliminary screening of treatable patients is also provided, as well as administration of a treatment agent to patients identified in accordance with the criteria disclosed herein. AAV can induce both cellular and humoral immune responses. As a consequence, a proportion of prospective patients for AAV-based gene therapy carry pre-existing anti-AAV antibodies. Jeune et al., "Pre-existing anti-Adeno-Associated Virus antibodies as a challenge in AAV gene therapy." Hum Gene Ther Methods , 24(2):59–67. Boutin et al., "Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors." Hum Gene Ther 21:704–712. Because even very low levels of antibodies can prevent successful transduction, pre-existing antibodies to AAV pose a major barrier to the widespread use of AAV-based gene therapy. In some embodiments, the patient's anti-AAV9 antibody titer levels are determined prior to administration of the AAV-based viral vector. In some embodiments, the patient's anti-AAV9 antibody titer levels are determined using an enzyme-linked immunosorbent assay ELISA. In some embodiments, prior to administration of the treatment, the patient has anti-AAV9 antibody titers of 1:100 or lower, as determined by enzyme-linked immunosorbent assay ELISA. In some embodiments, prior to administration of the treatment, the patient has anti-AAV9 antibody titers of 1:50 or lower, as determined by enzyme-linked immunosorbent assay ELISA. In some embodiments, the patient has anti-AAV9 antibody titers greater than 1:100, as determined by a post-treatment ELISA, and is monitored for 1-8 weeks or until titers decrease to below 1:100. In some embodiments, the patient has anti-AAV9 antibody titers greater than 1:100 as determined by a post-treatment ELISA and is monitored for 1-8 weeks or until titers decrease to below 1 :50.

One approach to overcome high AAV antibody titres is the use of immunosuppressive drugs. The anti-CD20 monoclonal antibody, rituximab, in combination with cyclosporine A has been shown to be effective in reducing anti-AAV antibody titers. Mingozzi et al., “Pharmacological modulation of humoral immunity in a nonhuman primate model of AAV gene transfer for hemophilia B.” Mol Ther 20:1410–1416. Another approach is to use plasmapheresis to deplete neutralizing antibodies to the administered vector. Monteilhet et al., "A 10 patient case report on the impact of plasmapheresis upon neutralizing factors against adeno-associated virus (AAV) types 1, 2, 6, and 8." Mol Ther , 19(11):2084–2091. During plasmapheresis, blood is drawn from the patient and the plasma and blood cells are separated using either centrifugation or hollow fiber filtration. The blood cells are then returned to the patient along with either treated plasma or replacement fluids such as 4.5% human albumin in saline. A common use of therapeutic apharesis is the removal of unwanted immunoglobulins, but in this case, plasmapheresis represents an attractive approach for depleting anti-AAV antibodies. In some embodiments, the patient has anti-AAV9 antibody titers greater than 1:100, as determined by an enzyme-linked immunosorbent assay (ELISA) before or after treatment, and is treated with plasmapheresis. In some embodiments, the patient has anti-AAV9 antibody titers greater than 1:50, as determined by a pre- or post-treatment enzyme-linked immunosorbent assay (ELISA), and is treated with plasmapheresis.

Pre-existing maternal antibodies to AAV9 can be transferred to the infant patient through breast milk or placental transfer in utero. In some embodiments, the patient has anti-AAV9 antibody titers greater than 1:100, as determined by enzyme-linked immunosorbent assay (ELISA) before or after treatment, and is formula-fed. In some embodiments, the patient has anti-AAV9 antibody titres greater than 1:50, as determined by enzyme-linked immunosorbent assay (ELISA) before or after treatment, and is formula-fed.

The patient's condition can be monitored before and after administration of the treatment agent. Some patients receiving AAV-based treatments may experience thrombocytopenia, which is a condition characterized by a low platelet count. Thrombocytopenia can be detected by a complete blood count using a diluted blood sample on a hemocytometer. Thrombocytopenia can also be detected by visually examining a microscopic specimen obtained from the patient's blood (blood smear or peripheral blood smear) under a microscope. Normal platelet counts in humans range from 150,000 cells/mL to approximately 450,000 cells/mL.

In some embodiments, prior to administration, the patient has platelet counts greater than about 67,000 cells/mL, or greater than about 100,000 cells/mL, or greater than about 150,000 cells/mL. In some embodiments, prior to administration, the patient has platelet counts below about 150,000 cells/mL, or below about 100,000 cells/mL, or below about 67,000 cells/mL, and is monitored for 1-8 weeks or until until the platelet count values increase to a value greater than about 67,000 cells/ml, or to a value greater than about 100,000 cells/ml, or to a value greater than about 150,000 cells/ml. In some embodiments, where platelet count values are below about 67,000 cells/ml after administration of the viral vector, the patient may be treated with a platelet transfusion. In some embodiments, the patient is not thrombocytopenic prior to administration of the viral vector. In some embodiments, the patient has thrombocytopenia after administration of the viral vector, and is monitored for approximately 1-8 weeks or until the patient's thrombocytopenia resolves. In some embodiments, the patient has thrombocytopenia following administration of the viral vector and is treated with a platelet transfusion.

Patient monitoring may also include routine blood tests that measure platelet levels, serum protein electrophoresis, serum gamma-glutamyltransferase (GGT), aspartate transaminase (AST) and alanine aminotransferase (ALT), total bilirubin, glucose, creatine kinase (CK), creatinine , blood urea nitrogen (BUN), electrolytes, alkaline phosphatase and amylase. Troponin I levels are a general indicator of heart health, and elevated levels reflect heart damage or heart-related conditions. In some embodiments, troponin I levels are monitored after administration of the viral vector. In some embodiments, patients may have troponin I levels of less than about 0.3, 0.2, 0.15, or 0.1 μg/ml before administration of the viral vector. In some embodiments, patients may have troponin I levels of less than about 0.176 μg/ml prior to administration of the viral vector. In some embodiments, patients may have troponin I levels greater than about 0.176 μg/ml after administration of the viral vector. In some embodiments, patients undergo cardiac monitoring after administration of the viral vector until troponin I levels are less than about 0.176 μg/ml.

Aspartate transaminase (AST), alanine aminotransferase (ALT), and total bilirubin levels are general indicators of liver function, while creatinine levels monitor kidney function. Elevated levels of AST, ALT, or total bilirubin may indicate liver dysfunction. In some embodiments, the patient has normal liver function prior to administration of the viral vector. In some embodiments, prior to administration of the viral vector, the patient's liver transaminase levels are less than about 8-40 IU/L. In some embodiments, prior to administration of the viral vector, the patient has AST or ALT levels of less than about 8-40 IU/L. In some embodiments, prior to administration of the viral vector, the patient has bilirubin levels of 3.0 mg/dL. In some embodiments, prior to administration of the viral vector, patients have creatinine levels of less than 1.8 mg/dL. In some embodiments, prior to administration of the viral vector, patients have hemoglobin (Hgb) levels between 8 and 18 g/dL. In some embodiments, prior to administration of the viral vector, the patient's white blood cell (WBC) counts are less than 20,000 per mm ³ .

The effectiveness of the treatment can be determined using a variety of motor skills tests before and after treatment. Specifically, the Children's Hospital of Philadelphia Test of Motor Function in Neonatal Neuromuscular Disorders (CHOP INTEND) was developed to assess the motor skills of patients with SMA type I. Glanzman et al., "The Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND): Test development and reliability." Neuromuscular Disorders , 20(3):155–161. The CHOP INTEND test was developed after evaluating 26 infants with SMA type I, mean age 11.5 months (range 1.4–37.9 months), using the Test of Infant Motor Performance (TIMP) and the Children's Hospital of Philadelphia SMA Strength Test (CHOP TOSS ), a newly developed motor function assessment for SMA. Treatment efficacy testing is not limited to the CHOP INTEND test, but may also include other tests of motor skills known in the art, including, but not limited to, TIMP, CHOP TOSS, Peabody Developmental Motor Activities Scale, Brazelton Neonatal Behavior Assessment Test, Motor Development Analysis using a distance indicator, Assessment of Apprehension with Interactive Video (ACTIVE), Bayley Scales of Infant Development, and Composite Motor Activity Potentials (CMAP).

In some embodiments, pre-treatment baseline testing is performed using the CHOP INTEND score. In one embodiment, treatment effectiveness is determined using the CHOP INTEND score at follow-up visits. In some embodiments, CHOP INTEND includes measures such as head support, righting reactions, supported sitting torso movements, supine and prone positions. In some embodiments, CHOP INTEND includes metrics such as gravity-defying assisted rollover, prone, and supported standing.

In many gene therapy studies involving AAV-based vectors, an antigen-specific T cell response to the AAV-based vector is observed and can be expected within 2-4 weeks of gene transfer. One possible consequence of this antigen-specific T cell response is clearance of transduced cells and loss of transgene expression. In an attempt to dampen the host immune response to AAV-based therapy, patients may be given immunosuppressive drugs. In some embodiments, patients may be provided with glucocorticoids prior to administration of the viral vector. In some embodiments, patients may be provided with a corticosteroid prior to administration of the viral vector. In some embodiments, patients may be provided with an oral steroid prior to administration of the viral vector. Examples of oral steroids include, but are not limited to, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, dexamethasone, and hydrocortisone. In some embodiments, the oral steroid is or contains prednisolone. In some embodiments, the patient begins taking a prophylactic steroid at least 24 hours before administration of the viral vector. In some embodiments, the patient is provided with an oral steroid for at least 30 days after administration of the viral vector. In some embodiments, the oral steroid is administered once daily. In some embodiments, the oral steroid is administered twice daily. In some embodiments, the oral steroid is provided at a dose of about 0.1-10 mg/kg, such as about 1 mg/kg. In some embodiments, the oral steroid is provided at a dose of about 0.1-10 mg/kg/day, such as about 1 mg/kg/day. In some embodiments, AST and ALT levels are monitored after administration of the viral vector. In such embodiments, the oral steroid treatment is administered when AST and ALT levels are twice the upper limit of normal, such as determined by clinical standards and methods known in the art, or approximately 120 IU/L. In some embodiments, the oral steroid treatment agent is administered for more than 30 days, provided that AST and ALT levels are twice the upper limit of normal, such as those determined using clinical standards and methods known in the art, or exceed approximately 120 IU/l. During long-term corticosteroid treatment, the adrenal glands naturally reduce the level of cortisol production. In cases where corticosteroid treatment is suddenly stopped, the body may become deficient in cortisol. In some embodiments, where an oral steroid is provided to the patient for at least 30 days, the dose of the steroid is slowly tapered according to a schedule. In some embodiments, the dose of the oral steroid is reduced when AST and ALT levels fall below twice the upper limit of normal, eg, as determined by clinical standards and methods known in the art, or approximately 120 IU/L. In some embodiments, the dose reduction involves a stepwise reduction to 0.5 mg/kg/day for 2 weeks and then to 0.25 mg/kg/day for an additional 2 weeks. In some other embodiments, the dose reduction of the oral steroid occurs at the discretion of the physician.

Kit

The present invention also provides a kit for the treatment of SMA in a patient in need thereof, wherein the kit contains one or more doses of a pharmaceutical composition containing an effective amount or dose of a viral vector containing the SMN polynucleotide disclosed herein, and depending on the type of SMA (as further disclosed herein), the kit further contains a contrast agent (eg, Omnipaque 180) and instructions for use of the pharmaceutical drug or composition and the contrast agent.

In some embodiments, the kit contains vials of a viral vector-based pharmaceutical composition. In some embodiments, the viral vector pharmaceutical composition is provided at a concentration of approximately 1.7-2.3×10 ¹³ g/mL. In some embodiments, the viral vector pharmaceutical composition is provided at a concentration of approximately 1.9-2.1×10 ¹³ g/mL. In some embodiments, the viral vector pharmaceutical composition is provided at a concentration of approximately 2.0 x 10 ¹³ g/mL. In some embodiments, the vials contain approximately 5.9 ml of the viral vector pharmaceutical composition. In some embodiments, the vials contain approximately 8.7 ml of the viral vector pharmaceutical composition. In some embodiments, the kit does not contain a 5.9 ml bottle, contains at least one 5.9 ml bottle, at least two 5.9 ml bottles, or at least three 5.9 ml bottles. In some embodiments, the kit does not contain an 8.7 ml bottle, contains at least one 8.7 ml bottle, at least two 8.7 ml bottles, at least three 8.7 ml bottles, at least at least four bottles with 8.7 ml, at least five bottles with 8.7 ml, at least six bottles with 8.7 ml.

In some embodiments, where the kit is used to treat SMA type I in a patient, the patient's weight is determined. In some embodiments, where the kit is used to treat SMA type I in a patient, the patient weighs at least about 2.6 kg. In some embodiments, where the kit is used to treat SMA type I in a patient, the patient weighs no more than about 8.5 kg. In some embodiments, where the kit is used to treat type I SMA in a patient, the patient weighs approximately 2.6-8.5 kg. In some embodiments, where the kit is used to treat SMA type I in a patient, the AAV-based viral vector from the vials in the kit is administered to the patient. In some embodiments, where the kit is used to treat SMA type I in a patient, the AAV-based viral vector from the vials in the kit is administered to the patient at a dose of approximately 1.0-2.5 x 10 ¹⁴ g/kg. In some embodiments, where the kit is used to treat SMA type I in a patient, the AAV-based viral vector from the vials in the kit is administered to the patient at a dose of approximately 1.1 x 10 ¹⁴ g/kg. In some embodiments, where the kit is used to treat SMA type I in a patient, the AAV-based viral vector from the vials in the kit is infused into the patient over approximately 45-70 minutes. In some embodiments, where the kit is used to treat SMA type I in a patient, the AAV-based viral vector from the vials in the kit is infused into the patient over approximately 60 minutes. In some embodiments, where the kit is used to treat SMA type I in a patient, the AAV-based viral vector from the vials in the kit is infused into the patient using an infusion pump, peristaltic pump, or any other equipment known in the art. In some embodiments, where the kit is used to treat SMA type I in a patient, the AAV-based viral vector from the vials in the kit is infused into the patient using a syringe pump.

In one embodiment, the vector is administered intravenously or intrathecally. In one embodiment, the vector is administered intravenously. In one embodiment, the vector is administered intravenously in conjunction with Omnipaque 180. In another embodiment, the vector is administered intrathecally in conjunction with Omnipaque 180.

In another aspect, provided herein are methods of transducing patient target cells (including but not limited to neural or glial cells) with rAAV.

Transduction of patient cells with the rAAV disclosed herein can result in sustained expression of the polypeptide or RNA encoded by the rAAV. Thus, the present invention provides methods for administering/delivering rAAV (eg, encoding the SMN protein) to an animal or human patient. Such methods include transduction of neural and/or glial cells with one or more rAAVs. Transduction can be accomplished using gene cassettes containing tissue-specific control elements. For example, promoters that promote expression specifically in neurons or specifically in astrocytes. Examples include the neuron-specific enolase and gliofibrillary acidic protein promoters. It is also possible to develop inducible promoters under the control of the absorbed drug.

In some aspects, it is believed that cell transduction is increased when a vector of the present invention is used in combination with a contrast agent as described herein, relative to the transduction of a vector of the present invention when used not in combination with a contrast agent. In various embodiments, cell transduction is increased by at least about 1%, or at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, according to 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 at least about 450%, at least about 500% or more when the vector of the present invention is used in combination with a contrast agent as described herein, relative to the transduction of the vector of the present invention when used not in combination with a contrast agent. In additional embodiments, cell transduction is increased by about 10% to about 50%, or by about 10% to about 100%, or by about 5% to about 10%, or by about 5% to about 50%, or from about 1% to about 500%, or from about 10% to about 200%, or from about 10% to about 300%, or from about 10% to about 400%, or from about 100% to about 500%, or from about 150% to about 300%, or from about 200% to about 500%, when the vector of the present invention is used in combination with a contrast agent that is described herein, relative to the transduction of the vector of the present invention the invention when it is not used in combination with a contrast agent.

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

In some aspects, it is believed that cell transduction is further enhanced when the vector of the present invention is used in combinations with a contrast agent and when the patient assumes the Trendelenberg position (head down position). In some embodiments, for example, patients are placed in a head down position at an angle of from about 1 degree to about 30 degrees, from about 15 to about 30 degrees, from about 30 to about 60 degrees, from about 60 to about 90 degrees, or from about 90 to approximately 180 degrees) during or after intrathecal infusion of vector. In various embodiments, cell transduction is increased by at least about 1%, or at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, according to 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 at least about 450%, at least about 500% or more when the vector of the present invention is used in combination with a contrast agent and the Trendelenberg position, which are described herein, relative to the transduction of the vector of the present invention when used not in combination with a contrast agent means and Trendelenberg position. In further embodiments, cell transduction is increased by about 10% to about 50%, or by about 10% to about 100%, or by about 5% to about 10%, or by about 5% to about 50%, or from about 1% to about 500%, or from about 10% to about 200%, or from about 10% to about 300%, or from about 10% to about 400%, or from about 100% to about 500%, or from about 150% to about 300%, or from about 200% to about 500%, when the vector of the present invention is used in combination with the contrast agent and Trendelenberg position as described herein with respect to transduction vector of the present invention when not used in combination with a contrast agent and Trendelenberg position.

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

As used in the present invention and the accompanying claims, the singular form includes references to the plural unless the context clearly dictates otherwise. “Optional” or “optional” means that the subsequently described event or circumstance may or may not occur, and that the description includes cases in which the event or circumstance occurs and cases in which it does not occur. For example, the phrase “the composition may optionally contain a combination” means that the composition may contain a combination of different molecules or may not include a combination, so that the description includes both combination and non-combination (ie, individual elements of the combination). Ranges may be expressed herein as from about one particular value and/or to about another particular value. When such a range is expressed, the other aspect includes from one specific value and/or to another specific value. Likewise, when values are expressed as approximations using a previous value, it will be understood that a particular value constitutes a different aspect. It will further be understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint.

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

EXAMPLES

The following examples are to be considered as illustrative and not limiting the scope of the invention described above.

Example 1 . Preparation of GMP Master Cell Bank Precursor

Ways

Defrosting. A vial of individual cells (1 x 10 ⁶ cells) was thawed in a water bath at 37°C for approximately 1 minute and the contents were diluted in 5 ml of pre-warmed complete culture medium. Cells were transferred to a T-mattress with a surface area of 25 cm ² and grown in an incubator at 37°C for 4 days, replacing the culture media daily with prewarmed complete growth medium.

Selection based on increased adhesiveness. To select highly adhesive cells, cells were cultured using the following procedure. Once the cells reached 95% confluency in a mattress with a surface area of 25 ^cm2, the cells were subcultured. Cells were washed with 5 ml PBS, then dissociated with 0.5-1 ml HyQTase for ~2 minutes at room temperature. Dissociation was stopped by adding 5 ml of complete nutrient medium and pipetting repeatedly to dissociate cell agglomerates. The cell suspension was then centrifuged for 4 minutes at 200 x g. The supernatant was discarded, and the cell pellet was resuspended in 10 ml of complete culture medium. Cells were transferred to a 75 cm ² surface area mattress after 4 hours of incubation in a 37°C incubator, and low-adherent cells were washed out by aspirating cell culture medium to remove low-adherent and non-adherent cells. The culture medium was replaced with 10 ml of prewarmed complete culture medium. Based on visual inspection, this process reduced cell mass by up to 35% of the cell number. Before subculture again, cells were incubated for an additional 2 days. This selection process, consisting of changing the medium 4 hours after seeding, was performed three times before allowing the selected cell population to expand (Figure 2 and Figure 3). At the last stage of selection, cells were seeded into 2 mats with a surface area of 175 cm ² with a final volume of 25 ml. It was noted that after the last change of medium 4 hours after seeding, there was a decrease in the level of cell loss.

Ensuring cell reproduction. Subsequently, after the cells in the 2 mattresses with a surface area of 175 cm ² reached a state of confluence, cell proliferation was ensured. Cells were washed with 15 ml PBS, then dissociated with 3 ml HyQTase and incubated for ~2 minutes at room temperature. Dissociation was stopped by adding 10 ml of complete nutrient medium. The cell suspension was then centrifuged to obtain 2 cell pellets after aspiration of the supernatant. Each pellet was resuspended in 8 ml of complete culture medium and 2 ml of this concentrated cell suspension was added to 8 mattresses with a surface area of 175 cm ² . The mattresses were prepared by adding 20 ml of complete culture medium, resulting in 22 ml of cell suspension and a split ratio of 1:4. In the next propagation stage, the same procedure was used with the following modifications: 4 mattresses with a surface area of 175 cm ² were propagated with a split ratio of 1:2, and 4 mattresses with a surface area of 175 cm ² were propagated with a split ratio of 1:3. This gave a total of 20 mattresses with a surface area of 175 cm ² .

Biomass collection. Cages were assembled from 20 mattresses with a surface area of 175 cm ² . Cells were washed with 15 ml PBS, then dissociated with 3 ml HyQTase as previously described. Cell dissociation was stopped by adding 10 ml of complete culture medium and collected in 50 ml tubes, while a cell suspension of 4 mattresses with a surface area of 175 cm ² was added to 1 50 ml tube. This resulted in 5 50 ml tubes containing 40 ml of cell suspension each. The tubes were centrifuged to obtain cell pellets, the supernatants were aspirated and the cells were resuspended with 10 ml of complete culture medium, resulting in 50 ml of cell suspension.

The volume was divided into 2 tubes of 50 ml each, with the total volume of the cell suspension in each tube being 25 ml. Samples diluted 1:2 were used to count the number of viable cells per tube using a hemocytometer and toludine (trypane) blue. The sample from tube 1 had a viable cell count of 1.99 x 10 ⁶ cells/mL, giving a cell concentration of 3.98 x 10 ⁶ cells/mL, and the sample from tube 2 had a viable cell count of 2.4 x 10 ⁶ cells/ml (total 1x10 ⁸ cells), which gave a cell concentration of 4.8x10 ⁶ cells/ml (total 1.2x10 ⁸ cells). Thus, a total of 2.2×10 ⁸ cells were collected. Both tubes were centrifuged again (6 minutes, 200 x g) and the pellets were resuspended in 10 ml (tube 1) and 12 ml (tube 2) of freezing medium, respectively, adjusting the cell concentration to 1 x 10 ⁷ cells/ml. The two cell suspensions were combined and 1 ml aliquots (each containing 1×10 ⁷ cells) were filled into 22 sterile cryofials (Table 3).

Table 3. Calculation of the total number of cells collected.

Cell concentration
in sample 1:2 Cell concentration
when collecting Total cells at
collection Test tube 1 1.99×10 ⁶ cells/ml 3.98×10 ⁶ cells/ml 1×10 ⁸ cells Test tube 2 2.4×10 ⁶ cells/ml 4.8×10 ⁶ cells/ml 1.2×10 ⁸ cells Total - - 2.2×10 ⁸ cells

The filled vials were then transferred to a freezer overnight with fresh isopropanol in a -80°C freezer for controlled rate freezing. The frozen vials were then transferred into liquid nitrogen gas into a liquid nitrogen tank. Ten vials were transferred to dry ice for storage in a GMP facility.

HEK293 cells from ATCC were thawed and successfully adapted to enhance adhesiveness in 3 subcultures before allowing expansion and successful seed bank establishment. The seed bank was tested for growth and the presence of introduced pathogens of infectious diseases (mycoplasmas, fungi and bacteria). The study showed that the seed bank was suitable for storage as a master cell bank in a GMP facility.

Example 2. Accumulation process

An accumulation process (see, for example, Figure 4) was used to obtain an intermediate product obtained from a working cell bank, the accumulation process comprising the steps of (a) culturing the adherent cells, (b) transfecting the cultured cells with three plasmids, as shown in Figure 1 (e.g., including the AAV SMN described herein), to ensure production of an AAV-based viral vector, (c) ensure lysis of adherent cells to release the AAV-based viral vector, (d) purify viral particles by filtration to remove any intact cells or cellular debris, and (e) subjecting the purified product from step (d) to tangential flow filtration and (f) freezing the resulting purified viral particle intermediate. In alternative embodiments, AAV produced by the accumulation process disclosed herein encodes an shRNA targeting SOD1 or MECP2 disclosed herein.

(a) Cultivation of adherent cells

HEK293 cells were thawed and expanded in seven subcultures in disposable mattresses using CO ₂ incubators. Thawed cells were washed with cell propagation medium, centrifuged, and resuspended with fresh cell propagation medium. The resuspended cells were seeded into a mattress containing a nutrient medium for cell propagation and incubated.

Once the cells were confluent, they were washed with DPBS and recovered from the mattresses using TrypLE Select enzyme solution. Cell expansion medium was added to neutralize the enzyme solution, and the suspended cells were separated and subcultured into new mats containing cell expansion medium. This process of ensuring reproduction was repeated 7 times. In the final iteration, the suspended cells were not subcultured into the mattresses, but instead the cell suspension was inoculated into a bioreactor for further propagation.

Before inoculation, the iCELLis 500/200 m ² or iCELLis 500/333 m ² adherent cell bioreactor was prepared for inoculation. Preparatory work included unpacking the disposable bioreactor, physical inspection, leak testing, connecting the tubing system, and balancing the sensors. To equilibrate the bioreactor, a nutrient medium was loaded for cell propagation. Once the pH (pH 6.9-7.5), temperature (35°C to 39°C) and dissolved oxygen (40-125%) values were confirmed to be within the specified ranges, the bioreactor was seeded at target seeding density of 4800-7000 cells/ ^cm2 (for a reactor measuring 200 ^m2 ) or 5000-12000 cells/ ^cm2 (for a reactor measuring 333 ^m2 ). The cell suspension from the previous step was added to the media in a recirculating media bag and circulated through the bioreactor.

(b) Transfection of adherent cells

At days 4, 5, or 6 from inoculation into the bioreactor, adherent HEK293 cells were transfected by co-precipitation with three DNA plasmids and PEI. The 3 plasmids used for this transfection were pSMN, pAAV2/9 and pHELP. The DMEM nutrient medium used for cell propagation was removed from the bioreactor and replaced with transfection medium. The scAAV9.CB.SMN vector was generated by transfecting three DNA plasmids into human embryonic kidney adherent cells (HEK293) by co-precipitation with polyethylenimine ("PEI") in a large-scale adherent cell bioreactor. The vector plasmid pSMN contained cDNA of the human survival motor neuron (SMN) protein. The 3 plasmids used for this transfection were pSMN (222 mg), pAAV2/9 (333 mg), and pHELP (444 mg). Plasmids could be transfected at a ratio of 1:1:1 mol. Before adding PEI and co-precipitation plasmids, the transfection medium was allowed to equilibrate in the bioreactor until the bioreactor temperature was >30°C. The process of co-precipitation of PEI and plasmids involved adding plasmids to the transfection medium and filtering through a 0.2 μm pore size filter into the reaction bag. PEI was added to the transfection medium and then to the reaction bag. The ratio of PEI to plasmid was approximately 1:1 by weight. The reaction medium of PEI and plasmids was mixed manually to obtain a homogeneous suspension, and the reaction took place over a period of 15-30 minutes. At the end of the reaction time, PEI and co-precipitation plasmids were transferred from the reaction bag to the bioreactor. PEI and co-precipitation plasmids were allowed to mix in the bioreactor for 1-2 hours (alternative durations are described in Example 7) before mixing was resumed. The transfection medium was recirculated in the bioreactor for 18-24 hours before being replaced with the next medium.

On bioreactor day 6, 18 to 24 hours after transfection, the bioreactor was emptied and the transfection medium in the recirculating media bag was replaced with post-transfection medium. The bioreactor was refilled with media for post-transfection use by recirculating the bioreactor. On day 7, 18 to 24 hours after the media change on day 6, the post-transfection media in the recirculating bag was replaced with a fresh bag of post-transfection media. The bioreactor was not emptied during this stage. Recirculation of the medium was continued until biomass was collected, usually on day 9.

(c) Ensure lysis of transfected adherent cells

After 9 days in the bioreactor, the last samples were taken from the reactor before collecting the biomass and the process of complete cell lysis began. Benzonase was added to the bioreactor to a final concentration of 100 IU/ml. The benzonase was left to stir in the reactor and lysis buffer was added to the reactor. The lysis buffer was stirred in the reactor at 15-25°C for 2 hours before the contents of the bioreactor were transferred to a biomass collection bag. Sucrose saline solution (SSS), which quenches the reaction with benzonase, was added to the biomass collection bag and stirred for 15 minutes. The bioreactor was then rinsed with bioreactor wash buffer for 15 minutes, and then the rinse was collected in a bag to accumulate the collected biomass along with the quenched cell lysis solution. After adding the rinse to the collection bag, the contents were stirred for 15 minutes and samples of the total biomass collected were collected.

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

The mixed total collected biomass was filtered through a POD depth filter into a collection bag. After all the total collected biomass was filtered, the TFF1 buffer was passed through a depth filter. The pool obtained using a depth filter was mixed and samples were taken from it. The pool obtained using the depth filter was then filtered through a 0.45 μm pore size filter to further clarify the total biomass material collected. TFF1 buffer was then passed through a 0.45 μm filter.

During the TFF1 step, 5.0 m ² regenerated cellulose membrane cassettes with a cutoff of MW 300 kDa were washed, disinfected with NaOH solution and equilibrated with TFF1 buffer. The concentration phase of this process was intended to reduce the volume of clarified harvested biomass by approximately 10 times. After reaching the target retentate volume, the diafiltration process began. The retentate was diafiltered with 6 dia volumes of TFF1 buffer. Alternatively, the retentate could be diafiltered with more than 6 dia volumes of TFF1 buffer, such as 10 dia volumes, 12 dia volumes, or 15 dia volumes. After reaching 6 diavolumes of the total permeate flow, the retentate was again concentrated and collected in a storage bag. Two successive membrane rinses were performed to maximize product recovery from the TFF system to obtain the drug intermediate.

(e) Freezing the intermediate product

The TFF1 intermediate was aliquoted into sterile 1 or 2 liter PETG bottles in an LFH fume hood and then frozen on dry ice or in a freezer and stored at -60°C.

Table 4. Buffers used in the accumulation process

Name Compound Process step(s) applied Nutrient medium for cell proliferation DMEM with 10% FBS, 4.5 g/L glucose, 4 mM L-glutamine Cell proliferation before transfection in the iCELLis bioreactor Transfection medium DMEM without FBS, without calcium,
without L-glutamine and 4.5 g/l glucose Transfection in the iCELLis bioreactor Medium for use after transfection OptiMEM with 2.3 g/l glucose,
4 mM L-glutamine and no FBS After transfection in the iCELLis bioreactor Lysis buffer 500 mM HEPES, 10% Tween 20, 20 mM MgCl ₂ , pH 8.0 Cell lysis in the iCELLis bioreactor Sucrose saline solution (SSS) 3700 mM NaCl, 10% sucrose Lightening Buffer for washing the bioreactor 20 mM Tris, 1 mM MgCl ₂ , 500 mM NaCl, 1% Tween 20, 1% sucrose Collection of biomass from the iCELLis bioreactor Buffer for TFF1 20 mM Tris, 1 mM _MgCl2 , 500 mM NaCl, 1% sucrose Lightening, TFF1 Disinfectant buffer for TFF1 0.5 M NaOH Membrane disinfection for TFF1

Example 3. Isolation and purification process

An isolation and purification process (see, for example, Figure 5) was used to process the intermediate product resulting from TFF1 to obtain the filtered drug substance. In some embodiments, the isolation and purification process disclosed herein can be used to process an intermediate containing AAV SMN, AAV MECP2, or AAV encoding shRNA targeting SOD1 described herein. The steps in the isolation and purification process include: (a) acidification and clarification of the intermediate (using filtration), (b) purification using cation exchange chromatography, (c) filtration using tangential flow filtration ("TFF2"), (d) ultracentrifugation using CsCl buffer to separate filled and empty viral capsids (e) collecting AAV viral vectors and (d) filtering the collected AAV viral vectors using a second tangential flow filtration step ("TFF3").

(a) Acidification and clarification

The TFF1 intermediate material from the accumulation process (thawed to room temperature if previously frozen) was pumped into a stirrer bag. The combined TFF1 intermediate was mixed and sampled for titer determination. Immediately thereafter, the combined TFF1 intermediate was treated by adding 11-14% Tween 20. Tween 20 was used to stimulate flocculation of host cell bulk proteins and DNA under acidic pH conditions. The mixture was left to incubate for 12-20 hours. The pH was then lowered by adding acidifying buffer (1 M glycine) to pH 3.3–3.7. The precipitate formed after lowering the pH was then removed by filtering the solution through Clarisolve depth filters with a size of 1.1 m ² and Millistak + C0HC with a size of 2.2 m ² and fine filters with a pore size of 0.45 μm. This process resulted in an acidified and clarified TFF intermediate.

(b) Purification by cation exchange chromatography

A cation exchange chromatography (CEX) step was used to separate viral capsids from proteins, DNA, and other process contaminants such as host cell lipids, TWEEN-20. A CIMmultus S03-8000 Advanced Composite Column (0.2 μm pore size) (8.0 L) was used for this step and was operated using a process-automated chromatography system. Buffers and solutions are described in following table.

Table 5. Buffers and solutions for one CEX run

Name of solution Compound Purpose Volume (l) per CEX cycle with 8 l volume WFI WFI Column washes 200 L Buffer A for CEX 50 mM glycine, 500 mM NaCl, 1.0% sucrose, 0.20% poloxamer 188,
pH 3.5 ± 0.1 at 20°C Equilibration, washing, elution 256 L Buffer B for CEX 50 mM glycine, 2.0 M NaCl, 1.0% sucrose, 0.20% poloxamer 188,
pH 3.5 ± 0.1 at 20°C Column equilibration and elution 40 L Monolithic Column Cleaning Solution 1 M NaOH, 2 M NaCl Column disinfection, CIP 96 L 1 M ammonium acetate 1 M ammonium acetate Restoring the pH of the Column 40 L Neutralization buffer, pH 9.0 1.0M Tris
pH 9.1 ± 0.1 at 20°C pH adjustment of CEX product 0.5 L Storage solution 20% ethanol in WFI Column storage 40 L

The acidified and clarified intermediate product resulting from TFF (i.e., CEX loading) was loaded onto a purified and equilibrated CEX column. Conditions were such that the viral vectors were bound to the monolithic column. Unbound material was washed from the column using CEX Buffer A. The product was eluted from the resin using a gradient of CEX Buffer B into CEX Buffer A. Accumulation of fraction 1 began at the beginning of the elution gradient over 10 column volumes (CV) to a target volume of 2.3-2.7 CV. The chromatography column was discarded after each batch (ie, the chromatography column was not reused). The CEX product eluate (fraction 2) was then neutralized using neutralization buffer to pH 7.7-8.3.

(c) Filtration by tangential flow filtration (TFF2)

At the TFF2 step, the viral vector was concentrated, protein contaminants were removed, and the buffer was replaced with a suitable buffer for the CsCl ultracentrifugation step. The neutralized CEX eluate was processed using a TFF system equipped with a 0.3 m ² MWCO 300 kDa regenerated cellulose membrane.

The volume of neutralized eluate resulting from CEX was reduced to the target retentate volume. After reaching the target retentate volume, diafiltration was started in batch TFF mode (batch mode). The retentate was diluted 2-fold with NaCl-based diafiltration buffer at TFF2 and the retentate was concentrated to its original volume. This was repeated until diafiltration was completed using NaCl-based diafiltration buffer at TFF2. The retentate was then diluted 2-fold with CsCl diafiltration buffer at TFF2 and the retentate was concentrated to its original volume. This was repeated until diafiltration was completed using NaCl-based diafiltration buffer at TFF2.

The retentate was further concentrated to a final mass based on the physical titer of neutralized CEX eluate, system retention volume, system wash volume, and retentate density to achieve the desired target vector concentration and recovered into a storage bag. One cycle of flushing the system with CsCl diafiltration buffer at TFF2 was followed by a product purge to maximize product recovery from the TFF system. To measure the physical titer, a sample of the retentate obtained from TFF2, which contained the retentate and the wash, was taken. TFF2 membrane cassettes were discarded after each batch (i.e., TFF membranes were not reused).

Table 6. Buffers for TFF2

Name of solution Compound NaCl-based buffer for diafiltration at TFF2 20 mM Tris, 2 mM MgCl ₂ , 150 mM NaCl, 0.2% poloxamer 188, 1% sucrose, pH 8.1 ± 0.1 at 20°C Buffer with CsCl for diafiltration at TFF2 20 mM Tris, 2 mM MgCl ₂ , 3 M CsCl, 0.2% poloxamer 188, pH 8.1 ± 0.1 at 20°C

(d) Ultracentrifugation with CsCl

The purpose of the ultracentrifugation step was to remove empty capsids from full capsids by applying cesium chloride gradient ultracentrifugation. The retentate resulting from TFF2 was added to ultracentrifugation tubes and the tubes were sealed. The tubes were placed in an ultracentrifuge, such as an Optima XPN 100 automatic ultracentrifugation system or equivalent system equipped with a 50.2 Ti rotor or equivalent rotor. The filled tubes were centrifuged at 45,000 rpm for 22 hours at 20°C.

(e) Collection of AAV-based viral vectors

After completion of the centrifugation step, the tubes were removed from the ultracentrifuge and placed in a microbiological safety cabinet. The tubes containing the product were placed on ring stands above the waste container. A lamp was placed directly below the tube to visualize the band of empty capsids (band A, the band occupying the topmost position), the paired bands of full capsids (band B and band C, the top and bottom band of a pair), and the band occupying the lowest position under the pair ( stripes D). The tubes were pierced with a needle attached to a syringe to release air from the tubes and strips B, C and D were removed using the needle. The collected material was transferred to a bag for accumulation. The collected ultracentrifuged pool (UC-pool) was diluted with TFF2 buffer to achieve the appropriate initial concentration of CsCl in the TFF2 loading material. The diluted UC pool was processed at the TFF3 stage. The buffer for the CsCl ultracentrifugation step is listed in the table below.

Table 7. Buffer for ultracentrifugation with CsCl

Name of solution Compound buffer with CsCl for diafiltration at TFF2 20 mM Tris, 2 mM MgCl ₂ , 3 M CsCl, 0.2% poloxamer 188, pH 8.1±0.10

(f) Filtration by tangential flow filtration (TFF3)

At TFF3 step, CsCl was removed and the complete vector was concentrated using buffer to obtain the final composition. A tangential flow filtration system was used in combination with 50 cm ² MWCO 300 kDa regenerated cellulose membranes. The viral vector was contained by membranes.

The volume of the diluted UC pool was reduced to the target retentate volume. Once the target volume was reached, continuous diafiltration was started at a constant volume of retentate. The retentate was diafiltered using TFF3 buffer. A sample of the diafiltered retentate was collected for

physical titer measurements. The retentate was further concentrated based on the mass of the permeate, which was calculated from 1) the volume of retentate in the TFF system at the end of diafiltration, 2) the physical titer of the diluted UC pool, 3) the target drug concentration (DS), 4) the combined volume of system washes and washes filters and 5) buffer density for TFF3. TFF3 membrane cassettes were discarded after each batch (ie, the cassettes were not reused).

Table 8. Buffers for TFF3

Name of solution Compound Buffer for TFF3, option 1 20 mM Tris, 1 mM MgCl ₂ , 200 mM NaCl, 0.001% poloxamer 188, pH 8.0 ± 0.1 at 20°C Buffer for TFF3, option 2 20 mM Tris, 1 mM MgCl ₂ , 200 mM NaCl, 0.005% poloxamer 188, pH 8.0 ± 0.1 at 20°C

To isolate the vector from the TFF system, two successive rinses of the TFF membranes were performed using 20 ml of TFF3 buffer. Washings were collected through a 0.2 mm Pall Supor® EKV sterilizing filter (Mini Kleenpak). Filter rinsing was performed with TFF3 buffer to recover vector remaining in the filter, if any, and to adjust the final volume of the filtered pool resulting from TFF3 (ie, drug substance, DS). DS was aliquoted into 125 or 250 mL PETG bottles and frozen at < -60°C.

Example 4. Compilation and filling

The drug product (DP) was a single dose of preservative-free, sterile, clear to slightly opaque, colorless to pale white solution for intravenous infusion of a non-replicating, self-complementary AAV9 vector at a target concentration of 2.0 x 10 ¹³ g.v./ml. DP contained 20 mM Tris, 1 mM MgCl ₂ , 200 mM NaCl, 0.005% w/v. poloxamer 188. The pH range of the solution was from 7.7 to 8.3.

Table 9. Typical process for obtaining a medicinal product - buffer composition

Name of solution Compound Drug Product Formulation Buffer (DP) Option 1 20 mM Tris, 1 mM MgCl ₂ , 200 mM NaCl, 0.001% poloxamer 188, pH 8.0 ± 0.1 Drug Product Formulation Buffer (DP) Option 2 20 mM Tris, 1 mM MgCl ₂ , 200 mM NaCl, 0.005% poloxamer 188, pH 8.0 ± 0.1

DP was filled into sterile, ready-to-use 10-mL Crystal Zenith (CZ) vials, sealed with sterile, ready-to-use chlorobutyl elastomer stoppers and sealed with sterile aluminum 20-mm tear-off caps. The vials were filled at a nominal fill volume of either 5.5 mL or 8.3 mL. The target overflow was 0.4 mL, and the vials were filled to 5.9 ± 0.1 mL or 8.7 ± 0.1 mL.

Example 5. Activity Analysis

The relative activity of the drug product was measured using an in vivo quantitative assay. The analysis used an established mouse model of SMA disease. Breeding pairs for the SMAΔ7 mouse strain (Jackson Laboratories, no. 005025) have a normal phenotype, but ∼25% of their offspring are homozygous for the target mutation in the SMN gene and exhibit an SMA-like phenotype. By day 5 they show signs of muscle weakness, and the following week they develop an abnormal gait and a tendency to fall. Jackson Laboratories reports that the mean survival of animals with the SMA-like phenotype is ~15 ± 2 days. In exploratory studies, the median survival time for animals with the SMA-like phenotype without treatment was demonstrated to be 16.3 days (geometric mean; n=3 studies; 10 mice per study).

A biologically active drug product administered by intravenous (IV) infusion results in increased survival time, which is dose dependent (g.v./kg). The activity of the drug product was measured relative to the reference material (previous batch of vector). The titer of drug product and reference material (vector genomes/ml; g.v./ml) was determined using droplet digital polymerase chain reaction (ddPCR). The vector was diluted in saline to achieve each of the three indicated dose levels to be administered to mice with an SMA-like phenotype.

Assay results were considered acceptable if the assay met the eligibility criteria. The eligibility criteria for the analysis were as follows.

1. Acceptance limit for negative control sample (15 ± 2 days, median survival value);

2. Acceptance limit for positive control sample (>40 days, median survival);

3. Acceptance limits on the dose response curve for median survival using the reference standard.

In this study, a previous batch of vector (hereinafter referred to as the “previous batch”) was used to determine the linear correlation between the median survival (in days) of the SMAΔ7 mouse upon dosing of the drug product (hereinafter referred to as the “test batch”) at five different dose levels, including 0 (zero) dose using 0.9% saline (no treatment group). The relative potency of the drug product from the test lot was determined by comparing the linear regression curve of the reference standard from the previous lot with the linear regression curve of the drug product from the test lot. This was accomplished by applying the relationship between the y-intercept and the slope of each linear regression line (i.e., reference standard and test drug). The calculation of the percentage of relative activity is described in equation (1) below.

%RP = [(y-intercept/test drug slope) ÷ (y-intercept/reference standard slope)] X 100 (1)

The previous batch used in the phase 1 clinical trial was used as the reference standard batch and its activity was taken as 100%.

A Δ7 mouse model was used to demonstrate the effectiveness of anti-SMA therapeutics, including the drug product. Control animals treated with TFF3 buffer (vehicle medium) provided a reliable baseline control against which product activity in terms of increased median survival could be measured. During drug product development, three (3) doses (excluding vehicle dose) determined by genomic titer using droplet digital PCR (ddPCR) were identified to influence survival in a mouse model with a linear correlation if the dose administered (g.v./kg) logarithmically transformed and plotted against the median survival (in days) of the treated neonatal SMAΔ7 mouse. See standard titers (g/mL) in Table 11 for low, medium, and high titer standards. In addition, TFF buffer solution (carrier medium) was used both as the zero point (0) of the calibration curve and as a negative control. The dose for which survival was observed to be ≥40 days (greater than the dose for which a doubling of median survival was observed) was also included as a positive control.

Table 11. Target doses

Dose (g.v./kg) Median survival (days) Standards and controls Buffer solution for TFF3 (carrier medium) 15±2 Negative control
(without processing) 1.10×10 ¹⁴ Median survival ≥40 days Positive control 1.00×10 ¹² 16±2 Standard-1 1.2×10 ¹³ 22 ± 3 Standard-2 7.5×10 ¹³ 31 ± 3 Standard-3

Dosage solution preparations (see example of dilution scheme in Table 12).

Negative control - buffer solution for TFF3 (buffer for obtaining the final composition of the drug product) was used as a negative control.

Positive control - a series of the test drug was obtained at a concentration of 1.10 x 10 ¹⁴ g/kg using a buffer solution for TFF3. For an example of a dilution scheme, see Table 12.

Reference Standard Solutions - A batch of reference standard was prepared at the three concentrations shown in Table 11 using TFF3 buffer solution.

Table 12. Dilution scheme for reference standard and test product (example)

Dose (g.v./kg) Titer of reference standard/test drug based on ddPCR results (g.v./ml) Transform
volume in g.v./µl Reference standard volume/
study drug for use (µl) Saline solution
(µl) Total dose volume
(µl) 1.2×10 ¹³ 5.0×10 ¹³ 5.0×10 ¹⁰ 2.6 47.4 50.0

Preparation of study drug - the study drug was diluted with TFF3 buffer solution. Dilutions were calculated to obtain the test doses (g.v./kg) indicated in Table 11 per mouse in a total final volume of 50 µl. Dilutions were made for 12 mice at a time with one additional volume as a positive control aimed at increasing the minimum lifespan of treated mice to a median survival (days) of ≥40 days.

Acceptance limits on control samples

Negative Control (Non-Treatment Mice)—The assay acceptance limit for the negative control group was that SMAΔ7 mice met the median survival criterion of 15 ± 2 days. In addition, any mouse that died after ≤10 days was excluded from the analysis.

Positive Control (group that was treated with the target clinical dose) - The assay acceptance limit for the positive control group was that the minimum lifespan of treated mice had to be ≥40 days, which was the median survival value. In addition, any mouse that died after ≤10 days was excluded from the analysis.

Acceptance limits on the dose response curve for the reference standard

Assay criteria for a linear dose response curve for a reference standard plotted as median survival (days) versus administered dose (g.i./mL) will be determined. Y-intercept/slope ratio - the linear regression curve of the dependence of the median survival value (in days) on the administered dose (g.v./kg) for the reference standard and the study drug was determined. For each case of linear regression, the ratio of the y-intercept to the slope was calculated.

Presentation of results

Qualitative presentation of relative activity results—assay eligibility criteria for each assay were assessed before determining the median point survival (in days) determined after ≥40 days from positive control material. If the median survival of the positive control group was ≥40 days, the study drug can be used if the criteria presented below are met.

Quantitative presentation of relative activity results - Before quantifying the relative activity of the test drug, assay suitability criteria were assessed for each assay. Relative activity for the test drug could be recorded when the positive control reached ≥40 days, and the median survival for the group of mice representing the upper standard dose of 7.5 x 10 ¹³ g.v./kg was 31 ± 3 days. Percentage relative potency (%RP) for the study drug was calculated using the y-intercept and the linear regression slope of the dose-response curve for median survival (in days) as follows:

%RP=100% * [(Y-intercept/test drug slope) ÷ (Y-intercept/reference standard slope)]

Example 6. Surfactant Inactivation Study

To separate the effects of low pH and Tween, a sample of the drug intermediate derived from TFF1, which had a pH of 7.6 and contained 4-8% Tween 20, was used for the surfactant inactivation study. Tween 20 concentration in the TFF1 intermediate was approximately 2.5 times lower than the full process when Tween 20 was added to the TFF1 intermediate before acidification. This lower concentration of Tween 20 was considered the worst condition for surfactant-activated inactivation during drug production. The test formulation for the virus inactivation step by surfactant treatment was a TFF-1 intermediate containing 4-8% Tween 20. In order to evaluate the ability of virus inactivation by the surfactant treatment step, both XMuLV and PRV to introduce the virus into the study drug in duplicate experiments. Viral quantification was performed using a plaque infectivity assay.

The TFF1 production step produced a surfactant concentration range of 4-8% Tween 20. The TFF1 intermediate was combined and an additional 12% Tween 20 was added to the TFF1 intermediate at an operating temperature of 16-20°C C with stirring for 12-20 hours. The virus removal process was carried out at a Tween 20 concentration of 4-8% and at a controlled temperature of 16.0 ± 0.1°C. The duration of the inactivation process was 120 minutes compared to the standard process time of 16 hours.

During the inactivation process, the inactivation load was obtained by measuring the volume of the test drug, equilibrating to the target temperature, and then adding the virus. Inactivation load samples were taken at six (6) time points to monitor inactivation kinetics over time: <1 minute, 15 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes. After collection, each sample was diluted in a nutrient medium to stop virus inactivation and analyzed for the presence of virus. To increase the sensitivity of the assay, larger volumes of samples collected at these time points were analyzed for virus at 90 min and 120 min. Due to the presence of Tween 20 in the test preparation at this stage, the titer of the inactivation load was calculated based on the titer of the introduced virus and the volume of the introduced virus.

Virus inactivation by surfactant (Tween 20) treatment was shown to be effective, as evidenced by LRV values greater than 4 log10 for both viruses at the 90 minute time point. The kinetics of inactivation by surfactant treatment are illustrated in Figure 6 and Figure 7 as graphs of the rate of virus inactivation over 120 minutes during treatment with Tween 20 surfactant.

Example 7 Effect of Higher Inoculation Density, Earlier Transfection, and Earlier Harvest and Time of DNA/PEI Mixing on Drug Production

The effects of higher inoculation density, transfection and harvest one day earlier, and DNA/PEI mixing time on DS production were assessed. Each condition was evaluated in duplicate in a 1.6 m ² bioreactor.

Materials and methods

Increase in cell mass

HEK 293 cells were thawed and resuspended in DMEM supplemented with 10% FBS. Cells were centrifuged at 209 x g, 5 min, at room temperature, then the supernatant was removed and fresh DMEM + 10% FBS was added. Cells were counted for viable cell density and viability, and seeded into 2 T pads with a surface area of 175 cm ² and incubated at 37.0°C, 5% CO ₂ for three days until cultures reached ~90% confluence. For each cell subculture, the used media was removed, the mattresses were washed with PBS (-CaCl ₂ , -MgCl ₂ ) at 0.08 ml/cm ² , and then the mattresses were treated with TrypLE Select (0.04 ml/cm ² ) and incubated at 37.0°C, 5% CO ₂ for 2-3 minutes. Trypsin was quenched using DMEM+10% FBS (0.04 ml/ ^cm2 ). Cells were propagated and seeded according to the diagram presented in Figure 8 .

Inoculation and cell monitoring

The bioreactors were inoculated in duplicate with HEK 293 cells at a target density of 8000 cells/ ^cm2 and 12000 cells/ ^cm2 in a nutrient medium (DMEM with high glucose content + 10% FBS from Australia + a mixture of 1:100 penicillin and streptomycin (Pen Strep) with stirring. The following processing parameters were set: pH 7.23, 37.0°C, 55% dissolved oxygen (DO) and linear velocity 2 cm/s. 24 hours after sowing, recirculation of the DMEM nutrient medium (0.188 ml/cm ² ) was started. at recirculation rate (12.5 ml/min.) Daily samples collected for determination of pH, metabolites and nutrients using a stand-alone device were read using Nova BioFlex on the fourth day (12,000 cells/ ^cm2 ), on the fifth day. (8000 cells/cm ² ) and on the ninth day after seeding, three fibers were selected and lysed using PBS, A100 and B100 solutions (ChemoMetec) in a 1:1:1 v/v ratio and the total was counted (NucleoCounter NC-200). number of cores to monitor crop growth.

Transfection

On the fourth day (12,000 cells/cm ² ) and the fifth day (8,000 cells/cm ² ) after cell inoculation, recycling was stopped and the cells in each bioreactor were transfected with plasmid DNA and polyethylenimine (PEI). DNA and PEI were mixed at a ratio of 1:1 mg/mg. Transfection with plasmid DNA was carried out in a mass ratio of 1:1.5:2, while plasmid pSMN, plasmid pAAV2/9 and plasmid pHELP were added to DMEM -/- medium with a high glucose content, -CaCl ₂ , -L-glutamine, and filtered through a 0.2 µm filter and mixed by inversion. PEI was added to the DMEM −/− medium and mixed by inversion. PEI was then added to the DNA, mixed by inversion and incubated at room temperature for 20 minutes for 8000 cells/ ^cm2 (control) and 12000 cells/ ^cm2 . DNA/PEI was incubated for 1 hour and 2 hours for the other two conditions at 8000 cells/cm ² . This DNA/PEI complex mixture was used to transfect two bioreactors for each of the four conditions. The DNA/PEI complex was added to each bioreactor and incubated at processing parameters for two hours. Two hours after transfection, the recirculation loop was turned on again.

Replacement of medium after transfection

24 hours after transfection, all media from the bioreactors and recirculation loops were removed and replaced with OptiMEM+1:100 Pen Strep (0.132 ml/cm ² ) and recirculation continued for 24 hours at treatment parameters. At 48 hours post-transfection, all media was removed from the recirculation system only and replaced with OptiMEM+1:100 Pen Strep (12 ml/min) and recirculation continued at treatment parameters.

Collection

On the eighth day (12,000 cells/cm ² ) and on the ninth day (8,000 cells/cm ² ) after cell inoculation, benzonase (100 IU/ml) was added, washed with lysis buffer (50 mM HEPES, 1% Tween 20) and incubated in for two hours at processing parameters. The bioreactors were emptied and sucrose saline solution (500 mM NaCl, 1% w/v sucrose) was added and mixed by inversion. Bioreactors were washed with bioreactor wash buffer (500 mM NaCl, 1% w/v sucrose, 20 mM Tris base, 1% v/v Tween 20, 1 _{mM MgCl2.6H2O} ) for approximately 15 min at processing parameters. The bioreactors were emptied and the bioreactor wash buffer was combined with the crude total collected biomass, mixed by inversion and sampled for ddPCR analysis.

Depth and tangential flow filtration

On the eighth day (12,000 cells/cm ² ) and the ninth day (control 8,000 cells/cm ² ) after cell inoculation, biomass was collected from the bioreactors, samples were taken, and the crude lysate of each condition was pooled (n=2 bioreactors). The pooled lysate was then clarified using a 270 cm ² Millistak C0HC Pod filter and a 200 cm ² Millipak 40 0.45 μm Durapore fine filter (EMD Millipore). After C0HC+0.45, samples were collected and frozen at -80.0°C. The clarified lysate was then concentrated using tangential flow on a Pellicon® 2 Ultrafiltration Module PLCMK C 0.1 m ² filter (EMD Millipore). At least 6 diavolumes were used to diafiltrate the final product. After TFF1 filtration, samples were obtained and frozen at -80.0°C. All samples were titrated against AAV2/9 and host cell protein.

Plasmids were used to produce DS in bioreactors. The data represents each condition in duplicate, corresponding to the bioreactor number and condition specified in Table 14.

Table 14. Bioreactor numbers and corresponding conditions

Bioreactor number Seeding density
cells/cm ² Transfection Day PEI/DNA Incubation Time
(min) Collection day 221, 222 12000 4 20 8 223, 224* 8000 5 20 9 225, 226 8000 5 60 9 227, 228 8000 5 120 9

Cell growth. Cells were counted on the day of plating (day 0), and nuclei were counted on day 4 (12,000 cells/cm ² ), day 5 (8,000 cells/cm ² ) and day 9 after plating. Data indicated that cells in all reactors grew exponentially from day 0 to day 5. After transfection, nuclear count results on day 9 indicated that the total number of nuclei in bioreactor 221 increased 2.0-fold from day 5 to day 9. All the remaining reactors (222 to 228) did not show significant growth from day 5 to day 9. The increase in growth in bioreactor 221 could be an artifact based on the uneven distribution of cells among the individual fibers used to count the total number of nuclei. It is possible that the cells in all bioreactors grew in a similar manner based on the metabolite data shown in Figures 9A-E.

pH, nutrients and metabolites. Glucose consumption varied similarly in all bioreactor cultures, suggesting that despite the increase in the growth curve for bioreactor 221, cells consumed glucose at similar rates. The pH for bioreactors seeded at 12,000 cells/cm ² averaged 7.06 and those seeded at 8,000 cells/cm ² averaged 7.18 for the first three days. The pH value decreased slightly with increasing nutrient metabolism and increased at day 9 along with increasing ammonium ion levels. Lactate levels increased until day 5 (bioreactors 221, 223, 224, 225, 227) and day 6 (bioreactors 222, 226, 228) and then leveled off towards the end of production, indicating the use of lactate as an energy source at this stage.

Product titles

Viral genomes from collected material were measured using digital droplet PCR (ddPCR). Titers were approximately 1.5 times higher in bioreactors seeded at 12,000 cells/ ^cm2 , with an average titer of 6.37E+10 g/mL (n=2), compared to control bioreactors seeded at 8,000 cells/cm ² , with an average titer of 4.33E+10 g/ml (n=2). Titer data suggested that seeding at higher densities, transfecting, and harvesting biomass one day earlier resulted in higher yields of DS production. The output titer for DNA/PEI incubated for one hour was characterized by a 1.4-fold decrease in the measured mean titer (3.17E+10 g.v./ml, n=2), and for a two-hour incubation the measured mean titer decreased 1.6 times (2.67E+10 g/ml, n=2) compared to the control, in which DNA/PEI was incubated for 20 minutes (4.33E+10 g/ml, n=2). The data suggested that longer incubation times resulted in lower titers. This could be due to DNA and PEI forming large complexes that were not able to effectively transfect HEK293 cells. Virus production per ml and surface area values are shown in Figure 10 in comparison with production in a known process as a positive control.

The virus titer measured at each stage of the clarification and concentration steps is shown in Figure 11. Residual host cell protein at each stage during the TFF1 stage is shown in Figures 12A-B.

Example 8 Effect of Inoculation Density on Drug Substance Production

The effect of seeding density on DS production was assessed. Four inoculum densities were assessed, and each inoculum density was presented in duplicate in the bioreactor.

Increase in cell mass

HEK 293 cells were thawed and resuspended in DMEM supplemented with 10% FBS. Cells were centrifuged at 209 x g, 5 min, at room temperature, then the supernatant was removed and fresh DMEM + 10% FBS was added. Cells were counted for viable cell density and viability, and seeded into 2 T pads with a surface area of 175 cm ² and incubated at 37.0°C, 5% CO ₂ for four days until the cultures reached ~90% confluency . For each cell subculture, the used media was removed, the mattresses were washed with PBS (-CaCl ₂ , -MgCl ₂ ) at 0.08 ml/cm ² , and then the mattresses were treated with TrypLE Select (0.04 ml/cm ² ) and incubated at 37.0°C, 5% CO ₂ for 2-3 minutes. Trypsin was quenched with DMEM+10% FBS (0.04 ml/ ^cm2 ). Cells were propagated and seeded according to the diagram presented in Figure 13 .

Inoculation and cell monitoring

Bioreactors were inoculated with HEK 293 cells at four target densities, each in duplicate: 8000 cells/ ^cm2 , 9350 cells/ ^cm2 , 10700 cells/ ^cm2 and 12050 cells/ ^cm2 in 700 ml of nutrient medium (DMEM with high glucose content + 10% FBS from Australia + 1:100 Pen Strep) with stirring. The treatment parameters were set as follows: pH 7.23, 37.0°C, 55% dissolved oxygen (DO), and linear velocity 2 cm/s. 24 hours after sowing, recirculation of the nutrient medium was started DMEM (total volume now 0.188 ml/cm ² ) at recirculation speed (12.5 ml/min). Collected daily samples for pH, metabolites and nutrients using a stand-alone instrument and read them using Nova BioProfile 400. On the fifth day and on the ninth day after plating, three fibers were collected and lysed using PBS, A100 and B100 solutions (ChemoMetec) in a 1:1:1 v/v ratio. and counted (NucleoCounter NC-200) the total number of nuclei to monitor culture growth.

Transfection

On the fifth day after cell inoculation, recirculation was stopped and the medium inside each bioreactor chamber (bottles without recirculation) was replaced with 600 ml of DMEM -/- medium (high glucose, - CaCl2, - L-glutamine). The contents of each reactor were transfected with plasmid DNA and polyethylenimine (PEIpro) in a 1:1 weight ratio. Plasmid DNA was mixed in a weight ratio of 1:1.5:2 (pSMN - 3.56 mg, pAAV2/9-5.34 mg and pHELP - 7.1 mg), added to 300 ml of DMEM -/- medium, filtered through filter with a pore size of 0.2 μm and mixed by inversion. PEI (16 mL) was added to 300 mL of DMEM −/− medium and mixed by inversion. The PEI and DNA mixtures were combined, mixed by inversion, and incubated at room temperature for 20 minutes. Every 600 ml of PEI/DNA complex mixture was used to transfect the contents of two bioreactors, repeated for each corresponding seeding density. The PEI/DNA complex was added to each bioreactor and incubated at processing parameters for two hours. The recirculation loop was turned on again two hours after transfection (12.5 ml/min).

Replacement of medium after transfection

At 24 hours post-transfection, all media in the bioreactors and recirculation loops were removed and replaced with OptiMEM (0.132 ml/ ^cm2 ) and recirculation was continued (12.5 ml/min) for 24 hours at treatment parameters. 48 hours after transfection, the medium in the recirculating bottle was replaced with fresh OptiMEM and recirculation continued at processing parameters (12 ml/min).

Collection

On the ninth day after cell inoculation, benzonase (100 IU/ml) was added, purified with lysis buffer (50 mM HEPES, 1% Tween 20), and incubated for two hours at treatment parameters. The bioreactors were emptied and sucrose saline solution (500 mM NaCl, 1% w/v sucrose) was added and mixed by inversion. The bioreactor was washed with bioreactor wash buffer (500 mM NaCl, 1% w/v sucrose, 20 mM Tris base, 1% v/v Tween 20, 1 mM _MgCl2.6H2O ) for 15 min at processing parameters. The bioreactor was emptied and the bioreactor wash buffer was combined with the crude total collected biomass, mixed by inversion, and sampled for ddPCR analysis.

Plasmids were used to obtain the drug in bioreactors. Reactors were seeded at different densities, transfected, and biomass harvested according to the same schedule (day 5, day 9 after seeding, respectively). The data represents two replicates of each seeding density and is shown in Table 15 below.

Table 15. Bioreactor number with initial seeding density

8000 cells/ ^cm2 221, 222 9350 cells/ ^cm2 223, 224 10700 cells/ ^cm2 225, 226 12050 cells/ ^cm2 227, 228

Cell growth. Cells were counted on the day of plating (day 0), and nuclei were counted on day 5 and day 9 after plating. Data indicated that cells in all reactors grew exponentially from day 0 to day 5. Despite differences in initial seeding densities, nuclear counts did not indicate significant differences in cell numbers among groups on day 5. After transfection, nuclear count results on day 9 showed that five reactors (221, 224, 225, 226, 228) doubled the total number of cores from day 5 to day 9. Two reactors (222, 223) increased the total number of cores by a factor of 1.4, as seen in fig. 14A. In reactor 227, the total number of nuclei increased 3.8-fold from day 5 to day 9. This difference could be an artifact based on the uneven distribution of cells among the individual fibers used for counting. It is possible that the cells in all reactors grew in a similar manner based on the metabolite data shown in Figures 14B-E.

pH, nutrients and metabolites. Glucose consumption (Figure 14B) varied similarly across all bioreactor cultures, suggesting that despite differences in initial seeding densities, cultures consumed glucose at similar rates. The pH measured by the off-line meter (Figure 14C) remained constant (pH 7.25) for the first three days in all crops, decreased as nutrient metabolism increased, and increased after day 8 concomitant with increasing ammonium ion levels (Figure 14C). . 14E). Lactate levels (Fig. 14D) increased until day 6 and then leveled off towards the end of production, suggesting the use of lactate as an energy source at this stage. No significant differences in metabolite profiles were observed between reactors seeded at different initial densities. This could be due to the fact that the difference in the initial values of sowing density was minimal, i.e. <1.2-fold difference.

Product titles

Viral genomes from collected material were measured using digital droplet PCR (ddPCR). Titers were comparable between initial seeding densities of 8000 cells/ ^cm2 and 10070 cells/ ^cm2 , averaging 3.99E+10 ± 2.1E+09 g/mL (n=2) and 3.70E+ 10 ± 7.4E+09 g/ml (n=2), respectively. For an unidentified reason, reactors seeded at 9350 cells/cm2 had a mean titer of 5.02E+08 g/mL (n=2), approximately 2 logs lower than the mean titers of reactors seeded at adjacent densities. . This difference is likely a consequence of an unidentified operational error during transfection or biomass harvest rather than insufficient productivity at this inoculation density. Duplicate reactors seeded at 12050 cells/cm ² showed a twofold difference between each other, with one reactor observed in the range for lower seeding densities, 3.2E+10 g/mL, while the second gave the titer is only 1.4E+10 g/ml. Virus production per ml and surface area values are shown in Figure 15A.

Seeding density and DS production were assessed as shown in FIG. 15B. HEK 293 cells seeded between 8E+03 and 10E+03 cells/cm ² showed consistent profiles of growth, pH, glucose consumption, lactate and ammonium production. In addition, comparable titers were obtained, indicating that slightly higher inoculum densities do not have a negative effect on production. In contrast, reactors seeded at the higher density of 12 x 10 ³ cells/cm ² had greater variability between replicates, including a lower mean titer than the other conditions, indicating that the approach used in this experiment may not be optimal for production. These results justified cell seeding at densities ranging from 8 x 10 ³ and 1 x 10 ⁴ cells/cm ² for bioreactor experiments.

Example 9: Comparability assessment

Comparability between the AVXS-101 drug product used in the Phase 1 clinical studies (Method A) and the drug product used in the pivotal clinical studies (Method B) was assessed as a primary objective with the secondary objective of assessing the uniformity of the manufacturing process using Method B by comparison series 600156 and 600307 of the medicinal product. Figure 16 shows the phase 1 (method A) and phase 3 (method B) production process sequences and the differences between them.

The comparability assessment was performed using the Phase 1 clinical drug product series NCHAAV9SMN0613 manufactured by Nationwide Children's Hospital (NCH) and the clinical drug product series 600156 manufactured by AveXis.

Product

The following batches of material were evaluated and are summarized in Table 16. The evaluation included a direct comparison of the quality attributes obtained between the Phase 1 clinical drug product series NCHAAV9SMN0613 obtained using Method A and the drug product AVXS-101 series 600156 obtained using Method B. In addition , the test results from the 600156 Series and 600307 Series products were evaluated overall with respect to large scale process reproducibility and uniformity.

Table 16. Drug product AVXS-101 to be assessed for comparability between Method A (Phase 1) and Method B (Phase 3) and for homogeneity of the manufacturing process in the case of Method B

Application Series number date of manufacture date of manufacture Storage condition Phase 1 study NCHAAV9SMN0613 December 10, 2013 Method A ≤ -60°C Phase 3 study AVXS-101 series 600156 November 7, 2017 Method B ≤ -60°C AVXS-101 series 600307 December 4, 2017 Method B ≤ -60°C

Production Process Overview

Figure 17A-B provides a summary of the homogeneity results.

Assessment of comparability and homogeneity of the production process

Materials from Method A and Method B were rated as comparable, and materials from Method B were rated as compatible. It was also determined that the materials from Method B have additional advantages, for example, for industrial scale production.

Testing methods

pH

pH analysis was performed on series NCHAAV9SMN0613 (method A), series 600156 (method B) and series 600307 (method B). The results of both processes ranged from 7.9 to 8.0. This demonstrated that the pH values of the materials from Method A and Method B were comparable. and that the materials from Method B were compatible.

Appearance

Visual assessment of appearance was performed on the NCHAAV9SMN0613 series (method A), series 600156 (method B) and series 600307 (method B). The apparent differences in appearance scores between Method A and Method B were due to different vector concentrations (genomic titer). The NCH AAV9SMN0613 series had a lower vector concentration than the Method B series. As a result, the NCH AAV9SMN0613 series was more dilute, resulting in a solution that was more clear and colorless, whereas the Method B series observed colorless to white and with a slight degree of opacity were the result of an approximately 4-fold increase in the concentration of viral particles in the solution per ml.

Taking into account the difference in concentration, the appearance of the materials from Method A and Method B were assessed as comparable, and the materials from Method B were assessed as compatible.

Osmolality

Estimation of osmolality by freezing point depression was carried out on the NCHAAV9SMN0613 series (method A), series 600156 (method B) and series 600307 (method B). The results of both processes ranged from 410 to 415 mOsm/kg. This demonstrated that the osmolality values of the materials from Method A and Method B were comparable and that the materials from Method B were compatible.

Particles invisible to the naked eye

The assessment of particles invisible to the naked eye using the chiaroscuro method was carried out on the NCHAAV9SMN0613 series (method A), series 600156 (method B) and series 600307 (method B). The results of both processes were significantly below the recommended limits in the USP monograph for injectable drug products. This demonstrated that the amounts of particles not visible to the naked eye for the materials from Method A and Method B were comparable, and that the materials from Method B were compatible.

Genomic titer

Genomic titer assessment using ddPCR was performed on series NCHAAV9SMN0613 (method A), series 600156 (method B) and series 600307 (method B). The genomic titer for the AVXS-101 batches was expected to fluctuate depending on production target concentrations. The genomic titer obtained using method B (3.7 × 10 ¹³ g./ml and 4.0 × 10 ¹³ g./ml) was at least 3 times higher than in the case of method A ( 1.1 x 10 ¹³ g/ml), so Method B was the best method for large-scale production of AVXS-101.

Infectious titer

Infectious titer assessment using TCID50 was performed on series NCHAAV9SMN0613 (method A), series 600156 (method B) and series 600307 (method B). Method B (1.3 × 10 ¹⁰ IU/ml and 6.7 × 10 ⁹ IU/ml) provided an average of 66% higher infectious titer than method A (5.9 × 10 ¹⁰ IU/ml), which may be advantageous, for example, in the case of large-scale production of rAAVs, such as AVXS-101.

Total protein

Total protein assessment by BCA microplate assay was performed on series NCHAAV9SMN0613 (method A), series 600156 (method B) and series 600307 (method B). Normalized to 1.0 x 10 ¹³ g/mL, with results obtained for both processes ranging from 167 to 179 μg/mL. From the normalized total protein values, it was evident that Method A and Method B were comparable, and that the materials from Method B were compatible.

Identity by Western blot

Identity assessment by Western blotting was performed on series NCHAAV9SMN0613 (method A), series 600156 (method B) and series 600307 (method B). The blot profile and apparent molecular weight values for the major bands (VP1, VP2 and VP3) were judged to be comparable between the materials from Method A and Method B, and the materials from Method B were judged to be compatible.

% Empty Capsids by AUC

Estimation of % empty capsids by AUC was carried out on series NCHAAV9SMN0613 (method A), series 600156 (method B) and series 600307 (method B). The result for the NCHAAV9SMN0613 series (method A) was 7%. The results for series 600156 and 600307 (method B) were 2% and 4%, respectively. Method B (2% and 4%) provided approximately half as many empty capsids, as measured by AUC, than method A (7%). Therefore, Method B provided an improved composition containing a lower concentration of empty capsids.

Identity and purity by SDS-PAGE

Identity and purity assessment by SDS-PAGE was carried out on the NCHAAV9SMN0613 series (method A),

series 600156 (method B) and series 600307 (method B). The % Total Purity in both processes was ≥98%, and the banding pattern as well as the apparent molecular weight for each of the three capsid proteins were highly consistent. From these results, it was clear that Method A and Method B were comparable, and that the materials from Method B were compatible.

Residual host cell protein

Evaluation of residual host cell protein by ELISA was performed on the NCHAAV9SMN0613 series (method A), series 600156 (method B) and series 600307 (method B). For this assay, results for all batches tested were <LOQ (8 ng/mL). From these results, it was clear that Method A and Method B were comparable, and that the materials from Method B were compatible.

Residual bovine serum albumin (BSA)

Residual bovine serum albumin (BSA) was assessed on series NCHAAV9SMN0613 (method A), series 600156 (method B) and series 600307 (method B). For this assay, results for all batches tested were <LOQ (0.50 ng/mL). From these results, it was clear that Method A and Method B were comparable, and that the materials from Method B were compatible.

Residual benzonase

Evaluation of residual benzonase by ELISA was performed on series NCHAAV9SMN0613 (method A), series 600156 (method B) and series 600307 (method B). For this assay, results for all batches tested were <LOQ (0.20 ng/mL). From these results, it was clear that Method A and Method B were comparable, and that the materials from Method B were compatible.

Residual host cell DNA

Evaluation of residual host cell DNA using qPCR was performed on the NCHAAV9SMN0613 series (method A),

series 600156 (method B) and series 600307 (method B). Normalized to 1.0 x 10 ¹³ g/ml, the result for method A was 3.7 x 10 ⁵ pg/ml, while the results for method B were 0.76 x 10 ⁵ pg/ml and 0.68 ×10 ⁵ pg/ml, respectively. Therefore, method B provided viral vectors with significantly less residual hcDNA, which may be advantageous, for example, in the case of large-scale production of rAAV, such as AVXS-101.

Statistical analysis

Statistical analysis was carried out using quantitative and qualitative criteria. Comparisons were made in pairs between a Method A lot (NCHAAV9SMN0613) and each Method B lot (600156 and 600307) as follows. These results are shown in Figures 18 and 19.

From the results of these studies, it is clear that Method B is an excellent method for producing viral vectors. Method B consistently provided more viral vectors (as measured by genomic titer and infectious titer) with few contaminants (lower residual hcDNA content) with fewer empty capsids.

Next Generation Sequencing

Next generation sequencing (NGS) was also performed to establish identity (determine and/or confirm the genomic sequence) and to assess whether sequence variants (subpopulations) exist in the case of the AVXS-101 drug product phase 3 material from Method B. As a result of the alignment of the data set sequences relative to the sponsor-provided reference sequence (pscSMN) revealed full (100%) breadth and sufficient depth of coverage across the entire length of the genome to enable variant detection. A total of four minor position variants were observed, but these appeared to represent sequencing errors in difficult-to-sequence regions (e.g., inverted terminal repeats (ITRs) in AAV, which are notoriously difficult to sequence due to their high abundance GC and palindromic sequences) rather than true variants. See Table 17 for sequencing results.

Table 17: Phase 3 Series 600156 AVXS-101 DNA Sequencing Results Obtained by Method B

Total quantity
readings,
used for
mapping Reference
subsequence Reference sequence length (bases) Total number of mapped reads % mapped population Average coverage depth Length of consensus sequence generated by mapping % reference sequence coverage % similarity to the reference sequence Total number of unmapped or low quality positions 44705268 AVXS-101 5991 48854239 98.1 16069957 5,991 100 100 0

NCHAAV9SMN0613 Series Phase 1 Stability Profile

Batch NCHAAV9SMN0613 was stored for 12 months at ≤ -60°C. This series was analyzed at each time point. No adverse trends were noted. Figure 20 shows the stability results to date.

A comparability study was conducted on AVXS-101 used in Phase 1 clinical studies. The evaluation was conducted using Phase 1 clinical drug product series NCHAAV9SMN0613 manufactured by Nationwide Children's Hospital and AVXS-101 drug product series 600156 manufactured by AveXis. Additionally, manufacturing process uniformity was assessed using batches 600156 and 600307 from Method B. For both the comparability assessment (Method A vs. Method B) and the manufacturing process homogeneity assessment (batch 600156 vs. Method B batch 600307), the study assessed the identity, quality, purity of the AVXS-101 clinical trial material using newly improved process and analytical techniques to provide a reliable assessment of the comparability and uniformity of the manufacturing process.

Statistical analysis was carried out using quantitative and qualitative criteria. Comparisons were made in pairs between Method A lot NCHAAV9SMN0613 and Method B lot 600156. Method B was the better method, which produced more viral vectors with higher purity than Method A. For example, compared to Method A, viral vectors produced by Method B , had an infectious titer of 48% higher, a genomic titer of 8% higher, 92% fewer particles invisible to the naked eye larger than 10 microns, 50% fewer particles invisible to the naked eye larger than 25 microns, 100% fewer empty capsids and 11% less residual hcDNA. All results were consistent with respect to the test limit for each quality attribute.

Moreover, to establish the homogeneity of the production process using Method B, a pairwise comparison was performed using series 600156 and 600307. The result obtained from this initial pairwise comparison between the series 600156 and 600307 from Method B demonstrates the homogeneity in the production process. All results were also consistent with respect to the test limit for each quality attribute.

Based on the evaluation results, the obtained quality attributes of the phase 1 clinical drug product batch NCHAAV9SMN0613 obtained using method A and the drug product batch 600156 AVXS-101 obtained

using Method B, demonstrated that Method B provided higher quantities of viral vector and improved purity, which may be advantageous, for example, in the case of large-scale production of rAAV. In addition, it was found that two batches of material produced using Method B (batch 600156 and 600307) were reproducible, further demonstrating the uniformity of the manufacturing process.

Example 10 Analysis Using Analytical Ultracentrifugation (AUC)

Material from phase 1 (method A, series NCHAAV9SMN0613) and phase 3 (method B, series 600156 and 600307) were analyzed using the AUC method. AUC profiles (assayed in duplicate) for NCHAAV9SMN0613, 600156 and 600307 are shown in Figure 21, Figure 22 and Figure 23, respectively.

AUC analysis of each material provided similar sedimentation coefficients for empty and full capsids with phase 1 material (method A, series NCHAAV9SMN0613), while demonstrating an increased content of empty capsids (7%) compared to phase 3 material (method B, series 600156 and 600307) with empty capsid contents of 2% and 4%, respectively. This is due to the ability of the CsCl gradient ultracentrifugation in the preparation step of Method B to more effectively separate empty capsids from full capsids compared to the iodixanol gradient ultracentrifugation in the preparation step used in Method A.

The AVXS-101 product batches, using clinical and commercial presentation, were comparably characterized by three visible capsid bands when subjected to a CsCl gradient purification process using ultracentrifugation, both in the phase 1 clinical trial material produced at Nationwide Children's Hospital (NCHAAV9SMN0613) and and in each of the subsequent product series manufactured by AveXis. Based on the AUC profiles for Phase 1 clinical drug product series NCHAAV9SMN0613 produced using Method A and drug product AVXS-101 series 600156 produced using Method B, the materials were judged to be comparable. In addition, the AUC profiles for two batches of material obtained using Method B (batch 600156 and 600307) were judged to be consistent.

Example 11. Accumulation process

The accumulation process was used to obtain an intermediate product obtained from a working cell bank, where the accumulation process includes the steps of (a) culturing the cells, (b) transfecting the cultured cells with three plasmids, which are shown in figure 1, (c) collecting an increased number of viral particles from cells after a culture period, (d) purifying the viral particles by filtration to remove any intact cells or cellular debris, (e) subjecting the eluent from step (d) to tangential flow filtration, and (f) freezing the resulting intermediate preparation of purified viral particles.

Before transfection, cells were expanded in suitable culture media, in mattresses or in a suitable bioreactor, or both. One culture medium was DMEM with 10% FBS, 4.5 g/L glucose, 4 mM L-glutamine. In one embodiment, adhesive cells were first grown in mattresses and then transferred to an iCELLis bioreactor for further propagation of adhesive cells in the bioreactor.

After cell expansion, adherent HEK293 cells were transfected by co-precipitation with three DNA plasmids and PEI. The 3 plasmids used for this transfection were pSMN, pAAV2/9 and pHELP. The DMEM medium used for cell propagation was replaced with modified DMEM transfection medium. DMEM transfection medium was free of FBS, calcium, L-glutamine and contained 4.5 g/L glucose. The scAAV9.CB.SMN vector was produced by transfecting three DNA plasmids into adherent HEK293 cells by co-precipitation with PEI in a large-scale adherent cell bioreactor. The vector plasmid pSMN contained cDNA for human SMN. The 3 plasmids used for this transfection were pSMN (222 mg), pAAV2/9 (333 mg), and pHELP (444 mg). Before adding PEI and co-precipitation plasmids, the transfection medium was allowed to equilibrate in the bioreactor until the bioreactor temperature was >30°C. The process of co-precipitation of PEI and plasmids involved adding plasmids to the transfection medium and filtering through a 0.2 μm pore size filter into the reaction bag. PEI was added to the transfection medium and then to the reaction bag. The reaction medium of PEI and plasmids was mixed manually to obtain a homogeneous suspension, and the reaction took place over a period of 15-30 minutes. At the end of the reaction time, PEI and co-precipitation plasmids were transferred from the reaction bag to the bioreactor. Allow PEI and plasmids to co-sediment in the bioreactor for 1-2 hours before recirculating again. The DMEM growth medium was recirculated in the bioreactor for 18-24 hours before being replaced with the next medium.

The rAAV SMN genome (nucleotides 980-3336 of SEQ ID NO: 1) has in its ITR sequence AAV2, chicken β-actin promoter with cytomegalovirus enhancer, SV40 intron, DNA encoding SMN, presented under (GenBank accession number NM_000344.2), polyadenylation signal sequence from bovine growth hormone and another AAV2 ITR. Conservative nucleotide substitutions in SMN DNA are also considered (for example, the substitution of guanine for adenine at position 625 listed in GenBank under accession number NM_000344.2). The genome lacks AAV rep and cap DNA, i.e. there is no rep or cap AAV DNA between the genome ITRs. Subject SMN polypeptides include, but are not limited to, the human SMN1 polypeptide listed in the NCBI Protein Database under NP_000335.1. The rAAV9 SMN vector is described in Foust et al., Nature Biotechnology 28(3): 271-274 (2010), with the vector genome insert sequence shown as nucleotides 980-3336 of SEQ ID NO: 1).

On day 6 in the bioreactor, 18 to 24 hours after transfection, the bioreactor was emptied and the DMEM in the recirculating media bag was replaced with 200 liters of fresh OptiMEM for post-transfection use. The bioreactor was refilled with 64 liters and recirculation was resumed in the bioreactor. On day 7, 18-24 hours after the change on day 6, OptiMEM for post-transfection use in a recirculating bag (~135 liters) was replaced with a fresh bag of OptiMEM media. The bioreactor was not emptied during this stage. Recirculation of the medium was continued until biomass collection on day 9.

After 9 days in the bioreactor, the last samples were taken from the reactor before collecting the biomass and the process of cell lysis began. Benzonase was added to the bioreactor to a final concentration of 100 IU/ml. The benzonase was then stirred in the reactor and 7.1 liters of lysis solution was added to the reactor. The lysis solution was mixed in the reactor for 2 hours before the first stage of biomass collection. At the end of the 2-hour lysis, the contents of the bioreactor were transferred to a biomass collection bag. 8.9 liters of sucrose saline solution (SSS) was added to the biomass collection bag and mixed for 15 minutes. The SSS solution quenched benzonase in the collected medium. The bioreactor was then rinsed with bioreactor wash buffer. To rinse the bioreactor, 64 liters of bioreactor rinse buffer was added to the bioreactor and mixed for 15 minutes. Then the wash was transferred to a common bag to accumulate the collected biomass. After adding the rinse to the collection bag, the contents were stirred for 15 minutes and samples of the total biomass collected were collected.

The mixed total collected biomass was filtered through a depth filter into a storage bag. After all the total collected biomass was filtered, 50 liters of diafiltration buffer was passed through the depth filter at TFF1. The pool obtained using a depth filter was mixed and samples were taken from it. The pool obtained using the depth filter was then filtered through a 0.45 μm pore size filter to further clarify the total biomass material collected. Six liters of TFF1 buffer was then passed through a 0.45 μm filter.

During the TFF1 step, 5.0 m ² regenerated cellulose membrane cassettes with a cutoff of MW 300 kDa were washed, disinfected with NaOH solution and equilibrated with TFF1 buffer. The concentration phase of this process was intended to reduce the volume of clarified harvested biomass by approximately 10 times. After reaching the target retentate volume, the diafiltration process began. The retentate was diafiltered with 6 dia volumes of TFF1 buffer. After reaching 6 diavolumes of the total permeate flow, the retentate was again concentrated and collected in a storage bag. Two successive membrane rinses were performed to maximize product recovery from the TFF system to obtain the drug intermediate. The TFF1 intermediate was aliquoted into sterile 1 or 2 liter PETG bottles in an LFH fume hood and then frozen on dry ice or in a freezer and stored at -60°C.

Table 18. Buffers used in the accumulation process

Name Compound Process step(s) applied Nutrient medium for cell proliferation DMEM with 10% FBS, 4.5 g/L glucose, 4 mM L-glutamine Cell proliferation before transfection in the iCELLis bioreactor Transfection medium DMEM without FBS, without calcium,
without L-glutamine and 4.5 g/l glucose iCELL is a bioreactor transfection Medium for use after transfection OptiMEM with 2.3 g/l glucose,
4 mM L-glutamine and no FBS After transfection in the iCELLis bioreactor Lysis buffer 500 mM HEPES, 10% Tween 20, 20 mM MgCl ₂ , pH 8.0 Cell lysis in the iCELLis bioreactor Sucrose saline solution (SSS) 3700 mM NaCl, 10% sucrose Lightening Buffer for washing the bioreactor 20 mM Tris, 1 mM MgCl ₂ , 500 mM NaCl, 1% Tween 20, 1% sucrose Collection of biomass from the iCELLis bioreactor Buffer for TFF1 20 mM Tris, 1 mM _MgCl2 , 500 mM NaCl, 1% sucrose Lightening, TFF1 Disinfectant buffer for TFF1 0.5 M NaOH Membrane disinfection for TFF1

Example 12. Isolation and purification process

An isolation and purification process was used to process the intermediate to obtain the filtered drug substance. The isolation and purification process steps included an acidification and clarification step (using filtration) followed by cation exchange chromatography, tangential flow filtration ("TFF2"), CsCl ultracentrifugation and an additional tangential flow filtration step ("TFF3") to obtain the filtered drug substances, wherein the purified AAV particles are suspended in a pharmaceutically acceptable carrier. Specifically, the isolation and purification process included the following production steps following the production of the TFF1 intermediate: thawing and combining the TFF1 intermediate, acidification and clarification, cation exchange chromatography (CEX), tangential flow filtration (TFF2) , ultracentrifugation with CsCl to separate full/empty capsids, tangential flow filtration (TFF3) for concentration/buffer exchange, filtration of TFF3 resulting pooled material to form drug substance, dilution and filtration of drug substance to obtain drug product, storage of drug product and filling bottles with the medicinal product.

The intermediate material resulting from TFF1 was thawed and mixed gently. Tween 20 was used to stimulate flocculation of host cell bulk proteins and DNA at acidic pH. The pH of the combined TFF1 intermediate containing 15% Tween 20 was reduced for CEX chromatography (pH 3.5). The precipitate formed after lowering the pH was then removed by filtering the solution through depth filters and filters with a pore size of 0.45 μm.

Tween 20 (36% solution of Tween 20 in 20 mM Tris, 1 mM MgCl ₂ , 500 mM NaCl, 1% sucrose w/v, pH 8.1) was slowly added to the TFF1 intermediate solution over 4 hours to achieve a final concentration of 20% Tween 20. After overnight incubation at room temperature (RT), the pH of the TFF1 intermediate containing Tween 20 was reduced by adding approximately 4 g of 1 M glycine, pH 2.5, per kg of combined TFF1/Tween-supplied intermediate to achieve the target pH of 3.5 ± 0.1. Once the pH value was within the acceptable range, the solution was passed through a Clarisolve POD depth filter along with a 0.45 µm Opticap XL10 Durapore filter or a 0.8/0.45 µm PES filter, followed by a rinse of the filters with double the POD filter retention volume plus one fine filter delay volume with buffer A for CEX.

A cation exchange chromatography (CEX) capture step was used to separate viral capsids from protein, DNA, and other process contaminants. A CIMmultus S03-8000 Advanced Composite Column (2 μm pore size) (8.0 L) was used for this step and was operated using a process-automated chromatography system. Buffers and solutions are described in following table.

Table 19. Buffers and solutions for one CEX run

Name of solution Compound Purpose Volume (l) per CEX cycle with 8 l volume WFI WFI Column washes 200 L Buffer A for CEX 50 mM glycine, 500 mM NaCl, 1.0% sucrose, 0.20% poloxamer 188,
pH 3.5 ± 0.1 at 20°C Equilibration, washing, elution 256 L Buffer B for CEX 50 mM glycine, 2.0 M NaCl, 1.0% sucrose, 0.20% poloxamer 188,
pH 3.5 ± 0.1 at 20°C Column equilibration and elution 40 L Monolithic Column Cleaning Solution 1 M NaOH, 2 M NaCl Column disinfection, CIP 96 L 1 M ammonium acetate 1 M ammonium acetate Restoring the pH of the Column 40 L Neutralization buffer, pH 9.0 1.0M Tris
pH 9.1 ± 0.1 at 20°C pH adjustment of CEX product 0.5 L Storage solution 20% ethanol in WFI Column storage 40 L

The CEX column loading was determined by the protein content of the clarified acidified intermediate resulting from TFF1. The protein loading on the CEX column was set at 70% of the maximum column capacity.

The elution peak was collected manually, starting with a sharp increase in OD280. OD280 increases at a conductivity coefficient of 80-85 mS/cm. The approximate volume of CEX eluate (product) was ~20 liters or 2.5 CV (column volume). The eluate resulting from CEX was collected in two fractions. The first fraction started with a sharp increase in OD280 and was collected at 1.5 CV. The second fraction began immediately after the first fraction and was collected at 1.0 CV. The two fractions were neutralized to pH 8.0 ± 0.30 using pH 9.0 neutralization buffer.

The TFF2 step product was concentrated, protein contaminants were removed, and the buffer was replaced with a suitable buffer for the CsCl ultracentrifugation step. The tangential flow filtration system was used in combination with 300 kDa MWCO regenerated cellulose membranes of size 0.4 m ² (two CEX cycles) or 0.2 m ² (one CEX cycle).

During the concentration phase of this process, the volume of eluates resulting from CEX was reduced. After reaching the target retentate volume, diafiltration was started in batch TFF mode (batch mode). The retentate was diluted 2-fold and subjected to diafiltration with 8 diavolumes of NaCl-based diafiltration buffer at TFF2 and then 8 diavolumes of CsCl diafiltration buffer TFF2 in batch TFF mode. After completion of diafiltration with CsCl, the retentate was concentrated to a predetermined volume, which depended on the retention volume in the system. Two successive membrane rinses were performed to maximize product recovery from the TFF2 system.

The retentate flow rate was set to 5 l/m ² /min (500 ml/min per 0.1 m ² cassette) with a 20% conversion rate to permeate (100 ml permeate flow rate per 500 ml retentate flow rate). The permeate flow rate was controlled using a clamp on the permeate tube, maintaining the permeate flow rate at 20% of the retentate feed flow rate.

Table 20. Buffers for TFF2

Name of solution Compound NaCl-based buffer for diafiltration at TFF2 20 mM Tris, 2 mM MgCl ₂ , 150 mM NaCl, 0.2% poloxamer 188, 1% sucrose, pH 8.1 ± 0.1 at 20°C Buffer with CsCl for diafiltration at TFF2 20 mM Tris, 2 mM MgCl ₂ , 3 M CsCl, 0.2% poloxamer 188, pH 8.1 ± 0.1 at 20°C

Ultracentrifugation could be used to remove empty capsids from hollow capsids by applying cesium chloride gradient ultracentrifugation. For the CsCl ultracentrifugation step, an Optima XPN 100 automated ultracentrifugation system or equivalent system equipped with a 50.2 Ti rotor or equivalent rotor was used. The purified filtered TFF2 material was slowly added to the ultracentrifuge tubes along the inside of the tube wall without introducing bubbles into the solution. The filled tubes were sealed with a manual heat sealer and centrifuged at 302,000 g (50,000 rpm in a 50.2 Ti rotor) for 17 hours at 20°C. After completion of the centrifugation step, the tubes were removed from the ultracentrifuge and placed in a microbiological safety cabinet. The tubes containing the product were placed on ring stands above the waste container. A lamp was placed directly below the tube and the tubes were marked with bands of empty capsids (band A being the band occupying the highest position), paired bands of full capsids (bands B and bands C, the top and bottom bands of the pair), and the band occupying the lowest position under the pair . Bands B, C, and D were removed with an 18-gauge needle attached to a 30-mL syringe inserted directly below band D in the middle of the tube. The collected material was transferred into a sterile 1 L PETG bottle. Material from all centrifuge tubes was collected into a sterile 1 L PETG bottle to form an ultracentrifuge (UC) pool. The buffer for the CsCl ultracentrifugation step is listed in the table below.

Table 21. Buffer for ultracentrifugation with CsCl

Name of solution Compound Buffer with CsCl for diafiltration at TFF2 20 mM Tris, 2 mM MgCl2, 3 M CsCl, 0.2% poloxamer 188, pH 8.1 ± 0.10

At TFF3 step, CsCl was removed and the complete vector was concentrated using buffer to obtain the final composition. A tangential flow filtration system was used in combination with two 50 cm ² MWCO 300 kDa regenerated cellulose membranes. The concentration phase of the TFF3 process was intended to reduce the concentration of residual CsCl and the volume of the UC pool. After reaching the target retentate volume, diafiltration was started. The retentate was diafiltered with 10 dia volumes of TFF3 buffer. Upon completion of diafiltration, the concentrated retentate was transferred to a secondary conical tube through a Pall Supor® EKV 0.2 μm sterilizing filter (Mini Kleenpak).

Sequential membrane rinses were performed to extract the vector from the TFF3 system. Add TFF3 buffer to the primary conical tube that previously contained the TFF3 retentate. This material was recycled through cellulose membranes. After recirculation, the wash was transferred to a secondary conical tube through a 0.2 μm Pall Supor® EKV sterilizing filter (Mini Kleenpak). TFF3 concentrate and partial pool were mixed to achieve a final vector concentration of ≥ 4.5 X 10 ¹³ g/mL drug (pooled TFF3 retentate + two rinses).

Sequential membrane rinses were performed to maximize product recovery from the TFF3 system. The TFF3 buffer was added to the primary conical tube that previously contained the TFF3 retentate and the initial wash material. This material was recycled through cellulose membranes. The secondary wash was transferred to a secondary conical tube through a Pall Supor® EKV 0.2 μm sterilizing filter (Mini Kleenpak) until a specified weight was reached in the secondary conical tube. The final concentrated solution was called drug substance (DS).

Table 22. Buffers for TFF3

Name of solution Compound Buffer for TFF3 20 mM Tris, 1 mM MgCl2, 200 mM NaCl, 0.001% poloxamer 188, pH 8.0 ± 0.1 at 20°C

DS was filtered using a Pall Supor® EKV sterilizing filter (Mini Kleenpak) into a sterile 1 L glass bottle using a sterilized disposable assembly. Before filtering the TFF3 pool, the filter was washed by passing the TFF3 buffer through the filter using a peristaltic pump and discarding it into a washout bag. The drug substance (DS) was then filtered through a wash filter using a peristaltic pump and collected in a sterile 1 L glass bottle. Based on a target DS concentration of 5 x 10 ¹³ g/mL, add TFF3 buffer to a secondary conical tube that contained DS and pass through a filter to obtain a diluted drug product (“DP”) with a target concentration of 3.5 ×10 ¹³ g./ml.

The TFF3 buffer used to wash the filter and dilute DS to obtain DP had the following composition.

Table 23. Typical process for obtaining a medicinal product - buffer composition

Name of solution Compound Buffer for TFF3 20 mM Tris, 1 mM MgCl2, 200 mM NaCl, 0.001% poloxamer 188, pH 8.0 ± 0.1

DP was filled into sterile, ready-to-use 5 ml Crystal Zenith (CZ) vials, capped with sterile, ready-to-use stoppers, and sealed with sterile, ready-to-use caps.

Example 13. Activity Analysis

The relative activity of the drug product was measured using an in vivo quantitative assay. The analysis used an established mouse model of SMA disease. Breeding pairs for the SMAΔ7 mouse strain (Jackson Laboratories, no. 005025) have a normal phenotype, but ∼25% of their offspring are homozygous for the target mutation in the SMN gene and exhibit an SMA-like phenotype. By day 5 they show signs of muscle weakness, and the following week they develop an abnormal gait and a tendency to fall. Jackson Laboratories reports that the mean survival of animals with the SMA-like phenotype is ~15 ± 2 days. In exploratory studies, the median survival time for animals with the SMA-like phenotype without treatment was demonstrated to be 16.3 days (geometric mean; n=3 studies; 10 mice per study).

A biologically active drug product administered by intravenous (IV) infusion results in increased survival time, which is dose dependent (g.v./kg). The activity of the drug product was measured relative to the reference material (previous batch of vector). The titer of drug product and reference material (vector genomes/ml; g.v./ml) was determined using droplet digital polymerase chain reaction (ddPCR). The vector was diluted in saline to achieve each of the three indicated dose levels to be administered to mice with an SMA-like phenotype.

Assay results were considered acceptable if the assay met the eligibility criteria. The eligibility criteria for the analysis were as follows.

4. Acceptance limit for negative control sample (15 ± 2 days, median survival value);

5. Acceptance limit for positive control sample (>40 days, median survival);

6. Acceptance limits on the dose response curve for median survival using the reference standard.

This study used a previous batch of vector (hereinafter referred to as “previous batch”) to determine the linear correlation between the median survival (in days) of a SMAΔ7 mouse dosed with drug product at five different dose levels, including 0 (zero) dose using 0 .9% saline (no treatment group). This was accomplished by applying the relationship between the y-intercept and the slope of each linear regression line (i.e., reference standard and test drug). The calculation of the percentage of relative activity is described in equation (1) below.

%RP = [(y-intercept/test drug slope) ÷ (y-intercept/reference standard slope)] X 100 (1)

A Δ7 mouse model was used to demonstrate the effectiveness of anti-SMA therapeutics, including the drug product. Saline-treated or untreated control animals provided a reliable baseline control against which product activity in terms of increased median survival could be measured. During drug product development, three (3) doses (excluding vehicle dose) determined by genomic titer using droplet digital PCR (ddPCR) were identified to influence survival in a mouse model with a linear correlation if the dose administered (g.v./kg) logarithmically transformed and plotted against the median survival (in days) of the treated neonatal SMAΔ7 mouse. See standard titers (g/mL) in Table 26 for low, medium, and high titer standards. In addition, TFF buffer solution (carrier medium) was used both as the zero point (0) of the calibration curve and as a negative control. The dose for which survival was observed to be ≥40 days (greater than the dose for which a doubling of median survival was observed) was also included as a positive control.

Table 25. Target doses

Dose (g.v./kg) Median survival (days) Standards and controls Saline solution 15±2 Negative control
(without processing) 1.50×10 ¹⁴ Median survival ≥ 40 days Positive control 0 (saline) 15±2 Standard-1 1.2×10 ¹³ 22 ± 3 Standard-2 7.5×10 ¹³ 31 ± 3 Standard-3

Dosage solution preparations (see example of dilution scheme in Table 26).

Negative Control - 0.9% saline solution was used as a negative control.

Positive control - a series of the test drug was prepared at a concentration of 1.5 × 10 ¹⁴ g/kg using saline solution.

Reference Standard Solutions - A batch of reference standard was prepared at the three concentrations shown in Table 25 using saline solution.

Table 26. Dilution scheme for reference standard and test product (example)

Dose (g.v./kg) Reference standard titer/
study drug by ddPCR (g.v./ml) Conversion to g/mL Reference standard volume/
study drug for use (µl) Saline solution
(µl) Total dose volume
(µl) 1.2×10 ¹³ 5.0×10 ¹³ 5.0×10 ¹⁰ 2.6 47.4 50.0

Study Drug Preparation - The study drug was diluted in saline solution. Dilutions were calculated to obtain the test doses (g.v./kg) indicated in Table 26 per mouse in a total final volume of 50 µl. Dilutions were made for 10 mice at a time with one additional volume as a positive control aimed at increasing the minimum lifespan of treated mice to a median survival (days) of ≥40 days.

Acceptance limits on control samples

Negative Control (Non-Treatment Mice) - The assay acceptance limit for the negative control group was that SMAΔ7 mice met the median survival criterion of 15 ± 2 days. In addition, any mouse that died after ≤ 10 days was excluded from the analysis. If more than 7 mice were used in a group, a maximum of 2 mice could be excluded due to death within <= 10 days.

Positive Control (group that was treated with the target clinical dose) - The assay acceptance limit for the positive control group was that the minimum lifespan of treated mice had to be ≥ 40 days, which was the median survival value. In addition, any mouse that died after ≤ 10 days was excluded from the analysis. If more than 7 mice were used in a group, a maximum of 2 mice could be excluded due to death within <= 10 days.

Acceptance limits on the dose response curve for the reference standard

Assay criteria for a linear dose response curve for a reference standard plotted as median survival (days) versus administered dose (g.i./mL) will be determined. Y-intercept/slope ratio - the linear regression curve of the dependence of the median survival value (in days) on the administered dose (g.v./kg) for the reference standard and the study drug was determined. For each case of linear regression, the ratio of the y-intercept to the slope was calculated.

Presentation of results

Qualitative presentation of relative activity results—assay eligibility criteria for each assay were assessed before determining the median point survival (in days) determined after ≥40 days from positive control material. If the median survival of the positive control group was ≥ 40 days, the study drug can be used if the criteria presented below are met.

Quantitative presentation of relative activity results - Before quantifying the relative activity of the test drug, assay suitability criteria were assessed for each assay. Relative activity for the test drug could be recorded when the positive control reached ≥ 40 days, and the median survival for the group of mice representing the upper standard dose of 7.5 x 10 ¹³ g.v./kg was 31 ± 3 days. Percentage relative potency (%RP) for the study drug was calculated using the y-intercept and the linear regression slope of the dose-response curve for median survival (in days) as follows:

%RP=100% * [(Y-intercept/test drug slope) ÷ (Y-intercept/reference standard slope)]

Example 14. Single-dose gene replacement therapy for spinal muscular atrophy: a dose study

Spinal muscular atrophy (SMA) is a severe childhood monogenic disorder resulting from loss or dysfunction of the gene encoding survival motor neuron factor 1 ( SMN1 ). The incidence of this disease is approximately 1 in 10,000 live births, and the carrier rate is 1 in 54. SMA is characterized by degeneration and loss of lower motor neurons, leading to muscle atrophy. The disease is divided into four subtypes (1-4), based on age and achievement of key points. SMA type 1 (SMA1) is the most severe form and the most common genetic cause of death in infants. There are two forms of SMN ; SMN1 is the main gene responsible for the functional production of SMN protein. SMN2 preferentially excludes exon 7 during splicing and, as a result, produces only a small proportion of functional SMN protein compared to SMN1 . Therefore, the copy number of SMN2 modifies the disease phenotype, and the presence of two copies of SMN2 is associated with SMA1.

Infants with biallelic deletions of SMN1 and two copies of SMN2 have a 97% risk of developing SMA1.

Recent studies on the natural history of SMA1 (disease course group) have shown that the median age

at onset of symptoms among infants with the disease was 1.2 months (range, 0 to 4 months), and

the disease was characterized by hypotension, severe weakness from infancy onwards, and inability to sit without support. In infants with SMA1 who have two copies of SMN2 , the median age at death or requirement of noninvasive ventilation for at least 16 hours per day for at least 14 consecutive days (considered equivalent to continuous ventilation) was 10.5 months. In one group of affected children, at 13.6 months, only 25% were alive without continuous mechanical ventilation and 8% were alive without this support at 20 months. Another prospective, multicenter, National Institutes of Health-sponsored disease progression study (NeuroNEXT) involving patients with two copies of SMN2 showed a median tracheostomy-free survival of 8 months (95% confidence interval, 6-17). All patients with SMA1 are characterized by a dramatic decline in respiratory and swallowing function after birth and ultimately require mechanical parenteral nutrition (via a nasogastric or gastrostomy tube) to maintain adequate nutrition and reduce the respiratory risks associated with aspiration. For SMA1 patients whose onset of symptoms occurs at 3 months of age, most patients require feeding until 12 months.

Patients with SMA1 also fail to achieve milestones in motor function and have decreased function as measured by the CHOP INTEND (Children's Hospital of Philadelphia Test of Motor Function in Neonatal Neuromuscular Disorders) score, which ranges from 0 to 64, with higher scores indicate better motor function, an instrument that is sensitive to subtle changes in motor function such as antigravity movements of the limbs. In a retrospective analysis of 34 patients with SMA1, all but 1 patient failed to achieve a score of at least 40 after 6 months of age. In the NeuroNEXT group, CHOP INTEND scores decreased by an average of 10.7 points from 6 to 12 months of age.

Therapeutic strategies to increase SMN protein levels in motor neurons focus on increasing the effectiveness of SMN2 . One approach has been to deliver central nervous system nusinersen (Ionis Pharmaceuticals/Biogen), an antisense oligonucleotide that was designed to inhibit splicing of exon 7 of SMN2 . The drug was shown to reduce weakness in a mouse model of severe SMA and increase the median lifespan of affected mice from 16 to 25 days. In December 2016, the Food and Drug Administration approved nusinersen for the treatment of SMA. This drug is administered by multiple intrathecal injections after four loading doses during the first 2 months of life.

A potential alternative treatment for SMA1 is gene therapy, administered as a single intravenous injection that delivers a copy of SMN in self-complementary adeno-associated virus serotype 9 (scAAV9). (The coding region of this recombinant virus forms an intramolecular double-stranded DNA [or self-complementary] template.) Using this approach, SMN expression was induced in motor neurons and peripheral tissues, which counteracted the effects of SMA in a mouse model and increased median survival in this model from 15 days to 28 days. .5 days at a low dose (6.7×10 ¹³ g.v. per kilogram of body weight) and more than 250 days at higher doses of the vector (2.0×10 ¹⁴ and 3.3×10 ¹⁴ g.v. per kilogram).

In addition to crossing the blood-brain barrier and targeting central nervous system neurons in all regions of the spinal cord, systemic administration of AAV9-mediated gene therapy may be advantageous given the fact that SMN protein is ubiquitously expressed and SMA1 targets multiple systems (eg, autonomic and enteric nervous systems, cardiovascular system, and pancreas), along with many cell types (eg, heart, pancreas, and skeletal muscle). The vector's self-complementarity feature in combination with the hybrid cytomegalovirus enhancer-promoter of the chicken beta-actin gene ensures rapid and stable expression of SMN . In April 2014, we began a gene replacement therapy study in infants with SMA1 who received a single dose of scAAV9 delivering the human motor neuron survival gene (h SMN ) under the control of the chicken beta actin gene promoter (scAAV9.CB.h SMN ) ( AVXS-101).

Ways

Study procedures and patient . For this study, all patients had a genetically confirmed diagnosis of SMA1, homozygous SMN1 exon 7 deletions, and two copies of SMN2 . Patients with the c.859G→C modifier in exon 7 of SMN2 were excluded. The patients who were recruited had an onset from birth to 6 months of age, characterized by hypotonia, as determined by clinical assessment, accompanied by delayed motor skills, poor head support, slouched posture, and joint laxity. Patients with active viral infections (including HIV or hepatitis B or C positive serology) or comorbidity that posed unnecessary risks of gene transmission were excluded from the study. Patients requiring invasive assisted mechanical ventilation (positive pressure tracheotomy) or pulse oximetry <95% saturation at the screening visit were also excluded.

Patients were included in two groups according to the dose of gene therapy that was administered. Patients in group 1 received a low dose (6.7 x 10 ¹³ g.v. per kilogram) and participated in the study for five months; patients in group 2 received a high dose (2.0 x 10 ¹⁴ g.v. per kilogram) and participated in the study for one year. At day 30 post-dose, IFN-γ ELISpot assay of Patient 1 of Group 1 revealed a T cell response and showed a sudden surge in the number of halo forming cells (SFC) per 10 ⁶ peripheral blood mononuclear cells (PBMCs), which were >50 are directed against the AAV9 capsid (normally <50 SFC per 10 ⁶ PBMC). Prednisolone was started at 2 mg/kg and continued for 35 days until the T-cell response and serum transaminase levels decreased. As a result, the experimental protocol was changed and patients 2-15 received oral prednisolone at a dose of 1 mg per kilogram per day for approximately 30 days, starting 24 hours before gene vector administration. Treatment was continued with prednisolone until AST and ALT enzyme levels fell below 120 IU/L and the T-cell response fell below 100 SFC per 10 ⁶ PBMC, after which the dose of prednisolone was reduced based on the discretion of the treating physician. .

The vector was delivered in normal saline (approximately 10-20 ml per kilogram), which was administered by intravenous infusion over approximately 60 minutes. During participation, some patients required enteral nutrition via gastrostomy tube or nasogastric tube, the choice of which was based on the preference of the parents or primary care provider. After enrollment in the study, all patients requiring parenteral nutrition underwent gastrostomy tube placement, and the tubes were not removed during the study.

Results . The primary outcome was a safety determination based on any grade 3 or higher treatment-related adverse events. Secondary outcome was time to death or need for continuous ventilatory support. The latter was defined as at least 16 hours of continuous respiratory support per day for at least 14 days in the absence of acute reversible disease or perioperative conditions. Exploratory outcomes included achievement of mobility milestones (specifically sitting without assistance) and CHOP INTEND scores.

Maintaining scores greater than 40 points was considered clinically significant in SMA when using the CHOP INTEND scale. Unassisted sitting was assessed and classified according to the following criteria: sitting unassisted for at least 5 seconds according to section 22 of the gross motor subtest using the Bayley Developmental Assessment Scales for Infants and Children 1–3 years of age (“unassisted sitting”). help"); sitting unassisted for at least 10 seconds in accordance with World Health Organization (WHO) criteria ("sitting unassisted in accordance with WHO criteria") and sitting unassisted for at least 30 seconds in accordance with section 26 Bailey scales mentioned above (“independent functional sitting”). Key mobility milestones were confirmed by reviewing patient videos by an independent assessor using the Ability Captured Through Interactive Video Evaluation-mini (ACTIVE-mini). Electrically evoked muscle responses (CMAP) were recorded from surface electrodes at baseline and every 6 months after infusion. Pathological muscle status was quantified using electrical impedance myography (EIM).

Statistical analysis . Safety analyzes were performed on all patients who were also included in the primary survival analysis (as defined above and in the protocol) and in the analyzes of change in CHOP INTEND score from baseline to 1 month and 3 months. Such changes from baseline to each study visit were analyzed using a mixed-effects model for repeated measures. The mixed model included group and visit fixed effects, as well as a baseline covariate. In group 2, achievement of key points was analyzed, as well as parenteral nutrition and assisted artificial ventilation. Statistical analysis was performed using SAS software, version 9.4. All comparisons with disease course groups were purely descriptive.

results

Patients . Of the 16 patients who were screened, 1 was excluded due to persistently elevated anti-AAV9 antibody titers (>1:50). Of the 15 patients who were included in the study, 3 were included in low-dose group 1 and 12 were included in high-dose group 2. The mean age of patients at the time of treatment was 6.3 months (range, 5.9 to 7.2) in group 1 and 3.4 months (range, 0.9 to 7.9) in group 2 (Table 28).

Table 28. Demographic and clinical characteristics of 15 patients

Characteristic Group 1 (N=3) Group 2 (N=12) Average age (range) - months. 6.3 (5.9-7.2) 3.4 (0.9-7.9) Average weight (range) - kg 6.6 (6.0-7.1) 5.7 (3.6-8.4) Gender - quantity (%) Male 1 (33) 5 (42) Female 2 (67) 7 (58) Race - count (%) Caucasians 3 (100) 5 (42) Rest 0 18) Average age of onset of symptoms (range) - months. 1.7 (1.0-3.0) 1.4 (0-3.0) Median age at genetic diagnosis (range) - days 33 (4-85) 60 (0-136) Average CHOP INTEND (range) 16 (2-27) 28 (12-50) Patients with clinical support - number (%) Parenteral nutrition 3 (100) 5 (42) Ventilation 3 (100) 2 (17)

Survival and continuous ventilation . At the end of the study, all patients were at least 20 months old and did not require continuous mechanical ventilation; the median age at last pulmonary assessment was 30.8 months in group 1 and 25.7 months in group 2. In contrast, only 8% of patients in the disease course group did not require continuous mechanical ventilation. At 29 months of age, one patient in Group 1 required continuous ventilation due to hypersalivation. After salivary gland ligation, the need for non-invasive ventilation was reduced by 25% to 15 hours per day.

Motor Function Assessments . All patients in groups 1 and 2 had increased CHOP INTEND scores compared to baseline and maintained these changes throughout the study. For patients in group 2, the mean increase was 9.8 points at month 1 and 15.4 points at month 3 (P < 0.001 for both comparisons); 11 patients achieved and maintained scores greater than 40 points. At the final day of the study, August 7, 2017, patients in Group 1 had a mean increase of 7.7 points from the baseline mean of 16.3 points, and patients in Group 2 had a mean increase of 24.6 points from the mean at baseline. 28.2 points.

Motor activity-related checkpoints in group 2 . A total of 11 of 12 patients in Group 2 were able to sit unassisted for at least 5 seconds, 10 for at least 10 seconds, and 9 for at least 30 seconds (Table 31). A total of 11 achieved head support, 9 were able to roll over, and 2 were able to crawl, reach to stand, stand independently, and walk independently. Eleven patients achieved the ability to speak. None of the patients in the disease course groups reached any of these motor milestones, and rarely achieved speech ability.

Table 29. Adverse event-free survival and activity-related and other endpoints for 12 patients in group 2.

Variable Age at start of study participation Adverse event-free survival Movement-related checkpoints Other achievements Raises hand to mouth Holds your head Rolls over Sits with help Sits without assistance Speaks It's swallowing Without using NIV Without parenteral nutrition months ≥5 s ≥10 s ≥30 s Number of patients 4 5.6 31.1 + + + + + + + 5 4.2 28.5 + + + + + + + + + + + 6 1.9 26.1 + + + + + + + + + + + 7 3.6 28.1 + + + + + + + + + 8 7.9 32.4 + 9 4.9 28.9 + + + + + + + + + + + 10 0.9 25.3 + + + + + + + + + + + eleven 2.3 23.8 + + + + + + + + + 12 2.6 23.9 + + + + + + + + + + + 13 0.9 22.1 + + + + + + + + 14 4.1 22.0 + + + + + + + + + + + 15 2.1 20.6 + + + + + + + + Patients with result (%) This study 100 100 92 75 92 92 83 75 92 92 58 50 Natural history studies 8 to 20 months N. d. 0 0 0 0 0 0 N. d. N. d. N. d. 8 to 20 months

Pulmonary function and nutritional status in group 2 . Among the 12 patients in group 2, 10 did not require noninvasive ventilation at baseline compared with 7 who were not dependent on mechanical ventilation at the last follow-up visit (Table 29). At baseline, 7 patients did not require enteral nutrition, including 1 patient who later required gastrostomy tube placement after gene replacement therapy, possibly due to surgery for scoliosis. Of the 5 patients who received enteral nutrition before gene replacement therapy, at the last follow-up visit, 11 of 12 patients achieved or maintained the ability to swallow independently, and 4 were able to feed orally.

Safety . At the end of the study, a total of 56 serious adverse events were reported in 13 patients in the two groups. Of these events, the investigators determined that 2 events were grade 4 treatment-related events based on laboratory values according to the Common Terminology Criteria for Adverse Events (Table 30). Patient 1 in group 1 was characterized by elevations in serum aminotransferase levels (31 times the upper limit of the normal range for alanine aminotransferase (ALT) and 14 times the upper limit of the aspartate aminotransferase (AST)) without other liver function abnormalities (i.e., total and free bilirubin and alkaline phosphatase) and without clinical manifestations. As described above, these increases were mitigated by prednisolone treatment, which was subsequently administered to the remaining patients. One patient in group 2 required additional prednisolone to reduce elevated serum ALT and AST levels (35 times the upper limit of normal range for ALT and 37 times the upper limit of normal range for ALT and 37 times for AST). Of the 241 non-serious adverse events, 3 were considered treatment related and consisted of asymptomatic elevations in serum aminotransferase levels in 2 patients (ALT and AST, both less than 10 times the upper limit of the normal range) that resolved without additional treatment with prednisone ( Table 0. No other liver function abnormalities were detected. Of the 15 patients, 14 had respiratory disease, which often leads to death or the need for tracheostomy in children with SMA1.

Table 30. Adverse events.

Phenomenon Group 1 (N=3) Group 2 (N=12) All patients (N=15 Phenomena Patients Phenomena Patients Phenomena Patients quantity quantity (%) quantity quantity (%) quantity quantity (%) Any adverse event 44 3 (100) 253 12 (100) 297 15 (100) Any serious adverse event 7 3 (100) 49 10 (83) 56 13 (87) Treatment-related adverse event 1 1 (33) 4 3 (25) 5 4 (27) General Adverse Event Upper respiratory tract infection 3 1 (33) 26 10 (83) 29 11 (73) Vomit 0 0 eleven 8 (67) eleven 8 (53) Constipation 4 4 (33) 9 7 (58) 10 8 (53) Pyrexia 1 1 (33) 10 6 (50) eleven 7 (47) Nasal congestion 0 0 8 6 (50) 8 6 (40) Gastroesophageal reflux 1 1 (33) 6 5 (42) 7 6 (40) Enterovirus infection 1 1 (33) 7 4 (33) 8 5 (33) Pneumonia 0 0 eleven 5 (42) 9 5 (33) Rhinovirus infection 1 1 (33) 10 4 (33) eleven 5 (33) Cough 0 0 9 5 (42) 9 5 (33) Otitis media 6 2 (67) 3 2 (17) 9 4 (27) Elevated aminotransferase levels 1 1 (33) 3 3 (25) 4 4 (27) Respiratory failure 1 1 (33) 5 3 (25) 6 4 (27) Parainfluenza virus infection 1 1 (33) 4 3 (25) 5 4 (27) Rash 0 0 5 4 (33) 5 4 (27) Lung collapse 0 0 4 4 (33) 4 4 (27) Viral gastroenteritis 0 0 4 4 (33) 4 4 (27) Rhinorrhea 0 0 4 3 (25) 4 3 (20) Bronchiolitis 0 0 3 3 (25) 3 3 (20) Diarrhea 0 0 3 3 (25) 3 3 (20) Ear infection 1 1 (33) 2 2 (17) 3 3 (20) Injury from a fall 0 0 3 3 (25) 3 3 (20) Human rhinovirus 0 0 3 3 (25) 3 3 (20) Streptococcal pharyngitis 1 1 (33) 2 2 (17) 3 3 (20) Respiratory syncytial virus Pneumonia 1 1 (33) 2 2 (17) 3 3 (20) Bronchiolitis 1 1 (33) 2 2 (17) 3 3 (20) Viral upper respiratory tract infection 0 0 3 3 (25) 3 3 (20)

A single intravenous infusion of an adeno-associated virus vector containing DNA encoding SMN in patients with SMA1 resulted in longer survival than in disease groups with this disease. All 15 patients exceeded the previously reported median age of survival without continuous ventilation of 10.5 months for patients with SMA1 with two copies of SMN2 . All patients also passed the 20-month milestone, with only 8% of patients with the disease typically surviving without continuous ventilation. Of the 12 patients in group 2, all but 1 reached key points associated with motor activity, which was not noted in the disease course groups. The motor function achieved was clinically significant, as reflected in feeding (bringing hand to mouth), sitting and speaking. The majority of patients who did not require supportive care at study entry did not have parenteral nutrition (6 of 7 patients) or ventilator support (7 of 10 patients) at the last follow-up visit. In two groups, patients had an increase in their CHOP INTEND score relative to baseline. Over the first month, group 2 had a mean increase of 9.8 points, in contrast to a mean decrease of more than 10 points between 6 and 12 months in the disease progression group of the NeuroNEXT study.

Preclinical studies of SMN gene replacement therapy in the SMNΔ7 mouse model demonstrated improved survival and motor function with early treatment, possibly while motor neurons were still intact. The clinical results obtained in the inventors' early treatment study reflected the direction of the results obtained in the preclinical studies. After early treatment, two patients were able to crawl, stand, and walk without support. Both of these patients had a family history of SMA, which likely contributed to early diagnosis. Although all patients in the two groups in our study continued to improve motor function, preclinical and clinical data suggested the benefits of early treatment and newborn screening for SMA.

Approximately 3 weeks after treatment, in two patients, serious adverse events caused by AAV-based gene replacement therapy were limited to elevated serum aminotransferase levels without disturbances of other liver enzymes; the other two patients had elevations that did not reach the threshold to meet the definition of serious adverse events (ie, >10 times the normal range). Elevations in liver enzyme levels were reduced by treatment with prednisolone. One patient was not screened due to the presence of an anti-AAV9 antibody, which is consistent with population-based studies showing a low incidence of AAV9 seropositivity in children and young adults and an increased incidence of AAV9 seropositivity among individuals over 40 years of age. . However, the presence of antibodies to the virus may be a limitation of AAV-based gene replacement therapy.

This study used a single-arm design with a disease course group as a control, which is one of a limited number of options when the natural history of the disease is well characterized and is fatal. To include a homogeneous sample in the study that was similar to samples from published disease studies, we limited study enrollment to include only symptomatic SMA1 patients who had biallelic SMN1 mutations and two copies of SMN2 and did not include patients with genetic modifier c.859G→C in exon 7 of SMN2 , since this genetic modifier determines a milder disease phenotype. However, this gene replacement therapy should not be limited to symptomatic patients or patients with a specific genome subtype.

In conclusion, a single high-dose intravenous infusion of an adeno-associated virus vector containing DNA encoding SMN in patients with SMA1 resulted in prolonged survival, improved motor function, and increased CHOP INTEND scores to levels not previously observed in this disease. These improvements resulted in a lower percentage of patients requiring maintenance therapy than in disease progression studies. At follow-up of up to 2 years, no weakening of the effect or clinical regression of motor function was observed. Several patients had transient and asymptomatic increases in aminotransferase levels. Additional studies are needed to evaluate the long-term safety and durability of gene replacement therapy results in patients with SMA1.

Example 15. Pharmacokinetics of scAAV9.CB.h SMN

Traditional clinical pharmacokinetic studies are not applicable to gene replacement therapy products. However, the results of scAAV9.CB.h SMN vector deactivation studies, which assessed the amount of vector excreted from the body through fluids and secretions, are an indicator that can be used in place of traditional pharmacokinetic studies in the case of gene replacement therapy.

Reduction of vector activity following infusion by scAAV9.CB.h SMN was examined at multiple time points during the clinical study. Saliva, urine, and stool samples were collected weekly for 30 days, then monthly for 12 months and every 3 months thereafter. Samples from 5 patients obtained at the 18-month visit were used to analyze the downregulation of the scAAV9.CB.h SMN vector using droplet digital polymerase chain reaction. All 5 patients assessed for downregulation of the SMN vector scAAV9.CB.h were administered a therapeutic dose of 1.1 x 10 ¹⁴ g i.v./kg.

scAAV9.CB.h SMN was detectable in postinfusion downregulation studies. Concentrations of scAAV9.CB.h SMN in urine and saliva ranged from 0.1 to 0.01% of the initial body concentration on day 1 postinfusion, after which concentrations fell below the limit of quantitation. Levels ranging from 10% to 30% of the initial body concentration were detected in stool on day 1 after infusion. One patient had a peak fecal concentration on day 14 post-infusion that was 280% of the initial body concentration. In contrast, in the 3 patients for whom data were available, concentrations <1% of initial body concentrations were observed at day 14 postinfusion, with concentrations decreasing by approximately 4 log (10,000-fold) through 30 days postinfusion. Overall, scAAV9.CB.h SMN was primarily excreted in feces and was below the limit of fecal quantitation by day 60 postinfusion.

Example 16. Non-clinical toxicology tests

Pharmacology in animals. Following infusion of the scAAV9.CB.hSMN vector in the delta 7 SMA mouse disease model (SMN Δ7 mice), body weight increased, righting reflexes improved, survival was significantly increased in a dose-dependent manner, and SMA-related heart failure returned to normal compared with SMN mice Δ7 without treatment.

Animal toxicology. Following intravenous infusion into mice, the vector and transgene were widely distributed, with the highest expression typically observed in the heart and liver and significant expression in the brain and spinal cord. In the 3-month GLP-compliant baseline toxicology studies in mice, the primary target organs for toxicity assessment were the heart and liver. scAAV9.CB.hSMN vector-associated findings in the ventricles included dose-related inflammation, edema, and fibrosis, and in the atrium, inflammation and thrombosis. Liver findings included hepatocellular hypertrophy, Kupffer cell activation, and disseminated hepatocellular necrosis. No Noxious Effect Level (NoAEL) was identified for the scAAV9.CB.hSMN vector-associated mouse liver and heart results, and the maximum tolerated dose was determined to be 1.5 x 10 ¹⁴ g i.v./kg, which provided the safety margin is approximately 1.4 times relative to the recommended therapeutic dose of 1.1 × 10 ¹⁴ g/kg. The transferability of the observed results in mice to primates is currently unknown.

Example 17. Spinal muscular atrophy in pediatric patients

This trial was a phase 1 study evaluating the safety and efficacy of the scAAV9.CB.hSMN vector in patients with SMA type 1 genetically tested to confirm biallelic deletions of SMN1 , 2 copies of the survival motor neuron 2 ( SMN2 ) gene, modification c negative .859G>C in exon 7 and with the onset of clinical symptoms before the age of 6 months. The scAAV9.CB.hSMN vector was delivered intravenously during a single dose infusion to patients aged 0.9 to 7.9 months. Doses were administered to two groups: group 1 (n=3) received the low dose used in this study, and group 2 (n=12) received the high dose (therapeutic dose: 1.1 x 10 ¹⁴ g/kg), used in this study. The published study results reflect Cohort 2 and include follow-up of all patients for up to 24 months after scAAV9.CB.hSMN vector infusion.

Mortality and event-free survival

The results of survival and time-to-event assays confirmed the effectiveness of the scAAV9.CB.hSMN vector. In group 2, all 12 patients (100%) were older than 24 months and had no events, in contrast to only 8% of patients in the natural history study. This indicated a significant and clinically meaningful increase in overall survival in patients infused with the scAAV9.CB.hSMN vector compared with patients without treatment. Two years after the infusion, no patient deaths were reported.

Developmental milestones associated with motor activity

We studied developmental milestones associated with motor activity; Assessments for all 15 patients were videotaped to provide confirmation of achievement of motor-related developmental milestones. Patients in group 2 consistently achieved key developmental milestones and maintained their progress. At 24 months post-dose follow-up, 11 patients (91.7%) were able to hold their head upright for ≥ 3 seconds and sit without support for ≥ 5 seconds, 10 patients (83.3%) were able to sit without support for ≥ 10 seconds, 9 patients (75.0%) were able to sit without support for ≥ 30 seconds, and each of 2 patients (16.7%) was able to stand independently, walk with assistance, and walk independently. Patients in Group 2, who are currently participating in long-term empirical follow-up in this study, maintained their movement-related milestones throughout development, and some achieved additional movement-related milestones.

Table 31. Patients who met developmental milestones based on independent central review at 24 months post-dose follow-up (full set of analyses) Vector scAAV9.CB.hSMN
Group 2
(N=12)
n (%) Rolling (back to side on both sides) 9 (75.0) Holds head straight for ≥ 3 seconds without support 11 (91.7) Sits with support, dependent sitting 11 (91.7) Sits without support ≥ 5 seconds 11 (91.7) Sits without support ≥ 10 seconds 10 (83.3) Sits without support ≥ 30 seconds 9 (75.0) Worth using 2 (16.7) Worth it on its own 2 (16.7) Walks with help 2 (16.7) Walks independently 2 (16.7)

Lungs

Of the 10 patients in group 2 who did not use noninvasive ventilation (NIV) at baseline, 7 did not require daily NIV use at 24 months of follow-up. Almost all patients had suffered from common childhood respiratory diseases, which usually lead to tracheostomy or death in children with SMA type 1. All patients survived respiratory hospitalizations without tracheostomy or the need for continuous ventilation.

Parenteral nutrition

The need for parenteral nutrition was also monitored. In group 2, seven patients did not receive enteral nutrition before gene replacement therapy. One (1) of these 7 patients had parenteral nutrition to facilitate wound healing after a difficult recovery from scoliosis surgery, but also had oral feeding. Four (4) of 5 patients in group 2 who received enteral nutrition before gene replacement therapy were able to feed orally at the end of the study; thus, a total of 11 of 12 patients in group 2 were able to feed orally, with the exception of the 6th patient.

Motor function (CHOP-INTEND)

Patients receiving the therapeutic dose achieved statistically significant improvements in motor function at month 1 and month 3; The mean increase in Children's Hospital of Philadelphia Test of Motor Function in Neonatal Neuromuscular Disorders (CHOP-INTEND) scores was 9.8 points (n=12, P < 0.001) and 15.4 points (n=12, P < 0.001), respectively. .

In patients who received an infusion of the scAAV9.CB.hSMN vector, improvements in motor function were maintained over time. Eleven of twelve (91.7%) patients in group 2 achieved a CHOP-INTEND score ≥ 50 points at 24 months. Early intervention and dose appeared to have a positive effect on response. In general clinical practice, untreated children with SMA type 1 aged 6 months and older do not exceed a CHOP-INTEND score of 40. Moreover, among untreated infants, a mean reduction of 10.7 points was reported between 6 and 12 months of age, which was partly consistent with the expected natural history of the disease.

Example 18. qPCR measurement of residual host cell DNA in AAV9 virus vectors

Way

This method was used to quantify, by qPCR, residual host cell DNA in an AAV drug substance, such as AVXS-101, and in in-process samples. Up to six samples were tested on one plate. qPCR analysis was performed using a TaqMan probe. The TaqMan probe has a fluorogenic reporter dye bound to the 5' end and a non-fluorescent quencher bound to the 3' end. While the probe is in an intact state, the proximity of the quencher to the reporter dye significantly reduces the fluorescence emitted by the reporter dye. Cleavage of the probe separates the reporter dye and the quencher, increasing the fluorescence of the reporter.

Flanking forward and reverse primers designed to bind to a repeat sequence in the human genome were added to the reaction mixture and hybridized with the target sequence present in the sample and standards. The TaqMan fluorogenic probe hybridized between the primer regions. Through successive cycles of template denaturation, primer hybridization, and product elongation, the target sequence was amplified. During the elongation step of the amplification cycle, the exonuclease activity of Taq DNA polymerase released the reporter dye from the probe, releasing the dye from the quencher, resulting in fluorescent emission proportional to the amount of template.

The fluorescence of each well of a 96-well plate was measured using a qPCR instrument. Additional PCR cycles produced increasing amounts of the target sequence, resulting in more reporter dye being released from the probe and higher fluorescence in each subsequent PCR cycle. The number of amplification cycles required for the fluorescent signal to reach a predetermined threshold value was measured. This cycle is called the threshold cycle or CT value. The higher the initial DNA concentration in the sample or standard well, the fewer the number of PCR cycles required to reach the fluorescence threshold level, and the lower the CT value. A calibration curve was created by plotting log10 (DNA concentration) versus the CT value measured for each standard point. The CT value was used to determine the amount of DNA present in the sample by using the individual CT value for the sample and determining the DNA value.

Sample preparation was first performed using the Wako DNA Extractor kit (Wako, 295-50201). Briefly, test samples were mixed well and diluted 1000-fold. The diluted samples were divided into four tubes (500 µl each) and 50 µl of water was added to two tubes (spike control) while 50 µl of 30,000 pg/ml DNA standard was added to the other two tubes (spiked control). . Protein solubilization was performed by adding 20 μl of sodium N-lauroyl sarcosinate solution to each tube, vortexing for 5 seconds, then briefly centrifuging. A NaI solution containing glycogen and Pellet Paint (Novagen, 70748) was prepared in a ratio of 2000:5:4 NaI:glycogen:Pellet Paint. 500 μl of NaI mixture was added to each tube and incubated at 53°C ± 1°C for 15 minutes. The tubes were removed from heating, the contents of the tubes were mixed with 900 μl of isopropanol and incubated at room temperature for 15 minutes. The tubes were then centrifuged at 10,000 g for 15 minutes at 18°C and the supernatant was discarded. The remaining sediment was washed with 800 μl of wash solution A, centrifuged, and the precipitation procedure was repeated twice more. Finally, the pellet was washed with 1500 μl of chilled isopropanol wash solution containing glycogen, centrifuged, and the sedimentation procedure was repeated. The final centrifuged mass was resuspended in 500 μl of nuclease-free water.

The qPCR reaction was performed using the resDNA SEQ Human Quantitative kit (Applied Biosystems, A26366). The reaction mixture was prepared by combining 2x Environmental Master Mix, 10x Human DNA Mix and Negative Control as specified in the kit instructions. 20 μl of the reaction mixture was mixed with 10 μl of the prepared sample and added to each well of the PCR plate. Each sample was added to the plate in triplicate. The tablet was hermetically sealed with optical adhesive film. During thermal cycling, melting was first performed at 95°C for 10 minutes, and then the samples were cycled from 95°C for 15 s to 60°C for 1 minute for 40 cycles.

The calibration curve was obtained by plotting the CT value versus the amount of DNA in log([pg/ml]). The data was fitted to a straight line given by the following equation:

CT value=m x log10 (x) + b,

where x=concentration of standard in pg/mL, m denotes the slope, and b denotes the y-intercept. The host cell DNA concentration was calculated back from the CT value in the well using the above equation, then corrected using the dilution factor.

results

Residual host cell DNA measured by qPCR was 3.7 x ¹⁰⁵ pg/ml for the previous vector batch, 0.76 x ¹⁰⁵ pg/ml for AVXS-101 series 600156, 0.68 x ¹⁰⁵ pg/ ml for AVXS1-101 series 600307 and 1.3×10 ⁵ pg/ml for AVXS-201 DS.

Example 19. ELISA measurement of residual host cell protein (HCP) in AAV9 virus vectors

Way

Host cell protein (HCP) concentrations in AVXS-101 samples were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The Cygnus Technologies Human Embryonic Kidney 293 HCP ELISA Kit is a two-step enzyme-linked immunosorbent assay kit. It is based on a direct sandwich method in which two polyclonal antibodies are directed against separate antigenic determinants in HCP. During incubation, HCP in the sample was bound to anti-HCP antibodies bound to the microplate well and to peroxidase-conjugated anti-HCP antibodies in solution.

After the incubation period, the wells were washed to remove any unbound enzyme-conjugated antibody. Substrate solution 3,3',5,5'-tetramethylbenzidine (TMB) was then added to the wells. The peroxidase-bound conjugate catalyzed the reaction to change the color of the substrate. The reaction was stopped by adding acid, which gave a colorimetric end point that could be read on a spectrophotometer at 450 nm. The amount of substrate hydrolyzed was directly proportional to the concentration of HCP present.

The samples to be tested were diluted to match the range of the given method, i.e. from 4 ng/ml to 200 ng/ml. Each sample was then diluted 2-fold in SDB (110 μL sample and 110 μL SDB) and mixed. Supplementation controls were also obtained to check consistency. For spiked controls, 110 μL of each sample was mixed with 27.5 μL of 200 ng/mL HCP standard and 82.5 μL of SDB. Finally, 50 μl of each standard, control, or test sample was added to a well of a 96-well plate and mixed with 100 μl of anti-HEK 293-HRP conjugate. All conditions were added to the plate in triplicate. The plate was sealed with sealing tape and shaken at 400-600 rpm for 2 hours at room temperature. After incubation, the solutions in the wells were removed by shaking the plate upside down and blotting it with an absorbent towel. The wells were washed with a solution from a wash bottle and blotted quickly, preventing the wash solution from being absorbed into the wells. The washing was repeated 4 times and left upside down for approximately 20 seconds. to drain the solution after the last rinse. Finally, 100 μl of TMB substrate was added to each well of the plate and incubated for 20-30 minutes at room temperature without stirring. The reaction was stopped by adding 100 μl of stop solution to each well. The plate was loaded into the plate reader for 45 min. after adding stop solution, the plate was read at 450 nm and 650 nm.

The average absorbance values of the standards were plotted against the theoretical HCP concentration of the standards on a semi-log plot to obtain a four-parameter logistic (4PL) curve fit based on the following equation:

Y = [(A - D) / (1+(X/C)^B)] + D,

where A denotes the lower asymptote, B denotes the slope of the curve, C denotes the concentration corresponding to the absorbance values at the midpoint between the two asymptotes (ng/ml), D denotes the upper asymptote, X denotes the sample concentration (ng/ml), and Y denotes optical density. A calibration curve was then used to determine the concentration of HCP in the spiked control and unspiked test samples using SoftMax Pro software. The test was considered valid only if the r ² of the standard curve was ≥ 0.98, the mean adjusted absorbance of the 200 ng/mL standard was ≥1.0 OD, and the mean adjusted absorbance of the 0 ng/mL standard was ≤ 0.2 OD, and the coefficient of variation of adjusted optical density for triplicate wells was ≤15%. The final HCP concentration for each sample was calculated using the equation:

_sample HCP concentration (ng/mL) = dilution factor x average measured HCP concentration (ng/mL).

results

Residual host cell protein measured by ELISA was below the limit of quantification (8 ng/ml) for the previous batch of vector, AVXS-101 series 600156, AVXS1-101 series 600307, and AVXS-201 DS.

Example 20. ELISA measurement of residual benzonase in AAV9 virus vectors

Way

The concentration of residual benzonase in an AAV product, such as AVXS-101, was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The Merck Benzonase Endonuclease ELISA Kit II is a two-step enzyme-linked immunosorbent assay kit. It is based on a direct sandwich method in which two polyclonal antibodies are directed against separate antigenic determinants of benzonase. During incubation, benzonase in the sample was coupled to benzonase antibodies bound to the microplate well and to peroxidase-conjugated benzonase antibodies in solution.

After the incubation period, the wells were washed to remove any unbound enzyme-conjugated antibody. Substrate solution 3,3',5,5'-tetramethylbenzidine (TMB) was then added to the wells.

The peroxidase-bound conjugate catalyzed the reaction to change the color of the substrate. The reaction was stopped by adding acid, which gave a colorimetric end point that could be read on a spectrophotometer at 450 nm. The amount of substrate hydrolyzed was directly proportional to the concentration of benzonase present.

Briefly, samples were diluted 2-fold by combining 175 μL of sample with 175 μL of PBST. In parallel, a benzonase control sample was also prepared by combining 175 μl of the sample with 35 μl of 10 ng/ml benzonase standard and 140 μl of PBST. The pre-coated ELISA kit strips were placed in the strip holder and 100 µl of each test mixture was loaded into each well. For blank samples, 100 μL of PBST was loaded in place of the sample. A sample of each condition was loaded into the plate in triplicate. The plate was sealed and incubated at room temperature for 2 hours ± 5 minutes with stirring on a plate shaker (450 rpm). After incubation, the contents were discarded and the plate was washed by adding ~350 μl of PBST using a microplate washer and incubating for 1 minute, then inverting and tapping on an absorbent towel. A total of 3 washes were performed before 100 μl of diluted HRP-conjugated antibody was added to each well. The plate was sealed and incubated at room temperature for 1 hour ± 5 minutes with stirring on a plate shaker (450 rpm). After incubation, the contents were discarded and the plate was washed by adding ~350 μl of PBST using a microplate washer and incubating for 1 minute, then inverting and tapping on an absorbent towel. A total of 3 washes were performed before 100 μL of TMB substrate was added to each well. The plate was sealed and the contents were incubated for 15-40 minutes at room temperature without stirring in the dark. The reaction was stopped by adding 100 μl of 0.2 N stop solution to each well. _{H2SO4 .} The optical density of the plate was measured using a spectrophotometer at 450 nm within 45 minutes after the addition of stop solution.

The average absorbance values of the standards were plotted against the theoretical concentration of benzonase in the standards on a semi-logarithmic plot to obtain a four-parameter logistic curve (4PL) fit based on the following equation:

Y = [(A - D) / (1+(X/C)^B)] + D,

where A denotes the lower asymptote, B denotes the slope of the curve, C denotes the concentration corresponding to the absorbance values at the midpoint between the two asymptotes (ng/ml), D denotes the upper asymptote, X denotes the sample concentration (ng/ml), and Y denotes optical density. A calibration curve was then used to determine the concentration of HCP in the spiked control and unspiked test samples using SoftMax Pro software. The test was considered valid only if the r ² of the standard curve was ≥ 0.98, the mean adjusted absorbance of the 2.5 ng/mL standard was ≥ 1.0 OD, the mean adjusted absorbance of the 0.10 ng standard /ml was greater than the mean OD of the PBST blank, and the coefficient of variation of the corrected absorbance for triplicate wells was ≤ 15%. The final HCP concentration for each sample was calculated using the equation:

_sample benzonase concentration (ng/ml) = dilution factor x average measured benzonase concentration (ng/ml).

results

The residual benzonase concentration measured by ELISA was below the limit of quantitation (0.2 ng/mL) for the previous batch of vector, AVXS-101 series 600156, AVXS1-101 series 600307, and AVXS-201 DS.

Example 21. BCA microplate assay measurement of protein concentration in AAV9 virus vectors

Way

Proteins in in-process drug and drug product samples, such as AVXS-101, were measured using a microplate BCA assay using 2 mg/mL bovine serum albumin (Thermo Fisher Scientific, 23209) and a Micro BCA kit as the reference protein. Protein Assay (Thermo Fisher Scientific, 23235). The assay is based on a detergent-compatible formulation with bicinchoninic acid (BCA) for colorimetric detection and quantification of total protein. BCA detects Cu ¹⁺ , which is formed when Cu ²⁺ is reduced by protein in an alkaline environment. The purple reaction product is formed by the chelation of two BCA molecules with one copper ion (Cu ¹⁺ ), which is characterized by a strong absorption at 562 nm that is linear with increasing protein concentrations.

Briefly, standards were prepared by performing serial dilutions of 2 mg/mL BSA in diluent (20-fold dilutions of formulation buffer, 200 mM NaCl, 20 mM Tris, 1 mM MgCl2, 0.001% w/v Pluronic F-68, pH 8 ,0). Test samples of AVXS-101 were also diluted 20 times with water and serial dilutions were made with diluent. The final concentration was approximately 7.5 μg/ml. Working reagent (WR) was prepared by mixing 25 parts BCA microplate assay reagent A, 24 parts reagent B, and 1 part reagent C from the kit. 150 μl of each of the standard and test sample was loaded in triplicate into a 96-well plate and mixed with 150 μl of WR. The plate was sealed and shaken at 300 rpm on a plate shaker for 30 seconds. The plate was then incubated without shaking at 37°C ± 2°C for 2 hours. After incubation, the plate was centrifuged at 1000 rpm for 2 minutes to collect condensate and the plate was cooled for 15-60 minutes. after incubation. The plate was read in a plate reader at 562 nm and data analyzed using SoftMax Pro.

The average absorbance of the standards relative to the theoretical concentration of protein in the standards was plotted on a semi-logarithmic plot and a quadratic fit was obtained. The quadratic approximation is based on the equation:

Y=A+Bx+Cx ² ,

where A, B, C denote the curve fitting parameters, x denotes the sample concentration in μg/mL, and Y denotes the absorbance in OD. The test was considered valid only if the r ² of the standard curve was ≥0.98, the average absorbance of the blank was less than that of the lowest standard (1 μg/ml), and the coefficient of variation of the absorbance for each standard in 3 replicates was ≤10%. A calibration curve was then used to determine the protein concentration of the test samples. The final protein concentration was calculated using the equation:

total protein concentration (µg/ml) = dilution factor x average measured protein concentration (µg/ml).

results

Total protein concentration measured by BCA microplate assay was 167 μg/mL at 1.0 x 10 ¹³ g/mL for the previous vector batch, 179 μg/mL at 1.0 x 10 ¹³ g/mL. ml for AVXS-101 series 600156, 176 μg/ml at 1.0×10 ¹³ g./ml for AVXS1-101 series 600307, 182 μg/ml at 1.0 × 10 ¹³ g./ml for AVXS-201 DP and 418 µg/ml at 1.0×10 ¹³ g/ml for AVXS-301 DP.

Example 22. Purity and specification when releasing AVXS-101, AVXS-201 and AVXS-301

The drug substance AVXS-101 and the drug product AVXS-101 from Examples 1-4 were tested for purity. Tables 32 and 33 provide the specification and release criteria for these products.

Table 32 Release Specification for Drug Substance AVXS-101

Process related impurities Origin Eligibility Criteria Host cell protein (HCP) Cell substrate ≤ 4 ng at 1.0E13 yr. Host cell DNA Cell substrate ≤ 1.15E5 pg at 1.0E13 g.v. Bovine serum albumin (BSA) Cell culture ≤ 0.22 ng at 1.0E13 yr. Plasmid DNA (pDNA) Cell culture ≤ 6.8E5 pg at 1.0E13 year. Polyethylimine (PEI) Cell culture Not tested upon release Benzonase Post-processing ≤ 0.09 ng at 1.0E13 yr. Tween 20 Post-processing Not tested upon release Poloxamer 188 Post-processing 20-80 ppm Cesium (Cs) Post-processing ≤ 30 µg/g (ppm) Ethanol Post-processing Not tested upon release

Table 33 Release Specifications for Drug Product AVXS-101

Category Index Eligibility Criteria General Appearance according to <631> USP and <855> USP Clear to slightly opaque solution, colorless to pale white, no visible particles pH according to <791> USP 7.7-8.3 Osmolality according to <785> USP 390-430 mOsm/kg Number of particles invisible to the naked eye according to <787> USP ≤ 600 particles ≥ 25 µm per container
≤ 6000 particles ≥ 10 µm per container Quantity Genomic titer by ddPCR 1.7E13-2.3E13 g/ml Infectious titer according to TCID ₅₀ TaqMan 3.9E8-8.4E10 IU at 1.0E13. Total protein by BCA microplate assay 100-300 mcg at 1.0E13 g. Pluronic F-68 content according to HPLC-ELSD 20-80 ppm Activity In vivo functional test using the Δ7SMA mouse model Median survival for 7.5E13 g/kg dose is ≥ 24 days Relative in vitro activity based on cell-based assays 70-130% Identity Vector genome identity by ddPCR Confirmed Identity (protein) by SDS-PAGE Basebands VP1, VP2, VP3 co-migrate with AVXS-101 reference standard Identity (of protein) by Western blotting Test positive for AAV capsid protein Purity % distribution of capsids by SV-AUC % empty ≤ 5% % peak 1+peak 2 ≥ 91.9% % full (peak 1) 37.4-70.3% % full (peak 2) 24.9-60.1% % of all other peaks ≤ 5% % Total Purity by SDS-PAGE % Total Purity (VP1, VP2, VP3) ≥ 95.0% % Total impurities by SDS-PAGE % Total impurities ≤ 5% Amount of individual related unnamed impurity > 2.0% Related Named Impurities: Specify value (%) up to 0.1% (up to LOQ)
- Impurity 1A (~71-73 kDa)
- Impurity 1 (~61-67 kDa)
- Impurity 2 (~56-64 kDa)
- Impurity 3 (~48-58 kDa)
- Impurity 4 (~33-38 kDa)
- Impurity 5 (~30-34 kDa) Safety Endotoxin according to <85> USP ≤ 0.75 EU/ml Sterility according to <71> USP No growth Container closure integrity according to <1207> USP, vacuum reduction Passed

AVXS-201 and AVXS-301 can be prepared and purified using the same process as described for AVXS-101, for example, as described in Examples 1-4. Specification and release criteria for batches of each product produced in bioreactors are shown in Tables 34-36.

Table 34. Medicinal substance AVXS-201 series 283-0218-005

Description of the test Result Bioburden < 1 CFU/ml pH 7.8 Appearance during visual inspection Transparent colorless solution Osmolality by freezing point depression 411 mOsm/kg Genomic titer by ddPCR 4.0×10 ¹³ g/ml Vector genome identity by ddPCR Confirmed Vector identity by SDS-PAGE Main bands VP1, VP2, VP3
co-migrate with AVXS-201 control Purity according to SDS-PAGE 98% % Empty Capsids by AUC TBD Total impurity content by SDS-PAGE Total % of impurities: 2%
% Impurities with MW:
53.2 kDa, <1.0%
36.9 kDa, <1.0%
29.9 kDa, <1.0% Residual BSA by ELISA < LOQ (< 0.50 ng/ml) Residual plasmid DNA by qPCR 1.2×10 ⁶ pg/ml per 1.0×10 ¹³ g/ml Residual host cell DNA by qPCR 1.3×10 ⁵ pg/ml per 1.0×10 ¹³ g/ml Residual benzonase by ELISA < LOQ (< 0.20 ng/ml) Host cell protein by ELISA < LOQ (< 8 ng/ml)

Table 35. Medicinal product AVXS-201 series 283-0218-006

Description of the test Result Sterility No growth Endotoxin 0.02 EU/ml per 1.0×10 ¹³ g/ml Replication of competent AAV TBD pH 7.8 Appearance during visual inspection Transparent colorless solution without visible particles Osmolality by freezing point depression 409 mOsm/kg Particles invisible to the naked eye using the chiaroscuro method • 0 particles measuring 25 microns per container or larger
• 0 particles measuring 10 microns per container or larger Genomic titer by ddPCR 3.6×10 ¹³ g/ml Infectious titer according to TCID ₅₀ 8.26×10 ¹⁰ TCID50/ml Total protein by BCA microplate assay 182 mcg/ml at 1.0×10 ¹³ g/ml Pluronic by HPLC-ELSD 46 ppm Vector genome identity by ddPCR Confirmed Identity by Western blot Test positive for AAV capsid protein
Baseband MW
VP1: 79.0 kDa
VP2: 65.0 kDa
VP3: 57.7 kDa Identity by SDS-PAGE Main bands VP1, VP2, VP3
co-migrate with AVXS-201 control % Empty Capsids by AUC 2% % Total Purity by SDS-PAGE 98% % Total impurities by SDS-PAGE Total % of impurities: 2%
% Impurities with MW:
52.5 kDa, <1.0%
36.4 kDa, <1.0%
29.1 kDa, <1.0% Cesium by ICP-MS 28 µg/g (ppm)

Table 36. Purity of the test batch of medicinal product AVXS-301

Description of the test Result Sterility No growth Endotoxin 0.09 EU/ml Replication of competent AAV Negative pH 8.0 Appearance during visual inspection Transparent and colorless solution Osmolality by freezing point depression 408 mOsm/kg Particles invisible to the naked eye using the chiaroscuro method • 0 particles measuring 25 microns per container or larger
• 0 particles measuring 10 microns per container or larger Genomic titer by ddPCR 2.5×10 ¹³ g/ml Infectious titer according to TCID ₅₀ 5.62×10 ¹⁰ TCID ₅₀ /ml Total protein by results
BCA microplate assay 418 µg/ml Pluronic by HPLC-ELSD 53.2 ppm Vector genome identity by ddPCR Confirmed Identity (of protein) by Western blotting Test positive for AAV capsid protein Identity (protein) by
SDS-PAGE Main bands VP1, VP2, VP3
co-migrate with AVXS-301 control % Empty Capsids by AUC 3% % Total Protein by
SDS-PAGE 98% % Total impurities by SDS-PAGE Total impurities: 2%
Impurities with MW:
- Impurity 1 (~61-67 kDa) 2% Cesium by ICP-MS Below LOQ (< 20 µg/g (ppm))

The high purity achieved for AVXS-201 and AVXS-301 demonstrated that the methods for preparing and purifying AAV viral vectors described herein have broad applicability to AAV viral vectors with a variety of payloads.

When reading the described embodiments with reference to the accompanying drawings, it should be understood that the present invention is not limited to the exact embodiments, and that various changes and modifications may be made thereto by those skilled in the art without departing from the scope or spirit of the present invention and embodiments , defined in the attached claims.

List of Illustrative Embodiments

1. Pharmaceutical composition containing:

(a) 1-8 X 10 ¹³ vector genomes/ml parvovirus constructed to include a transgene;

(b) less than 5.0% empty capsids;

(c) less than 40 ng/ml of residual host cell protein per 1 X 10 ¹³ g/ml;

(d) less than 1.2 X 10 ⁶ pg/ml of residual host cell DNA per 1 X 10 ¹³ g/ml and at the same time

(e) at least 80% of the 1-8 X 10 ¹³ vector genomes/ml are functional.

2. The composition according to embodiment 1, wherein the amount of residual plasmid DNA is less than 1.7 X 10 ⁶ pg per 1 X 10 ¹³ g.

3. The composition according to embodiment 1, wherein the number of vector genomes/ml of parvovirus is 1.8-2.2 X 10 ¹³ vector genomes/ml.

4. A method for purifying AAV particles from a culture of mammalian host cells to obtain a frozen drug intermediate, comprising the steps of:

(a) culturing cells that have been transfected with a recombinant AAV virion;

(b) collecting increased amounts of viral particles from the cells after the culture period;

(c) purifying the viral particles by filtration to remove any intact cells or cellular debris;

(d) subjecting the eluent from step (c) to tangential flow filtration and

(e) freezing the resulting intermediate preparation of purified viral particles.

5. The method according to embodiment 4, wherein an endonuclease is used during the collection step.

6. The method according to embodiment 5, wherein the endonuclease is a benzonase.

7. The method of embodiment 4, wherein the purification step employs depth filtration followed by filtration through a filter that removes macromolecular contaminants and cellular debris.

8. The method according to embodiment 4, wherein the tangential flow filtration step uses cellulose membranes.

9. A method for purifying a sample of AAV particles from a mammalian host cell culture to obtain a medicinal product, comprising the steps of:

(a) an acidification and clarification step;

(b) a step of performing cation exchange chromatography;

(c) a tangential flow filtration step;

(d) a CsCl ultracentrifugation step to remove empty capsids; and

(e) a tangential flow filtration step.

10. The method of embodiment 9, wherein the acidification and clarification step involves adjusting the sample to pH 3.5 and removing host cell proteins and DNA by detergent flocculation.

11. The method of embodiment 9, wherein the cation exchange column contains a resin having sulfonyl groups.

12. The method according to embodiment 9, wherein the tangential flow filtration step (c) uses cellulose membranes with a molecular weight cutoff of MW 300 kDa and reduces the volume of the eluate from the cation exchange step by at least six times.

13. The method of embodiment 9, wherein the ultracentrifugation step with CsCl removes at least 80% of the empty capsids in the sample, depth filtration is used, followed by filtration through a 0.45 micron pore size filter.

14. The method according to embodiment 9, wherein in step (e) of tangential flow filtration, cellulose membranes with a molecular weight cutoff of MW 300 kDa are used.

15. A method of treating a neurological disease in a patient in need thereof, comprising intravenous or intrathecal delivery of a pharmaceutical composition according to any of embodiments 1-3, wherein the parvovirus comprises a self-complementary AAV9 genome, and wherein the engineered transgene comprises an SMN polynucleotide, and wherein the disease is an SMA .

16. A method of treating a neurological disease in a patient in need thereof, comprising intrathecal delivery of a pharmaceutical composition according to any of embodiments 1-3 with a contrast agent, wherein the parvovirus comprises a self-complementary AAV9 genome, and wherein the engineered transgene comprises an SMN polynucleotide, wherein the disease is an SMA , and where the contrast agent is Omnipaque 180.

17. The method according to any one of embodiments 15-16, wherein the SMA is a Type II, Type III, or Type IV SMA.

18. A method of treating type II, III or IV SMA in a patient in need thereof, comprising intrathecal delivery of a pharmaceutical composition according to any one of embodiments 1-3 with a contrast agent, wherein the parvovirus contains a self-complementary AAV9 genome, wherein the engineered transgene contains an SMN polynucleotide, and where the contrast agent is Omnipaque 180.

19. A method of treating type I SMA in a patient in need thereof, comprising intravenous delivery of a pharmaceutical composition according to any one of embodiments 1-3, wherein the parvovirus comprises a self-complementary AAV9 genome, and wherein the engineered transgene comprises an SMN polynucleotide.

20. The method according to embodiment 19, wherein the age of the patient is 0-9 months.

21. The method according to embodiment 20, wherein the age of the patient is 0-6 months.

22. The method according to embodiment 19, wherein the weight of the pediatric patient is no more than about 8 kg.

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LIST OF SEQUENCES

<110> AveXis Inc.

<120> MEANS AND METHOD FOR OBTAINING VIRAL VECTORS AND OPTIONS

THEIR APPLICATIONS

<130>AVEX-003/001WO

<150> 62/583,035

<151> 2017-11-08

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 2359

<212> DNA

<213> Artificial

<220>

<223> vector design

<400> 1

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cctcccatat gtccagattc tcttgatgat gctgatgctt tgggaagtat gttaatttca 1800

tggtacatga gtggctatca tactggctat tatatggggtt ttagacaaaa tcaaaaagaa 1860

ggaaggtgct cacattcctt aaattaagga gaaatgctgg catagagcag cactaaatga 1920

caccactaaa gaaacgatca gacagatcta gaaagcttat cgataccgtc gactagagct 1980

cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 2040

gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 2100

attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 2160

agcaaggggg aggattggga agacaatagc aggcatgctg gggagagatc gatctgagga 2220

acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg 2280

gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 2340

gcgcagagag ggagtggcc 2359

<210> 2

<211> 294

<212> PROTEIN

<213> Homo sapiens

<400> 2

Met Ala Met Ser Ser Gly Gly Ser Gly Gly Gly Val Pro Glu Gln Glu

1. 5 10 15

Asp Ser Val Leu Phe Arg Arg Gly Thr Gly Gln Ser Asp Asp Ser Asp

20 25 30

Ile Trp Asp Asp Thr Ala Leu Ile Lys Ala Tyr Asp Lys Ala Val Ala

35 40 45

Ser Phe Lys His Ala Leu Lys Asn Gly Asp Ile Cys Glu Thr Ser Gly

50 55 60

Lys Pro Lys Thr Thr Pro Lys Arg Lys Pro Ala Lys Lys Asn Lys Ser

65 70 75 80

Gln Lys Lys Asn Thr Ala Ala Ser Leu Gln Gln Trp Lys Val Gly Asp

85 90 95

Lys Cys Ser Ala Ile Trp Ser Glu Asp Gly Cys Ile Tyr Pro Ala Thr

100 105 110

Ile Ala Ser Ile Asp Phe Lys Arg Glu Thr Cys Val Val Val Tyr Thr

115 120 125

Gly Tyr Gly Asn Arg Glu Glu Gln Asn Leu Ser Asp Leu Leu Ser Pro

130 135 140

Ile Cys Glu Val Ala Asn Asn Ile Glu Gln Asn Ala Gln Glu Asn Glu

145 150 155 160

Asn Glu Ser Gln Val Ser Thr Asp Glu Ser Glu Asn Ser Arg Ser Pro

165 170 175

Gly Asn Lys Ser Asp Asn Ile Lys Pro Lys Ser Ala Pro Trp Asn Ser

180 185 190

Phe Leu Pro Pro Pro Pro Met Pro Gly Pro Arg Leu Gly Pro Gly

195 200 205

Lys Pro Gly Leu Lys Phe Asn Gly Pro Pro Pro Pro Pro Pro Pro Pro

210 215 220

Pro Pro His Leu Leu Ser Cys Trp Leu Pro Pro Phe Pro Ser Gly Pro

225 230 235 240

Pro Ile Ile Pro Pro Pro Pro Pro Ile Cys Pro Asp Ser Leu Asp Asp

245 250 255

Ala Asp Ala Leu Gly Ser Met Leu Ile Ser Trp Tyr Met Ser Gly Tyr

260 265 270

His Thr Gly Tyr Tyr Met Gly Phe Arg Gln Asn Gln Lys Glu Gly Arg

275 280 285

Cys Ser His Ser Leu Asn

290

<210> 3

<211> 736

<212> PROTEIN

<213> Adeno-associated virus 9

<400> 3

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1. 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 4

<211> 1621

<212> DNA

<213> Homo sapiens

<400> 4

ccacaaatgt gggagggcga taaccactcg tagaaagcgt gagaagttac tacaagcggt 60

cctcccggcc accgtactgt tccgctccca gaagccccgg gcggcggaag tcgtcactct 120

taagaaggga cggggcccca cgctgcgcac ccgcgggttt gctatggcga tgagcagcgg 180

cggcagtggt ggcggcgtcc cggagcagga ggattccgtg ctgttccggc gcggcacagg 240

ccagagcgat gattctgaca tttgggatga tacagcactg ataaaagcat atgataaagc 300

tgtggcttca tttaagcatg ctctaaagaa tggtgacatt tgtgaaactt cgggtaaacc 360

aaaaaccaca cctaaaagaa aacctgctaa gaagaataaa agccaaaaga agaatactgc 420

agcttcctta caacagtgga aagttgggga caaatgttct gccatttggt cagaagacgg 480

ttgcatttac ccagctacca ttgcttcaat tgattttaag agagaaacct gtgttgtggt 540

ttacactgga tatggaaata gagaggagca aaatctgtcc gatctacttt ccccaatctg 600

tgaagtagct aataatatag aacagaatgc tcaagagaat gaaaatgaaa gccaagtttc 660

aacagatgaa agtgagaact ccaggtctcc tggaaataaa tcagataaca tcaagcccaa 720

atctgctcca tggaactctt ttctccctcc accacccccc atgccagggc caagactggg 780

accaggaaag ccaggtctaa aattcaatgg cccaccaccg ccaccgccac caccaccacc 840

ccacttacta tcatgctggc tgcctccatt tccttctgga ccaccaataa ttcccccacc 900

acctcccata tgtccagatt ctcttgatga tgctgatgct ttgggaagta tgttaatttc 960

atggtacatg agtggctatc atactggcta ttatatgggt ttcagacaaa atcaaaaaga 1020

aggaaggtgc tcacattcct taaattaagg agaaatgctg gcatagagca gcactaaatg 1080

acaccactaa agaaacgatc agacagatct ggaatgtgaa gcgttataga agataactgg 1140

cctcatttct tcaaaatatc aagtgttggg aaagaaaaaa ggaagtggaa tgggtaactc 1200

ttcttgatta aaagttatgt aataaccaaa tgcaatgtga aatattttac tggactcttt 1260

tgaaaaacca tctgtaaaag actggggtgg gggtgggagg ccagcacggt ggtgaggcag 1320

ttgagaaaat ttgaatgtgg attagatttt gaatgatatt ggataattat tggtaatttt 1380

atggcctgtg agaagggtgt tgtagtttat aaaagactgt cttaatttgc atacttaagc 1440

atttaggaat gaagtgttag agtgtcttaa aatgtttcaa atggtttaac aaaatgtatg 1500

tgaggcgtat gtggcaaaat gttacagaat ctaactggtg gacatggctg ttcattgtac 1560

tgtttttttc tatcttctat atgtttaaaa gtatataata aaaatattta attttttttt 1620

a 1621

<---

Claims (58)

1. A method for producing a viral vector based on AAV, including: a. cultivation of adherent cells; b. transfecting adherent cells with plasmid(s) to produce an AAV-based viral vector; c. lysis of adhesive cells with release of an AAV-based viral vector; d. acidification and clarification of the cell lysate from (c); e. purifying the product from (d) using cation exchange chromatography (CEX); f. filtering the product from (e) using tangential flow filtration (TFF); g. ultracentrifugation of the product from (f) in cesium chloride (CsCl) buffer; And h. collection of AAV-based viral vectors from the product in (g). 2. The method according to claim 1, where AAV is AAV9. 3. The method according to claim 1 or 2, where AAV is self-complementary (scAAV). 4. Method according to any one of paragraphs. 1-3, where the adherent cells are HEK293 cells. 5. The method of claim 4, wherein step (a) comprises seeding the cells into a large-scale bioreactor that can provide continuous circulation of the cell culture medium. 6. Method according to claim 5, where the seeding density is 8000-12000 cells/ ^cm2 . 7. Method according to any one of paragraphs. 1-6, wherein the transfection step (b) involves contacting the adherent cells with a helper plasmid (pHELP) for the adenovirus, and a plasmid encoding the AAV rep and AAV cap genes. 8. The method of claim 7, wherein the AAV rep gene is rep2 and the AAV cap gene is cap9. 9. Method according to any one of paragraphs. 1-8, wherein the transfection step (b) includes contacting the adherent cells with a plasmid containing a polynucleotide encoding a survival motor neuron (SMN) protein, a polynucleotide encoding MeCP2, or a polynucleotide encoding SOD1 shRNA. 10. The method of claim 9, wherein the plasmid containing the polynucleotide encoding the SMN protein contains a modified AAV2 ITR, a chicken beta-actin (CB) promoter, a cytomegalovirus (CMV) immediate-early enhancer, a modified SV40 late 16s intron, a hormone polyadenylation signal bovine growth growth (BGH) and unmodified AAV2 ITR. 11. The method according to claim 9 or 10, where the polynucleotide encodes the SMN protein under SEQ ID NO: 2. 12. Method according to any one of paragraphs. 1-11, wherein the AAV9-based viral vector contains SEQ ID NO: 1. 13. Method according to any one of paragraphs. 1-12, wherein the transfection step comprises contacting the adherent cell with a polyethylenimine (PEI) transfection agent. 14. Method according to any one of paragraphs. 1-13, where the transfection step includes contacting the adherent cell with a transfection medium that does not contain serum, calcium or glutamine. 15. Method according to any one of paragraphs. 1-14, where the lysis step includes the use of a lysis buffer with the addition of benzonase and TWEEN. 16. Method according to any one of paragraphs. 1-15, further comprising freezing the cell lysate from step (c) to the acidification step (d). 17. A method for obtaining a viral vector based on AAV from a cell culture lysate, including the stages: a. acidification and clarification of cell lysate; b. purifying the product from (a) using cation exchange chromatography (CEX); c. filtering the product from (b) by tangential flow filtration (TFF); d. ultracentrifuging the product from (c) using 2 - 4 M cesium chloride (CsCl) buffer; e. collecting AAV-based viral vectors from the product of (d); f. filtering the product from (e) by means of a second tangential flow filtration. 18. Method according to any one of paragraphs. 1-17, where the acidification step includes acidifying the cell lysate to a pH value of 3.0 - 4.0. 19. Method according to any one of paragraphs. 1-18, where ultracentrifugation is carried out at 40,000 - 50,000 rpm. 20. Method according to any one of paragraphs. 1-19, where the CsCl buffer contains 3 M CsCl, Tris, MgCl ₂ and poloxamer 188, and the pH is pH 7.5 to 8.5. 21. Method according to any one of paragraphs. 1-20, where the cell lysate is incubated with Tween until the acidification step. 22. Method according to any one of paragraphs. 1-21, where the clarification step includes filtering the cell lysate through a depth filter. 23. Method according to any one of paragraphs. 1-22, where CEX includes a resin with sulfonyl groups. 24. Method according to any one of paragraphs. 1-23, wherein at least one tangential flow filtration (TFF) step includes the use of cellulose membranes with a molecular weight cutoff of MW 300 kDa. 25. Method according to any one of paragraphs. 1-24, wherein the amount of empty viral capsids is less than 7%, less than 5%, less than 3%, or less than 1% of the total amount of viral capsids after collection of AAV-based viral vectors from ultracentrifuged cell lysate. 26. Method according to any one of paragraphs. 17-25, wherein the AAV-based viral vectors collected after the second TFF step are stored in a solution containing Tris, MgCl ₂ , NaCl and poloxamer 188, and the pH is pH 7.5 to 8.5. 27. Method according to any one of paragraphs. 17-26, wherein the AAV-based viral vectors collected after the second TFF contain less than 30 μg/g or less than 20 μg/g CsCl. 28. Method according to any one of paragraphs. 17-27, where the concentration of AAV-based viral vectors collected after the second TFF is 3×10 ¹³ g/ml or more. 29. Method according to any one of paragraphs. 1-28, where the resulting AAV yield is greater than 5×10 ¹⁵ g.v., or greater than 8×10 ¹⁵ g.v., or greater than 1×10 ¹⁶ g.v. per production batch. 30. Method according to any one of paragraphs. 1-29, wherein the AAV-based viral vector comprises a polynucleotide encoding a survival motor neuron (SMN) protein, a polynucleotide encoding a MeCP2 protein, or a polynucleotide encoding a SOD1 shRNA. 31. A method of treating spinal muscular atrophy (SMA) type I in a patient in need thereof, comprising administering a composition containing an AAV-based vector obtained in accordance with the method of any one of claims. 1-30, to a patient, wherein the AAV-based vector contains a polynucleotide encoding a survival motor neuron (SMN) protein, and where the patient: a. characterized by an age of nine months or less; b. has a body weight of at least about 2.6 kg; c. has biallelic null mutations or deletions in SMN1 and d. has at least one functional copy of SMN2 . 32. The method according to claim 31, where the viral vector is administered at a dose of 1-2.5×10 ¹⁴ g/kg. 33. The method according to claim 31 or 32, where the patient’s body weight is no more than 8.5 kg. 34. Method according to any one of paragraphs. 31-33, where the patient does not have the c.859G>C substitution in exon 7 of at least one copy of the SMN2 gene. 35. Method according to any one of paragraphs. 31-34, wherein the treatment agent is administered to the patient before 6 months of age or until the onset of one or more SMA symptoms selected from hypotonia, delayed motor development, poor head support, slouched posture, and joint hypermobility. 36. Method according to any one of paragraphs. 31-35, wherein the viral vector is injected into 5-20 ml/kg, 10-20 ml/kg or 5.5-6.5 ml/kg Tris-buffered saline. 37. Method according to any one of paragraphs. 31-36, where efficiency is determined using the CHOP-INTEND scale. 38. Method according to paragraphs. 31-37, including the administration of a dose volume selected from the group consisting of a dose of 16.5 ml for a patient with a body weight of 2.6-3.0 kg, a dose of 19.3 ml for a patient with a body weight of 3.1-3, 5 kg, doses of 22.0 ml for a patient weighing 3.6-4.0 kg, doses of 24.8 ml for a patient weighing 4.1-4.5 kg, doses of 27.5 ml for a patient with weight body weight 4.6-5.0 kg, doses 30.3 ml for a patient weighing 5.1-5.5 kg, doses 33.0 ml for a patient weighing 5.6-6.0 kg, doses 35 .8 ml for a patient with a body weight of 6.1-6.5 kg, doses of 38.5 ml for a patient with a body weight of 6.6-7.0 kg, doses of 41.3 ml for a patient with a body weight of 7.1- 7.5 kg, 44.0 ml dose for a patient weighing 7.6-8.0 kg and 46.8 ml dose for a patient weighing 8.1-8.5 kg. 39. Method according to any one of paragraphs. 31-38, wherein the composition is administered by intravenous or intrathecal infusion to a patient in need thereof. 40. The method according to claim 39, where the infusion of the viral vector is carried out for 45-75 minutes.