AAV viral vectors and their uses

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 April 17, 2019. Priority is claimed from application Ser. No. 42, the contents of which are incorporated by reference in their entirety. and is incorporated herein by reference.

[0002] Array List This application is incorporated herein by reference in its entirety. 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 contains a cis-acting inverted terminal repeat (ITR) sequence, approximately 145 nucleotides in length. and can fold into a hairpin structure that functions as a primer during the initiation of DNA replication. Interrupted palindromic sequences. In addition to their role in DNA replication, ITR sequences The virion nucleus is responsible for viral integration, rescue from the host genome, and assembly into mature virions. It has been shown to play a role in capsid formation of the acid (Muzyczka, 1999). 92)Curr.Top.Micro.Immunol.158:97-129).

[0005] There are multiple serotypes of AAV, which offer different tissue tropisms. Known serotypes include: , e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11 are included. Nos. 7,198,951 and 7,198,951, 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 enabled 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 demonstrated. 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 is 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 coding sequence and cis-acting sequences that direct host cell chromosomal integration are contained within the ITRs. AAV promoters (p5, p19, and p40 based on their relative map positions) (named 'AAV') are the sequences of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19) drive expression of a single AAV vector. In conjunction with differential splicing of the nucleotides (nucleotides 2107 and 2227), the rep gene The gene contains four rep proteins (rep78, rep68, rep52, and rep4 0) is produced. The rep protein 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 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 eliminating natural host cell responses to viral infection, such as interferon-mediated responses. (iii) the wild-type virus has never been associated with any pathology in humans; (iv) In contrast to wild-type AAV, which can integrate into the host cell genome, replication-deficient AAV vectors They generally persist episomally and therefore are resistant to insertional mutagenesis or oncogene activation. (v) In contrast to other vector systems, AAV vectors have significant (See ii) and therefore (their gene products are rejected) (unless otherwise specified) provides long-term expression of the therapeutic transgene.

[0008] Self-complementary adeno-associated vectors (scAAV) are naturally occurring vectors that are used in gene therapy. It is a viral vector engineered from the adeno-associated virus (AAV) found in the sc AAVs are designed so 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 together 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 SMN protein levels and selective motor neuron activation. 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 deletion of the survival motor neuron 1 (SMN1) gene Humans also have a second mutation in the SMN1 gene, called SMN2. Both the SMN1 and SMN2 genes encode the SMN transcription factor. Although SMN2 expresses the protein, the amount of functional full-length protein produced by SMN2 is SMN2 is produced at a much lower rate (10-15%) than SMN1. Although the loss of the N1 gene cannot be fully compensated for, it is generally more prevalent in patients with milder forms of SMA. The large-scale first study by Feldkotter et al. In a phase 2 study, two copies of SMN2 predicted the development of type 1 SMA by 97%, and SMN2 Three copies of SMN2 predict 83% of developing SMA type II, and four copies of SMN2 predict 83% of developing SMA type III. 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 under 11 months of age, and 2 with two SMN2 copies were under 11 months of age. In the case of 1, 88 / 94 patients survived for less than 21 months, and in the case of 3, 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 most common and severe form (type I) develops within the first few months of life. Hypotonia and progressive weakness are recognized in the early stages of childhood and diagnosed by 6 months of age, with respiratory failure by 2 years of age. SMA type 1 is the leading genetic cause of infant death. Motor neuron loss is evident early after birth (or may even begin prenatally), and the patient Individuals with type 1 SMA typically have the SMN2 gene. In contrast, type 2 SMA becomes apparent 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 2 SMA typically have three copies of the SMN2 gene. Patients with Type III SMA gain the ability to walk unassisted. In the Type III classification: Patients with type IIIa usually present with disease onset before age 3, whereas patients with type IIIb present with disease onset after age 3. In patients with SMA types II and III, motor neurons adapt and replace themselves 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 embryonic This shows an early loss of motor neurons during the natal 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 contributes to the development of motor neurons. Type 1 SMA patients in whom loss of the receptor is evident early after birth (or especially within the first 3 months of life) This contrasts with type 1 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 against human SMN. If there is a high copy number of SMN2, there is no risk (types II and III with a high copy number of SMN2 For example, SMA patients such as SMA types II and III may have a more severe form of SMA than those with SMA types II and III. Increasing SMN levels in individuals presents a therapeutic option.

[0012] For example, previous treatment efforts in SMA, such as SMA types II and III, have primarily focused on The potential for small molecules to increase SMN levels has been highlighted. Deacetylases such as protease, sodium butyrate, phenylbutyrate, and trichostatin A Inhibitors are used to reduce the disease to a milder form 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 hydroxybenzoates Clinical trials with thiourea have not provided sufficient 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 Increases SMN protein production by regulating ATP synthesis, thereby Antisense oligonucleotide (ASO) drugs designed to compensate for genetic defects approved one drug, nusinersen. Summary of the Invention [Problem to be solved by the invention]

[0014] Clinical studies have shown some modest promise for improving motor function; However, treatment must be administered quarterly via intrathecal injection indefinitely to be effective. Safety considerations requiring a long induction period and clinical monitoring before 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 for treating SMA, for example, in patients with SMA types II and III. In some embodiments, the methods include detecting SMA phenotypes, such as SMA type II and SMA type III. This includes intrathecal injection of an AAV9 viral vector capable of modifying the resulting in a slower course of disease progression, halting disease progression, and / or improved functional development . [Means for solving the problem]

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

[0017] The present disclosure provides polynucleotides encoding survival motor neuron (SMN) proteins. Intrathecal administration of an AAV9 viral vector comprising the viral vector However, about 1 × 10 13 vg~5×10 14 vg dose, for patients who need it In the treatment of spinal muscular atrophy (SMA), for example, 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, a nucleotide sequence similar to that of the unmodified AAV2 ITR, and a nucleotide sequence similar to that of the 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 6 months of age or older at the time of administration. 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 and 24 months of age. are 60 months of age or younger at the time of administration, and optionally are between 24 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 100 mg / kg. The maximum value is approximately 6.0 x 10 13 In some embodiments, A is administered at a dose of 0.05 mg / kg. The AV9 viral vector was approximately 6.0 × 10 13 It is administered at a dose of vg. In embodiments, the AAV9 viral vector is up to about 1.2 x 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 approximately 2.4 x 10 14 It is administered at a dose of vg.

[0018] In some embodiments, the AAV9 viral vector is about 1.0 x 10 13 vg~9 .9×10 14 In some embodiments, the AAV9 virus is administered in a unit dose of 1000 mg / kg. The spectrum is approximately 1.0 × 10 13 vg~5.0×10 14 Administered in a unit dose 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 mg / kg. The spectrum is approximately 6.0 × 10 13 The dose is administered in a unit dose containing vg. In this state, the AAV9 viral vector is expressed at 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. A 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 screened for one or more genomic tests. In some embodiments, the patient has disease onset before about 12 months of age. In some embodiments, the patient demonstrates the ability to sit unassisted for about 10 seconds or more at the time of administration. In some embodiments, the patient has a muscular dystrophy, 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, as defined by the Bayley Scales of Infant Development®, and In some embodiments, the patient has the ability to stand for at least about 3 seconds on the floor, e.g., For example, the Bayley Infant and Child Health Index (BMI) was assessed approximately 1-24 months after administration, e.g., 12 months after administration. Ability to walk unassisted after administration as defined by the Child Development Test®. In this embodiment, the patient is treated with steroids after about 1 to 24 months, e.g., 12 months, after administration. Independence after administration 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. The results were assessed approximately 1-24 months after birth, e.g., 12 months after birth, using the Bayley Scales of Infant Development ( ) and indicates the change from the baseline measurement at the time of treatment after administration.

[0020] In some embodiments, the patient is evaluated for serotonin levels after about 1 to 24 months, e.g., 12 months after administration. In the evaluation of the patient, there was no significant side effect, e.g., a spinal curvature of 50 degrees or more, evident on radiography after administration. In some embodiments, the patient does not have a spinal tap procedure or administration of intrathecal therapy. In some embodiments, the patient has not had a previous scoliosis repair surgery or procedure. and optionally, the patient has not received the In some embodiments, the patient is not undergoing scoliosis repair surgery or treatment. In some embodiments, the patient does not require the use of invasive ventilatory support after administration. In some embodiments, the patient has no history of standing or walking independently prior to administration. In some embodiments, a gastric feeding tube is not used before and / or after administration. Patients must have an active viral infection (human immunodeficiency virus (HIV); or type 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 administration. In some embodiments, the patient has not had any previous symptoms of flu-like symptoms (e.g., 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 comorbid conditions, such as symptomatic cardiomyopathy, prior to administration. In some embodiments, patients have 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 does not have any allergies or hypersensitivity to iodine or iodine prior to administration. have no known allergies or hypersensitivities to element-containing products. The patient is not taking any medications to treat myopathy or neuropathy. In some embodiments, the patient has received immunosuppressive therapy, plasma exchange, adalimumab, or other anticoagulant therapy within three months prior to administration. Not receiving immunomodulators such as mabs.

[0021] In some embodiments, the patient is tested for HIV infection prior to administration, for example, 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. Tamiltransferase level, less than about 3.0 mg / dL, bilirubin level, about 1. Creatinine levels below 0 mg / dL, Hgb levels between approximately 8 and 18 g / dL, and / or approximately 20,000 / mm 3 having one or more white blood cell counts less than In this setting, patients must be aware of any investigational or approved compounds 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 approximately 1.0 to 2.0 mL, for example, approximately 1.5 mL. Optionally, the imaging agent is administered within, e.g., less than 24 hours, less than 12 hours, less than 6 hours, or 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 can be administered sequentially, for example, with the contrast agent administered first (e.g., intrathecally). ) is administered, and following administration of the contrast agent, the AAV9 viral vector is administered (e.g., intrathecally). 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 fluoroscopy 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 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 this embodiment, the patient may experience a trendy 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 left there.

[0022] In some embodiments, the patient is administered, for example, about 2 doses of the AAV9 viral vector. Oral steroids are administered at least approximately 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. Oral steroids are administered for at least about 10 to 60 days. In some embodiments, the oral steroid is administered once daily. In some embodiments, the patient receives a viral vector twice daily. Afterward, patients were monitored for ALT and / or AST levels, and oral steroids were administered to and / or ALT levels less than twice the upper limit of normal or less than approximately 120 IU / L In some embodiments, the patient continues to receive the AAV9 virus for up to 30 days. 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 for a T cell response in a patient sample, such as a blood sample containing less than 0.00 cells It continues to be administered afterwards.

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

[0024] In some embodiments, the oral steroid is administered to maintain AST and ALT levels below 2 times the upper limit of normal. In some embodiments, the dose is gradually decreased after the dose reaches or falls below about 120 IU / L. The dose should be tapered to approximately 0.5 mg / kg / day over 2 weeks, followed by approximately 0. In some embodiments, oral administration includes gradual reduction to 25 mg / kg / day. Steroids were administered at a dose of approximately 1 mg / kg for 30 days, followed by 0.5 mg / kg over 2 weeks. The dose is 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 measured by the Bayley Scale of Infant Development® scale. and / or Hammersmith Functional Motor Scale Extended (HFMSE). In some embodiments, the method comprises 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 other such embodiments, 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 In some embodiments, the therapeutic agent comprises nusinersen and / or stamumab. The amount of AV9 viral vector genome is measured using ddPCR. In embodiments, patients receive a 1:25, 2:30 or 3:40 dose of 1:25 or 2:40 or 3:5 ... Anti-AAV9 antibody titers of 1:50, 1:75, or 1:100 or greater were observed 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, patients are 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, and the antibody titer has 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 blood 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 blood count of less than about 67,000 cells / ml, or less than about 100,000 cells / ml after administration. Patients with a platelet count of less than 0 cells / ml or less than about 150,000 cells / ml and who have been diagnosed with a disease for about 1 to 8 weeks or platelet counts of approximately 67,000 cells / ml or approximately 100,000 cells / m The cells are monitored until they increase to above 1 / 1 or above 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 platelet transfusions. In some embodiments, the patient has normal liver function prior to administration of the agent. 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 oral 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. To provide.

[0028] The present disclosure provides an AAV9 viral vector and a pharmaceutically acceptable carrier suitable for intrathecal administration. The AAV9 viral vector comprises a modified AAV2ITR, a chicken 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 a nucleotide sequence comprising an unmodified AAV2 ITR and a nucleotide sequence comprising an unmodified AAV2 ITR. The otide encodes the SMN protein of SEQ ID NO: 2. In some embodiments, A The AV9 viral vector comprises SEQ ID NO: 1. In some embodiments, the pharmaceutical composition In some embodiments, the contrast agent is present in a volume of, for example, about 1.5 mL. It is present in an amount of about 1.0 to 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 contains no more than about 9 mL, or no more than about 8 mL. In some embodiments, the pharmaceutical composition further comprises a therapeutic agent. This document is intended for use in any of the following areas:

[0030] In some embodiments, the pharmaceutical composition contains about 1.0×10 13 vg~9.9×10 14 In some embodiments, the pharmaceutical composition comprises a unit dose of about 1.0 x 10 vg. 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 contains about 6.0 x 10 13 The unit dose contains 1000mg of In some embodiments, the pharmaceutical composition comprises about 1.2 x 10 14 In unit doses containing vg In some embodiments, the pharmaceutical composition contains about 2.4 x 10 14 unit dose containing vg is.

[0032] In some embodiments, the pharmaceutical composition comprises at least one of the following: (a) about pH 7.7-8.3, (b) approximately 390-430 mOsm / kg, (c) less than 25 μm per container (d) less than about 600 particles of size 10 μm or larger per container; less than 0 particles, (e) approximately 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) approx. 20 to 80 ppm Pluronic® F-68 content, (i) about 70 to 130% Relative titer of (j) 7.5 × 10 13 In the SMNΔ7 mouse model, (k) less than about 5% empty capsids; (l) and about 95% empty capsids; % total purity or greater, and (m) endotoxin less than or equal to 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, and (b) about 30 μg / g (ppm) less than cesium, (c) approximately 20-80 ppm poloxamer 188, (d) 1 .0×10 13less than approximately 0.22 ng of BSA per vg, (e) 1.0 × 10 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 less than pg, (g) 1.0 × 10 13 Approximately per vg Less than 4 ng of rHCP, (h) approximately pH 7.7 to 8.3, (i) approximately 390 to 430 mOsm / kg, (j) less than about 600 particles 25 μm or larger in size per container, (k) per container (l) less than about 6,000 particles of 10 μm or larger 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 approximately 70–130%, and (p) less than approximately 5% empty capsids.

[0034] In some embodiments, the use of the methods or compositions described herein provides a method for determining whether a patient has 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(R), Third Edition (Bayley(R)-III) resulting in improved scores. [Brief explanation of the drawings]

[0035] [Figure 1] The body weights of treated and control mice following AAV administration are shown. [Figure 2] Initial study design for a Phase 1, open-label, single-dose trial in infants and children with SMA Type II or III. Patients will receive AVXS-101 in a dose-comparison safety study. [Figure 3] Figure 1 shows a waterfall plot of the change from baseline in the Hammersmith Functional Motor Scale Extended (HFMSE), ranked from 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 by gray bars; black bars indicate patients between 2 and 5 years of age at the time of infusion. [Figure 4] 1 shows the HFMSE scores of individual patients with SMA type II. [Figure 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 plot of change from baseline in HFMSE scores to month 12 for the 24 to <60 month age group (primary PNCR analysis) - ITT set. [Figure 8] Spaghetti plot of change from baseline in HFMSE score to month 12 for the 24 to <60 month 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 after 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 score as assessed by the Bayley Scale® at each visit through 12 months after baseline for patients under 24 months of age at the time of dosing - ITT set. [Figure 11] Shown is a spaghetti plot of the change from baseline in 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 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 visit for patients under 24 months of age at the time of dosing who continued on study past 24 months of age are shown - ITT set. DETAILED DESCRIPTION OF THE INVENTION

[0036] To better understand the present disclosure, certain exemplary embodiments are discussed herein. Additionally, certain terms are provided to aid understanding.

[0037] In some embodiments, a "vector" refers to a molecule that can self-assemble when associated with appropriate control elements. Plasmids, phages, and transposons are capable of self-replicating and transferring genetic sequences between cells. It refers to 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 and AAV-9 An AAV vector may contain one or more genes, such as 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 contain sequences that provide for replication and packaging of the vector (e.g., functional ITRs). ITRs are defined herein. ITRs do not have to be wild-type nucleotide sequences, but rather may be sequences that function. As long as it provides efficient rescue, replication, and packaging, e.g., nucleotide insertion In one embodiment, the vector may be modified by insertion, deletion, or substitution. It is an AAV-9 vector with ITRs derived from V-2. , which provides an efficient vehicle for delivering vector nucleic acid to the nucleus of target cells. The term "capsid" refers to a solid shell or capsid.

[0039] In some embodiments, "scAAV" refers to naturally occurring AAVs for use in gene therapy. Autologous, a viral vector engineered from adeno-associated virus (AAV) found in This refers to a complementary 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 double-stranded DNA templates within the do.

[0040] In some embodiments, a "recombinant virus" refers to, for example, a virus that contains a heterologous nucleic acid construct inserted into a particle. "Recombinant" means a virus that has been genetically modified 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" encompasses "recombinant AAV" or "rAAV." is intended.

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

[0042] In some embodiments, "recombinant AAV virions," "rAAV virions," "AA The terms "V vector particle," "full capsid," and "full particle" are used herein. encapsidating a heterologous nucleotide sequence of interest flanked on both sides by AAVITR Defined as an infectious, replication-defective virus containing an AAV protein shell. V virions contain the AAV vector, AAV helper functions, and accessors introduced into them. The resulting product is then produced in a suitable host cell containing sequences that specify the desired transcription function. The cells are then transfected with an AAV vector (carrying a recombinant nucleotide sequence of interest) for subsequent gene delivery. and packaging the AAV polypeptide (including the virion fragment) into infectious recombinant virion particles. You will be able to code the code.

[0043] Unless otherwise specified, all technical and scientific terms used herein are defined by the principles 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 the discussion conflicts with this disclosure, the latter shall control.

[0044] As used herein, the singular form of a word applies to the plural form of the word unless the context clearly indicates otherwise. For example, the terms "a," "an," and "the" refer to 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 any listed elements or integers or steps, or any elements, integers or steps. implies the inclusion of a group of steps, but does not include other elements or integers or steps, or elements or integers or It is understood that the exclusion of steps is not implied. The word "consists 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. The words "consisting essentially of" or "or Variations such as "consists essentially of" An option is an inclusion of a stated element or integer or step, or group of elements or integers or steps. and that do 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. It is understood to indicate

[0046] Approximately means ±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% or within ±0.5% (for example, "about 5%" is 4.5% to 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 vector In one aspect, disclosed herein is an rAAV genome. In the present invention, the rAAV genome contains a polynucleotide sequence encoding an SMN polypeptide. In some embodiments, the polynucleotide comprises one or more AAVITRs comprising: For example, promoter DNA, one or more enhancer DNAs, and / or gene A polyadenylation signal sequence that functions in target cells to form a transfected DNA The gene cassette may also contain intron sequences. may also include a nucleotide sequence that facilitates processing of the RNA transcript when expressed in mammalian cells. .

[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, among others. 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. The complete genome of AAV-2 is available in GenBank under the 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 under 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 AAV-7 and AAV-8 genomes are identified under GenBank accession numbers 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 described 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 encoding the SMN cDNA, i.e., an SMN cDNA expression cassette, For example, the "left" and "right" of the polynucleotide encoding the SMN gene, In some embodiments, the nucleotide sequence is flanked by associated virus inverted terminal repeat (ITR) sequences. 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 native, mutated, or modified AAVITR sequence. In the AAV2 ITR gene, at least one ITR sequence is a native, mutant, 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 for 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 a CMV enhancer / chicken β - further comprises the actin ("CB") promoter. In some embodiments, pSMN The plasmid further contains a Simian Virus 40 (SV40) intron. In embodiments, the pSMN plasmid contains a bovine growth hormone (BGH) polyadenylation (polyadenylation). A) Further comprising a termination signal. One or more of the components discussed above may be used. Exemplary sequences are shown in Table 1 below. In some embodiments, all of the sequences shown in Table 1 below are In some embodiments, "AVXS-101" refers to all of the sequences in Table 1. Non-limiting examples of vector constructs within the term pSMN include those using the sequence These vector embodiments and methods for preparing and purifying them are described, for example, in PCT / US2018 / 058744, the entire contents of which are incorporated herein by reference. No. 6,499,499.

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

[0051] [Table 1]

[0052] In some embodiments, the vector construct sequence is a sequence similar to that of the AAV9 virion, e.g., In these embodiments, encapsidation can occur, for example, in 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 an AAV9 capsid, e.g., VP2 and V P3 is a truncated form of VP1, all with a common C-terminal sequence. The 60 viral proteins (V) produced by lysis were separated in a ratio of 1:1:10. In some embodiments, the present invention is directed to a manufacturing process for, for example, a pharmaceutical product. Process's products contain non-replicating recombinant AAV9 capsids and are stable, fully functional human In some embodiments, the capsid comprises VP2 and The alternative sequences are such that VP3 is a truncated version of VP1, all of which share a common C-terminal sequence. 60 viral proteins in a ratio of 1:1:10 ( VP1, VP2, VP3). Methods for preparing and purifying them are described, for example, in , provided in PCT / US2018 / 058744.

[0053] In various embodiments, p, 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 with 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 set forth in SEQ ID NO:3. It is derived from the encoding transcript.

[0057] In various embodiments, disclosed herein are DNA fragments containing the rAAV genome. The DNA plasmid encodes the rAAV genome and the AAV9 capsid protein. AAV helper virus (e.g., adenovirus) is used to assemble into infectious viral particles containing Adenovirus, E1-deleted adenovirus, or herpesvirus) The AAV genome, rep and cap genes, and hepatocytes 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 production of rAAV is achieved by single cell (as described herein). The rAAV is present in the packaging cells (referred to as packaging cells 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 production of pseudotyped rAAV involves, for example, the complete expression of the International Publication No. WO 01 / 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. Modification of the capsid protein is known in the art. See, for example, U.S. Pat. No. 6,229,999, the entire contents of which are incorporated herein by reference. US Patent Application Publication No. 2005 / 0053922 and US Patent Application Publication No. 2009 / See specification 0202490.

[0058] The general principles of rAAV production 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 ,658,776 specification; WO 95 / 13392 pamphlet; WO 9 6 / 17947 pamphlet; PCT / US98 / 18600 specification; International Publication No. 7 / 09441 pamphlet (PCT / US96 / 14423 specification); International Publication No. 97 / 08298 pamphlet (PCT / US96 / 13872 specification); International Publication No. 97 / 21825 pamphlet (PCT / US96 / 20777 specification); International Publication Patent No. 97 / 06243 (PCT / FR96 / 01064 specification); International Publication No. 99 / 11764 Brochure; 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 Prepared, purified, manufactured, and / or formulated in accordance with the disclosure of S2018 / 058744. The foregoing 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. rAAV containing the polynucleotide is referred to as "rAAV SMN." In embodiments, the rAA SMN genome comprises the first AAV2 ITR, the cytomegalovirus genome, and the second AAV2 ITR. Chicken β-actin promoter with enhancer, SV40 intron, 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, for example, those listed 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. Examples include human SMN1 polypeptides described in database number NP_000335.1. In embodiments, the SMN DNA is a polypeptide encoding a human SMN polypeptide. containing ribonucleotides (e.g., Uniprot accession number Q16637, isoform 1 (Q Human SMN protein identified by SMN1-modifier polynucleotides (SEQ ID NO: 16637-1). Peptiplastin-3 (PLS3) is also envisioned [Oprea et al., S Science 320(5875):524-527(2008)]. Other polypeptides The sequence encoding the SMN DNA may replace the SMN DNA.

[0061] Pharmaceutical Composition In various embodiments, the viral particles of the present disclosure (referred to as viral particles) are delivered to the intrathecal cavity. 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. and formulated into a formulation suitable for intrathecal administration in a mammalian subject, such as a human, using obtain.

[0062] In some embodiments, the pharmaceutical preparation 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. The AAV9 viral vector contains a modified AAV2 ITR, a chicken β-actin (CB) promoter, and promoter, cytomegalovirus (CMV) immediate / early enhancer, modified SV40 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 10-30 nM, such as 20 mM. H8.0, about 7.7 to about 8.3 (e.g., 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 embodiments, the sodium chloride concentration is about 100-300 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 × 10 1 3 In some embodiments, the formulation comprises about vg / mL of AAV9 viral vector. 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 0 13 vg / mL, and the AAV9 viral vector was approximately 6.0 × 10 13 vg units In some embodiments, the formulation is administered in a dose of about 1-8 x 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 It is administered in a unit dose of vg. In embodiments, the formulation contains about 1-8 x 10 13 vg / mL, containing AAV9 viral vector - is approximately 2.4 x 10 14 In some embodiments, the formulation is administered in a unit dose of 0.05 mg / kg. is approximately 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 a tonicity agent to make it isotonic or isotonic. surfactants such as polysorbate 20 and polysorbate 80, The composition may be stabilized against shear and stress. Buffered using citrate, histidine, phosphate, or Tris buffers for optimal pH These compositions may be sterilized using sterilization techniques and may maintain their stability. 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 additives such as pH adjusting and buffering agents; tonicity adjusting agents; Sodium lactate, sodium chloride, potassium chloride, calcium chloride, molybdenum chloride, Pharmaceutically useful wetting agents such as sorbitan laurate 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., one or more additional active or inactive components, such as PEG-100, PEG-10 ... In some embodiments, the pharmaceutical composition comprises an SMN polynucleotide disclosed herein. a viral vector containing a nucleotide; and an imaging agent (e.g., Omnipaque (trademark) ), or iohexol-containing agents). 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. In some embodiments, a contrast agent (e.g., Omnipaque ( Some drugs, such as iohexol and iodine, increase motor neuron transduction. In embodiments, the contrast agent (e.g., Omnipaque™, iohexol, etc.) , to aid in guiding the intrathecal needle into the subarachnoid space.

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

[0067] In some embodiments, the pharmaceutical compositions are, for example, those described herein, the entire contents of which are incorporated by reference. and the methods described in PCT / US2018 / 058744, which is incorporated by reference herein. In some embodiments, the hydroxybenzoates may be prepared and purified according to methods known in the art. For example, the pharmaceutical composition may have less than about 7% empty cells as assessed by, for example, 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 the following purity: 1.0×10 13 Less than 0.09ng of benzodiazepine per vg enzymes, less than 30 μg / g (ppm) cesium, approximately 20-80 ppm poloxamer 1 88, 1.0×10 13 <0.22ng BSA per vg, 1.0 × 10 13 vg a 6.8 x 10 5 Less than pg of residual plasmid DNA, 1.0 × 10 13 1 per vg. 1×10 5 Residual hcDNA less than pg, and 1.0 × 10 13 Less than 4ng r per vg HCP.

[0068] In various embodiments, the pharmaceutical composition is within ±20%, ±15%, ±20% of the reference standard. In one embodiment, the titer is maintained between ±10%, or ±5%. 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 x 10 13 given a dose of vg / kg Mice tested were >15 days, >20 days, >22 days, or >24 days old. 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 70-130% potency.

[0069] In some embodiments, the pharmaceutical composition comprises, for example, about 1-8×10 13 Between vg / mL etc., about 1 x 10 13 vg / mL ~ 1 × 10 15 rAAV virus at a concentration between 0.1 mg / mL and 0.2 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. Then, 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 × 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 viral vector genome / mL that is functional. , 1×10 13 1.7 × 10 per vg / ml6 pg / ml or less, or 1 x 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 Less than 0.2 ng per vg, 1.0 × 10 13 Less than 0.1 ng per vg, or 1.0 x 10 13 Benzonase concentration of less than 0.09 ng per vg. In some embodiments, the pharmaceutical composition comprises 1.0 x 10 13 Less than 0.5ng per vg, 1.0 x10 13 Less than 0.3 ng per vg or 1.0 x 10 13 Less than 0.22ng per vg In some embodiments, the pharmaceutical composition has a bovine serum albumin (BSA) concentration of less than 100 mg / mL. is 1.0 x 10 13 Less than approximately 1 EU / mL per vg / mL, 1.0 × 10 13 vg / m Less than approximately 0.75 EU / mL per L, 1.0 × 10 13 Approximately 0.5 EU / vg / mL Less than 1.0 × 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 0.3EU / 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 Less than approximately 0.13 EU / mL per 1.0 x 10 13Approximately 0.1 EU / m vg / mL Less than L, 1.0 × 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 saturation level 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 may include adding about 10 to 100 ppm, 15 to 90 ppm, or about 20 to 80 ppm of polyisoprene. In some embodiments, the rAAV viral vector comprises oxamer 188. The pharmaceutical composition contains less than 2,000 particles, less than 1,500 particles, each having a size of 25 μm or more. In some embodiments, the pharmaceutical composition has fewer than 1000 particles, or fewer than 600 particles. The number of particles per container is less than 10,000, less than 8,000, or 100, with a size of 10 μm or more. In some embodiments, the pharmaceutical composition has fewer than 0 or fewer 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 a blood glucose concentration between 330 and 490 mOsm / kg, between 360 and 490 mOsm / kg, 60 mOsm / kg, or have an osmolality between 390 and 430 mOsm / kg. In some embodiments, the pharmaceutical composition contains 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 assay. Based on the assay, approximately 30-150%, approximately 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 50-400 μg 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 mg / 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 one or more (e.g., all) of the above criteria. Fill the mix.

[0070] The present disclosure also provides a method for treating a variety of conditions, including, for example, SMA type II or SMA type III, in a patient in need thereof. A kit for treating SMA is provided, the kit comprising a pharmaceutical composition disclosed herein. and / or a combination of the SMN polynucleotides disclosed herein. and optionally, for example, an imaging agent (e.g., one or more active or inactive ingredients, such as PEG-100, PEG-10 ... 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 an SMN polynucleotide disclosed herein. and optionally a contrast agent (e.g., Omnipaque™, or ionizing radiation). 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 of contrast media and one or more additional containers of medicinal agents. 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 viral vector pharmaceutical compositions. In some embodiments, each vial contains up to or about 6.0 x 10 13 v g of the viral vector pharmaceutical composition. In this embodiment, each vial (e.g., each unit dose) of viral vector in the kit contains about 6.0x 10 13 In some embodiments, the kit comprises a pharmaceutical composition at a dose of 0.05 mg / kg. Each vial (e.g., each unit dose) of the drug contains up to or about 1.2 x 10 14 vg dose 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 200 mg of the drug at a dose of 100 mg. 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 x 10 14 In some embodiments, the viral vector pharmaceutical composition comprises a pharmaceutical composition at a dose of 0.05 mg / kg. is approximately 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 In some embodiments, the aliquot contains more than a single dose of the rAAV viral vector. 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 SMA type III. In some embodiments, the delivery comprises administering a rAAV9 as disclosed herein. In some embodiments, the delivery is intrathecal delivery to the central nervous system of the patient, comprising: 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 first, followed by administration of the contrast agent. In this case, rAAV9 is administered first, and the contrast agent is administered following the administration of the AAV9 viral vector. In an embodiment in which the AAV9 viral vector and the imaging agent are administered sequentially, The administration of the AAV9 viral vector and the contrast agent can be performed, for example, within about 2 hours, within 1 hour, or within 45 minutes. It may be done 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 rAAV9 are administered intrathecally (either simultaneously or sequentially). (regardless of the next dose).

[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 also help target delivery directly to the subarachnoid space. In some embodiments, the rAAV9 genome is a self-complementary genome. In this 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 some cases, rAAV viral vectors are delivered to the motor cortex and / or brainstem. In some embodiments, the rAAV viral vector is delivered to the spinal cord. In one embodiment of the present disclosure, the rAAV viral vector is delivered to the lower motor neuron. Using rAAV9, rAAV viral vectors are delivered to neurons and glial cells. In some embodiments, the glial cells are microglia, oligodendrocytes, or astrocytes. In some embodiments, rAAV9 is used to express rAAV viral vectors. is delivered to Schwann cells.

[0076] The titer of the administered rAAV viral vector will 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 measured at 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 in this application 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 dose 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 vg / kg or more vectors -may be genome-wide.

[0077] In some embodiments, rAAV9 is 1.0 x 1013 vg~9.9×10 14 v In some embodiments, the rAAV9 is administered at a dose of 5.0 x 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 1000 mg / kg. is approximately 1.2 x 10 14 In some embodiments, the rAAV9 is administered at a dose of 1000 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 at a dose of vg.

[0078] In some embodiments, the rAAV9 is about 1.0 x 10 13 vg~9.9×10 14 In some embodiments, the rAAV9 is administered at a unit dose of about 1.0 x 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 a unit dose of vg In some embodiments, the rAAV9 is about 1.2 x 10 14 Administered in a unit dose of vg In some embodiments, the rAAV9 is about 2.4 x 10 14 Administered in a unit dose of vg can be.

[0080] The dose can be determined by any suitable method, for example, a dose specific for the viral vector. While PCR using large primers can provide a relative measurement, qPCR uses smaller primers. It may be used for small samples and absolute measurements. In some embodiments, ddPCR 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 assessed. Compared to traditional PCR-based techniques, ddPCR is faster because it does not require the use of new primers. Generally, large sample volumes are not used. Examples of commercially available ddPCR instruments are Although not specified, BioRad Qx100dd PCR and RainDanc e Raindrop Digital PCR. In one embodiment, the dose is In another embodiment, the dose is determined using qPCR. In another embodiment, the dose is determined using digital droplet PCR (ddPCR). In some embodiments, multiple methods are used. In some embodiments, P The CR-based method uses specially designed primers and probes that target the SMN gene. The probe is used to detect and quantify encapsidated AAV9 viral genomes. In another embodiment, the PCR-based method targets the chicken β-actin promoter. Using specially designed primers and probes to identify encapsidated AAV9 In other embodiments, 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. PCR-based methods use specially designed primers and primers that target ITR sequences. The probe is used to detect and quantify encapsidated AAV9 viral genomes. In other embodiments, the PCR-based method comprises the step of amplifying the bovine growth hormone polyadenylation signal. Using specially designed primers and probes that target the encapsidated In some embodiments, the titer is determined by detecting and quantifying the AAV9 viral genome. This can be measured using appropriate in vitro cellular assays or in vivo animal models. For example, SMNΔ Using animal models of SMA, such as SMAΔ7 mice, or from the cortex of SMAΔ7 mice, Quantitative cell-based analysis using appropriate cell lines, such as primary neural progenitor cells (NPCs) isolated from Using this assay, for example, titer or % functional AAVSMN viral particles can be measured. In one embodiment, the titer may be determined according to the method of Foust et al., Nat. Biotech. hnol.,28(3),pp.271-274(2010) method, Any suitable reference standard may be used. The dosage, purity, and yield of functional rAAV viral vectors of the disclosed An exemplary method for determining the fraction is described in , 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 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 to patients lacking a functional copy of SMN1 has been shown to treat SMA. Humans also have a second, nearly identical copy of the SMN gene, called SMN2, which can be used in the treatment of 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. Although SMN2 expresses the protein, it contains a translationally silent mutation in exon 7, resulting in This results 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 The truncated form is the predominant form. Consequently, the functional full-length 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 compensates for 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. Over 95% of SMA patients 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 modifier. 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 used may contain more than 1 copy, more than 2 copies, more than 3 copies, more than 4 copies, or more than 5 copies. Extra copies of the SMN2 gene and / or c.85 on exon 7 of the SMN2 gene In some embodiments, the present invention is administered to patients with SMA type II who lack 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. SMN2 gene mutations greater than 100% and / or c.859 on exon 7 of the SMN2 gene In some embodiments, the present invention is administered to patients with type III SMA who lack the G>C mutation. The rAAV SMN disclosed herein is administered intrathecally to patients with SMA type II. In some embodiments, the rAAV SMN disclosed herein is administered intrathecally to patients with SMA type III. It is administered internally.

[0083] Type 1 SMA (also known as 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 difficulty swallowing and sucking. Infants often have difficulty sitting up and do not reach the developmental milestone of being able to sit up unassisted. Select from hypotonia, delayed motor skills, poor head control, hunched posture, and joint hypermobility. These infants often present with one or more of the SMA symptoms. All new SMA cases have two copies of the SMN2 gene, one on each chromosome. More than half of patients have type 1 SMA. In type 1 SMA, approximately 80% of patients have a mutation in one of the SMN2 genes. It has one or two copies.

[0084] Type II or intermediate SMA is when SMA develops until a child is able to stand independently between the ages of 7 and 18 months. This occurs before a child is able to walk. Approximately 82% of patients with type II SMA have at least three SMN2 genes. It has three copies. Late-onset SMA (Type III and IV SMA, mild SMA, adult-onset SMA) SMA (also known as Kugelberg-Welander disease) is a condition characterized by various levels of Type III SMA develops after 18.5 months and may require assistance. However, children are able to stand and walk independently. Approximately 96% of people with type III SMA They 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 the two, from which adequate amounts of full-length SMN protein can be produced.

[0085] In one embodiment, for treating SMA, e.g., SMA Type II or III, For example, rAAV, such as the rAAV9 vector disclosed herein, can be administered intrathecally. The terms "treat," "treatment," and other related forms of the term a composition comprising the rAAV disclosed herein 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 before the onset of the disorder / 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 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 against, for example, type II or type III SMA. It is administered intrathecally to patients needing treatment for SMA.

[0087] In some embodiments, the patient is 0 to 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 old. 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 non-functional In some embodiments, the patient has a mutation in the SMN1 gene. The two copies of the gene have one or more mutations, such as null mutations. In this embodiment, the patient has one or more mutations, e.g., null mutations, 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 functional copies of the SMN2 gene. In some embodiments, the patient has at least one functional copy of the SMN2 gene. In some embodiments, the patient has four functional copies of the SMN2 gene. In some embodiments, the patient has at least five functional copies. Some children have an SMN1 null mutation or deletion and three copies of SMN2. In embodiments, the patient has a c.8 mutation in exon 7 of at least one copy of the SMN2 gene. 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, or the like. and / or may be determined by partial or complete chromosome or genome 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 , may be determined by Sanger sequencing. In some embodiments, SMN1 or 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 to identify, for example, SMA type II or III, In some embodiments, the type II or type III SMA is: For example, CHOP INTEND, Bayley Scales of Infant Development®, or HAMMAS It is diagnosed by clinical assessment of symptoms such as the MI Functional Motor Scale Extended (HFMSE). In some embodiments, Type II or Type III SMA is diagnosed by physical examination. In some embodiments, 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 before showing symptoms (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 needed. In some embodiments, the patient is assessed, for example, using one of the tests described herein. 1. The patient exhibited symptoms (e.g., one or more symptoms) of SMA Type I or III, as described in In some embodiments, the patient is symptomatic of SMA Type II or III and subsequently treated. In some embodiments, patients are treated before they become symptomatic. A diagnosis of type II or type III SMA is made based on the following:

[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 their ability to perform the Spontaneous movement is observed and motor skills include the patient lifting elbows, knees, hands, and feet off surfaces. In some embodiments, a patient's grip strength is assessed by the ability to place fingers in the palm of the patient's hand. This is measured by lifting the patient 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 if they bend their elbows and shoulders to reach for a stimulus placed at shoulder height. In some embodiments, shoulder posture may also be assessed by observing the patient's With the patient in a lateral position, whether or not they could reach for a stimulus placed at shoulder height by bending their elbows and shoulders. In some embodiments, motor skills may also be assessed by observing footwork. Determine whether the patient bends their hips or knees when you stroke, tickle, or pinch their In some embodiments, the assessment is performed by observing shoulder flexion, elbow flexion, hip flexion, and Spine adduction, neck flexion, head extension, neck extension, and / or spinal curvature may be performed, e.g., CHOP IN Other SMA symptoms may be assessed by known clinical measures such as TED. 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 and walk. In some embodiments, the patient is unable to hold their head upright for at least 10 seconds 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, it indicates the ability to sit independently.

[0092] Without being bound by theory, intrathecal administration is thought to allow the drug to circumvent the blood-brain barrier. As a result, for drugs that target the central nervous system, direct delivery by intrathecal administration is possible. This allows for a reduction in the total dose and / or volume of pharmaceutical composition required (e.g., IV administration). (compared to ) may thereby reduce the risk of hepatotoxicity. Direct delivery allows for better delivery to cells of the central nervous system, e.g., lower motor neurons, glial cells, etc. The amount of cerebrospinal fluid (CSF) in the subarachnoid space may allow for high transduction efficiency. The effective dose concentration selected for intravenous delivery may influence the CSF volume in humans before the age of 3 years. Since the dose remains relatively constant after administration, patient dosing can be more easily and uniformly distributed across different patients. In some embodiments, intrathecal administration can be performed in a controlled manner to cross the blood-brain barrier. In some embodiments, the rAAV9 viral vector disclosed herein is used. The investigators are, for example, patients identified as needing treatment for SMA type II or III. In some embodiments, 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 (i.e., a general practitioner). After administration, the patient's vital signs will be 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, intrathecal administration of the rAAV9 viral vector is administered to the patient. It is performed with the patient in the prone, knee-chest, lateral, Sims, or lateral positions. In some embodiments, the rAAV9 viral vector may be injected with a syringe or syringe. In some embodiments, the catheter is L1-L2, L2-L It may also be inserted into the subarachnoid space through 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 patient should be given an appropriate radiopaque contrast solution (e.g., metrizamide, iopamidol, iohexol). Premixed with acetaminophen, ioversol, Omnipaque™, etc., and administered intrathecally. In some embodiments, a contrast agent solution (e.g., metrizamide, iopamine) is injected directly into the spleen. Midol, Iohexol, Ioversol, Omnipaque™, etc.) In some embodiments, the intrathecal administration is prior to intrathecal administration of the AV9 viral vector. The contrast agent solution was administered within 2 hours, 1 hour, or 3 hours before intrathecal administration of the rAAV9 viral vector. Intrathecal administration 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 In some embodiments, the contrast agent solution is administered intrathecally after the intrathecal administration of the vector. Within 2 hours, within 1 hour, within 45 minutes, and within 3 hours after intrathecal administration of rAAV9 viral vectors The drug is administered intrathecally within 0, 15, 10, or 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 receives the rAAV9 viral vector following administration of the rAAV9 viral vector. 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 an angle of 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 should be given a trendy dose over a period of 10 to 30 minutes, e.g., about 15 minutes. The patient is placed in the Dehlenburg position or tilted head down at, for example, 30 degrees.

[0094] In some embodiments, treatment is for SMA, such as type II or type III SMA. Prevent, alleviate, relieve, delay, and / or partially or completely relieve one or more symptoms The effectiveness of the treatment was evaluated by 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. “Bay 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) 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 evaluated to determine whether or not they intentionally put their hand in their mouth. In some embodiments, the patient is assessed for whether they attempt the task. To make sure you keep your hands open most of the time when you're 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 blocks and see if they pick them up with one hand or both hands, and if they pick them 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 blocks with partially opposable thumbs and fingers. The form involves presenting a patient with food pellets and determining whether the patient grasps the pellet with the pad of their thumb or fingertip. The patient is assessed to see if they can grasp the mass with partially opposable thumb and fingers. In one embodiment, a patient is given a book and asked whether they wish to turn one or more pages at a time. In some embodiments, the patient is evaluated to see if the crayon or pencil While the patient is marking the paper, the crayon or pencil is held in a palmar grip and a static three-finger grip. The subject is assessed 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 test was conducted to see if the subject held the device in this position and scribbled or drew with the other hand. do.

[0095] In some embodiments, the patient is asked to determine whether they thrust their arms or legs out repeatedly during play. In some embodiments, the patient is evaluated to determine if they are able to hold their head intermittently without support. In some embodiments, the method is evaluated to see if it can raise the The patient is asked to hold his 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 they also have the ability to take five steps. The patient is assessed for coordination and balance according to item 43 of the Bayley®-III-Gross Motor Examination. They are assessed to determine whether they have the ability to walk at least five steps with sensation. In some embodiments, the patient is assessed for their ability to stand without assistance or a support surface, and The robot is evaluated to see if it has feedback control. In an embodiment, the patient is provided with supplementary exercises according to item 40 of the Bayley®-III-Gross Motor Exercise. In some embodiments, the subject is evaluated to determine whether or not they have the ability to stand unaided. The study was conducted to determine whether the patient was able to stand unsupported at approximately 24 months, 12 months, 9 months, or 6 months after administration of the treatment. A patient is considered to have received effective treatment if they achieve the required ability. In this study, patients showed improvements in cooperation and sympathetic nervousness at approximately 24, 12, 9, or 6 months after treatment administration. Achieved 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 assesses the abilities of non-ambulatory individuals with SMA, the HFMSE have demonstrated additional methods that successfully differentiate motor skills between individuals with SMA types II and III. Thirteen add-ons were provided. In some embodiments, patients were able to sit unsupported in a chair or The patient is assessed for their ability to sit on the floor. In some embodiments, the patient is assessed for their ability to sit on a chair or floor without support. The patient is assessed for their ability to touch their head with their hands while seated on the floor. The patient is asked to rate their ability to touch their head with both hands while sitting unsupported in a chair or on the floor. In some embodiments, the patient is able to roll over while lying down. In some embodiments, the patient is assessed for whether they are lying face up. The ability to roll from the top to the bottom or vice versa is evaluated. 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 their forearms. In some embodiments, the patient is assessed for ability to perform the procedure while in the prone position. In some embodiments, the patient is assessed for ability to lift their head. The question is whether the patient can support themselves with straight arms while lying prone for a count of three. In some embodiments, the patient is evaluated by rolling from a lying position to a prone position. In some embodiments, the patient is assessed for their ability to sit up. You will be assessed on your ability to get down on your hands and knees while keeping your head up. In some embodiments, the patient is assessed for ability to crawl forward on hands and knees. In some embodiments, the patient is assessed in a supine position with arms folded across their chest. During this time, the patient is assessed for ability to lift their head. The patient is asked 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 ill while lying in a supine position. They are assessed on whether they can bring either knee to their chest. In this embodiment, the patient is tested to see if they can go from a kneeling position to a semi-kneeling position without using their arms. In some embodiments, the patient is assessed for whether they are able to stand 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 their ability to move from standing to sitting without using their arms. In embodiments, the patient is assessed for ability to move from a standing to a squatting position without using their arms. In some embodiments, the patient is assessed for: In some embodiments, the patient is assessed for their ability to jump. is whether you can go up and down four flights of stairs without assistance or with the help of one handrail. In some embodiments, the patient is evaluated for a 24-month, 12-month, or 30-month follow-up period after administration of the treatment. , 9 months, or 6 months, an increase of, say, 8 points from baseline on the HFMSE If the patient shows an increase of 5 to 10 points, the patient is considered to have received effective treatment. In some embodiments, the patient is treated within about 24 months, 12 months, 9 months, or 6 months of administration of the treatment. Patients were considered to have 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 for about 24 months, 1 year, or 2 years after administration of the treatment. A 10-point increase from baseline in the HFMSE score at 2, 9, or 6 months If so, the patient is 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 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 baseline measurement includes measuring the ability 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®. In some embodiments, the method includes measuring the ability to stand 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 treatment is assessed by assessing the Bayley Infant Development Item 43 of the gross motor component of the Test (registered trademark) (walking at least five steps unassisted) was measured. In some embodiments, treatment is assessed by determining a baseline level and comparing it to a baseline level. Validity was assessed using item 40 (supportive) of the gross motor component of the Bayley Scales of Infant Development®. (stand for at least 3 seconds without touching the floor) and comparing it to a baseline. In some embodiments, the efficacy of treatment is assessed by assessing the patient on the HFMSE and comparing the baseline score with the baseline score before treatment. In some embodiments, the baseline The efficacy of treatment is established by measurements within 30 days prior to treatment. Efficacy is assessed within 30 days of treatment. In some embodiments, the efficacy of treatment is assessed within 30 days of treatment. The efficacy is assessed 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 milestones are assessed by a standard motor milestone development survey, such as that shown in The effectiveness of treatment is expected to last at least 12 months, at least 24 months, and at least 48 months after treatment. months, at least 72 months, or up to 10 years after treatment.

[0098] [Table 2]

[0099] In some embodiments, the test to assess the efficacy of treatment is the Bayley Scales of Infant Development. (registered trademark), Hammersmith Functional Motor Scale Extended (HFMSE), or Motor Miles Tone development studies include, but are not limited to, CHOP INTEND, TIMP, CHOP TO SS, Peabody Developmental Motor Scale, Brazelton Neonatal Assessment Behavioral evaluation and interactive video evaluation (ACTIVE) including, but not limited to, measurements of exercise capacity and compound motor action potential (CMAP) Other motor skill tests known in the art may also be included, not limited to those described herein.

[0100] For example, methods for identifying SMA, such as Type II or Type III SMA, disclosed herein Pre-screening of patients suitable for treatment with Administration of treatment to patients 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-mediated transduction, even at low antibody levels, which 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 are the patients 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 has 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 higher IL-16 ... In some embodiments, after treatment, the patient has an anti-AAV9 antibody titer of: have an anti-AAV9 antibody titer of greater than 1:100 as measured by ELISA binding immunoassay The patient is monitored for 1 to 8 weeks or until the titer drops below 1:100. In some embodiments, the patient has, after treatment, a 1% or lower IL-12 level as measured by an ELISA-linked immunosorbent assay. Anti-AAV9 antibody titers of >1:100 for 1-8 weeks or titers less than 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 may be administered, such as rituximab in combination with cyclosporine A. 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 Antibodies against HIV-1 resistant strains exceeding 1:100 as measured by ELISA binding immunoassay before or after treatment AAV9 antibody titer and one or more In some embodiments, the patient is treated with an immunosuppressant. 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 immunosuppressive drugs, such as steroids like prednisolone.

[0103] In some embodiments, patients with high anti-AAV antibody titers are treated with IgG4-associated antibodies 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 processed plasma or The fluid is returned to the patient along with replacement fluids such as 4.5% human albumin in normal saline. A common use of apheresis is the removal of unwanted immunoglobulins, such as anti-AAV antibodies. In some embodiments, the patient is tested using an ELISA-linked immunoassay 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 tested for IL-16 by ELISA binding immunoassay before or after treatment. Patients have a baseline anti-AAV9 antibody titer of greater than 1:50 and are treated with plasma exchange.

[0104] Pre-existing maternal antibodies against AAV9 can be transferred to young patients through breast milk or intrauterine placental transfer. In some embodiments, patients may be tested with ELISA binding assays before or after treatment. had an anti-AAV9 antibody titer of greater than 1:100 as measured by immunoassay and were able to switch to artificial nutrition. In some embodiments, the patient is administered an ELISA binding immunoglobulin (ELISA) before or after treatment. The patient had an anti-AAV9 antibody titer of more than 1:50 as measured by the assay, and was switched to formula feeding. can be done.

[0105] The patient's condition may be monitored before and after administration of the 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 a 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 approximately 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 or greater than about 67,000 cells / ml prior to administration. Platelet counts greater than 100,000 cells / ml or greater than approximately 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 to 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 is monitored until it reaches approximately 67,000 cells / ml. In some embodiments, where the number of platelets is less than 1, the patient may be treated with platelet transfusions. In embodiments, the patient does not have thrombocytopenia prior to administration of the viral vector. In some embodiments, the patient has thrombocytopenia after administration of the viral vector and is at least 100% thrombocytopenic for about 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 counts, 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 phosphate This involves standard blood tests that measure levels of one or more of: phospholipase A, phospholipase B, 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 drug. In some embodiments, the patient has a serum IL-1 level 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 indicative of a viral infection. Following vector administration, cardiac troponin-I levels were monitored until the troponin-I level was below 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 suggest liver dysfunction. In some embodiments, the patient may have normal liver function prior to administration of the viral vector. In some embodiments, the patient has been in the hospital for about 8 to 40 days prior to administration of the viral vector. 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 before administration of the viral vector In some embodiments, the patient is screened according to standards known in the art, such as CLIA standards. γ-glutamyltransferase less than three 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 1.0 mg / dL before administration of the viral vector. In some embodiments, the patient has a creatinine level of less than 0.0 mg / dL. Prior to viral vector administration, patients should have a hemoglobin (Hgb) level between 8 and 18 g / dL. In some embodiments, the patient has a HIV infection rate of 20,000 / mL or higher prior to administration of the viral vector. mm 3 Have a white blood cell (WBC) count of less than

[0109] In various embodiments, gene therapy using the AAV vectors described herein includes: For example, antigen-specific T cells against the AAV vector may be expressed within 2–4 weeks following gene transfer. One possible consequence of such an antigen-specific T cell response is The elimination of transduced cells and loss of transgene expression are the main challenges facing host defense against AAV-based therapies. Patients may be given immunosuppressants in an attempt to suppress the immune response. In embodiments, T cell responses may be measured by an ELISPOT assay. In some embodiments, the T cell response prior to administration of the vector is 10 6 Peripheral blood mononuclear cells (P In some embodiments, the patient's BMC is 100 spot-forming cells (SFC) per 100 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 receives oral steroids prior to administration of the viral vector. Examples of oral steroids include, but are not limited to, propranolol, steroids, and steroids. 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 administered, for example, at least two doses of the viral vector. Start prophylactic steroids at least 12 to 48 hours beforehand, such as 4 hours before. In some embodiments, the patient may experience, for example, at least Oral steroids are administered for at least 10 to 60 days, with 30 days being the most common. In some embodiments, the oral steroid is administered once daily. In some embodiments, the oral steroid is administered twice daily. For example, it is administered at a dose of about 0.1 to 10 mg / kg, such as about 1 mg / kg. In some embodiments, oral steroids are administered at a dose of about 0.1 to 1 mg / kg / day, such as 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-stage treatment is indicated when AST and ALT levels are below the clinical threshold, e.g., as defined by clinical criteria known in the art. It is administered when blood levels measured by this method exceed twice the upper limit of normal, or approximately 120 IU / L. In some embodiments, oral steroid treatment is for more than 30 days and is effective in reducing AST and A LT levels are above normal, e.g., as measured by clinical criteria and methods known in the art. The dose is administered as long as the level exceeds twice the normal limit, or as long as the level exceeds approximately 120 IU / L. In some embodiments, oral steroid treatment may be used to treat T cell responses of 10 6 1 per PBMC In some embodiments, oral administration is performed for more than 30 days, as long as the SFC is above 0.00. Steroid treatment increases T cell responses by 10 6 30 minutes until the number of SFCs per PBMC is 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. 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. In some embodiments, the taper is at or below about 120 IU / L. 0.5 mg / kg / day for 2 weeks, followed by 0.25 mg / kg for approximately another 2 weeks In some other embodiments, oral steroids are administered in a gradual manner, with a reduction in the dose to 100 mg / day. The tapering of the steroid is at the discretion of the physician. In some embodiments, blood samples are taken. Serum antibodies against AAV9 by ELISA and serum antibodies against SMN by ELISA were also detected. 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 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 of administration. Have undergone any prior, planned, or anticipated scoliosis corrective surgery or procedure within the past 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 independent standing or walking. In some embodiments, the patient does not have active AAV9 at the time of administration of the rAAV9 viral vector. In a further embodiment, 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 disorders, diabetes mellitus, idiopathic hypocalcaemia, In some embodiments, the patient does not have a concomitant disease such as calciumuria or symptomatic cardiomyopathy. Subjects must have a severe non-pulmonary or respiratory tract infection within 4 weeks of receiving 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 steroids, or their excipients In some embodiments, the patient has no hypersensitivity to iodine or iodine-containing products. In some embodiments, the patient has no known allergies or hypersensitivities to the drug. Not taking concurrent medications to treat encephalopathy or neuropathy. In this case, patients were required to have received 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 they alter binding to pre-mRNAs and alter their splicing patterns. Examples include antisense oligonucleotides (ASOs), which stimulate the immune system. 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 It is a recognized ASO that regulates the splicing of SMN2 to produce the full-length SMN protein. In some embodiments, the AAV9 viral vector is In some embodiments, the disclosed methods of treatment are administered in combination with a muscle-building agent. The proposed treatment involves administering an AAV9 viral vector in combination with a neuroprotective agent. In some embodiments, the disclosed therapeutic methods include administering an anti-sense oligonucleotide to a subject. Administration of AAV9 viral vectors in combination with anti-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 administering to a subject in combination with stamula umab. In some embodiments, the method comprises administering an AAV9 viral vector in combination with the administration of the AAV9 viral vector. The indicated treatment method involves administering an AAV9 viral vector in combination with two or more additional treatments. 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 preparing contaminants from host cells. It attempts to remove contaminants and chemicals added during collection of the viral vector. In some embodiments, the entire contents of PCT / US2002 / 003994 are incorporated herein by reference. In some embodiments, the methods disclosed in US Pat. No. 5,644,444 are used. , the method may, for example, involve about 1 to 8 x 10 13 vg / mL, e.g., approximately 1 x 10 13 vg / mL ~1×10 15 yield rAAV viral vectors at concentrations between 1000 and 1000 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 dose. is approximately 1.0 x 10 13 vg~5.0×10 14 rA in vg dose (e.g., unit dose) In some embodiments, the method results in about 5.0 x 10 AV viral vectors. 1 3 vg~3.0×10 14 rAAV viral vector at a dose (e.g., unit dose) of 1000 mg / vg In some embodiments, the method results in about 6.0 x 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 1000 mg / kg. In some embodiments, the method comprises administering a 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 saturation level by less than about 10%, less than about 8%, less than about 7%, or less than about 5%. This results in rAAV viral vectors with less than % empty viral capsids. In an embodiment, the method comprises: 13 less than approximately 100 ng / mL of host per vg / mL In some embodiments, the rAAV viral vector comprises a cellular protein. , this method is 1 x 10 13 vg / mL, approximately 5 × 10 6 Less than pg / mL, approximately 1 × 1 0 6 Less than pg / mL, approximately 7.5 × 10 5 pg / mL or less than 6.8 × 10 5 pg / mL yielding rAAV viral vectors with less than residual host cell DNA (hcDNA) In some embodiments, the method comprises: 13 Approximately 10ng 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 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 per vg / ml x10 6 yielding rAAV viral vectors with pg / ml residual plasmid DNA In some embodiments, the method 13 Less than 0.2ng per vg, 1 .0×10 13 Less than 0.1 ng per vg, or 1.0 x 10 13 0.09n per vg Some experiments have yielded rAAV viral vectors with benzonase concentrations below 1000 mg. In an embodiment, the method comprises: 13 Less than 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 containing albumin (BSA). 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 Less than approximately 0.5 EU / mL, 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 Less than approximately 0.1 EU / mL, 1.0 x 10 13 Less than approximately 0.05 EU / mL per vg / mL , or 1.0 × 10 13 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 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 may include adding about 10 to 100 ppm, 15 to 90 ppm, or about 20 to 80 ppm of polyisoprene. In some embodiments, the rAAV viral vector comprises oxamer 188. The method is to count less than 2000 particles, less than 1500 particles, and less than 10 particles of 25 μm or more in size per container. This results in an rAAV viral vector with fewer than 600 particles. In some embodiments, the method comprises producing fewer than 10,000 particles per container that are 10 μm or greater in size; rAAV virus vectors with fewer than 8,000, fewer than 1,000, or fewer than 6,000 particles In some embodiments, the method provides a method for producing a β-actin derivative having a β-actin derivative of between 7.5 and 8.5, between 7.6 and 8. rAAV viral vectors with a pH between 0.4 and 7.8 or between 7.8 and 8.3. In some embodiments, the method comprises administering a blood glucose level between 330 and 490 mOsm / kg, between 360 and 46 rAA with an osmolality between 0 mOsm / kg or 390-430 mOsm / kg In some embodiments, the method results in 1.0 x 10 V viral vectors. 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 1.1U. In some embodiments, the method comprises determining a reference standard and a target protein based on an in vitro cell-based assay. and / or relative to an appropriate control, about 30-150%, about 60-140%, or about 70-1 In some embodiments, the resulting 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 50-400 μg per dose, or 1.0 × 10 13 Approximately 100-300 μg of total protein per vg In some embodiments, the rAAV viral vector has a high protein level. , the method is 7.5 x 10 13 Vg / kg dose in SMNΔ7 mice As judged by the median survival time, 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 1000kJ / mL.

[0115] In any of the above embodiments, the preparation and / or purification method may be formulated for administration. and / or about 6.0 x 10 13 vg of the pharmaceutical composition, In any of the above embodiments, the preparation may result in a rAAV viral vector. The production and / or purification method may be formulated for administration and / or about 1.2 x 10 1 4 rAAV viral vectors present in the pharmaceutical composition in a unit dose of .gt; In any of the above embodiments, the preparation and / or purification method may be used to prepare the compound for administration. and / or about 2.4 x 10 14 present in the pharmaceutical composition in a unit dose of vg. This may result in an rAAV viral vector.

[0116] For example, in some embodiments, the method provides for a reduction in the oxidative stress 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 dose of about 6.0 x 10 13 v In some embodiments, the method comprises administering to the patient a pharmaceutical composition containing at least about 10% of the active ingredient in a unit dose of about 100 mg of the active ingredient in a pharmaceutical composition. 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 0.15 mg. In some embodiments, the method includes removing less than about 10%, less than about 8%, less than about 7%, or less than about 5% empty viruses. 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 a unit dose of vg In some embodiments, the formulation or pharmaceutical composition is present in about 6.0 x 10 13 v The rAAV viral vector comprises a unit dose of about 1 g of the rAAV viral vector. 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 contains about 1.2 x 10 14 vg rAAV The rAAV viral vector contains a unit dose of the viral vector, and the rAAV viral vector is less than about 10%, about 8 %, less than about 7%, or less than about 5% empty viral capsids. In the form, the formulation or pharmaceutical composition contains about 2.4 x 10 14 vg rAAV viral vector and the rAAV viral vector comprises less than about 10%, less than about 8%, or less than about 7% have less than, or less than about 5% empty viral capsids.

[0117] In some embodiments, the method comprises: 13 Approximately 100ng / mL per vg / mL and resulting in an rAAV viral vector having less than 100% host cell proteins. The vector is formulated for administration and / or contains approximately 6.0 x 10 13 vg unit dose In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising administering to said subject a pharmaceutical composition comprising at least 1×10 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 x10 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 Producing rAAV viral vectors and formulating the rAAV viral vectors for administration and / or approximately 2.4 x 10 14 vg of unit dose in the pharmaceutical composition. In some embodiments, the formulation or pharmaceutical composition contains about 6.0 x 10 13 vg rAAV virus The unit dose of vector contains 1 x 10 rAAV viral vectors. 13 vg / mL In some embodiments, the host cell protein is less than about 100 ng / mL. The formulation or pharmaceutical composition may contain about 1.2 x 10 14 vg unit dose of rAAV viral vector 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 1 mL of host cell protein. is approximately 2.4 x 10 14 vg of rAAV viral vector, The viral vector was 1 × 10 13 Less than approximately 100 ng / mL of host cells per vg / mL It has proteins.

[0118] In some embodiments, the method comprises: 13 pg / mL, approximately 5 × 10 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 the rAAV viral vector is formulated for administration, and / or approximately 6.0 × 1013 vg of the pharmaceutical composition. In this state, this method 13 pg / mL, approximately 5 × 10 6 Less than pg / mL, approx. 1×10 6 Less than pg / mL, approximately 7.5 × 10 5 pg / mL or less than 6.8 × 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 in a volume of about 1.2 x 1 0 14 In some embodiments, the method comprises the step of: , 1×10 13 pg / mL, approximately 5 × 10 6 Less than pg / mL, approximately 1 × 10 6 pg / m Less than L, approximately 7.5 x 10 5 pg / mL or less than 6.8 × 10 5 Less than pg / mL of residual The rAAV viral vector containing host cell DNA (hcDNA) is obtained. The virus vector is formulated for administration and / or contains approximately 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 viral vector unit dose of 1000 mg / kg, The kuta is 1 x 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 pg / mL or less than 6.8 × 10 5 Residues less than pg / m In some embodiments, the formulation or pharmaceutical composition comprises a recombinant host cell DNA (hcDNA). The object is approximately 1.2 x 10 14 vg of rAAV viral vector, 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 pg / mL or less than 6.8 × 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 x 10 14 vg unit dose of rAAV viral vector Contains 1 x 10 rAAV viral vectors 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 x10 5 Has residual host cell DNA (hcDNA) less than pg / mL.

[0119] In some embodiments, the method comprises: 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 ) and the rAAV viral vector has and / or about 6.0 x 10 13 present in the pharmaceutical composition in a unit dose of vg. In some embodiments, the method comprises: 13 Approximately 10 ng per 1000 mg / 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 approximately 1.2 x 10 14 Present in the pharmaceutical composition in a unit dose of vg In some embodiments, the method comprises: 13 Approximately 10ng 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 CP), and the rAAV viral vector and / or approximately 2.4 x 10 14 The amount of the compound present in the pharmaceutical composition is determined by a unit dose of vg. In some embodiments, the formulation or pharmaceutical composition contains about 6.0 x 10 13 vg's r The unit dose of the AAV viral vector is 1.0 x 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 contains about 1.2 x 10 14 Contains a unit dose of rAAV viral vector (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 x 10 14 vg The rAAV viral vector comprises a unit dose of 1.0 x10 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 contains approximately 6.0 x 10 13 vg unit In some embodiments, the method comprises administering to a subject a functional AAV9 At least about 50%, at least about 60%, at least about 10% About 70%, at least about 80%, at least about 90%, at least about 95%, or less and / or rAAV viral vectors formulated for administration, resulting in approximately 100% or approximately 1.2 x 10 14 In some embodiments, the pharmaceutical composition is present in a unit dose of 0.05 mg / vg. In one embodiment, the method comprises: 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%, and 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 about 6.0 x 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 approximately 100% rAAV (e.g., rAAV9) viral vector genomes / mL In some embodiments, the formulation or pharmaceutical composition contains about 1.2 x 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 contains about 2.4 x 10 mL. 14 a 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: 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 pg / ml residual plasmid DNA the rAAV viral vector is formulated for administration, and / or or approximately 6.0 x 10 13 In some embodiments, the pharmaceutical composition is present in a unit dose of 0.05 mg / vg. So, the method is 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 per vg / ml x10 6yielding rAAV viral vectors with pg / ml residual plasmid DNA , the rAAV viral vector is formulated for administration, and / or about 1.2 x 10 14 In some embodiments, the method comprises administering a pharmaceutical composition containing 1×10 hydroxybenzoates to a patient in a unit dose of 1×10 vg. 13 1.7 × 10 per vg / ml 6 pg / ml or less, or 1 x 10 13 vg / ml 1×10 5 pg / ml to 1×10 13 1.7 × 10 per vg / ml 6 pg / ml residual A rAAV viral vector containing the plasmid DNA is obtained, and the rAAV viral vector is The tar is formulated for administration and / or contains approximately 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 containing about 6.0 x 10 1 3 vg of a unit dose of an rAAV viral vector, the rAAV viral vector comprising: 1×10 13 1.7 × 10 per vg / ml 6 pg / ml or less, or 1 x 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. In some embodiments, the formulation or pharmaceutical composition is approximately 1.2 x 10 14 vg of rAAV viral vector, The viral vector was 1 × 10 13 1.7 × 10 per vg / ml 6 pg / ml or less, or is 1 x 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 x 10 14 vg of rAAV viral vector The dose of rAAV viral vectors was 1 x 10 13 1.7 × 10 per vg / ml 6 pg / ml or less, or 1 x 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 0.2ng per vg, 1. 0×10 13 Less than 0.1 ng per vg, or 1.0 x 10 13 0.09ng per vg and resulting in an rAAV viral vector having a benzonase concentration of less than The vector is formulated for administration and / or contains about 6.0 x 10 13 In unit doses of vg In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising 1.0×10 13 vgata Less than 0.2 ng, 1.0 × 10 13 Less than 0.1 ng per vg, or 1.0 x 10 13 rAAV viral vectors with benzonase concentrations of less than 0.09 ng per vg. The rAAV viral vector is formulated for administration and / or administered in a volume of about 1.2 x 1 0 14 In some embodiments, the method comprises the steps of: .0×1013 Less than 0.2 ng 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 the rAAV viral vector is formulated for administration, and / or approximately 2.4 × 10 14 In some embodiments, the compound is present in a unit dose of 0.05 mg. In embodiments, the formulation or pharmaceutical composition contains about 6.0 x 10 13 vg rAAV viral 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 x 10 13 vg a In some embodiments, the formulation or The pharmaceutical composition contains about 1.2 x 10 14 Contains a unit dose of rAAV viral vector (vg) , rAAV viral vector 1.0 × 10 13 Less than 0.2ng per vg, 1.0x 10 13 Less than 0.1 ng per vg, or 1.0 x 10 13 Less than 0.09ng per vg In some embodiments, the formulation or pharmaceutical composition has a benzonase concentration of about 2. 4×10 14 rAAV viral vector unit dose of 1000 mg / kg, The culprit is 1.0 x 10 13 Less than 0.2 ng per vg, 1.0 × 10 13 0 per vg Less than 0.1ng or 1.0 x 10 13 Benzonase concentrations below 0.09 ng per vg Has.

[0123] In some embodiments, the method comprises: 13 Less than 0.5ng per vg, 1. 0×10 13 Less than 0.3 ng per vg or 1.0 x 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 about 6.0 x 10 13 In some embodiments, the method comprises administering a 1.0× unit dose of 1.00 mg of 1.0 x 1 ... 10 13 Less than 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 approximately 1.2 x 10 14 It is present in the pharmaceutical composition in a unit dose of vg. In some embodiments, the method comprises: 13 Less than 0.5ng per vg, 1.0x 10 13 Less than 0.3 ng per vg or 1.0 x 10 13 Less than 0.22ng per vg rAAV viral vectors with a bovine serum albumin (BSA) concentration of r The AAV viral vector is formulated for administration and / or administered in an amount of 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 viral vector unit dose of 1000 mg / kg, The virus vector is 1.0 × 10 13 Less than 0.5 ng per vg, 1.0 × 1013 vg Less than 0.3 ng per unit volume, or 1.0 x 10 13 Bovine serum albumin less than 0.22ng per vg In some embodiments, the formulation or pharmaceutical composition has a concentration of about 1.2×10 14 rAAV virus vector unit dose of 1000 mg / kg, The spectrum is 1.0×10 13 Less than 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 0.22ng per vg In some embodiments, the formulation or pharmaceutical composition has a BSA concentration of about 2. 4×10 14 rAAV viral vector unit dose of 1000 mg / kg, The culprit is 1.0 x 10 13 Less than 0.5 ng per vg, 1.0 × 10 13 0 per vg Less than 0.3ng 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 x 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 approximately 0.35 EU / mL per vg / mL, 1.0 x10 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 x 10 13 Less than approximately 0.2 EU / mL per vg / mL Full, 1.0 x 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 × 10 13 Approximately 0.05 per vg / mL Less than EU / mL or 1.0 x 10 13 Less than approximately 0.02 EU / mL per vg / mL The rAAV viral vector has a toxin level, and the rAAV viral vector , and / or about 6.0 x 10 13 in a pharmaceutical composition at a unit dose of vg In some embodiments, the method comprises: 13 Approximately 1 per vg / mL Less than 1.0 × 10 EU / mL 13 Less than approximately 0.75 EU / mL per vg / mL, 1.0 x10 13 Less than approximately 0.5 EU / mL per vg / mL, 1.0 × 10 13 vg / mL Approximately less than 0.4 EU / mL, 1.0 × 10 13 Approximately 0.35 EU / mL per vg / mL Full, 1.0 x 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 viral vectors having endotoxin levels of less than L, The vector is formulated for administration and / or contains approximately 1.2 x 10 14 Medications in vg unit doses In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising 1.0×10 13 vg / mL Less than approximately 1 EU / mL per 1.0 x 10 13 Approximately 0.75 EU / mL per vg / mL Full, 1.0 x 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 Less than approximately 0.2 EU / mL, 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 vg / Less than approximately 0.05 EU / mL per mL, or 1.0 x 10 13 Approximately 0.0 per vg / mL rAAV viral vectors with endotoxin levels of less than 2 EU / mL, The V viral vector is formulated for administration and / or contains approximately 2.4 x 10 14 vg unit 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 1000 mg / kg, 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 0.5EU / 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 Less than approximately 0.3 EU / mL per 1.0 × 10 13 Approximately 0.25 EU / m vg / mL Less than L, 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 Approximately 0.0 ... Less than 1 EU / mL, 1.0 × 10 13 less than about 0.05 EU / mL per vg / mL, or 1.0×10 13 and endotoxin levels of less than about 0.02 EU / mL per vg / mL. In some embodiments, the formulation or pharmaceutical composition contains about 1.2 x 10 14 vg rAAV The unit dose of the rAAV viral vector is 1.0 x 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 0.35 EU / 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 × 10 13 Approximately 0.13 EU / vg / mL Less than 1.0 × 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 x 10 13 per vg / mL In some embodiments, the formulation or The pharmaceutical composition contains about 2.4 x 10 14 Contains a unit dose of rAAV viral vector (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 / Less than approximately 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 a method for determining whether a saturation level is less than 100 μg / g (ppm), 50 μg / g (ppm), or 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 approximately 6.0 x 10 13 In some embodiments, the pharmaceutical composition is present in a unit dose of 0.05 mg / kg. The method is to achieve a concentration of less than 100 μg / g (ppm), less than 50 μg / g (ppm), or less than 30 μg / g (ppm). resulting in an rAAV viral vector having a cesium concentration of less than g / g (ppm), The rAAV viral vector is formulated for administration and / or administered in an amount of about 1.2 x 10 14 v In some embodiments, the method comprises administering a pharmaceutical composition containing 100 μg of the compound to a patient 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 high concentration of cesium, - is formulated for administration and / or 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 volume of about 6.0 x 10 13 The rAAV viral vector contains 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 1000 mg / kg, 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 contains about 2.4 x 10 14 Contains a unit dose of rAAV viral vector (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 dose of about 6.0 x 10 13 v In some embodiments, the method comprises administering a pharmaceutical composition containing 10 to 100 mg of ethanol to the patient in a unit dose of about 10 to 100 mg of ethanol. 00 ppm, 15-90 ppm, or about 20-80 ppm of poloxamer 188 Producing rAAV viral vectors and formulating the rAAV viral vectors for administration and / or about 1.2 x 10 14 vg of unit dose in the pharmaceutical composition. In some embodiments, the method comprises 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 contains approximately 2.4 x 10 14 vg units In some embodiments, the formulation or pharmaceutical composition is present in a dosage of about 6 .0×10 13 rAAV virus containing a unit dose of 1000 mg of rAAV virus vector The vector may be present in a concentration of about 10 to 100 ppm, 15 to 90 ppm, or about 20 to 80 ppm of polysaccharide. In some embodiments, the formulation or pharmaceutical composition has about 1.2x 10 14 vg of rAAV viral vector, - about 10 to 100 ppm, 15 to 90 ppm, or about 20 to 80 ppm of poloxamer In some embodiments, the formulation or pharmaceutical composition has about 2.4 x 10 1 4 vg of a unit dose of an rAAV viral vector, the rAAV viral vector comprising: Poloxamer 18 at approximately 10-100 ppm, 15-90 ppm, or approximately 20-80 ppm It has 8.

[0127] In some embodiments, the method comprises the step of: providing less than 2000 cells per container that are 25 μm or larger in size; rAAV virus with 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 0.05 mg / vg. The method is to count less than 2000 particles, less than 1500 particles, and less than 10 particles of 25 μm or more in size per container. rAAV viral vectors having less than 00 or less than 600 particles, The AV viral vector is formulated for administration and / or contains approximately 1.2 x 10 14 vg In some embodiments, the method comprises administering a single dose of the compound to a patient in a pharmaceutical composition per container. The number of particles is less than 2000, less than 1500, less than 1000, or less than 600 with a size of 25 μm or more. and resulting in an rAAV viral vector having a particle size of 100 μm, 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 contains about 6.0 x 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 1.2x 10 14 vg of rAAV viral vector, - Less than 2000 particles, less than 1500 particles, less than 1000 particles, each with a size of 25 μm or more per container In some embodiments, the formulation or pharmaceutical composition has fewer than 100 or fewer than 600 particles. is approximately 2.4 x 10 14 vg of rAAV viral vector, Viral vectors are limited to 2,000 or less, 1,500 or more, with a size of 25 μm or more per container. less than 1000 particles or less than 600 particles.

[0128] In some embodiments, the method comprises: rAAV viruses having less than 8000, less than 1000, or less than 6000 particles the rAAV viral vector is formulated for administration, and / or or approximately 6.0 x 10 13 In some embodiments, the pharmaceutical composition is present in a unit dose of 0.05 mg / vg. The method is to limit the number of particles per container to less than 10,000 particles with a size of 10 μm or more, or less than 8,000 particles. resulting in rAAV viral vectors having less than 1,000 or less than 6,000 particles. and the rAAV viral vector is formulated for administration and / or contains approximately 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 viral vector having less than 6,000 particles, and The vector is formulated for administration and / or contains approximately 2.4 x 10 14 Medications in vg unit doses 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, is less than 10,000 particles, less than 8,000 particles, and 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 approximately 1.2 x 10 14 vg of rAAV viral vector, V viral vectors are contained in a container containing less than 10,000 cells with a size of 10 μm or more, and 800 In some embodiments, the particle size is less than 0, less than 1000, or less than 6000 particles. , the formulation or pharmaceutical composition is about 2.4 x 10 14 vg of rAAV viral vector The rAAV viral vectors were placed in 100 tubes of 10 μm or larger size per tube. 00, less than 8000, less than 1000 or less than 6000 particles.

[0129] In some embodiments, the method comprises a step of: The rAAV virus vector has a pH between 0.8 and 8.3. The vector is formulated for administration and / or contains about 6.0 x 10 13 In unit doses of vg In some embodiments, the method comprises administering a 200 mg / kg / day or 200 mg / kg / day dose of 7.5 to 8.5 mg / kg / day of acetone in a pharmaceutical composition. 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 in a volume of about 1.2 x 1 0 14 In some embodiments, the method comprises administering to a subject a pharmaceutical composition comprising administering to said subject a pharmaceutical composition comprising: rAA with a pH between 0.5 and 8.5, between 7.6 and 8.4, or between 7.8 and 8.3 the rAAV viral vector is formulated for administration, and / or approximately 2.4 × 10 14 In some embodiments, the compound is present in a unit dose of 0.05 mg. In embodiments, the formulation or pharmaceutical composition contains about 6.0 x 10 13 vg rAAV viral 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 contains about 1.2 x 10 14 Contains a unit dose of rAAV viral vector (vg) , the rAAV viral vector is between 7.5 and 8.5, between 7.6 and 8.4, or 7.8 In some embodiments, the formulation or pharmaceutical composition has a pH between about 2. 4×10 14 rAAV viral vector unit dose of 1000 mg / kg, 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. Has.

[0130] In some embodiments, the method comprises administering a blood glucose level between 330 and 490 mOsm / kg, between 360 and 46 rAA with an osmolality between 0 mOsm / kg or 390-430 mOsm / kg the rAAV viral vector is formulated for administration, and / or approximately 6.0 × 10 13 In some embodiments, the compound is present in a unit dose of 0.05 mg. In embodiments, the method includes administering a blood glucose level between 330 and 490 mOsm / kg, between 360 and 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 manner that is approximately 1 .2×10 14 In some embodiments, the method comprises administering to the subject a dose of 0.05 mg of the compound of formula (I) to the subject. The method is between 330 and 490 mOsm / kg, between 360 and 460 mOsm / kg, or 3 rAAV viral vectors with osmolality between 90 and 430 mOsm / kg , the rAAV viral vector is formulated for administration, and / or about 2.4 x 10 14 In some embodiments, the formulation or pharmaceutical composition is present in a unit dose of vg. The object is approximately 6.0 x 10 13 vg of rAAV viral vector, V viral vectors were administered between 330 and 490 mOsm / kg and 360 and 460 mOsm / kg, or osmolality between 390 and 430 mOsm / kg. In this embodiment, the formulation or pharmaceutical composition contains about 1.2 x 10 14 vg rAAV viral vector The unit dose of rAAV viral vector is between 330 and 490 mOsm / kg. Osmolality between 360 and 460 mOsm / kg or between 390 and 430 mOsm / kg In some embodiments, the formulation or pharmaceutical composition has about 2.4 x 10 14 vg's r The unit dose of AAV viral vector is 330-4 Between 90 mOsm / kg, between 360 and 460 mOsm / kg, or between 390 and 430 mO It has an osmolality between 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 are then injected with a rAAV virus vector having a high viral titer. and / or approximately 6.0 x 10 13 The amount of the compound present in the pharmaceutical composition is determined by a unit dose of vg. 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 rAAV viral vectors with an infectious titer of U. is formulated for administration, and / or about 1.2 x 10 14 Pharmaceutical composition in a unit dose of vg In some embodiments, the method comprises: 13 Approximately 1.0 per vg x108 ~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 drug is formulated for administration and / or contains approximately 2.4 x 10 14 Drug in vg unit dose In some embodiments, the formulation or pharmaceutical composition is present in a composition containing about 6.0 x 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 x 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. In this embodiment, the formulation or pharmaceutical composition contains about 1.2 x 10 14 vg rAAV viral vector The unit dose of rAAV viral vectors contains 1.0 x 10 13 Approximately 1.0 per vg x10 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 viral vector unit dose of 1000 mg / kg, The culprit is 1.0 x 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 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%, and The vector is formulated for administration and / or contains approximately 6.0 x 10 13 Medications in vg unit doses In some embodiments, the method comprises administering to the subject an antibody or antibody fragment present in a pharmaceutical composition. Based on (a), approximately 30 to 150%, approximately 60 to 150%, relative to a reference standard and / or appropriate control rAAV viral vectors with a relative titer of 140%, or approximately 70-130% and the rAAV viral vector is formulated for administration and / or contains approximately 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, rAAV viruses with relative titers of about 150%, about 60-140%, or about 70-130% the rAAV viral vector is formulated for administration, and / or or approximately 2.4 x 10 14 In some embodiments, the pharmaceutical composition is present in a unit dose of 0.05 mg / vg. In this case, the formulation or pharmaceutical composition is about 6.0 x 10 13 vg rAAV viral vector single The rAAV viral vectors are 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. In some embodiments, the formulation or pharmaceutical composition The object is approximately 1.2 x 10 14 vg of rAAV viral vector, V viral vectors are expressed in vitro in cell-based assays 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 x 10 1 4 vg of a unit dose of an rAAV viral vector, the rAAV viral vector comprising: Based on in vitro cell-based assays in conjunction with reference standards and / or appropriate controls; The relative potency is about 30 to 150%, about 60 to 140%, or about 70 to 130%.

[0133] In some embodiments, the method comprises: 13 Approximately 10-500 μg 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 dose of about 6.0 x 10 13 v In some embodiments, the method comprises administering a 1.0×100 mg / kg unit dose of 1.0 x 100 mg of ethanol to the pharmaceutical composition. 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 Producing rAAV viral vectors and formulating the rAAV viral vectors 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 10-500 μg per vg, 1.0× 10 13 Approximately 50-400 μg per vg, or 1.0 × 10 13 Approximately 100-3 per vg rAAV viral vectors with a total protein level of 0.1 μg, The viral vector is formulated for administration and / or contains approximately 2.4 x 10 14 vg units In some embodiments, the formulation or pharmaceutical composition is present in a dosage of about 6 .0×10 13 rAAV virus containing a unit dose of 1000 mg of rAAV virus vector Vector is 1.0 x 10 13 Approximately 10-500 μg per vg, 1.0 × 10 13 vg a Approximately 50-400 μg 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 vg of rAAV viral vector, - is 1.0 x 10 13 Approximately 10-500 μg per vg, 1.0 × 10 13 Approximately per vg 50 to 400 μg or 1.0 × 10 13 Approximately 100-300 μg of total protein per vg In some embodiments, the formulation or pharmaceutical composition has a quality level of about 2.4 x 10 1 4 vg of a unit dose of an rAAV viral vector, the rAAV viral vector comprising: 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 S administered a dose of vg / kg As judged by median survival time in MNΔ7 mice, the survival time was >15 days, >20 days, and >2 days. The present invention provides rAAV viral vectors with in vivo efficacy of more than 2 days or more than 24 days. and the rAAV viral vector is formulated for administration and / or contains approximately 6.0 x 10 1 3 In some embodiments, the method comprises the step of: x10 13 Median survival time in SMNΔ7 mice receiving a dose of 1000 mg / kg In vivo efficacy of 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 and / or approximately 1.2 x 10 14 present in the pharmaceutical composition in a unit dose of vg In some embodiments, the method comprises: 13 S administered a dose of vg / kg As judged by median survival time in MNΔ7 mice, the survival time was >15 days, >20 days, and >2 days. The present invention provides rAAV viral vectors with in vivo efficacy of more than 2 days or more than 24 days. and the rAAV viral vector is formulated for administration and / or contains approximately 2.4 x 10 1 4 In some embodiments, the formulation or pharmaceutical composition is present in a unit dose of 0.05 mg / kg. The product is approximately 6.0 × 10 13 vg of rAAV viral vector, AV viral vectors were 7.5 × 10 13 SMNΔ7 mice administered a dose of 1000 mg / kg As judged by the median survival time in mice, the survival rates were more than 15 days, more than 20 days, and more than 22 days. or has an in vivo efficacy of greater than 24 days. The object is approximately 1.2 x 10 14 vg of rAAV viral vector, V viral vectors were 7.5 × 10 13 SMNΔ7 mice administered a dose of 1000 mg / kg As judged by the median survival time in the study, the survival rates were more than 15 days, more than 20 days, more than 22 days, or has an in vivo efficacy of greater than 24 days. is approximately 2.4 x 10 14 vg of rAAV viral vector, The viral vector was 7.5 × 10 13 SMNΔ7 mice administered a dose of vg / kg As judged by the median survival time in the has an in vivo efficacy of more than 24 days.

[0135] In some embodiments, the formulation or pharmaceutical composition contains about 6.0 x 10 13 vg rAAV A unit dose of viral vector and one or more of the following shipping criteria: less than about 8%, less than about 7%, or less than about 5% empty viral capsids; 1 x 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 pg / mL or less than 6.8 × 10 5 Less than pg / mL of residual host cell DNA (hcDNA );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 At least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least 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 / ml 1×10 13 1.7 × 10 per vg / mL 6 pg / Residual plasmid DNA less than 1.0 x 10 mL 13 Less than 0.2ng per vg, 1.0 x10 13 Less than 0.1 ng per vg, or 1.0 x 10 13 Less than 0.09ng per vg Benzonase concentration: 1.0 x 10 13 Less than 0.5 ng per vg, 1.0 × 1013 Less than 0.3 ng per vg or 1.0 x 10 13 Less than 0.22ng per vg of bovine blood 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 / Less than approximately 0.5 EU / mL, 1.0 × 10 13 Approximately 0.4 EU / vg / mL Less than 1.0 × 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 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 / m Less than approximately 0.1 EU / mL per L, 1.0 × 10 13 Approximately 0.05EU / vg / mL Less than mL or 1.0 x 10 13 Endotoxin levels of less than approximately 0.02 EU / mL per 1000 mg / mL Bel: Less than 100 μg / g (ppm), less than 50 μg / g (ppm), or less than 30 μg / 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 or larger per container Less than, 1500, 1000, or 600 particles per container; size are 10 μm or larger and less than 10,000 particles, less than 8,000 particles, less than 1,000 particles, or 6,000 particles particles; 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 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 related to 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 contains about 6.0 x 10 13 vg rAAV A unit dose of viral vector, and one or more of the following shipping standards: about 7.7 to pH 8.3; osmolality approximately 390-430 mOsm / kg; 25 μm or more per container Less than approximately 600 particles of 10 μm or larger size per container; Less than approximately 6000 particles of 10 μm or larger size per container Child: Approximately 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 an 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 approximately 5% empty capsids; greater than or equal to approximately 95% total purity; approximately 0.13 EU / mL The following endotoxins:

[0137] In some embodiments, the formulation or pharmaceutical composition contains about 6.0 x 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 of 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 <0.22ng BSA; 1.0 x 10 13 Approximately 6.8 × 10 per vg 5 Sub-pg residues Plasmid DNA: 1.0 × 10 13 Approximately 1.1 × 10 per vg 5 Less than pg of residual hcD NA: 1.0×10 13 Less than approximately 4 ng of rHCP per vg; pH of approximately 7.7 to 8.3; Osmolality of approximately 390-430 mOsm / kg; approximately 600 per container with a size of 25 μm or more Less than 6000 particles of 10 μm or larger size per container; Less than 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 approximately 70-130% relative to an appropriate control; less than approximately 5% empty caps Sid.

[0138] In some embodiments, the formulation or pharmaceutical composition contains about 1.2 x 10 14 vg rAAV A unit dose of viral vector and one or more of the following shipping criteria: less than about 8%, less than about 7%, or less than about 5% empty viral capsids; 1 x 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 pg / mL or less than 6.8 × 10 5 Less than pg / mL of residual host cell DNA (hcDNA );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 At least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least 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 / ml 1×10 13 1.7 × 10 per vg / mL 6 pg / Residual plasmid DNA less than 1.0 x 10 mL 13 Less than 0.2ng per vg, 1.0 x10 13 Less than 0.1 ng per vg, or 1.0 x 10 13 Less than 0.09ng per vg Benzonase concentration: 1.0 x 10 13 Less than 0.5 ng per vg, 1.0 × 10 13 Less than 0.3 ng per vg or 1.0 x 10 13 Less than 0.22ng per vg of bovine blood 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 / Less than approximately 0.5 EU / mL, 1.0 × 10 13 Approximately 0.4 EU / vg / mL Less than 1.0 × 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 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 / m Less than approximately 0.1 EU / mL per L, 1.0 × 10 13 Approximately 0.05EU / vg / mL Less than mL or 1.0 x 10 13 Endotoxin levels of less than approximately 0.02 EU / mL per 1000 mg / mL Bel: Less than 100 μg / g (ppm), less than 50 μg / g (ppm), or less than 30 μg / 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 or larger per container Less than, 1500, 1000, or 600 particles per container; size are 10 μm or larger and less than 10,000 particles, less than 8,000 particles, less than 1,000 particles, or 6,000 particles particles; 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 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 related to 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 contains about 1.2 x 10 14 vg rAAV A unit dose of viral vector, and one or more of the following shipping standards: about 7.7 to pH 8.3; osmolality approximately 390-430 mOsm / kg; 25 μm or more per container Less than approximately 600 particles of 10 μm or larger size per container; Less than approximately 6000 particles of 10 μm or larger size per container Child: Approximately 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 an 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 approximately 5% empty capsids; greater than or equal to approximately 95% total purity; approximately 0.13 EU / mL The following endotoxins:

[0140] In some embodiments, the formulation or pharmaceutical composition contains about 1.2 x 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 of 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 <0.22ng BSA; 1.0 x 10 13 Approximately 6.8 × 10 per vg 5 Sub-pg residues Plasmid DNA: 1.0 × 10 13 Approximately 1.1 × 10 per vg 5 Less than pg of residual hcD NA: 1.0×10 13 Less than approximately 4 ng of rHCP per vg; pH of approximately 7.7 to 8.3; Osmolality of approximately 390-430 mOsm / kg; approximately 600 per container with a size of 25 μm or more Less than 6000 particles of 10 μm or larger size per container; Less than 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 approximately 70-130% relative to an appropriate control; less than approximately 5% empty caps Sid.

[0141] In some embodiments, the formulation or pharmaceutical composition contains about 2.4 x 10 14 vg rAAV A unit dose of viral vector and one or more of the following shipping criteria: less than about 8%, less than about 7%, or less than about 5% empty viral capsids; 1 x 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 pg / mL or less than 6.8 × 10 5 Less than pg / mL of residual host cell DNA (hcDNA );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 At least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least 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 / ml 1×10 13 1.7 × 10 per vg / mL 6 pg / Residual plasmid DNA less than 1.0 x 10 mL 13 Less than 0.2ng per vg, 1.0 x10 13 Less than 0.1 ng per vg, or 1.0 x 10 13 Less than 0.09ng per vg Benzonase concentration: 1.0 x 10 13 Less than 0.5 ng per vg, 1.0 × 10 13 Less than 0.3 ng per vg or 1.0 x 10 13 Less than 0.22ng per vg of bovine blood 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 / Less than approximately 0.5 EU / mL, 1.0 × 1013 Approximately 0.4 EU / vg / mL Less than 1.0 × 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 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 / m Less than approximately 0.1 EU / mL per L, 1.0 × 10 13 Approximately 0.05EU / vg / mL Less than mL or 1.0 x 10 13 Endotoxin levels of less than approximately 0.02 EU / mL per 1000 mg / mL Bel: Less than 100 μg / g (ppm), less than 50 μg / g (ppm), or less than 30 μg / 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 or larger per container Less than, 1500, 1000, or 600 particles per container; size are 10 μm or larger and less than 10,000 particles, less than 8,000 particles, less than 1,000 particles, or 6,000 particles particles; 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 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 related to 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 contains about 2.4 x 10 14 vg rAAV A unit dose of viral vector, and one or more of the following shipping standards: about 7.7 to pH 8.3; osmolality approximately 390-430 mOsm / kg; 25 μm or more per container Less than approximately 600 particles of 10 μm or larger size per container; Less than approximately 6000 particles of 10 μm or larger size per container Child: Approximately 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 an 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 approximately 5% empty capsids; greater than or equal to approximately 95% total purity; approximately 0.13 EU / mL The following endotoxins:

[0143] In some embodiments, the formulation or pharmaceutical composition contains about 2.4 x 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 of 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 <0.22ng BSA; 1.0 x 10 13 Approximately 6.8 × 10 per vg 5 Sub-pg residues Plasmid DNA: 1.0 × 10 13 Approximately 1.1 × 10 per vg 5 Less than pg of residual hcD NA: 1.0×10 13 Less than approximately 4 ng of rHCP per vg; pH of approximately 7.7 to 8.3; Osmolality of approximately 390-430 mOsm / kg; approximately 600 per container with a size of 25 μm or more Less than 6000 particles of 10 μm or larger size per container; Less than 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 approximately 70-130% relative to an appropriate control; less than approximately 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 hereby incorporated by reference. , the contents of which are incorporated herein by reference in their entirety for all purposes. [Example]

[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 x 10 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. scAAV9.CB.SMN-treated mice Approximately 42±2% of lumbar motoneurons were transduced in the 2-HT2B1 / 2 mice. SMN levels were also significantly higher in the 2-HT2B1 / 2 mice. 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 at P1 was increased compared to that of the WT control. Recovery of SMA animals treated with either CB.SMN or scAAV9.CB.GFP The ability to catch the serotonin was assessed and compared with wild-type (WT) control mice and untreated mice. The mice recovered 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 significantly improved in the open field test and wheel running test. The running assay was nearly identical to the WT control.

[0146] Survival of SMN-treated SMA animals was significantly improved compared with 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 sharply until death, whereas SMN Mice showed steady weight gain until around P40, stabilizing at 17 g (approximately half the weight of WT controls). The smaller corrected animal size reflects the tropism and incomplete expression of scAAV9. Transduction resulted in "chimeric" animals in which some cells were not transduced. Furthermore, the smaller size may explain the role of SMN in fetal development. Most strikingly, SMN-treated mice survived well beyond 250 days of age. was doing.

[0147] Toxicological biodistribution was also studied. In a non-clinical (non-GLP) study, 24 mice were and four non-human primates (NHPs) administered scAAV9.CB.SMN 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 mice (6 males / 9 females) along with B.SMN (6 males / 9 females). This dose was injected into SMA16 SMNΔ7 mice. P1 mice have previously been shown to be the most effective in this model. Simulate clinical trials in infants, the intended target population for in-human clinical trials All mice survived the injection procedure without any signs of distress or weight loss. All rats 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 tests and clinical chemistry evaluations (ALT, AST, 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, with the blood collection site being 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 autopsied. All organs were normal; in particular, none of the organs (heart, liver, kidney) No inflammation was observed in any part of the body (spleen, muscles, gonads, brain, lungs, lymph nodes, and 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 dose 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 animals were observed for six months until they were killed at approximately nine months of age. All clinical chemistries were normal. Peripheral blood mononuclear cells (PBMCs) were analyzed using ELISpot. When T cell immune responses were tested in the mice, all were 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 transfusion 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 a 99.9% improvement in schizophrenia at 24 weeks post-treatment. No test article-related mortality or evidence of toxicity was observed. Treatment-related mean body weight and mean body weight gain The decrease in the activated partial thromboplastin time (APTT) value was due to the mild effects of the treatment. However, no toxicity was observed.

[0152] The activity of scAAV9.CB.SMN was investigated by biodistribution and 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 In males and females given 1000 mg / kg (Group 3), the results were as follows: 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 is 3.3 × 10 14 in vg / kg It is considered to be there.

[0153] In these studies, intrathecal administration of scAAV9.CB.SMN into the CSF significantly improved the response of mice (1 It was safe and well tolerated in naive rats (up to 2 weeks after injection) and 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 transgene expression. The results show that the amount of Trendelenburo was higher in the cervical and lumbar regions compared to the thoracic region. The subjects were kept in the diaphragmatic position for 5 minutes 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 were observed in the brain or spinal cord. , was not observed.

[0154] In a central GLP (Good Laboratory Practice) 3-month mouse toxicity study, Following intravenous injection into mice, the vector and transgene were administered intravenously. 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 hepatocellular hypertrophy, Kupffer cells, and fibrosis, and inflammation and thrombosis in the atria. AVXS-101-associated cardiac events in mice were characterized by hepatocyte activation and scattered hepatocyte necrosis. No NOAEL was identified for liver and pulmonary findings, and the maximum tolerated dose was 1.5 x 10 14 vg / kg, and the recommended therapeutic dose is 1.1 × 10 14 vg / kg, and This provides a 1.4-fold safety margin. The 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 was in a head-down Trendelenburg position for 5 or 10 minutes, A detailed biodistribution analysis was performed on human primate tissues (n=5). These animals were tested for improved brain distribution, making this approach advantageous 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 deoxyribonucleic acid (DNA) and RNA characterization. Biodistribution analysis was performed. scAAV9.CBA.GFP was distributed to most tissues except the spleen and liver. The levels were low in all peripheral tissues compared with high levels in the brain and spinal cord. These findings are consistent with those of other groups of studies. This is consistent with previous reports from the 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. whereas in soft tissues and glands, the level of RNA was higher than expected for the detected viral genome. In particular, the testes, intestine, and spleen are 1.0 times lower than the DNA. Despite the detection of AAV in peripheral organs, the whole body The amount of vector detected in the periphery was significantly reduced compared to that in the control group. al.; Gray et al. Furthermore, at P1 after 24 weeks of treatment, intravenous or intravenous Similar observations were made when comparing mice injected intravenously with 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 When administered at a dose 10 times lower than that administered intravenously, Widespread transgene expression was observed throughout the spinal cord of mice and non-human primates. In rhesus monkeys, lower doses can be used than in mice, resulting in similar motor neuron targeting efficiency. Maintaining the subject in the Trendelenburg position to facilitate vector spread It was found that the effect of introducing the 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-minute tilt increased the transduction of motor neurons in the cervical, thoracic, and lumbar regions by 55 minutes, respectively. , 62, and 80%, which is comparable to the levels observed in mouse models. According to Skew, this means a significant benefit for patients. correlates closely with the quantification of GFP transcripts in each spinal cord segment.

[0158] Example 1 - Clinical Trial Protocol The Phase 1 open-label, single-dose clinical trial was conducted in patients with a genetic diagnosis consistent with SMA, biallelic SMN1 The patient had a deletion of the gene and three copies of SMN2 without genetic modifiers, and was at locus 1 at the time of study enrollment. It is performed on infants and children who can stand but cannot stand or walk. Patients will be randomized to receive the study in a dose-comparison safety study of up to three potential therapeutic doses, as described below. Patients will be administered AVXS-101. Patients will be between 6 and 24 months of age at the time of administration. The group will be stratified into two groups: one group (aged 24 months or older and under 60 months at the time of administration) and the other group (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 will be enrolled, and at least 60 patients aged 24 months to over will be enrolled. Twelve patients under the age of 1 month will be enrolled.

[0159] The first cohort consisted of 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 were All potential, probable, or certain Consult with the Data Safety Monitoring Board (DSMB) at the time of enrollment of the first three patients. Subsequently, based on the available safety data, the drug is either a) discontinued due to toxicity or b) discontinued. Amount B is used to determine whether to proceed to cohort 2.

[0160] For dose B, 1.2 × 10 14 vg of AVXS-101 (Dose B), 6 Three patients under 0 months of age will be enrolled. Again, between dosing of the three patients in the cohort Three patients in Cohort 2 and all patients in Cohort 1 had at least a 4-week interval between treatments. Available safety data suggests an additional 4-week interval between doses between patients is necessary. The investigator will consider any 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, the study was discontinued if: a) the study was discontinued due to toxicity; or b) the study was discontinued for 6 months. 12 patients aged 24 months or older and under 24 months and 12 patients aged 24 months or older and under 60 months were randomly assigned to the A decision will be made on whether to continue enrolling 21 more patients until they receive 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 under 60 months of age will be included. , up to 2.4 × 10 administered intrathecally 14 Cohort 1 and Cohort 2 will receive dose C of 1000 mg / kg. 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 under 24 months and 24 months Twenty-one more 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 effectiveness 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 vg / kg are safe for systemic exposure in children weighing up to 8.4 kg. (intravenous) administered (total dose 9.24 × 10 14 vg). Furthermore, in preclinical studies 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 is monitored by monitoring adverse event (AE) reports and concomitant medication use. by conducting a physical examination, vital signs assessment, cardiovascular assessment, and laboratory evaluation Patients will be observed in the hospital for 48 hours after the intrathecal injection. Patients will return for follow-up visits on days 21 and 30. After the 30-day visit, Dose administration will be followed by monthly returns for 12 months. Upon completion of the study, study patients will receive AVXS Patients are being asked to enroll in a pivotal long-term follow-up study examining the sustained 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 administered in the dose-escalation schedule specified herein. Allocate 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 monitor the trial throughout the trial. The DSMB will continue to monitor safety data. 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 be related to Enrollment in the study will be stopped by the investigator if toxicity is experienced. These include: , patient death, significant clinical laboratory findings, or serious local injection site reactions associated with the administration of the investigational drug. 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 upon recommendation of regulatory authorities. Finally, patients may be required to undergo gene replacement therapy. Possibly, probably, or definitely related to therapy; related to clinical symptoms and / or unexpected CTCAE Grade III or higher AE / toxicity requiring treatment The study may be terminated if an unacceptable level of toxicity occurs, defined as an incidence .

[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 confirmation during the screening period for genotype, the patient must be 6 months or older and 60 months of age (1 Patients up to 800 days of age Genotypic confirmation of diagnosis includes homozygous deletion of SMN1 exon 7; SMN2 This includes experimental evidence of exactly three copies of the 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. is defined as the unsupported head position according to the World Health Organization (WHO)-MGRS standard. This is defined as being able to sit upright for at least 10 seconds. should not be used for balancing or postural support (Wijnhoven 2004). . 5. Age-appropriate institutional standards for the use of anesthesia and sedation as deemed necessary by the investigator. The following conditions must be met. 6. Up-to-date childhood vaccinations. According to the American Academy of Pediatrics (AAP 2009), Palivizumab prophylaxis (Sina) for preventing respiratory syncytial virus (RSV) infection Seasonal vaccinations, including flu-like flu (also known as flu-like flu), 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 tap 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-draining shunt or an implanted CNS catheter. 3. Functional measures (e.g., interferes with ability to achieve / demonstrate physical activity (e.g., standing, walking) or interferes with ability to receive IT administration; Severe contracture. Radiographically evident severe scoliosis (defined as a spinal curvature of 50 degrees or more). (This is the case.) 4. Preceding, planned, or anticipated scoliosis within 1 year of dose administration Reparative surgery / procedure. 5. Use of invasive ventilatory support (tracheostomy with positive pressure) or patient awareness at screening Pulse oximetry saturation less than 95% in the state or at altitudes above 1000 m Oxygen saturation below 92% while the patient is awake - Pulse oximetry saturation must be within 4% between screening and the highest value on the day of administration It should not decrease by more than this point. 6. Patients who have used or required non-invasive ventilatory support for 12 hours or more daily within the 2 weeks prior to administration are. 7. Gastric feeding tube where the majority of nutritional support is given parenterally (i.e., nasogastric) medical need for a tube (nasojejunal or nasojejunal tube) or weight for age not in line with the WHO Child Gr The 3rd percentile was calculated based on the Lowth Standards (Onis 2006). Patients with a permanent gastrostomy tube placed before screening are not excluded. Not possible. 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 study enrollment Respiratory infections requiring increased supportive care. 11. Severe non-pulmonary / respiratory tract infection (e.g., pyelonephritis or meningitis) within 4 weeks prior to study administration 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 tumor, or LP procedure or C MRI or CT abnormalities that interfere with SF circulation. 13. Prednisolone or other glucocorticosteroids or their excipients Known allergies or hypersensitivities. 14. Known allergy or hypersensitivity to iodine or iodine-containing products. 15. Medications for the treatment of myopathy or neuropathy, or for the treatment of diabetes mellitus within 3 months of the study administration or ongoing immunosuppressive therapy, plasmapheresis, adalimumab, etc. Concomitant use of any immunomodulatory or immunosuppressive therapy (e.g., corticosteroids, cyclosporine, tacrolimus, methotrexate, cyclophosphamide, intravenous immunoglobulin Brin, rituximab). 16. Unable to refrain from use of laxatives or diuretics within 24 hours prior to dose administration. 17. Anti-AAV9 antibody titer greater than 1:50 as measured by ELISA binding immunoassay - If the patient candidate demonstrates an anti-AAV9 antibody titer of greater than 1:50, the patient will be considered for inclusion in the study from the screening period. Retesting may be performed within 30 days, provided 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 greater than 18 g / Dl, 20,000 / cmm (More than WBC). 19. Participation in a recent SMA treatment clinical trial or any of the following prior to screening for this study At the time of writing, 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 administration -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 consent forms.

[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 toxicity, defined as the occurrence of adverse events / toxicities of I or higher If a patient dies, the trial may be terminated. If a patient dies, the trial will be terminated. In that case, untreated patients will be excluded. In cases where patients die after participating in a gene transfer study, an autopsy is required. Comply with three or more consecutive visits and study procedures required by the protocol, except as required by If the patient does not change their plan, they 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-actin It contains the cDNA of the human SMN gene under the control of the cytochrome-hybrid promoter (CB). The AAV is a non-replicating recombinant self-complementary adeno-associated virus serotype 9 (AAV9). The inverted terminal repeats (ITRs) are modified to promote intramolecular annealing of the transgene. thus forming a double-stranded transgene ready for transcription. This modified ITR, called (sc)ITR, allows the transgene to be transcribed and the resulting transcript to be 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] Pre-existing and concomitant medications The reference drug and concomitant medications must be submitted to the electronic case report form (eCFR) from 2 weeks before the study administration until the end of the clinical trial visit. Record the results on the CRF.

[0176] Prophylactic administration of prednisolone Antigen-specific T cell responses to AAV vectors were measured using AVXS-10 via intravenous infusion. This was observed in an ongoing Phase 1 clinical trial investigating treatment with 2 A response to such antigen-specific T cell responses is expected within 4 weeks. A possible consequence is the elimination of transduced cells and loss of transgene expression.

[0177] AVXS-10 in an attempt to suppress the host immune response to AAV-based therapies Patients were given prophylactic prednisolone (glucocorticoid) (approximately 1 mg / kg) 24 hours before administration. g / day). 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 If ALT levels exceed 2 times the upper limit of normal (ULN) or after 30 days of treatment T cell response was 100SFC / 10 6 If the value is higher than PBMC, the values ​​of AST and ALT Maintain prednisolone dose until T cell responses decrease below the 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 drugs 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, but only if such medications are administered at clinically appropriate levels , may be used to treat respiratory complications of SMA. - Ongoing immunosuppressive therapy, plasmapheresis, or adalimumab within 3 months of the start of the clinical trial immunomodulatory agents such as steroids, 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 part of routine clinical management. The use of prednisone in this situation is permitted at the discretion of the supervising physician. The drug should be appropriately documented, and the event that triggered its use should be reported as an AE. should be properly documented.

[0181] As part of care during the tapering of prednisolone, corticosteroids (bronchial If inhaled corticosteroids (excluding inhaled corticosteroids for seizures) are considered, the medical management of this The sponsor's medical monitor should be consulted regarding the tapering. Responsible for 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] Test product dose 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 x 10 as a third dose 14 vg of AVXS-101 was administered intrathecally. Direct delivery to the CSF via intrathecal injection delivers approximately 1000 mg of viral vector. The virus vector is a 1 / 1000 effective marker, which distributes evenly throughout the CNS. The burden on the patient can be reduced and further optimized. The justification for the trial is based on clinical findings from patients receiving prior treatment, 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 dose Amount 9.24×10 14 vg). Furthermore, in preclinical studies, 2 × 10 13 vg / kg dose Intrathecal administration of scAAV9.CB.SMN in large non-human primates was safe and The injections were well tolerated up to 14 months after treatment.

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

[0186] AVXS-101 is indicated 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 should be delivered to designated small pediatric intensive care unit (PICU) rooms or to acute care units. Other appropriate settings with immediate access to critical care (e.g., interventional rooms, operating rooms, specialized The patient is then delivered to the appropriate treatment room.

[0188] Patients should be placed in a PICU unit or other appropriate facility with immediate access to acute critical care. AVXS-101 intrathecally in a suitable setting (e.g., interventional room, operating room, dedicated procedure room) under sterile conditions. Patients were admitted to the hospital and given a 15 (+ / - 5) day follow-up period following AVXS-101 administration. 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, resulting in improved cerebrospinal fluid quality after lumbar puncture, including in children. It has been shown to reduce the risk of fluid leakage. 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 is at the discretion of the local anesthesiologist, but Trendelenburg positioning during and after the procedure is recommended. Incorporate a sufficient degree of sedative or anxiolytic to ensure analgesia and lack of movement. Patients should be placed in a 30° head-down position for 15 minutes following vector administration. The patient is placed in Trendelenburg position to facilitate distribution to the cervical and cerebral regions.

[0191] AVXS-101 will be administered by investigators, interventionalists, and other Fluorescence imaging is performed by a cardiologist or other appropriately trained and experienced physician. It is administered under sterile conditions under fluoroscopic / radiographic guidance. The patient is placed in the lateral position and the L3- Lumbar puncture into the L4 or L4-L5 interspinous space is used to insert a catheter with a proboscis needle into the arachnoid membrane. Subarachnoid cannulation allows the clear cerebrospinal fluid (CSF) to escape from the catheter. ) flow. Approximately 4 mL of CSF is removed for doses A and B, and approximately 4 mL of CSF is removed for dose C. removed a volume of CSF very close to the injected volume of AVXS-101 and contrast agent (maximum 7 mL), Dispose of according to your institution's guidelines. AVXS-101 in premixed contrast media Inject directly into the subarachnoid space. Administer 0.5 mL of saline according to institutional standards / guidelines. It is permissible to flush the needle with saline.

[0192] Post-administration procedures Following AVXS-101 administration, patients will be admitted to a designated PICU bed or other appropriate setting. Patients should be placed back on a regular schedule and their vital signs monitored closely. Concomitant medications and any AEs / serious AEs should also be monitored. Monitor and document administration procedures.

[0193] The patient was admitted to the PICU unit for 48 hours for closer monitoring of his mental status. other appropriate settings with immediate access to acute critical care (e.g., interventional wards, operating rooms, etc.) During the patient's hospitalization, staff will follow appropriate institutional standards for infection control. 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 agitation, neck pain, light sensitivity, and vomiting. Standardized IRB-approved handouts regarding the target will be provided. If the following criteria are met: The patient may be discharged if: Afebrile No hypersensitivity reactions No meningism No abnormal laboratory test results suggesting possible CNS infection or complications

[0194] Dose escalation All patients in Cohort 1 had a 4-week interval between doses before receiving the next dose. Safety analysis results at six time points (1, 2, 7, 14, 21, and 30 days) were available. To do so.

[0195] The investigator was responsible for determining whether any Grade III or higher AEs occurred within 48 hours of recognition. All potentially, probably, or definitely related to the investigational drug Discuss this with the DSMB before continuing enrollment. Age 6 months or older but under 24 months at the time of administration Based on the available safety data following enrollment of the first three patients, a) no adverse events were reported due to toxicity; or b) to proceed to cohort 2 using dose B.

[0196] For Dose B, during the first 3 doses in a cohort of patients younger than 60 months at the time of dosing The first 3 patients in Cohort 2 and all patients in Cohort 1 were randomly assigned to receive the IVF therapy, with at least 4 weeks between the two. Available safety data from patients suggests an additional 4-week gap between doses between patients. Investigators should consider Grade III thromboembolism occurring within 48 hours. Among the above AEs, those that are possibly, probably, or definitely related to the study drug All cases will be discussed with the DSMB before continuing enrollment. Enrollment of the first 6 patients Following enrollment, based on available safety data, the study will be discontinued a) 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 12 years of age have received Dose B Decide whether to

[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 will receive up to 2.4 x 10 14 vg of dose C. Cohort As with 1 and 2, there was also a 4-week period between doses in 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 children aged 6 months to 24 months 12 patients under 60 months of age and 12 patients aged 24 months to 60 months will receive dose C. A decision will be made on whether to continue enrolling 21 more patients until further notice.

[0198] Physiotherapy assessment: Hammersmith Functional Movement Scale Extension Hammersmith Functional Motor Scale Extended with Type 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 of the procedure. and monthly for all patients 24 months of age and older through month 12. Patients under 24 months of age will be assessed by the Hammersmith Functional Motor Scale at 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® 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 must be completed within 30 days prior to baseline administration. Then, monthly assessments are completed up to month 12. The Bayley Scales® assessments are videotaped. can be.

[0201] Physical Therapy Evaluation: Motor Milestone Development Survey Achievement of important motor milestones is assessed by the Bayley Scale® at each mile. 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 achieved each milestone on the Motor Milestone Development Study. Once observed, a movement 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 will complete an assessment of baseline milestone achievement according to Table 4. The assessment will be videotaped and findings will be documented. ) does not necessarily require a child to repeat previously achieved milestones, 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 Physical therapy 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 may also provide the research facility with home videotapes demonstrating achievement of functional skills. May be shared.

[0204] Videos are independently and centrally reviewed for unbiased assessment of milestone achievement. Using the Motor Milestone Development Survey, an independent judge will assess whether the video is 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] Other Clinical Assessment: Demographics / Medical History Patient demographic and medical history information was collected at baseline and included on the case report form (CRF). Medical history throughout the study will be collected at each visit. Spinal muscular atrophy, including but not limited to affected siblings 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 assessments: 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. Vital signs, including pulse oximetry and heart rate, should be monitored. During the injection, team members continuously monitored and recorded. Following the administration treatment, the patient was monitored every 15 (+ / - 5) minutes for 4 hours, then every 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.

[0207] Other clinical assessments: weight and length / height Weight, length and / or height will be measured at the times specified in Table 4, as appropriate.

[0208] Other clinical evaluations: Physical examination The physical examination includes a review of the following systems: head, eyes, ears, nose, and throat ( HEENT), lungs / thorax, cardiovascular system, abdomen, musculoskeletal system, nervous system, skin system, lymphatic system, and secretory system Genitourinary. Head circumference is measured at each physical examination. To measure head circumference, the examiner measures the circumference between the eyebrows. Measure a flexible tape measure around your head at 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 highest measurement recorded to the nearest 0.1 cm. A baseline physical examination will be performed according to the time points specified in Table 4 within 30 days of treatment. Complete.

[0209] Other Clinical Evaluations: Vaccination Recommendations Patients should continue to attend all regularly scheduled appointments as recommended by the Centers for Disease Control (CDC). Vaccination compliance is encouraged. According to the American Academy of Pediatrics (AAP 2009), respiratory Palivizumab prophylaxis (also known as Synagis) for preventing respiratory syncytial virus (RSV) infection Seasonal vaccinations, including those against the known strains of flu, 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. Conducted at months 6, 9, and 12 (or early termination). ECG tracings or ECG device data for centralized review by attending physicians 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 monitor will remain on for 48 hours (Day 3). Serial ECG data will be collected at the following times: Obtain data from the filter monitor 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 Echocardiography was performed at screening / baseline and at 3, 6, and 9 months. The study will be conducted at the first and 12-month visits (or early termination).

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

[0214] Other Clinical Evaluations: 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 require non-invasive ventilatory support will be monitored by study staff to record actual use data. You will be asked to bring the device to each study visit so that you can remove the SD card. The data will be transferred to a clinical database. Patients requiring 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 an interventional cardiologist in accordance with institutional guidelines. Fluoroscopy should be performed by a cardiologist or other appropriately trained and experienced physician. This procedure is performed under sterile conditions under visual inspection. X-ray image acquisition may not be necessary. .

[0216] Other clinical evaluations: Injection site photographs Photographs of the injection site were taken at the time points specified in Table 4 up to 30 days to monitor healing of the injection wound. -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 Act (CLIA)-certified local laboratory. In some cases, samples are processed for immediate results or other safety or regulatory reasons. May be harvested locally for gistic concerns.

[0218] [Table 5]

[0219] If there is not enough blood available from a patient, blood will be given to the recipient with the highest first priority and the recipient with the lowest last priority. Lowest priority, 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 completed genetic reconfirmation testing.

[0221] Other Clinical Evaluations: Hematology Hematology analysis included a CBC with differential and a platelet count with smear. Samples were collected from a central The samples will be collected and sent according to the laboratory manual provided. Immediate / same-day hematology analysis of the medication during administration 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 by a local 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; GGT, AST / ALT, serum total bilirubin, direct bilirubin, albumin Creatinine, 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 the FDA's 6.0 standard, CK-MB was used instead of Instead, troponin I was measured. Screening and enrollment were performed, but protocol revision 5 At the time of the implementation of Protocol Version 6.0, patients who had not yet received gene replacement therapy (Visit 2) Participants without a baseline troponin I test prior to treatment with AVXS-101 Troponin I will be tested instead of CK-MB. CK-MB will be tested in all other participants. The investigator receives test results for all study visits from the central laboratory ( -Except for the first day).

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

[0227] Other Clinical Evaluations: Coagulation Tests Coagulation tests include prothrombin time (PT), partial prothrombin time (PTT), and The intravenous normalized ratio (INR) was collected according to the test manual provided by the central laboratory. Coagulation tests 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. Day 1 and immediate / same-day urine tests during hospitalization as determined by the investigator The urine test will be performed in a local laboratory according to 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 Subclinical casts, crystals, bacteria, yeast.

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

[0230] Other clinical evaluations: ELISA: Anti-AAV9 antibody Blood samples were tested for AAV9 at screening and according to the time points specified in Table 4. 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 test 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 testing laboratory.

[0233] Other Clinical Assessments: Maternal Baseline Screening The mothers of 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 the mother for circulating antibodies against AAV9, Once informed consent is obtained, the mother's peripheral vein is used to administer the drug. Blood will be drawn from the patient and sent to a central laboratory for screening for anti-AAV9 antibodies. If any body parts are identified, the investigator will discuss with the mother whether to continue or discontinue breastfeeding. Intake of banked breast milk from donors who cannot be tested for anti-AAV9 antibodies Patients on breastfeeding should be transitioned to formula feeding before enrollment.

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

[0235] Other clinical evaluations: saliva, urine, and stool collection 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 occurs in the blood for up to one week after injection. They can be found in urine, saliva and faeces. The risks associated with shed vectors are 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. The patient is prohibited from donating blood for two years after the vector injection. can be.

[0236] Saliva, urine, and feces samples were collected according to Table 4, including at 24 and 48 hours after dosing. Viral shedding tests are collected according to the laboratory manual. Patients aged 48 months or older who stopped using the drug should be at least 18 years old on days 7, 14, and 30. Provide a complete urine and complete stool sample for each void and bowel movement. Prepare the pool according to the laboratory manual, store it in a -80°C freezer, and then resuspend it in the Therefore, the samples will be sent to the central laboratory. The test set consisted of 24-hour total urine and fecal samples collected from 24 to 48 hours after administration. Collect (including all excretion during 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. Spinal muscular atrophy (SMA) - able to sit but unable to stand or walk AVXS-101 was administered intrathecally to patients with biallelic deletion of SMN1. In addition, the patient had three copies of the SMN2 gene. Two groups were divided into two groups: one under 24 months of age and one aged 24 to 60 months at the time of administration. The patients were stratified into 16 patients aged >6 months and <24 months, and 16 patients aged >24 months. Twelve patients under 60 months of age were enrolled. In the younger group, three patients had a 6.0× 10 13 The remaining young patients and all older patients received 200 mg of AVXS-101 (dose A). Patients received 1.2 × 10 14 vg of AVXS-101 (Dose B).

[0238] Patients will receive a single dose via lumbar intrathecal (IT) injection for radiographic monitoring. AVXS-101 was administered premixed with 1.5 mL of 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. For patients aged >6 months but <24 months, the efficacy measure was patients achieving the ability to stand independently. (Bayley Scales of Infant Development®-Gross Motor Subset #40). Rolling from back to side, crawling, standing with support, pulling up to standing, with or without assistance World Health Organization Multicenter Growth Standards Study, including walking ganization Multicentre Growth Reference Defined by the WHO-MGRS criteria (Wijnhoven 2004) Additional milestones were also evaluated. In patients under 1 month of age, baseline Hammersmith Functional Motor Scale Extended (HFMSE) The outcome measure was change from baseline. Percentage of responders (HFMSE score >3 points) The achievement of core goals (defined as achievement of core goals; 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 Bayley 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 improved from baseline Seven of the 16 patients had at least one new Bayley ( Two patients, one in each dose group, achieved the study endpoint of "f independent achieved standing before 20 months of age (E02, E24); one patient (E24) achieved standing 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 will receive dose B of intrathecal AVXS-101. Then (1.2 × 10 14 vg; n = 12) were assessed 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 to 1.3 points, with 9 of 12 (75%) patients Five of 12 patients (42%) showed improvement from baseline. At least one new Bayley® item was achieved in two 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] For patients after reaching 2 years of age (6-24 months old group) and older patients (2-5 years old group), The Hammersmith Functional Motor Scale Extended (HFMSE) was administered. The increase ranged from -4 to 14 points (mean increase + SD was 4.3 + 5.3 points), with 19 patients Twelve (63.1%) of the patients showed improvement from baseline. Older group (2-5 years old) Seven of 12 patients (58%) in the younger group (6-12 years) showed improvement in HFMSE, whereas At age 24 months, 5 of 7 patients (71%) improved. One patient (E02) achieved the ability to stand unsupported. 63% were considered responders (achieved a 3-point or greater 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, HFMSE testing did not begin until they reached 24 months of age. 63% (12 of 19) of patients showed improvement in HFMSE. 13 One patient in the vg group showed an 8-point improvement after 8 months of treatment; the second patient patients showed a 2-point decrease after the 7-month evaluation.

[0246] Patients who achieved at least a 3-point improvement in HFMSE were considered responders in this study. In the older cohort (2-5 years), HFMSE was performed on 12 patients at baseline. From the beginning of treatment to the fifth month, 10 and 5 patients were evaluated at six and seven months, respectively. In the younger cohort (6 months to 2 years of age), one patient developed a pulmonary embolism after AVXS-101 treatment. 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 2 years of age or older are shown in Figure 5. After one month, a 50% rapid responder rate was observed. The responder rate remained constant throughout the seven-month study. The responder rate remained above 50% and tended to increase over time.

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

[0248] Overall, 23 new cases occurred in 11 of 24 patients during the 4- to 12-month observation 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%) experienced 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 with a score of >3 points); The responder rate gradually increased over time. Treatment with AVXS-101 was significantly higher than standard treatment, e.g. These results are more effective than those reported for other treatments, such as steroid therapy. Demonstrated that a number of patients had an early response to a single dose of intrathecal AVXS-101 The rapid onset of response and the sustained response of intrathecally administered AVXS-101 throughout the study period were 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 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 1 month of age, a baseline assessment was performed before treatment using the Bayley Scales (registered trademark). For the age group aged 24 months or older and younger than 60 months, the HFMSE was used. Additional baseline assessments were performed.

[0250] Within these two age groups, three different treatment doses were administered as described: Three patients aged 6 months or older but less than 24 months at the time of administration had a 6.0 × 10 13 vg AVXS- Thirteen patients aged 6 months to under 24 months received a single IT dose of 101 (Dose A). and 12 patients aged 24 months or older but less than 60 months, 1.2 × 10 14 AVXS for vg Three mice aged 6 months or older but under 24 months at the time of administration received a single IT dose of 101 (Dose B). 2.4 × 10 14 Received a single IT administration of 100 mg of AVXS-101 (Dose C) In future studies, 21 additional patients will receive Dose C, 9 of whom had a 6% chance of survival at the time of administration. The patients were from the age group of ≥ 24 months and < 24 months, and 12 were ≥ 24 months at the time of administration. 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 12 were between 6 and 24 months of age, and 12 were between 24 and 60 months of age. Four patients (three dose A and one dose B) were followed up for 12 months after treatment. Efficacy completer analysis set (ECA) was defined as all patients who completed the period. All efficacy analyses were performed using the intention-to-treat set as the primary population for interim results. , was conducted using ECAS as a supportive population.

[0252] Data from patients treated with AVXS-101 will be presented in the Pediatric Neuromuscular Clinical Study (PNCR) Network-collected, peer-reviewed, and widely cited natural history datasets 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. Three large, internationally recognized tertiary care centers with knowledge (Harvard University) university / Boston Children's Hospital,Col Columbia University and University of Pennsylvania ania / Children's Hospital of Philadelphia ) was developed from a cohort of 337 patients with all forms of SMA who were followed This is a large-scale natural history study. The data include the Bayley Scales of Infant Development (registered trademark). ) 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.,“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 under 24 months, A cohort of 51 individuals from the PNCR natural history study served as a "population-matched" control cohort. This comparison cohort included 51 patients enrolled in the PNCR trial who met the following criteria: All patients are included: (1) with type II or type III SMA; (2) with the three SMN2 (3) symptoms with onset before 12 months of age, and (4) at least one symptom with onset before 36 months of age Of this cohort, 7 / 51 (13.74%) patients lost the ability to stand independently. This is achieved by 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. The ability to walk independently was achieved in 5 / 51 (10%) patients. This is based on item #20 of the HFMSE at 36 months of age or any time before. A score of 2 was defined as having been achieved.

[0254] PNCR n=15 natural history control group: patients aged 24 months to under 60 months , patient-level data from a cohort of 15 patients extracted from the PNCR natural history study The data were selected as a "population-matched" control cohort. This control group was used for the primary analysis. The natural history control group had: (1) SMA type II or III, (2) 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. One patient had a recorded HFMSE score of 0 at baseline and all follow-up visits. In 5 / 15 (33%) 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 (18%). The median survival time was 42 months in 1 / 15 patients (13%) and 48 months in 1 / 15 patients (7%).

[0255] PNCR N=17 Natural history control group: Patients aged 24 months to under 60 months To improve matching of the patient group with the natural history control group, we extracted data from the PNCR study. Patient-level data were identified from a cohort of 17 patients who underwent randomized controlled trials. This control group was used for sensitivity analyses. Twelve patients originally in the PNCR N=15 control group were included in the PNCR N=1 control group. 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 whose last visit was more than 12 months ago) These 17 individuals were matched as closely as possible to the study group in terms of age, clinical characteristics, and The baseline age was between 24 and 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 unable to sit independently. Genetically, the patient had biallelic SMN. The patient had a PNCR natural history control and three 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 breakdown by treatment and age is detailed in Table 9. Safety Summary demographic and baseline characteristics by treatment and age group are presented for the sex analysis set. 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 for 6 months or more but 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®-Gross Motor Subset item #40 was achievement of "standing unsupported for at least 3 seconds." If the milestone is achieved at any time during the 2-month follow-up, the patient will be considered for this milestone. The milestone was considered achieved. A video of the laboratory evaluation of the milestone was released independently. 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 old 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 with Support to the Side (Bailey® Climb) Registered trademark #38).

[0263] Dose B (1.2×10 14 In the case of AVXS-101 (vg), 1 in 13 (7.7%) ) Patient 007-002 achieved unassisted standing within 3 months of treatment. The patient was approximately 7 months old when administered the drug. According to the investigator, the patient had neurological problems. 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%), patient 007-002( (above) achieved the milestone of standing unsupported three months after treatment.

[0266] [Table 13]

[0267] Natural history pairs with Type II and 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 variance and Kaplan-Meier test for supportive efficacy endpoints Analysis was performed according to protocol using the year analysis. 12 months after the baseline visit The primary efficacy endpoint of achieving the ability to stand independently at any time point is summarized in Table 11. To promise.

[0269] The time to achieve independent standing was measured for all patients in the PNCR group, as well as for IT patients. 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 assessed using a standardized model, the hazard ratio (95% CI) was 0.43 (0.05, 3.93) in group B, 0 (0, unevaluable) in group C, and 0 (0, inevaluable) in groups B and C. and 0.37 (0.04, 3.39), and the p-values ​​were 0.4576 and 0.995, respectively. At the time of reporting the interim results, most of the patients were independent. have not achieved the milestone of standing, and are at the 25th percentile, median, and 75th percentile. Values ​​such as centiles could not be calculated.

[0270] Interim results: Interim evaluation of the primary efficacy endpoint in the 24- to 60-month-old group (Dose B; Total n=12) a. Primary efficacy analysis using PNCR N=15 natural history control group The primary efficacy endpoint for this age group was change from baseline in HFMSE at 12 months. The baseline value of HFMSE, post-baseline value, and difference from baseline value were Changes will be summarized and analyzed using the intention-to-treat 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 Change from baseline in HFMSE scores to 12 months The plot is shown in Figure 7. Descriptive statistics for treated patients and controls are shown in Table 12.

[0272] PNCR N=15 natural history controls showed ± standard deviation of baseline HFMSE scores ( The mean mean (SD) of HFMSE scores was 11.8 ± 7.34. The change from baseline was -0.6±1.35 at 2 months, 0.4±0.9 at 4 months, and 8), 6 months (0.2±1.72), 9 months (1.0±2.16), 12 months (0 The calculated values ​​were 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 up to 8 months of HFMSE data (11 HFMSE scores from baseline at 2, 4, 6, 9, and 12 months The changes in α were 3.5±4.38, 3.6±5.07, 3.9±5.85, and 5. The dose B treatment group was compared with the PNCR N=15 natural history control group. showed a strong increase in HFMSE scores compared with placebo.

[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, the baseline HFMSE score was 12.1 ± 9 The mean change from baseline in HFMSE scores was 0.21 at 2 months (-0.05). 0.2±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. 41% (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 2, 4, 6, 9, and 12 months 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, respectively.

[0279] The Dose B treatment group had a significant improvement in HFMSE scores compared to the PNCR N=17 natural history control group. showed a strong increase.

[0280] [Table 16]

[0281] [Table 17]

[0282] Interim Outcome: Secondary Efficacy Endpoint - Motor Milestones, walking at least 5 steps independently Secondary efficacy endpoints were the age groups 6 months to under 24 months and the age groups 24 months to 60 months. The Bayley Scales of Infant Development®-Gross Motor Skills were used for both the under-12 months age group and the under-12 months age group. The first milestone was set item #43 ("walked 5 or more steps independently"). Scores were collected at all study visits up to 12 months after treatment. The video evidence was reviewed and confirmed by an independent central reviewer.

[0283] For patients aged 6 months or older but under 24 months at the time of administration, dose B (1.2 × 10 14 vg) One patient (007-002) was able to walk 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) 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 teriyaki group walked independently.

[0284] For patients aged 24 months or older but less than 60 months at the time of administration, all patients received 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 natural history control group.

[0285] Interim Results: Exploratory Efficacy Endpoints - 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(R), Third Edition (Bayley(R)-III) Changes from baseline in motor and gross motor components were assessed. In the under-24-month age group, a second exploratory endpoint will be continued 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 under 24 months of age are provided because RA (Registered Trademark) was not assessed. .

[0286] People with Type 1 SMA have severe fine motor impairments that prevent babies from grasping with their whole hand. However, in SMA types II and III, the Bayley® 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 development Proximal muscle dysfunction was associated with distal muscle dysfunction as reflected in their Bayley® scores. is significantly larger than

[0287] a. Patients aged 6 months or older but 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 (registered trademark) at 12 months The change from baseline was 12.3 ± 6.51 for the fine motor subtest and 5.5 ± 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, it was available in all 13 patients (5.4 ± 3.57). Incomplete data were available 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), 10th month (n=3; 11.7±3.06), 11th month (n=2; 12.5±4.95 At 12 months, there was one patient with 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. For subsequent months, available data were incomplete. The results were: 7 months (n=12; 4.7±4.29), 8 months (n=10; 4.9±6 .45), 9 months (n=6; 3.5±2.07), and 10 months (n=3; 5.7±4. 73), 11th month (n=2; 8.0±4.24), and 12th month (n=1; 11.0 ) Patients continued to achieve gross motor milestones. Patients who lost milestones There was no one there.

[0290] Dose C(2.4×10 14 vg): Change from baseline in fine motor subtests Only limited data were available for the 2nd month (n=3; 0.7±0.58), 3rd month (n=3; 0.7±0.58), and 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 4 months. The mean values ​​were 0.3±1.53 at 2 months (n=3; 0.3±1.53), 0.5±3.5 at 3 months (n=2; 0.5±3.5 4), and month 4 (n=1; 4.0).

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

[0292] [Table 18]

[0293] b. Patients aged 4 months or older but under 60 months at the time of administration Age groups aged 24 months or older and younger than 60 months were given 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 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 values ​​were 9.5±0.71. There was one patient with 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. For subsequent months, no data were available. Complete: 7th month (n=11; 2.0±4.36), 8th month (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] Changes 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 significantly different. The change in the gross motor function of patient 008-003 was "0" rather than "-8". The change in ability score from baseline measurement 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 represent the clinical data described in Example 1 at 12 months post-treatment. The efficacy results from the study are summarized. Descriptive statistics for the Bayley Scales® are shown in Table 15. Shown below.

[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 higher among patients in the 6-month to under 24-month age group at 24 months of age. Pre-treatment baseline data were not available for any patient, so H The first recorded FMSE is defined as the baseline. The months specified below are test months. This study was not related to 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 initial 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), 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 the first recording 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] Change from baseline in HFMSE scores through Month 12 of Dose B The time plot is shown in Figure 13. As shown in Table 16, at dose B, 12 months after baseline 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 aged 24 months or older and had their first HFMSE Records were 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 diagnosed with spinal muscular atrophy (SMA). An ongoing Phase 1, open-label, single-dose intrathecal (IT) study in infants and children under 0 months of age The data obtained to date on treated patients are from the following age groups: The progress of the study, including skill development, milestone improvement, and disease stabilization, is outlined in the summary. The study demonstrates clinically meaningful changes in motor function.

[0305] 6 months to under 24 months old 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) of 1.2 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) at 24 h. Four patients, including one patient, completed the 12-month post-treatment evaluation.

[0306] The primary efficacy outcome measure for this age group was the Bayley Scales of Infant Development®-Gross Motor Subset #40 was the 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 a mean survival of 11 days after treatment. At month 1, patients achieved unsupported standing for at least 3 seconds. 7-002 achieved unaided standing three months after treatment.

[0307] Secondary efficacy endpoints were the Bayley Scales of Infant Development®-Gross Motor Subset #43 ("walked 5 or more steps independently"). One patient (0 07-002) walked at least five steps unassisted four months after treatment.

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

[0309] 24 months to under 60 months old 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-assessment 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 ≥3-point improvement in HFMSE score was Good is something that is considered meaningful and important to stakeholders, such as caregivers and clinicians, and is relevant to clinical trials. It is used as a threshold for detecting meaningful changes in clinical trials. 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 a 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 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 change from baseline in the fine and gross motor components of the Bayley®-III. Similar to younger patients, patients continued to achieve gross motor milestones. No patients lost milestones. The inventions described in the original claims of this application are listed below. [Invention 1] the step of intrathecally administering an AAV9 viral vector comprising a polynucleotide encoding a survival motor neuron (SMN) protein, wherein the viral vector is administered intrathecally to approximately 1 x 10 13 vg~5×10 14 vg of a method for treating spinal muscular atrophy (SMA) in a patient in need thereof. [Invention 2] The method described in Invention 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. [Invention 3] The method according to any one of claims 1 to 2, wherein the polynucleotide encodes the SMN protein of SEQ ID NO:2. [Invention 4] 4. The method according to any one of Inventions 1 to 3, wherein the AAV9 viral vector comprises SEQ ID NO: 1. [Invention 5] 5. The method according to any one of Inventions 1 to 4, wherein the patient is 6 months of age or older at the time of administration. [Invention 6] 6. The method according to any one of claims 1 to 5, wherein said patient is 24 months of age or younger at the time of administration, and optionally between 6 and 24 months of age. [Invention 7] 6. The method according to any one of Inventions 1 to 5, wherein the patient is 24 months of age or older at the time of administration. [Invention 8] 6. The method according to any one of claims 1 to 5, wherein said patient is 60 months of age or younger at the time of administration, and optionally between 24 and 60 months of age. [Invention 9] The AAV9 viral vector is about 5.0 × 10 13 vg~3.0×10 14 9. The method according to any one of claims 1 to 8, wherein the compound is administered at a dose of vg. [Invention 10] The AAV9 viral vector is up to about 6.0 × 10 13 10. The method according to any one of claims 1 to 9, wherein the compound is administered at a dose of 0.5 mg / kg. [Invention 11] The AAV9 viral vector is about 6.0 × 10 13 11. The method according to any one of claims 1 to 10, wherein the compound is administered at a dose of 0.5 mg / kg. [Invention 12] The AAV9 viral vector is up to about 1.2 × 10 14 10. The method according to any one of claims 1 to 9, wherein the compound is administered at a dose of 0.5 mg / kg. [Invention 13] The AAV9 viral vector is about 1.2 × 10 14 10. The method according to any one of claims 1 to 9, wherein the compound is administered at a dose of 0.5 mg / kg. [Invention 14] The AAV9 viral vector is up to about 2.4 × 10 14 10. The method according to any one of claims 1 to 9, wherein the compound is administered at a dose of 0.5 mg / kg. [Invention 15] The AAV9 viral vector is about 2.4 × 10 14 10. The method according to any one of claims 1 to 9, wherein the compound is administered at a dose of 0.5 mg / kg. [Invention 16] 16. The method of any one of inventions 1 to 15, wherein said patient comprises a biallelic SMN1 null mutation or an inactivating deletion, optionally wherein said mutation comprises a deletion of exon 7 of SMN1. [Invention 17] 17. The method according to any one of claims 1 to 16, wherein the patient has three copies of SMN2. [Invention 18] 18. The method according to any one of claims 1 to 17, wherein said patient does not have the c.859G>C substitution in exon 7 on at least one copy of the SMN2 gene. [Invention 19] 19. The method according to any one of claims 1 to 18, wherein said patient in need thereof is determined by one or more genomic tests. [Invention 20] 20. The method according to any one of claims 1 to 19, wherein said patient shows disease onset before about 12 months of age. [Invention 21] 21. The method according to any one of claims 1 to 20, wherein said patient, at the time of administration, has the ability to sit unassisted for about 10 hours or more, but is unable to stand or walk. [Invention 22] 22. The method according to any one of claims 1 to 21, wherein said patient, at the time of administration, has the ability to sit unassisted, for example as defined by the World Health Organization Multicenter Growth Reference Study (WHO-MGRS) criteria. [Invention 23] 23. The method of any one of claims 1 to 22, wherein the patient has the ability to stand unsupported for at least about 3 seconds after administration, e.g., as defined by the Bayley Scales of Infant Development®, assessed at about 1 to 24 months, such as 12 months after administration. [Invention 24] 23. The method of any one of claims 1 to 22, wherein said patient has the ability to walk unassisted following administration, e.g., as defined by the Bayley Scales of Infant Development®, assessed at about 1 to 24 months, such as 12 months after administration. [Invention 25] 25. The method of any one of claims 1 to 24, wherein the patient has the ability to take at least 5 steps independently after administration, e.g., as defined by the Bayley Scales of Infant Development®, assessed at about 1 to 24 months, e.g., 12 months after administration. [Invention 26] 26. The method of any one of inventions 1 to 25, wherein the patient exhibits a change from the baseline measurement at the time of treatment after administration, e.g., as defined by the Bayley Scales of Infant Development®, assessed at about 1 to 24 months, e.g., 12 months after administration. [Invention 27] 27. The method according to any one of claims 1 to 26, wherein the patient does not have severe scoliosis, e.g., a spinal curvature of 50 degrees or more, as evident by radiography after the administration, at an evaluation about 1 to 24 months, e.g., 12 months after the administration. [Invention 28] 28. The method according to any one of claims 1 to 27, wherein said patient has no contraindications for a spinal tap procedure or the administration of intrathecal therapy. [Invention 29] 29. The method of any one of claims 1 to 28, wherein said patient has not previously undergone scoliosis repair surgery or procedure, and optionally said patient has not undergone scoliosis repair surgery or procedure within 6 months to 3 years, e.g., within 1 year of administration. [Invention 30] 30. The method according to any one of claims 1 to 29, wherein said patient does not require the use of invasive ventilatory support before and / or after administration. [Invention 31] 31. The method according to any one of Inventions 1 to 30, wherein the patient has no history of standing or walking independently prior to administration. [Invention 32] 32. The method according to any one of claims 1 to 31, wherein said patient does not use a gastric feeding tube before and / or after administration. [Invention 33] 33. The method according to any one of claims 1 to 32, wherein said patient does not have an active viral infection (including seropositive for human immunodeficiency virus (HIV); or hepatitis B or C or Zika virus) at the time of treatment. [Invention 34] 34. The method according to any one of inventions 1 to 33, wherein the patient has not had a severe non-pulmonary / respiratory infection (e.g., pyelonephritis or meningitis) within 4 weeks prior to administration. [Invention 35] 35. The method according to any one of claims 1 to 34, wherein said patient does not have any concomitant diseases, such as, for example, major renal or hepatic disorders, known seizure disorders, diabetes mellitus, idiopathic hypocalciuria or symptomatic cardiomyopathy, prior to administration. [Invention 36] 36. The method according to any one of claims 1 to 35, wherein said patient has no history of bacterial meningitis or brain or spinal cord disease prior to administration. [Invention 37] 37. The method according to any one of claims 1 to 36, wherein said patient has no known allergies or hypersensitivities to prednisolone or other glucocorticosteroids or excipients. [Invention 38] 38. The method according to any one of claims 1 to 37, wherein said patient has no known allergies or hypersensitivities to iodine or iodine-containing products prior to administration. [Invention 39] 39. The method according to any one of inventions 1 to 38, wherein the patient is not taking any medication to treat myopathy or neuropathy. [Invention 40] 40. The method according to any one of claims 1 to 39, wherein the patient has not received immunosuppressive therapy, plasma exchange, or immunomodulators such as adalimumab within 3 months prior to administration. [Invention 41] 41. The method of any one of Inventions 1 to 40, wherein the patient has an anti-AAV9 antibody titer of 1:25, 1:50, 1:75, or 1:100 or less prior to administration, as measured, for example, by an ELISA binding immunoassay. [Inven...

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 after 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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