AAV viral vectors and uses thereof

Abstract

To provide a method of treating spinal muscular atrophy (SMA).SOLUTION: Provided is a method comprising the step for administering intrathecally an AAV9 viral vector comprising a polynucleotide encoding a survival motor neuron (SMN) protein, where the viral vector is administered at a dose of about 1×1013 vg to 5×1014 vg.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application is a continuation of U.S. Provisional Patent Application No. 62 / 773,89, filed November 30, 2018. No. 4, and U.S. Provisional Patent Application No. 62 / 835,2 filed on April 17, 2019. No. 42, the contents of which are incorporated by reference in their entireties. No. 6,399,433, which is incorporated herein by reference.

[0002] Array List This application is incorporated herein by reference in its entirety in electronic form. The above ASCII copy was created on November 12, 2019. The file is named 14452_0025-00304_SL.txt and is 14, It is 833 bytes.

[0003] The present disclosure relates to compositions and uses of viral particles. [Background technology]

[0004] Adeno-associated viruses (AAVs) are members of the Parvoviridae family. The AAV genome is a linear, single-stranded genome approximately 4.7 kilobases (kb) in length. It contains a DNA molecule and encodes the nonstructural Rep (replication) and structural Cap (capsid) proteins. It has two major open reading frames that flank the AAV coding region. It has a cis-acting inverted terminal repeat (ITR) sequence of approximately 145 nucleotides in length. and can fold into a hairpin structure that functions as a primer during the initiation of DNA replication. In addition to their role in DNA replication, ITR sequences The virion nucleus is involved in the integration of the virus, its rescue from the host genome, and its assembly into mature virions. It has been shown that it plays a role in the encapsidation of glycine (Muzyczka, (1999) 92)Curr.Top.Micro.Immunol.158:97-129).

[0005] AAV exists in multiple serotypes, providing different tissue tropism. Known serotypes include: , e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. AAV9 is one of them. No. 7,198,951 and U.S. Pat. No. 6,219,951, the entire contents of which are incorporated herein by reference. Gao et al., J. Virol., 78:6381-6388 (2004). Advances in AAV6 and AAV8 delivery have allowed for simple systemic intravenous or intraperitoneal delivery. Following intravenous injection, skeletal and cardiac muscle can be transduced with these serotypes. Pacak et al., Circ. Res., 99(4):3-9(2006) and Wang et al.,Nature Biotech.23(3):321-8(2 However, the use of AAV to target cell types within the central nervous system has not been shown to be For use, surgical intraparenchymal injection is required. Kaplitt et al., “Safe ty and tolerability of gene therapy with an adeno-associated virus(AAV)borne GAD gene for Parkinson's disease: an open la bel,phase I trial.”Lancet,369:2097-2105; Marks et al., “Gene delivery of AAV2-neur turin for Parkinson's disease: a double-b lind,randomized,controlled trial.”Lancet Neurol 9:1164-1172; and Worgall et al., “Tr eating of late infantile neuronal ceroi d lipofuscinosis by CNS administration o fa serotype 2 adeno-associated virus ex pressing CLN2 cDNA.”Hum Gene Ther,19(5): See 463-74.

[0006] The nucleotide sequence of the AAV serotype 2 (AAV2) genome was reported by Ruffing et al. Corrected by I., J Gen Virol, 75:3385-3392 (1994) Srivastava et al., J Virol, 45:555-564 ( 1983) Viral DNA replication (rep), encapsidation / packaging The cis-acting sequences that direct the transcription factor, the transcription factor sequence, and the host cell chromosomal integration are contained within the ITRs. AAV promoters (p5, p19, and p40 based on their relative map positions) The rep and cap genes are encoded by two AAV internal reading frames, which are named Two rep promoters (p5 and p19) drive expression of a single AAV insert. In conjunction with differential splicing of the rep gene (nucleotides 2107 and 2227), Four rep proteins (rep78, rep68, rep52, and rep4) were identified from the gene. 0), which results in the production of the rep protein, which is ultimately responsible for replicating the viral genome. It possesses multiple enzymatic properties. The cap gene is expressed from the p40 promoter, which is It encodes three capsid proteins, VP1, VP2, and VP3. Consensus translation initiation sites and non-consensus translation initiation sites are involved in the generation of three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetic properties of AAV are described in Muzyczka, Current Topics in Microbiology and Immunology,1 58:97-129 (1992).

[0007] AAV-derived vectors are particularly attractive for delivery of genetic material for the following reasons: ) They infect a wide variety of non-dividing and dividing cell types, including muscle fibers and neurons. (ii) they lack viral structural genes, thereby e.g. For example, by abrogating natural host cell responses to viral infection, such as the interferon-mediated response. (iii) the wild-type virus has never been associated with any pathology in humans; (iv) In contrast to wild-type AAV, which can integrate into the host cell genome, replication-defective AAV vectors They generally persist episomally and therefore have no risk of insertional mutagenesis or oncogene activation. (v) in contrast to other vector systems, AAV vectors have significant (see ii) and therefore (their gene products are not likely to cause rejection) The GFP gene confers long-term expression of the therapeutic transgene (unless otherwise specified).

[0008] Self-complementary adeno-associated vectors (scAAV) are naturally occurring vectors that have been developed for use in gene therapy. It is a viral vector engineered from the adeno-associated virus (AAV) present in the sc AAVs are designed such that the coding region forms an intramolecular double-stranded DNA template. Since the typical AAV genome is a single-stranded DNA template, The rate-limiting step in the standard AAV genome life cycle involves second strand synthesis. This is not the case for the scAAV genome, which upon infection does not wait for cellular synthesis of the second strand. The two complementary halves of the scAAV bind and are ready for immediate replication and transcription. It forms one double-stranded DNA (dsDNA) unit.

[0009] Spinal muscular atrophy (SMA) is characterized by reduced levels of the SMN protein and selective induction of motor neurons. The survival motor neuron 1 gene (SMN1) on chromosome 5q13 leads to functional impairment. SMA is a neurogenetic disorder caused by loss or mutation. It is a childhood disease with an incidence of 1 in 10,000 births. rman et al., “Pan-ethnic carrier screening g and prenatal diagnosis for spinal musc ular atrophy: clinical laboratory analysis s of >72,400 specimens.”European journal of human genetics, 20(1):27-32. All forms of SMA is autosomal recessive in inheritance and is caused by a deletion of the survival motor neuron 1 (SMN1) gene. Humans also have a subfamily of SMN1 gene called SMN2. Both the SMN1 and SMN2 genes encode the SMN transcription factor α, β, and β-terminal domains. Although most of the mice express the same protein, the amount of functional full-length protein produced by SMN2 was The amount produced by SMN2 is much less than that produced by SMN1 (10-15%). Although they cannot fully compensate for the loss of the N1 gene, they are generally more prevalent in patients with milder forms of SMA. The large-scale first study by Feldkotter et al. In a phase 1 study, two copies of SMN2 predicted the development of type 1 SMA by 97%, and Three copies of SMN2 are 83% predictive of developing SMA type II, and four copies of SMN2 are 83% predictive of developing SMA type III. It predicted SMA 84% of the time. Feldkotter et al., “Quantitative ive analyzes of SMN1 and SMN2 based on r eal-time lightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. ”American Journal of Human Genetics,70(2 ):358-368. These percentages do not reflect the effects of modifier mutations; This may underestimate the relationship between copy number (in the absence of genetic modifications) and clinical phenotype. Of 113 patients with type 2 SMA, 9 with one SMN2 copy were diagnosed with pulmonary edema before 11 months of age, and 2 with two SMN2 copies were diagnosed with pulmonary edema before 11 months of age. In the first group, 88 / 94 patients survived for less than 21 months, and in the third group, 8 / 10 patients survived for 33-66 months. did.

[0010] Type 1 SMA is the leading genetic cause of infant death. Disease Severity and Clinical Prognosis The onset of glaucoma depends on the number of copies of SMN2. In the most common and severe form (type I), Hypotonia and progressive weakness are recognized in the early stages and diagnosed by 6 months of age, with respiratory failure by 2 years of age. Type I SMA is the leading genetic cause of infant death. Motor neuron loss is evident early after birth (or may even begin prenatally) and is associated with Individuals with type I SMA typically have the SMN2 gene. In contrast, type II SMA becomes evident within the first 18 months. The condition progresses and children affected are able to sit unaided but are unable to walk independently. Patients with type II SMA typically have three copies of the SMN2 gene. Individuals with Type III SMA acquire the ability to walk unaided. In the Type III classification, Patients with type IIIa usually show disease onset before age 3, whereas patients with type IIIb usually show disease onset after age 3. In patients with SMA types II and III, motor neurons adapt and are replaced during development. Patients with type III SMA typically have the SMN2 gene. They have three or four copies of the gene. Findings from various neurophysiological and animal studies suggest that embryos This shows an early loss of motor neurons during the embryonic and early postnatal period. t al.,“Natural history of denervation in SMA:relation to age,SMN2 copy number,an d function.”Annals of neurology 57(5):70 4-12;Le et al.,“Temporal requirement for high SMN expression in SMA mice.”Human molecular genetics,20(18):3578-91;Farrar et al., “Corticomotoneuronal integrity a nd adaptation in spinal muscular atrophy ”Archives of neurology,69(4):467-73.

[0011] Patients with types II and III SMA have a relatively stable clinical course. The difference in outcome may be due to the ability of motor neurons to adapt and compensate during childhood development and persist into adulthood. This suggests that the number of copies of SMN2 plays a key role in the regulation of motor function. In type I SMA, loss of the receptor is evident early after birth (or occurs within the first 3 months of life). This contrasts with type I SMA, which may even begin prenatally in some individuals. Currently, it has been shown to be well tolerated in both mice and non-human primates and has been shown to be effective in treating human SMN. If the copy number of SMN2 is high, there is no risk (types II and III with high copy number of SMN2 For example, in patients with SMA, such as SMA types II and III, Increasing SMN levels in individuals with glaucoma presents a therapeutic option.

[0012] For example, previous treatment efforts in SMA, such as types II and III, have primarily focused on We have focused on the possibility of small molecules to increase SMN levels. Deacetylases such as proacid, sodium butyrate, phenylbutyrate, and trichostatin A Inhibitors are included. The expression of the disease toward the milder features seen in patients with SMA type III. These drugs activate the SMN2 promoter and induce S Increased full-length SMN protein in MA animal models. t al.,“SAHA ameliorates the SMA phenotyp e in two mouse models for spinal muscula r atrophy.”Human molecular genetics,19(8 ):1492-506;Dayangac-Erden et al., “Carbox ylic acid derivatives of histone deacety lase inhibitors induce full length SMN2 transcripts:a promising target for spina l muscular atrophy therapeutics.”Arch Me d Sci,7(2):230-4 2011.

[0013] Some of these drugs, most notably phenylbutyrate, valproate, and hydroxybutyric acid, Clinical trials with thiourea have not yielded significant clinical benefit. t al.,“Evaluation of muscle strength and motor abilities in children with Type I I and III spinal muscle atrophy treated with valproic acid.”BMC Neurol,11:36;www ClinicalTrials.gov. The FDA recently announced that the splice variant of the SMN2 gene By regulating the production of SMN protein, the ultimate goal Antisense oligonucleotide (ASO) drugs designed to compensate for genetic defects One drug, nusinersen, was approved. Summary of the Invention [Problem to be solved by the invention]

[0014] Clinical studies have shown some modest promise for improving motor function; However, the treatment must be administered quarterly via intrathecal injection indefinitely to be effective. Safety considerations requiring a long induction period and clinical monitoring Therefore, alternatives such as those disclosed herein can be used to treat types II and I. There remains a need for improved treatments for SMA, including SMA type II.

[0015] Disclosed herein are compositions comprising AAV9 viral vectors and methods for using them. and methods of treating SMA, for example, in patients with SMA types II and III. In some embodiments, the methods include determining whether an SMA phenotype, such as, for example, SMA type II and SMA type III, is present. Intrathecal injection of an AAV9 viral vector capable of modifying, for example, resulting in a more gentle course of disease progression, halting disease progression, and / or improved functional development . [Means for solving the problem]

[0016] The present disclosure relates to compositions and methods for treating SMA, such as SMA Type II or SMA Type III. For example, the present invention provides a method for the production of scAA expressing the SMN transgene disclosed herein. Recombinant viral vectors such as V provide therapeutic approaches to increase SMN levels. Because the SMN transgene is small, it can be efficiently packaged with scAAV. and the viral titer compared to typical prototype single-stranded AAV viral vectors. However, patients with SMA types II and III tend to be diagnosed at a later age. Many patients are too large to receive safe and effective weight-based intravenous administration of rAAV. Therefore, it is possible that AAV viral vectors can cross the blood-brain barrier and reach the cerebrospinal Intrathecal administration delivered directly to the cerebrospinal fluid is safe and efficient for transmitting lower viral titers Alternatively, alternative methods may be provided.

[0017] The present disclosure relates to polynucleotides encoding survival motor neuron (SMN) proteins. The method comprises administering an AAV9 viral vector comprising administering to the spinal cavity an AAV9 viral vector comprising the steps of: However, about 1 × 10 13 vg~5×10 14 vg dose, for patients who need it In one embodiment, the present invention relates to a method for treating spinal muscular atrophy (SMA), such as SMA type II or SMA type III. In one such embodiment, the AAV9 viral vector comprises a modified AA V2ITR, chicken β-actin (CB) promoter, cytomegalovirus (CM V) immediate / early enhancer, modified SV40 late 16S intron, bovine growth hormone ( In another embodiment, the AAV2 ITR comprises a polyadenylation signal, and an unmodified AAV2 ITR. In another embodiment, the polynucleotide encodes the SMN protein of SEQ ID NO:2. In some embodiments, the AAV9 viral vector comprises SEQ ID NO: 1. In other embodiments, the patient is 24 months of age or older at the time of administration. In other embodiments, the patient is under 6 months of age and optionally between 6 months and 24 months of age. is 60 months of age or younger at the time of administration, and optionally is between 24 months and 60 months of age. In some embodiments, the AAV9 viral vector is about 5.0 x 10 13 vg~3. 0×10 14 In some embodiments, the AAV9 viral vector is administered at a dose of 1.5 mg / kg. The maximum resolution is approximately 6.0 x 10 13 In some embodiments, A is administered at a dose of 100 mg / kg. The AV9 viral vector is approximately 6.0 × 10 13 vg dose. In embodiments, the AAV9 viral vector is up to about 1.2×10 14 Administered at a dose of vg In some embodiments, the AAV9 viral vector is about 1.2 x 10 14 vg In some embodiments, the AAV9 viral vector is administered at a dose of up to about 2 .4×10 14 In some embodiments, the AAV9 viral vector is administered at a dose of 100 mg / kg. The culprit is about 2.4 x 10 14 It is administered in a dose of vg.

[0018] In some embodiments, the AAV9 viral vector is about 1.0×10 13 vg~9 .9×10 14 In some embodiments, the AAV9 virus is administered in a unit dose of 1000 μg. The spectrum is approximately 1.0×10 13 vg~5.0×10 14 Administered in unit doses of vg In some embodiments, the AAV9 viral vector is about 5.0 x 10 13 vg~3 .0×10 14 In some embodiments, the AAV9 virus is administered in a unit dose of 1000 μg. The spectrum is approximately 6.0 × 10 13 In some embodiments, the dose is administered in a unit dose containing 100 mg of vg. In this state, the AAV9 viral vector is approximately 1.2 × 10 14 Administered in unit doses containing vg In some embodiments, the AAV9 viral vector is about 2.4 x 10 14 vg The compound is administered in a unit dose comprising:

[0019] In some embodiments, the patient has a biallelic SMN1 null mutation or inactivating deletion. and optionally, the mutation comprises a deletion of exon 7 of SMN1. In some embodiments, the patient has three copies of SMN2. The c.859G>C substitution in exon 7 on at least one copy of the SMN2 gene In some embodiments, a patient in need thereof is not In some embodiments, the patient has onset of the disease before about 12 months of age. In some embodiments, the patient demonstrates the ability to sit unassisted for about 10 seconds or more upon administration. In some embodiments, the patient has a pulmonary embolism, but is unable to stand or walk. For example, at the time of administration, the World Health Organization Multicenter Growth Standards Study nization Multicentre Growth Reference St Ability to sit unassisted as defined by the WHO-MGRS criteria. In some embodiments, patients are evaluated, for example, about 1 to 24 months after administration, such as, for example, 12 months after administration. Evaluated, for example, by the Bayley Scales of Infant Development®, as defined by In some embodiments, the patient has the ability to stand for at least about 3 seconds. For example, the Bayley Infant and Child Health Scale (BAI-ESA) was used to assess efficacy and safety in patients with pulmonary embolism, for example, after approximately 1-24 months of treatment, such as 12 months after treatment. Ability to walk unassisted after dosing as defined by the Child Development Assessment Test. In embodiments, the patient may be treated with the medicament after about 1 to 24 months, such as, for example, 12 months, after administration. Independence after dosing as assessed, for example, as defined by the Bayley Scales of Infant Development® In some embodiments, the patient has the ability to walk at least five steps after administration of, for example, The study was conducted after approximately 1–24 months of age, e.g., 12 months after the Bayley Scales of Infant Development ( The change from baseline measurements during treatment after dosing is defined as a change from baseline measurements during treatment after dosing.

[0020] In some embodiments, the patient is evaluated for progression after about 1 to 24 months, e.g., 12 months after administration. In the evaluation of the patient after administration, there was no evidence of severe lateral sclerosis, e.g., spinal curvature of 50 degrees or more, as seen by radiography. In some embodiments, the patient is free of a curvature. In some embodiments, the patient has not had a prior scoliosis repair surgery or procedure. and optionally, the patient is In some embodiments, the patient is not undergoing scoliosis repair surgery or treatment prior to administration and / or In some embodiments, the patient does not require the use of invasive ventilatory support following administration. In some embodiments, the patient has no history of standing or walking independently prior to administration. In some embodiments, the patient does not require a gastric feeding tube before and / or after administration. Patients must have an active viral infection (human immunodeficiency virus (HIV); or hepatitis B or (including seropositivity for Hepatitis C or Zika virus). In this condition, patients must have had a severe non-pulmonary / respiratory infection (e.g., pyelonephritis) within 4 weeks prior to dosing. or meningitis). In some embodiments, the patient has not had a chronic inflammatory bowel disease (e.g., rheumatoid arthritis or meningitis) prior to administration. , major renal or hepatic impairment, known seizure disorder, diabetes mellitus, idiopathic hypocalciuria, or In some embodiments, the patient is free of co-morbid conditions, such as symptomatic cardiomyopathy, prior to administration. In some embodiments, the patient has no history of fungal meningitis or brain or spinal cord disease. Known allergies to prednisolone or other glucocorticosteroids or excipients prior to administration In some embodiments, the patient is free of allergies or hypersensitivity to iodine or iodine prior to administration. In some embodiments, the individual has no known allergies or hypersensitivity to products containing the element. The patient was not taking any medications to treat his myopathy or neuropathy. In some embodiments, the patient has received immunosuppressive therapy, plasma exchange, adalimumab, or other anticoagulant therapy within 3 months prior to administration. Not receiving immunomodulatory drugs such as mabs.

[0021] In some embodiments, the patient is tested for immunization prior to administration, e.g., by ELISA binding immunoassay. Anti-AAV9 antibody titers of 1:25, 1:50, 1:75, or 1:100 or less as measured by In some embodiments, the patient has a γ-glucose level less than about 3 times the upper limit of normal prior to administration. Tamyltransferase level less than 3.0 mg / dL, bilirubin level less than 1. A creatinine level of less than 0 mg / dL, an Hgb level between approximately 8 and 18 g / dL, and / or about 20000 / mm 3 In some embodiments, the patient has one or more of the following: In this setting, patients are required to receive treatment with an investigational or approved compound intended to treat SMA prior to administration. In some embodiments, the AAV9 viral vector is Administered with a contrast agent, optionally the contrast agent comprises iohexol. In this case, the amount of contrast agent administered is about 1.0 to 2.0 mL, for example, about 1.5 mL. Optionally, the imaging agent is administered for, e.g., less than 24 hours, less than 12 hours, less than 6 hours, Less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 30 minutes before administration Immediately prior to administration, the AAV9 viral vector is mixed with the imaging agent. AAV9 viral vectors may be administered sequentially, e.g., contrast agent first (e.g., intrathecally). ) is administered, and following administration of the contrast agent, the AAV9 viral vector is administered (e.g., intrathecally). ) are administered. In some embodiments, the imaging agent and the AAV9 viral vector are administered sequentially. For example, an AAV9 viral vector may be administered first (e.g., intrathecally) to induce AA Following administration of the V9 viral vector, a contrast agent is administered (e.g., intrathecally). In embodiments in which the AAV9 viral vector and the imaging agent are administered sequentially, The time for administration of the contrast agent and the CT scan was within 2 hours, 1 hour, 45 minutes, 30 minutes, or 15 minutes. In some embodiments, the A administered to the patient is The total volume of the AV9 viral vector and the contrast agent is greater than about 10 mL, about 9 mL, or about 8 mL. In some embodiments, the method further comprises sedation or anesthesia. In one embodiment, the patient is trendy during and / or after administration of the AAV9 viral vector. In some embodiments, the patient is placed in the St. John's position. After administration, tilt your head down at about 30° for about 10 to 60 minutes, for example, about 15 minutes. It will be placed there.

[0022] In some embodiments, the patient will be administered, for example, about 2 Oral steroids are administered at least about 1-48 hours before, such as 4 hours before. In this embodiment, the patient is administered the viral vector for a short period of time, such as about 30 days. In some embodiments, the oral steroid is administered for at least about 10 to 60 days. In some embodiments, the oral steroid is administered once a day. In some embodiments, the patient is administered the viral vector twice daily. After treatment, patients were monitored for ALT and / or AST levels, and oral steroids were administered to and / or ALT levels below twice the upper limit of normal or below approximately 120 IU / L In some embodiments, the patient continues to receive AAV9 virus up to 30 days after the initial administration. After administration of the vector, the level of T cell response is monitored, and oral steroids, e.g. For example, 10 6 1 spot forming cell (SFC) per PBMC 30 days to detect a T cell response in a patient sample, such as a blood sample with fewer than 0.00 cells The drug continues to be administered afterward.

[0023] In some embodiments, the oral steroid is administered at a dose of about 1 mg / kg.

[0024] In some embodiments, oral steroids are administered to maintain AST and ALT levels below 2x the upper limit of normal. or below about 120 IU / L. , tapering to approximately 0.5 mg / kg / day over 2 weeks, followed by approximately 0. In some embodiments, the oral administration of Steroids were administered at a dose of approximately 1 mg / kg for 30 days, followed by 0.5 mg / kg for 2 weeks. The dose is then tapered to 0.25 mg / kg / day for an additional 2 weeks. In some embodiments, the oral steroid is prednisolone or an equivalent.

[0025] In some embodiments, the therapeutic effect is evaluated by the Bayley Scale of Infant Development®. and / or the Hammersmith Functional Motor Scale Extended (HFMSE). In some embodiments, the method includes administering the AAV9 viral vector simultaneously or sequentially. In some such embodiments, the method further comprises administering a second therapeutic agent to the patient. In another such embodiment, the second therapeutic agent comprises a muscle building agent or a neuroprotective agent. The second therapeutic agent is an antisense oligonucleotide that targets SMN1 and / or SMN2. In some embodiments, the second The therapeutic agent comprises nusinersen and / or stamlumab. In some embodiments, The amount of AV9 viral vector genome is measured using ddPCR. In embodiments, patients receive a 1:25, 2:3, or 3:4 dose of 1:25, 2:4, or 3:5 dose of 1:10, as measured by ELISA binding immunoassay after administration. The patient has an anti-AAV9 antibody titer of 1:50, 1:75, or 1:100 or more and has been receiving treatment for approximately 1 to 8 weeks. or until the antibody titer decreases to less than 1:25, 1:50, 1:75, or 1:100 In some embodiments, the patient is monitored after administration, e.g., by ELISA binding. Anti-A at 1:25, 1:50, 1:75, or 1:100 or greater as measured by immunoassay AV9 antibody titer decreased to less than 1:25, 1:50, 1:75, or 1:100 For example, a steroid such as prednisolone is administered. In some embodiments, the patient has a total cell count of greater than about 67,000 cells / ml or greater than about 100,000 cells / ml prior to administration. / ml or greater than about 150,000 cells / ml. In embodiments, the patient has a serum erythrocyte count of less than about 67,000 cells / ml, or less than about 100,000 cells / ml after administration. A platelet count of less than 0 cells / ml or less than about 150,000 cells / ml and a patient receiving treatment for about 1 to 8 weeks or platelet counts of about 67,000 cells / ml or about 100,000 cells / m The cells are monitored until they increase to greater than 1.5 l or greater than about 150,000 cells / ml. In some embodiments, the patient has a platelet count of less than about 67,000 cells / ml after administration. In some embodiments, the patient is treated with a platelet transfusion. In some embodiments, the patient has normal liver function prior to administration of the ketamine. Have liver transaminase levels less than ~40 U / L.

[0026] In some embodiments, the hepatic transaminases include AST, ALT, and combinations thereof. In some embodiments, the AAV9 viral vector is selected from the group consisting of intrathecal It is a pharmaceutical formulation suitable for internal administration.

[0027] The present disclosure also provides methods for treating SMA, e.g., type II or type I, by the methods described herein. The use of AAV9 viral vectors in the treatment of type II spinal muscular atrophy (SMA) has also been proposed. Provide.

[0028] The present disclosure relates to an AAV9 viral vector and a pharma- ceutically acceptable carrier suitable for intrathecal administration. The AAV9 viral vector comprises a modified AAV2ITR, β-actin (CB) promoter, cytomegalovirus (CMV) immediate / early end Hanser, modified SV40 late 16S intron, bovine growth hormone (BGH) polyadenylation In some embodiments, the polynucleotide comprises an unmodified AAV2 ITR and a nucleotide sequence that confers a polynucleotide sequence similar to that of the unmodified AAV2 ITR. The otide encodes the SMN protein of SEQ ID NO:2. The AV9 viral vector comprises SEQ ID NO: 1. In some embodiments, the pharmaceutical composition In some embodiments, the contrast agent is about 1.5 mL, for example. is present in an amount of about 1.0-2.0 mL.

[0029] In some embodiments, the total volume of the AAV9 viral vector and imaging agent is about 10 mL, In some embodiments, the pharmaceutical composition comprises an additional In some embodiments, the pharmaceutical composition further comprises a therapeutic agent. This document is intended for use in any of the following legal proceedings:

[0030] In some embodiments, the pharmaceutical composition comprises about 1.0×10 13 vg~9.9×10 14 In some embodiments, the pharmaceutical composition comprises a unit dose of about 1.0×10 13 v g~5.0×10 14 In some embodiments, the pharmaceutical composition comprises a unit dose of: Approximately 5.0×10 13 vg~3.0×10 14 Contains a unit dose of vg.

[0031] In some embodiments, the pharmaceutical composition comprises about 6.0×10 13 The unit dose contains vg. In some embodiments, the pharmaceutical composition comprises about 1.2×10 14 In unit doses containing vg In some embodiments, the pharmaceutical composition is about 2.4×10 14 Unit dose containing vg It is.

[0032] In some embodiments, the pharmaceutical composition comprises at least one of the following: (a) about pH 7.7-8.3, (b) about 390-430 mOsm / kg, (c) less than 25 μm per container (d) less than about 600 particles of size 10 μm or more per container; 0 particles, (e) about 1.7 × 10 13 ~5.3×10 13 Genomic titer in vg / mL , (f) 1.0×10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 IU's infectiousness Value, (g) 1.0 x 10 13 Approximately 100-300 μg of total protein per vg, (h) approximately (i) about 70 to 130% Pluronic® F-68 content; Relative potency of (j) 7.5 × 10 13 In the SMNΔ7 mouse model, (k) less than about 5% empty capsids; (l) and median survival of 24 days or more in the presence of 100% or less empty capsids; % total purity or greater, and (m) endotoxin levels less than about 0.13 EU / mL.

[0033] In some embodiments, the pharmaceutical composition comprises at least one of the following conditions: (a) 1.0×10 13 (b) less than about 0.09 ng of benzonase per vg; (c) poloxamer 188 at about 20-80 ppm; (d) 1 .0×10 13(e) less than about 0.22 ng of BSA per vg; 13 vg a Approximately 6.8 x 10 5 Residual plasmid DNA less than pg, (f) 1.0 × 10 13 vg a Approximately 1.1 x 10 5 Residual hcDNA in pg or less (g) 1.0 × 10 13 Approximately per vg Less than 4 ng of rHCP, (h) approximately pH 7.7-8.3, (i) approximately 390-430 mOsm (j) less than about 600 particles 25 μm or larger in size per container; (k) less than about 600 particles 25 μm or larger in size per container (l) less than about 6,000 particles with a size of 10 μm or more per unit area, (l) about 1.7 × 10 13 ~5. 3×10 13 Genomic titer in vg / mL, (m) 1.0 × 10 13 Approximately 3.9 x 1 per vg 0 8 ~8.4×10 10 Infectious titer in IU, (n) 1.0 × 10 13 Approximately 100 per vg 300 μg total protein, (o) a relative titer of about 70-130%, and (p) less than about 5% of empty capsids.

[0034] In some embodiments, the use of the methods or compositions described herein includes administering a pre-administration score Associated with improved scores on the Hammersmith Functional Motor Scale Extended. In some embodiments, the use of the methods or compositions described herein is associated with a pre-administration score. Bayley Scales of Infant Development, Third Edition (Bayley-III) resulting in improved scores. [Brief description of the drawings]

[0035] [Figure 1] The body weights of treated and control mice following AAV administration are shown. [Diagram 2] Initial study design for a Phase 1, open-label, single-dose study in infants and children with SMA Type II or III. Patients will receive AVXS-101 in a dose-comparison safety study. [Diagram 3] Figure 1 shows a waterfall plot of the change from baseline in the Hammersmith Functional Motor Scale Extended (HFMSE), ranked best to worst, in patients with SMA type 2 receiving Dose A (6.0 x 10 vg; indicated by diamonds) or Dose B (1.2 x 10 vg) intrathecal AVXS-101, assessed after 24 months of age. Results for patients between 6 months and 2 years of age at the time of infusion are shown as grey bars; black bars indicate ages between 2 and 5 years at the time of infusion. [Figure 4] 4 shows HFMSE scores for individual patients with SMA type II. [Diagram 5] 1 shows the response to AVXS-101 treatment, as measured by HFMSE, in patients between 6 months and 5 years of age at the time of treatment. [Figure 6] 1 shows the response to AVXS-101 treatment, as measured by HFMSE, in patients between 2 and 5 years of age at the time of treatment who received a dose of 1.2×10 14 vg. [Figure 7] Spaghetti plots of change from baseline in HFMSE scores through Month 12 for the 24 to <60 months of age group (primary PNCR analysis) - ITT set. [Figure 8] Spaghetti plot of change from baseline in HFMSE scores through Month 12 for the 24 to <60 months of age group (sensitivity PNCR analysis) - ITT set. [Figure 9] Shown is a spaghetti plot of the change from baseline in fine motor scores as assessed by the Bayley Scale® at each visit through 12 months post-baseline for patients under 24 months of age at the time of dosing - ITT set. [Figure 10]Spaghetti plot of change from baseline in gross motor scores as assessed by the Bayley Scale® at each visit through 12 months post-baseline for patients under 24 months of age at time of dosing - ITT set. [Figure 11] Spaghetti plot of change from baseline in fine motor scores as assessed by the Bayley Scale® at each visit through 12 months post-baseline for patients aged 24 months or older and under 60 months at the time of dosing - ITT set. [Figure 12] Spaghetti plot of change from baseline in gross and fine motor scores as assessed by the Bayley Scale® at each visit up to 12 months after baseline for patients aged 24 months or older and under 60 months at the time of dosing - ITT set. [Figure 13] Spaghetti plots of the change from baseline in HFMSE at each post-baseline at each visit for patients under 24 months of age at the time of dosing who continued on study past 24 months of age - ITT set. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] In order to provide a better understanding of the present disclosure, certain exemplary embodiments are discussed herein. Additionally, certain terminology is provided to aid understanding.

[0037] In some embodiments, a "vector" refers to a molecule that can self-assemble when associated with the appropriate control elements. Plasmids, phages, transposons, capable of self-replicating and transferring genetic sequences between cells By "genetic element" is meant any genetic element, such as a cosmid, chromosome, virus, virion, etc. Thus, the term includes cloning and expression vehicles, as well as viral vectors. .

[0038] In some embodiments, an "AAV vector" includes, but is not limited to, AAV-1 , AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV Vectors derived from adeno-associated virus serotypes, including AAV-8, AAV-9, and AAV-10, An AAV vector can include, for example, one or more of the rep and / or cap genes. Multiple AAV wild-type genes may be deleted in whole or in part, but functional adjacent ITR sequences Functional ITR sequences are essential for the rescue, replication, and packaging of AAV virions. Therefore, AAV vectors are required for in situ (in cis) viral replication. and at least a sequence that provides for replication and packaging of the gene (e.g., functional ITRs). ITRs are defined herein. The ITRs need not be the wild-type nucleotide sequence, but may be any sequence that is functional. As long as it provides efficient rescue, replication, and packaging, e.g., insertion of nucleotides In one embodiment, the vector may be modified by insertion, deletion, or substitution. It is an AAV-9 vector that has the ITR derived from V-2. , which provides an efficient vehicle for delivering vector nucleic acid to the nucleus of a target cell. By "capsid" is meant the solid shell or capsid.

[0039] In some embodiments, "scAAV" refers to naturally occurring AAV for use in gene therapy. Autologous, a viral vector engineered from the adeno-associated virus (AAV) found in scAAV stands for complement adeno-associated virus (scAAV). scAAV is a virus in which the coding region is a molecule They are called "self-complementary" because they are designed to form a double-stranded DNA template within the do.

[0040] In some embodiments, a "recombinant virus" refers to, for example, a virus that is a vector that incorporates a heterologous nucleic acid construct into a particle. "Recombinant" means a virus that has been genetically altered by the addition or insertion of a nucleotide sequence. It may be abbreviated as "r", for example, rAAV may refer to recombinant AAV. As used herein, the term "AAV" includes "recombinant AAV" or "rAAV." is intended.

[0041] In some embodiments, an "AAV virion" refers to a wild-type (wt) AAV viral particle. A vector (containing a linear, single-stranded AAV nucleic acid genome bound to the AAV capsid protein coat) In this respect, the term "sense" or "antigen" refers to a complete virus particle, such as a A single-stranded AAV nucleic acid molecule of either the complementary sense, such as the "opponent" strand, can be used to identify any one of the AAV nucleic acid molecules. It can be packaged into AV virions and both strands are equally infectious.

[0042] In some embodiments, a "recombinant AAV virion," "rAAV virion," or "AA The terms "V vector particle," "complete capsid," and "complete particle" are used herein. and encapsidating a heterologous nucleotide sequence of interest flanked on both sides by AAVITR. It is defined as an infectious, replication-defective virus containing the AAV protein shell. V virions contain the AAV vector, AAV helper functions, and accessor functions introduced into them. The recombinant vector is then produced in a suitable host cell, which contains sequences that specify the transcription function. The cells are then transfected with AAV vectors (carrying the recombinant nucleotide sequence of interest) for subsequent gene delivery. The AAV polypeptide provides for packaging of the virion protein into an infectious recombinant virion particle. This will allow you to code the code.

[0043] Unless otherwise specified, all technical and scientific terms used herein are intended to be understood as being within the scope of the present disclosure. It has the same meaning as commonly understood by a person skilled in the art to which it pertains. All references used are incorporated by reference in their entirety. To the extent that discussion conflicts with this disclosure, the latter shall control.

[0044] As used herein, the singular form of a word refers to the plural form of the word, unless the context clearly indicates otherwise. By way of example, the terms "a," "an," and "the" refer to either singular or plural. By way of example, "an element" means one or more elements. The term "and / or" is intended to mean "and / or" unless the specific context indicates otherwise. do.

[0045] The term "comprising" or "comprises" Such variations include but are not limited to the listed elements or integers or steps, or the elements, integers or steps. implies the inclusion of a group of steps, but may include other elements or integers or steps, or elements or integers or It is understood that the exclusion of steps is not implied. "consisting of" or "consists of " and variations such as " " may be used to refer to any of the listed elements or integers or steps, or to any of the elements or integers or steps. is the inclusion of a set of steps and any other element or integer or step, or element or integer or It is understood that the phrase "essentially derived from" implies the exclusion of steps. "consisting essentially of" or "consisting essentially of" Variations such as "consists essentially of" An option is any inclusion of any stated element or integer or step, or group of elements or integers or steps. and does not materially affect the basic and novel characteristics of the present disclosure and / or claims. implies the inclusion of any other element or integer or step or group of elements or integers or steps not included in the It is understood to indicate

[0046] Approximately means, for example, ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the stated value , 1%, 0.5%, 0.1%, 0.05%, or 0.01%, etc., understood to be within ±10%. When used in reference to percentage values, "about" means within ±1% (e.g., "about 5%" is 4% to 6% (for example, "about 5%" can be understood as 4.5% to 6% or within ±0.5% Unless otherwise clear from the context, All numerical values ​​provided herein are modified by the term "about." All ranges given are inclusive of the endpoints.

[0047] rAAV viral vectors In one aspect, disclosed herein is a rAAV genome. In the present invention, the rAAV genome comprises a polynucleotide sequence encoding an SMN polypeptide, the sequence of which is flanked by a polynucleotide sequence encoding the SMN polypeptide. In some embodiments, the polynucleotide comprises one or more AAVITRs comprising: For example, a promoter DNA, one or more enhancer DNAs, and / or a gene catalogue. A polyadenylation signal sequence that functions in the target cell to form a set of DNA and other transcription factors. The gene cassette may also contain intron sequences. and may facilitate processing of the RNA transcript when expressed in a mammalian cell. .

[0048] In some embodiments, the rAAV genome disclosed herein comprises AAVrep and The AAV DNA in the rAAV genome (e.g., ITRs) is 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 Any AAV serum capable of inducing recombinant virus, including but not limited to: The nucleotide sequences of the genomes of AAV serotypes are known in the art. For example, the complete genome of AAV-1 is available under GenBank accession number NC_00207. 7 and the complete genome of AAV-2 is available under GenBank accession number NC001401. and Srivastava et al., Virol.,45:555-564{19 The complete genome of AAV-3 is available under GenBank accession number NC_1829. The complete AAV-4 genome is available at GenBank accession number NC_001829. The AAV-5 genome is available under GenBank accession number AF085716; The complete genome of AV-6 is provided under GenBank accession number NC_001862;AA At least a portion of the V-7 and AAV-8 genomes are designated GenBank Accession Nos. AX 753246 and AX753249; the AAV-9 genome is provided by Gao et al. l., J. Virol., 78:6381-6388 (2004); AAV- The 10 genome is provided in Mol. Ther., 13(1):67-76 (2006); A The AV-11 genome is published in Virology, 330(2):375-383 (2004). Provided.

[0049] As used herein, a "pSMN" vector plasmid is a vector encoding the SMN protein. The cassette comprises a polynucleotide that encodes an SMN cDNA expression cassette, For example, the "left" and "right" polynucleotides encoding the SMN gene are It is flanked by associated virus inverted terminal repeat (ITR) sequences. In some embodiments, A polynucleotide encoding SMN can be, for example, a naturally occurring human SMN sequence or In some embodiments, the SMN sequence is a human SMN sequence, such as an isoform, variant, or mutant thereof. In some embodiments, the ITR sequence is a naturally occurring, mutated, or modified AAVITR sequence. In the present invention, at least one ITR sequence is a native, mutated, or modified AAV2 ITR sequence. In some embodiments, the two ITR sequences are native, mutated, or modified AAV2I. In some embodiments, the "left" ITR is a nucleotide sequence that is used to generate a self-complementary genome. The "right" ITR is a modified AAV2 ITR sequence that allows In some embodiments, the "right" ITR allows for the generation of a self-complementary genome. The "left" ITR is the native AAV2 ITR sequence. In some embodiments, the pSMN plasmid contains the CMV enhancer / chicken beta -actin ("CB") promoter. In some embodiments, pSMN The plasmid further contains a Simian Virus 40 (SV40) intron. In an embodiment, the pSMN plasmid contains the bovine growth hormone (BGH) polyadenylation (poly A) Further comprising a termination signal. It can be used with one or more of the components discussed above. Exemplary sequences are shown in Table 1 below. In some embodiments, all sequences shown in Table 1 below are In some embodiments, "AVXS-101" is any one of the sequences in Table 1. A non-limiting example of a vector construct within the scope of the term pSMN is These vector embodiments and methods for preparing and purifying them are described, for example, in No. PCT / US2018 / 058744, the entire contents of which are incorporated herein by reference. No. 6,331,625.

[0050] In some embodiments, the pSMN vector comprises an SMN cDNA expression cassette, a modified AAV2ITR, chicken β-actin (CB) promoter, cytomegalovirus (CMV) immediate / early enhancer, modified SV40 late 16s intron, bovine growth hormone and an unmodified AAV2 ITR. The modified and unmodified ITRs can be oriented in either direction ( That is, it may come at the 5' or 3' position.

[0051] [Table 1]

[0052] In some embodiments, the vector construct sequence is a sequence that is encoded by, for example, an AAV9 virion. In these embodiments, the encapsidation can be, for example, a fully functional Non-replicative recombinant vectors capable of delivering stable functional transgenes, such as the human SMN transgene. In some embodiments, the capsid is, for example, an AAV9 capsid. The alternative splices P3 and P4 are two truncated forms of VP1, all with a common C-terminal sequence. The 60 viral proteins (V In some embodiments, the process for the production of, for example, a pharmaceutical product, The process's products contain non-replicating recombinant AAV9 capsids and are stable, fully functional human In some embodiments, the capsid comprises VP2 and Alternative sequences have been identified such that VP3 is a truncated form of VP1, all of which share a common C-terminal sequence. 60 viral proteins generated by purifying in a ratio of 1:1:10 ( VP1, VP2, VP3). Methods for preparing and purifying them are described, for example, in , as provided in PCT / US2018 / 058744.

[0053] In various embodiments, the pA vector construct, such as, for example, the AVXS-101 vector construct, The DNA sequence of the SMN vector construct comprises SEQ ID NO:1: [ka] [ka]

[0054] In some embodiments, the vector is controlled by a pSMN plasmid, such as AVx101. The amino acid sequence of the encoded SMN protein includes: [ka]

[0055] In some embodiments, the AAV capsid proteins VP1, VP2, and VP3 are identical. These are derived from transcripts that have alternative start sites but share a common carboxy terminus. In the figure, the amino acid sequence unique to VP1 is shown in bold black. The amino acid sequences of all three capsid proteins are underlined and italicized. Amino acids common to both are in bold italics. [ka]

[0056] In one embodiment, the AAV capsid protein has the amino acid sequence shown in SEQ ID NO:3. It is derived from the encoding transcript.

[0057] In various embodiments, disclosed herein is a DNA probe comprising an rAAV genome. The DNA plasmid encodes the rAAV genome and the AAV9 capsid protein. To assemble into infectious viral particles containing the AAV helper virus (e.g., adenovirus), Adenovirus, E1-deleted adenovirus, or herpesvirus) The AAV genome, rep and cap genes, and helper genes are packaged and transferred to the cells. Techniques for generating rAAV particles in which per-viral functions are provided to cells are described in the art. In some embodiments, the generation of rAAV is achieved by single cell (as described herein). The rAAV is a recombinant AAV that is present in a cellular environment (referred to as a packaging cell in the literature) and contains the following components: genome, AAVrep and cap genes separate from (i.e., not included in) the rAAV genome Genes, and helper virus functions. The generation of pseudotyped rAAV can be achieved by, for example, International Publication No. WO01 / 83692, the disclosure of which is incorporated herein by reference. In various embodiments, the AAV capsid protein is modified to: The delivery of the recombinant vector may be enhanced. Modifications of the capsid protein may be performed using techniques known in the art. See, for example, U.S. Pat. No. 6,393,623, the entire contents of which are incorporated herein by reference. US Patent Application Publication No. 2005 / 0053922 and US Patent Application Publication No. 2009 / Please refer to specification 0202490.

[0058] General principles of rAAV generation are described, for example, in Carter, 1992, Current Op inions in Biotechnology, 1533-539; and Muzyc zka,1992,CUM Topics in Microbial.and Imm Various approaches are reviewed in Ratsch et al. in et al.,Mol.Cell.Biol.4:2072(1984);Hen nonat et al.,Proc.Natl.Acad.Sci.USA,81:6 466(1984);Tratschin et al.,Mol.Cell.Biol .5:3251(1985);McLaughlin et al., J. Virol. , 62:1963 (1988); and Lebkowski et al., 1988 M ol.Cell.Biol.,7:349(1988).Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Patent No. 5,173 ,414; WO 95 / 13365 and corresponding U.S. Pat. No. 5,414,414; ,658,776; WO 95 / 13392; WO 9 No. 6 / 17947; PCT / US98 / 18600; International Publication No. No. 7 / 09441 (PCT / US96 / 14423 specification); International Publication No. 97 / 08298 pamphlet (PCT / US96 / 13872 specification); International Publication No. 97 / 21825 (PCT / US96 / 20777 specification); International Publication Patent No. 97 / 06243 (Patent No. PCT / FR96 / 01064); International Publication No. 99 / 11764; Perrin et al. (1995) Va ccine 13:1244-1250;Paul et al. (1993) Huma n Gene Therapy 4:609-615;Clark et al.(19 96) Gene Therapy 3:1124-1132; U.S. Patent No. 5,786,2 No. 11; U.S. Pat. No. 5,871,982; and U.S. Pat. No. 6,258,5 95. Additionally, the rAAV disclosed herein is

[0023] Preparation, purification, manufacture, and / or formulation according to the disclosure of S2018 / 058744. The aforementioned documents are incorporated herein by reference in their entirety. This document focuses on sections related to the preparation, purification, production, manufacturing, and formulation of rAAV. is placed there.

[0059] In another embodiment, a vector encoding an SMN protein, such as the rAAV9 discussed herein, is used. A rAAV containing a polynucleotide that encodes the rAAV SMN is referred to as a "rAAV SMN." In an embodiment, the rAA SMN genome comprises a first AAV2 ITR, a cytomegalovirus endonucleases (ITR), The chicken β-actin promoter with enhancer, SV40 intron, and SMN A polynucleotide encoding a polyadenylation signal sequence derived from bovine growth hormone, and a second AAV2 ITR in the sequence. Polynucleotides that encode the polypeptides are described, for example, in GenBank accession numbers MN_000344.2, GenBank accession number NM_017411, or any other suitable human SMN isoform Exemplary SMN sequences include the following: [ka]

[0060] Conservative nucleotide substitutions in SMN DNA are also contemplated (see, e.g., GenBank A guanine to adenine change at position 625 of accession number NM_000344.2) In some embodiments, the genome lacks AAV rep and cap DNA, That is, there is no AAV rep or cap DNA between the ITRs of the genome. MN polypeptides include, but are not limited to, those listed in the NCBI protein database. The human SMN1 polypeptide is described in database number NP_000335.1. In an embodiment, the SMN DNA comprises a polypeptide encoding a human SMN polypeptide. nucleotides (e.g., Uniprot accession number Q16637, isoform 1 (Q Human SMN protein identified by SMN1-modifier polyclonal antibody (SEQ ID NO: 16637-1). Peptideplastin-3 (PLS3) is also envisioned [Oprea et al., science 320(5875):524-527(2008)]. Other polypeptides The sequence encoding the tide may replace the SMN DNA.

[0061] Pharmaceutical Compositions In various embodiments, the viral particles of the present disclosure (referred to as viral particles) are The composition may be provided in a pharmaceutical composition suitable for intravenous administration. In addition to the viral particles, the composition may contain one or more It may be provided in a formulation containing multiple inactive ingredients and / or one or more additional active ingredients. In some embodiments, the compositions of the present disclosure may be prepared using ingredients and techniques known in the art. into a formulation suitable for intrathecal administration in a mammalian subject, such as a human. obtain.

[0062] In some embodiments, the pharmaceutical formulation comprises: (a) a survival motor neuron (SMN) protein; (b) an AAV9 viral vector containing a polynucleotide encoding a protein; solution, (c) magnesium chloride, (d) sodium chloride, and (e) poloxamer (e.g. , poloxamer 188), and the pharmaceutical composition is preservative-free. In one embodiment of the formulation, The AAV9 viral vector contains a modified AAV2ITR, chicken β-actin (CB) promoter, and promoter, cytomegalovirus (CMV) immediate / early enhancer, modified SV40 post 16s intron, bovine growth hormone (BGH) polyadenylation signal, and unmodified In one embodiment of the formulation, the Tris buffer concentration is, for example, about In one embodiment, the pH of the formulation is, for example, about p H8.0, etc., is about 7.7 to about 8.3 (for example, USP <791> Measured by (the entire contents of which are incorporated herein by reference). In one embodiment of the formulation, The magnesium concentration is, for example, about 0.5 to 1.5 mM, such as about 1 mM. In an embodiment, the sodium chloride concentration is about 100 to 300 mM, for example, about 200 mM. In one embodiment, the formulation contains, for example, about 0.005% w / v poloxamer 188. and the like, containing about 0.001-0.15% w / v poloxamer 188. In this embodiment, the formulation may contain, for example, about 1.9 to 4.2 × 10 13 vg / mL, approximately 1 to 8 x 10 1 3 vg / mL of AAV9 viral vector. In some embodiments, the formulation comprises about 1~8×10 13 vg / mL, and the AAV9 viral vector was approximately 6.0 × 10 13 In some embodiments, the formulation is administered in a unit dose of about 1.9 to 4.2 x 1 vg. 0 13 vg / mL, and the AAV9 viral vector was approximately 6.0 × 10 13 Unit of vg In some embodiments, the formulation is administered at a dose of about 1-8×10 13 vg / mL The AAV9 viral vector contains approximately 1.2 x 10 14 It is administered in a unit dose of vg. In some embodiments, the formulation comprises about 1.9 to 4.2 x 10 13 vg / mL, AA The V9 viral vector was approximately 1.2 × 10 14 vg unit dose. In embodiments, the formulation comprises about 1-8×10 13 vg / mL, AAV9 viral vector - is about 2.4 x 10 14 In some embodiments, the formulation is administered in a unit dose of 100 mg / kg. is about 1.9 to 4.2 × 10 13 vg / mL, and the AAV9 viral vector contains approximately 2 .4×10 14 It is administered in a unit dose of vg.

[0063] When formulated as a solution or suspension, the delivery system may be an acceptable carrier, such as an aqueous carrier. The solution may comprise a suitable carrier, such as water, buffered water, and / or saline. Various aqueous carriers may be used. The formulation may also contain, for example, NaCl, sugar, mannitol, etc. Any solution may comprise an isotonicity agent to make it isotonic or isotonic. For example, surfactants such as polysorbate 20 and polysorbate 80 are included, and the like are used for the interface and The composition may be stabilized against shear and stress. Buffered with citrate, histidine, phosphate or Tris buffer to obtain optimal pH. These compositions may be sterilized using sterilization techniques. The resulting aqueous solutions may be packaged for immediate use. or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.

[0064] For example, compositions, such as pharmaceutical compositions, may contain pH adjusting and buffering agents; tonicity adjusting agents; Sodium lactate, sodium chloride, potassium chloride, calcium chloride, molybdate, Pharmaceutically useful wetting agents such as sorbitan nolaurate and triethanolamine oleate Acceptable auxiliary agents may be included to approximate physiological conditions. In some other embodiments, the pharmaceutical composition comprises a preservative. Does not include.

[0065] In some embodiments, the pharmaceutical composition also optionally contains, for example, an imaging agent (e.g. , Omnipaque™ 180), In some embodiments, the pharmaceutical composition comprises an SMN polynucleotide as disclosed herein. a viral vector containing a nucleotide; In some such embodiments, the imaging agent also contains a iohexol-containing agent. In some other embodiments, the imaging agent is premixed with the pharmaceutical composition. In some embodiments, the imaging agent is not premixed with the composition immediately prior to intrathecal administration. In some embodiments, a contrast agent (e.g., Omnipaque ( Some drugs, such as iohexol and iometholone, increase the transduction of motor neurons. In embodiments, the contrast agent (e.g., Omnipaque™, iohexol, etc.) , aiding in guidance of the intrathecal needle into the subarachnoid space.

[0066] In some embodiments, the imaging agent comprises an SMN polynucleotide disclosed herein. and the imaging agent is administered in combination with a viral vector comprising the Not premixed or co-formulated. For example, in some embodiments, The contrast agents and viral vectors containing the SMN polynucleotides disclosed herein are In some embodiments, the imaging agent is administered as a single bolus. Prior to this, the cells are mixed with a viral vector containing the SMN polynucleotide.

[0067] In some embodiments, the pharmaceutical compositions are, for example, those described herein, the entire contents of which are hereby incorporated by reference. No. PCT / US2018 / 058744, incorporated by reference herein. In some embodiments, the compound may be prepared and purified according to methods known in the art. The pharmaceutical composition may have less than about 7% empty cells as assessed, for example, by qPCR or ddPCR. Psid (e.g., 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or any value in between) In some embodiments, the pharmaceutical composition has a purity of Having one or more of the following characteristics: 13 Less than 0.09ng of benzodiazepine per vg enzymes, less than 30 μg / g (ppm) cesium, and approximately 20-80 ppm poloxamer 1 88, 1.0×10 13 <0.22ng BSA per vg, 1.0×10 13 vg a 6.8×10 5 Residual plasmid DNA, less than pg, 1.0 × 10 13 1 per vg. 1×10 5 Residual hcDNA less than pg, and 1.0 × 10 13 R less than 4ng per vg HCPs.

[0068] In various embodiments, the pharmaceutical composition is within ±20%, within ±15%, or within ±20% of the reference standard. 10%, or ±5%. In one embodiment, the titer is determined by Foust et al.,Nat.Biotechnol.,28(3),pp.271-274( 2010) method against a reference standard. Any suitable reference standard may be used. In one embodiment, the pharmaceutical composition is tested in SMAΔ7 mice. In one embodiment, the in vivo potency is 7.5×10 13 vg / kg dose. Mice were tested for >15 days, >20 days, >22 days, or >24 days. In one embodiment, the pharmaceutical composition is administered in an in vitro cell-based assay. 50-150%, 60-140% of the reference standard and / or appropriate control tested by % or has a potency of 70-130%.

[0069] In some embodiments, the pharmaceutical composition comprises, for example, about 1-8×10 13 Between vg / mL etc., about 1 × 10 13 vg / mL ~ 1 × 10 15 rAAV virus at a concentration between 100 and 200 mg / mL In some embodiments, the pharmaceutical composition has less than about 10%, less than about 8% In some embodiments, the viral capsids have less than about 7% or less than about 5% empty viral capsids. So, the pharmaceutical composition is 1×10 13 Less than approximately 100 ng / mL of host cells per vg / ml In some embodiments, the pharmaceutical composition comprises 1×10 13 vg / ml Approximately 5 x 10 6 Less than pg / mL, approximately 1 × 10 6 Less than pg / mL, approximately 7.5 × 10 5 p g / mL or less than 6.8 x 10 5 Residual host cell DNA (hcDNA) less than pg / mL In some embodiments, the pharmaceutical composition has a molecular weight of 1.0×10 13 Per vg / mL Less than about 10 ng, less than about 8 ng, less than about 6 ng, or less than about 4 ng of residual host cell protein. In some embodiments, the pharmaceutical composition comprises at least about 50 %, at least about 60%, at least about 70%, at least about 80%, at least about 90 %, at least about 95%, or at least about 100% of the rAAV (e.g., AAV9) In some embodiments, the pharmaceutical composition comprises a vector having a viral vector genome / mL that is functional. , 1×10 13 1.7 × 10 per vg / ml6 pg / ml or less, or 1×10 13 vg 1 × 10 per ml 5 pg / ml to 1×10 13 1.7 × 10 per vg / ml 6 pg / ml of residual plasmid DNA. In some embodiments, the pharmaceutical composition comprises 1. 0×10 13 <0.2ng per vg, 1.0×10 13 Less than 0.1 ng per vg or 1.0 x 10 13 Has a benzonase concentration of less than 0.09 ng per vg. In some embodiments, the pharmaceutical composition comprises 1.0×10 13 <0.5ng per vg, 1.0 ×10 13 Less than 0.3 ng per vg or 1.0 × 10 13 Less than 0.22 ng per vg In some embodiments, the pharmaceutical composition has a bovine serum albumin (BSA) concentration of less than 100 mg / mL. is 1.0 × 10 13 Less than approximately 1 EU / mL per vg / mL, 1.0 × 10 13 vg / m Approximately 0.75 EU / mL per liter, less than 1.0 x 10 13 Approximately 0.5 EU / vg / mL Less than 1.0 x 10 mL 13 Less than approximately 0.4 EU / mL per vg / mL, 1.0 × 10 1 3 Less than approximately 0.35 EU / mL per vg / mL, 1.0 × 10 13 Approximately 0 per vg / mL Less than .3EU / mL, 1.0×10 13 Less than approximately 0.25 EU / mL per vg / mL, 1 .0×10 13 Less than approximately 0.2 EU / mL per vg / mL, 1.0 × 10 13 vg / mL Approximately 0.13 EU / mL per 1.0 × 10 13Approximately 0.1 EU / m per vg / mL Less than L, 1.0 x 10 13 Less than about 0.05 EU / mL per vg / mL, or 1.0 x 1 0 13 Some have endotoxin levels of less than about 0.02 EU / mL per vg / mL. In embodiments, the pharmaceutical composition has a pH of less than 100 μg / g (ppm), less than 50 μg / g (ppm), In some embodiments, the cesium concentration is less than 30 μg / g (ppm). The method includes adding about 10 to 100 ppm, 15 to 90 ppm, or about 20 to 80 ppm of porphyrin. In some embodiments, the rAAV viral vector comprises oxamer 188. The pharmaceutical composition contains less than 2,000 particles or less than 1,500 particles having a size of 25 μm or more per container. In some embodiments, the pharmaceutical composition has fewer than 1000 or fewer than 600 particles. The number of objects per container is less than 10,000, 8,000, or 100, with a size of 10 μm or more. In some embodiments, the pharmaceutical composition has less than 0 or less than 6000 particles. It has a pH between 7.5 and 8.5, between 7.6 and 8.4, or between 7.8 and 8.3. In some embodiments, the pharmaceutical composition has an oxidative stress response of between 330-490 mOsm / kg, between 360-4 60 mOsm / kg, or between 390 and 430 mOsm / kg. In some embodiments, the pharmaceutical composition is 1.0×10 13 Approximately 1.0 × 10 per vg 8 ~10.0×10 10 IU, 1.0×10 13 Approximately 2.5 × 10 per vg 8 ~9.0×1 0 10 IU or 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 IU In some embodiments, the pharmaceutical composition comprises an in vitro cell-based Based on the assay, about 30-150%, about 6% relative to the reference standard and / or appropriate control. In some embodiments, the relative potency is about 0-140%, or about 70-130%. The drug composition is 1.0 x 10 13 Approximately 10-500 μg per vg, 1.0 × 10 13 vg a Approximately 50-400μg per dose, or 1.0×10 13 Approximately 100-300 μg of total protein per vg In some embodiments, the pharmaceutical composition has a protein level of 7.5×10 13 v As determined by median survival time in SMNΔ7 mice receiving a dose of 1 g / kg, It has an in vivo efficacy of more than 5 days, more than 20 days, more than 22 days, or more than 24 days. In some embodiments, the pharmaceutical composition meets a combination of one or more (e.g., all) of the aforementioned criteria. Fill the mix.

[0070] The present disclosure also provides methods for treating a variety of conditions, such as SMA type II or III, in a patient in need thereof. A kit for treating SMA is provided, the kit comprising a pharmaceutical composition disclosed herein. and / or one or more doses of the compound, e.g., an SMN polynucleotide as disclosed herein. and optionally, for example, an imaging agent (e.g., , Omnipaque™ 180), and instructions on how to use the pharmaceutical formulation or composition. In some embodiments, the kit includes one or more doses of the pharmaceutical compositions disclosed herein, e.g., For example, an effective amount or dose of a viral vector comprising the SMN polynucleotide disclosed herein. and optionally a contrast agent (e.g., Omnipaque™, or ion exchange). It contains hexol-containing agents.

[0071] In some embodiments, the kit includes a contrast agent premixed in the same container as the pharmaceutical composition. In some embodiments, the kit comprises one of or a plurality of containers, and a medical agent provided in one or more additional containers. In some embodiments, the imaging agent is mixed with the pharmaceutical composition prior to intrathecal administration. are combined.

[0072] In some embodiments, the kit comprises one or more vectors of the viral vector pharmaceutical composition. In some embodiments, each vial contains up to or about 6.0 x 10 13 v g of the viral vector pharmaceutical composition. In one embodiment, each vial (e.g., each unit dose) of viral vector in the kit contains about 6.0× 10 13 In some embodiments, the kit comprises a pharmaceutical composition in a dose of 0.5 mg / kg. Each vial (e.g., each unit dose) of the drug contains up to or about 1.2 x 10 14 Dosage of vg In some embodiments, each vial of viral vector in the kit comprises a pharmaceutical composition. (e.g., each unit dose) is about 1.2 x 10 14 The pharmaceutical composition contains a dose of 100 mg / kg. In some embodiments, each vial (e.g., each unit dose) of viral vector in the kit contains: Maximum or approximately 2.4 x 10 14 In some embodiments, the pharmaceutical composition comprises a dose of Each vial (e.g., each unit dose) of viral vector in the kit contains approximately 2.4×10 14 In some embodiments, the pharmaceutical composition comprises a viral vector pharmaceutical composition in a dose of 0.05 mg / kg. is about 0.1 to 5.0 × 10 13 In some embodiments, each The vials contain a single dose of the rAAV viral vector. In some embodiments, each vial The vial contains more than a single dose of the rAAV viral vector. In some embodiments, Each vial contains less than a single dose of the rAAV viral vector.

[0073] Use of rAAV9 viral vectors In various embodiments, disclosed herein are rAAV SMN polynucleoside analogs. For example, type II or delivers polynucleotides to patients in need of treatment for SMA, including type III SMA. In some embodiments, the delivery comprises administering a rAAV9 as disclosed herein. In some embodiments, the method comprises administering the compound to the patient via intrathecal delivery to the patient's central nervous system. In some such embodiments, the rAAV9 and the AAV9 are administered together with an imaging agent. The imaging agents are, for example, co-administered in a single pharmaceutical composition. In some embodiments, the rAAV9 and the imaging agent are administered sequentially. For example, in some embodiments, the imaging agent is The rAAV9 is administered following administration of the imaging agent. In this case, rAAV9 was administered first, and the contrast agent was administered following administration of the AAV9 viral vector. In an embodiment in which the AAV9 viral vector and the imaging agent are administered sequentially, The AAV9 viral vector and the contrast agent can be administered, for example, within about 2 hours, within 1 hour, or within 45 The test may be conducted within 30 minutes, 15 minutes, 10 minutes, or 5 minutes. In some embodiments, at least one of the imaging agent and the rAAV9 is administered intrathecally. In some embodiments, both the imaging agent and the rAAV9 are administered intrathecally (either simultaneously or sequentially). (The next dose does not matter.)

[0074] In some embodiments, the contrast agent is a non-ionic, low osmolarity contrast agent. In embodiments, the imaging agent may increase transduction of target cells in the patient's central nervous system. In some embodiments, the imaging agent may help target delivery directly to the subarachnoid space. In some embodiments, the rAAV9 genome is a self-complementary genome. In one embodiment, the rAAV9 genome is a single-stranded genome.

[0075] In some embodiments, the rAAV viral vector is capable of reaching the cerebrospinal fluid (CSF). In some embodiments, the rAA is delivered intrathecally to the spinal canal or subarachnoid space. V viral vectors may diffuse within the CSF to areas distal to the delivery site. In embodiments, the rAAV viral vector is delivered to a brain region. In this study, rAAV viral vectors are delivered to the motor cortex and / or brainstem. In embodiments, the rAAV viral vector is delivered to the spinal cord. In one embodiment, the rAAV viral vector is delivered to a lower motor neuron. Using rAAV9, rAAV viral vectors are delivered to neuronal and glial cells In some embodiments, the glial cells are microglial cells, oligodendrocytes or astrocytes. In some embodiments, rAAV9 is used to express the rAAV viral vector. is delivered to Schwann cells.

[0076] The titer of an administered rAAV viral vector can vary depending on, for example, the particular rAAV, the mode of administration, Depending on the therapeutic goal, the age and other characteristics of the individual being treated, and the cell type being targeted. The titer may vary. The titer may be measured by known methods. The titer of rAAV is 1 Approximately 1 x 10 per ml 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 , about 1×10 15 The dosage may be in the range of DNase resistant particles (DRP) or more. Genomic titers may be expressed in units of (vg). Genomic titers are described herein and in Lock et al. al., or any other method known in the art. The dosage may also vary based on the timing of administration to humans. The dosage of these rAAVs is approximately 1× per kilogram of body weight for adults or newborns. 10 11 vg / kg, approximately 1×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 1×1 0 14 vg / kg, approximately 1×10 15 vg / kg, approximately 1×10 16 Vectors > vg / kg - genome-wide.

[0077] In some embodiments, rAAV9 is administered at 1.0×1013 vg~9.9×10 14 v In some embodiments, the rAAV9 is administered at a dose of 5.0×10 13 vg ~3.0×10 14 In some embodiments, the rAAV9 is administered at a dose of Max 6.0×10 13 In some embodiments, the rAAV9 is administered at a dose of 100 mg / kg. , about 6.0×10 13 In some embodiments, the rAAV9 is administered at a dose of 100 mg / kg. , up to 1.2×10 14 In some embodiments, the rAAV9 is administered at a dose of 100 mg / kg. is about 1.2 x 10 14 In some embodiments, the rAAV9 is administered at a dose of 100 mg / kg. is up to 2.4 x 10 14 In some embodiments, the rAAV is administered at a dose of 100 mg / kg. 9 is approximately 2.4 x 10 14 It is administered in a dose of vg.

[0078] In some embodiments, the rAAV9 is about 1.0×10 13 vg~9.9×10 14 In some embodiments, the rAAV9 is administered at a unit dose of about 1.0×10 13 vg~5.0×10 14 In some embodiments, the r AAV9 is approximately 5.0 × 10 13 vg~3.0×10 14 It is administered in a unit dose of vg.

[0079] In some embodiments, the rAAV9 is about 6.0 x 10 13 Administered in unit doses of vg In some embodiments, the rAAV9 is about 1.2×10 14 Administered in unit doses of vg In some embodiments, the rAAV9 is about 2.4×10 14 Administered in unit doses of vg can be.

[0080] The dosage can be determined by any suitable method, for example, a method specific for the viral vector. While PCR using large primers can provide a relative measurement, qPCR uses smaller primers. In some embodiments, ddPCR may be used for small samples and absolute measurements. ddPCR performs digital PCR based on water-oil emulsion droplet technology. Baker et al., “Digital PCR hit s its stride.”Nature Methods,9(6):541-54 4.Sykes et al., “Quantitation of targets for PCR by use of limiting dilution.”Bio Techniques, 13(3) 444-449. The sample is fractionated into tens of thousands of droplets, PCR amplification of template molecules is performed in each droplet. Standard curves can be generated and amplification efficiency can be evaluated. Compared to traditional PCR-based techniques, ddPCR is faster because it does not require new primers. Generally, large numbers of samples are not used. Examples of commercially available ddPCR instruments include Although not specified, BioRad Qx100dd PCR and RainDanc e Raindrop Digital PCR. In one embodiment, the dose is In another embodiment, the dosage is determined using qPCR. In another embodiment, the dose is determined using digital droplet PCR (ddPCR). In some embodiments, multiple methods are used. The PCR-based method uses specifically designed primers and probes that target the SMN gene. The probe is used to detect and quantify the encapsidated AAV9 viral genome. In another embodiment, the PCR-based method targets the chicken β-actin promoter. Using specially designed primers and probes to identify encapsidated AAV9 In another embodiment, the PCR-based method detects and quantifies the viral genome. Using specially designed primers and probes targeting the CMV enhancer, Encapsidated AAV9 viral genomes are detected and quantified. The PCR-based method uses specifically designed primers and primers that target the ITR sequences. The probe is used to detect and quantitate the encapsidated AAV9 viral genome. In other embodiments, the PCR-based method comprises the step of amplifying the bovine growth hormone polyadenylation signal. Using specifically designed primers and probes that target the encapsidated In some embodiments, the titer is determined by detecting and quantifying the AAV9 viral genome. The expression of SMNΔ is measured using appropriate in vitro cellular assays or in vivo animal models. Using animal models of SMA, such as SMAΔ7 mice, or from the cortex of SMAΔ7 mice, for example, Quantitative cell-based analysis using appropriate cell lines, such as primary neural progenitor cells (NPCs) isolated from mice Using this assay, for example, titer or percent functional AAVSMN viral particles can be measured. In one embodiment, the titer is determined according to the method of Foust et al., Nat. Biotech. Using the method of Hnol., 28(3), pp.271-274(2010), the reference Any suitable reference standard may be used. Dosage, purity, and percent functional viral vectors of the disclosed rAAV viral vectors. Exemplary methods for determining the fraction are described in US Pat. No. 6,393,363, the entire contents of which are incorporated herein by reference. , and the disclosure of PCT / US2018 / 058744.

[0081] The formulation of the rAAV viral vector to be administered may include, for example, the method of intrathecal administration, the dosage, and pharmaceutical excipients. Grouls et al., "General considerations in the formulation of dr ugs for spinal delivery.”Spinal Drug Del ivery,Chapter 15,Elsevier Science,Yaksh In some embodiments, the rAAV viral vector is administered intrathecally. It may be administered in a suitable therapeutic formulation. In some embodiments, the rAAV viral vector In some embodiments, the rAA may be administered intrathecally as a bolus injection. The V viral vector may be administered intrathecally as a slow infusion. In some embodiments, the rAAV viral vector may be formulated in a sterile isotonic drug solution. In some embodiments, the rAAV viral vector may be formulated in saline. In embodiments, the rAAV viral vector is delivered in an artificial CS solution, such as, for example, Elliot's B solution. F. In some embodiments, the therapeutic formulation is filtered prior to administration.

[0082] In various embodiments, the methods and materials disclosed herein are used to treat SMA. For example, intrathecal administration of SMN1 has been shown to be effective in treating SMA in patients who lack a functional copy of SMN1. Humans also have a second, nearly identical copy of the SMN gene, designated SMN2, which can be used in therapeutics. Lefebvre et al. “Identification of nd characterization of a spinal muscular atrophy-determining gene.”Cell,80(1):15 5-65;Monani et al. “Spinal muscular atrop hy:a deficiency in a ubiquitous protein; a motor-neuron specific disease.”Neuron, 48(6):885-896. Both the SMN1 and SMN2 genes encode the SMN protein. However, SMN2 contains a translationally silent mutation in exon 7, resulting in , resulting in inefficient inclusion of exon 7 in the SMN2 transcript. N2 produces both the full-length SMN protein and a truncated form of SMN lacking exon 7 As a result, the functional full-length ribonuclease protein produced by SMN2 is The amount of protein is much less than that produced by SMN1 (70-90%) ). Lorson et al. “A single nucleotide in t he SMN gene regulates splicing and is re sponsible for spinal muscular atrophy.”P NAS,96(11)6307-6311;Monani et al,“A sing le nucleotide difference that alters spl icing patterns distinguishes the SMA gen e SMN1 from the copy gene SMN2.”Hum Mol Genet 8(7):1177-1183. SMN2 completely reverses loss of the SMN1 gene. Although not compensatory, patients with milder forms of SMA generally have higher SMN2 copy numbers. Lefebvre et al., “Correlation between severity and SMN protein level in spinal column muscular atrophy.”Nat Genet 16(3):265-2 69;Park et al., “Spinal muscular atrophy: new and emerging insights from model mic e.”Curr Neurol Neurosci Rep 10(2):108-11 7. More than 95% of people with SMA carry at least one copy of the SMN2 gene. It is important to note that SMN2 copy number is not the only phenotypic modifier. The c.859G>C variant in exon 7 of the MN2 gene has been reported as a positive disease modifying factor. Patients with this mutation have a less severe disease phenotype. t al.,“A positive modified of spinal mus cular atrophy in the SMN2 gene.”Am J Hum Genet 85(3):408-413. In some embodiments, the methods disclosed herein The rAAV SMN to be administered may contain more than 1 copy, more than 2 copies, more than 3 copies, more than 4 copies, or more than 5 copies. Multiple copies of the SMN2 gene and / or c.85 in exon 7 of the SMN2 gene In some embodiments, the present invention is administered to a patient with SMA type II who lacks the 9G>C mutation. The rAAV SMN disclosed in the specification may contain more than 2 copies, more than 3 copies, more than 4 copies, or more than 5 copies. >100% SMN2 gene and / or c.859 in exon 7 of the SMN2 gene In some embodiments, the present invention is administered to a patient with SMA type III who lacks the G>C mutation. The rAAV SMN disclosed herein is administered intrathecally to SMA type II patients. In some embodiments, the rAAV SMN disclosed herein is administered intrathecally to patients with SMA type III. It is administered intravenously.

[0083] Type I SMA (also called infantile-onset or Werdnig-Hoffmann disease) is a type of SMA Symptoms may appear at birth or by age 6 months. In this form, infants typically Infants have low muscle tone (hypotonia), a weak cry, and difficulty breathing. Infants have difficulty swallowing and sucking. Infants often have difficulty sitting up and do not reach the developmental milestone of being able to sit unassisted. Select from hypotonia, delayed motor skills, poor head control, hunched posture, and hypermobility of joints. These infants often have one or more of the SMA symptoms that are associated with All new cases of SMA have two copies of the SMN2 gene, one on each chromosome. More than half of all cases are diagnosed with SMA type I. In type I SMA, approximately 80% of patients have a mutation in the SMN2 gene. It has one or two copies.

[0084] Type II or intermediate SMA is when SMA develops until a child is able to stand or stand independently between the ages of 7 and 18 months. Children with type II SMA generally have a low Approximately 82% of patients with type II SMA have at least three SMN2 genes. Has three copies. Late-onset SMA (Types III and IV SMA, Mild SMA, Adult Onset SMA) SMA, also known as Kugelberg-Welander disease, is a condition that affects people with various levels of Type III SMA develops after 18 1 / 2 months and may require assistance. However, children are able to stand and walk independently. Approximately 96% of people with type III SMA People with SMA have three or four copies of the MN2 gene. Type IV SMA develops in adulthood and people People with Type III or IV SMA typically have 4-8 The SMN2 gene is between 0 and 1, from which adequate amounts of the full-length SMN protein can be produced.

[0085] In one embodiment, for treating SMA, e.g., SMA Type II or III, For example, a rAAV, such as the rAAV9 vector disclosed herein, can be administered intrathecally. The terms "treat," "treatment," and other related terms refer to A composition comprising the rAAV disclosed herein for use in an animal (including a human) in need thereof. The method includes administering, for example, intrathecally, an effective dose or effective multiple doses of Administration is prophylactic if the dose is administered prior to the onset of a disease. In embodiments, an effective dose is administered to treat the disorder being treated. Partially or completely reduce (i.e., eliminate) at least one symptom associated with the harm / disease state slow or prevent the progression of a disorder / disease state; slow or prevent the progression of a disorder / disease state to delay or prevent the progression of the disease; to reduce the severity of the disease, resulting in remission (partial or complete) of the disease; and / or survival-prolonging doses. Examples of disease states contemplated for treatment include: As described herein.

[0086] In one embodiment, the rAAV9 compositions of the present disclosure are directed to a therapeutic targeting a particular SMA gene, such as, for example, SMA type II or SMA type III. It is administered intrathecally to patients in need of treatment for SMA.

[0087] In some embodiments, the patient is 0-72 months of age. In some embodiments, the patient is 6 to 60 months of age. In some embodiments, the patient is 6 to 24 months of age. In some embodiments, the patient is at least 6 months of age. In this case, the patient is over 24 months of age.

[0088] In some embodiments, the patient has a mutation in one copy of the SMN1 gene, e.g., a null mutation. have one or more mutations, such as a mutation that renders the encoded SMN1 protein nonfunctional In some embodiments, the patient has a mutation in the SMN1 gene. The two copies of the gene have one or more mutations, e.g., null mutations. In one embodiment, the patient has at least one mutation, e.g., a null mutation, in all copies of the SMN1 gene. In some embodiments, the patient has one or more mutations in 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 biallelic SMN1 mutation i.e., either a deletion or substitution of SMN1 in both alleles on 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 functions of the SMN2 gene. In some embodiments, the patient has at least three capable copies of the SMN2 gene. In some embodiments, the patient has at least one functional copy of the SMN2 gene. In some embodiments, the patient has four functional copies of the SMN2 gene. In some embodiments, the patient has at least five functional copies. Some children have a null mutation or deletion in SMN1 and three copies of SMN2. In embodiments, the patient has a c.8 In some embodiments, the patient does not have a biallelic SMN1 have a null mutation or deletion, have three copies of SMN2, and have at least one copy of the SMN2 gene In some embodiments, one copy does not have a c.859G>C substitution in exon 7. The gene sequence of the SMN1 or SMN2 gene can be determined by, for example, hybridization, PCR amplification, and / or by partial or complete chromosomal or genomic sequencing. In another embodiment, the gene sequence and copy number of the SMN1 or SMN2 gene are In some embodiments, the SMN1 or S The gene sequence and copy number of the MN2 gene may be determined by microarray analysis. In some embodiments, the gene sequence and copy number of the SMN1 or SMN2 gene are , may be determined by Sanger sequencing. The gene sequence and copy number of the SMN2 gene were determined by fluorescence in situ hybridization (FISH). It may be determined by:

[0089] In some embodiments, the patient may undergo, prior to or concurrently with, treatment, e.g., genomic testing and / or Or motor function tests and / or physical examinations may reveal, for example, SMA type II or III. In some embodiments, the patient has been diagnosed with SMA Type II or III. For example, CHOP INTEND, the Bayley Scales of Infant Development, or HAMMAS It is diagnosed by clinical assessment of symptoms such as the MIS Functional Motor Scale Extended (HFMSE). In some embodiments, SMA Type II or III is diagnosed by physical examination. In some embodiments, the type II SMA treated by the methods disclosed herein The patients were aged 24 months, 22 months, 20 months, 18 months, 16 months, 14 months, Disease before 12 months, 10 months, 8 months, or 6 months of age, or any age in between In some embodiments, the methods disclosed herein include Type III SMA patients treated with this method were aged 12, 14, 16 and 18 months. Disease onset after 20, 22, or 24 months of age, or any age in between. In some embodiments, the patient has or develops type II or type III S. Alternatively, the patient may be treated prior to exhibiting a symptom (e.g., one or more symptoms) of MA, e.g. For example, one of the genetic tests described herein is used to determine that treatment is required. In some embodiments, the patient is assessed, for example, using one of the tests described herein. As described above, the present invention relates to a method for treating SMA. In some embodiments, the patient is subsequently treated for symptoms of SMA Type II or III. In some embodiments, patients are treated prior to becoming symptomatic. A diagnosis of type II or type III SMA is made based on:

[0090] In some embodiments, the patient exhibits one or more symptoms of SMA. Symptoms include hypotonia, delayed motor skills, poor head control, hunched posture, and hypermobility of the joints. In some embodiments, poor head control may include shoulder (front and back) Head control is assessed by placing the patient in a ring position with the aid of a hand. In some embodiments, the patient is assessed by the ability of the patient to Spontaneous movement was observed and motor skills were assessed by the patient's ability to lift elbows, knees, hands, and feet off a surface. In some embodiments, a patient's grip strength is assessed by the ability to place fingers on the patient's palm. This is measured by lifting the patient up until the shoulder is off the surface. Force is measured by how fast / long the patient holds the grip. Head control measures the patient's ability to maximally rotate their head and return it to midline. In some embodiments, shoulder posture is assessed by supporting the head and trunk. The patient is seated and observed to see whether he or she bends their elbow or shoulder to reach for a stimulus placed at shoulder height. In some embodiments, shoulder posture may also be assessed by observing the patient's The patient was placed in a lateral position and asked to bend his or her elbow or shoulder to reach for a stimulus placed at shoulder height. In some embodiments, motor skills may also be assessed by observing footwork. Determine whether the patient bends their hips or knees when the patient is stroked, tickled, or pinched. In some embodiments, the shoulder flexion, elbow flexion, hip flexion, Spine adduction, neck flexion, head extension, neck extension, and / or spinal curvature may be, for example, Other SMA symptoms may be assessed by known clinical measures such as CHO P may be assessed according to known clinical measures such as INTEND.

[0091] In some embodiments, the patient exhibits the ability to sit, but not walk. In this embodiment, the patient demonstrates the ability to sit unassisted for at least 10 seconds, but is unable to stand or walk. In some embodiments, the patient is unable to hold the head upright for 10 seconds or more without assistance. In some embodiments, the patient demonstrates the ability to sit upright but is unable to walk or stand. World Health Organization Multicenter Growth Standards Study n Multicentre Growth Reference Study)(WH As defined by the O-MGRS criteria, indicates the ability to sit independently.

[0092] Without being bound by theory, it is believed that intrathecal administration allows the drug to avoid the blood-brain barrier. As a result, for drugs that target the central nervous system, direct delivery via intrathecal administration is possible. This allows for a reduction in the total dose and / or volume of pharmaceutical composition required (e.g., IV administration). ), thereby reducing the risk of hepatotoxicity. Direct delivery allows for greater access to cells of the central nervous system, e.g., lower motor neurons, glial cells, etc. This may allow for high transduction efficiency. The amount of cerebrospinal fluid (CSF) in the subarachnoid space may be increased by 100% compared to the amount in the intrathecal space. The amount of CSF in humans before the age of 3 years may affect the effective dose concentration selected for intravenous delivery. Since the dose remains relatively constant after administration, it is easier and more uniform to administer the dose to different patients. In some embodiments, intrathecal administration can be controlled in a uniform manner to cross the blood-brain barrier. In some embodiments, the rAAV9 viral vector disclosed herein is used to The investigators are, for example, patients identified as needing treatment for SMA type II or III. In some embodiments, the rAAV9 is delivered intrathecally to a patient in need thereof. is injected into the spinal canal. In some embodiments, rAAV9 is injected into the subarachnoid space. In some embodiments, the rAAV9 viral vector is administered in a PICU unit or in an acute setting. Other appropriate settings with immediate access to critical care (e.g., interventional rooms, operating rooms, specialized In some embodiments, the viral vector is injected under sterile conditions in a veterinary clinic (such as a veterinary clinic). After administration, patients' vital signs were monitored approximately every 15 ± 5 minutes for 4 hours and every 1 hour ± 15 minutes for 24 hours. In some embodiments, the rAAV9 viral vector is preservative-free. In some embodiments, sedation or anesthesia is administered prior to administration of the rAAV9 viral vector. In some embodiments, the rAAV9 viral vector is administered intrathecally to the patient. was performed with the patient in the prone, knee-chest, lateral, Sims, or lateral positions. In some embodiments, the rAAV9 viral vector can be delivered by syringe or catheter. In some embodiments, the catheter is L1-L2, L2-L It may also be inserted into the subarachnoid space via the L3, L3-L4, or L4-L5 interspinous spaces. In some embodiments, a lumbar puncture is performed and a maximum of 10 mL, a maximum of 9 mL, a maximum of 8 mL, up to 7mL, up to 6mL, up to 5mL, up to 4mL, up to 3mL, up to 2mL, or up to 1 mL of cerebrospinal fluid is collected. In some embodiments, the rAAV9 viral vector In some embodiments, the rAAV9 viral vector is injected directly into the subarachnoid space. The cardiologist should administer an appropriate X-ray contrast solution (e.g., metrizamide, iopamidol, iohexadecane, etc.). Premixed with sucrose, ioversol, Omnipaque, etc., and administered intrathecally. In some embodiments, a contrast agent solution (e.g., metrizamide, iopamine, Midol, Iohexol, Ioversol, Omnipaque (trademark, etc.) In some embodiments, the AV9 viral vector is administered intrathecally prior to intrathecal administration. The contrast agent solution was administered within 2 hours, 1 hour, or 24 hours before intrathecal administration of the rAAV9 viral vector. Administered intrathecally within 45 minutes, 30 minutes, 15 minutes, 10 minutes, or 5 minutes In some embodiments, a contrast agent solution (e.g., metrizamide, iopamidol, iodine Hexol, Ioversol, Omnipaque™, etc.) are used to inject rAAV9 viruses In some embodiments, the contrast agent solution is administered intrathecally after intrathecal administration of the vector. Within 2 hours, within 1 hour, within 45 minutes, and within 3 hours after intrathecal administration of rAAV9 viral vector The drug is administered intrathecally within 0 minutes, within 15 minutes, within 10 minutes, or within 5 minutes.

[0093] In some embodiments, the amount of contrast agent administered is up to about 0.5 mL, up to about 1.0 mL. mL, up to about 1.5 mL, up to about 2.0 mL, or up to about 2.5 mL. In embodiments, the total volume administered (rAAV9 viral vector and imaging agent) is about 5 mL less than about 6 mL, less than about 7 mL, less than about 8 mL, less than about 9 mL, or less than about 10 mL In some embodiments, the patient is In some embodiments, the patient is placed in a different position. Following administration of the drug, the patient is placed in Trendelenburg position or tilted at 20 degrees to 30 degrees, for example. In some embodiments, the patient is placed in a head-down position at a 40 degree angle. Following administration of the vector, the patient is given a trendline for 10 to 30 minutes, e.g., for about 15 minutes. The subject is placed in the Dehlenburg position or tilted head down, for example at 30 degrees.

[0094] In some embodiments, treatment includes treatment of SMA, such as type II or type III SMA. Prevent, alleviate, ameliorate, delay, and / or partially or completely relieve one or more symptoms The effectiveness of the treatment was evaluated using various tests of motor skills before and after treatment. In particular, the Bayley Scales of Infant Development® is used to assess the development of infants. Bayley N. “Bayley ley Scales of Infant and Toddler Development ment.”3rd edition,Harcourt Assessment In c., 2006. In particular, the Bayley Scales (registered trademark) version III (3rd edition) The motor scale components include gross and fine motor skills such as grasping, sitting, building blocks, and climbing stairs. In some embodiments, the patient measures whether the hand is clenched into a fist most of the time. In some embodiments, the patient is assessed for whether their eyes follow a moving person. In some embodiments, the patient is assessed to determine whether or not they intentionally put their hand in their mouth. In some embodiments, the patient is assessed for whether or not they attempt the task. To see if you keep your hands open most of the time when you are not In some embodiments, the patient is able to freely move their wrist when manipulating small objects. The ability to rotate the hand from palm down to palm up is assessed. So we give a patient a block and see if they pick it up with one hand or both hands, and if they pick it up with the other hand, Whether the blocks are moved by the hand, whether the blocks are grasped with the pad of the thumb or the fingertips, and whether the patient It is assessed whether the subject grasps the block with partially opposable thumb and fingers. The morphology involves presenting a patient with food pellets and determining whether the patient grasps the pellets with the pad of their thumb or the tips of their fingers. The patient is assessed to see if he or she can grasp the mass with partially opposable thumb and fingers. In one embodiment, a patient is given a book and the patient is asked whether they wish to turn one or more pages at a time. In some embodiments, the patient is assessed to see if the crayon or pencil and paper, and while the patient marks the paper, the patient is given a crayon or pencil in a palm grip, static three-finger grip, The subject is evaluated to see if they grasp with a four-finger grasp. So, while the patient is marking the paper, is their grip mature, controlled, and dynamic? In some embodiments, the patient is assessed to see if the paper is in place in one hand. The hand is evaluated to see if it is held in the same position and scribbled or drawn with the other hand. do.

[0095] In some embodiments, the patient is asked to determine whether or not they thrust their arms or legs out in play. In some embodiments, the patient is evaluated to determine whether the patient is able to hold their head intermittently without support. In some embodiments, the method is evaluated to see if the patient can be raised to a higher level. The patient is asked to hold his / her head upright for at least 3 seconds without support. In some embodiments, the patient is evaluated for at least one of the following: coordination and balance. In some embodiments, the subject is evaluated to see if he or she has the ability to take five steps. The patient is assessed for coordination and balance according to the Bayley®-III-Gross Motor Scale, item 43. The individual is assessed to see if he or she has the ability to walk at least five steps with sensation. In some embodiments, the patient is assessed for ability to stand without assistance or a support surface, and The robot is evaluated to see if it has feedback control over its posture. In an embodiment, the patient is provided with supplemental exercises according to item 40 of the Bayley®-III-Gross Motor Exercise. In some embodiments, the subject is evaluated to determine whether or not he or she has the ability to stand unaided. The study was conducted to determine whether patients were able to stand unsupported approximately 24 months, 12 months, 9 months, or 6 months after administration of treatment. A patient is considered to have received effective treatment if the patient achieves the required ability to: In this study, patients showed improvements in their cooperation and average Achieve the ability to walk unassisted, defined as taking at least five steps independently while demonstrating balance. If this is achieved, the patient is considered to have received effective treatment.

[0096] Another commonly used measure of infant development is the Hammersmith Functional Motor Scales (Extended). O'Hagen et al., "An expanded version of the Hammersmith Functional Mo tor Scale for SMA II and III patients.”N euromuscul Disord,17(9-10):693-7;Glanzma n et al., “Validation of the Expanded Ham mersmith Functional Motor Scale in spina l muscular atrophy type II and III.”J Ch ild Neurol,26(12):1499-1507. Hammersmith Functional Movement While Kale successfully assessed the ability of non-ambulatory individuals with SMA, the HFMSE have identified additional methods that successfully differentiate motor skills between individuals with SMA types II and III. Thirteen add-ons were provided. In some embodiments, the patient sits unsupported in a chair or The patient is assessed for their ability to sit on the floor. In some embodiments, the patient can sit on a chair or floor without support. The patient is assessed for the ability to touch his or her head with his or her hands while seated on the floor. Patients are asked to rate their ability to touch their head with both hands while sitting unsupported on a chair or on the floor. In some embodiments, the patient is able to roll over while lying down or In some embodiments, the patient is assessed for whether or not they are in a supine position while lying down. The ball is evaluated on its ability to roll downwards from the top or vice versa. In this embodiment, the patient is assessed for their ability to transition from a sitting position to lying down in a controlled manner. In some embodiments, the patient is assessed in a prone position, supported on the forearms. In some embodiments, the patient is assessed for ability to perform a pelvic floor exercise while in the prone position. In some embodiments, the patient is assessed for ability to lift their head. Can you support yourself on straight arms while lying on your stomach for a count of three? In some embodiments, the patient is evaluated by rolling from a side-lying position to a prone position. In some embodiments, the patient is assessed for ability to sit up. Participants will be assessed on their ability to get on their hands and knees while keeping their head up. In some embodiments, the patient is asked whether they can crawl forward on their hands and knees. In some embodiments, the patient is assessed in a supine position with arms folded across the chest. During this time, the patient is assessed for ability to lift their head. The patient is assessed for ability to stand for a count of three without using one or both hands for support. In some embodiments, the patient is assessed for their ability to walk unaided. In some embodiments, the patient is assessed for whether or not the patient is Each time, the athlete is assessed on whether they can bring either knee to their chest. In this embodiment, the patient is asked to see if he or she can go from a kneeling position to a semi-kneeling position without using their arms. In some embodiments, the patient is assessed for whether or not they are able to move from a kneeling position without using their arms. In some embodiments, the patient is assessed for ability to transition to a standing position. Patients are assessed on whether they can move from standing to sitting without using their arms. In an embodiment, the patient is assessed to see if they can go from a standing to a squatting position without using their arms. In some embodiments, the patient is assessed for 12 inches forward motion from a standing position. In some embodiments, the patient is evaluated for ability to jump. can walk up and down four flights of stairs without assistance or with the help of one handrail In some embodiments, the patient is evaluated for at least about 24 months, 12 months, or 24 months of administration of the treatment. , 9 months, or 6 months, an increase of, say, 8 points from baseline on the HFMSE If the patient showed an increase of 5 to 10 points, the patient was considered to have received effective treatment. In some embodiments, the patient is administered the treatment within about 24 months, 12 months, 9 months, or 6 months. Patients were considered to be on effective treatment if they subsequently demonstrated a 9-point increase from baseline in the HFMSE. In some embodiments, a patient is considered to have received the treatment within about 24 months of administration of the treatment. A 10-point increase from baseline in HFMSE at 2, 9, or 6 months If so, the patient was considered to have received effective treatment.

[0097] In some embodiments, the efficacy of treatment is measured by changes in developmental performance. In some embodiments, the baseline measurement is performed prior to administration of the rAAV9 viral vector. In some embodiments, the baseline measurement is the Bayley Scales of Infant Development (BAI). In some embodiments, the method includes measuring the fine and gross motor components of the The baseline measurement was performed on item 43 of the gross motor component of the Bayley Scales of Infant Development. In some embodiments, the method includes measuring the ability of the subject to walk at least five steps unassisted. The baseline measurement was performed on item 40 of the gross motor component of the Bayley Scales of Infant Development. (standing unsupported for at least 3 seconds). Baseline measurements were performed on patients according to the Hammersmith Functional Motor Scale Extended (HFMSE). In some embodiments, the efficacy of the treatment is assessed by evaluating the Bayley Infant Development. Item 43 of the gross motor component of the Test (walking at least five steps unassisted) was measured. In some embodiments, treatment is assessed by determining a baseline level of Validity was assessed using the Bayley Scales of Infant Development (registered trademark) gross motor component item 40 (supportive (stand without food for at least 3 seconds) and compare it to a baseline. In some embodiments, efficacy of treatment is assessed by assessing the patient on the HFMSE and comparing baseline baseline scores with baseline scores before treatment. In some embodiments, the baseline is evaluated by comparing the The efficacy of treatment is established by measurements within 30 days prior to treatment. Efficacy is evaluated within 30 days of treatment. In some embodiments, efficacy of treatment is evaluated within 30 days of treatment. The efficacy is evaluated monthly for 12 months after treatment. The assessment is videotaped. In some embodiments, important motor milestones are as set forth in Table 2. In some embodiments, the motor milestone development is assessed using a standard motor milestone development survey as shown in The efficacy of treatment is expected to continue for at least 12 months, at least 24 months, and at least 48 months after treatment. Patients will be evaluated for at least 72 months, or up to 10 years after treatment.

[0098] [Table 2]

[0099] In some embodiments, the test to assess efficacy of treatment is the Bayley Scales of Infant Development. (registered trademark), Hammersmith Functional Motor Scale Extended (HFMSE), or Motor Miles Not limited to tone development studies, CHOP INTEND, TIMP, CHOP TO SS, Peabody Development Motor Scale, Brazelton Neonatal Scale Obtained through behavior evaluation and interactive video evaluation (ACTIVE) These include, but are not limited to, the ability to perform exercises and compound motor action potential (CMAP) measurements. Other motor skill tests known in the art may also be included that are not intended to be included.

[0100] For example, methods for identifying SMA, such as SMA Type II or SMA Type III, disclosed herein Pre-screening of patients suitable for treatment with Administration of a treatment to a patient identified as such is also envisioned.

[0101] AAV may induce both cellular and humoral immune responses. A portion of potential patients for AAV-based gene therapy will have pre-existing antibodies to AAV. eune et al., “Pre-existing anti-Adeno-Ass associated Virus antibodies as a challenge in AAV gene therapy.”Hum Gene Ther Metho ds,24(2):59-67;Boutin et al.,“Prevalence of serum IgG and neutralizing factors a gainst adeno-associated virus (AAV) types 1,2,5,6,8,and 9 in the healthy population n:implications for gene therapy using AA V vectors.”Hum Gene Ther,21:704-712. Very low Prior anti-AAV antibodies have been shown to be effective against AAV-specific markers, because even low levels of antibodies can prevent successful transduction. , poses a serious obstacle to the universal application of AAV gene therapy. Prior to administration of the AAV viral vector, the patient's anti-AAV9 antibody titer level is measured, and the antibody Only if the titer is below a threshold level is the patient given AAV by intrathecal administration. In some embodiments, the level of anti-AAV9 antibody titer in the patient is determined by an ELISA binding immunoassay. In some embodiments, the patient is administered an ELISA result prior to administration of the treatment. have an anti-AAV9 antibody titer of 1:100 or less as measured by combined immunoassay. In embodiments, the patient has a 1 or 2 or more IgG4-associated IL-16 expression level as measured by an ELISA binding immunoassay prior to administration of the treatment. In some embodiments, following treatment, the patient has an anti-AAV9 antibody titer of 0.50 or less. have an anti-AAV9 antibody titer of greater than 1:100 as measured by ELISA binding immunoassay The patient is then monitored for 1-8 weeks or until the titer falls below 1:100. In some embodiments, the patient has, after treatment, a 1 Anti-AAV9 antibody titers of >100 for 1-8 weeks or titers of <1:50 It is monitored until it drops to zero.

[0102] In some embodiments, patients with high anti-AAV antibody titers are those with one or more immunosuppressants. Anti-inflammatory drugs such as rituximab in combination with cyclosporine A may also be administered. The monoclonal anti-CD20 antibody may reduce anti-AAV titers. i et al., “Pharmacological modulation of humoral immunity in a nonhuman primate m odel of AAV gene transfer for hemophilia B.” Mol Ther, 20:1410-1416. In some embodiments, the patient had an antibody titer of more than 1:100 as measured by ELISA binding immunoassay before or after treatment. AAV9 antibody titers and one or more In some embodiments, the patient is treated with an immunosuppressant drug. have an anti-AAV9 antibody titer of greater than 1:50 as measured by SA binding immunoassay, e.g. , and are treated with one or more immunosuppressant drugs, including steroids such as prednisolone.

[0103] In some embodiments, patients with high anti-AAV antibody titers are treated with IgG4 antibody therapy prior to and after vector administration. and / or subsequent plasma exchange may be performed to deplete neutralizing antibodies. al.,“A 10 patient case report on the im pact of plasmapheresis upon neutralizing factors against adeno-associated virus( AAV)types 1,2,6,and 8.”Mol Ther,19(11):2 In plasmapheresis, blood is drawn from the patient and centrifuged or The plasma and blood cells are separated by hollow fiber filtration. The blood cells are then separated into treated plasma or The fluid is then returned to the patient along with replacement fluids such as 4.5% human albumin in saline. A common use of apheresis is the removal of unwanted immunoglobulins, such as anti-AAV antibodies. In some embodiments, the patient is tested for ELISA binding immunoassays before or after treatment. The patient has an anti-AAV9 antibody titer of more than 1:100 as measured by ELISA and is treated with plasma exchange. In some embodiments, the patient is monitored by ELISA binding immunoassay before and after treatment. Patients with AAV9 antibody titers above 1:50 at baseline are treated with plasma exchange.

[0104] Pre-existing maternal antibodies against AAV9 may be transferred to young patients via breast milk or intrauterine placental transfer. In some embodiments, the patient may be administered an ELISA binding assay before or after treatment. The patient had an anti-AAV9 antibody titer of >1:100 as measured by immunoassay and was switched to artificial nutrition. In some embodiments, the patient is administered an ELISA binding immunoassay before or after treatment. The patient had an anti-AAV9 antibody titer of >1:50 as measured by the assay and was switched to artificial feeding. can be done.

[0105] The patient's condition may be monitored before and after administration of treatment. Patients receiving AAV-based therapy may also experience low platelet counts or thrombocytopenia. Thrombocytopenia is a condition characterized by a particularly low platelet count. Thrombocytopenia can also be detected by complete blood count using diluted blood. A slide made with the patient's blood (a thin blood film or peripheral smear) is examined under a microscope. Normal human platelet counts range from 150,000 cells / ml to about 45,000 cells / ml. The range is 0,000 cells / ml.

[0106] In some embodiments, the patient has greater than about 67,000 cells / ml prior to administration, or A platelet count greater than 100,000 cells / ml or greater than about 150,000 cells / ml In some embodiments, the patient has less than about 150,000 cells / ml prior to administration. or a platelet count of less than about 100,000 cells / ml, or less than about 67,000 cells / ml for 1-8 weeks, or a platelet count greater than about 67,000 cells / ml, or Increased to greater than about 100,000 cells / ml or greater than about 150,000 cells / ml After administration of the viral vector, the platelet count was monitored at approximately 67,000 cells / ml. In some embodiments, where the patient has less than 100% platelet count, the patient may be treated with platelet transfusions. In embodiments, the patient does not have thrombocytopenia prior to administration of the viral vector. In one embodiment, the patient has thrombocytopenia following administration of the viral vector and is at least 1 to 8 weeks In some embodiments, the patient is monitored for a period of time or until the patient is free of thrombocytopenia. The patient had thrombocytopenia after administration of the viral vector and was treated with platelet transfusions. .

[0107] Monitoring the patient's condition also involves platelet, serum protein electrophoresis, serum gamma- Glutamyltransferase (GGT), Aspartate transaminase (AST) ) and alanine aminotransferase (ALT), total bilirubin, glucose, Creatinine kinase (CK), creatinine, blood urea nitrogen (BUN), electrolytes, alkaline phosphatase This involves standard blood tests that measure levels of one or more of: amino acids, sphatase, and amylase. Troponin I levels are a general measure of cardiac health, and elevated levels are associated with cardiac In some embodiments, the viral vector Troponin-I levels are monitored after administration. less than about 0.3, 0.2, 0.15, or 0.1 μg / ml prior to administration of the virus vector. In some embodiments, the patient may have a roponin-I level. The patient may have a troponin-I level of less than about 0.176 μg / ml prior to administration of the antibody. In some embodiments, the patient has a IgG antibody titer of greater than about 0.176 μg / ml after administration of the viral vector. In some embodiments, the patient may have a troponin-I level that is Following administration of the vector, cardiac troponin-I levels were monitored until the troponin-I level was less than approximately 0.176 μg / ml. Be monitored.

[0108] Aspartate transaminase (AST) and alanine aminotransferase ALT and total bilirubin are common measures of liver function, while creatinine is a measure of kidney function. Elevated levels of AST, ALT, or total bilirubin indicate liver dysfunction. In some embodiments, the patient has normal liver function prior to administration of the viral vector. In some embodiments, the patient has been administered the viral vector for about 8 to 40 days. In some embodiments, the patient has liver transaminase levels of less than 1 U / L. have an AST or ALT level of less than about 8-40 U / L prior to administration of the viral vector In some embodiments, the patient is screened according to known in the art criteria, such as CLIA criteria. Gamma-glutamyltransferase less than 3 times the upper limit of normal as measured by clinical criteria and methods In some embodiments, the patient has GGT prior to administration of the viral vector. In some embodiments, the patient has a bilirubin level of less than 3.0 mg / dL. , less than 1.8 mg / dL, less than 1.4 mg / dL, or less than 1. In some embodiments, the patient has a creatinine level of less than 0.0 mg / dL. Prior to administration of the viral vector, patients were required to maintain hemoglobin (Hgb) levels between 8 and 18 g / dL. In some embodiments, the patient has a HIV infection rate of 20,000 / mL prior to administration of the viral vector. mm 3 Have a white blood cell (WBC) count less than

[0109] In various embodiments, gene therapy using the AAV vectors described herein includes: For example, antigen-specific T cells directed against the AAV vector may be produced within 2–4 weeks following gene transfer. One possible consequence of such an antigen-specific T cell response is Elimination of transduced cells and loss of transgene expression. In an attempt to suppress the immune response, the patient may be given immunosuppressants. In embodiments, the T cell response may be measured by an ELISPOT assay. In some embodiments, the T cell response prior to administration of the vector is at least 10 6 Peripheral blood mononuclear cells (P In some embodiments, the patient is treated with 100 spot forming cells (SFC) per BMC. The subject may be given glucocorticoids prior to administration of the viral vector. In some embodiments, the patient may be given corticosteroids prior to administration of the viral vector. In some embodiments, the patient is given oral steroids prior to administration of the viral vector. Examples of oral steroids include, but are not limited to, progesterone. Rednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone bethamethasone, dexamethasone, and hydrocortisone. In some embodiments, the oral steroid is or comprises prednisolone. nothing.

[0110] In some embodiments, the patient is, for example, administered at least two doses of the viral vector. Begin prophylactic steroids at least 12-48 hours prior, such as 4 hours prior. In some embodiments, the patient, after administration of the viral vector, e.g., at least Oral steroids are administered for at least 10 to 60 days, with the most common being 30 days. In some embodiments, the oral steroid is administered once a day. In some embodiments, the oral steroid is administered twice daily. For example, the drug is administered at a dose of about 0.1 to 10 mg / kg, such as about 1 mg / kg. In some embodiments, the oral steroid is administered at a dose of about 0.1 to 1 mg / kg / day, for example, about 1 mg / kg / day. In some embodiments, the viral vector is administered at a dose of 0 mg / kg / day. AST and ALT levels are monitored after administration. Late-phase treatment may be initiated when AST and ALT levels meet, for example, clinical criteria and procedures known in the art. It is administered when blood levels exceed twice the upper limit of normal, or approximately 120 IU / L, as measured by the method. In some embodiments, oral steroid treatment is for more than 30 days and AST and A The level of LT is above normal, e.g., as measured by clinical criteria and methods known in the art. The drug is administered as long as it exceeds twice the limit or as long as the level exceeds about 120 IU / L. In some embodiments, oral steroid treatment reduces T cell responses by 10 6 1 per PBMC In some embodiments, the oral dose is 0.00 SFC or higher and is administered for more than 30 days. Steroid treatment increased T cell responses by 10 6 30, until the SFC per PBMC falls below 100. During continuous treatment with corticosteroids, the adrenal glands produce cortisol. If corticosteroid treatment is suddenly stopped, the body will naturally decrease production of corticosteroids. Patients may also experience thyroid deficiency. Oral steroids should be given to patients for at least 30 days. In some embodiments, the steroid dose is slowly tapered according to a schedule. In some embodiments, the oral steroid dose is adjusted to reduce AST and ALT levels, e.g. For example, less than two times the upper limit of normal as measured by clinical criteria and methods known in the art. or below about 120 IU / L. In some embodiments, the taper is about 0.5 mg / kg / day for 2 weeks, followed by 0.25 mg / kg for another 2 weeks In some other embodiments, oral steroids are administered in a gradual manner, down to 10 g / day. The tapering of the steroid is at the discretion of the physician. In some embodiments, blood samples are taken. The serum antibody to AAV9 was detected by ELISA, and the serum antibody to SMN was detected by ELISA. Serum antibodies or interferon gamma (IFN-g) by ELISpot are tested. .

[0111] Methods for selecting patients who will benefit from the treatments disclosed herein are also contemplated herein. In some embodiments, the patient is prescribed a spinal tap procedure or administration of an intrathecal therapy. In some embodiments, the patient does not have scoliosis or is not contraindicated for, e.g., , and does not have severe scoliosis, defined as a spinal curvature of 50 degrees or greater. In this study, patients were randomly assigned to receive rAAV9 viral vectors within 2 years, 1 year, or 6 months after the administration of the vector. Have had prior, planned or anticipated scoliosis corrective surgery or procedure within the last 12 months In some embodiments, the patient does not require invasive ventilatory support or a gastric feeding tube. In some embodiments, the patient has no history of standing or walking independently. In some embodiments, the patient does not have active AAV9 virus at the time of administration of the rAAV9 viral vector. In further embodiments, these viral infections include: including, but not limited to, human immunodeficiency virus (HIV); or types B or C In some embodiments, the patient is seropositive for hepatitis C or Zika virus. Patients may have, for example, major renal or hepatic impairment, known seizure disorder, diabetes mellitus, idiopathic hypocalcaemia, In some embodiments, the patient is free of coexisting conditions such as luciduria or symptomatic cardiomyopathy. Participants will develop a severe non-pulmonary or airway infection within 4 weeks of administration of the rAAV9 viral vector. In some embodiments, the patient is free of infection (e.g., pyelonephritis or meningitis). In some embodiments, the patient has no history of bacterial meningitis, brain disease, or spinal cord disease. Glucocorticosteroids, such as prednisone or prednisolone Known allergy or toxicity to ocorticosteroids or their excipients In some embodiments, the patient does not have hypersensitivity to iodine or iodine-containing products. In some embodiments, the patient has no known allergies or hypersensitivity to the drug. Not taking medications to treat chronic kidney disease or neuropathy at the same time. In the present study, the patient was randomly assigned to receive immunosuppressive therapy within 3 months prior to administration of the rAAV9 viral vector. , plasma exchange, and immunomodulatory agents such as adalimumab.

[0112] Combination therapy is also contemplated herein. This includes either simultaneous or sequential treatment. Combinations of therapies may be used in combination with certain standard therapies. (e.g., riluzole in ALS) and / or in combination with novel therapies. For example, other treatments for SMA that may be used in the disclosed combination therapies. As such, they alter the binding to pre-mRNAs and their splicing patterns. Examples include antisense oligonucleotides (ASOs), which stimulate the immune system to react with certain proteins. Singh.et al.,“A multi-exon-skipping detection ass. ay reveals surprising diversity of splic e isoforms of spinal muscular atrophy ge nes." Plos One, 7(11):e49595. In some embodiments, Synersen (U.S. Pat. No. 8,361,977, incorporated herein by reference) and U.S. Patent No. 8,980,853) may be used. Targeting intron 6, exon 7, or intron 7 of the MN2 pre-mRNA ASO that regulates the splicing of SMN2 to synthesize the full-length SMN protein In some embodiments, the AAV9 viral vector is In some embodiments, the disclosed method of treatment is administered in combination with a muscle-building agent. The proposed treatment method involves administering AAV9 viral vectors in combination with neuroprotective agents. In some embodiments, the disclosed therapeutic methods include administering an anti-SMN-targeting anti-SMN inhibitor to a patient. Administration of AAV9 viral vectors in combination with antisense oligonucleotide-based drugs In some embodiments, the disclosed methods of treatment include administering in combination with nusinersen. In some embodiments, the disclosed The proposed treatment involves administering an AAV9 viral vector in combination with a myostatin inhibitor. In some embodiments, the disclosed methods of treatment include combining stamulamulab. In some embodiments, the method further comprises administering an AAV9 viral vector to the patient. The indicated therapeutic methods involve administering an AAV9 viral vector in combination with two or more additional therapies. This includes giving.

[0113] The rAAV viral vectors disclosed herein can be prepared and purified using methods known in the art. In some embodiments, the purification method can be prepared according to a method for the purification of contaminants from host cells. It is intended to remove any contaminating substances and chemicals added during the collection of the viral vector. In some embodiments, the entire contents of PCT / US2005 / 013666 are incorporated herein by reference. In some embodiments, the methods disclosed in US Pat. No. 5,999,333 are used. The method may, for example, involve measuring about 1 to 8 x 10 13 vg / mL, e.g., approximately 1 × 10 13 vg / mL ~1×10 15 The resulting rAAV viral vectors are at a concentration between 100 and 200 mg / mL. In an embodiment, the method comprises: 13 vg~9.9×10 14 vg dose (e.g. In some embodiments, the method provides a rAAV viral vector in a single unit dose. is approximately 1.0 x 10 13 vg~5.0×10 14 vg dose (e.g., unit dose) of rA In some embodiments, the method results in about 5.0×10 1 3 vg~3.0×10 14 rAAV viral vector at a dose (e.g., unit dose) of vg In some embodiments, the method results in about 6.0×10 13 vg dose (e.g. In some embodiments, the method provides a rAAV viral vector in a single unit dose. , about 1.2×10 14 rAAV viral vectors at a dose (e.g., unit dose) of . In some embodiments, the method comprises administering about 2.4×10 14 vg dose (e.g., single The rAAV viral vector is delivered at a dose of approximately 100 mg / kg.

[0114] In some embodiments, the method comprises reducing the creatinine concentration by less than about 10%, by less than about 8%, by less than about 7%, or by less than about 5%. % empty viral capsids. In one embodiment, the method comprises: 13 Less than approximately 100 ng / mL of host per vg / mL In some embodiments, the rAAV viral vector is provided with a cellular protein. , this method is 1 x 10 13 Approximately 5 × 10 per vg / mL 6 Less than pg / mL, approximately 1×1 0 6 Less than pg / mL, approximately 7.5 × 10 5 Less than pg / mL or 6.8 x 10 5 pg / mL yielding rAAV viral vectors with less than 10% residual host cell DNA (hcDNA) In some embodiments, the method comprises: 13 Approximately 10 ng per vg / mL less than about 8 ng, less than about 6 ng, or less than about 4 ng of residual host cell protein (rH In some embodiments, the method provides an rAAV viral vector having a CP. , functional rAAV (e.g., AAV9) viral vector genomes / mL at least approximately 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100%. So, the method is 1×10 13 1.7 × 10 per vg / ml 6 pg / ml or less, or 1x 10 13 1 × 10 per vg / ml 5 pg / ml to 1×10 13 1.7 per vg / ml ×10 6 yielding rAAV viral vectors with pg / ml residual plasmid DNA In some embodiments, the method comprises: 13 Less than 0.2 ng per vg, 1 .0×10 13 Less than 0.1 ng per vg or 1.0 × 10 13 0.09n per vg Several studies have demonstrated that rAAV viral vectors have benzonase concentrations of less than 1 g. In an embodiment, the method comprises: 13 <0.5 ng per vg, 1.0 × 10 13 v Less than 0.3 ng per g or 1.0 x 10 13 Bovine serum less than 0.22ng per vg Several experiments have been carried out to produce rAAV viral vectors with albumin (BSA) concentrations. In an embodiment, the method comprises: 13 Less than approximately 1 EU / mL per vg / mL, 1.0× 10 13 Less than approximately 0.75 EU / mL per vg / mL, 1.0 × 10 13 vg / mL Approximately 0.5 EU / mL or less, 1.0×1013 Less than approximately 0.4 EU / mL per vg / mL , 1.0×10 13 Less than approximately 0.35 EU / mL per vg / mL, 1.0 × 10 13 vg / mL, less than approximately 0.3 EU / mL, 1.0 × 10 13 Approximately 0.25E per vg / mL U / mL, less than 1.0 × 10 13 Less than approximately 0.2 EU / mL per vg / mL, 1.0 x 1 0 13 Less than approximately 0.13 EU / mL per vg / mL, 1.0 × 10 13 Per vg / mL Approximately less than 0.1 EU / mL, 1.0 x 10 13 Less than approximately 0.05 EU / mL per vg / mL , or 1.0×10 13 Has endotoxin levels of less than approximately 0.02 EU / mL per vg / mL In some embodiments, the method results in a rAAV viral vector that is Less than g / g (ppm), less than 50 μg / g (ppm), or less than 30 μg / g (ppm) In some embodiments, the rAAV viral vector has a cesium concentration of The method includes adding about 10 to 100 ppm, 15 to 90 ppm, or about 20 to 80 ppm of porphyrin. In some embodiments, the rAAV viral vector comprises oxamer 188. The method is to have less than 2000 particles, less than 1500 particles, or less than 10 particles with a size of 25 μm or more per container. The resulting rAAV viral vector has fewer than 600 particles. In some embodiments, the method comprises: rAAV virus vectors having less than 8,000, 1,000, or 6,000 particles In some embodiments, the method provides a method for producing a β-actin ester having a pH between 7.5 and 8.5, 7.6 and 8. 4, or between 7.8 and 8.3. In some embodiments, the method includes administering a blood glucose level between 330 and 490 mOsm / kg, between 360 and 46 rAA with an osmolality between 0 mOsm / kg or between 390 and 430 mOsm / kg In some embodiments, the method results in a 1.0×10 V virus vector. 13 v Approximately 1.0 x 10 per g 8 ~10.0×10 10 IU, 1.0×10 13 Approximately 2 per vg .5×10 8 ~9.0×10 10 IU or 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 The resulting rAAV virus vector has an infectious titer of 1.1 IU. In some embodiments, the method comprises determining the presence or absence of a reference standard and a method comprising determining the presence or absence of a reference standard and a method comprising determining the presence or absence of a reference standard and a method comprising determining the presence or absence of a reference standard and a method comprising determining the presence or absence of a reference standard and a method for ... and / or about 30-150%, about 60-140%, or about 70-150%, relative to an appropriate control. In some embodiments, the rAAV viral vector has a relative titer of 30%. The method is 1.0 x 10 13 Approximately 10-500 μg per vg, 1.0 × 10 13 vg a Approximately 50-400μg per dose, or 1.0×10 13 Approximately 100-300 μg of total protein per vg In some embodiments, the present invention provides a rAAV viral vector having a high protein level. , the method is 7.5 x 10 13 Vg / kg dose of SMNΔ7 mice The median survival time was determined to be more than 15 days, more than 20 days, more than 22 days, or more than 24 days. This results in rAAV viral vectors with in vivo potency exceeding

[0115] In any of the above embodiments, the preparation and / or purification methods may be formulated for administration. and / or about 6.0×10 13 vg of unit dose of the pharmaceutical composition, In any of the above embodiments, the preparation may provide a rAAV viral vector. The preparation and / or purification method may be formulated for administration and / or may comprise about 1.2×10 1 4 rAAV viral vectors present in the pharmaceutical composition in a unit dose of .vg. In any of the above embodiments, the preparation and / or purification methods may be processed for administration. and / or about 2.4×10 14 vg of unit dose of the pharmaceutical composition. This may result in a rAAV viral vector.

[0116] For example, in some embodiments, the method provides for a reduction in the oxidative stress profile of less than about 10%, less than about 8%, less than about 7%, or resulting in an rAAV viral vector having less than about 5% empty viral capsids, The rAAV viral vector is formulated for administration and / or administered in a volume of about 6.0 x 10 13 v In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising administering to the subject a pharmaceutical composition comprising at least about 10% by weight of the compound. rAAV with less than about 8%, less than about 7%, or less than about 5% empty viral capsids. The rAAV viral vector is formulated for administration, and and / or approximately 1.2 x 10 14 In some embodiments, the pharmaceutical composition is present in a unit dose of 100 mg / kg. In some embodiments, the method includes reducing the amount of empty virus particles to less than about 10%, less than about 8%, less than about 7%, or less than about 5%. and providing an rAAV viral vector having a rus capsid. is formulated for administration and / or about 2.4 x 10 14 Pharmaceutical composition in unit doses of vg In some embodiments, the formulation or pharmaceutical composition is present in about 6.0×10 13 v The unit dose of the rAAV viral vector is about 1 g. have less than 0%, less than about 8%, less than about 7%, or less than about 5% empty viral capsids. In some embodiments, the formulation or pharmaceutical composition has a concentration of about 1.2×10 14 vg rAAV The rAAV viral vector comprises a unit dose of the virus vector, and the rAAV viral vector is about 10% or less, about 8% or less. %, less than about 7%, or less than about 5% empty viral capsids. In the form, the formulation or pharmaceutical composition may have a dosage form of about 2.4×10 14 rAAV viral vectors of vg and the rAAV viral vector comprises less than about 10%, less than about 8%, or less than about 7% less than, or less than about 5% empty viral capsids.

[0117] In some embodiments, the method comprises administering 1×10 13 Approximately 100 ng / mL per vg / mL and resulting in an rAAV viral vector having less than 100% host cell protein. The vector is formulated for administration and / or has a concentration of about 6.0 x 10 13 Unit dose in vg In some embodiments, the method comprises administering to the patient a pharmaceutical composition comprising administering to the patient a pharmaceutical composition comprising at least one of: 13 vg / mL rAAV viral vectors with less than about 100 ng / mL of host cell protein per and the rAAV viral vector is formulated for administration and / or provides about 1.2 ×10 14 In some embodiments, the method comprises the step of: , 1×10 13 Has less than about 100 ng / mL of host cell protein per vg / mL Providing a rAAV viral vector, the rAAV viral vector being formulated for administration and / or about 2.4 x 10 14 vg of unit dose in the pharmaceutical composition. In some embodiments, the formulation or pharmaceutical composition comprises about 6.0×10 13 vg rAAV virus The unit dose of vector contained 1 × 10 rAAV viral vector. 13 vg / mL In some embodiments, the host cell has less than about 100 ng / mL of host cell protein. The formulation or pharmaceutical composition may be about 1.2×10 14 Unit dose of rAAV viral vector in vg The rAAV viral vector contained 1 x 10 13 Approximately 100 ng / vg / mL In some embodiments, the formulation or pharmaceutical composition has less than mL of host cell protein. is about 2.4 x 10 14 vg of rAAV viral vector unit dose, The viral vector was 1 × 10 13 Less than approximately 100 ng / mL of host cells per vg / mL Has protein.

[0118] In some embodiments, the method comprises the steps of: 13 Approximately 5 × 10 per pg / mL 6 Less than pg / mL, approximately 1 × 10 6 Less than pg / mL, approximately 7.5 × 10 5 Less than pg / mL, or 6.8×10 5 rAAV with residual host cell DNA (hcDNA) below pg / mL providing a rAAV viral vector, the rAAV viral vector being formulated for administration, and / or about 6.0 × 1013 In some embodiments, the pharmaceutical composition is present in a unit dose of 1000 mg / kg. In this case, the method 13 Approximately 5 × 10 per pg / mL 6 Less than pg / mL, approx. 1×10 6 Less than pg / mL, approximately 7.5 × 10 5 Less than pg / mL or 6.8 x 10 5 pg rAAV viral vectors with residual host cell DNA (hcDNA) of less than 1 / mL The rAAV viral vector is formulated for administration and / or administered at approximately 1.2×1 0 14 In some embodiments, the method comprises the step of: , 1×10 13 Approximately 5 × 10 per pg / mL 6 Less than pg / mL, approximately 1 × 10 6 pg / m Less than L, about 7.5 x 10 5 Less than pg / mL or 6.8 x 10 5 Residual amount less than pg / mL The rAAV virus vector containing host cell DNA (hcDNA) is obtained. The virus vector is formulated for administration and / or has a concentration of about 2.4 x 10 14 For vg units In some embodiments, the formulation or pharmaceutical composition is present in the pharmaceutical composition in an amount of about 6. 0×10 13 rAAV virus vector unit dose of 10 ... Kutar is 1×10 13 Approximately 5 × 10 per vg / ml 6 Less than pg / mL, approximately 1 × 10 6 p Less than 7.5×10 g / mL 5 Less than pg / mL or 6.8 x 10 5 Residuals less than pg / m In some embodiments, the formulation or pharmaceutical composition comprises a recombinant host cell DNA (hcDNA). The object is about 1.2 x 10 14 rAAAV viral vector unit dose of 10 ... V viral vectors were 1 × 10 13 Approximately 5 × 10 per vg / ml 6 Less than pg / mL, approx. 1×10 6 Less than pg / mL, approximately 7.5 × 10 5 Less than pg / mL or 6.8 x 10 5 pg In some embodiments, the formulation has residual host cell DNA (hcDNA) of less than 1 / ml. Or the pharmaceutical composition is about 2.4×10 14 Unit dose of rAAV viral vector in vg The rAAV viral vector contains 1 x 10 13 Approximately 5 × 10 per vg / mL 6 pg / Less than mL, approximately 1 × 10 6 Less than pg / mL, approximately 7.5 × 10 5 Less than 6.8 pg / mL ×10 5 Has residual host cell DNA (hcDNA) less than pg / mL.

[0119] In some embodiments, the method comprises: 13 Approximately less than 10 ng per vg / mL , less than about 8 ng, less than about 6 ng, or less than about 4 ng of residual host cell protein (rHCP The rAAV viral vector has a and / or about 6.0 x 10 13 vg of unit dose of the pharmaceutical composition. In some embodiments, the method comprises: 13 Approximately 10 ng per vg / mL less than about 8 ng, less than about 6 ng, or less than about 4 ng of residual host cell protein (rHC P), and the rAAV viral vector is administered and / or about 1.2 x 10 14 Present in the pharmaceutical composition in a unit dose of vg In some embodiments, the method comprises: 13 Approximately 10 ng per vg / mL less than about 8 ng, less than about 6 ng, or less than about 4 ng of residual host cell protein (rH The rAAV viral vector has a CP. and / or approximately 2.4 x 10 14 The pharmaceutical composition contains 100 mg of In some embodiments, the formulation or pharmaceutical composition comprises about 6.0×10 13 vgr The unit dose of the AAV viral vector is 1.0×1 0 13 Less than about 10 ng, less than about 8 ng, less than about 6 ng, or about 4 ng per vg / mL In some embodiments, the formulation or The pharmaceutical composition comprises about 1.2×10 14 Contains a unit dose of rAAV viral vector of 10000 vg The rAAV viral vector was 1.0 × 10 13 Less than approximately 10 ng per vg / mL , less than about 8 ng, less than about 6 ng, or less than about 4 ng of residual host cell protein (rHCP In some embodiments, the formulation or pharmaceutical composition has about 2.4×10 14 vg The rAAV viral vector comprises a unit dose of 1.0 ×10 13 Less than about 10 ng, less than about 8 ng, less than about 6 ng, or less than about 4 ng per vg / mL with less than ng of residual host cell protein (rHCP).

[0120] In some embodiments, the method comprises producing functional AAV9 viral vector genomes / mL. At least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100%, The V viral vector is formulated for administration and / or has a concentration of about 6.0×10 13 vg single In some embodiments, the method comprises the step of: At least about 50%, at least about 60%, at least about About 70%, at least about 80%, at least about 90%, at least about 95%, or less and / or the rAAV viral vector is formulated for administration, and / or Or about 1.2 x 10 14 In some embodiments, the pharmaceutical composition is present in a unit dose of 100 mg / kg. The method comprises: providing at least about 50% functional AAV9 viral vector genomes / mL; At least about 60%, at least about 70%, at least about 80%, at least about 90%, At least about 95%, or at least about 100%, of the rAAV viral vector , and / or about 2.4 x 10 14 in a pharmaceutical composition at a unit dose of vg In some embodiments, the formulation or pharmaceutical composition is present in an amount of about 6.0×10 13 vg and about 50%, at least about 60%, or less of a unit dose of rAAV viral vector. at least about 70%, at least about 80%, at least about 90%, at least about 95%, or At least about 100% rAAV (e.g., rAAV9) viral vector genomes / mL In some embodiments, the formulation or pharmaceutical composition comprises about 1.2×10 14 vg of a unit dose of rAAV viral vector, about 50%, at least about 60%, At least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% of the rAAV (e.g., rAAV9) viral vector genome / In some embodiments, the formulation or pharmaceutical composition is about 2.4×10 mL. 14 rAAV viral vector, comprising a unit dose of 1000 mg of rAAV viral vector; About 50%, at least about 60%, at least about 70%, at least about 80%, at least About 90%, at least about 95%, or at least about 100% of the rAAV (e.g., rAA V9) viral vector genomes / mL are functional.

[0121] In some embodiments, the method comprises administering 1×10 13 1.7 × 10 per vg / ml 6 pg / ml or less, or 1×10 13 1 × 10 per vg / ml 5 pg / ml to 1×10 13 v 1.7 x 10 per g / ml 6 rAAV virus with residual plasmid DNA of pg / ml the rAAV viral vector is formulated for administration, and / or Or about 6.0 x 10 13 In some embodiments, the pharmaceutical composition is present in a unit dose of 100 mg / kg. So, the method is 1×10 13 1.7 × 10 per vg / ml 6 pg / ml or less, or 1x 10 13 1 × 10 per vg / ml 5 pg / ml to 1×10 13 1.7 per vg / ml ×10 6yielding rAAV viral vectors with pg / ml residual plasmid DNA , the rAAV viral vector is formulated for administration and / or has a concentration of about 1.2 x 10 14 In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising administering to the subject a unit dose of 1×10 13 1.7 × 10 per vg / ml 6 pg / ml or less, or 1×10 13 vg / ml 1×10 5 pg / ml to 1×10 13 1.7 × 10 per vg / ml 6 pg / ml residual Providing a rAAV viral vector having a plasmid DNA, The turpentine is formulated for administration and / or at about 2.4 x 10 14 Pharmaceutical composition in unit dose of vg In some embodiments, the formulation or pharmaceutical composition is present in a composition having about 6.0×10 1 3 vg of a rAAV viral vector, 1×10 13 1.7 × 10 per vg / ml 6 pg / ml or less, or 1×10 13 vg / 1 x 10 per ml 5 pg / ml to 1×10 13 vg / ml per 1.7 x 10 6 pg / ml of residual plasmid DNA. is about 1.2 x 10 14 vg of rAAV viral vector unit dose, The viral vector was 1 × 10 13 1.7 × 10 per vg / ml 6 pg / ml or less, or is 1×10 13 1 × 10 per vg / ml 5pg / ml to 1×10 13 Per vg / ml .1.7×10 6 pg / ml of residual plasmid DNA. , the formulation or pharmaceutical composition is about 2.4×10 14 vg unit dose of rAAV viral vector The rAAV viral vector contained a dose of 1 x 10 13 1.7 × 10 per vg / ml 6 pg / ml or less, or 1×10 13 1 × 10 per vg / ml 5 pg / ml to 1×10 13 1.7 × 10 per vg / ml 6 pg / ml of residual plasmid DNA.

[0122] In some embodiments, the method comprises: 13 Less than 0.2 ng per vg, 1. 0×10 13 Less than 0.1 ng per vg or 1.0 × 10 13 0.09ng per vg and providing an rAAV viral vector having a benzonase concentration of less than 100 mg / mL. The vector is formulated for administration and / or has a concentration of about 6.0×10 13 in unit doses of vg In some embodiments, the method comprises administering to the patient a dose of 1.0×10 13 vgata Less than 0.2 ng, 1.0 × 10 13 Less than 0.1 ng per vg or 1.0 × 10 13 The rAAV viral vectors have a benzonase concentration of less than 0.09 ng per vg. The rAAV viral vector is formulated for administration and / or administered at approximately 1.2×1 0 14 In some embodiments, the method comprises the steps of: .0×1013 <0.2ng per vg, 1.0×10 13 <0.1ng per vg , or 1.0×10 13 rAA with benzonase concentration less than 0.09 ng per vg and a rAAV viral vector is formulated for administration, and / or about 2.4 x 10 14 In some embodiments, the pharmaceutical composition is present in a unit dose of 0.1 mg / kg. In embodiments, the formulation or pharmaceutical composition comprises about 6.0×10 13 vg rAAV virus vector The rAAV viral vector contains a unit dose of 1.0 × 10 13 0 per vg. Less than 2 ng, 1.0 × 10 13 Less than 0.1 ng per vg or 1.0 × 10 13 vg a In some embodiments, the formulation or The pharmaceutical composition comprises about 1.2×10 14 Contains a unit dose of rAAV viral vector in vg , rAAV viral vector, 1.0 × 10 13 <0.2ng per vg, 1.0x 10 13 Less than 0.1 ng per vg or 1.0 × 10 13 <0.09ng per vg In some embodiments, the formulation or pharmaceutical composition has a benzonase concentration of about 2. 4×10 14 rAAV virus vector unit dose of 10 ... The culprit is 1.0×10 13 <0.2ng per vg, 1.0×10 13 0 per vg Less than .1ng or 1.0×10 13 Benzonase concentration of less than 0.09 ng per vg Yes.

[0123] In some embodiments, the method comprises: 13 Less than 0.5 ng per vg, 1. 0×10 13 Less than 0.3 ng per vg or 1.0 × 10 13 0.22ng per vg yielding rAAV viral vectors with bovine serum albumin (BSA) concentrations of less than , the rAAV viral vector is formulated for administration, and / or has a concentration of about 6.0 x 10 13 In some embodiments, the method comprises administering a 1.0× unit dose of 1.0× 10 13 <0.5 ng per vg, 1.0 × 10 13 Less than 0.3 ng per vg, or 1.0×10 13 Contains a bovine serum albumin (BSA) concentration of less than 0.22 ng per vg. The rAAV viral vector is Formulated and / or about 1.2 x 10 14 vg of unit dose of the pharmaceutical composition. In some embodiments, the method comprises: 13 <0.5ng per vg, 1.0x 10 13 Less than 0.3 ng per vg or 1.0 × 10 13 <0.22ng per vg rAAV viral vectors having a bovine serum albumin (BSA) concentration of r The AAV viral vector is formulated for administration and / or is about 2.4 x 10 14 vg In some embodiments, the formulation or pharmaceutical composition is present in the pharmaceutical composition in a unit dose of , about 6.0×10 13 rAAV virus vector unit dose of 1.0001vg, The virus vector is 1.0 × 10 13 <0.5 ng per vg, 1.0 × 1013 vg Less than 0.3 ng per unit or 1.0 x 10 13 Bovine serum albumin less than 0.22 ng per vg In some embodiments, the formulation or pharmaceutical composition has a BSA concentration of about 1.2×10 14 rAAV virus vector unit dose of 1.0001 vg, The spectrum is 1.0×10 13 <0.5 ng per vg, 1.0 × 10 13 vgata Less than 0.3 ng or 1.0 x 10 13 Bovine serum albumin less than 0.22 ng per vg In some embodiments, the formulation or pharmaceutical composition has a BSA concentration of about 2. 4×10 14 rAAV virus vector unit dose of 10 ... The culprit is 1.0×10 13 <0.5 ng per vg, 1.0 × 10 13 0 per vg Less than .3ng or 1.0×10 13 Bovine serum albumin less than 0.22ng per vg (BSA) concentration.

[0124] In some embodiments, the method comprises: 13 Approximately 1 EU / mL per vg / mL Less than 1.0×10 13 Less than approximately 0.75 EU / mL per vg / mL, 1.0 × 10 13 Less than approximately 0.5 EU / mL per vg / mL, 1.0 × 10 13 Approximately 0.4 per vg / mL Less than 1.0×10 EU / mL 13 Less than about 0.35 EU / mL per vg / mL, 1.0 ×10 13 Less than approximately 0.3 EU / mL per vg / mL, 1.0 × 10 13 vg / mL Approximately 0.25 EU / mL or less than 1.0 × 10 13 Approximately 0.2 EU / mL per vg / mL Full, 1.0×10 13 Less than approximately 0.13 EU / mL per vg / mL, 1.0 × 10 13 v Less than approximately 0.1 EU / mL per g / mL, 1.0 x 10 13 Approximately 0.05 per vg / mL Less than EU / mL or 1.0 x 10 13 Within approximately 0.02 EU / mL per vg / mL The rAAV viral vector has a toxin level, , and / or about 6.0 x 10 13 in a pharmaceutical composition at a unit dose of vg In some embodiments, the method comprises the steps of: 13 Approximately 1 per vg / mL Less than 1.0×10 EU / mL 13 Less than about 0.75 EU / mL per vg / mL, 1.0 ×10 13 Less than approximately 0.5 EU / mL per vg / mL, 1.0 × 10 13 vg / mL Approximately 0.4 EU / mL or less than 1.0 × 10 13 Approximately 0.35 EU / mL per vg / mL Full, 1.0×10 13 Less than approximately 0.3 EU / mL per vg / mL, 1.0 × 10 13 vg / mL, less than approximately 0.25 EU / mL, 1.0 × 10 13 Approximately 0.2E per vg / mL U / mL, less than 1.0 × 10 13 Less than approximately 0.13 EU / mL per vg / mL, 1.0× 10 13 Less than approximately 0.1 EU / mL per vg / mL, 1.0 × 10 13 Per vg / mL Less than about 0.05 EU / mL or 1.0 x 10 13 Approximately 0.02 EU / m per vg / mL rAAV virus vectors having endotoxin levels of less than L, The vector is formulated for administration and / or has a concentration of about 1.2×10 14 Medications in unit doses of vg In some embodiments, the method comprises administering to the patient a dose of 1.0×10 13 vg / mL Approximately 1 EU / mL per 1.0 × 10 13 Approximately 0.75 EU / mL per vg / mL Full, 1.0×10 13 Less than approximately 0.5 EU / mL per vg / mL, 1.0 × 10 13 vg / mL, less than approximately 0.4 EU / mL, 1.0 × 10 13 Approximately 0.35E per vg / mL U / mL, less than 1.0 × 10 13 Less than approximately 0.3 EU / mL per vg / mL, 1.0 x 1 0 13 Less than approximately 0.25 EU / mL per vg / mL, 1.0 × 10 13 Per vg / mL Approximately 0.2 EU / mL or less, 1.0 x 10 13 Less than approximately 0.13 EU / mL per vg / mL , 1.0×10 13 Less than approximately 0.1 EU / mL per vg / mL, 1.0 × 10 13 vg / Less than about 0.05 EU / mL or 1.0 x 10 13 Approximately 0.0 per vg / mL Resulting in rAAV viral vectors with endotoxin levels of less than 2EU / mL, The V viral vector is formulated for administration and / or has a concentration of about 2.4×10 14 vg single In some embodiments, the formulation or pharmaceutical composition is present in the pharmaceutical composition in a dose of about 6.0×10 13 rAAV virus vector unit dose of 1.0001 vg, The spectrum is 1.0×10 13Less than approximately 1 EU / mL per vg / mL, 1.0 × 10 1 3 Less than approximately 0.75 EU / mL per vg / mL, 1.0 × 10 13 Approximately 0 per vg / mL Less than .5EU / mL, 1.0×10 13 Less than approximately 0.4 EU / mL per vg / mL, 1. 0×10 13 Less than approximately 0.35 EU / mL per vg / mL, 1.0 × 10 13 vg / mL Approximately 0.3 EU / mL per 1.0 × 10 13 Approximately 0.25 EU / m per vg / mL Less than L, 1.0 x 10 13 Less than approximately 0.2 EU / mL per vg / mL, 1.0 × 10 13 Less than approximately 0.13 EU / mL per vg / mL, 1.0 × 10 13 Approximately 0.0 ... Less than 1EU / mL, 1.0×10 13 Less than about 0.05 EU / mL per vg / mL, or 1.0×10 13 has an endotoxin level of less than about 0.02 EU / mL per vg / mL. In some embodiments, the formulation or pharmaceutical composition has a concentration of about 1.2×10 14 vg rAAV The unit dose of the rAAV virus vector is 1.0 × 10 13 v Less than approximately 1 EU / mL per g / mL, 1.0 × 10 13 Approximately 0.75 EU per vg / mL / mL, less than 1.0 × 10 13 Less than approximately 0.5 EU / mL per vg / mL, 1.0 × 10 13 Less than approximately 0.4 EU / mL per vg / mL, 1.0 × 10 13 Approximately 0 per vg / mL Less than .35EU / mL, 1.0×10 13 Less than approximately 0.3 EU / mL per vg / mL, 1 .0×1013 Less than approximately 0.25 EU / mL per vg / mL, 1.0 × 10 13 vg / m Less than approximately 0.2 EU / mL per L, 1.0 x 10 13 Approximately 0.13 EU / vg / mL Less than 1.0 x 10 mL 13 Less than approximately 0.1 EU / mL per vg / mL, 1.0 × 10 1 3 Less than about 0.05 EU / mL per vg / mL, or 1.0 × 10 13 Per vg / mL In some embodiments, the formulation or The pharmaceutical composition comprises about 2.4×10 14 Contains a unit dose of rAAV viral vector in vg , rAAV viral vector, 1.0 × 10 13 Less than approximately 1 EU / mL per vg / mL , 1.0×10 13 Less than approximately 0.75 EU / mL per vg / mL, 1.0 × 10 13 vg / mL, less than approximately 0.5 EU / mL, 1.0 × 10 13 Approximately 0.4 EU per vg / mL / mL, less than 1.0 × 10 13 Less than approximately 0.35 EU / mL per vg / mL, 1.0 x 1 0 13 Less than approximately 0.3 EU / mL per vg / mL, 1.0 × 10 13 Approximately per vg / mL Less than 0.25EU / mL, 1.0×10 13 Less than approximately 0.2 EU / mL per vg / mL 1.0×10 13 Less than approximately 0.13 EU / mL per vg / mL, 1.0 × 10 13 vg / Approximately less than 0.1 EU / mL, 1.0 x 10 13 Approximately 0.05 EU per vg / mL / mL or less than 1.0 × 10 13 Less than approximately 0.02 EU / mL of endotoxin per vg / mL It has a level.

[0125] In some embodiments, the method provides for a concentration of less than 100 μg / g (ppm), 50 μg / g (ppm), rAAV virus with a cesium concentration of less than 30 μg / g (ppm) or less than 30 μg / g (ppm) The rAAV viral vector is formulated for administration, and / or is about 6.0 x 10 13 In some embodiments, the pharmaceutical composition is present in a unit dose of 1000 mg / kg. The method is to determine whether the concentration is less than 100 μg / g (ppm), less than 50 μg / g (ppm), or less than 30 μg / g (ppm). providing an rAAV viral vector having a concentration of cesium of less than g / g (ppm); The rAAV viral vector is formulated for administration and / or administered at a concentration of about 1.2×10 14 v In some embodiments, the pharmaceutical composition is present in a unit dose of 100 μg. / g(ppm), less than 50μg / g(ppm), or less than 30μg / g(ppm) Providing an rAAV viral vector having a cesium concentration, is formulated for administration and / or is about 2.4 x 10 14 Pharmaceutical composition in unit doses of vg In some embodiments, the formulation or pharmaceutical composition is present in a volume of about 6.0×10 13 The rAAV viral vector comprises a unit dose of 1 vg of rAAV viral vector. Less than 00μg / g(ppm), less than 50μg / g(ppm), or less than 30μg / g(ppm In some embodiments, the formulation or pharmaceutical composition has a concentration of cesium of less than about 1.2×10 14 rAAV virus vector unit dose of 1.0001 vg, The vector is less than 100 μg / g (ppm), less than 50 μg / g (ppm), or less than 30 In some embodiments, the formulation or The pharmaceutical composition comprises about 2.4×10 14 Contains a unit dose of rAAV viral vector in vg , rAAV viral vectors are less than 100 μg / g (ppm), 50 μg / g (ppm ) or has a cesium concentration of less than 30 μg / g (ppm).

[0126] In some embodiments, the method comprises adding about 10 to 100 ppm, 15 to 90 ppm, or about providing an rAAV viral vector having 20 to 80 ppm of poloxamer 188; The rAAV viral vector is formulated for administration and / or administered in a volume of about 6.0 x 10 13 v In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising ... 00 ppm, 15-90 ppm, or about 20-80 ppm of poloxamer 188. Providing a rAAV viral vector, the rAAV viral vector being formulated for administration and / or about 1.2 x 10 14 vg of unit dose in the pharmaceutical composition. In some embodiments, the method includes adding about 10 to 100 ppm, 15 to 90 ppm, or about 20 to 80 ppm. 0 ppm poloxamer 188, resulting in a rAAV viral vector The viral vector is formulated for administration and / or has a concentration of about 2.4×10 14 Unit of vg In some embodiments, the formulation or pharmaceutical composition is present in a dosage of about 6 .0×10 13 rAAV virus vector containing a unit dose of 1000 μg of rAAV virus vector The vector is about 10 to 100 ppm, 15 to 90 ppm, or about 20 to 80 ppm of pol. In some embodiments, the formulation or pharmaceutical composition has about 1.2× 10 14 rAAV viral vector unit dose of 10 ... - Approximately 10 to 100 ppm, 15 to 90 ppm, or approximately 20 to 80 ppm of poloxamer In some embodiments, the formulation or pharmaceutical composition has about 2.4×10 1 4 vg of a rAAV viral vector, About 10-100 ppm, 15-90 ppm, or about 20-80 ppm of poloxamer 18 Has 8.

[0127] In some embodiments, the method comprises: rAAV virus having less than 1500, less than 1000, or less than 600 particles vector, the rAAV viral vector is formulated for administration, and / or Approximately 6.0×10 13 In some embodiments, the pharmaceutical composition is present in a unit dose of 100 mg / kg. The method is to have less than 2000, 1500, or 10 particles with a size of 25 μm or more per container. 00 or less than 600 particles, resulting in an rAAV viral vector having rAAV particles The AV viral vector is formulated for administration and / or is about 1.2 x 10 14 vg In some embodiments, the method comprises administering a single dose of the pharmaceutical composition to a patient in a single container. The number of particles is less than 2000, 1500, 1000 or less than 600 with a size of 25 μm or more. and resulting in a rAAV viral vector having 100,000 particles, the rAAV viral vector being and / or about 2.4 x 10 14 in a pharmaceutical composition at a unit dose of vg In some embodiments, the formulation or pharmaceutical composition comprises about 6.0×10 13 vg The rAAV viral vector contains a unit dose of the rAAV viral vector. and less than 2000 particles, less than 1500 particles, less than 1000 particles, or less than 60 particles with a size of 25 μm or more. In some embodiments, the formulation or pharmaceutical composition has less than about 1.2× 10 14 rAAV viral vector unit dose of 10 ... - Less than 2000, 1500, or 1000 particles of size 25 μm or more per container In some embodiments, the formulation or pharmaceutical composition has fewer than 100 or fewer than 600 particles. is about 2.4 x 10 14 vg of rAAV viral vector unit dose, Viral vectors are limited to less than 2,000 cells with a size of 25 μm or more, and 1,500 cells per container. less than 1000 or less than 600 particles.

[0128] In some embodiments, the method comprises: rAAV virus having less than 8000 particles, less than 1000 particles, or less than 6000 particles the rAAV viral vector is formulated for administration, and / or Or about 6.0 x 10 13 In some embodiments, the pharmaceutical composition is present in a unit dose of 100 mg / kg. The method is to have less than 10,000 particles with a size of 10 μm or more per container, or less than 8,000 particles. resulting in an rAAV viral vector having less than 1000 or less than 6000 particles. and the rAAV viral vector is formulated for administration and / or has a concentration of about 1.2 x 10 1 4In some embodiments, the method further comprises the steps of: Or, less than 10,000 particles, less than 8,000 particles, less than 1,000 particles, or particles with a size of 10 μm or more Resulting in an rAAV virus vector having less than 6000 particles, The vector is formulated for administration and / or has a concentration of about 2.4×10 14 Medications in unit doses of vg In some embodiments, the formulation or pharmaceutical composition is present in a pharmaceutical composition having a molecular weight of about 6.0×1 0 13 vg of rAAV viral vector unit dose, is less than 10,000 particles, less than 8,000 particles, or less than 1,000 particles with a size of 10 μm or more per container. In some embodiments, the formulation or pharmaceutical composition has fewer than 6,000 particles. The object is about 1.2 x 10 14 rAAAV viral vector unit dose of 10 ... V virus vectors are contained in a container with less than 10,000 cells of 10 μm or more in size, and 800 In some embodiments, the nanoparticles have fewer than 0, fewer than 1000, or fewer than 6000 particles. , the formulation or pharmaceutical composition is about 2.4×10 14 vg unit dose of rAAV viral vector The rAAV viral vector is 100 pieces per container, each of which is 10 μm or larger in size. 00, less than 8000, less than 1000 or less than 6000 particles.

[0129] In some embodiments, the method comprises a pH between 7.5 and 8.5, between 7.6 and 8.4, or between 7. The rAAV virus vector has a pH between 0.8 and 8.3. The vector is formulated for administration and / or has a concentration of about 6.0×10 13 in unit doses of vg In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising: The rAAV viral vector has a pH between 6 and 8.4, or between 7.8 and 8.3. The rAAV viral vector is formulated for administration and / or administered at approximately 1.2×1 0 14 In some embodiments, the method comprises the steps of: rAA with a pH between 7.5 and 8.5, between 7.6 and 8.4, or between 7.8 and 8.3 and a rAAV viral vector is formulated for administration, and / or about 2.4 x 10 14 In some embodiments, the pharmaceutical composition is present in a unit dose of 0.1 mg / kg. In embodiments, the formulation or pharmaceutical composition comprises about 6.0×10 13 vg rAAV virus vector The rAAV viral vector contains a unit dose of 7.5 to 8.5, 7.6 to 8 In some embodiments, the formulation or The pharmaceutical composition comprises about 1.2×10 14 Contains a unit dose of rAAV viral vector in vg , the rAAV viral vector is between 7.5 and 8.5, between 7.6 and 8.4, or between 7.8 In some embodiments, the formulation or pharmaceutical composition has a pH of between about 2. 4×10 14 rAAV virus vector unit dose of 10 ... The culprits are those with a pH between 7.5 and 8.5, between 7.6 and 8.4, or between 7.8 and 8.3. Yes.

[0130] In some embodiments, the method comprises administering between 330 and 490 mOsm / kg, between 360 and 46 rAA with an osmolality between 0 mOsm / kg or between 390 and 430 mOsm / kg and a rAAV viral vector is formulated for administration, and / or about 6.0 x 10 13 In some embodiments, the pharmaceutical composition is present in a unit dose of 0.1 mg / kg. In an embodiment, the method includes administering between 330-490 mOsm / kg, 360-460 mOsm / kg g or osmolality between 390 and 430 mOsm / kg. The rAAV viral vector is formulated for administration and / or administered in a dose of about 1 .2×10 14 In some embodiments, the method comprises administering to the subject a unit dose of 0.1 mg of the compound. The method is between 330 and 490 mOsm / kg, between 360 and 460 mOsm / kg, or Resulting in rAAV viral vectors with osmolality between 90 and 430 mOsm / kg , the rAAV viral vector is formulated for administration, and / or has a concentration of about 2.4 x 10 14 In some embodiments, the formulation or pharmaceutical composition is present in a unit dose of 1000 mg / kg. The object is about 6.0 x 10 13 rAAAV viral vector unit dose of 10 ... V viral vectors were administered at a concentration between 330 and 490 mOsm / kg and 360 and 460 mOsm / kg. kg, or between 390 and 430 mOsm / kg. In one embodiment, the formulation or pharmaceutical composition comprises about 1.2×10 14 vg rAAV viral vector The unit dose of rAAV viral vector is between 330 and 490 mOsm / kg. Osmolality between 360-460mOsm / kg or 390-430mOsm / kg In some embodiments, the formulation or pharmaceutical composition has about 2.4×10 14 vgr The unit dose of the AAV viral vector is 330 to 4 Between 90mOsm / kg, between 360-460mOsm / kg, or between 390-430mO sm / kg.

[0131] In some embodiments, the method comprises: 13 Approximately 1.0 × 10 per vg 8 ~1 0.0×10 10 IU, 1.0×10 13 Approximately 2.5 × 10 per vg 8 ~9.0×10 1 0 IU or 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 IU's Feelings The rAAV viral vectors obtained were then administered to the recipient organism. and / or about 6.0 x 10 13 The pharmaceutical composition contains 100 mg of In some embodiments, the method comprises: 13 Approximately 1.0 × 10 per vg 8 ~10.0×10 10 IU, 1.0×10 13 Approximately 2.5 × 10 per vg 8 ~9.0× 10 10 IU or 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 I and obtaining an rAAV viral vector having an infectious titer of U. is formulated for administration and / or about 1.2 x 10 14 Pharmaceutical composition in unit doses of vg In some embodiments, the method comprises the step of: 13 Approximately 1.0 per vg ×108 ~10.0×10 10 IU, 1.0×10 13 Approximately 2.5 × 10 per vg 8 ~9 .0×10 10 IU or 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 The rAAV virus vector had an infectious titer of IU. The vector is formulated for administration at about 2.4 x 10 14 Drug in unit dose of vg In some embodiments, the formulation or pharmaceutical composition is present in a composition having about 6.0×10 13 vg of rAAV viral vector, , 1.0×10 13 Approximately 1.0 × 10 per vg 8 ~10.0×10 10 IU, 1.0×1 0 13 Approximately 2.5 × 10 per vg 8 ~9.0×10 10 IU or 1.0 x 10 13 vg Approximately 3.9 x 10 8 ~8.4×10 10 IU of infectious titer. In one embodiment, the formulation or pharmaceutical composition comprises about 1.2×10 14 vg rAAV viral vector The unit dose of the rAAV viral vector is 1.0 × 10 13 Approximately 1.0 per vg ×10 8 ~10.0×10 10 IU, 1.0×10 13 Approximately 2.5 × 10 per vg 8 ~9 .0×10 10 IU or 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 IU. In some embodiments, the formulation or pharmaceutical composition has an infectious titer of about 2. 4×10 14 rAAV virus vector unit dose of 10 ... The culprit is 1.0×10 13 Approximately 1.0 × 10 per vg 8 ~10.0×10 10 IU, 1 .0×10 13 Approximately 2.5 × 10 per vg 8 ~9.0×10 10 IU or 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 It has an infectious titer of IU.

[0132] In some embodiments, the method comprises the step of: determining whether a reference standard is present based on an in vitro cell-based assay; About 30-150%, about 60-140%, or about 70% relative to a reference and / or appropriate control. The rAAV virus vector had a relative titer of ~130%, The vector is formulated for administration and / or has a concentration of about 6.0×10 13 Medications in unit doses of vg In some embodiments, the method comprises administering to the subject an antibody or antibody fragment thereof in an in vitro cell-based assay. Based on (a), about 30 to 150%, about 60 to 150%, relative to the reference standard and / or appropriate control 140%, or about 70-130% relative titer of rAAV viral vectors. and the rAAV viral vector is formulated for administration and / or has a concentration of about 1.2 x 10 1 4 In some embodiments, the method comprises the step of: Based on a cell-based assay, in conjunction with a reference standard and / or appropriate control, about 30 rAAV viruses having a relative titer of about 150%, about 60% to 140%, or about 70% to 130%. the rAAV viral vector is formulated for administration, and / or or about 2.4 x 10 14 In some embodiments, the pharmaceutical composition is present in a unit dose of 100 mg / kg. In the formulation or pharmaceutical composition, 13 vg rAAV viral vector single The rAAV viral vector is administered in association with a reference standard and / or appropriate control. Based on in vitro cell-based assays, the results are approximately 30-150%, approximately 60-140%, and or about 70-130% relative potency. The object is about 1.2 x 10 14 rAAAV viral vector unit dose of 10 ... V viral vectors are expressed in vitro, in cell-based, and in association with reference standards and / or appropriate controls. Based on the assay, about 30-150%, about 60-140%, or about 70-130% In some embodiments, the formulation or pharmaceutical composition has a relative potency of about 2.4×10 1 4 vg of a rAAV viral vector, Based on in vitro cell-based assays in conjunction with reference standards and / or appropriate controls; The relative potency is about 30-150%, about 60-140%, or about 70-130%.

[0133] In some embodiments, the method comprises: 13 Approximately 10-500μg per vg 1.0×10 13 Approximately 50-400 μg per vg, or 1.0 × 10 13 Approximately 1 per vg resulting in rAAV viral vectors with total protein levels of 00-300 μg, The rAAV viral vector is formulated for administration and / or administered in a volume of about 6.0 x 10 13 v In some embodiments, the method comprises administering a 1.0×100 mg / kg unit dose of the pharmaceutical composition to a patient in need thereof. 0 13 Approximately 10-500 μg per vg, 1.0 × 10 13 Approximately 50-400μg per vg , or 1.0×10 13 Has a total protein level of approximately 100-300 μg per vg Providing a rAAV viral vector, the rAAV viral vector being formulated for administration and / or about 1.2 x 10 14 vg of unit dose in the pharmaceutical composition. In some embodiments, the method comprises: 13 Approximately 10-500μg per vg, 1.0x 10 13 Approximately 50-400 μg per vg, or 1.0 × 10 13 Approximately 100-3 per vg 100 μg total protein level, resulting in rAAV viral vectors The viral vector is formulated for administration and / or has a concentration of about 2.4×10 14 Unit of vg In some embodiments, the formulation or pharmaceutical composition is present in a dosage of about 6 .0×10 13 rAAV virus vector containing a unit dose of 1000 μg of rAAV virus vector Vector is 1.0×10 13 Approximately 10-500 μg per vg, 1.0 × 10 13 vg a Approximately 50-400μg per dose, or 1.0×10 13 Approximately 100-300 μg of total protein per vg In some embodiments, the formulation or pharmaceutical composition has a protein level of about 1.2x. 10 14 rAAV viral vector unit dose of 10 ... - is 1.0 x 10 13 Approximately 10-500 μg per vg, 1.0 × 10 13 Approximately per vg 50-400μg or 1.0×10 13 Approximately 100-300μg total protein per vg In some embodiments, the formulation or pharmaceutical composition has a quality level of about 2.4×10 1 4 vg of a rAAV viral vector, 1.0×10 13 Approximately 10-500 μg per vg, 1.0 × 10 13 Approximately 50 per vg 400 μg or 1.0 × 10 13 Total protein level of approximately 100-300 μg per vg It has a

[0134] In some embodiments, the method comprises: 13 vg / kg dose of S As judged by the median survival time in MNΔ7 mice, the survival time was >15 days, >20 days, and >2 days. The present invention provides rAAV viral vectors having in vivo efficacy of more than 2 days or more than 24 days. and the rAAV viral vector is formulated for administration and / or has a concentration of about 6.0 x 10 1 3 In some embodiments, the method comprises the step of: ×10 13 Median survival time in SMNΔ7 mice receiving a dose of 1000 mg / kg In vivo efficacy for more than 15 days, more than 20 days, more than 22 days, or more than 24 days as determined by the and providing an rAAV viral vector having the nucleic acid sequence for administration. and / or approximately 1.2 x 10 14 vg of unit dose of In some embodiments, the method comprises: 13 vg / kg dose of S As judged by the median survival time in MNΔ7 mice, the survival time was >15 days, >20 days, and >2 days. The present invention provides rAAV viral vectors having in vivo efficacy of more than 2 days or more than 24 days. and the rAAV viral vector is formulated for administration and / or has a concentration of about 2.4 x 10 1 4 In some embodiments, the formulation or pharmaceutical composition is present in a unit dose of 1000 mg / kg. The product is about 6.0 × 10 13 rAAV virus vector unit dose of 10 ... AV viral vectors were 7.5 × 10 13 SMNΔ7 mice were administered a dose of 1000 mg / kg. The median survival time in mice was >15 days, >20 days, and >22 days. or has an in vivo efficacy of more than 24 days. The object is about 1.2 x 10 14 rAAAV viral vector unit dose of 10 ... V viral vector, 7.5 × 10 13 SMNΔ7 mice administered a dose of 1000 mg / kg The median survival time in the study was more than 15 days, more than 20 days, more than 22 days, or has an in vivo efficacy of more than 24 days. is about 2.4 x 10 14 vg of rAAV viral vector unit dose, The viral vector was 7.5 × 10 13 SMNΔ7 mice administered a dose of vg / kg The median survival time in the study was more than 15 days, more than 20 days, more than 22 days, or has an in vivo efficacy of more than 24 days.

[0135] In some embodiments, the formulation or pharmaceutical composition has a concentration of about 6.0×10 13 vg rAAV A unit dose of viral vector and one or more of the following shipping standards: less than about 8%, less than about 7%, or less than about 5% empty viral capsids; 1×10 13 Less than approximately 100 ng / mL of host cell protein per vg / mL; 1 × 10 13 vg / m Approximately 5 x 10 per L 6 Less than pg / mL, approximately 1 × 10 6 Less than pg / mL, approximately 7.5 × 10 5 Less than pg / mL or 6.8 x 10 5 Residual host cell DNA (hcDNA) less than pg / mL );1.0×10 13 Less than about 10 ng, less than about 8 ng, less than about 6 ng per vg / mL or less than about 4 ng residual host cell protein (rHCP); at least about 50%, At least about 60%, at least about 70%, at least about 80%, at least about 90%, At least about 95%, or at least about 100%, functional rAAV viral vector genome / mL; 1×10 13 1.7 × 10 per vg / mL 6 pg / mL or 1 x 10 13 vg 1 x 10 per mL 5 pg / ml1×10 13 1.7 × 10 per vg / mL 6 pg / Residual plasmid DNA less than 1.0 x 10 13 Less than 0.2ng per vg, 1.0 ×10 13 Less than 0.1 ng per vg or 1.0 × 10 13 Less than 0.09 ng per vg Benzonase concentration: 1.0 x 10 13 <0.5 ng per vg, 1.0 × 1013 Less than 0.3 ng per vg or 1.0 × 10 13 Bovine blood less than 0.22ng per vg Serum albumin (BSA) concentration: 1.0 x 10 13 Less than approximately 1 EU / mL per vg / mL 1.0×10 13 Less than approximately 0.75 EU / mL per vg / mL, 1.0 × 10 13 vg / Approximately 0.5 EU / mL or less than 1.0 × 10 13 Approximately 0.4 EU / vg / mL Less than 1.0 x 10 mL 13 Less than approximately 0.35 EU / mL per vg / mL, 1.0 × 10 13 Less than approximately 0.3 EU / mL per vg / mL, 1.0 × 10 13 Approximately 0 per vg / mL Less than .25EU / mL, 1.0×10 13 Less than approximately 0.2 EU / mL per vg / mL, 1 .0×10 13 Less than approximately 0.13 EU / mL per vg / mL, 1.0 × 10 13 vg / m Less than approximately 0.1 EU / mL per L, 1.0 x 10 13 Approximately 0.05 EU / vg / mL mL or less than 1.0 x 10 13 Endotoxin levels below approximately 0.02 EU / mL per vg / mL Bel; less than 100μg / g (ppm), less than 50μg / g (ppm), or less than 30μg / g Cesium concentration less than (ppm); about 10-100 ppm, 15-90 ppm, or about 2 0-80 ppm Poloxamer 188; 2000 particles ≥ 25 μm in size per container Less than, 1500, 1000, or 600 particles per container; size Less than 10,000 particles, less than 8,000 particles, less than 1,000 particles, or 6,000 particles of 10 μm or more particles less than 7.5; pH between 7.5 and 8.5, between 7.6 and 8.4, or between 7.8 and 8.3 between 330 and 490 mOsm / kg, between 360 and 460 mOsm / kg, or between 390 Osmolality between ~430 mOsm / kg; 1.0 × 10 13 Approximately 1.0 × 10 per vg 8 ~ 10.0×10 10 IU, 1.0×10 13 Approximately 2.5 × 10 per vg 8 ~9.0×10 10 IU or 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 IU's Infectious titer; based on an in vitro cell-based assay and correlated with a reference standard and / or appropriate controls Relative potency of about 30-150%, about 60-140%, or about 70-130%;1. 0×10 13 Approximately 10-500 μg per vg, 1.0 × 10 13 Approximately 50-40 per vg 0 μg or 1.0 × 10 13 Total protein levels of approximately 100-300 μg per vg; 7.5×10 13 Median survival time in SMNΔ7 mice receiving a dose of vg / kg Judging by the value, the living body is more than 15 days, more than 20 days, more than 22 days, or more than 24 days old. Internal effectiveness.

[0136] In some embodiments, the formulation or pharmaceutical composition has a concentration of about 6.0×10 13 vg rAAV A unit dose of the viral vector, and one or more of the following shipping standards: about 7.7 to 100 mg / mL. pH of 8.3; osmolality of approximately 390-430 mOsm / kg; sieve size of 25 μm or more per container Less than about 600 particles of 10 μm or more in size per container; Less than about 6,000 particles of 10 μm or more in size per container Child: Approx. 1.7 x 10 13 ~5.3×10 13 Genomic titer in vg / mL; 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 Infectious titer in IU: 1.0 x 10 13 vg Total protein levels of approximately 100-300 μg per serving; approximately 20-80 ppm Pluronics (R) F-68 Content, Based on In Vitro Cell-Based Assay, Reference Standard and / or or a relative titer of approximately 70-130% relative to an appropriate control; 7.5 x 10 13 vg / kg The SMNΔ7 mouse model was characterized by a median survival time of more than 24 days at a dose of In vivo potency; less than 5% empty capsids; greater than 95% total purity; approximately 0.13 EU / mL The following endotoxins:

[0137] In some embodiments, the formulation or pharmaceutical composition has a concentration of about 6.0×10 13 vg rAAV A unit dose of viral vector and one or more of the following shipping standards: 1.0 x 1 0 13 Less than about 0.09 ng benzonase per vg; less than about 30 μg / g (ppm) Cesium, approximately 20-80 ppm Poloxamer 188; 1.0 × 10 13 Approximately 0 per vg < .22ng BSA; 1.0 x 10 13 Approximately 6.8 × 10 per vg 5 Sub-picogram residues Plasmid DNA: 1.0 × 10 13 Approximately 1.1 × 10 per vg 5 Sub-pg hcD residue NA;1.0×10 13 Less than approximately 4 ng rHCP per vg; pH of approximately 7.7 to 8.3; Osmolarity of about 390-430 mOsm / kg; about 600 per vessel with a size of 25 μm or more Less than 1000 particles per container; Less than about 6000 particles of 10 μm or more in size; Approximately 1.7× 10 13 ~5.3×10 13 Genomic titer in vg / mL; 1.0 x 10 13 Approximately 3 per vg .9×10 8 ~8.4×10 10 Infectious titer in IU: 1.0 x 10 13 Approximately 100 per vg ~300μg total protein level; based on in vitro cell-based assay, reference standard and / or a relative potency of about 70-130% relative to an appropriate control; less than about 5% empty caps Sid.

[0138] In some embodiments, the formulation or pharmaceutical composition has a concentration of about 1.2×10 14 vg rAAV A unit dose of viral vector and one or more of the following shipping standards: less than about 8%, less than about 7%, or less than about 5% empty viral capsids; 1×10 13 Less than approximately 100 ng / mL of host cell protein per vg / mL; 1 × 10 13 vg / m Approximately 5 x 10 per L 6 Less than pg / mL, approximately 1 × 10 6 Less than pg / mL, approximately 7.5 × 10 5 Less than pg / mL or 6.8 x 10 5 Residual host cell DNA (hcDNA) less than pg / mL );1.0×10 13 Less than about 10 ng, less than about 8 ng, less than about 6 ng per vg / mL or less than about 4 ng residual host cell protein (rHCP); at least about 50%, At least about 60%, at least about 70%, at least about 80%, at least about 90%, At least about 95%, or at least about 100%, functional rAAV viral vector genome / mL; 1×10 13 1.7 × 10 per vg / mL 6 pg / mL or 1 x 10 13 vg 1 x 10 per mL 5 pg / ml1×10 13 1.7 × 10 per vg / mL 6 pg / Residual plasmid DNA less than 1.0 x 10 13 Less than 0.2ng per vg, 1.0 ×10 13 Less than 0.1 ng per vg or 1.0 × 10 13 Less than 0.09 ng per vg Benzonase concentration: 1.0 x 10 13 <0.5 ng per vg, 1.0 × 10 13 Less than 0.3 ng per vg or 1.0 × 10 13 Bovine blood less than 0.22ng per vg Serum albumin (BSA) concentration: 1.0 x 10 13 Less than approximately 1 EU / mL per vg / mL 1.0×10 13 Less than approximately 0.75 EU / mL per vg / mL, 1.0 × 10 13 vg / Approximately 0.5 EU / mL or less than 1.0 × 10 13 Approximately 0.4 EU / vg / mL Less than 1.0 x 10 mL 13 Less than approximately 0.35 EU / mL per vg / mL, 1.0 × 10 13 Less than approximately 0.3 EU / mL per vg / mL, 1.0 × 10 13 Approximately 0 per vg / mL Less than .25EU / mL, 1.0×10 13 Less than approximately 0.2 EU / mL per vg / mL, 1 .0×10 13 Less than approximately 0.13 EU / mL per vg / mL, 1.0 × 10 13 vg / m Less than approximately 0.1 EU / mL per L, 1.0 x 10 13 Approximately 0.05 EU / vg / mL mL or less than 1.0 x 10 13 Endotoxin levels below approximately 0.02 EU / mL per vg / mL Bel; less than 100μg / g (ppm), less than 50μg / g (ppm), or less than 30μg / g Cesium concentration less than (ppm); about 10-100 ppm, 15-90 ppm, or about 2 0-80 ppm Poloxamer 188; 2000 particles ≥ 25 μm in size per container Less than, 1500, 1000, or 600 particles per container; size Less than 10,000 particles, less than 8,000 particles, less than 1,000 particles, or 6,000 particles of 10 μm or more particles less than 7.5; pH between 7.5 and 8.5, between 7.6 and 8.4, or between 7.8 and 8.3 between 330 and 490 mOsm / kg, between 360 and 460 mOsm / kg, or between 390 Osmolality between ~430 mOsm / kg; 1.0 × 10 13 Approximately 1.0 × 10 per vg 8 ~ 10.0×10 10 IU, 1.0×10 13 Approximately 2.5 × 10 per vg 8 ~9.0×10 10 IU or 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 IU's Infectious titer; based on an in vitro cell-based assay and correlated with a reference standard and / or appropriate controls Relative potency of about 30-150%, about 60-140%, or about 70-130%;1. 0×10 13 Approximately 10-500 μg per vg, 1.0 × 10 13 Approximately 50-40 per vg 0 μg or 1.0 × 10 13 Total protein levels of approximately 100-300 μg per vg; 7.5×10 13Median survival time in SMNΔ7 mice receiving a dose of vg / kg Judging by the value, the living body is more than 15 days, more than 20 days, more than 22 days, or more than 24 days old. Internal effectiveness.

[0139] In some embodiments, the formulation or pharmaceutical composition has a concentration of about 1.2×10 14 vg rAAV A unit dose of the viral vector, and one or more of the following shipping standards: about 7.7 to 100 mg / mL. pH of 8.3; osmolality of approximately 390-430 mOsm / kg; sieve size of 25 μm or more per container Less than about 600 particles of 10 μm or more in size per container; Less than about 6,000 particles of 10 μm or more in size per container Child: Approx. 1.7 x 10 13 ~5.3×10 13 Genomic titer in vg / mL; 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 Infectious titer in IU: 1.0 x 10 13 vg Total protein levels of approximately 100-300 μg per serving; approximately 20-80 ppm Pluronics (R) F-68 Content, Based on In Vitro Cell-Based Assay, Reference Standard and / or or a relative titer of approximately 70-130% relative to an appropriate control; 7.5 x 10 13 vg / kg The SMNΔ7 mouse model was characterized by a median survival time of more than 24 days at a dose of In vivo potency; less than 5% empty capsids; greater than 95% total purity; approximately 0.13 EU / mL The following endotoxins:

[0140] In some embodiments, the formulation or pharmaceutical composition has a concentration of about 1.2×10 14 vg rAAV A unit dose of viral vector and one or more of the following shipping standards: 1.0 x 1 0 13Less than about 0.09 ng benzonase per vg; less than about 30 μg / g (ppm) Cesium; approximately 20-80 ppm Poloxamer 188; 1.0 x 10 13 Approximately 0 per vg < .22ng BSA; 1.0 x 10 13 Approximately 6.8 × 10 per vg 5 Sub-picogram residues Plasmid DNA: 1.0 × 10 13 Approximately 1.1 × 10 per vg 5 Sub-pg hcD residue NA;1.0×10 13 Less than approximately 4 ng rHCP per vg; pH of approximately 7.7 to 8.3; Osmolarity of about 390-430 mOsm / kg; about 600 per vessel with a size of 25 μm or more Less than 1000 particles per container; Less than about 6000 particles of 10 μm or more in size; Approximately 1.7× 10 13 ~5.3×10 13 Genomic titer in vg / mL; 1.0 x 10 13 Approximately 3 per vg .9×10 8 ~8.4×10 10 Infectious titer in IU: 1.0 x 10 13 Approximately 100 per vg ~300μg total protein level; based on in vitro cell-based assay, reference standard and / or a relative potency of about 70-130% relative to an appropriate control; less than about 5% empty caps Sid.

[0141] In some embodiments, the formulation or pharmaceutical composition has a concentration of about 2.4×10 14 vg rAAV A unit dose of viral vector and one or more of the following shipping standards: less than about 8%, less than about 7%, or less than about 5% empty viral capsids; 1×10 13 Less than approximately 100 ng / mL of host cell protein per vg / mL; 1 × 10 13 vg / m Approximately 5 x 10 per L 6 Less than pg / mL, approximately 1 × 10 6 Less than pg / mL, approximately 7.5 × 10 5 Less than pg / mL or 6.8 x 10 5 Residual host cell DNA (hcDNA) less than pg / mL );1.0×10 13 Less than about 10 ng, less than about 8 ng, less than about 6 ng per vg / mL or less than about 4 ng residual host cell protein (rHCP); at least about 50%, At least about 60%, at least about 70%, at least about 80%, at least about 90%, At least about 95%, or at least about 100%, functional rAAV viral vector genome / mL; 1×10 13 1.7 × 10 per vg / mL 6 pg / mL or 1 x 10 13 vg 1 x 10 per mL 5 pg / ml1×10 13 1.7 × 10 per vg / mL 6 pg / Residual plasmid DNA less than 1.0 x 10 13 Less than 0.2ng per vg, 1.0 ×10 13 Less than 0.1 ng per vg or 1.0 × 10 13 Less than 0.09 ng per vg Benzonase concentration: 1.0 x 10 13 <0.5 ng per vg, 1.0 × 10 13 Less than 0.3 ng per vg or 1.0 × 10 13 Bovine blood less than 0.22ng per vg Serum albumin (BSA) concentration: 1.0 x 10 13 Less than approximately 1 EU / mL per vg / mL 1.0×10 13 Less than approximately 0.75 EU / mL per vg / mL, 1.0 × 10 13 vg / Approximately 0.5 EU / mL or less than 1.0 × 1013 Approximately 0.4 EU / vg / mL Less than 1.0 x 10 mL 13 Less than approximately 0.35 EU / mL per vg / mL, 1.0 × 10 13 Less than approximately 0.3 EU / mL per vg / mL, 1.0 × 10 13 Approximately 0 per vg / mL Less than .25EU / mL, 1.0×10 13 Less than approximately 0.2 EU / mL per vg / mL, 1 .0×10 13 Less than approximately 0.13 EU / mL per vg / mL, 1.0 × 10 13 vg / m Less than approximately 0.1 EU / mL per L, 1.0 x 10 13 Approximately 0.05 EU / vg / mL mL or less than 1.0 x 10 13 Endotoxin levels below approximately 0.02 EU / mL per vg / mL Bel; less than 100μg / g (ppm), less than 50μg / g (ppm), or less than 30μg / g Cesium concentration less than (ppm); about 10-100 ppm, 15-90 ppm, or about 2 0-80 ppm Poloxamer 188; 2000 particles ≥ 25 μm in size per container Less than, 1500, 1000, or 600 particles per container; size Less than 10,000 particles, less than 8,000 particles, less than 1,000 particles, or 6,000 particles of 10 μm or more particles less than 7.5; pH between 7.5 and 8.5, between 7.6 and 8.4, or between 7.8 and 8.3 between 330 and 490 mOsm / kg, between 360 and 460 mOsm / kg, or between 390 Osmolality between ~430 mOsm / kg; 1.0 × 10 13 Approximately 1.0 × 10 per vg 8 ~ 10.0×10 10 IU, 1.0×10 13 Approximately 2.5 × 10 per vg 8 ~9.0×10 10IU or 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 IU's Infectious titer; based on an in vitro cell-based assay and correlated with a reference standard and / or appropriate controls Relative potency of about 30-150%, about 60-140%, or about 70-130%;1. 0×10 13 Approximately 10-500 μg per vg, 1.0 × 10 13 Approximately 50-40 per vg 0 μg or 1.0 × 10 13 Total protein levels of approximately 100-300 μg per vg; 7.5×10 13 Median survival time in SMNΔ7 mice receiving a dose of vg / kg Judging by the value, the living body is more than 15 days, more than 20 days, more than 22 days, or more than 24 days old. Internal effectiveness.

[0142] In some embodiments, the formulation or pharmaceutical composition has a concentration of about 2.4×10 14 vg rAAV A unit dose of the viral vector, and one or more of the following shipping standards: about 7.7 to 100 mg / mL. pH of 8.3; osmolality of approximately 390-430 mOsm / kg; sieve size of 25 μm or more per container Less than about 600 particles of 10 μm or more in size per container; Less than about 6,000 particles of 10 μm or more in size per container Child: Approx. 1.7 x 10 13 ~5.3×10 13 Genomic titer in vg / mL; 1.0 x 10 13 Approximately 3.9 × 10 per vg 8 ~8.4×10 10 Infectious titer in IU: 1.0 x 10 13 vg Total protein levels of approximately 100-300 μg per serving; approximately 20-80 ppm Pluronics (R) F-68 Content, Based on In Vitro Cell-Based Assay, Reference Standard and / or or a relative titer of approximately 70-130% relative to an appropriate control; 7.5 x 10 13 vg / kg The SMNΔ7 mouse model was characterized by a median survival time of more than 24 days at a dose of In vivo potency; less than 5% empty capsids; greater than 95% total purity; approximately 0.13 EU / mL The following endotoxins:

[0143] In some embodiments, the formulation or pharmaceutical composition has a concentration of about 2.4×10 14 vg rAAV A unit dose of viral vector and one or more of the following shipping standards: 1.0 x 1 0 13 Less than about 0.09 ng benzonase per vg; less than about 30 μg / g (ppm) Cesium, approximately 20-80 ppm Poloxamer 188; 1.0 × 10 13 Approximately 0 per vg < .22ng BSA; 1.0 x 10 13 Approximately 6.8 × 10 per vg 5 Sub-picogram residues Plasmid DNA: 1.0 × 10 13 Approximately 1.1 × 10 per vg 5 Sub-pg hcD residue NA;1.0×10 13 Less than approximately 4 ng rHCP per vg; pH of approximately 7.7 to 8.3; Osmolarity of about 390-430 mOsm / kg; about 600 per vessel with a size of 25 μm or more Less than 1000 particles per container; Less than about 6000 particles of 10 μm or more in size; Approximately 1.7× 10 13 ~5.3×10 13 Genomic titer in vg / mL; 1.0 x 10 13 Approximately 3 per vg .9×10 8 ~8.4×10 10 Infectious titer in IU: 1.0 x 10 13 Approximately 100 per vg ~300μg total protein level; based on in vitro cell-based assay, reference standard and / or a relative potency of about 70-130% relative to an appropriate control; less than about 5% empty caps Sid.

[0144] The present disclosure is further illustrated by the following examples, which should not be construed as limiting. All references, patents, and published patent applications cited throughout this application, as well as figures, are incorporated herein by reference in their entirety. , the contents of which are incorporated by reference in their entireties for all purposes. EXAMPLES

[0145] Preclinical Examples SMNΔ7 mice are a suitable model for studying gene transfer. ch et al., “Abnormal motor phenotype in t he SMNΔ7 mouse model of spinal muscular atrophy.”Neurobiology of disease,27(2):2 07-19. Inject 5 × 10 of scAAV9.CB.SMN into the facial vein of 1-day-old mice. 11 pieces Injection of the viral genome rescues the SMNΔ7 mouse model. t al.,“Rescue of the spinal muscular atr. ophy phenotype in a mouse model by early postnatal delivery of SMN.”Nature biote chnology, 28(3):271-4. Approximately 42±2% of lumbar motoneurons were transduced in the 14-HT100 mice. SMN levels were similarly high. Furthermore, the brain, spinal cord, and muscle of animals injected with scAAV9.CB.SMN were significantly higher than those of untreated SMA mice. The expression of scAAV9 in P1 mice was increased compared to that in WT controls (although lower than in WT controls). Recovery of SMA animals treated with either CB.SMN or scAAV9.CB.GFP The ability of mice to absorb the IgG antibody was assessed and compared with that of wild-type (WT) control mice and untreated mice. The mice were able to recover quickly, but were treated with SMN and green fluorescent protein (GFP). SMA animals showed difficulty at P5, but by P13, 90% of SMN-treated animals , compared with 20% of GFP-treated controls and 0% of untreated SMA animals. At P18, SMN-treated animals were larger than GFP-treated animals, but not WT controls. The motor abilities of SMN-treated mice were evaluated in the open field test and wheel running test. Running assays were nearly identical to the WT control.

[0146] Survival of SMN-treated SMA animals was significantly improved compared to GFP-treated SMA animals GFP-treated control animals did not survive beyond P22, with a median lifespan of 15.5 days. The body weight of FP mice peaked at P10 and then declined rapidly until death, whereas the body weight of SMN mice Mice showed steady weight gain until around P40, when they stabilized at 17 g (approximately half the weight of WT controls). The smaller corrected animal size may be due to the tropism and incomplete expression of scAAV9. Transduction resulted in "chimeric" animals in which some cells were not transduced. Furthermore, the smaller size of SMN may explain its role in fetal development. Most strikingly, SMN-treated mice survived well beyond 250 days of age. was doing.

[0147] The toxicological biodistribution was also examined. In a non-clinical (non-GLP) study, 24 mice were We administered scAAV9.CB.SMN to four non-human primates (NHPs) via vascular delivery. To evaluate toxicity and safety, the vehicle (P BS) (3 males / 6 females) or 3.3 × 10 14 vg / kg scAAV9.C scAAV9.CB.SMN was transfected with P1 wild-type flanking vector B.SMN (6 males / 9 females). The dose was 1.2 mg / kg / day for SMA16 SMNΔ7 mice. The P1 mouse model has previously been shown to be the most effective. Simulate clinical trials in infants, the proposed target population for in-human clinical trials All mice survived the injection procedure without any signs of distress or weight loss. All animals survived the initial 24-hour observation period and were weighed and examined weekly for the remainder of the study. Field observations were performed; no differences were found between the control and treatment groups (Figure 1).

[0148] At 60, 90, and 180 days after injection, hematological and clinical chemistry evaluations (ALT, AST, ALT, and A Blood was collected from the mice for LK, Phos, creatinine, BUN, electrolytes, and CK. At 90 days, all but one variant was normal. This difference was due to This was likely due to a technical issue, as the blood collection site was different from all other mice. Thirteen mice were necropsied 120 days after injection and 8 mice were necropsied 180 days after injection for histopathological diagnosis. Mice were necropsied. All organs were normal; in particular, none of the organs (heart, liver, kidney) No inflammation was observed in any part of the body (spleen, muscle, gonads, brain, lungs, lymph nodes, or intestines). .

[0149] In a safety study of four male cynomolgus monkeys, subjects were injected at 90 days of age and developed type 1 SMA infants. The scAAV9.CB.SMN vector closely mimics the age at which infants are expected to receive treatment. 6.7 × 10 13 The drug was administered once at a dose of 1 / kg. This corresponds to the lowest dose tested at which SMN-Δ7 mice showed a significant increase in survival. The rats were observed for 6 months until they were killed at approximately 9 months of age. No side effects were observed. All clinical chemistries were normal. Peripheral blood mononuclear cells (PBMCs) were analyzed using ELISpot. Tests for T cell immune responses in the mice were all negative six months after injection.

[0150] In these non-GLP studies, serum chemistry and hematology were unremarkable, and histopathology The clinical evaluation was similar. NHP subjects demonstrated an appropriate immune response to the capsid (but (not shown for transgenes), and sustained very high transgene expression 6 months after injection These studies demonstrated that systemically delivered scAAV9.CB.SMN penetrates the blood-brain barrier. provides strong evidence that even the high doses used for penetration are safe and well tolerated. Ru. Foust et al.Nat.Biotechnol.,28(3),pp.2 71-274(2010).

[0151] Up to 3.3 × 10 14 At the vg / kg level, scAAV9. A single intravenous injection of CB.SMN administered on day 1 resulted in No test article-related mortality or evidence of toxicity was observed. Treatment-related mean body weight and mean body weight gain The decrease in thromboplastin time (APTT) and the decrease in activated partial thromboplastin time (APTT) were consistent with mild treatment. However, no toxicity was observed.

[0152] The activity of scAAV9.CB.SMN was evaluated in terms of biodistribution and its expression in the primary target tissues, the brain and This was demonstrated by the presence of specific transgene ribonucleic acid (RNA) expression in the spinal cord. 3. 3×10 14 vg / kg (Group 3) in males and females at 12 and 24 weeks Low levels of antibodies against the AAV9 capsid were observed. Clinical and histopathological analysis Based on these results, no changes were observed in the s The no observed adverse effect level (NOAEL) of cAAV9.CB.SMN was 3.3 × 10 14 In vg / kg It is considered to be.

[0153] In these studies, intrathecal administration of scAAV9.CB.SMN into the CSF significantly improved the survival of mice (1 The study was safe and well tolerated in patients with bronchitis (up to 2 weeks after injection) and in macaques (up to 14 months after injection). CSF delivery in mice reduced peripheral exposure of scAAV9.CB.SMN Qualitative polymerase chain reaction (qPCR) results may indicate expression of the transgene. The results show that the amount of Trendelenbuvir in the cervical and lumbar regions was higher than that in the thoracic region. The subjects were kept in a lumbar position for 5 min and confirmed to be seronegative for anti-AAV9 antibodies before injection. All non-human primates were highly positive for AAV9 antibodies up to 6 months after injection. Six months after injection, cells responded to the AAV9 capsid or SMN transgene. No cytotoxic T-lymphocyte responses were observed. No tissue degradation or reactive responses in the brain or spinal cord were observed. , was not observed.

[0154] In a central GLP-compliant 3-month mouse toxicity study, Following intravenous injection into mice, the vector and transgene were The gene is widely distributed, with the highest expression generally observed in the heart and liver, and significant expression in the brain and spinal cord. AVXS-101-related ventricular findings included dose-related inflammation, edema, and The liver showed signs of inflammation and thrombosis, including hepatocellular hypertrophy, Kupffer cells, and fibrosis. The AVXS-101-associated cardiac events in mice were characterized by hepatocyte activation and diffuse hepatocyte necrosis. No NOAEL was identified for organ and liver findings, and the maximum tolerated dose was 1.5 × 10 14 vg / kg, with a recommended therapeutic dose of 1.1 × 10 14 vg / kg, and This provides a 1.4-fold safety margin. Translatability of findings observed in mice to primates is not known at present.

[0155] These data support moving forward to clinical trials.

[0156] Whether CSF delivery can reduce transduction of peripheral organs compared to intravenous (IV) injection To determine whether the patient had a normal head-down Trendelenburg position for 5 or 10 minutes, A detailed biodistribution analysis was performed on human primate tissues (n=5). These animals were given a much improved brain distribution, making this approach favorable for clinical trials. were chosen in preference to non-human primates that did not lay head down. The animals were killed and various tissues were collected for detailed DNA and RNA characterization. Biodistribution analysis was performed. scAAV9.CBA.GFP was distributed in most tissues except for the spleen and liver. The levels were low in all peripheral tissues compared with the high levels in the brain and spinal cord. These findings are consistent with those of other groups. This is consistent with previous reports from Dirren et al., “Intrac erebrobeventricular injection of adeno-ass associated virus 6 and 9 vectors for cell t ype specific transgene expression in the spinal cord.”Hum Gene Ther 25:109-120;G ray et al., “Global CNS gene delivery and evasion of anti-AAV-neutralizing antibo dies by intrathecal AAV administration i n non-human primates.”Gene Ther 20:450-4 59. In skeletal muscle and the central nervous system, there is a strong correlation between DNA and RNA levels. On the other hand, in soft tissues and glands, the levels of RNA were not predictive of the viral genome detected. In particular, the testis, intestine, and spleen are 1.0 times lower than DNA. 00-fold fewer RNA molecules. Despite detection of AAV in peripheral organs, The amount of vector detected in the periphery was significantly reduced compared to that in the control group. al.; Gray et al. In addition, intravenous or intravenous Similar observations were made when comparing mice injected into CSF. Delivery adds an important potential safety component to future clinical trials with AVXS-101 do.

[0157] In some embodiments, the Trendelenburg position improves CSF delivery. A single injection of scAAV9-SMN delivered directly to the CSF in mice and non-human primates The researchers assessed the dose and efficacy of the drug. When administered at a dose 10 times lower than that administered intravenously, Diffuse transgene expression was observed throughout the spinal cord of mice and non-human primates. In mice, lower doses can be used to achieve similar motor neuron targeting efficiency than in humans. Maintaining the subject in the Trendelenburg position to enhance vector spread resulted in increased transduction It was found that the effect of introducing the stimuli was further improved. ing single injection CSF delivery of AAV 9-mediated gene therapy for SMA:a dose-r esponse study in mice and nonhuman primaries tes.”Molecular therapy:the journal of th e American Society of Gene Therapy 23,47 7-487. Immunofluorescence and GFP / ChAT double-positive motor neurons after tilting the animal Transduction of the thoracic and cervical regions of the spinal cord was significantly improved as demonstrated by quantification of A 10-min tilt increased the transduction of motor neurons by 55-59% in the cervical, thoracic, and lumbar regions, respectively. , 62, and 80%, which is comparable to the levels observed in mouse models. According to Skew, this could mean big benefits for patients. correlates closely with the quantification of GFP transcripts in each spinal cord segment.

[0158] Example 1 - Clinical Trial Protocol Phase 1 open-label, single-dose clinical trial: A randomized controlled trial of a patient with a genetic diagnosis consistent with SMA, biallelic SMN1 The patient had a deletion of the gene and three copies of SMN2 without any genetic modifiers and was randomly assigned to the locus at study enrollment. It is performed on infants and children who can stand but cannot stand or walk. Patients will be randomized to receive 100 mg of ... Patients will be administered AVXS-101. The patients will be in the group aged 6 months to under 24 months at the time of administration. The study will be stratified into two groups: those aged 24 months or older and under 60 months at the time of administration, and those aged 24 months or older and under 60 months at the time of administration. At least 15 patients aged 6 months to under 24 months were enrolled, and 24 months to over 60 months were enrolled. Twelve patients under the age of one month will be enrolled.

[0159] The first cohort was 6.0 × 10 13 vg of AVXS-101 (dose A) Three patients aged 6 months to 24 months (Cohort 1) will be enrolled. There will be an interval of at least 4 weeks between doses for each patient. Within 48 hours, grade III or higher AEs that may be related to the study drug All potential, probable or certain The Data Safety Monitoring Board (DSMB) will be consulted at the following address: Subsequently, based on the available safety data, a) the study will be discontinued due to toxicity or b) the study will be discontinued. Amount B is used to determine whether to proceed to cohort 2.

[0160] For dose B, 1.2 × 10 14 6 vg of AVXS-101 (dose B) Enroll 3 patients under 0 months of age. Again, between dosing of 3 patients in the cohort Three patients in cohort 2 and all patients in cohort 1 had at least a 4-week interval between the Available safety data suggests an additional 4-week interval between doses is needed between patients. Investigators must report Grade III or greater A events occurring within 48 hours. E: Possibly, probably, or definitely related to the investigational drug All will be discussed with the DSMB before continuing enrollment. Following enrollment of the first six patients, Based on the available safety data, patients were advised to either a) discontinue due to toxicity or b) discontinue treatment for 6 months. Twelve patients aged 24 months or older and younger than 24 months and 12 patients aged 24 months or older and younger than 60 months were randomly assigned to A decision will be made whether to continue enrolling 21 more patients until they receive dose B.

[0161] 1.2×10 14 Ongoing evaluation of safety and efficacy data from patients treated with the vg dose Based on the efficacy of the drug, a third dose (Dose C) will be considered for testing. Three patients aged 60 months or younger will be , up to 2.4 × 10 administered intrathecally 14 vg of dose C. As with 2, there was also a 4-week interval between doses for the first three patients receiving dose C. Following enrollment of the first three Dose C patients, and based on available safety data, a) discontinued due to toxicity or b) 12 patients aged 6 months to less than 24 months and 24 months An additional 21 patients were enrolled until 12 patients aged ≥60 months received dose C. Decide whether to continue recording.

[0162] The selection and justification of the appropriate dose to test dose C was performed using dose B (1.2 × 10 14 vg (supported by ongoing safety and efficacy review of clinical findings from patients receiving The dose selected is 2.4 x 10 delivered intrathecally. 14 Up to vg 1.1×10 14 Doses up to 1000 mg / kg have been shown to be safe for whole body administration in children weighing up to 8.4 kg. (intravenous) administered (total dose 9.24 × 10 14 vg). In addition, in preclinical trials is 2 x 10 13 scAAV9.CB.SM in large non-human primates at a dose of vg / Intrathecal administration of N was safe and well tolerated up to 14 months after injection.

[0163] The overall study design is summarized in Figure 2.

[0164] Safety was monitored by monitoring adverse event (AE) reports and concomitant medication use. by performing a physical examination, vital signs assessment, cardiovascular assessment, and laboratory evaluation Patients will be observed in the hospital for 48 hours after intrathecal injection. Patients returned for follow-up visits on days 21 and 30. After the 30-day visit, Dose administration will be followed up monthly for 12 months. Upon completion of the study, study patients will Patients are being asked to enroll in a pivotal long-term follow-up study examining the continued safety of -101 up to 15 years. It is requested.

[0165] Number of patients At least 27 patients will be enrolled; if escalation to Dose C is deemed necessary, A maximum of 51 patients may be enrolled.

[0166] Treatment Allocation This is an open-label, comparative, single-dose study. Treatment will be in the dose escalation schedule specified herein. Allocation will be done according to schedule.

[0167] Dose adjustment criteria This study will investigate a single intrathecal injection of AVXS-101.

[0168] Exam Completion Criteria An independent Data Safety Monitoring Board (DSMB) and medical monitors will continue to serve the trial throughout the trial. The DSMB will continue to monitor safety data periodically. The DSMB may recommend early termination of the study for safety reasons. If any patient develops clinical symptoms and requires medical treatment, Unexpected Grade III or higher A that may, probably, or definitely is related to Enrolment in the study will be halted by the investigator if toxicity is experienced. These include: , patient death, significant clinical laboratory findings, or serious localized injection site injury associated with administration of the investigational product. Complications include:

[0169] If the DSMB recommends early termination of the trial for safety reasons, the trial may be terminated. Also, trials may be terminated at the recommendation of regulatory authorities. Possibly, probably, or definitely related to the therapy; related to the clinical condition and / or unexpected CTCAE grade III or higher AEs / toxicities requiring treatment The study may be terminated if an unacceptable level of toxicity occurs, defined as .

[0170] Patient Inclusion Criteria Patients met all of the following inclusion criteria: 1. Ability to sit unassisted for more than 10 seconds, but unable to stand or walk; genetic At the time of administration after diagnosis during the screening period for fetal type, the age of 6 months to 60 months (1 Patients up to 800 days of age -Genotypic confirmation of diagnosis includes homozygous deletion of SMN1 exon 7; SMN2 Contains experimental evidence of exactly three copies of 2. Negative genetic testing for SMN2 gene modifier mutation (c.859G>C). 3. Onset of clinical signs and symptoms consistent with SMA before 12 months of age. 4. Able to sit independently, but unable to stand or walk independently. Able to sit independently is defined as the World Health Organization (WHO)-MGRS standard for head support without support. is defined as being able to sit still for at least 10 seconds. Children should be able to use their arms or hands to support themselves. should not be used for balancing or postural support (Wijnhoven 2004). . 5. Age-specific facility standards for the use of anesthesia and sedation as deemed necessary by the investigator The following conditions are met. 6. Up-to-date childhood vaccinations. According to the American Academy of Pediatrics (AAP 2009), According to the study, palivizumab prophylaxis (Sina Seasonal vaccinations, including against rabies (also known as rabies), are recommended. 7. Parent / legal guardian willing and able to complete the informed consent process.

[0171] Patient exclusion criteria Patients must not meet any of the following exclusion criteria: 1. Current or past ability to stand and walk independently. 2. Contraindications to spinal puncture procedures or intrathecal therapy (e.g., spina bifida, meningitis, disorders, or coagulation disorders) or obstructive spinal hardware that prevents effective access to the CSF space, or Presence of an implanted F-drainage shunt or an implanted CNS catheter. 3. Functional measures (e.g. , stand, walk) or interfere with the ability to receive IT administration; Severe contracture. Severe scoliosis (defined as a spinal curvature of 50 degrees or more) evident on radiography. (I). 4. Preceding, planned, or anticipated scoliosis within 1 year of dose administration Repair surgery / procedure. 5. Use of invasive ventilatory support (tracheotomy with positive pressure) or patient awareness at screening Pulse oximetry saturation less than 95% in the state or at altitudes of 1000 m or more. Patient is awake and has an oxygen saturation of less than 92% - Pulse oximetry saturation was greater than 4 percent between screening and peak on day of treatment It should not decrease by more than this point. 6.Has used or required non-invasive ventilatory support for 12 hours or more per day within the 2 weeks prior to treatment. is. 7. Gastric feeding tube where the majority of nutritional support is given via parenteral methods (i.e., nasogastric medical need for a tube or nasojejunal tube, or weight for age not in line with the WHO Child Gr. The 3rd percentile was calculated based on the first standard (Onis 2006). Patients with a permanent gastrostomy tube placed before screening were not excluded. No. 8. Active viral infection (human immunodeficiency virus (HIV); or hepatitis B or C or includes seropositivity for Zika virus). 9. Severe non-respiratory illness requiring systemic treatment and / or hospitalization within 2 weeks prior to study enrollment. 10. Have had any medical consultation, medical intervention, or other medical procedure within 4 weeks prior to enrollment in the study. Respiratory infections requiring increased supportive care. 11. Severe non-pulmonary / respiratory tract infection (e.g., pyelonephritis or meningeal ulcers) within 4 weeks prior to study dosing. or any combination of the following that the PI determines poses unnecessary risk to gene transfer: Symptoms: -Major renal or hepatic dysfunction - Known seizure disorder -diabetes mellitus -Idiopathic hypocalciuria - Symptomatic cardiomyopathy 12. History of bacterial meningitis or brain or spinal cord disease, including tumors, or LP procedures or C MRI or CT abnormalities that interfere with SF circulation. 13. Prednisolone or other glucocorticosteroids or their excipients Known allergies or hypersensitivity. 14. Known allergy or hypersensitivity to iodine or iodine-containing products. 15. Use of any medication for myopathy or neuropathy, or for the treatment of diabetes mellitus within 3 months of the study administration. Medications used in the treatment of rheumatoid arthritis or ongoing immunosuppressive therapy, plasma apheresis, adalimumab, etc. Concomitant use of any immunomodulatory or immunosuppressive therapy (e.g., corticosteroids, Cyclosporine, tacrolimus, methotrexate, cyclophosphamide, intravenous immunoglobulin Brin, rituximab). 16. Inability to refrain from use of laxatives or diuretics within 24 hours prior to dose administration. 17. Anti-AAV9 antibody titer of greater than 1:50 as measured by ELISA binding immunoassay - If a patient candidate demonstrates an anti-AAV9 antibody titer of >1:50, the patient will be considered for inclusion in the study at the end of the screening period. Retesting may be performed within 30 days, provided that the anti-AAV9 antibody titer is 1:50 or less. If you qualify, you will be eligible to participate. 18. Abnormal laboratory values ​​(INR>1.4) considered clinically significant before study administration, 3 GGT above XULN, bilirubin above 3.0 mg / dL, and bilirubin above 1.0 mg / dL Creatinine, less than 8 g / Dl or Hgb, more than 18 g / Dl, 20,000 / cmm (More than WBC). 19. Participation in a recent SMA treatment clinical trial or prior to screening for this study At the time of writing, any investigational or approved compound products or treatments administered with the intent to treat SMA Taking medications (e.g., valproic acid, nusinersen) Oral beta-agonists must be discontinued 30 days prior to dosing -Inhaled albuterol specifically prescribed for respiratory (bronchodilator) management is acceptable and is not contraindicated at any time prior to screening for this study. 20. Anticipation of major surgical procedures (e.g., spinal surgery or Tracheotomy). 21. Unable or unwilling to comply with study procedures or travel to follow-up appointments I can't. 22. Keeping research results / observations confidential or posting confidential testing on social media sites Unwillingness to refrain from posting results / observations. 23. Refusal to sign a consent form.

[0172] Patient Withdrawal Criteria and Discontinuation The patient has a clinical condition that is possibly, probably, or definitely related to gene replacement therapy. Unexpected CTCAE grade II associated with floor symptoms and / or requiring treatment Unacceptable levels of toxicity, defined as the occurrence of adverse events / toxicity of I or higher If a patient dies, the trial may be terminated. In that case, untreated patients will be removed. Autopsies will be required for patients who die after participating in a gene transfer study. Complying with all visits and study procedures required by the protocol on at least three consecutive occasions, except as required by law If the patient does not change his / her schedule or does not change his / her schedule, the patient may be withdrawn. Patients who withdraw will also be withdrawn from the study. Finally, patients may be withdrawn at the discretion of the investigator. Patients who discontinue the study prematurely for any reason may be terminated early. The order must be completed within 14 days.

[0173] Test Product Description The biological product is a cytomegalovirus (CMV) enhancer / chicken β-activator The cDNA of the human SMN gene was generated under the control of the CB-hybrid promoter. The AAV virus is a non-replicating, recombinant, self-complementary adeno-associated virus serotype 9 (AAV9). The inverted terminal repeats (ITRs) were modified to promote intramolecular annealing of the transgene. thus forming a double-stranded transgene ready for transcription. This modified ITR, called the (sc)ITR, regulates the transcription of the transgene and the resulting transcriptional AVXS-101 has been shown to significantly increase the rate at which proteins are produced. Cells transduced with scAAV9.CB.hSMN express human SMN protein do.

[0174] [Table 3]

[0175] Previous and concomitant medications The reference drug and concomitant medications must be recorded in electronic case report form (eCFR) from 2 weeks before the study administration until the end of the study visit. Record the results on the CRF.

[0176] Prophylactic prednisolone Antigen-specific T cell responses to AAV vectors were determined using AVXS-10 via i.v. This was observed in an ongoing Phase 1 clinical trial investigating a treatment that follows gene transfer,2 A single response to such an antigen-specific T cell response is expected within ~4 weeks. A possible consequence is the elimination of the transduced cells and loss of transgene expression.

[0177] In an attempt to suppress the host immune response to AAV-based therapies, AVXS-10 Patients were given prophylactic prednisolone (glucocorticoid) (approximately 1 mg / kg) 24 hours prior to administration of g / day) will be administered. Treatment will continue for approximately 30 days according to the following treatment guidelines: . - For at least 30 days after injection: 1 mg / kg / day Weeks 5 and 6: 0.5 mg / kg / day Weeks 7 and 8: 0.25 mg / kg / day Week 9: Prednisolone is discontinued.

[0178] Aspartate aminotransferase (AST) or alanine aminotransferase ALT levels >2x upper limit of normal (ULN) or after 30 days of treatment T cell response was 100SFC / 10 6 If PBMC or higher, AST and ALT values Maintain prednisolone dose until T cell responses are reduced below threshold after day 60 If prednisolone is to be continued, the investigator should consider the risks and benefits of continuing prednisolone. Variations from these recommendations should be based on the potential safety of each patient. At the investigator's discretion based on the issue.

[0179] Prohibited Substances The following drug combinations are prohibited: Drugs for treating myopathy or neuropathy Drugs used to treat diabetes mellitus • Therapies aimed at treating SMA (e.g., valproic acid, nusinersen). -Oral beta-agonists must be discontinued at least 30 days prior to gene therapy administration. -Inhaled beta-agonists are permitted only if such medications are administered at clinically appropriate levels. , may be used to treat respiratory complications of SMA. - Ongoing immunosuppressive therapy, plasma apheresis, or adalimumab within 3 months of the start of the clinical trial immunomodulatory agents such as cyclosporine, or immunosuppressive therapy (e.g., corticosteroids, cyclosporine , tacrolimus, methotrexate, cyclophosphamide, intravenous immunoglobulin, Ritz ximab)

[0180] Following completion of prednisolone tapering, the use of corticosteroids is a routine part of clinical management. The use of prednisone in this situation is permitted at the discretion of the managing physician. The drug should be appropriately documented and the event that triggered its use should be reported as an AE. should be appropriately documented.

[0181] As part of care during the tapering of prednisolone, corticosteroids (bronchial If inhaled corticosteroids (excluding inhaled corticosteroids for seizures) are being considered, this medical management should be The sponsor's medical monitor should be consulted regarding any recommendations regarding tapering. Responsible for any indicated medication adjustments.

[0182] Treatment Compliance AVXS-101 is administered as a single intrathecal injection.

[0183] Randomization and blinding This is an open-label study.

[0184] Dosage of test product and dose justification The patient had a 6.0 × 10 13 vg, 1.2×10 14 vg, or if deemed necessary , up to 2.4 × 10 as a third dose 14 vg of AVXS-101 was administered intrathecally. Direct delivery of viral vectors into the CSF via intrathecal injection delivers approximately 1 10-fold reduction in the amount of HIV-1-associated virions and their even distribution throughout the CNS allows for effective viral vector delivery. The burden on the patient is reduced and further optimization is possible. Selection of appropriate dose and any dose escalation trials are considered. The justification for the trial was based on clinical findings from previously treated patients, as described. This is further supported by ongoing safety and efficacy review of the maximum dose selected. 2.4 x 10 delivered intrathecally 14 vg. 1.1×10 14 vg / kg This dose has been safely administered systemically (intravenously) to children weighing up to 8.4 kg (total Amount 9.24×10 14 vg). In addition, in preclinical trials, 13 Dosage in vg / kg Intrathecal administration of scAAV9.CB.SMN in large non-human primates was safe and The injections were well tolerated up to 14 months after administration.

[0185] Preparation of test products Preparation of AVXS-101 is performed aseptically under sterile conditions by a pharmacist.

[0186] AVXS-101 is being investigated for radiographic monitoring of injection via lumbar intrathecal injection. Premix with an appropriate pediatric approved and labeled contrast agent. The total volume should not exceed 8 mL.

[0187] Dose delivery containers will be delivered to designated small pediatric intensive care unit (PICU) rooms or to acute care facilities. Other appropriate settings with immediate access to critical care (e.g., interventional rooms, operating rooms, specialized The patient is then delivered to the designated treatment room.

[0188] Patients should be placed in a PICU room or other appropriate facility with immediate access to acute critical care. The AVXS-101 intrathecal catheter was administered under sterile conditions in a suitable setting (e.g., intervention room, operating room, dedicated procedure room). Patients were admitted to the hospital and following AVXS-101 administration treatment, 15(+ / -5) days were administered. Vital signs will be monitored every 4 hours (+ / - 15 minutes) and every hour (+ / - 15 minutes) for 24 hours. - to do.

[0189] The institution was instructed to use an atraumatic needle inserted at a bevel parallel to the dural fibers; This significantly reduces damage to the dura mater and, as a result, reduces cerebrospinal fluid levels after lumbar puncture, including in children. It has been shown that the risk of leakage is reduced. Ebinger et al., “He adache and backache after lumbar punctur e in children and adolescents: a prospect ive study.”Pediatrics,113:1588-1592;Kiec hl-Kohlendorfer et al., “Cerebrospinal fl. uid leakage after lumbar puncture in neo nates:incidence and sonographic appearance ce.”Am J Roentgenol,181:231-234.

[0190] All patients receiving AVXS-101 will require sedation / anesthesia. The procedure and post-procedure Trendelenburg positioning are at the discretion of the local anesthesiologist. Incorporate a sufficient degree of sedative or anxiolytic to ensure analgesia and lack of movement. Patients should be placed in a 30 degree head down position for 15 minutes following vector administration. The subject is placed in Trendelenburg position to facilitate distribution to the cervical and brain regions.

[0191] AVXS-101 will be administered by investigators, interventional cardiologists, and clinical investigators in accordance with institutional guidelines. The procedure is performed by a neuroradiologist or other appropriately trained and experienced physician. It is administered under sterile conditions with fluoroscopic / radiographic guidance. The patient is placed in the lateral position and Lumbar puncture into the L4 or L4-L5 interspinous space was used to insert a catheter with a proboscis into the arachnoid membrane. Subarachnoid cannulation allows the clear cerebrospinal fluid (CSF) to escape from the catheter. ) flow. Approximately 4 mL of CSF was removed for doses A and B, and 1 mL for dose C. The volume of CSF removed was very close to the volume of AVXS-101 and contrast agent injected (maximum 7 mL). Dispose of according to your institution's guidelines. AVXS-101 in premixed contrast media Inject directly into the subarachnoid space. Inject with 0.5 mL of saline according to institutional standards / guidelines. It is permitted to flush the needle with saline.

[0192] Post-administration procedures Following administration of AVXS-101, patients will be admitted to a designated PICU bed or other appropriate setting. Patients should be placed on a strict daily schedule and their vital signs monitored closely. Concomitant medications and any AEs / serious AEs should also be monitored. The administration procedure will be followed and documented.

[0193] The patient was admitted to the PICU for 48 hours for closer monitoring of his / her mental status. other appropriate settings with immediate access to acute critical care (e.g., interventional wards, operating rooms, etc.). During the patient's stay, staff will follow appropriate infection control measures in accordance with institutional standards. Appropriate safety precautions must be followed; standards include gowns, gloves, masks, glasses, and gloves. Personal protective equipment (PPE), such as closed-toe shoes, is required. Monitor for changes in mental status, including monitoring for arousal, neck pain, photosensitivity, and vomiting. Standardized IRB-approved handouts regarding the subject will be provided if the following criteria are met: The patient may be discharged if: Afebrile No hypersensitivity reactions No meningitis - No abnormal laboratory test results suggesting possible CNS infection or complications

[0194] Dose escalation There was a 4-week interval between doses for all patients in cohort 1 before the next patient was dosed. Safety analysis results at six time points (1, 2, 7, 14, 21, and 30 days) to.

[0195] Investigators were required to report any grade III or higher AEs occurring within 48 hours of recognition. All those potentially, probably, or definitely related to the investigational drug. The DSMB will be consulted regarding this matter before continuing enrollment. Based on the available safety data following enrollment of the first three patients, or b) to proceed to cohort 2 using dose B.

[0196] For Dose B, during dosing of the first 3 patients younger than 60 months at the time of dosing within a cohort The first 3 patients in cohort 2 and all patients in cohort 1 were randomly assigned to receive the immunization Available safety data from patients suggests an additional 4-week gap between doses in patients. Investigators should consider Grade III events occurring within 48 hours. Among the above AEs, those that are possibly, probably, or definitely related to the study drug were All studies will be discussed with the DSMB before continuing enrollment. Enrollment of the first 6 patients Following enrollment, based on available safety data, a) the study will be discontinued due to toxicity, or b) ) 12 patients aged 6 months to under 24 months at the time of administration and 12 patients aged 24 months to 60 months at the time of administration Continue enrolling 21 more patients until 12 patients under 2 years of age have received Dose B Decide whether or not to do so.

[0197] 1.2×10 14 Ongoing evaluation of safety and efficacy data from patients treated with the vg dose Based on the efficacy of the drug, testing of a third dose (Dose C) may be considered. Patients were randomly assigned to receive up to 2.4 × 10 14 vg of dose C. As with 1 and 2, there was also a 4-week period between dosing of the first 3 patients receiving dose C. Based on available safety data, following enrollment of the first three Dose C patients, a) Dose C is discontinued due to safety concerns, or b) in animals aged 6 months to 24 months Twelve patients aged 12 months or younger and 12 patients aged 24 months or older but younger than 60 months will receive dose C. The study will determine whether to continue enrolling an additional 21 patients until the end of the study.

[0198] Physiotherapy assessment: Hammersmith Functional Movement Scale Extension Hammersmith Functional Motor Scale Extended has types II and III spinal muscular atrophy Designed for use in children to provide objective information on motor skills and clinical progression .

[0199] The Hammersmith Functional Movement Scale-Extended was administered by a physiotherapist according to Table 4 within 30 days after and monthly for all patients aged 24 months or older through month 12. Patients under 24 months of age will be assessed using the Hammersmith Functional Motor Scale once they reach 24 months of age. Begin the extended assessment. The Hammersmith Functional Movement Scale extended session will be videotaped. can be.

[0200] Physical Therapy Assessment: Bayley Scales of Infant Development(R) Bayley Scales of Infant and Toddler Dev elopment(registered trademark),Third Edition is a standard rdized, norm-referenced infant assessment Gross and fine motor subsets were completed within 30 days prior to baseline dosing. Then, monthly assessments are completed through month 12. Bayley Scales® assessments are videotaped. can be.

[0201] Physical Therapy Evaluation: Motor Milestone Development Survey Achievement of important motor milestones is tracked at each mile on the Bayley Scale® Using the standard motor milestone development survey shown in Table 2 along with the definition of tone, physical therapy The physical therapist will assess the patient (see the Physical Therapy Manual). The physical therapist will: Record whether the patient has achieved each milestone on the Motor Milestone Development Study. Once observed, an athletic milestone is considered achieved. The milestone achievement date is determined by the date of the visit at which the milestone was observed. During the training visit, the physical therapist completed an assessment of baseline milestone achievement according to Table 4. The assessment is videotaped and findings are documented. ) does not necessarily require a child to repeat milestones that he or she has previously achieved, Each milestone may be videotaped. Developmental milestone assessment sessions are conducted in a written format. It is written down.

[0202] [Table 4]

[0203] Video Evidence Physiotherapy evaluations at each study visit were convincing, as measured by change in functional ability. Videotaping as part of an effort to generate meaningful, demonstrable, documented evidence of effectiveness The parent / legal guardian will also provide the research facility with a home video that demonstrates achievement of functional skills. You may share.

[0204] Videos are reviewed independently and centrally for unbiased assessment of milestone achievement. Using the Motor Milestones Development Survey, an independent panel will evaluate the videos to determine Document whether evidence of athletic milestone achievement is provided. The milestone achievement date is calculated as the earliest video date on which the milestone achievement is demonstrated. will be done.

[0205] Additional Clinical Assessment: Demographics / Medical History Patient demographic and medical history information was collected at baseline and recorded on a case report form (CRF). Medical histories throughout the study will be collected at each visit. Spinal muscular atrophy, including but not limited to affected sibs or parental carriers family history; gestational age at birth; length / height / head circumference at birth; number, duration, and reasons for hospitalizations; hospitalization information from birth, including ICD-10 codes if available; previous ventilatory assistance; previous feeding assistance, if any.

[0206] Other Clinical Assessment: Vital Signs Vital signs included blood pressure, respiratory rate, pulse, and blood pressure at the time points specified in Table 4 within 30 days of administration. and axillary temperature. Vital signs, including pulse oximetry and heart rate, should be monitored. During the shoot, team members continuously monitored and recorded. Following dosing, subjects were administered 15 (+ / - 5) minutes for 4 hours, 1 hour (+ / - 15 minutes) for 2 hours, and 1 hour (+ / - 15 minutes) for 2 hours. Over a 4-hour period, blood pressure, respiratory rate, pulse rate, axillary temperature, pulse oximetry, and heart rate were monitored. Monitor vital signs to assess risk of heart failure.

[0207] Other clinical evaluations: weight and length / height If necessary, weight, length and / or height will be measured at the times specified in Table 4.

[0208] Other Clinical Evaluation: Physical Examination The physical examination includes a review of the following systems: head, eyes, ears, nose, and throat ( HEENT), pulmonary / thoracic, cardiovascular, abdominal, musculoskeletal, nervous, cutaneous, lymphatic, and secretory systems. Genitourinary. Head circumference is measured at each physical examination. To measure head circumference, the examiner must measure Measure your head with a flexible tape measure around the top, widest part of your forehead, above your ears, and at the most prominent part of the back of your head. Wrap tightly. Measurements should be taken three times and the maximum measurement recorded to within 0.1 cm. A baseline physical examination will be performed as specified in Table 4 within 30 days of treatment. Completed.

[0209] Other Clinical Evaluations: Vaccination Recommendations Patients should continue to attend all regularly scheduled appointments as recommended by the Centers for Disease Control and Prevention (CDC). Compliance with vaccinations is encouraged. According to the American Academy of Pediatrics (AAP 2009), Palivizumab prophylaxis (also known as Synagis) for preventing respiratory syncytial virus (RSV) infection Seasonal vaccinations, including those against the well-known rabies virus, are recommended.

[0210] Other clinical evaluations: 12-lead electrocardiogram (ECG) 12-lead ECGs were performed at screening / baseline, days 1, 2, 3, and 3 months. The study will be conducted at months 3, 6, 9, and 12 (or early termination). ECG tracings or ECG device data for centralized review by clinicians 12-lead ECGs were collected on the day of gene delivery and on the second and third days after gene delivery. Additional electrophysiological monitoring should be performed at the local facility. This is at the discretion of the investigator, in accordance with the institution's guidelines.

[0211] Other clinical evaluations: 12-lead Holter 24 hours prior to dosing on Day 1, patients will be fitted with a 12-lead continuous Holter monitor. The filter monitor will remain on for 48 hours (Day 3). Serial ECG data will be collected at the following times: Obtain luther monitor data three times: pre-dose, 2 hours, 4 hours, 6 hours, 8 hours, and 1 hour. 2 hours, 24 hours, 36 hours, and 48 hours. 24-hour Holter monitoring is At the time of leaning and at visits at 1, 2, 3, 6, 9, and 12 months (or early termination) .

[0212] Other clinical evaluations: Echocardiogram Echocardiograms were performed at screening / baseline and at 3, 6, and 9 months. The study will be performed at the first and 12-month visits (or early termination).

[0213] Other clinical evaluations: spine x-ray Patients with severe scoliosis who had a screening / baseline spine x-ray Patients will be excluded if they have a history of cerebrovascular disease or require major spinal surgery during the one-year study evaluation period.

[0214] Other Clinical Evaluation: Pulmonary Examination Patients were evaluated by a pulmonologist at the time points specified in Table 4 and were monitored by a pulmonologist and / or At the investigator's discretion, non-invasive positive pressure ventilation (e.g., BiPAP) may be administered. Patients who required non-invasive ventilatory support were asked to provide a written record of their actual use by the study staff. You will be asked to bring the device to each study visit so that the SD card can be removed. Data from the study will be transferred to a clinical database. Patients who require non-invasive ventilatory support will be admitted to the study. If you miss a session, you will be asked to remove the SD card and send it to a research facility.

[0215] Fluoroscopy / X-ray Guidance for AVXS-101 Injection The AVXS-101 intrathecal injection procedure was performed by the interventional cardiologist in accordance with the institution's guidelines. The fluoroscopy should be performed by a neuroradiologist or other appropriately trained and experienced physician. This procedure is performed under sterile conditions under visual inspection. The acquisition of x-ray images may not be necessary. .

[0216] Other clinical evaluations: Photographs of injection sites Photographs of the injection site were taken at the time points specified in Table 4 up to 30 days to monitor wound healing. - to do.

[0217] Other Clinical Evaluations: Laboratory Evaluations Biological samples will be collected throughout the study at the time points specified in Table 4. Samples will be collected and sent to the central laboratory. Laboratory samples on the day before dosing (day -1) was collected prior to administration and administered by the facility's Clinical Laboratory Improvement Actions (CLIA)-certified local laboratory. In some cases, samples are processed for immediate results or other safety or regulatory purposes. May be harvested locally for gistic concerns.

[0218] [Table 5]

[0219] If not enough blood is available from a patient, blood will be given first priority and then last priority. It has the lowest priority and is used in the following order of precedence: 1. Safety Hematology Lab: Chemistry > Hematology > Coagulation > CK-MB or Troponin 2. IFN-γ ELISpot to detect T cell responses 3. Serum antibodies against AAV9 and SMN 4. Genetic reconfirmation testing

[0220] If there is not enough blood volume at the screening visit to include a genetic reconfirmation sample, If so, patients return before Visit 2. All patients have had genetic reconfirmation testing completed.

[0221] Other Clinical Evaluations: Hematology Hematology analyses included CBC with differential and platelet count with smear. Samples were collected from a central The samples will be collected and sent according to the laboratory manual provided by the laboratory. Immediate / same-day hematology analysis of the current dose will be performed in a local laboratory according to standard site procedures. It will be carried out.

[0222] Other clinical evaluations: Serum chemistry Samples will be collected and sent according to the laboratory manual provided by the central laboratory.

[0223] Immediate / same-day chemistry analysis of medications administered during hospitalization as determined by the investigator will be performed at a local clinical laboratory. This will be carried out in accordance with the facility's standard procedures.

[0224] Chemistry analyses included the following at all study visits: serum gamma-glutamyltransferase; ferase (GGT), AST / ALT, serum total bilirubin, direct bilirubin, albumin Creatine, glucose, total creatine kinase, creatinine, BUN, electrolytes, alkaline phosphatase Fatase.

[0225] CK-MB or troponin I were measured at screening, days 7, 30, 60, and and at months 6, 9, and 12 / end of study. In new patients screened and enrolled after the implementation of IFN-Cal 6.0, Instead, troponin I was measured. Screening and registration were performed, but protocol revision 5 Those who have not yet received gene replacement therapy (visit 2) at the time (Protocol Version 6.0) came into effect Participants not yet diagnosed with idiopathic pulmonary disease should have a baseline troponin I test prior to treatment with AVXS-101. Troponin I was tested instead of CK-MB, which was the same as all other participants. The investigator will receive test results for all study visits from the central laboratory ( -Except for the 1st day).

[0226] Other clinical evaluations: Viral serology Administration of AAV vectors carries the risk of causing immune-mediated hepatitis. or for patients with hepatitis B or C or Zika virus seropositivity, Administration of an AAV vector may represent an unreasonable risk; therefore, prior to treatment Negative serological tests are confirmed by screening. These samples are provided by a central laboratory. Collect and send samples according to the provided experimental manual.

[0227] Other Clinical Evaluations: Coagulation Tests Coagulation tests include prothrombin time (PT), partial prothrombin time (PTT), and The INR was collected according to the test manual provided by the central laboratory. Coagulation testing will be performed according to the time points specified in Table 4.

[0228] Other Clinical Evaluations: Urinalysis Urine samples were collected according to the time points specified in Table 4 and the test manual provided by the central laboratory. Urine samples will be collected according to the guidelines set forth in the protocol. Day 1 and immediate / same-day urine samples will be collected during hospitalization as determined by the investigator. Urine testing will be performed in a local laboratory following standard site procedures. Meters included: Color, Clarity / Turbidity, pH, Specific Gravity, Glucose, Ketones, Nitrite, White Blood cell esterase, bilirubin, blood, protein, red blood cells, white blood cells, squamous epithelial cells, nitrite Subcrine casts, crystals, bacteria, yeast.

[0229] Other clinical evaluations: Capillary blood gas Capillary blood gases are completed according to the time points specified in Table 4. The device is used to stimulate the patient's skin layer in highly vascularized areas (heels, fingers, toes). A puncture or small incision is made in the vein to accelerate blood flow and reduce the difference in gas pressure between the artery and vein. Warm the area before puncturing. When blood flows freely from the puncture site, collect the sample into a capillary tube. Take.

[0230] Other clinical evaluations: ELISA: Anti-AAV9 antibody Blood samples were collected at screening and at the time points specified in Table 4 to determine whether the vaccine was effective against AAV9. Serum antibodies are collected according to the testing manual and sent to the central laboratory.

[0231] Other clinical evaluations: ELISA: Anti-SMN antibody Blood samples will be tested for serum antibodies to SMN according to the time points specified in Table 4. The samples are collected according to the testing manual and sent to the central laboratory.

[0232] Other clinical evaluations: IFN-γ ELISpot Blood was examined for T cell responses to AAV9 and SMN according to the time points specified in Table 4. A test to perform interferon gamma (IFN-γ) ELISpot to detect The samples will be collected according to the testing manual and sent to the central laboratory.

[0233] Other Clinical Assessments: Maternal Baseline Screening The mothers of the enrolled patients may have pre-existing antibodies to AAV9. It may be transferred to the patient in utero, across the placenta, or theoretically through breast milk. To screen mothers for circulating antibodies against AAV9, Informed consent is sought. Once informed consent is obtained, the embryo is transferred from the mother's peripheral vein. Blood will be drawn from each patient and sent to a central laboratory for screening for anti-AAV9 antibodies. If any breastfeeding is identified, the investigator will discuss with the mother whether to continue or discontinue breastfeeding. Ingestion of banked breast milk from donors who cannot be tested for anti-AAV9 antibodies Patients on feeding should be transitioned to formula feeding prior to enrollment.

[0234] Other Clinical Evaluations: Blood for Diagnostic Confirmatory Testing Blood samples were collected during the screening visit to determine SMN1 deletion, SMN2 copy number, and and to reconfirm the absence of exon 7 gene modifier mutations (c.859G>C), The diagnostic tests are sent to a central laboratory according to a manual to ensure consistency in the performance of the tests. This is carried out to

[0235] Other clinical evaluations: saliva, urine, and stool collections Studies have shown that some vectors can be excreted from the body for up to several weeks after injection; This is called "viral shedding." Vector shedding can occur in the blood for up to one week after injection. They can be found in urine, saliva and faeces. The risk associated with shed vectors is currently However, because the vector is non-infectious and cannot replicate, it is possible In any case, direct contact with the patient's body fluids and / or excretions for at least 2 weeks after injection is required. IRB-approved instructions regarding the use of protective gloves and good hand hygiene during contact are provided. Patients are also prohibited from donating blood for two years after vector injection. can be.

[0236] Saliva, urine, and feces samples were collected at 24 and 48 hours post-dose, including: Viral shedding tests will be collected according to the laboratory manual. Patients aged 48 months or older who stopped using the drug should be at least Provide a complete urine and complete stool sample for each void and defecation. Prepare the pool according to the experiment manual, store it in a -80 °C freezer, and then resuspend it in a cool, dark place. The samples of patients from facilities that have chosen to participate in the viral shedding substudy will be sent to the central laboratory. The subset consisted of 24-hour urine and fecal samples collected from 24 to 48 hours after dosing. Collect (including all excreta from these times).

[0237] Example 2 - AVXS-101 Study in SMA Patients (Clinical Trial Interim Results I) Patients were identified, treated, and evaluated according to the protocol described in Example 1. A patient with spinal muscular atrophy (SMA) is able to sit but is unable to stand or walk at the time of recording. The patient had a biallelic deletion of SMN1 and received intrathecal AVXS-101. In addition, the patient had three copies of the SMN2 gene. Two groups were administered: one under 24 months of age, and the other aged 24 to 60 months at the time of administration. Sixteen patients aged >6 months and <24 months, and 16 patients aged >24 months. Twelve patients aged 60 months or younger were enrolled. In the younger group, three patients had a 6.0× 10 13 The remaining young patients and all older patients were administered 100 mg of AVXS-101 (dose A). Patients received 1.2 × 10 14 vg of AVXS-101 (Dose B).

[0238] Patients were administered a single dose via lumbar intrathecal (IT) injection for radiographic monitoring. The subjects were administered 1.5 mL of AVXS-101 premixed with the appropriate contrast agent. Patients were given prophylactic prednisolone for the first 2 months after treatment to suppress the response. Safety and efficacy will be evaluated periodically for 12 months after treatment. In patients aged >6 months and <24 months, the efficacy measure was patients who achieved the ability to stand independently. (Bayley Scales of Infant Development®-Gross Motor Subset #40). Rolling from back to side, crawling, standing with support, standing with being pulled up, with or without assistance World Health Organization Multicenter Growth Standards Study Including Walking ganization Multicentre Growth Reference Defined according to the WHO-MGRS criteria (Wijnhoven 2004) Additional milestones that were assessed were also evaluated. In patients under 1 month of age, baseline Hammersmith Functional Motor Scale Extended (HFMSE) The outcome measure was change from baseline in HFMSE score of >3 points. The achievement of core goals (defined as achievement of core competencies; Swoboda, et al. 2010) was assessed monthly. .

[0239] Dose A(6.0×10 13 vg; n = 3) or Dose B (1.2 × 10 14 vg;n=1 3) After intrathecal administration of AVXS-101, mice aged 6 to 24 months with type II SMA were Patients between the ages of 5 and 12 months were evaluated. As shown in Table 6, The change in gross motor scale score was -1 to 14 points (mean increase + SD was 3.6 +3.5pt), and 14 of 16 (87.5%) patients had a baseline Seven of 16 patients had at least one new Bayley ( Two patients, one in each dose group, achieved the study endpoint of "f-independence." All patients achieved "stand on their own" (E02, E24); one patient (E24) achieved "stand on their own" before 20 months of age. He achieved this and is now walking independently.

[0240] [Table 6]

[0241] Patients between 2 and 5 years of age with Type II SMA received dose B of intrathecal AVXS-101. Then (1.2×10 14 vg; n = 12) were evaluated between 5 and 9 months. Thus, the change in Bayley® Gross Motor Scale score was -8 to 10 points ( The mean increase + SD ranged from 2.1 + 1.3 pts, with 9 of 12 (75%) patients Five of 12 patients (42%) showed improvement from baseline. At least one new Bayley® item was achieved in 2 patients (E07; E13). demonstrated the ability to stand with support after treatment. One patient (E07) was able to walk with assistance. It became possible to do so.

[0242] [Table 7]

[0243] After patients reached 2 years of age (6-24 months of age group) and for older patients (2-5 years of age group), The Hammersmith Functional Motor Scale Extended (HFMSE) was administered. Changes in HFMSE scores The mean increase + SD was 4.3 + 5.3 points, and 19 Twelve of the patients (63.1%) showed improvement from baseline. Older group (2-5 years) Seven of 12 patients (58%) in the younger group (6–10 years) showed improvement in HFMSE, whereas At age 24 months, 5 of 7 patients (71%) improved. One patient (E02) achieved the ability to stand without support. 63% were considered responders (achieved a ≥3-point improvement on the HFMSE) (Figure 3). No correlation was found between HFMSE scores and patient age at the time of treatment. Swoboda et al. (2010) “SMA CARNI-VAL Trial Part I:Double-Blind,Randomized,Placebo- Controlled Trial of L-Carnitine and Valp roic Acid in Spinal Muscular Atrophy,”PL OS ONE 5(8):e12140.

[0244] [Table 8]

[0245] Figure 3 shows the HFMSE scores of individual patients as a function of the patient's age. In patients in the 4-month age group, testing with HFMSE did not begin until they reached 24 months of age. Sixty-three percent of patients (12 of 19) showed improvement in HFMSE. 13 One patient in the vg) group showed an 8-point improvement after 8 months of treatment; the second patient of patients had a 2 point decrease after the 7-month evaluation.

[0246] Patients who achieved at least a 3-point improvement in HFMSE were identified as responders in this study. In the older cohort (2–5 years), HFMSE was performed on 12 patients at baseline. From the first to the fifth month of treatment, 10 and 5 patients were evaluated at the sixth and seventh months, respectively. In the younger cohort (ages 6 months to 2 years), one patient developed a pulmonary embolism after treatment with AVXS-101. 1 patient was evaluated at 3 and 4 months, and 5 patients were evaluated at 6 and 7 months. All patients in the cohort and patients in the younger cohort who reached age 2 years or older are shown in Figure 5. A rapid responder rate of 50% was seen after one month. The responder rate continued throughout the seven-month study. The responder rate remained above 50% and tended to increase over time.

[0247] For the entire cohort (n=12) from baseline to month 5 of treatment, HFMSE Monthly responder rates for the older cohort of patients (ages 2-5 years) evaluated are shown in Figure 6. After one month, a 50% responder rate was seen. Except for the sixth month after treatment, the responder rates were The response rate was maintained at 50% or higher throughout the 7-month study. One early responder was assessed at 6 months. The HFMSE declined and the responder rate dropped to 50% at this point.

[0248] Overall, 23 new cases were identified in 11 of 24 patients during the 4- to 12-month follow-up period. Motor milestones were observed (Tables 6-8). In the older cohort, mean HFMSE scores were A increased by 4.3 points during the 5- to 9-month study period (Table 8). The majority of patients (63%) showed improvement in HFMSE scores after treatment, regardless of dose. Fifty percent of patients in this study were clinically unresponsive after only 1 month of treatment. demonstrated meaningful HFMSE improvement (i.e., responders who scored >3 points); The responder rate gradually increased over time. Treatment with AVXS-101 was, for example, These results are more effective than those reported for other treatments, such as bronchodilator therapy. Demonstrated that a number of patients had early responses to a single dose of intrathecal AVXS-101 The rapid onset of response and the sustained efficacy of intrathecal AVXS-101 over the course of the study This indicates that the effect was maintained.

[0249] Example 3 - AVXS-101 Study in SMA Patients (Clinical Trial Interim Results II) Further interim results from the clinical trial detailed in Examples 1 and 2 are presented here. Able to sit for more than 10 seconds without lifting, but unable to stand or walk independently. AVXS-101 was administered intrathecally to patients with spinal muscular atrophy (SMA). In addition to the biallelic deletion of N1, the patient had three copies of the SMN2 gene. The groups were aged between 6 months and under 24 months at the time of administration, and aged between 24 months and over 60 months at the time of administration. All patients (aged 6 months or older) were stratified into two groups: For children under 18 months of age, baseline assessment was performed using the Bayley Scales (registered trademark) before treatment. For the age group aged 24 months to 60 months, the HFMSE was used to evaluate the Additional baseline assessments were performed.

[0250] Within these two age groups, three different treatment doses were administered as described: Three patients aged 6 months to 24 months at time of administration had a 6.0 × 10 13 AVXS- of vg Thirteen patients aged 6 months to 24 months received a single IT dose of 101 (Dose A). and 12 patients aged 24 months to 60 months, 1.2 × 10 14 AVXS in vg Three mice aged 6 months or older and under 24 months at the time of administration received a single IT dose of -101 (Dose B). In patients, 2.4 × 10 14 Received a single IT dose of AVXS-101 (Dose C) at 100 mg / kg In future studies, 21 additional patients will receive dose C, 9 of whom had a 6-month follow-up. The patients were from the age group ≥24 months and <24 months, and 12 were ≥24 months at the time of treatment. The patients were from the age group under 60 months.

[0251] The current study population was defined as all patients who received IT AVXS-101. Thirty-one patients from the intent-to-treat (ITT) set were also included, of which 19 patients 11 were between 6 and 24 months of age, and 12 were between 24 and 60 months of age. Four patients (three in Dose A and one in Dose B) were followed up for 12 months after treatment. Efficacy completer analysis set (ECA) All efficacy analyses were included in the ITT set as the primary population for interim outcomes. , was conducted using ECAS as a supportive population.

[0252] Data from patients treated with AVXS-101 are included in the Pediatric Neuromuscular Clinical Study (PNCR) A peer-reviewed and widely cited natural history dataset collected by the network We compared patient-level data extracted from Kaufmann et al., “P prospective cohort study of spinal musculature ar atrophy types 2 and 3.”(2012)Neurolog y,79(18):1889-1897. PNCR is an important expert in the management of SMA. The study was conducted at three internationally recognized large tertiary care centers (Harvard Medical School, University of Maryland, USA) with a total of 10,000 patients. university / Boston Children's Hospital,Col umbia university and university of pennsylvania ania / Children's Hospital of Philadelphia The study was developed from a cohort of 337 patients with any form of SMA who were followed in the This is a large-scale natural history study. The data include the Bayley Scales of Infant Development (BAI) ) is not included in the evaluation, so the use of PNCR data is limited to ≥ 6 months and ≥ 24 months of age. The SMN2 modifier mutations described by Prior et al. c.859G>C) was not evaluated in the PNCR study cohort. Prior et al. al.,“A positive modifier of spinal musc ular atrophy in the SMN2 gee.”(2009)AJ Hum. Genet.,85(3):408-441.

[0253] PNCR n=51 Natural history control group: For patients aged 6 months to 24 months, A cohort of 51 patients from the PNCR natural history study served as a “population-matched” control cohort. The comparison cohort included 51 patients enrolled in the PNCR study who met the following criteria: All patients with SMA type II or III and SMN2 (3) symptoms that began before 12 months of age; and (4) at least one copy before 36 months of age. Of this cohort, 7 / 51 (13.74%) patients lost the ability to stand independently. This is achieved by scoring on HFMSE item #19 at 36 months of age or any time before. Achieving a score of 2 was defined as achieving the ability to walk independently in 5 / 51 (10%) patients. This was done at 36 months of age or earlier, as determined by HFMSE item #20. Achieving a score of 2 was defined as

[0254] PNCR n=15 natural history control group: Patients aged 24 months to 60 months , patient-level data from a cohort of 15 patients drawn from the PNCR natural history study. The data were selected as a "population-matched" control cohort. This control group was included in the primary analysis. The natural history control group had: (1) SMA type II or III, 2) three copies of SMN2; (3) symptoms onset before 12 months of age; (4) symptoms onset before 24 months of age diagnosis of SMA, and (5) inability to stand or walk at the time of enrollment in the PNCR study. 24 Cohort members who underwent HFMS or HFMSE assessment between the ages of 60 months and 80 months were included in the follow-up This was used as a baseline for comparison of the evaluations. The PNCR group of 15 patients included baseline One patient had a documented HFMSE score of 0 at baseline and all follow-up visits. In 5 / 15 (33%) of individuals in the study, HFMSE scores improved over a 12-month period. The final visit was 18 months in 2 / 15 patients (13%) and 2 / 15 patients (16%). The median follow-up time was 42 months in 1 / 15 patients (7%) and 48 months in 1 / 15 patients (7%).

[0255] PNCR N=17 Natural history control group: Patients aged 24 months to 60 months To improve matching of the patient group with the natural history control group, a randomized controlled trial was drawn from the PNCR study. Patient-level data were identified from a cohort of 17 patients who were included in the study. This control group was used for sensitivity analyses. Twelve patients originally from the PNCR N=15 control group were included in the PNCR N=1 7 became the natural history control group. Three patients who were originally in the PNCR N=15 control group were not included. (1 patient with HFMSE=0 at baseline and follow-up visits, 1 patient with last visit >12 months These 17 individuals were age-, clinically, and genetically matched to the study group. The baseline age range was 24 months to 60 months. Subsequent visits within a 12-month interval were used to assess the baseline score of the HFMSE. Clinically, these individuals were able to sit but were not able to sit independently. Genetically, the patient had biallelic SMN. The study participants had a 1-fold deletion and 3 copies of SMN2. The limitation was that the assessment intervals were not consistent across participants. Some individuals had 12 months or less of data (see, for example, Table 13).

[0256] For all enrolled patients, the patient disposition by treatment and age is detailed in Table 9. Safety Summary demographic and baseline characteristics by treatment and age group for the gender analysis set are presented. Provided at 10.

[0257] [Table 9]

[0258] [Table 10]

[0259] [Table 11]

[0260] [Table 12]

[0261] Interim results: Interim evaluation of the primary efficacy endpoint over 6 months and less than 24 months (Dose A, B, and C; total n=19) The primary efficacy outcome measure for this age group was the Bayley Scales of Infant Development(R)-Gross Motor Subset item #40 was achievement of "standing for at least 3 seconds without support." If the milestone is achieved at any time during the two-month follow-up, the patient will be considered for this treatment. The milestone was deemed to have been achieved. A video of the laboratory evaluation of the milestone was released on Independent The results were confirmed by a central reviewer.

[0262] The primary efficacy results by dose for the ITT set are summarized below and in Table 11: Dose A(6.0×10 13 In the case of AVXS-101 in vg, 1 in 3 (33.3%) Patient 007-001 achieved supported standing 11 months after treatment. The patient was approximately 20 months of age when administered the drug. Participants achieved the following skills at the time of study enrollment: weight bearing (Bailey® #33) , Walking with Support (Bailey® #37), Walking Sideways with Support (Bailey® Climbing) Registered trademark #38).

[0263] Dose B (1.2×10 14 In the case of AVXS-101 in vg, 1 in 13 (7.7%) Patient 007-002 of the 4-year-old group achieved standing without support within 3 months of treatment. The patient was approximately 7 months of age when administered the drug. According to the investigator, the patient had No SMA manifestations were identified by biochemical testing. The patient was diagnosed at a young age by genetic testing and followed up with nerve conduction studies. The compound muscle action potential (CMAP) of the patient was abnormal.

[0264] Dose C(2.4×10 14 For the AVXS-101 study, evaluation was performed up to 12 months after treatment. None of the patients achieved the milestone of standing without support (0 out of 3) Table 11).

[0265] At dose B + dose C, 1 of 16 patients (6.3%) had a 007-002 (above) achieved the milestone of standing unsupported three months after treatment.

[0266] [Table 13]

[0267] Natural history pairs with type II and type III SMA from the PNCR N=51 dataset In the study, 7 of 51 patients (13.7%) achieved the milestone of standing without support. was achieved (see Table 11).

[0268] Statistical analysis was performed to compare the proportion of patients who achieved milestones (primary efficacy endpoints) between groups. Fisher's exact test for and Kaplan-Meier test for supportive efficacy outcomes. Analysis was performed according to protocol using the 12-month baseline visit. The primary efficacy endpoint of achieving the ability to stand independently at any time point is summarized in Table 11. Make a promise.

[0269] The time to achieve independent standing was measured for all patients in the PNCR group, as well as for all patients in the IT group. The data were summarized for each dose of the T-set. Cox proportional hazards analysis was performed using patient age at baseline as a covariate. When the treatment difference was evaluated using a standardized model, the hazard ratio (95% CI) was in group B, 0.43 (0.05, 3.93), in group C, 0 (0, not estimable), and in groups B + C. and 0.37 (0.04, 3.39), with p-values ​​of 0.4576 and 0.995, respectively. At the time of reporting the interim results, most of the patients were independent. The 25th percentile, median, and 75th percentiles have not achieved the milestone of standing up. Values ​​such as centiles could not be calculated.

[0270] Interim results: Interim evaluation of the primary efficacy endpoint in the 24 months to less than 60 months age group (Dose B; Total n=12) a. Primary efficacy analysis using PNCR N=15 natural history control group The primary efficacy outcome measure for this age group was change from baseline in HFMSE at 12 months. The baseline, post-baseline, and post-baseline HFMSE values ​​were Changes will be summarized and analyzed using the ITT set. PNCR N=15 natural history control group: To be used as the primary "population-matched" control cohort for protocol-specified analyses.

[0271] Individuals treated with AVXS-101 at dose B and PNCR N=15 natural history controls Changes from baseline in HFMSE scores to 12 months in The plot is shown in Figure 7. Descriptive statistics for treated patients and controls are shown in Table 12.

[0272] In PNCR N=15 natural history controls, baseline HFMSE scores ± standard deviation ( The mean mean HFMSE score was 11.8 ± 7.34. The change from baseline was -0.6±1.35 at 2 months and 0.4±0.9 at 4 months. 8), 6 months (0.2±1.72), 9 months (1.0±2.16), 12 months (0 The calculated mean ± SEM image was 0.8±2.86.

[0273] In the AVXS-101 Dose B treatment group, baseline HFMSE values ​​were 14.8 ± 9.98 Most treated patients had HFMSE data for up to 8 months (11 HFMSE scores from baseline at months 2, 4, 6, 9, and 12 The changes in a were 3.5±4.38, 3.6±5.07, 3.9±5.85, and 5. The mean mean ± SD was 7 ± 6.72 and 7 ± 6.72. The dose B treatment group was compared with a PNCR N=15 natural history control group. showed a strong increase in HFMSE scores in comparison.

[0274] [Table 14]

[0275] [Table 15]

[0276] b. Sensitivity analysis using PNCR N=17 Natural history control group Descriptive statistics and spaghetti plots for dose B and PNCR N=17 natural history controls are shown in Table 1 3 and 8.

[0277] In the PNCRN=17 natural history control group, baseline HFMSE score was 12.1 ± 9 The mean change from baseline in HFMSE scores at month 2 (-0.21) was 0.01; .2±1.56), 4th month (0.5±1.05), 6th month (-0.4±5.32), The calculated values ​​were 1.1±2.03 at 9 months and -0.2±8.11 at 12 months. Forty-one percent (7 / 17) of PNCR patients had no 12-month HFMSE score.

[0278] The AVXS-101 Dose B treatment group had a baseline HFMSE score of 14.8 ± 9.98. Mean HFM from baseline at months 2, 4, 6, 9, and 12 The changes in SE scores were 3.5±4.38, 3.6±5.07, and 3.9±5.8, respectively. 5, 5.7±6.72, and 7.

[0279] Dose B treatment group had a stronger HFMSE score than the PNCR N=17 natural history control group It showed a strong increase.

[0280] [Table 16]

[0281] [Table 17]

[0282] Interim Outcome: Secondary Efficacy Endpoints - Motor Milestones, walking at least 5 steps independently Secondary efficacy endpoints were the age groups 6 months to under 24 months and 24 months to 60 months. The Bayley Scales of Infant Development (R)-Gross Motor Score was used for both the pre- and post-month age groups. The milestone was set as #43 ("walked 5 or more steps independently"). The first milestone assessment was performed at all study visits up to 12 months after treatment. The video evidence was reviewed and verified by an independent central reviewer.

[0283] For patients aged 6 months or older but less than 24 months at the time of administration, dose B (1.2 × 10 14 vg) One treated patient (007-002) walked unassisted by the 4-month visit. (See patient description above.) The proportion of patients who achieved the ability to walk unassisted was Amount A(6.0×10 13 vg), 0% (0 / 3), Dose B (1.2 × 10 14 vg) 7 .7% (1 / 13), and dose C (2.4 × 10 14 vg), the rate was 0% (0 / 3). The PNCR N=51 natural history control group was used for this analysis. Five of the patients (9.8%) walked independently at baseline. None of the patients in the control group walked independently.

[0284] For patients aged 24 months or older and younger than 60 months at the time of dosing, all patients were administered dose B (1.2 × 1 0 14 vg). No patients in this age group received dose C. None of the treated patients walked independently. Primary PNCR N=15 Natural history control group No patients walked independently in the sensitivity PNCR N=17 or natural history control group.

[0285] Interim Results: Exploratory Efficacy Outcomes - Bayley Scales of Infant Development® Assessment In the age groups 6 months to under 24 months and 24 months to under 60 months, Details of the Bayley Scales of Infant Development, Third Edition (Bayley III) Changes from baseline in motor and gross motor components were assessed. In the <2 months age group, the second exploratory endpoint was to continue the study beyond 24 months of age. Baseline in patients with at least 6 months of recorded post-baseline HFMSE assessments The change in HFMSE from the Bayley scale ( Only descriptive statistics for patients younger than 24 months of age are provided because ANOVA was not evaluated. .

[0286] People with Type I SMA have severe fine motor impairments that prevent infants from grasping with their whole hand. However, in SMA types II and III, the Bayley® test for fine motor development As reflected in the scores, fine motor function is relatively well preserved. ctis et al., “Developmental milestones in type I spinal muscular atrophy.”(2016)N euromuscul.Disord.26(11):754-759;Chabano n et al., “Prospective and longitudinal n natural history study of patients with Ty pe 2 and 3 spinal muscular atrophy:Basel ine data NatHis-SMA study.”(2018)PLoS ON E,13(7):e0201004. In SMA types II and III, gross motor symptoms are Proximal muscle dysfunction was associated with distal muscle dysfunction as reflected by the subjects' Bayley® scores. is significantly greater than

[0287] a. Patients aged 6 months or older and under 24 months at the time of administration Dose A(6.0×10 13 vg): All three patients in this group were The evaluation period was completed. The baseline Bayley Scales® at 12 months The change from baseline was 12.3 ± 6.51 for the fine motor subtest and 5.0 ± 6.51 for the gross motor subtest. .7±1.15.

[0288] Dose B (1.2×10 14 vg): Change from baseline in fine motor subtests was At 6 months, data were available for all 13 patients (5.4 ± 3.57). Data available were incomplete for: 7 months (n=11; 7.8±3.03); , 8th month (n=10; 7.4±3.60), 9th month (n=6; 8.2±3.25), At 10 months (n=3; 11.7±3.06), at 11 months (n=2; 12.5±4.95 At 12 months, one patient had a change from baseline of 16.0. As predicted by the study, fine motor skills continued to improve in these patients. anon et al., “Prospective and longitudina l natural history study of patients with Type 2 and 3 spinal muscular atrophy:Ba seline data NatHis-SMA study.”(2018)PLoS ONE.13(7):e0201004.

[0289] Changes from baseline in gross motor subtests were available for all 13 patients at 6 months. The available data for the following months were incomplete. The results were: 7 months (n=12; 4.7±4.29), 8 months (n=10; 4.9±6 .45), 9th month (n=6; 3.5±2.07), and 10th month (n=3; 5.7±4. 73), 11 months (n=2; 8.0±4.24), and 12 months (n=1; 11.0 ). Patients continued to achieve gross motor milestones. Patients who lost milestones There was no one.

[0290] Dose C(2.4×10 14 vg): Change from baseline in fine motor subtests Only limited data were available for the second month (n=3; 0.7±0.58), the third month (n=3; 0.7±0.58), and the third month (n=3; 0.7±0.58). At month 4 (n=2; 3.5±0.71), the change from baseline was 6.0 Changes from baseline in gross motor subtests were available through Month 4. At 2 months (n=3; 0.3±1.53), at 3 months (n=2; 0.5±3.5 4), and at 4 months (n=1; 4.0).

[0291] Dose B + Dose C: Bayley Scale® scores at 12 months for Dose B + Dose C Spaghetti plots of the change from baseline in fine motor control and coarse motor control are shown in Figure 9 and Figure 10. Descriptive statistics for the Bayley Scales® are shown in Table 14.

[0292] [Table 18]

[0293] b. Patients aged 4 months to 60 months at the time of administration Age groups aged 24 months or older and younger than 60 months were treated with dose B (1.2 × 1014 vg) The study consisted of 12 patients who received the treatment. Increases in fine and gross motor subsets were observed. Changes from baseline in fine motor subtests were statistically significant in all 12 patients at 6 months. For the following months, available data were Incomplete: 7th month (n=11, 6.6±5.33), 8th month (n=11, 8. 0±5.74), 9th month (n=10, 7.9±5.53), and 10th month (n=2, 10.5±0.71). At 11 months (n=1) and 12 months (n=1), the mean age was 9.5±0.71. One patient had data for scores of .0 and 10.0.

[0294] In the gross motor subset, change from baseline was 0.01 in all 12 patients at 6 months. The mean age of the 19-month follow-up was 1.8 ± 4.47. For the following months, limited data were available. Complete: 7 months (n=11; 2.0±4.36), 8 months (n=11; 2.3 ±4.47), 9th month (n=10; 2.4±5.08), 10th month (n=2; 5.5 ±6.36). No patients missed the Bayley® gross motor milestones. .

[0295] Change from baseline in the Bayley Scale® to Month 12 of Dose B The spaghetti plots of the changes are shown in Figures 11 and 12. The curves for patients 008-003 are incorrect. Patient 008-003 had a baseline score of 20 instead of 28 (maximum Therefore, gross motor scores between baseline and month 1 were The change in gross motor function of patient 008-003 was "0" rather than "-8". The change in ability scores from baseline measurements was "0" in the second and third months, and "0" in the fourth month. "+1", 5th and 6th months are "0", 7th to 11th months are "+1", 12th month was "+2".

[0296] These interim data are from the clinical trial described in Example 1 at 12 months post-treatment. The efficacy results from the study are summarized in Table 15. Descriptive statistics for the Bayley Scales® As shown in.

[0297] [Table 19]

[0298] Interim results: Patients aged 6 months to under 24 months who will continue the study beyond 24 months of age Change in HFMSE score HFMSE scores were significantly higher among patients in the 6 months to 24 months age group than among those who reached 24 months of age. Pretreatment baseline data were not available for any patient, so H The first recorded FMSE is defined as the baseline. The months specified below are the test months. This study was conducted on the first recording of HFMSE at age 24 months or older, not on the first recording of HFMSE at age 24 months or older.

[0299] Dose A(6.0×10 13 vg): Two patients reached 24 months of age. Changes from the first recording are shown: 1 month (n=2; -0.5±4.95), 2 months (n= 2; 4.0 ± 0.00), 3 months (n = 2; 3.5 ± 0.71), and 4 months (n = 2; 3.0±2.83), 5th month (n=1; 5.0), and 6th month (n=2; 2.0±5 .66).

[0300] Dose B (1.2×10 14vg): Eight patients reached 24 months of age. Changes from initial recordings are shown: 1 month (n=7, 2.0±2.83), 2 months (n=7 , 2.7±2.69), 3 months (n=6, 1.3±4.97), 4 months (n=3, 4 .7±4.51), and at 5 months (n=2, 7.5±0.71).

[0301] Spatial correlation coefficient (SCI) of change from baseline in HFMSE scores to Month 12 of Dose B The t-test plot is shown in Figure 13. As shown in Table 16, for Dose B, the t-test plot was The maximum change from baseline in HFMSE score (mean) at any visit up to month 1 The mean ± SD was 17.7 ± 5.28 (n = 7).

[0302] Dose C(2.4×10 14 vg): One patient was ≥24 months of age and had a first HFMSE A record was reached. Only this single "baseline" data point was available.

[0303] [Table 20]

[0304] Interim conclusion The clinical trials described herein were conducted in children aged 6 months or older who were diagnosed with spinal muscular atrophy (SMA). An ongoing Phase 1, open-label, single-dose intrathecal study in infants and children less than 0 months of age The data obtained so far on treated patients are as follows: The progress of the study, including skill improvement, milestone advancement, and disease stabilization, is described in the abstract. The study showed clinically meaningful changes in motor function.

[0305] Ages 6 months to under 24 months Nineteen patients aged 6 months to 24 months were enrolled in the clinical trial. Three patients had a 6. 0×10 13 A single dose of AVXS-101 (Dose A) at 1.2 × 1 vg in 13 patients 0 14 vg of AVXS-101 (Dose B), 2.4 × 10 14 v Three patients in dose group A and three in dose group B were given a single dose of AVXS-101 (dose C). Four patients, including one patient, completed the 12-month evaluation after treatment.

[0306] The primary efficacy outcome measure for this age group was the Bayley Scales of Infant Development(R)-Gross Motor Subset #40 was achievement of "standing unsupported for at least 3 seconds." Two patients The primary efficacy endpoint was met. Patient 007-001, who received Dose A, achieved At month 1, patients achieved unsupported standing for at least 3 seconds. 7-002 achieved unaided standing 3 months after treatment.

[0307] Secondary efficacy outcomes were the Bayley Scales of Infant Development (R)-Gross Motor Subset #43 ("walked ≥5 steps independently"). One patient who received dose B (0 07-002) walked at least five steps unassisted four months after treatment.

[0308] Exploratory outcomes were assessed using the Bayley Scales of Infant Development, Third Edition (Bayley The changes from baseline in fine and gross motor components of the P The Bayley Scales® were not evaluated in the NCR dataset, so 24 Only descriptive statistics for patients under 1 month of age are provided. However, patients achieved gross motor milestones. No patients lost any milestones.

[0309] Ages 24 months to 60 months Twelve patients aged 24 months to 60 months were enrolled in the clinical trial and given dose B. No patients in this age group received dose C. One patient died at 12 months of treatment. Post-evaluation was completed.

[0310] The primary efficacy outcome measure for this age group was change from baseline in HFMSE. To put the changes observed in dose B into context, the improvement of ≥3 points in HFMSE score was Goods are those that are considered meaningful and important to stakeholders, such as caregivers and clinicians, and that are relevant to clinical trials. It is used as a threshold for detecting meaningful changes in clinical trials. Mercuri et al. al., “Nusinersen versus sham control in later-onset spinal muscular atrophy.”NE ngl J Med.378(7):625-635. Dose B treatment group: PNCRN=1 5 showed a stronger increase in HFMSE scores than the natural history control group. In the natural control group, the maximum change in HFMSE score of 1.0 ± 2.16 was observed at 9 months (n = 7 ) was observed when a sensitivity analysis was performed using the natural history control group of PNCRN=17. Similar results were observed in , with the maximum change in HFMSE score at 9 months (n=8) being 1. The mean mean ± S.D. value was 1±2.03.

[0311] The dose B treatment group showed a clinically significant change in HFMSE score of 5.7 ± 6.72 at 9 months. A significant increase was observed (n=10).

[0312] Exploratory endpoints were the fine and gross motor components of the Bayley-III. Changes from baseline were consistent. Similar to younger populations, patients achieved gross motor milestones. No patients lost any milestones.

Claims

1. A composition for use in treating spinal muscular atrophy (SMA) in a patient in need of treatment, the composition comprising an AAV9 viral vector comprising a polynucleotide encoding a survival motor neuron (SMN) protein, the patient being 2 years of age or older, and the AAV9 viral vector being formulated for intrathecal administration at a dose of about 5 x 10 13 vg to 3 x 10 14 vg.

2. The composition described in claim 1, wherein the AAV9 viral vector 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 an unmodified AAV2 ITR.

3. The composition described in claim 1 or 2, wherein the SMN protein comprises the amino acid sequence of SEQ ID NO: 2 and / or the AAV9 viral vector comprises the nucleic acid sequence of SEQ ID NO:

1.

4. A composition described in any one of claims 1 to 3, wherein the AAV9 viral vector comprises a capsid comprising the amino acid sequence of SEQ ID NO:

3.

5. A composition described in any one of claims 1 to 4, wherein the composition further comprises a pharmaceutically acceptable carrier suitable for intrathecal administration.

6. A composition described in any one of claims 1 to 5, wherein the composition further contains a contrast agent.

7. A composition described in any one of claims 1 to 6, wherein the contrast agent comprises iohexol.

8. A composition described in any one of claims 1 to 7, wherein the composition is in a container and comprises at least one of the following characteristics a) to s): a) pH of approximately 7.7 to 8.3; b) Osmolality is approximately 390-430 mOsm / kg; c) less than approximately 600 particles 25 μm or larger per container; d) less than approximately 6,000 particles 10 μm or larger per container; e) genome titer of approximately 1.7×10 13 to 5.3×10 13 vg / mL; f) an infectious titer of approximately 3.9×10 8 to 8.4×10 10 IU per 1.0×10 13 vg; g) total protein approximately 100-300 μg per 1.0 × 10 13 vg; h) Pluronic F-68 (Poloxamer 188) content of approximately 20-80 ppm; i) a relative potency of about 70-130% relative to a reference standard and / or appropriate control based on an in vitro cell-based assay; j) titer with a median survival time of 24 days or more at a dose of 7.5 x 1013 vg / kg in the SMNΔ7 mouse model; k) empty capsid content less than about 5%; l) total purity of approximately 95% or more; m) endotoxin content of approximately 0.13 EU / mL or less; n) benzonase content less than approximately 0.09 ng per 1.0 × 10 13 vg; o) Cesium content less than approximately 30 μg / g (ppm); p) less than approximately 0.22 ng of bovine serum albumin (BSA) per 1.0 × 10 13 vg; q) less than approximately 6.8 × 10 5 pg of residual plasmid DNA per 1.0 × 10 13 vg; r) less than approximately 1.1 × 10 5 pg of residual hcDNA per 1.0 × 10 13 vg, and s) Less than approximately 4 ng of rHCP per 1.0 × 10 13 vg.

9. A composition described in any one of claims 1 to 8, wherein the SMA is type II or type III SMA.

10. A composition described in any one of claims 1 to 9, wherein the patient is between 24 months and 60 months of age at the time of administration of the composition.

11. A composition described in any one of claims 1 to 10, wherein the patient is in at least one of the following conditions a) to z) before or at the time of administration of the composition. a) have biallelic null mutations or inactivating deletions of SMN1; b) SMN1 has a deletion in exon 7, c) have three or more copies of SMN2; d) absence of the c.859G>C substitution in exon 7 of at least one copy of the SMN2 gene; e) disease onset before approximately 12 months of age; f) able to sit unaided for at least about 10 seconds, but unable to stand or walk, as defined by the World Health Organization Multicenter Growth Reference Study (WHO-MGRS), at the time of administration of the composition; g) having one or more of the following: a gamma-glutamyltransferase level less than about three times the upper limit of normal; a bilirubin level less than about 3.0 mg / dL; a creatinine level less than about 1.0 mg / dL; a hemoglobin level between about 8 and 18 g / dL; and / or a white blood cell count less than about 20,000 / mm 3 ; h) a platelet count greater than about 67,000 cells / ml, greater than about 100,000 cells / ml, or greater than about 150,000 cells / ml; i) normal liver function; j) liver transaminase levels of approximately 8 to less than 40 U / L; k) Anti-AAV9 antibody titers by ELISA (enzyme-linked immunosorbent assay) of 1:25, 1:50, 1:75, or 1:100 or less; l) X-rays do not reveal severe scoliosis (defined as a curvature of the spine greater than 50 degrees); m) There are no contraindications to spinal tap or intraspinal therapy; n) No previous scoliosis repair surgery or procedure; o) does not require the use of invasive ventilatory support; p) No experience of standing or walking independently; q) Not using a gastric feeding tube; r) no active viral infection; s) have not had a severe non-pulmonary and / or respiratory infection within the past four weeks; t) no comorbidities, significant renal or hepatic dysfunction, known seizure disorder, diabetes, idiopathic hypocalciuria, or symptomatic cardiomyopathy; u) no history of bacterial meningitis, brain disease, or spinal cord disease; v) no known allergies or hypersensitivities to prednisolone or other glucocorticosteroids or excipients; w) no known allergies or hypersensitivities to iodine or iodine-containing products; x) not taking medication for muscle or neuropathy; y) not receiving or receiving immunosuppressive therapy, plasma exchange therapy, immunomodulatory drugs, or adalimumab within the past 3 months; and z) Not receiving any investigational or approved compound products or treatments intended to treat SMA.

12. A composition described in any one of claims 1 to 11, wherein the patient is placed in the Trendelenburg position during and / or after administration of the composition.

13. A composition described in any one of claims 1 to 12, wherein the composition is administered at least about 1 to 48 hours after administration of an oral steroid, and the patient is administered the oral steroid under at least one of the following conditions: a) to g) a) administered at a dose of about 1 mg / kg; b) administered once or twice daily; c) administered at a dose of approximately 1 mg / kg, then tapered to 0.5 mg / kg / day over 2 weeks, and then tapered to 0.25 mg / kg / day over another 2 weeks; d) administered for at least about 10-60 days; e) administered until aspartate transaminase (AST) and / or alanine aminotransferase (ALT) levels are less than twice the upper limit of normal or less than about 120 IU / L for more than 30 days; f) administered for more than 30 days and until the T cell response in a blood sample from the patient is below 100 spot-forming cells (SFC) per 10 6 peripheral blood mononuclear cells (PBMC); and g) administered until the patient's anti-AAV9 antibody titer, as measured by ELISA, falls below 1:25, 1:50, 1:75, or 1:

100.

14. The composition of claim 13, wherein the oral steroid is prednisolone or an equivalent.

15. A composition described in any one of claims 1 to 14, wherein the composition is administered simultaneously or sequentially with a second therapeutic agent.

16. The composition described in claim 15, wherein the second therapeutic agent comprises an antisense oligonucleotide targeting SMN1 and / or SMN2, a muscle-building agent, and / or a neuroprotective agent.

17. A composition described in any one of claims 1 to 16, wherein the patient after administration of the composition is in at least one of the following conditions a) to g): a) No radiographic evidence of severe scoliosis (defined as a spinal curvature of 50 degrees or greater); b) have not undergone scoliosis repair surgery or procedures within the past 6 months to 3 years; c) does not require the use of non-invasive ventilatory support; d) not using a gastric feeding tube; e) Anti-AAV9 antibody titers measured by ELISA are ≥ 1:25, 1:50, 1:75, or 1:100 and are monitored for approximately 1-8 weeks or until the titer decreases to < 1:25, 1:50, 1:75, or 1:100; f) a platelet count of less than about 67,000 cells / ml, less than about 100,000 cells / ml, or less than about 150,000 cells / ml and being monitored for about 1 to 8 weeks or until the platelet count reaches about 67,000 cells / ml, about 100,000 cells / ml or more, or about 150,000 cells / ml or more; and g) Platelet count is less than approximately 67,000 / ml and is being treated with platelet transfusions.

18. A composition according to any one of claims 1 to 17, wherein the composition is effective in improving the patient's score on the Hammersmith Functional Motor Scale-Expanded in relation to their pre-administration score, and / or the patient achieves an improved score on the Bayley Scales of Infant and Toddler Development (registered trademark) in relation to their pre-administration score.

19. The composition according to any one of claims 1 to 18, wherein the AAV9 viral vector is formulated for administration at a dose of 6 x 10 13 vg to 2.4 x 10 14 vg.

20. The composition according to any one of claims 1 to 9, wherein the composition is used so that the AAV9 viral vector is administered at a dose of about 1.2 x 10 14 vg.

21. The composition according to any one of claims 1 to 19, wherein the composition is used so that the AAV9 viral vector is administered at a dose of about 2.4 x 10 14 vg.

22. A composition described in any one of claims 18 to 21, wherein the patient achieves at least one of the following a) to e) 1 to 24 months after administration of the composition: a) Ability to stand unsupported for at least approximately 3 seconds as defined by the Bayley Scales of Infant and Toddler Development®; b) Ability to walk unassisted as defined by the Bayley Scales of Infant and Toddler Development®; c) Ability to walk independently for at least five steps as defined by the Bayley Scales of Infant and Toddler Development®; d) Post-treatment change from baseline measurements at treatment as defined by the Bayley Scales of Infant and Toddler Development®; and e) At least a 3-point improvement in the gross motor component of the Bayley Scales of Infant and Toddler Development® in relation to the pre-treatment score.

23. A composition described in any one of claims 18 to 22, wherein the patient achieves at least one of the following a) to f): a) at least a 3-point improvement on the Hammersmith Functional Motor Scale-Expanded relative to the pre-treatment score by 9 months post-treatment; b) at least a 4-point improvement on the Hammersmith Functional Motor Scale-Extended relative to the pre-treatment score by 9 months after treatment; c) at least a 5-point improvement on the Hammersmith Functional Motor Scale-Extended relative to the pre-treatment score by 9 months after treatment; d) at least a 3-point improvement in the gross motor component of the Bayley Scales of Infant and Toddler Development® after treatment relative to the pre-treatment score; e) Ability to stand unsupported for at least 3 seconds by 12 months after administration; and f) Ability to walk at least five steps independently by 12 months after treatment.

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