Methods of treating spinal muscular atrophy

Abstract

The invention features biomarkers for use in, for example, the diagnosis and prognosis of spinal muscular atrophy (SMA) and the identification of responders to SMA treatment. Methods of treating a subject with SMA are also provided.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of January 25, 2019, the priority date of January 25, 2018, the application number 201980016968.4, and the invention name “Method for treating spinal muscular atrophy”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 622,027 filed on January 25, 2018, U.S. Provisional Application No. 62 / 684,507 filed on June 13, 2018, and U.S. Provisional Application No. 62 / 738,134 filed on September 28, 2018. The contents of the prior applications are incorporated herein by reference in their entirety. Technical Field

[0004] The present disclosure relates generally to biomarkers for spinal muscular atrophy. Background Art

[0005] Spinal muscular atrophy (SMA) is an autosomal recessive disease resulting in a deficiency of the SMN protein, which in turn results in a phenotype of anterior horn motor neuron loss, axonal degeneration, and progressive muscle wasting, mobility impairment, and respiratory failure. Disease severity is divided into five types (0-4), with patients with type 0 having the most severe (neonatal lethal) phenotype and patients with type 4 having only the mildest symptoms and a normal life span.

[0006] Timely and appropriate treatment of subjects with SMA requires the ability to select presymptomatic subjects who need treatment with a therapy to treat SMA and to determine whether the SMA therapy is effective. Therefore, there is a need for biomarkers for SMA. Summary of the invention

[0007] Neurofilaments are a major component of the neuronal cytoskeleton, particularly in axons, where they are essential for growth and maintenance. Structurally, neurofilaments consist of three intertwined core subunits: neurofilament heavy chain (NF-H), medium chain / intermediate chain (NF-M), and light chain (NF-L) polypeptides that form a "neurofilament triad". The present disclosure is based, at least in part, on the discovery that neurofilament levels serve as an effective biomarker for spinal muscular atrophy (SMA).

[0008] In one aspect, the disclosure features a method of treating SMA in a human subject in need thereof. The method includes administering to the human subject a therapeutically effective amount of an SMA therapy, wherein the human subject has previously been determined in a biological sample obtained from the human subject to have neurofilament levels above a control prior to starting the SMA therapy. For example, the method can be used to treat a pre-symptomatic subject.

[0009] In another aspect, the present disclosure provides a method of treating SMA in a human subject in need thereof. The method comprises measuring neurofilament levels in a biological sample obtained from the human subject prior to initiating SMA treatment, and administering a therapeutically effective amount of SMA therapy to the human subject.

[0010] In some embodiments of the above two aspects, the neurofilament level (e.g., phosphorylated neurofilament heavy chain (pNF-H) level) in the biological sample is higher than the control level. In some embodiments, the neurofilament level (e.g., pNF-H level) in the biological sample is higher than 300 pg / mL. In some embodiments, the neurofilament level (e.g., pNF-H level) in the biological sample is higher than 400 pg / mL. In some embodiments, the neurofilament level (e.g., pNF-H level) in the biological sample is higher than 500 pg / mL. In some embodiments, the neurofilament level (e.g., pNF-H level) in the biological sample is higher than 600 pg / mL. In some embodiments, the neurofilament level (e.g., pNF-H level) in the biological sample is higher than 700 pg / mL. In some embodiments, the neurofilament level (e.g., pNF-H level) in the biological sample is higher than 800 pg / mL. In some embodiments, the neurofilament level (e.g., pNF-H level) in the biological sample is higher than 900 pg / mL. In some embodiments, the level of neurofilaments (e.g., pNF-H levels) in the biological sample is higher than 1,000 pg / mL. In some embodiments, the level of neurofilaments (e.g., pNF-H levels) in the biological sample is higher than 1,100 pg / mL. In some embodiments, the level of neurofilaments (e.g., pNF-H levels) in the biological sample is higher than 1,200 pg / mL. In some embodiments, the level of neurofilaments (e.g., pNF-H levels) in the biological sample is higher than 1,300 pg / mL. In some embodiments, the level of neurofilaments (e.g., pNF-H levels) in the biological sample is higher than 1,400 pg / mL. In some embodiments, the level of neurofilaments (e.g., pNF-H levels) in the biological sample is higher than 1,500 pg / mL. In some embodiments, the level of neurofilaments (e.g., pNF-H levels) in the biological sample is higher than 2,000 pg / mL. In some embodiments, the neurofilament level (e.g., pNF-H level) in the biological sample is above 3,000 pg / mL. In some embodiments, the neurofilament level (e.g., pNF-H level) in the biological sample is above 4,000 pg / mL. In some embodiments, the neurofilament level (e.g., pNF-H level) in the biological sample is above 5,000 pg / mL. These neurofilament levels can be obtained using the assays described in the Examples section of this application. It should be understood that the values ​​of neurofilament levels may be different if different neurofilament assays (e.g., OD or international units) that provide different readouts are used.

[0011] In another aspect, the disclosure features a method of treating SMA in a human subject in need thereof. The method includes: measuring neurofilament levels (e.g., pNF-H levels) in a first biological sample obtained from the human subject before initiating SMA therapy; administering SMA therapy (e.g., a therapeutically effective amount of SMA therapy) to the human subject; and after initiating the SMA therapy, measuring neurofilament levels (e.g., pNF-H levels) in a second biological sample obtained from the human subject.

[0012] In another aspect, the disclosure features a method of treating SMA in a human subject in need thereof. The method includes: measuring the level of neurofilaments (e.g., pNF-H levels) in a first biological sample obtained from the human subject before administering a candidate amount of the SMA therapy; measuring the level of neurofilaments (e.g., pNF-H levels) in a second biological sample obtained from the human subject after administering the candidate amount of the SMA therapy, wherein the level of neurofilaments in the second biological sample is lower than the level of neurofilaments in the first biological sample, thereby indicating that the candidate amount of the SMA therapy is a therapeutically effective amount; and administering the therapeutically effective amount of the SMA therapy to the human subject after measuring the reduced level of neurofilaments in the second biological sample.

[0013] In certain embodiments, the neurofilament level (e.g., pNF-H level) in the first biological sample is higher than 300 pg / mL, higher than 400 pg / mL, higher than 500 pg / mL, higher than 600 pg / mL, higher than 700 pg / mL, higher than 800 pg / mL, higher than 900 pg / mL, higher than 1,000 pg / mL, higher than 1,500 pg / mL, higher than 2,000 pg / mL, higher than 3,000 pg / mL, higher than 4,000 pg / mL, or higher than 5,000 pg / mL. In certain embodiments, the neurofilament level (e.g., pNF-H level) measured in the second biological sample is lower than the neurofilament level measured in the first biological sample. In some embodiments, the neurofilament level (e.g., pNF-H level) measured in the second biological sample shows a decrease of greater than 30% relative to the neurofilament level measured in the first biological sample. In some embodiments, the neurofilament level (e.g., pNF-H level) measured in the second biological sample is between 10% and 80% of the neurofilament level measured in the first biological sample. In some embodiments, the neurofilament level (e.g., pNF-H level) measured in the second biological sample is between 20% and 95% of the neurofilament level measured in the first biological sample. In some embodiments, the neurofilament level (e.g., pNF-H level) measured in the second biological sample is between 30% and 90% of the neurofilament level measured in the first biological sample. In some embodiments, the neurofilament level (e.g., pNF-H level) measured in the second biological sample is between 30% and 95% of the neurofilament level measured in the first biological sample. In such cases, the SMA therapy is continued. In certain embodiments, the neurofilament level (e.g., pNF-H level) measured in the second biological sample is higher than the neurofilament level measured in the first biological sample. In such cases, the SMA therapy is stopped.

[0014] In some embodiments, the second biological sample is obtained from the human subject 40 to 90 days after starting the SMA therapy. In some embodiments, the second biological sample is obtained from the human subject 50 to 80 days after starting the SMA therapy. In some embodiments, the second biological sample is obtained from the human subject 60 to 70 days after starting the SMA therapy. In some embodiments, the second biological sample is obtained from the human subject about 64 days after starting the SMA therapy.

[0015] In some embodiments of the second biological sample obtained from the human subject 40 to 90 days, 50 to 80 days, 60 to 70 days, or about 64 days after the start of the SMA therapy, the neurofilament levels measured in the second biological sample are reduced by at least 50% compared to the neurofilament levels measured in the first biological sample. In some embodiments of the second biological sample obtained from the human subject 40 to 90 days, 50 to 80 days, 60 to 70 days, or about 64 days after the start of the SMA therapy, the neurofilament levels measured in the second biological sample are reduced by at least 60% compared to the neurofilament levels measured in the first biological sample. In some embodiments of the second biological sample obtained from the human subject 40 to 90 days, 50 to 80 days, 60 to 70 days, or about 64 days after the start of the SMA therapy, the neurofilament levels measured in the second biological sample are reduced by at least 70% compared to the neurofilament levels measured in the first biological sample.

[0016] In some embodiments where the second biological sample is obtained from the human subject 40 to 90 days, 50 to 80 days, 60 to 70 days, or about 64 days after initiation of the SMA therapy, the neurofilament levels measured in the second biological sample are reduced by less than 50% compared to the neurofilament levels measured in the first biological sample. In some embodiments where the second biological sample is obtained from the human subject 40 to 90 days, 50 to 80 days, 60 to 70 days, or about 64 days after initiation of the SMA therapy, the neurofilament levels measured in the second biological sample are reduced by less than 40% compared to the neurofilament levels measured in the first biological sample.

[0017] In some embodiments, the second biological sample is obtained from the human subject 40 to 90 days, 50 to 80 days, 60 to 70 days, or about 64 days after initiating the SMA therapy, and the dose of the SMA therapy is altered for subsequent administration to the human subject based on the percent decrease in the neurofilament levels measured in the second biological sample compared to the neurofilament levels measured in the first biological sample.

[0018] In another aspect, the present disclosure relates to a method for predicting the prognosis of SMA. The method includes measuring the level of neurofilaments (e.g., pNF-H levels) in a biological sample obtained from a human subject, wherein the human subject has a mutation in both copies of the SMN1 gene that results in a lack of functional SMN protein (the mutation may be homozygous or heterozygous). The method also includes comparing the level of neurofilaments (e.g., pNF-H levels) measured in the biological sample with a control. Compared to the control, the level of neurofilaments (e.g., pNF-H levels) measured in the biological sample can predict the severity or type of SMA that the subject will suffer from.

[0019] In certain embodiments, the biological sample is obtained from the human subject prior to initiating SMA therapy, and the neurofilament level (e.g., pNF-H level) measured in the biological sample compared to the control is predictive of the severity or type of SMA that the human subject will develop in the absence of treatment.

[0020] In some embodiments, the biological sample is obtained from the human subject after starting SMA therapy, and the level of neurofilaments (e.g., pNF-H level) measured in the biological sample compared to the control can predict the severity or type of SMA that the subject will suffer from when receiving the SMA therapy. In some cases, the biological sample is obtained from the human subject at least two weeks after starting the SMA therapy. In some cases, the biological sample is obtained from the human subject at least four weeks after starting the SMA therapy. In some cases, the biological sample is obtained from the human subject at least six weeks after starting the SMA therapy. In some cases, the biological sample is obtained from the human subject at least eight weeks after starting the SMA therapy. In some cases, the biological sample is obtained from the human subject at least ten weeks after starting the SMA therapy. In some cases, the biological sample is obtained from the human subject at least twelve weeks after starting the SMA therapy. In some cases, the biological sample is obtained from the human subject at least two months after starting the SMA therapy. In some cases, the biological sample is obtained from the human subject at least three months after starting the SMA therapy. In some cases, the biological sample is obtained from the human subject at least four months after starting the SMA therapy. In some cases, the biological sample is obtained from the human subject at least five months after starting the SMA therapy. In some cases, the biological sample is obtained from the human subject at least six months after starting the SMA therapy.

[0021] On the other hand, the present disclosure relates to a method for predicting the prognosis of SMA. The method includes measuring the level of neurofilaments (e.g., pNF-H levels) in a first biological sample obtained from a human subject before starting SMA therapy, wherein the human subject has a mutation (the mutation may be homozygous or heterozygous) in two copies of the SMN1 gene that causes a lack of functional SMN protein. The method also includes measuring the level of neurofilaments (e.g., pNF-H levels) in a second biological sample obtained from the human subject after starting the SMA therapy. The method also includes comparing the level of neurofilaments (e.g., pNF-H levels) measured in the second biological sample with the level of neurofilaments (e.g., pNF-H levels) measured in the first biological sample. Compared with the level of neurofilaments (e.g., pNF-H levels) measured in the first biological sample, the level of neurofilaments (e.g., pNF-H levels) measured in the second biological sample can predict the severity or type of SMA that the subject will suffer from. Generally speaking, the greater the percentage decrease in neurofilament levels (e.g., pNF-H levels) in the second biological sample compared to the neurofilament levels (e.g., pNF-H levels) measured in the first biological sample, the better the prognosis for future motor function in the human subject.

[0022] In some embodiments, the second biological sample is obtained from the human subject 40 to 90 days after starting the SMA therapy. In some embodiments, the second biological sample is obtained from the human subject 50 to 80 days after starting the SMA therapy. In some embodiments, the second biological sample is obtained from the human subject 60 to 70 days after starting the SMA therapy. In some embodiments, the second biological sample is obtained from the human subject about 64 days after starting the SMA therapy.

[0023] In some embodiments of the second biological sample obtained from the human subject 40 to 90 days, 50 to 80 days, 60 to 70 days, or about 64 days after the start of the SMA therapy, the neurofilament levels measured in the second biological sample are reduced by at least 50% compared to the neurofilament levels measured in the first biological sample. In some embodiments of the second biological sample obtained from the human subject 40 to 90 days, 50 to 80 days, 60 to 70 days, or about 64 days after the start of the SMA therapy, the neurofilament levels measured in the second biological sample are reduced by at least 60% compared to the neurofilament levels measured in the first biological sample. In some embodiments of the second biological sample obtained from the human subject 40 to 90 days, 50 to 80 days, 60 to 70 days, or about 64 days after the start of the SMA therapy, the neurofilament levels measured in the second biological sample are reduced by at least 70% compared to the neurofilament levels measured in the first biological sample.

[0024] In some embodiments where the second biological sample is obtained from the human subject 40 to 90 days, 50 to 80 days, 60 to 70 days, or about 64 days after initiation of the SMA therapy, the neurofilament levels measured in the second biological sample are reduced by less than 50% compared to the neurofilament levels measured in the first biological sample. In some embodiments where the second biological sample is obtained from the human subject 40 to 90 days, 50 to 80 days, 60 to 70 days, or about 64 days after initiation of the SMA therapy, the neurofilament levels measured in the second biological sample are reduced by less than 40% compared to the neurofilament levels measured in the first biological sample.

[0025] The following embodiments apply to any of the above aspects. In some cases, the SMA therapy comprises nusinersen or a nusinersen salt. In some cases, the SMA therapy comprises nusinersen sodium. In some cases, nusinersen sodium is administered by intrathecal injection of 5 mL of a 2.4 mg / mL solution. In some cases, the SMA therapy comprises SPINRAZA , olesoxime, AVX-101, CK-2127107, RG7916, RG7800, RO7034067, LMI070 or SRK-015. In some cases, the SMA therapy includes a small molecule. In some cases, the SMA therapy includes a gene therapy. In some cases, the SMA therapy includes a p38aDMAPK inhibitor. In some cases, the SMA therapy includes a DcpS inhibitor. In some cases, the SMA therapy includes a JNK inhibitor. In some cases, the control is a pre-established neurofilament cutoff. In some cases, the control is a neurofilament level (e.g., pNF-H level) in one or more biological samples obtained from one or more human subjects who do not suffer from SMA.

[0026] In another aspect, the disclosure features a method for measuring neurofilament levels (e.g., pNF-H levels). The method includes providing a biological sample obtained from a human subject having a mutation in both copies of the SMN1 gene that results in a lack of functional SMN protein; and measuring neurofilament levels (e.g., pNF-H levels) in the biological sample.

[0027] These embodiments are applicable to any of the above aspects. In some cases, the neurofilament is a neurofilament heavy chain (e.g., phosphorylated NF-H). In some cases, the neurofilament is a neurofilament medium chain / intermediate chain. In some cases, the neurofilament is a neurofilament light chain. In some cases, the neurofilament is an internexin. In some cases, the neurofilament is a peripheral protein. In some cases, the biological sample is blood, serum, plasma, or cerebrospinal fluid. In some cases, NF-H is detected using a polyclonal anti-NF-H antibody. In some cases, NF-H is detected using a polyclonal anti-NF-H antibody (e.g., Encor Biotechnology catalog number RPCA-NF-H; and / or catalog number CPCA-NF-H) that specifically detects a hyperphosphorylated form of NF-H. In some cases, NF-H is detected using a monoclonal anti-NF-H antibody. In certain embodiments, the human subject is a fetus. In certain embodiments, the human subject is an infant. In certain embodiments, the human subject is less than 6 months old. In certain embodiments, the human subject is greater than 6 months old. In certain embodiments, the human subject is a child less than 12 years old. In certain embodiments, the human subject is a child less than 18 years of age. In certain embodiments, the human subject is an adult 18 years of age or older.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used for the practice or test of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present application (including definitions) shall prevail. The materials, methods and embodiments are illustrative only and are not intended to be restrictive.

[0029] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Shown are levels of phosphorylated neurofilament heavy chain (pNF-H) in plasma and CSF samples from the EMBRACE and NURTURE clinical studies at the same time point and in the same subject.

[0031] Figure 2 Shown are pNF-H levels in plasma samples from subjects with two, three, or four copies of SMN2 from the NURTURE, ENDEAR, EMBRACE, and CHERISH clinical studies.

[0032] Figure 3 is a depiction of pNF-H levels in subjects of increasing age with two copies of SMN2.

[0033] Figure 4 is a depiction of pNF-H levels in subjects of increasing age with three copies of SMN2.

[0034] Figure 5 is a depiction of pNF-H levels and age of symptom onset in subjects with two, three, or four copies of SMN2.

[0035] Figure 6 is a table providing baseline characteristics and SMA medical history by baseline pNF-H level.

[0036] Figure 7 is a table providing baseline characteristics and SMA medical history by baseline pNF-H level.

[0037] Figure 8 is a table providing baseline characteristics and SMA medical history by baseline pNF-H level.

[0038] Fig. 9 Shown is the correlation of baseline log(pNF-H) levels with baseline CHOP INTEND scores.

[0039] Fig.10 is a table showing the ability to correlate phenotype at baseline by pNF-H levels in ENDEAR.

[0040] Fig.11 is a graphical depiction of pNF-H levels in ENDEAR.

[0041] Fig.12 is a graphical depiction of the percent change in NF-H levels in ENDEAR.

[0042] Fig.13 is a graphical depiction of the effect of SMA drug therapy on pNF-H levels in ENDEAR.

[0043] Fig.14 is a graphical depiction of the effect of SMA drug therapy on the percent change in NF-H levels in ENDEAR.

[0044] Fig.15 is a depiction of the changes in pNF-H levels at different time points.

[0045] Fig.16 is a graph depicting the correlation of HINE-2 score on day 183 with pNF-H level on day 64.

[0046] Fig.17 Shown is the association of HINE-2 score at day 183 with pNF-H level at day 64 (with covariates).

[0047] Fig.18 is a graph depicting the correlation of CHOP INTEND score on day 183 with pNF-H level on day 64.

[0048] Fig.19 Shown is the association of CHOP INTEND score at day 183 with pNF-H levels at day 64 (with covariates).

[0049] Fig. 20 is a depiction of HINE-2 (responder / non-responder) categories versus pNF-H levels at day 183.

[0050] Fig.21 is a graphical depiction of HINE-2 (responder / non-responder) categories versus percent change in pNF-H levels at Day 183.

[0051] Fig. 22 is a graphical depiction of CHOP INTEND (responder / non-responder) categories versus pNF-H levels at Day 183.

[0052] Fig.23is a graphical depiction of the CHOP INTEND (responder / non-responder) categories versus percent change in pNF-H levels at Day 183.

[0053] Fig.24 is a graphical depiction of the maintenance of drug effects based on pNF-H (pg / mL) levels in NURTURE.

[0054] Fig.25 is a graphical depiction of the maintenance of drug effects based on pNF-H (pg / mL) levels in EMBRACE.

[0055] Fig.26 is a graphical depiction of the effect of drug on the percent change in pNF-H (pg / mL) levels in NURTURE.

[0056] Fig. 27 is a graphical depiction of the effect of drugs on the percent change in pNF-H (pg / mL) levels in EMBRACE.

[0057] Fig.28 are graphs depicting the association of HINE-2 score on day 183 with pNF-H level on day 64. For the top graph: root MSE: 2.87069; R-square: 0.0184; adjusted R-square: 0.0002. For the bottom graph: root MSE: 1.70576; R-square: 0.224; adjusted R-square: 0.1831. DF = degrees of freedom; MSE = mean square error.

[0058] Fig.29 are graphs depicting the association of CHOP INTEND score at day 183 with pNF-H level at day 64. For the top graph: root MSE: 10.09399; R-square: 0.0754; adjusted R-square: 0.0583. For the bottom graph: root MSE: 9.41166; R-square: 0.2176; adjusted R-square: 0.1764. DF = degrees of freedom; MSE = mean square error.

[0059] Fig.30 is a graphical representation of pNF-H levels over time in surviving infants with non-missing values ​​on day 302.

[0060] Fig.31 is a graphical depiction of the percent change in pNF-H levels over time among surviving infants with non-missing values ​​at 302 days.

[0061] Fig.32 is a graphical depiction of the correlation of peroneal CMAP amplitude on day 183 with pNF-H levels on day 64.

[0062] Fig.33 Graphs depicting the association of peroneal CMAP amplitude on day 183 with pNF-H levels on day 64. For the top graph: root MSE: 0.56401; R-square: 0.0164; adjusted R-square: -0.0029. For the bottom graph: root MSE: 0.18933; R-square: 0.2906; adjusted R-square: 0.2512. DF = degrees of freedom; MSE = mean square error.

[0063] Fig.34 is a graphical depiction of the association of ulnar CMAP (compound muscle action potential) amplitude on day 183 with pNF-H levels on day 64. Root MSE: 0.24904; R-square: 0.1746; Adjusted R-square: 0.1633. DF = degrees of freedom; MSE = mean square error.

[0064] Fig.35 Graphs depicting the association of ulnar CMAP amplitude on day 183 with pNF-H levels on day 64. For the top graph: root MSE: 0.2824; R-square: 0.0517; adjusted R-square: 0.0338. For the bottom graph: root MSE: 0.11118; R-square: 0.2406; adjusted R-square: 0.1984. DF = degrees of freedom; MSE = mean square error.

[0065] Figures 36A-36C is a receiver operating characteristic (ROC) graph depicting the measurement of day 29 ( Fig.36A )、Day 64( Fig.36B ) and Day 183 ( Fig.36C ) in predicting motor function at day 302.

[0066] Figures 37A-37B is to depict the percentage change in measured pNF-H levels as CHOP INTEND responders in ENDEAR ( Fig.37A ) and Motor Milestone Responders ( Fig.37B ) of the predictive indicators.

[0067] Figures 38A-38C is a graph depicting the percentage change in measured pNF-H levels as HFMSE responders in CHERISH ( Fig.38A ),RULM responder( Fig.38B ) and WHO responders ( Fig.38C ) of the predictive indicators.

[0068] Figures 39A-39B is to characterize the Fig.39A ) and SMN2 copy number ( Fig.39B) versus age at first dose.

[0069] Fig.40 is a graph depicting the decrease in cerebrospinal fluid pNF-H levels after treatment with nussenelsen.

[0070] Figures 41A-41D is a depiction of CSF in subjects before symptom onset after treatment with Nussenelsen ( Fig.41A ), CSF of subjects with infantile onset ( Fig.41B ), plasma from subjects with infantile onset ( Fig.41C ) and plasma from late-onset subjects ( Fig.41D ) are graphs showing the percent changes in pNF-H and NF-L levels in the rats.

[0071] Figures 41E-41F is a depiction of plasma levels in infantile-onset subjects after treatment with nussenelsen ( Fig.41E ) and plasma from late-onset subjects ( Fig.41F ) is a graph showing the absolute changes in pNF-H and NF-L levels in the rats. DETAILED DESCRIPTION

[0072] The present disclosure is based, in part, on the unexpected discovery that neurofilament (NF) levels can serve as an effective biomarker for SMA.

[0073] 1. Spinal muscular atrophy

[0074] After cystic fibrosis, spinal muscular atrophy (SMA) is the second most common lethal autosomal recessive disorder in Caucasians. This disease is characterized by progressive degeneration of the alpha motor neurons in the anterior horns of the spinal cord, which leads to muscle atrophy, paralysis, and sometimes even death. The most common form of SMA is caused by the 5q13 survival mutation of the motor neuron (SMN1) gene. This disease affects 1 in 6,000 to 10,000 infants, with a carrier frequency of 1 in 40. For SMA, several clinical types have been described, grouping the disease according to the age of onset and the progression of symptoms.

[0075] According to the above classification, there are five types of SMA: Type 0 (embryonic form), Type I (Wedening-Hoffmann), Type II (intermediate), Type III (Kugleberg-Weiland) and Type IV (adult form). The most severe Type 0 is characterized by decreased fetal movement between 30 and 36 weeks of gestation and a very short life expectancy. Type I is the second most severe form, with onset before 6 months of age and a life expectancy of about 2 years. Types II and III are called chronic forms and are less severe, with onset between 6 and 18 months (Type II) and after 18 months (for Type III), respectively. In many cases, Type IV mimics the symptoms of Type III, but with onset after 18 years of age (usually around 30 years of age). A normal life expectancy is characteristic of this adult form.

[0076] The SMA determining gene, SMN1, is mapped to the 5q11.2-13.3 region. Homozygous deletion of SMN1 exon 7 is the most common mutation in SMA patients; however, there are several cases of compound heterozygous patients in which deletions and different point mutations have been detected. In humans, there are two forms of the SMN gene on each allele: a telomeric form (SMN1) and a centromeric form (SMN2). Transcription of the SMN1 gene produces a full-length messenger RNA (mRNA) transcript encoding the SMN protein. The SMN2 gene is identical to the SMN1 gene, except that a C to T substitution at position 840 results in the exclusion of exon 7 during transcription. The resulting truncated protein is inactive and rapidly degraded. Importantly, the exclusion of exon 7 from the SMN2 mRNA is not complete, and therefore a small fraction (approximately 10% to 15%) of the total mRNA transcripts produced from the SMN2 gene contain exon 7, which encodes normal SMN protein. However, the full-length SMN protein is synthesized in such a small amount that it cannot maintain motor neuron survival.

[0077] All patients with SMA lack a functioning SMN1 gene and are therefore dependent on their SMN2 gene to produce the SMN protein necessary for survival. Therefore, SMA is caused by a deficiency in the SMN protein, which results in selective motor neuron loss. Several genotype / phenotype analyses have shown a positive correlation between SMN2 copy number and a milder SMA phenotype. Although SMN2 copy number is the main determinant of SMA severity, it is clearly not the only phenotypic modifier. At least three adult patients with a mild 3b phenotype and only 2 copies of SMN2 have been described in the art. This seemingly inconsistent finding is explained by the fact that these individuals have a c.859G>C exon 7 mutation, which produces an exonic splicing enhancer element that increases the production of full-length SMN protein and a milder phenotype. Other modifiers have been described, and more are expected as the understanding of the molecular pathogenesis of SMA improves. These findings suggest that the SMA phenotype cannot always be inferred from the SMN2 copy number determination alone.

[0078] 2. Therapies for SMA

[0079] One therapy used to treat SMA is SPINRAZA , which is a nussenal compound (also known as ASO-10-27 / ISIS 396443 / ISIS SMNRx / ISIS-396443 / ISIS-SMNRx). Nussenal is a modified antisense oligonucleotide compound (modified in that the 2'-hydroxyl group of the furanosyl ring is replaced by a 2'-O-2-methoxyethyl group and the phosphate linkage is replaced by a phosphorothioate linkage) that binds to a specific sequence of the SMN2 transcript in the intron downstream of exon 7. The sodium salt of nussenal (nussenal sodium) is sold under the trade name SPINRAZA Sale. Nussenelsen is intended to treat SMA caused by mutations in chromosome 5q that result in SMN protein deficiency. Using in vitro assays and studies in transgenic animal models of SMA, Nussenelsen was shown to increase exon 7 incorporation in SMN2 messenger RNA (mRNA) transcripts and production of full-length SMN protein. Nussenelsen counteracts the SMN protein deficiency that causes SMA by increasing the splicing efficiency of SMN2 pre-mRNA.

[0080] Nussenelsen is an 18-mer 2'-MOE phosphorothioate antisense oligonucleotide. Nussenelsen is designed to pair with a specific target sequence on SMN2 pre-mRNA to replace the heterogeneous nuclear ribonucleoprotein (hnRNP) at the intronic splicing silencer site-1 (ISS-1) between exons 7 and 8 to allow more complete translation of SMN protein from the paralogous gene SMN2. Nussenelsen is injected intrathecally into the cerebrospinal fluid (CSF) so that it is distributed from the CSF to the target central nervous system (CNS) tissue. The complete sequence of Nussenelsen is [2'-O-(2-methoxyethyl)](3'-5')(P-thio) (T-5mC-A-5mC-TTT-5mC-ATAATG-5mC-TGG) (SEQ ID NO:4). Antisense oligonucleotides contain 2'-O-(2-methoxyethyl) (2'-MOE)-oligoribonucleotides to reduce nuclease degradation and enhance binding affinity to complementary RNA.

[0081] There are also several other therapies for the treatment of SMA. These include compounds that increase SMN levels, such as histone deacetylase inhibitors, aminoglycosides and quinazoline derivatives. Histone deacetylase inhibitors, such as valproic acid, sodium butyrate, phenyl butyrate and trichostatin A activate the SMN2 promoter, thereby causing an increase in full-length SMN protein. Other therapies include CK-2127107 (rapid skeletal muscle troponin activator), LMI070 (branaplan, formerly known as NVS-SM1), olesoxime (a member of the cholesterol oxime family), RG7916 (splicing modifier), SMN gene therapy (AAV9-SMN1; AVXS-101 (AAV-human SMN transgenic)) and SRK-015 (a selective and local inhibitor antibody of the latent form of myostatin). In some cases, one or more of these agents are used in combination to treat SMA. The method of the present invention is intended to cover any treatment of SMA.

[0082] 3. Biomarkers of SMA

[0083] In order to identify subjects who will benefit from treatment with SMA therapy (such as one or more of the above-mentioned SMA therapies), or to determine whether the therapy is effective, it is helpful to have a biomarker. A biomarker is a property that can be objectively measured and evaluated as an indicator of a normal biological process, a pathological process, or a pharmacological response to a therapeutic intervention. A biomarker can be a biological measure (e.g., a small molecule, a metabolite, a peptide, a protein, an RNA, a DNA), a physiological measure (e.g., blood pressure, electromyography, respiratory function), or a structural measure (e.g., ultrasound, magnetic resonance imaging, or histological assessment). A biomarker can be a prognostic biomarker that predicts future clinical outcomes; a disease progression biomarker that indicates the severity of the disease impact; a predictive biomarker that predicts future clinical responses to therapy and helps to classify the therapy; a pharmacodynamic biomarker that monitors or quantifies the effect of a treatment; and a surrogate endpoint biomarker that predicts future clinical responses to therapy, wherein changes in the endpoint are associated with future clinical responses.

[0084] There are several known biomarkers for SMA. These include prognostic biomarkers, such as SMN2 copy number as an indicator of disease severity; disease progression biomarkers, such as compound muscle action potential (CMAP) amplitude, which serves as an indicator of motor neuron loss; predictive biomarkers, such as reduced CMAP amplitude, which indicates a less responsiveness to SMN restoration therapy; pharmacokinetic biomarkers, such as increased full-length SMN transcripts and / or increased SMN protein as indicators of effective induction of the SMN2 gene; and surrogate endpoint biomarkers, such as increased motor unit estimate (MUNE) as an indicator of improved physical function.

[0085] The present disclosure illustrates the use of neurofilament levels as a novel biomarker for SMA. Neurofilaments (NF) are the main cytoskeletal elements in nerve cells and play a role not only in imparting mechanical stability but also in determining the caliber of axons. Human NF consists of three protein subunits NF-L, NF-M and NF-H. These proteins share the same basic structure with other intermediate filament subunit proteins. Neurofilaments in the mammalian nervous system also contain protein interconnectin, and neurofilaments in the peripheral nervous system may also contain protein peripherin. Therefore, as used herein, "neurofilament protein" refers to neurofilament heavy chain (NF-H), neurofilament middle chain / intermediate chain (NF-M), neurofilament light chain (NF-L), interconnectin or peripherin. SMA biomarkers can be one or more of NF-H, NF-M, NF-L, interconnectin and peripherin. In some cases, the SMA biomarker is phosphorylated NF-H (pNF-H). In some cases, the SMA biomarker is phosphorylated NF-L. RNA (e.g., mRNA) or protein can be used to assess the level of neurofilament biomarkers.

[0086] The amino acid sequence of human NF-H is provided in SEQ ID NO: 1 and SEQ ID NO: 5 and in Lees et al., EMBO J, 7(7); 1947-1955 (1988), UniProtKB - P12036, NCBI Reference Sequence: NG_008404.1, NCBI Reference Sequence: NP_066554.2.

[0087] SEQ ID NO:1

[0088]

[0089] SEQ ID NO:5

[0090]

[0091] The amino acid sequence of human NF-L is provided in SEQ ID NO:2 and in Julien et al., Biochimica et Biohysica Acta, 909:10-20 (1987), UniProtKB - P07196, NCBI Reference Sequence: NP_006149.2, and NCBI Reference Sequence: NG_008492.1.

[0092] SEQ ID NO:2

[0093] MSSFSYEPYYSTSYKRRYVETPRVHISSVRSGYSTARSAYSSYSAPVSSSLSVRRSYSSSSGSLMPSLENLDLSQVAAISNDLKSIRTQEKAQLQDLNDRFASFIERVHELEQQNKVLEAELLVLRQKHSEPSRF RALYEQEIRDLRLAAEDATNEKQALQGEREGLEETLRNLQARYEEEVLSREDAEGRLMEARKGADEAALARAELEKRIDSLMDEISFLKKVHEEEIAELQAQIQYAQISVEMDVTKPDLSAALKDIRAQYEKLAAK NMQNAEEWFKSRFTVLTESAAKNTDAVRAAKDEVSESRRLLKAKTLEIEACRGMNEALEKQLQELEDKQNADISAMQDTINKLENELRTTKSEMARYLKEYQDLLNVKMALDIEIAAYRKLLEGEETRLSFTSVGS ITSGYSQSSQVFGRSAYGGLQTSSYLMSTRSFPSYYTSHVQEEQIEVEETIEAAKAEEAKDEPPSEGEAEEEEKDKEEAEEAAEEEEAAKEESEEAKEEEEGGEGEEGEETKEAEEEEKKVEGAGEEQAAKKKD

[0094] The amino acid sequence of human NF-M is provided in SEQ ID NO:3 and SEQ ID NO:6 and in Myers et al., EMBO J., 6(6):1617-1626 (1987) and UniProtKB-P07197.

[0095] SEQ ID NO:3

[0096] MSYTLDSLGNPSAYRRVTETRSSFSRVSGSPSSGFRSQSWSRGSPSTVSSSYKRSMLAPRLAYSSAMLSSAESSLDFSQSSSLLNGGSGPGGDYKLSRSNEKEQLQGLNDRFAGYIEKVHYLEQQNKEIEAEIQALRQKQASHAQLGDAYDQEIRELRATLEMVNHEKAQVQLDSDHLEEDIHRLKERFEEEARLRDDTEAAAIRALRKDIEEASLVKVELDKKVQSLQD EVAFLRSNHEEEVADLLAQIQASHITVERKDYLKTDISTALKEIRSQLESHSDQNMHQAEEWFKCRYAKLTEAAEQNKEAIRSAKEEIAEYRRQLQSKSIELESVRGTKESLERQLSDIEERHNHDLSSYQDTIQQLENELRGTKWEMARHLREYQDLNLVKMALDIEIAAYRKKLLEGEETRFSTFAGSITGPLYTHRPPITISSKIQKPKVEAPKLKVQHKFVEEIIE ETKVEDEKSEMEEALTAITELAVSMKEEKKEAAEEKEEPEAEEEVAAKKSPVKATAPEVKEEGEKEEEGQEEEEEEDEGAKSDQAEEGGSEKGSSEKEEGQEEGETEAAEEGEEAEAKEEKKEEKVEEKSEEVATKEELVADAKVEKPEKAKSPVPKSPVEEKGKSPVPKSPVEEKGKSPVPKSPVEEKGKSPVSKSPVEEAAKSPVPKSPVEEAKSKAEVGKGEQKEEEKEVKEAPKEEKVEKKEKPKDVPEKKKAESPVKEEAVAEVVTITKSVKVHLEKETKEEGKPLQQEKEKEKEKKAGGEGGSEEEGSDKGAKGSRKEDIAVNGEVEGKEEVEQETKEKGGSGREEKGVVTNGLDLSPADEKKGGDKSEEKVVVTKTVEKITSEGGDGATKYITKSVTVTQKVEEHEETFEEKLVSTKKVEKVTSHAIVKEVTQSD

[0097] SEQ ID NO:6

[0098] MARHLREYQDLLNVKMALDIEIAAYRKLLEGEETRFSTFAGSITGPLYTHRPPITISSKIQKPKVEAPKLKVQHKFVEEIIEETKVEDEKSEMEEALTAITEELAVSMKEEKKEAAEEKEEEPEAEEEEVAAKKSPVKATAPEVKEEEGEKEEEEGQEEEEEEDEGAKSDQAEEGGSEKEGSSEKEEGEQEEGETEAEAEGEEAEAKEEKKVEEKSEEVATKEELVADAKVEKPEKAKSPVPKSPVEEKGKSPVPKSPVEEKGKSPVPKSPVEEKGKSPVPKSPVEEKGKSPVSKSPVEEKAKSPVPKSPVEEAKSKAEVGKGEQKEEEEKEVKEAPKEEKVEKKEEKPKDVPEKKKAESPVKEEAVAEVVTITKSVKVHLEKETKEEGKPLQQEKEKEKAGGEGGSEEEGSDKGAKGSRKEDIAVNGEVEGKEEVEQETKEKGSGREEEKGVVTNGLDLSPADEKKGGDKSEEKVVVTKTVEKITSEGGDGATKYITKSVTVTQKVEEHEETFEEKLVSTKKVEKVTSHAIVKEVTQSD

[0099] The amino acid sequence of the human internexin is provided in SEQ ID NO:7.

[0100] SEQ ID NO:7

[0101] MSFGSEHYLCSSSSYRKVFGDGSRLSARLSGAGGAGGFRSQSLSRSNVASSAACSSASSLGLGLAYRRPPASDGLDLSQAAARTNEYKIIRTNEKEQLQGLNDRFAVFIEKVHQLETQNRALEAELAALRQRHAEPSRVGELFQRELRDLRAQLEEASSARSQALLERDGLAEEVQRLRARCEEESRGREGAERALKAQQRDVDGATLARLDLEKKVESLLDELAFVRQVHDEEVAELLATLQASSQAAAEVDVTVAKPDLTSALREIRAQYESLAAKNLQSAEEWYKSKFANLNEQAARSTEAIRASREEIHEYRRQLQARTIEIEGLRGANESLERQILELEERHSAEVAGYQDSIGQLENDLRNTKSEMARHLREYQDLLNVKMALDIEIAAYRKLLEGEETRFSTSGLSISGLNPLPNPSYLLPPRILSATTSKVSSTGLSLKKEEEEEEASKVASKKTSQIGESFEEILEETVISTKKTEKSNIEETTISSQKI

[0102] The amino acid sequence of human peripherin is provided in SEQ ID NO:8.

[0103] SEQ ID NO:8

[0104] MSHHPSGLRAGFSSTSYRRTFGPPPSLSPGAFSYSSSSRFSSSRLLGSASPSSSVRLGSFRSPRAGAGALLRLPSERLDFSMAEALNQEFLATRSNEKQELQELNDRFANFIEKVRF LEQQNAALRGELSQARGQEPARADQLCQQELRELRRELELLGRERDRVQVERDGLAEDLAALKQRLEEETRKREDAEHNLVLFRKDVDDATLSRLELERKIESLMDEIEFLKKLHEEE LRDLQVSVESQQVQQVEVEATVKPELTAALRDIRAQYESIAAKNLQEAEEWYKSKYADLSDAANRNHEALRQAKQEMNESRRQIQSLTCEVDGLRGTNEALLRQLRELEEQFALEAG GYQAGAARLEEELRQLKEEMARHLREYQELLNVKMALDIEIATYRKLLEGEESRISVPVHSFASLNIKTTVPEVEPPQDSHSRKTVLIKTIETRNGEVVTESQKEQRSELDKSSAHSY

[0105] In certain instances, the level of NF (e.g., pNF-H) is used in combination with one or more other SMA biomarkers.

[0106] 4. Diagnosis of SMA

[0107] The present disclosure features a method of diagnosing whether a subject (e.g., a presymptomatic subject) has a biologically active disease (i.e., whether SMA is active and its severity). The method includes measuring the level of neurofilaments in a biological sample obtained from the subject. If the subject's neurofilament level is higher than a control level, SMA is diagnosed. Neurofilament levels can also predict the severity of the disease: the higher the NF level relative to the control, the more severe the SMA.

[0108] In some cases, the method includes measuring the level of NF-H in a biological sample obtained from the subject. In some cases, the method includes measuring the level of pNF-H in a biological sample obtained from the subject. In some cases, the method includes measuring the level of NF-M in a biological sample obtained from the subject. In some cases, the method includes measuring the level of NF-L in a biological sample obtained from the subject.

[0109] The biological sample can be, for example, blood, serum, plasma, or cerebrospinal fluid. In some cases, the biological sample is plasma.

[0110] In some cases, the subject is a human fetus. In some cases, the subject is a newborn. In some cases, the subject is less than or equal to 6 months of age (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months or 6 months). In some cases, the subject is greater than 6 months and less than or equal to 18 months of age (e.g., 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months or 18 months). In some cases, the subject is a person older than 18 months. In some cases, the subject is a person older than 18 years.

[0111] In some cases, NF levels are measured by assessing the level of NF RNA (e.g., mRNA) in a biological sample.

[0112] In some cases, the NF level is measured by assessing the level of NF protein (NF-H, NF-M or NF-L protein) in a biological sample. In some cases, the NF protein is pNF-H. The concentration of one or more target proteins can be measured using any method known in the art, such as immunoassay. Non-limiting examples of such methods include enzyme immunoassay, radioimmunoassay, chemiluminescent immunoassay, electrochemiluminescent immunoassay, latex turbidimetric immunoassay, latex photometric immunoassay, immunochromatographic assay and Western blot. In certain embodiments, the concentration of one or more target proteins is measured by mass spectrometry.

[0113] In some embodiments, the level of neurofilaments (e.g., pNF-H) in a biological sample is higher than a control level. In some embodiments, the level of neurofilaments in a biological sample is higher than 300 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is higher than 400 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is higher than 500 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is higher than 600 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is higher than 700 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is higher than 800 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is higher than 900 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is higher than 1,000 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is higher than 1,100 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is higher than 1,200 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is higher than 1,300 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is above 1,400 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is above 1,500 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is above 2,000 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is above 3,000 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is above 4,000 pg / mL. In some embodiments, the level of neurofilaments in a biological sample is above 5,000 pg / mL.

[0114] Any SMA therapy can be administered to a human subject diagnosed with SMA. In some cases, an SMA therapy is administered to a human subject who has been pre-determined to have SMA (e.g., by measuring NF levels in a biological sample from the subject). In some cases, the therapy is SPINRAZA In some cases, SPINRAZA was administered intrathecally at a dose of 12 mg per administration. In some cases, an SMA therapy is a combination therapy.

[0115] Exemplary levels of pNF-H in subjects with SMA are provided in the following table:

[0116]

[0117] 5. Response to treatment

[0118] The level of NF can also be used to determine whether a subject receiving SMA therapy is responding to the treatment. This can be assessed by obtaining a first biological sample from the subject before the SMA therapy is administered to the subject and obtaining a second biological sample from the subject after the SMA therapy is administered to the subject, and measuring the level of NF (e.g., NF-H, NF-M, or NF-L) in such samples. In one example, the level of NF is the level of pNF-H. In some cases, the therapy is SPINRAZA In some cases, SPINRAZA was administered intrathecally at a dose of 12 mg per administration. In some cases, an SMA therapy is a combination therapy.

[0119] In some cases, the first one or more biological samples may be collected from the subject at any time before treatment, such as one week before, a few days before, a day before, a few hours before, an hour before, or less than an hour before the administration of the SMA therapy. Similarly, a second biological sample or multiple second biological samples may be collected from the subject at any time after the administration of the SMA therapy, such as less than one hour after the administration of the SMA therapy, one hour after, a few hours after, a day after, a few days after, a week after, a few weeks after, a month after, two months after, three months after, four months after, five months after, six months after, seven months after, or eight months after. A reduction in NF levels after the start of SMA therapy indicates the effectiveness of the SMA therapy. In such cases, it is indicated to continue the SMA therapy. Failure to reduce NF levels after the start of SMA therapy indicates the need to change the dosage of the SMA therapy (e.g., increase the dosage), or lack of effectiveness of the specific SMA therapy. In the latter case, it may be recommended to stop the specific SMA therapy and consider using one or more different SMA therapies.

[0120] NF levels can be assessed by measuring RNA or protein levels. In some cases, the level of pNF-H is determined. The concentration of one or more target NF proteins can be measured using any method known in the art, such as an immunoassay. Non-limiting examples of such methods include enzyme immunoassays, radioimmunoassays, chemiluminescent immunoassays, electrochemiluminescent immunoassays, latex turbidimetric immunoassays, latex photometric immunoassays, immunochromatographic assays, and Western blots. In certain embodiments, the concentration of one or more target proteins is measured by mass spectrometry.

[0121] In some cases, the level of neurofilaments (e.g., pNF-H) in the first biological sample is greater than 300 pg / mL, greater than 400 pg / mL, greater than 500 pg / mL, greater than 600 pg / mL, greater than 700 pg / mL, greater than 800 pg / mL, greater than 900 pg / mL, greater than 1,000 pg / mL, greater than 1,500 pg / mL, greater than 2,000 pg / mL, greater than 3,000 pg / mL, greater than 4,000 pg / mL, or greater than 5,000 pg / mL. In certain embodiments, the level of neurofilaments measured in the second biological sample is lower than the level of neurofilaments measured in the first biological sample. In some embodiments, the neurofilament levels measured in the second biological sample show a decrease of greater than 30% (e.g., greater than 31%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, or 95%) relative to the neurofilament levels measured in the first biological sample. In some embodiments, the neurofilament levels measured in the second biological sample are between 10% and 80% of the neurofilament levels measured in the first biological sample. In some embodiments, the neurofilament levels measured in the second biological sample are between 20% and 80% of the neurofilament levels measured in the first biological sample. In some embodiments, the neurofilament levels measured in the second biological sample are between 20% and 85% of the neurofilament levels measured in the first biological sample. In some embodiments, the neurofilament levels measured in the second biological sample are between 20% and 90% of the neurofilament levels measured in the first biological sample. In some embodiments, the level of neurofilaments measured in the second biological sample is between 20% and 95% of the level of neurofilaments measured in the first biological sample. In some embodiments, the level of neurofilaments measured in the second biological sample is between 30% and 80% of the level of neurofilaments measured in the first biological sample. In some embodiments, the level of neurofilaments measured in the second biological sample is between 30% and 85% of the level of neurofilaments measured in the first biological sample. In some embodiments, the level of neurofilaments measured in the second biological sample is between 30% and 90% of the level of neurofilaments measured in the first biological sample. In some embodiments, the level of neurofilaments measured in the second biological sample is between 30% and 95% of the level of neurofilaments measured in the first biological sample.

[0122] In some cases, subject is the people before symptom onset.In some cases, subject is a newborn.In some cases, subject is less than or equal to 6 months of age (for example, 2 days of age, 3 days of age, 4 days of age, 5 days of age, 6 days of age, 1 week of age, 2 weeks of age, 3 weeks of age, 1 month of age, 2 months of age, 3 months of age, 4 months of age, 5 months of age or 6 months of age).In some cases, subject is greater than 6 months of age and less than or equal to 18 months of age (for example, 7 months of age, 8 months of age, 9 months of age, 10 months of age, 11 months of age, 12 months of age, 13 months of age, 14 months of age, 15 months of age, 16 months of age, 17 months of age or 18 months of age).In some cases, subject is greater than 18 months of age.In some cases, subject is greater than 18 years of age.

[0123] 6. Predicting disease progression

[0124] NF levels in biological samples from subjects can also be used to predict future phenotypes. For example, NF levels can predict future motor function. As an illustration, pNF-H levels one to two months after starting therapy can predict motor function ten months after starting therapy. The lower the pNF-H level relative to the control one to two months after starting therapy, the more likely the subject will have improved motor function relative to a subject with a higher pNF-H level one to two months after starting therapy. This predictable possibility allows healthcare practitioners to modify or adjust the dosing and treatment of the subject.

[0125] 7. Comparison

[0126] As described above, the methods of the present disclosure may include measuring the expression level (e.g., mRNA or protein concentration) of one or more NF genes or proteins in a biological sample from a subject (e.g., a presymptomatic human subject), wherein the expression level of one or more of the NF genes or proteins compared to a control predicts whether the subject has SMA; the severity of SMA; the future phenotype; and whether the subject is a candidate for a treatment that includes SMA therapy (e.g., SPINRAZA ) of the treatment.

[0127] In certain embodiments, when diagnosing whether a subject has SMA, the subject is identified as possibly having SMA when the concentration of NF protein (e.g., pNF-H) in a biological sample from the subject is higher than that of a control. In this context, the term "control" includes samples obtained from subjects of the same or similar age who are known not to have SMA (from the same source, such as blood, plasma, serum, CSF). For example, if a subject who is a newborn is being tested, the control is also from a newborn who does not have SMA; if a subject of 6-18 months of age is being tested, the control is also from a subject of 6-18 months of age who does not have SMA. The term "control" also includes samples (from the same tissue) obtained from subjects known not to have SMA in the past and used as references for comparison with test samples obtained from subjects predicting SMA in the future. A "control" expression level / concentration of a particular NF protein may also be pre-established by analyzing one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, 15, 20, 25, 30, 35, or 40 or more) human subjects of similar age who do not suffer from SMA. Such a pre-established reference value (which may be an average or median expression level / concentration taken from multiple subjects who do not suffer from SMA) may then be used for the "control" concentration / expression level of the protein or nucleic acid in comparison with the test sample. In such a comparison, if the expression level of the analyzed NF is higher than the pre-established reference, then the subject is predicted to suffer from SMA.

[0128] The methods of the present disclosure may include measuring the expression level (e.g., mRNA or protein concentration) of one or more genes (e.g., one or more NF genes) in a biological sample from a subject having or suspected of having SMA, wherein the expression level of one or more of the NF genes compared to a control predicts the subject's responsiveness to a therapy comprising SMA (e.g., SPINRAZA ) treatment. In certain embodiments, when the concentration of NF protein (e.g., pNF-H) in a biological sample from a subject suffering from or suspected of suffering from SMA is lower than that of a control, the subject is identified as likely to respond to a therapy including an SMA therapy. In this context, the term "control" includes samples (from the same source, e.g., plasma, blood, serum, CSF) obtained from subjects of the same or similar age who are known to be unresponsive to the SMA therapy. The term "control" also includes samples (from the same tissue) obtained in the past from subjects known to be unresponsive to the SMA therapy and used as a reference for comparison with test samples obtained from subjects predicted to be responsive to treatment in the future. The "control" expression level / concentration of a specific NF protein in a specific cell type or tissue can be determined by analyzing one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, or 40 or more) of the expression level / concentration of a specific NF protein in a specific cell type or tissue for a SMA therapy (e.g., SPINRAZA ) treatment did not respond to the therapy. This pre-established reference value (which can be an average or median expression level / concentration taken from multiple subjects who did not respond to the therapy) can then be used for the "control" concentration / expression level of the protein or nucleic acid in comparison with the test sample. In such a comparison, if the expression level of the NF gene analyzed is lower than the pre-established reference, then the subject is predicted to be responsive to a SMA therapy including SMA therapy (e.g., SPINRAZA ) therapy.

[0129] Alternatively, the use of SMA therapies (e.g., SPINRAZA ) treatment to pre-establish a "control" concentration of a particular protein (e.g., NF-H) in a particular biological fluid, cell type, or tissue. This pre-established reference value (which can be an average or median expression level taken from multiple subjects who responded to the therapy) can then be used as the "control" expression level in comparison with the test sample. In this comparison, if the concentration of the analyzed protein is the same or comparable (at least 85% but less than 100%) to the pre-established reference, then the subject is predicted to be responsive to the SMA therapy (e.g., SPINRAZA ) has a response.

[0130] In certain embodiments, a "control" is a predetermined cutoff value.

[0131] In some embodiments, the methods described herein include determining whether the concentration of one or more target NF proteins is above or below a predetermined cutoff value.

[0132] A cutoff value is typically a concentration of a protein above or below which a concentration is considered predictive of something, such as the likelihood of developing SMA or the responsiveness of a subject to a therapy of interest. Thus, according to the methods described herein, a reference concentration of a NF protein (e.g., pNF-H) is identified as a cutoff value above or below which a concentration is considered predictive of a subject having SMA or demonstrating responsiveness to a therapy for SMA (e.g., SPINRAZA ) of subjects with responsiveness to the test. Some cutoffs are not absolute, as clinical relevance remains significant over a range of values ​​on either side of the cutoff; however, it is possible to select an optimal cutoff for NF protein concentration (e.g., a varying H-score) for a particular sample type. The cutoffs determined for use in the methods described herein can be compared to, for example, published NF concentration ranges, but can be individualized based on the method and patient population used. It should be understood that improvements in the optimal cutoff can be determined based on the complexity of the statistical methods used and the number and source of samples used to determine reference level values ​​for different proteins, genes, and sample types. Therefore, established cutoffs can be adjusted upward or downward based on periodic reassessment or changes in the method or population distribution.

[0133] A reference concentration of one or more NF proteins can be determined by a variety of methods. For example, a reference concentration of one or more NF proteins can be determined by comparing, for example, ) or do not respond to SMA therapy. This can be achieved, for example, by a histogram analysis, in which the entire patient group is presented graphically, wherein the first axis represents the concentration of the target protein and the second axis represents the number of subjects in the group whose samples contain one or more concentrations. The reference concentration of the protein can then be determined based on the amount or concentration that best distinguishes these individual groups. The reference level can be a single number, equally applicable to each subject, or the reference level can vary according to a specific subgroup of the subject. For example, older subjects may have different reference levels than younger subjects. In addition, subjects with more severe disease may have different reference values ​​than subjects with milder disease (e.g., type I vs. type IV SMA; type I vs. type III SMA; type I vs. type II SMA).

[0134] The pre-established cutoff value can be a NF protein concentration (e.g., pNF-H) determined based on a receiver operating characteristic (ROC) analysis. The ROC curve is used to determine the cutoff value for clinical testing. Considering the situation where there are two groups of patients and by using established standard techniques, one group is known to respond to SMA therapy, and the other group is known to be unresponsive to SMA therapy. Measurements made using biological samples from all members of the two groups are used to test the responsiveness to SMA therapy. The test will find some (but not all) responders who respond to SMA therapy. The ratio of responders found by the test to the total number of responders (known by established standard techniques) is the true positive rate (also known as sensitivity). The test will find some (but not all) non-responders who do not respond to SMA therapy. The ratio of non-responders found by the test to the total number of non-responders (known by established standard techniques) is the true negative rate (also known as specificity). It is hoped that the ROC curve analysis of the SMA therapy responsiveness test will find a cutoff value that will minimize the number of false positives and false negatives. ROC is a chart that illustrates the performance of a binary classifier system when its discrimination threshold changes. It is created by plotting the fraction of true positives against the fraction of false negatives at various threshold settings.

[0135] In one embodiment, the NF protein concentration is determined based on ROC analysis and positive predictive value for predicting response to SMA therapy, wherein a concentration of a target protein (e.g., pNF-H) equal to or below a pre-established cutoff value is a low concentration of the target protein, and a value above the pre-established cutoff value is a high concentration of the target protein. The positive predictive value is the proportion of positive test results that are true positives; it reflects the likelihood that a positive test reflects the underlying condition being tested. Methods for constructing ROC curves and determining positive predictive values ​​are well known in the art.

[0136] In another embodiment, the pre-established cutoff value can be a NF protein concentration determined based on a simulation model that predicts responsiveness to an SMA therapy, and wherein a concentration of the target protein (e.g., pNF-H) equal to or below the pre-established cutoff value is a low concentration of the target protein, and a value above the pre-established cutoff value is a high concentration of the target protein.

[0137] 8. Biological samples

[0138] Suitable biological samples for methods described herein include any biological fluid, cell, tissue or part thereof, including target analyte biomolecules, such as NF protein or nucleic acid (e.g., RNA (mRNA)). Biological samples can be specimens obtained from human subjects, for example, or can be derived from such subjects. For example, the sample can be a tissue section obtained by biopsy, a biofluid archived, or a cell placed in a tissue culture or suitable for tissue culture. In some cases, the biological sample is a biological fluid, such as blood, plasma, serum, cerebrospinal fluid (CSF), urine, or such a sample absorbed on a matrix (e.g., glass, polymer, paper). If desired, the biological sample can be further fractionated into fractions containing specific cell types. For example, blood sample fractionation can be serum or fractions containing specific types of blood cells such as red blood cells or white blood cells (leukocytes). If desired, the sample can be a combination of samples from a subject, such as a combination of tissue and fluid samples.

[0139] The biological sample may be obtained from a subject having SMA, suspected of having SMA, or at risk of developing SMA. In certain embodiments, the subject is a human fetus. In certain embodiments, the subject is a presymptomatic human infant. In certain embodiments, the subject is a presymptomatic human child. In certain embodiments, the subject is a presymptomatic human adult.

[0140] Any suitable method for obtaining a biological sample may be employed, but exemplary methods include, for example, phlebotomy, fine needle aspiration biopsy procedures. Samples may also be collected, for example, by microdissection (eg, laser capture microdissection (LCM) or laser microdissection (LMD)).

[0141] Methods for obtaining and / or storing samples that retain the activity or integrity of molecules (e.g., nucleic acids or proteins) in the sample are well known to those skilled in the art. For example, the biological sample can be further contacted with one or more additional agents, such as buffers and / or inhibitors, including one or more of nuclease, protease and phosphatase inhibitors, which retain molecules (e.g., nucleic acids or proteins) in the sample or minimize changes in the molecules. Such inhibitors include, for example, chelating agents such as ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis (p-aminoethyl ether) N,N,N1,N1-tetraacetic acid (EGTA); protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, antiproteases, etc.; and phosphatase inhibitors such as phosphates, sodium fluoride, vanadates, etc. Suitable buffers and conditions for separating molecules are well known to those skilled in the art, and may vary depending, for example, on the type of molecule in the sample to be characterized (see, e.g., Ausubel et al. Current Protocols in Molecular Biology (Appendix 47), John Wiley & Sons, New York (1999); Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press (1988); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1999); Tietz Textbook of Clinical Chemistry, 3rd Edition Burtis and Ashwood, ed. W. B. Saunders, Philadelphia, (1999)). The sample may also be processed to eliminate or minimize the presence of interfering substances. For example, the biological sample may be fractionated or purified to remove one or more non-target materials. Methods for fractionating or purifying biological samples include, but are not limited to, chromatography, such as liquid chromatography, ion exchange chromatography, size exclusion chromatography, or affinity chromatography. For use in the methods described herein, the sample may be in a variety of physical states. For example, the sample may be a liquid or solid, may be dissolved or suspended in a liquid, may be in an emulsion or gel, or may be absorbed onto a material.

[0142] 9. Determine the expression level / concentration of biomarkers

[0143] Gene expression can be detected as, for example, protein or RNA expression of a target gene. That is, the presence or expression level (amount) of a gene can be determined by detecting and / or measuring the level of mRNA or protein expression of the gene. In some embodiments, gene expression can be detected as the activity of a protein encoded by a NF gene.

[0144] In one embodiment, the expression of the gene can be determined by detecting and / or measuring the expression or concentration of the protein encoded by the gene. The method for determining protein expression / concentration is well known in the art. Commonly used methods include using antibodies that are specific to the target protein. For example, methods for determining protein expression include but are not limited to Western blot or dot blot analysis, immunohistochemistry (e.g., quantitative immunohistochemistry), immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT; Coligan, JE, et al., editor (1995) Current Protocols in Immunology. Wiley, New York), radioimmunoassay, chemiluminescent immunoassay, electrochemiluminescent immunoassay, latex turbidity immunoassay, latex photometric immunoassay, immuno-chromatographic assay and antibody array analysis (see, e.g., U.S. Publication Nos. 2003 / 0013208 and 2004 / 171068, the disclosures of which are hereby incorporated by reference in their entirety). Further descriptions of the above-mentioned many methods and other methods for detecting protein expression can be found in, e.g., Sambrook et al. (ibid.).

[0145] In one example, the presence or amount of NF protein expression of a NF gene (e.g., NF-H) can be determined using a Western blotting technique. For example, a lysate can be prepared from a biological sample, or the biological sample itself can be contacted with Laemmli buffer and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE resolved proteins separated by size can then be transferred to a filter membrane (e.g., nitrocellulose) and immunoblotting techniques can be performed using a detectably labeled antibody specific for the target protein. The presence or amount of the bound detectably labeled antibody indicates the presence or amount of the protein in the biological sample.

[0146] In one embodiment, the SimplePlex platform is used to measure the level of NF-H (e.g., phosphorylated NF-H). SimplePlex is commercially available from Protein Simple (San Jose, CA, USA) (see Dysinger M, et al. J. Immunol. Methods. 451: 1-10, 2017).

[0147] In one embodiment, an assay for measuring NF-L (e.g., phosphorylated NF-L) is used. Assays for measuring NF-L in serum have been described (see, e.g., Gaiottino et al., PLoS ONE 8: e75091, 2013; Kuhle et al., J. Neurol. Neurosurg. Psychiatry 86(3): 273-279, 2014). In one example, serum from a subject is centrifuged at 1000 g for 10 minutes at room temperature and stored at -80°C within 2 hours of collection. Serum NF-L concentrations can be measured (e.g., in duplicate) using a ready-to-use enzyme-linked immunosorbent assay (ELISA) (Mabtech AB, Nacka Strand, Sweden) or an electrochemiluminescence (ECL) immunoassay as described in Gaiottino et al., PLoS ONE 8: e75091, 2013, or a single molecule array (SIMOA) method as described in Disanto et al., Ann. Neurol. 81(6): 857-870, 2017. Assay methods have been compared in Kuhl et al., Clinical Chemistry and Laboratory Medicine 54(10): 1655-1661, 2016. The SIMOA assay (specifically, the Simoa NF-light Advantage kit) is commercially available from Quanterix Corp. (Lexington, MA, USA).

[0148] In another example, immunoassay can be used for detecting and / or measuring the protein expression of gene (for example, NF-H gene).As mentioned above, for the purpose of detection, immunoassay can be performed with antibodies with detection moieties (for example, fluorescent agents or enzymes).Protein from biological sample can be directly conjugated with solid phase matrix (for example, porous assay plate, nitrocellulose, agarose, sepharose, coded particles or magnetic beads), or it can be conjugated with first member of specific binding pair (for example, biotin or streptavidin), which is attached to solid phase matrix after being combined with second member of described specific binding pair (for example, streptavidin or biotin).This attachment with solid phase matrix allows protein to be purified from other interferences or irrelevant components of biological sample before contacting with detection antibody, and also allows unbound antibodies to be washed subsequently.Here, as mentioned above, the presence or amount of the antibody of the detectable label combined indicates the presence or amount of protein in biological sample.

[0149] There is no particular limitation on the form of the antibody, and the present disclosure includes polyclonal antibodies as well as monoclonal antibodies. It also includes antisera obtained by immunizing an animal (such as a rabbit) with the protein of the present invention or a fragment thereof (i.e., a protein of the NF protein or an immune fragment thereof), and all types of polyclonal antibodies and monoclonal antibodies, human antibodies, and humanized antibodies produced by genetic recombination.

[0150] The entire protein or its partial peptide can be used as an antigen for immunization. As a partial peptide of a protein, for example, an amino (N)-terminal fragment and a carboxyl (C)-terminal fragment of a protein can be given.

[0151] The gene encoding the target protein or its fragment (e.g., immune fragment) is inserted into a known expression vector, and the host cell is transformed with the vector described herein, and the desired protein or its fragment is recovered from the outside or inside of the host cell using a standard method. Such a protein can be used as a sensitizing antigen. In addition, cells expressing the protein, cell lysates, or chemically synthesized proteins of the present invention can also be used as sensitizing antigens.

[0152] Mammals to be immunized with sensitizing antigens are not limited; however, it is preferred to select animals by considering compatibility with parent cells used in cell fusion. In general, animals belonging to the order Rodentia, Lagomorpha, or Primates are used. Examples of animals belonging to the order Rodentia that can be used include, for example, mice, rats, and hamsters. Examples of animals belonging to the order Lagomorpha that can be used include, for example, rabbits. Examples of animals belonging to the order Primates that can be used include, for example, monkeys. Examples of monkeys to be used include the order Catarrhalis (Old World Monkeys), such as cynomolgus monkeys, rhesus monkeys, baboons, and chimpanzees.

[0153] Well-known methods can be used to immunize animals with sensitizing antigens. For example, the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is appropriately diluted and suspended in physiological saline, phosphate buffered saline (PBS), etc., and mixed with an appropriate amount of general adjuvant, such as Freund's complete adjuvant, as required. Then, the solution is emulsified and injected into a mammal. Thereafter, the sensitizing antigen appropriately mixed with Freund's incomplete adjuvant is preferably administered several times every 4 to 21 days. When an animal is immunized with a sensitizing antigen, a suitable carrier may also be used. After immunization, the increase in serum antibody levels is detected by a common method.

[0154] Polyclonal antibodies to proteins of the present disclosure can be prepared as follows. After confirming that the desired serum antibody level has been reached, blood is drawn from a mammal sensitized with an antigen. Serum is separated from the blood using conventional methods. The serum containing the polyclonal antibodies can be used as the polyclonal antibodies, or, as desired, fractions containing the polyclonal antibodies can be further separated from the serum. For example, fractions of antibodies that specifically recognize proteins of the present invention can be prepared by using an affinity column coupled to a protein. The fractions can then be further purified using a protein A or protein G column to prepare immunoglobulin G or M.

[0155] In order to obtain monoclonal antibodies, after confirming that the desired serum antibody level has been reached in a mammal sensitized with the above antigen, immune cells are obtained from the mammal and used for cell fusion. For this purpose, spleen cells can be mentioned as preferred immune cells. As parent cells fused with the above immune cells, mammalian myeloma cells are preferably used. More preferably, myeloma cells with characteristics are used as parent cells, and the myeloma cells can be used to distinguish fused cells by agents.

[0156] The cell fusion between the above-mentioned immune cells and myeloma cells can be performed according to a known method, for example, the method of Milstein et al. (Galfre et al., Methods Enzymol. 73:3-46, 1981).

[0157] The hybridoma obtained from cell fusion is selected by culturing cells in a standard selection medium, such as a HAT medium (a medium containing hypoxanthine, aminopterin and thymidine). The cultivation in this HAT medium continues for a sufficiently long time to kill cells (non-fused cells) other than the target hybridoma, usually several days to several weeks. Then, the common limiting dilution method is performed to screen and clone the hybridoma that produces the target antibody.

[0158] In addition to the above-mentioned method for obtaining hybridomas, hybridomas producing the desired human antibodies having the activity of binding to proteins can be obtained by immunizing animals other than humans with antigens by the following method: human lymphocytes, such as human lymphocytes infected with Epstein-Barr virus, are sensitized in vitro with proteins, cells expressing proteins, or their lysates, and the sensitized lymphocytes are fused with myeloma cells (e.g., U266) derived from humans and having the ability of permanent cell division.

[0159] The monoclonal antibody obtained by transplanting the obtained hybridoma into the peritoneal cavity of a mouse and extracting ascites can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, affinity column coupled with the protein of the present disclosure, and the like.

[0160] Monoclonal antibodies can also be obtained as recombinant antibodies produced using genetic engineering techniques (see, for example, Borrebaeck CAK and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD (1990)). Recombinant antibodies are produced by cloning encoding DNA from immune cells such as hybridomas or sensitized lymphocytes that produce antibodies, incorporating them into suitable vectors, and introducing such vectors into hosts to produce the antibodies. Such recombinant antibodies are also encompassed by the present disclosure.

[0161] Antibodies or antibody fragments specific for proteins encoded by one or more biomarkers can also be generated by in vitro methods, such as phage display.

[0162] In addition, the antibodies of the present disclosure may be antibody fragments or modified antibodies, as long as they bind to the protein encoded by the biomarker of the present invention. For example, Fab, F (ab') 2, Fv or single-chain Fv (scFv) can be given as antibody fragments, wherein the H chain Fv and the L chain Fv are appropriately connected by a linker (Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883, (1988)). Specifically, antibody fragments are produced by treating antibodies with enzymes such as papain or pepsin. Alternatively, they can be produced by constructing genes encoding antibody fragments, introducing the genes into expression vectors, and expressing the vectors in appropriate host cells (see, e.g., Co et al., J. Immunol., 152:2968-2976, 1994; Better et al., Methods Enzymol., 178:476-496, 1989; Pluckthun et al., Methods Enzymol., 178:497-515, 1989; Lamoyi, Methods Enzymol., 121:652-663, 1986; Rousseaux et al., Methods Enzymol., 121:663-669, 1986; Bird et al., Trends Biotechnol., 9:132-137, 1991).

[0163] Antibodies can be conjugated to various molecules, such as fluorescent substances, radioactive substances, and luminescent substances. Methods for attaching such moieties to antibodies are established and routine in the art (see, for example, US Pat. Nos. 5,057,313 and 5,156,840).

[0164] Examples of methods for determining the antigen binding activity of the antibody include, for example, measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) and / or immunofluorescence. For example, when using ELISA, the protein encoded by the biomarker of the present invention is added to a plate coated with the antibody of the present disclosure, and then the antibody sample is added, such as the culture supernatant of cells producing the antibody or the purified antibody. Then, a second antibody that recognizes the first antibody is added, the second antibody is labeled with alkaline phosphatase and such enzymes, the plate is incubated and washed, and the absorbance is measured after adding an enzyme substrate (such as p-nitrophenyl phosphate) to evaluate the antigen binding activity. As a protein, a protein fragment, such as a fragment comprising a C-terminus or a fragment comprising an N-terminus, can be used. In order to evaluate the activity of the antibody of the present invention, BIAcore (Pharmacia) can be used.

[0165] By using these methods, the antibodies of the present invention are contacted with a sample presumed to contain the protein of the present invention, and the protein encoded by the biomarker of the present invention is detected or measured by detecting or measuring the immune complex formed between the above-mentioned antibody and the protein.

[0166] Quantitative determination methods based on mass spectrometry (such as, but not limited to, a combination of methods based on multiple reaction monitoring (MRM) and stable isotope labeled internal standards) are alternative methods for immunoassays for quantitative measurement of proteins. These methods do not require the use of antibodies, and therefore the analysis can be performed in a cost- and time-effective manner (see, for example, Addona et al., Nat. Biotechnol., 27: 633–641, 2009; Kuzyk et al., Mol. Cell Proteomics, 8: 1860–1877, 2009; Paulovich et al., Proteomics Clin. Appl., 2: 1386-1402, 2008). In addition, MRM also provides excellent multiplexing capabilities, allowing multiple proteins to be quantified simultaneously in parallel. The basic theory of these methods has been well established and widely used in drug metabolism and pharmacokinetic analysis of small molecules.

[0167] In another embodiment, the expression level of the target NF gene is determined by measuring RNA levels. A variety of suitable methods can be used to detect and / or measure the mRNA expression level of a gene. For example, RNA or dot blot analysis, reverse transcriptase-PCR (RT-PCR; e.g., quantitative RT-PCR), in situ hybridization (e.g., quantitative in situ hybridization) or nucleic acid array (e.g., oligonucleotide array or gene chip) analysis can be used to determine mRNA expression. Details of such methods are described below and in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Edition Vols. 1, 2 and 3. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, USA, Nov. 1989; Gibson et al. (1999) Genome Res., 6(10):995-1001; and Zhang et al. (2005) Environ. Sci. Technol., 39(8):2777-2785; U.S. Publication No. 2004086915; European Patent No. 0543942; and U.S. Patent No. 7,101,663; the disclosures of each of which are incorporated herein by reference in their entirety.

[0168] In one example, the presence or amount of one or more discrete mRNA populations in the biological sample can be determined by isolating total mRNA from the biological sample (see, for example, Sambrook et al. (supra) and U.S. Patent No. 6,812,341) and subjecting the isolated mRNA to agarose gel electrophoresis to separate mRNA according to size. The size-separated mRNA is then transferred (e.g., by diffusion) to a solid support such as a nitrocellulose membrane. The presence or amount of one or more mRNA populations in the biological sample can then be determined using one or more detectably labeled polynucleotide probes complementary to the target mRNA sequence, which are combined with their corresponding mRNA populations and thus make the corresponding mRNA populations detectable. Detectable labels include, for example, fluorescent labels (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine amine fluorescein, dansyl chloride, allophycocyanin (APC) or phycoerythrin), luminescent labels (e.g., europium, terbium, Qdot™ nanoparticles supplied by QuantumDot Corporation, Palo Alto, CA), radioactive labels (e.g., 125 I. 131 I. 35 S. 32 P. 33 P or 3H) and enzymatic (horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase) labeling.

[0169] In another example, the presence or amount of a discrete population of mRNA (e.g., mRNA encoded by one or more NF genes) in a biological sample can be determined using a nucleic acid (or oligonucleotide) array. For example, the RT-PCR of the first chain synthesis mediated by, for example, random hexamer or oligo (dT)-primer can be used to amplify the separated mRNA from the biological sample. The amplicon can be fragmented into shorter segments. The RT-PCR step can be used to detectably mark the amplicon, or optionally, the amplicon can be detectably marked after the RT-PCR step. For example, any one of a variety of suitable techniques can be used to enzymatically (e.g., by nick translation or kinase, such as T4 polynucleotide kinase) or chemically conjugated to the amplicon (see, for example, Sambrook et al., supra). The detectably labeled amplicon is then contacted with a plurality of polynucleotide probe groups, each group containing one or more polynucleotide (e.g., oligonucleotide) probes that are specific to the corresponding amplicon (and can be bound to the corresponding amplicon), and wherein the plurality of polynucleotide probe groups contain many probe groups that each correspond to different amplicons. In general, the probe set is bound to a solid support, and the position of each probe set is predetermined on the solid support. The binding of the detectably labeled amplicon to the corresponding probe of the probe set indicates the presence or amount of the target mRNA in the biological sample. Other methods for detecting mRNA expression using nucleic acid arrays are described in, for example, U.S. Patent Nos. 5,445,934; 6,027,880; 6,057,100; 6,156,501; 6,261,776; and 6,576,424; the disclosures of each of the patents are incorporated herein by reference in their entirety.

[0170] The method for detecting and / or quantifying the detectable label depends on the nature of the label. The product of the reaction catalyzed by the appropriate enzyme (wherein the detectable label is an enzyme; see above) can be, but is not limited to, fluorescent, luminescent or radioactive, or they can absorb visible or ultraviolet light. Examples of detectors suitable for detecting such detectable labels include, but are not limited to, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, photometers, and densitometers.

[0171] Methods for detecting or measuring gene expression (e.g., protein or mRNA expression) can optionally be performed in a form that allows rapid preparation, processing, and analysis of multiple samples. For example, this can be in a multi-well assay plate (e.g., 96-well or 386-well) or array (e.g., a nucleic acid chip or a protein chip). Stock solutions of various reagents can be provided manually or robotically, and subsequent sample preparation (e.g., RT-PCR, labeling, or cell fixation), pipetting, dilution, mixing, distribution, washing, incubation (e.g., hybridization), sample readout, data collection (optical data), and / or analysis (computer-assisted image analysis) can be performed robotically using commercially available analysis software, robotics, and detection instruments capable of detecting signals generated from the assay. Examples of such detectors include, but are not limited to, spectrophotometers, luminometers, fluorometers, and devices for measuring radioisotope decay. Exemplary high-throughput cell-based assays (e.g., detecting the presence or level of a target protein in a cell) can be performed using ArrayScan VTI HCS Reader or KineticScan HCS Reader technology (Cellomics Inc., Pittsburg, PA).

[0172] In some embodiments, the expression level of one NF gene, two NF genes, or three NF genes may be assessed and / or measured.

[0173] To help detect the presence or expression level of one or more NF genes, any portion of the nucleic acid sequence of the gene can be used, for example, as a hybridization polynucleotide probe or primer (e.g., for amplification or reverse transcription). Probes and primers can be oligonucleotides of sufficient length to provide specific hybridization with RNA, DNA, cDNA or fragments thereof isolated from a biological sample. Depending on the specific application, different hybridization conditions can be used to achieve different degrees of selectivity of the probe or primer to the target sequence. Primers and probes can be detectably labeled with reagents that facilitate detection (e.g., fluorescent labels, chemical labels (see, e.g., U.S. Patent Nos. 4,582,789 and 4,563,417) or modified bases).

[0174] Standard stringent conditions are described by Sambrook et al. (supra) and Haymes et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). In order for a nucleic acid molecule to act as a primer or probe, it is only necessary that there is sufficient complementarity in sequence to form a stable double-stranded structure under the particular hybridization conditions (e.g., solvent and salt concentrations) employed.

[0175] Hybridization can be used to assess the homology between two nucleic acid sequences. According to standard hybridization techniques, nucleic acid sequences as described herein or fragments thereof can be used as hybridization probes. The hybridization of a target probe (e.g., a probe containing a portion of a nucleotide sequence as described herein or its complementary sequence) with a DNA, RNA, cDNA or fragment from a test source indicates the presence of a DNA or RNA corresponding to the probe in the test source. Hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY, 6.3.1-6.3.6, 1991. Moderate hybridization conditions are defined as hybridization at 30°C in 2X sodium chloride / sodium citrate (SSC), followed by washing at 50°C in 1X SSC, 0.1% SDS. High stringency conditions are defined as hybridization at 45°C in 6X SSC, followed by washing at 65°C in 0.2X SSC, 0.1% SDS.

[0176] Primers can be used for methods of various PCR types. For example, polymerase chain reaction (PCR) technology can be used to amplify specific sequences from DNA and RNA, including sequences from total genomic DNA or total cellular RNA. PCR primers are designed to flank the region of interest amplification. Primers can be located near the 5' end, the 3' end, or anywhere within the nucleotide sequence to be amplified. The length of the amplicon is determined by the experimental goal. For qPCR, the target length is close to 100 base pairs, and for standard PCR, the target length is close to 500 base pairs. In general, oligonucleotide primers are designed using sequence information from the end or further regions of the target region, and the oligonucleotide primers are identical or similar in sequence to the opposite strand of the template to be amplified. PCR primers can be chemically synthesized as single nucleic acid molecules (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology), or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized, the oligonucleotides containing the desired sequence, wherein each pair contains a short complementary segment (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pairs are annealed. A DNA polymerase is used to extend the oligonucleotides, thereby producing a single double-stranded nucleic acid molecule per oligonucleotide pair.

[0177] Additionally, nucleic acid sequences or fragments thereof (eg, oligonucleotide probes) can be used in nucleic acid arrays to detect and / or quantify gene expression.

[0178] 10. Treatment

[0179] The methods disclosed herein enable the assessment of whether a subject having or suspected of having SMA is likely to respond to an SMA therapy (e.g., SPINRAZA SMA therapy (e.g., SPINRAZA) may be administered to subjects with SMA or suspected of having SMA who may respond to SMA therapy. ). Conversely, a subject having or suspected of having SMA who is unlikely to respond to an SMA therapy may be administered a different SMA therapy suitable for treating SMA.

[0180] The disclosed methods can also stratify subjects who have or are suspected of having SMA into those who are more likely to benefit from a treatment that includes an SMA therapy (e.g., SPINRAZA The ability to select such subjects from a pool of SMA subjects being considered for treatment with SMA therapies is beneficial in administering effective treatments to the subjects.

[0181] Subjects considered for treatment including SMA therapy include, but are not limited to, subjects who have, are suspected of having, or are likely to have SMA. In one embodiment, the subject to be treated with SMA therapy has, is suspected of having, or is likely to have SMA type 0. In one embodiment, the subject to be treated with SMA therapy has, is suspected of having, or is likely to have SMA type I. In one embodiment, the subject to be treated with SMA therapy has, is suspected of having, or is likely to have SMA type II. In one embodiment, the subject to be treated with SMA therapy has, is suspected of having, or is likely to have SMA type III. In one embodiment, the subject to be treated with SMA therapy has, is suspected of having, or is likely to have SMA type IV.

[0182] If a subject with SMA is more likely to respond to an SMA therapy (based on the concentration of one or more of the above biomarkers (e.g., pNF-H protein)), an effective amount of an SMA therapy (e.g., SPINRAZA ). The effective amount of the compound can be appropriately determined by a health care practitioner taking into account, for example, the patient's characteristics (age, sex, weight, race, etc.), the progression of the disease, and previous exposure to the drug. If a subject is unlikely to respond to one SMA therapy, a different SMA therapy may optionally be administered to the subject.

[0183] Subjects of all ages can be affected by SMA. Thus, biological samples for use in the methods described herein can be obtained from human subjects of any age, including fetuses, infants, children, adolescents, or adults, such as adults suffering from or suspected of suffering from SMA.

[0184] The methods can also be applied to patients who are undergoing SMA therapy (e.g., SPINRAZA ) individuals at risk for treatable SMA. Such individuals include those who have (i) a family history of such a disorder (genetic susceptibility) or (ii) one or more risk factors for developing such a disorder.

[0185] The subjects were divided into groups based on whether they were more or less likely to respond to SMA therapies (e.g., SPINRAZA ) After stratifying or selecting subjects for response, a medical practitioner (e.g., a doctor) can administer an appropriate treatment modality to the subjects. Methods for administering SMA therapies are known in the art.

[0186] It is understood that any of the therapies described herein (e.g., including SPINRAZA Therapy that does not include SPINRAZA Therapies described herein may include one or more additional therapeutic agents. That is, any of the therapies described herein may be co-administered (administered in combination) with one or more additional therapeutic agents, such as, but not limited to, other SMA therapies described herein. In addition, any of the therapies described herein may include one or more therapeutic agents for treating SMA including therapies (e.g., SPINRAZA )'s side effects.

[0187] Combination therapy (e.g., including (e.g., SPINRAZA ) and one or more additional SMA therapies or co-administration of an SMA therapy of an additional therapeutic agent) can be, for example, simultaneous or sequential. For example, the SMA therapy and the one or more additional therapeutic agents can be administered simultaneously or at different times. In some embodiments, the one or more additional therapeutic agents can be administered at a first time, and the SMA therapy (e.g., SPINRAZA) can be administered at a second time. ).

[0188] Patients with SMA who have been previously treated for SMA and are expected to respond to SMA therapy (e.g., SPINRAZA In the case of SMA therapy administered to a subject who is responsive to SPINRAZA, the therapy may replace or augment a previously or currently administered therapy. After treatment, non-SPINRAZA may be discontinued or reduced Administration of therapy, such as at lower levels. In some embodiments, the previous therapy may be maintained until SPINRAZA to levels sufficient to provide a therapeutic effect.

[0189] In some cases, the method of treatment includes treating the fetus in utero. Based on the high NF level (e.g., pNF-H or NF-L) relative to the control, it is determined that the fetus to be treated is in need of treatment with SMA therapy. For example, if the fetus has been identified as having SMA based on genetic testing, and the fetus has been identified as having elevated NF levels, then an SMA therapy (e.g., SPINRAZA) may be used. ) to treat the fetus. In some cases, the treatment method involves a type 0 patient. If the fetus is identified as having very high NF levels and one SMN2 copy, the fetus has a higher likelihood of having type 0 SMA. ) treating such a fetus can effectively treat the fetus.

[0190] 11. Medicine Box

[0191] The present disclosure also provides a medicine kit. In certain embodiments, the medicine kit may include one or more antibodies that can be used to detect one or more biomarkers disclosed herein or their concentration or expression level. For example, the medicine kit may include an antibody that specifically binds NF-H (e.g., pNF-H). The antibody in the medicine kit may be a monoclonal antibody or a polyclonal antibody, and may be further conjugated with a detectable label. In some embodiments, the medicine kit includes a probe that can be used to identify or detect any biomarker disclosed herein. In some embodiments, the medicine kit includes any nucleic acid array. In some embodiments, the medicine kit includes probes and antibodies that can be used to identify or detect any biomarker disclosed herein or its expression or expression level. The medicine kit may optionally contain instructions for detecting and / or measuring the concentration of one or more proteins in a biological sample or the level of mRNA.

[0192] The kit may optionally include, for example, a control (e.g., a concentration standard of the protein being evaluated) or a control labeled amplicon set containing a known amount of one or more amplicons recognized by the nucleic acid probes of the array. In some cases, the control may be a marker containing a marker that predicts SMA or is sensitive to SMA therapy (e.g., SPINRAZA ) or RNA that is responsive to a protein (e.g., pNF-H) or RNA that is expressed at a level or a range of levels thereof (e.g., a paper insert or an electronic medium such as a CD, DVD, or floppy disk).

[0193] In some embodiments, the kit may include one or more reagents for processing a biological sample (e.g., calibration reagents, buffers, dilutions, colored reagents, reagents for terminating reactions). For example, a kit may include reagents for isolating proteins from a biological sample and / or reagents for detecting the presence and / or amount of proteins in a biological sample (e.g., antibodies that bind to a protein that is the subject of a detection assay and / or antibodies that bind to an antibody that binds to the protein).

[0194] In certain embodiments, the kit includes at least one microplate (e.g., a 96-well plate; i.e., 12 strips of 8-well plates). The microplate may be provided with its corresponding plate cover. The microplate may be polystyrene or have any other suitable material. The microplate may have antibodies for identifying the presence of a specific biomarker coated inside each well. The antibodies may be conjugated with a detectable label. The kit may also include at least one adhesive strip.

[0195] In some embodiments, the kit can include a software package for analyzing the results of, for example, an expression profile or microarray analysis.

[0196] The kit may also include one or more antibodies for detecting protein expression of any gene described herein (e.g., NF-H). For example, the kit may include one or more antibodies (or in some cases, composed thereof), which are capable of specifically binding to one or more proteins encoded by any gene described herein; and optionally, instructions for detecting and / or measuring the concentration of one or more proteins; and / or detection antibodies, which include detectably labeled antibodies capable of binding to at least one of a plurality of antibodies. In some embodiments, the kit may include antibodies that recognize NF-H, NF-L, and / or NF-M. In some embodiments, the kit may include antibodies that recognize pNF-H.

[0197] In certain embodiments, the kit may also optionally include one or more unit doses of an SMA therapy (e.g., SPINRAZA ).

[0198] The kits described herein may also optionally include instructions for administering an SMA therapy, wherein the concentration of one or more proteins or the expression level of one or more RNAs can predict that a subject having or suspected of having SMA will respond to an SMA therapy (e.g., SPINRAZA ) has a response.

[0199] In a specific embodiment, the kit includes one or more of the following:

[0200] (i) A microplate (e.g., a 96-well plate). The microplate may be coated with an anti-NF-H antibody conjugated to a detectable label. The anti-NF-H antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may, for example, be from a mouse, a rabbit, a rat, or a guinea pig. The detectable label may be, for example, horseradish peroxidase, biotin, a fluorescent moiety, a radioactive moiety, a histidine tag, or a peptide tag. The microplate may be provided with a cover and optionally one or more adhesive strips.

[0201] (ii) A vial containing anti-NF-H conjugated to a detectable label. The detectable label can be, for example, horseradish peroxidase, biotin, a fluorescent moiety, a histidine tag, a peptide tag. The vial may also include a preservative.

[0202] (iii) a vial containing a NF-H standard of known concentration. The NF-H may be recombinant human NF-H.

[0203] (iv) Vial containing assay diluent.

[0204] (v) Vial containing calibrant diluent.

[0205] (vi) A vial containing a wash buffer. The buffer may be provided in the form of a concentrate.

[0206] (vii) One or more vials containing a developer.

[0207] (viii) A vial containing stop solution to terminate the colorimetric reaction.

[0208] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without exercising the ability of the present invention and without departing from the scope of the invention.

[0209] Example

[0210] The following examples relate to several clinical studies designated: NURTURE, ENDEAR, EMBRACE, and CHERISH.

[0211] ENDEAR was a Phase 3, multicenter, randomized, double-blind, sham-controlled study of ISIS 396443 in symptomatic subjects with infantile-onset SMA. Results from the final analysis of this study provided clear evidence that subjects treated with intrathecal (IT) ISIS 396443 achieved statistically significant and clinically meaningful improvements in motor milestones, as well as sustained and clinically meaningful improvements in event-free survival, overall survival, motor function, and motor neuron health, compared to control subjects who received a sham procedure. Improvements relative to control were observed as early as 2 months after the start of treatment (end of the loading dose period), with clear separation from the control group at 6 months after the start of treatment.

[0212] During final analysis, 121 subjects received at least 1 dose of ISIS 396443 administered as IT injection by lumbar puncture (LP) [n=80] or sham surgery (n=41). Among these subjects, 89 subjects (65 of ISIS 396443 group and 24 of control group) completed the study. When the study terminated, a total of 42 subjects (32 subjects of ISIS 396443 group and 10 subjects of control group) remained in the study, and were recorded as stopping treatment due to early research termination. Two subjects in ISIS 396443 group and 1 subject in control group voluntarily withdrew from the study before transitioning to open label treatment, and 29 subjects (13 of ISIS 396443 group and 16 of control group) died.

[0213] The demographics and SMA history of the 121 subjects in this study were consistent with the SMA type I population.

[0214] • There were 45% males and 55% females; 86% of the subjects were white.

[0215] • Most participants (86%) had symptom onset at less than 12 weeks of age. The median age at SMA symptom onset was 8.0 weeks (range: 1 to 20 weeks), the median age at SMA diagnosis was 12.0 weeks (range: 0 to 30 weeks), and the median age at screening was 166 days (range: 20 to 211 days). The median disease duration at enrollment was 13.1 weeks (range: 0.0 to 25.86 weeks).

[0216] • Most subjects (99%) had 2 copies of the SMN2 gene at study entry based on local laboratory testing.

[0217] • Of the 80 subjects in the ISIS 396443 group, 73 (91%) received at least 4 doses of ISIS 396443 (i.e., completed the loading dose phase); 32 subjects (40%) received all 6 planned doses. Of the 41 subjects in the control group, 34 subjects (83%) underwent at least 4 sham procedures, of which 14 subjects (34%) underwent all 6 procedures.

[0218] CHERISH is a Phase 3, double-blind, randomized, sham-controlled study of ISIS 396443 in subjects with late-onset SMA. Approximately 117 subjects were enrolled in the study and assigned to treatment with ISIS 396443 or sham control in a 2:1 ratio. ISIS 396443 was administered IT using a loading regimen (dosing on Days 1, 29, and 85), followed by a maintenance dose (dosing on Day 274) 6 months later.

[0219] Results from the interim analysis provided clear evidence that subjects treated with ITISIS 396443 achieved statistically significant gains in motor function and sustained, clinically meaningful improvements in motor milestones compared with control subjects who received a sham procedure. Although improvements in motor function were observed at all time points, separation from the control group was evident at 6 months after the start of treatment. Based on the risk-benefit assessment of ISIS 396443, the sponsor decided to terminate the study early and give subjects the opportunity to enroll in an open-label extension study.

[0220] As of the interim analysis, 126 subjects received at least 1 dose of ISIS 396443 (n=84) or sham surgery (n=42). No subject stopped treatment or withdrew from the study. As of the data cutoff date, 49 (58%) of the 84 subjects in the ISIS 396443 group and 23 (55%) of the 42 subjects in the control group are continuing the study. The main analysis of the change of HFMSE score relative to baseline at 15 months is based on the ITT set composed of 126 subjects (84 treated with ISIS 396443 and 42 sham surgery). The main analysis of WHO motor milestones is carried out using the interim efficacy set, which is composed of 54 subjects (35 subjects treated with ISIS 396443 and 19 subjects who underwent sham surgery) who have the opportunity to be evaluated at 456 days of follow-up (i.e., the 15th month). All other secondary and tertiary endpoint analyses are carried out on the ITT set.

[0221] The demographic and baseline disease characteristics of the 126 subjects in this study, including SMA and medical history, were consistent with a population at high risk for developing type II or type III SMA.

[0222] • 47% males and 53% females; 75% white.

[0223] • The median age at SMA symptom onset was 11 months (range: 6 to 20 months), the median age at SMA diagnosis was 18 months (range: 0 to 48 months), and the median age at screening was 3.0 years (range: 2 to 9 years). The median disease duration at recruitment was 35.7 months (range: 8 to 94 months).

[0224] • Most subjects (88%) had 3 copies of the SMN2 gene based on laboratory testing at study entry, and 8% had 2 copies.

[0225] • Of the 84 subjects in the ISIS 396443 group, all received at least 3 doses of ISIS 396443 (i.e., completed the loading dose); 76 subjects (90%) received all 4 doses. Of the 42 subjects in the control group, all received at least 3 sham surgeries, and 40 subjects (95%) received all 4 surgeries.

[0226] • The median duration of the study was similar between the 2 treatment groups (i.e., 412.5 days and 419.5 days for ISIS 396443 and control groups, respectively). The total age of subjects studied was 134.06 (88.84 subject ages in the ISIS 396443 group and 45.22 subject ages in the control group).

[0227] Subjects treated with ISIS 396443 achieved clinically meaningful benefits compared to subjects who received sham surgery. These benefits included statistically significant increases in motor function as measured by HFMSE, as well as improvements in upper limb functional capacity.

[0228] NURTURE is an ongoing Phase 2, open-label, multicenter, single-arm study evaluating the efficacy, safety, tolerability, and PK of ISIS 396443 in presymptomatic SMA. The study is conducted in subjects ≤6 weeks of age at recruitment with genetic documentation of 5qSMA, 2 or 3 copies of the SMN2 gene, CMAP ≥1 mV, and no signs or symptoms of SMA. Up to 25 subjects are planned. Efficacy data available to date indicate that the onset and achievement of motor milestones in most subjects are more consistent with normal outcomes compared to the natural history of Type I SMA.

[0229] At the time of data cutoff for NURTURE, 20 subjects had been enrolled and received at least 1 dose of ISIS 396443. All subjects are continuing in the study. The 18 subjects who received all 4 loading doses or had the opportunity to complete the Day 64 follow-up constituted the efficacy set.

[0230] • The majority of subjects were male (55%) and white (50%).

[0231] • Age at first dose ranged from 3 to 42 days, with a median of 19 days.

[0232] • Of the 18 subjects, 13 subjects (72%) had 2 copies of the SMN2 gene and 5 subjects (28%) had 3 copies.

[0233] Efficacy data were available for 18 subjects at Day 64, 16 subjects at Day 183, 11 subjects at Day 302, 9 subjects at Day 365, and 5 subjects at Day 421. Results at these later follow-up periods suggest that the experiences of subjects with SMA Type I and affected siblings are not consistent and are consistent with age-matched expectations for healthy infants.

[0234] EMBRACE is a Phase 2, randomized, sham-controlled, multicenter study of ISIS 396443 in subjects with SMA who are not eligible for ENDEAR or CHERISH. This study evaluated a unique group of subjects who presented with symptoms of infantile-onset SMA at an age too young to qualify for CHERISH, or who presented with late-onset symptoms of SMA at an age too old to qualify for ENDEAR and were screened at an age too old or with too many copies of SMN2 to qualify for ENDEAR. Therefore, the population in this study provides an opportunity to explore the safety, tolerability, and efficacy of ISIS 396443 in the setting of infantile-onset and late-onset SMA in subjects with up to 3 copies of SMN2.

[0235] This study was originally designed as a double-blind study, but after observing positive and robust efficacy results from the pivotal study of the ISIS 396443 clinical development program, it developed into a 2-part study including a double-blind period (Part 1) and an open-label extension period (Part 2). Part 1 is a randomized, double-blind, sham-controlled study. 21 subjects from 7 sites in the United States and Germany were recruited to the study. The randomization of subjects was stratified according to the age of SMA (infant-onset [≤6 months] vs. late-onset [>6 months]). Subjects are scheduled to receive a total of 6 intrathecal (IT) injections or 6 sham surgeries over a dosing period of approximately 10 months. However, due to the observation of positive and robust efficacy results in the interim analysis of a pivotal study in the ISIS 396443 clinical development program, Part 1 of the study was terminated early. Since the study sponsor decided to terminate Part 1 of the study early based on the risk-benefit assessment of ISIS 396443, all subjects were invited to complete the end-of-Part 1 evaluation as soon as possible and participate in Part 2 of the study. The total duration for subjects in Part 1 was planned to be approximately 15 months, but due to the early termination of Part 1 of the study, eligible subjects had the opportunity to be recruited into Part 2 of the study immediately following their end-of-Part 1 assessments.

[0236] A total of 21 subjects were enrolled in this study. Subjects were randomized in a 2:1 ratio to ISIS 396443 or control (sham surgery). Randomization was stratified according to age of onset of clinical signs and symptoms consistent with SMA (≤6 months [infantile-onset] vs. >6 months [late-onset]). In part 1 of the study, 14 subjects received ISIS 396443 and 7 subjects received sham surgery.

[0237] A total of 21 subjects were screened and enrolled in Part 1 of this study. All subjects were randomized in a 2:1 ratio: 14 subjects received ISIS 396443 and 7 subjects received control treatment (sham surgery). In addition, randomization was stratified according to age of onset of clinical signs and symptoms consistent with infantile-onset (≤6 months) and late-onset (>6 months) SMA. The first subject was treated on August 19, 2015, and the study (Part 1) ended on December 20, 2016. All subjects received study treatment according to their randomized group assignment.

[0238] 21 subjects who were randomized and dosed to form the ITT set were recruited at 7 research sites in 2 countries. 16 subjects (76%) were recruited at 6 sites in the United States, and 5 subjects (24%) were recruited at 1 site in Germany. Subjects were randomized across sites. Of the 21 randomized subjects, 13 developed SMA at ≤6 months, and the remaining 8 developed SMA at >6 months.

[0239] Twenty-one subjects received treatment, and of these, 14 completed due to premature study termination (six subjects [86%] in the control group and eight subjects [57%] in the ISIS 396443 group). As of Day 302, nine subjects (43%) completed treatment (two subjects [29%] in the control group and seven subjects [50%] in the ISIS 396443 group). Six subjects (29%) (all in the ISIS 396443 group) completed the final follow-up assessment of Part 1 (Day 422). One subject (5%) assigned to the control group died on Study Day 289 due to brain death.

[0240] Demographics and baseline disease characteristics:

[0241] Of the 21 subjects in the study, 11 (52%) were male and 10 (48%) were female. The age at the first dose ranged from 7 to 53 months (median: 17 months). 11 subjects (52%) were between 7 and 18 months of age, and 10 subjects (48%) were greater than 18 months of age. 9 subjects (43%) were white, 5 subjects (24%) were Asian, and 2 subjects (10%) were other.

[0242] Efficacy and Pharmacokinetics

[0243] • Participants in the ISIS 396443 group required less ventilator use compared to participants in the control group.

[0244] • The proportion of HINE motor milestone responders was greater in the ISIS 396443 group than in the control group, and there was at least 1 responder in each HINE motor milestone category from the ISIS 396443 group, with the categories with the largest number of subjects showing improvements in head control, rolling, and sitting.

[0245] Security

[0246] In this study, ISIS 396443 was well tolerated when administered as multiple IT injections (4 loading doses followed by maintenance doses every 4 months).No new safety concerns were identified in the overall safety profile of ISIS 396443.

[0247] The results from the first part of this phase 2, randomized, sham-controlled study of ISIS 396443 show that there is clear evidence that intrathecal administration of ISIS 396443 is effective in a population with infantile onset or late-onset SMA. When compared to a control group that only received sham administration, subjects treated with ISIS 396443 achieved and maintained growth in motor milestones, and generally appeared to be growing and thriving according to the assessments of investigators and caregivers. A positive correlation was observed between CSF ISIS 396443 concentrations and HINE motor milestone total scores, and increased over time. If ISIS 396443 is safe and well tolerated in this study (because no new safety considerations were identified), the dosing regimen, and the safety results are consistent with the results of other studies from the ISIS 396443 clinical development program. Based on the results observed in all major safety and exploratory efficacy parameters, and in contrast to the results observed in the untreated control group, ISIS 396443 improves motor function in subjects with infantile onset and late-onset SMA.

[0248] method

[0249] Plasma at selected time points was assayed for pNF-H concentration. All plasma samples were frozen at -70°C until ready for use. Plasma samples at selected protocol times were diluted to the minimum required dilution (MRD) using assay dilution buffer.

[0250] NF-H was measured using polyclonal anti-NF-H antibodies from Encor Biotechnology (Cat. No. RPCA-NF-H; and / or Cat. No. CPCA-NF-H). These antibodies specifically detect the hyperphosphorylated form of NF-H.

[0251] Prior to assaying any patient samples, a qualification experiment was performed to evaluate the potential of the protein to be expressed in the presence of The Ella technology platform developed to generate pNF-H concentrations with reproducibility and variability. The Ella technology utilizes a microfluidic cartridge containing pre-loaded reagents, so only the sample and wash buffer are loaded into the cartridge. Once the cartridge is loaded, the platform automatically calculates the pNF-H concentration by using its built-in factory calibration standard curve.

[0252] To assess the quality of each cartridge and any unforeseen potential confounding effects in real time, four quality control (QC) samples are included in each cartridge: three QCs spiked with buffer (high, medium, and low), and an endogenous quality control sample (EQC) from a commercially available multiple sclerosis (MS) patient (Bioreclamation) is run. These four QCs are used to test the performance of the bioanalytical method and to assess the integrity and validity of the results of unknown samples analyzed in a single batch.

[0253] Samples from the same subject were analyzed on the same box. Two aliquots of each sample were placed in the same box in random order to ensure that the sample analysis took into account possible placement effects.

[0254] Example 1: Correlation of CSF versus plasma pNF-H levels

[0255] Plasma and CSF samples from the clinical studies NURTURE and EMBRACE were analyzed at the same time points and in the same subjects to determine the correlation of pNF-H levels. A high correlation (R=0.88) between CSF and plasma levels of pNF-H was identified. Figure 1 ). This result indicates that plasma levels of pNF-H and other subunits of neurofilaments are highly predictive of CSF levels of these same proteins.

[0256] Example 2: Plasma levels of pNF-H as a predictor of disease and severity

[0257] Plasma samples from clinical studies NURTURE, ENDEAR, EMBRACE, and CHERISH were analyzed for pNF-H levels at baseline (before treatment with ISIS 396443 (NURTURE, ENDEAR, EMBRACE, and CHERISH) or before sham surgery (ENDEAR, EMBRACE, and CHERISH). pNF-H levels were also analyzed in plasma samples from healthy volunteers (4-18 years old).

[0258] Results from healthy volunteers show pNF-H <300 pg / mL for all ages. Results from NURTURE, ENDEAR, EMBRACE, CHERISH show that plasma levels of pNF-H in clinical trial subjects with SMA are almost all >300 pg / mL. Some subjects have levels as high as approximately 50,000 pg / mL. In general, subjects with two copies of SMN2 have higher levels than those with three copies of SMN2. In addition, subjects with symptom onset ≤6 months of age (ENDEAR) have higher levels than those with symptom onset >6 months of age (CHERISH). See Figure 2 .

[0259] However, important exceptions were observed. Within NURTURE, one subject had 2 copies of SMN2, and pNF-H levels were more consistent with subjects with 3 copies of SMN2. This subject had a sibling with type II SMA who also had two copies of SMN2. Therefore, although this subject's SMN2 copy number indicated that the subject should suffer from type I SMA, the subject's family history indicated that the subject would suffer from type II SMA. In addition to SMN1 deletion and SMN2 copy number, pNF-H levels can also provide additional diagnostic acuity. Within ENDEAR, one subject had 3 copies of SMN2, and pNF-H levels were more consistent with subjects with 2 copies of SMN2. This subject had symptom onset at ≤6 months of age, and was therefore more consistent with type I SMA (most commonly 2 SMN2 copies) than type II SMA (most commonly 3 SMN2 copies).

[0260] Overall, the results show that in subjects with SMA, the plasma levels of pNF-H are significantly elevated compared to healthy volunteers. And, in subjects with SMA, subjects with a more severe phenotype have higher plasma pNF-H levels than subjects with a less severe phenotype.

[0261] Example 3: Correlation of pNF-H versus age (SMN2 copy number = 2)

[0262] Plasma samples from clinical studies NURTURE, ENDEAR, EMBRACE, and CHERISH were analyzed for levels of pNF-H at baseline (before treatment with ISIS 396443 (NURTURE, ENDEAR, EMBRACE, and CHERISH) or before sham surgery (ENDEAR, EMBRACE, and CHERISH). For this analysis, subjects were restricted to those with two copies of SMN2. Baseline log-transformed pNF-H levels were plotted versus age at first dose (or sham surgery). In general, younger subjects had higher pNF-H levels than older subjects. See Figure 3 .

[0263] These results suggest that pNF-H levels decrease with age in subjects with 2 copies of SMN2.

[0264] Example 4: Correlation of pNF-H versus age (SMN2 copy number = 3)

[0265] Plasma samples from clinical studies NURTURE, ENDEAR, EMBRACE, and CHERISH were analyzed for levels of pNF-H at baseline (before treatment with ISIS 396443 (NURTURE, ENDEAR, EMBRACE, and CHERISH) or before sham surgery (ENDEAR, EMBRACE, and CHERISH). For this analysis, subjects were restricted to those with three copies of SMN2. Baseline log-transformed pNF-H levels were plotted versus age at first dose (or sham surgery). In general, younger subjects had higher pNF-H levels than older subjects. See Figure 4 .

[0266] These results suggest that pNF-H levels decrease with age in subjects with three copies of SMN2.

[0267] Example 5: Correlation of pNF-H with age

[0268] Plasma samples from clinical studies NURTURE, ENDEAR, EMBRACE, and CHERISH were analyzed for levels of pNF-H at baseline (before treatment with ISIS 396443 (NURTURE, ENDEAR, EMBRACE, and CHERISH) or before sham surgery (ENDEAR, EMBRACE, and CHERISH). For this analysis, subjects were stratified according to age at symptom onset (presymptomatic (NURTURE), <6 months, ≥6 months) and SMN2 copy number (2, 3, 4, or NA). In general, within each stratum of age at symptom onset and SMN2 copy number, younger subjects had higher pNF-H levels than older subjects. See Figure 5 .

[0269] These results suggest that pNF-H levels in subjects decrease with age, independent of the age of symptom onset or SMN2 copy number.

[0270] Example 6: Correlation of pNF-H versus age

[0271] Plasma samples from the clinical study ENDEAR were analyzed for pNF-H levels at baseline (prior to treatment with ISIS 396443 or sham surgery). In contrast to this analysis, quartiles of pNF-H were identified across the study population. The quartiles were defined as 2390 to 10,900 pg / mL; 10,900 to 15,400 pg / mL; 15,400 to 21,600 pg / mL; and 21,600 to 50,100 pg / mL. In general, subjects with higher pNF-H levels at baseline appeared to be younger at the first dose, younger at symptom onset, and younger at SMA diagnosis. In addition, subjects with higher pNF-H levels at baseline appeared to have lower mean CHOP INTEND (Children's Hospital of Philadelphia Infant Neuromuscular Disorders Examination) scores and peroneal CMAP amplitudes at baseline. See Figure 6-8 .

[0272] These results suggest that higher pNF-H levels at the first dose are associated with a more severe phenotype as evidenced by younger age at symptom onset; lower motor function (CHOP INTEND); and poorer motor neuron health (CMAP amplitude).

[0273] Example 7: pNF-H Levels and Motor Function

[0274] Plasma samples from the clinical study ENDEAR were analyzed for pNF-H levels at baseline (before treatment with ISIS 396443 or sham surgery). For this analysis, log-transformed pNF-H levels were plotted relative to baseline CHOP INTEND. In general, subjects with higher pNF-H levels had lower CHOP INTEND, and subjects with lower pNF-H levels had higher CHOP INTEND, with a correlation of -0.3. See Fig. 9 .

[0275] This result suggests that pNF-H levels are negatively correlated with motor function. This relationship appears to be linear.

[0276] Example 8: Prediction of motor function based on pNF-H levels

[0277] Plasma samples from the clinical study ENDEAR were analyzed for pNF-H levels at baseline (before treatment with ISIS 396443 or sham surgery). For this analysis, a linear regression model was constructed to examine the relationship between various potential predictors of baseline CHOP INTEND. The final model was identified to include only those variables that remained statistically significant (baseline NF-H; treatment group, disease duration (weeks); sex; age at first dose (days); age at SMA symptom onset (weeks); age at SMA diagnosis (weeks); gestational age (weeks); baseline weight (kg); (gestational age + age at first dose) x (gestational age + age at SMA symptom onset); (gestational age + age at SMA diagnosis). In this final model, baseline log-transformed pNF-H levels were statistically significant. See Fig.10 .

[0278] These results suggest that pNF-H levels are an important predictor of baseline motor function.

[0279] Example 9: pNF-H (pg / mL) Levels in ENDEAR

[0280] pNF-H levels were analyzed in plasma samples from the clinical study ENDEAR on study days 1, 2, 29, 64, 183, and 302 (sham surgery). For this analysis, absolute values ​​of pNF-H were log-transformed and plotted by study date. Over time, mean pNF-H levels decreased almost linearly from approximately 18,000 pg / mL on study day 1 to approximately 5,000 pg / mL on study day 302. See Fig.11 .

[0281] Example 10: Percent Change in pNF-H (pg / mL) Levels in ENDEAR

[0282] pNF-H levels were analyzed in plasma samples from the clinical study ENDEAR on study days 1, 2, 29, 64, 183, and 302 (sham surgery). For this analysis, the percent change from baseline in absolute values ​​of pNF-H was plotted by study date. Over time, the mean percent change in pNF-H levels decreased almost linearly from approximately 0% on study day 1 to approximately -60% on study day 302. See Fig.12 .

[0283] Example 11: Effect of Drugs on pNF-H (pg / mL) Levels in ENDEAR

[0284] pNF-H levels were analyzed in plasma samples from the clinical study ENDEAR on study days 1, 2, 29, 64, 183, and 302 (ISIS 396443). For this analysis, absolute values ​​of pNF-H were log-transformed and plotted by study date. Over time, mean pNF-H levels decreased from approximately 18,000 pg / mL on study day 1 to approximately 5,000 pg / mL on study day 64 and then to approximately 1,000 pg / mL on study day 302. See Fig.13 .

[0285] This result suggests that subjects treated with ISIS 396443 experienced a different pattern of decrease in pNF-H levels compared to subjects receiving sham-operated controls.

[0286] Example 12: Effect of Drug on Percent Change in pNF-H (pg / mL) Levels in ENDEAR

[0287] pNF-H levels in plasma samples from the clinical study ENDEAR were analyzed on study days 1, 2, 29, 64, 183, and 302 (ISIS 396443). For this analysis, the percent change from baseline in the absolute value of pNF-H was plotted by study date. Over time, the mean percent change in pNF-H levels decreased almost linearly from approximately 0% on study day 1 to approximately -70% on study day 64 and -90% on study day 302. See Fig.14 .

[0288] This result suggests that subjects treated with ISIS 396443 experienced a different pattern of decrease in pNF-H levels compared to subjects receiving sham-operated controls.

[0289] Example 13: Changes in pNF-H levels at different time points

[0290] pNF-H levels in plasma samples from the clinical study ENDEAR on study days 1, 2, 29, 64, 183, and 302 (ISIS 396443 and sham control) were analyzed. For this analysis, the percentage of subjects who achieved a specific percentage change in absolute pNF-H values ​​relative to baseline on each study day in subjects receiving ISIS 396443 was compared to subjects receiving sham control. Statistical significance between the two study groups was first identified on study day 64 (p<0.05). The maximum difference between the percentages of each group achieving a specific change in pNF-H occurred on study day 183 for a change of -80%. See Fig.15 .

[0291] This result indicates that differences between groups could be identified as early as study day 64, and that the best separation between groups occurred using -80% as the change threshold.

[0292] Example 14: Correlation of HINE-2 score on day 183 with pNF-H level on day 64

[0293] Plasma samples from the clinical study ENDEAR on study day 64 were analyzed for pNF-H levels and total motor milestone scores (HINE-2 (Hammersmith Infant Neurological Examination Section 2)) were assessed on study day 183 (ISIS 396443 and sham controls). For this analysis, a linear regression model was constructed to determine if pNF-H levels on study day 64 were associated with total HINE-2 scores on study day 183. pNF-H levels on study day 64 were significantly associated with total HINE-2 scores on study day 183, p=0.0006. See Fig.16 .

[0294] These results suggest that current pNF-H levels may predict future motor milestones in infants with infantile-onset SMA.

[0295] Example 15: Association of HINE-2 score at day 183 with pNF-H level at day 64 (with covariates)

[0296] pNF-H levels were analyzed in plasma samples from Study CS3B (ENDEAR) at Study Day 64, and total motor milestone scores (HINE-2 (Hammersmith Infant Neurological Examination Section 2)) were assessed at Study Day 183 (ISIS 396443 and sham controls). For this analysis, a linear regression model was constructed to determine if pNF-H levels at Study Day 64 were associated with total HINE-2 scores at Study Day 183 after accounting for multiple potential confounders. The final model included log-transformed pNF-H at Day 64 (p<0.0001) and disease duration (p=0.0029) after controlling for multiple potential confounders. See Fig.17 .

[0297] These results suggest that current pNF-H levels predict future motor milestones after controlling for treatment and other potential confounders in infants with infantile-onset SMA.

[0298] Example 16: Correlation of CHOP INTEND on Day 183 with pNF-H Levels on Day 64

[0299] pNF-H levels were analyzed in plasma samples from the clinical study ENDEAR on study day 64 and CHOP INTEND on study day 183 (ISIS 396443 and sham controls). For this analysis, a linear regression model was constructed to determine whether pNF-H levels on study day 64 were associated with CHOP INTEND scores on study day 183. pNF-H levels on study day 64 were significantly associated with CHOP INTEND scores on study day 183, p<0.0001. See Fig.18 .

[0300] These results suggest that current pNF-H levels may predict future levels of gross motor function in infants with infantile-onset SMA.

[0301] Example 17: Association of CHOP INTEND score at day 183 with pNF-H level at day 64 (with covariates quantity)

[0302] pNF-H levels were analyzed in plasma samples from the clinical study ENDEAR at study day 64, and CHOP INTEND scores were assessed at study day 183 (ISIS 396443 and sham controls). For this analysis, a linear regression model was constructed to determine whether pNF-H levels at study day 64 were associated with CHOP INTEND scores at study day 183 after accounting for multiple potential confounders. The final model included log-transformed pNF-H at day 64 (p=0.0001), treatment group (p=0.005), and disease duration (p=0.0003) after controlling for multiple potential confounders. See Fig.19 .

[0303] These results suggest that current pNF-H levels predict future levels of overall motor function after controlling for treatment and other potential confounders in infants with infantile-onset SMA.

[0304] Example 18: HINE-2 (Responder / Non-Responder) Classification Versus pNF-H Levels at Day 183

[0305] pNF-H levels were analyzed in plasma samples from clinical study ENDEAR on Study Day 183, and HINE-2 (Hammersmith Infant Neurological Examination Section 2) responder status was assessed on Study Day 183; between Study Day 183 and Study Day 302; and after Study Day 302 (ISIS 396443 and sham controls). If a subject became a responder according to the protocol definition of a HINE-2 responder, the time to reach this point was recorded. If the subject did not become a responder in Study CS3B, please note. No subject in the sham control group became a HINE-2 responder during Study CS3B. The majority of subjects who received ISIS 396443 and became responders reached this point on Study Day 183. Among sham control subjects, pNF-H levels were above the overall median (2130 pg / mL) for almost all subjects. Among subjects who received ISIS 396443 and became responders on Study Day 183, the majority of subjects had pNF-H levels below the study median on Study Day 183. Among subjects who received ISIS 396443 and became responders between Study Day 183 and 302, the majority of subjects had pNF-H levels below the study median on Study Day 183. Among subjects who received ISIS 396443 and became responders between Study Day 302 and the end of the study, the majority of subjects had pNF-H levels below the study median on Study Day 183. Among subjects who received ISIS 396443 and did not become responders in ENDEAR, approximately half of the subjects had pNF-H levels below the study median on Study Day 183 (2130 pg / mL). See Fig. 20 .

[0306] These results suggest that reaching a certain pNF-H threshold 183 days after the start of treatment is able to distinguish treatment from sham controls and predict eventual responders to treatment.

[0307] Example 19: HINE-2 (responder / non-responder) categories versus pNF-H levels at day 183 Percent Change

[0308] pNF-H levels in plasma samples from clinical study ENDEAR on study day 183 were analyzed, and HINE-2 responder status was assessed on study day 183; between study day 183 and study day 302; after study day 302 (ISIS 396443 and sham control). The percent change in pNF-H between baseline and study day 183 was calculated. If a subject became a responder according to the protocol definition of a HINE-2 responder, the time to reach this point was recorded. If the subject did not become a responder in study CS3B, please note. No subject in the sham control group became a HINE-2 responder during study CS3B. The majority of subjects who received ISIS 396443 and became responders reached this point on study day 183. Among sham control subjects, pNF-H levels decreased by <80% for all subjects. Among subjects who received ISIS 396443 and became responders on Study Day 183, almost half of the subjects had a >80% decrease in pNF-H levels on Study Day 183. Among subjects who received ISIS 396443 and became responders between Study Day 183 and Study Day 302, the majority of subjects had a >80% change in pNF-H levels on Study Day 183. Among subjects who received ISIS 396443 and became responders between Study Day 302 and the end of the study, the majority of subjects had a >80% change in pNF-H levels on Study Day 183. Among subjects who received ISIS 396443 and did not become responders within ENDEAR, approximately half of the fingers had a >80% change in pNF-H levels on Study Day 183. See Fig.21 .

[0309] These results suggest that reaching a certain threshold of pNF-H decline 183 days after the start of treatment is able to distinguish treatment from sham controls and predict eventual responders to treatment.

[0310] Example 20: CHOP INTEND (Responder / Non-Responder) Classification Versus pNF-H Levels at Day 183

[0311] pNF-H levels in plasma samples from clinical study ENDEAR on study day 183 were analyzed, and CHOP INTEND responder status was assessed on study day 183; between study day 183 and study day 302; after study day 302 (ISIS 396443 and sham control). If a subject became a responder according to the protocol definition of a CHOP INTEND responder, the time to reach this point was recorded. If the subject did not become a responder in study CS3B, please note. During study CS3B, one subject in the sham control group became a CHOP INTEND responder. Most subjects who received ISIS 396443 and became responders reached this point on study day 183. Among sham control subjects, almost all subjects had pNF-H levels above the overall median. Among subjects who received ISIS 396443 and became responders on study day 183, most subjects had pNF-H levels below the study median on study day 183. Among subjects who received ISIS 396443 and became responders between Study Days 183 and 302, the majority of subjects had pNF-H levels below the study median on Study Day 183. Among subjects who received ISIS 396443 and became responders between Study Day 302 and the end of the study, the majority of subjects had pNF-H levels below the study median on Study Day 183. Among subjects who received ISIS 396443 and did not become responders in ENDEAR, approximately half of the subjects had pNF-H levels below the study median on Study Day 183. See Fig. 22 .

[0312] These results suggest that reaching a certain pNF-H threshold 183 days after the start of treatment is able to distinguish treatment from sham controls and predict eventual responders to treatment.

[0313] Example 21: CHOP INTEND (responder / non-responder) categories versus pNF-H levels at day 183 Percent change in H level

[0314] pNF-H levels in plasma samples from clinical study ENDEAR on study day 183 were analyzed, and CHOP INTEND responder status was assessed on study day 183; between study day 183 and study day 302; after study day 302 (ISIS 396443 and sham control). The percent change in pNF-H between baseline and study day 183 was calculated. If the subject became a responder according to the protocol definition of a HINE-2 responder, the time to reach this point was recorded. If the subject did not become a responder in study CS3B, please note. During study CS3B, one subject in the sham control group became a CHOP INTEND responder. The majority of subjects who received ISIS 396443 and became responders reached this point on study day 183. Among sham control subjects, pNF-H levels decreased by < 80% in all but one subject. Among subjects who received ISIS 396443 and became responders on Study Day 183, almost half of the subjects had a >80% decrease in pNF-H levels on Study Day 183. Among subjects who received ISIS 396443 and became responders between Study Day 183 and Study Day 302, the majority of subjects had a >80% change in pNF-H levels on Study Day 183. Among subjects who received ISIS 396443 and became responders between Study Day 302 and the end of the study, the majority of subjects had a >80% change in pNF-H levels on Study Day 183. Among subjects who received ISIS 396443 and did not become responders within ENDEAR, approximately half of the fingers had a >80% change in pNF-H levels on Study Day 183. See Fig.23 .

[0315] These results suggest that reaching a certain threshold of pNF-H decline 183 days after the start of treatment is able to distinguish treatment from sham controls and predict eventual responders to treatment.

[0316] Example 22: Maintenance of drug effect: pNF-H (pg / mL) levels in NURTURE

[0317] pNF-H levels were analyzed in plasma samples from clinical study NURTURE on study days 1, 64, 183, 302, and 421 (ISIS 396443). In this analysis, the absolute levels of pNF-H were plotted according to study day. Fig.24 .

[0318] Results from this analysis showed a significant decrease by study day 64, and this new level appeared to stabilize through study day 421.

[0319] Example 23: Maintenance of drug effect: pNF-H (pg / mL) levels in EMBRACE

[0320] pNF-H levels were analyzed in plasma samples from the clinical study EMBRACE on study days 1, 64, and 183 (ISIS 396443). In this analysis, the absolute levels of pNF-H were plotted according to study day. See Fig.25 .

[0321] Results from this analysis showed a significant decrease by study day 64, and this new level appeared to stabilize through study day 183.

[0322] Example 24: Maintenance of drug effect: Percent change in pNF-H (pg / mL) levels in NURTURE

[0323] pNF-H levels were analyzed in plasma samples from Study 232SM201 (NURTURE) on Study Days 1, 64, 183, 302, and 421 (ISIS 396443). In this analysis, the levels of pNF-H as a percentage of change from baseline were plotted according to study day. Fig.26 .

[0324] Results from this analysis showed a significant decrease (-80%) by Study Day 64, and this new level appeared to stabilize through Study Day 421.

[0325] Example 25: Maintenance of drug effect: pNF-H (pg / mL) levels in EMBRACE

[0326] pNF-H levels were analyzed in plasma samples from the clinical study EMBRACE on study days 1, 64, and 183 (ISIS 396443). In this analysis, the absolute levels of pNF-H were plotted according to study day. See Fig. 27 .

[0327] Results from this analysis showed a significant decrease (-50%) by Study Day 64, and this new level appeared to stabilize through Study Day 183.

[0328] Example 26: Correlation of HINE-2 score on day 183 with pNF-H level on day 64

[0329] pNF-H levels in plasma samples from the clinical study ENDEAR on study day 64 were analyzed, and total motor milestone scores (HINE-2) on study day 183 (ISIS 396443 and sham controls) were assessed. For this analysis, a linear regression model was constructed to determine whether pNF-H levels on study day 64 were associated with total HINE-2 scores on study day 183. A separate analysis was performed in subjects receiving ISIS 396443 and sham controls. In subjects receiving ISIS 396443, log-transformed pNF-H levels on study day 64 were not statistically significantly associated with total HINE-2 scores on study day 183, p=0.3186, but a trend was such that higher pNF-H levels were associated with lower total HINE-2 scores. In subjects who received sham controls, log-transformed pNF-H levels on Study Day 64 were statistically significantly correlated with total HINE-2 scores on Study Day 183, p=0.0302, such that higher pNF-H levels were associated with lower total HINE-2 scores. Fig.28 .

[0330] These results suggest that current pNF-H levels may predict future motor milestones in infants with infantile-onset SMA.

[0331] Example 27: Correlation of CHOP INTEND score at day 183 with pNF-H level at day 64

[0332] pNF-H levels in plasma samples from the clinical study ENDEAR on study day 64 were analyzed, and CHOP INTEND scores on study day 183 (ISIS 396443 and sham control) were assessed. For this analysis, a linear regression model was constructed to determine whether pNF-H levels on study day 64 were associated with CHOP INTEND scores on study day 183. A separate analysis was performed in subjects receiving ISIS 396443 and sham control. In subjects receiving ISIS 396443, the logarithmic transformed pNF-H levels on study day 64 were statistically significantly correlated with the CHOP INTEND scores on study day 183, p=0.0406, such that higher pNF-H levels were associated with lower total CHOP INTEND scores. In subjects who received sham controls, log-transformed pNF-H levels on Study Day 64 were statistically significantly correlated with CHOPINTEND scores on Study Day 183, p=0.0330, such that higher pNF-H levels were associated with lower CHOPINTEND scores. Fig.29 .

[0333] These results suggest that current pNF-H levels may predict future motor function levels in infants with infantile-onset SMA.

[0334] Example 28: pNF-H levels over time in surviving infants with non-missing values ​​at day 302

[0335] Analyze the pNF-H level in the plasma samples from the clinical study ENDEAR at the 1st day, 2nd day, 29th day, 64th day, 183rd day and 302nd day (ISIS 396443 and sham operation).For this analysis, the colony is limited to the subjects who survived to the 302nd day of the study and measured the pNF-H level at the 302nd day of the study.The absolute value of pNF-H is drawn according to the study day.Over time, in the subjects who received sham operation, the average pNF-H level almost linearly decreased from about 16,000 pg / mL of the 1st day of the study to about 7,000 pg / mL of the 302nd day of the study.Over time, in the subjects who received ISIS 396443, the average pNF-H level decreased from about 18,000 pg / mL of the 1st day of the study to about 4,000 pg / mL of the 64th day of the study, and then remained stable at about 1,000 pg / mL at the 302nd day of the study. See also Fig.30 .

[0336] The results of this analysis suggest that the decline in both groups is real and not due to survivorship bias.

[0337] Example 29: Changes in pNF-H levels over time in surviving infants with non-missing values ​​at day 302 percentage

[0338] pNF-H levels in plasma samples from the clinical study ENDEAR on study days 1, 2, 29, 64, 183, and 302 (ISIS 396443 and sham surgery) were analyzed. For this analysis, the population was limited to subjects who survived to study day 302 and had pNF-H levels measured on study day 302. Changes from baseline were plotted according to study day. Over time, in subjects who received sham surgery, the change in pNF-H levels decreased almost linearly to approximately -50% on study day 302. Over time, in subjects who received ISIS 396443, the change in pNF-H decreased to approximately -70% on study day 64 and then remained stable at approximately -90% on study day 302. See Fig.31 .

[0339] The results of this analysis suggest that the decline in both groups is real and not due to survivorship bias.

[0340] Example 30: Correlation of Peroneal CMAP Amplitude on Day 183 with pNF-H Levels on Day 64

[0341] Plasma samples from the clinical study ENDEAR on study day 64 were analyzed for pNF-H levels, and peroneal CMAP amplitude was assessed on study day 183 (ISIS 396443 and sham controls). For this analysis, a linear regression model was constructed to determine if pNF-H levels on study day 64 were associated with peroneal CMAP amplitude on study day 183. pNF-H levels on study day 64 were significantly correlated with peroneal CMAP amplitude on study day 183, p=0.0021. See Fig.32 .

[0342] These results suggest that current pNF-H levels may predict future overall motor health in infants with infantile-onset SMA.

[0343] Example 31: Correlation of Peroneal CMAP Amplitude on Day 183 with pNF-H Levels on Day 64

[0344] pNF-H levels in plasma samples from the clinical study ENDEAR on study day 64 were analyzed, and the peroneal CMAP amplitude on study day 183 (ISIS 396443 and sham controls) were assessed. For this analysis, a linear regression model was constructed to determine whether pNF-H levels on study day 64 were associated with peroneal CMAP amplitude on study day 183. A separate analysis was performed in subjects receiving ISIS 396443 and sham controls. In subjects receiving ISIS 396443, log-transformed pNF-H levels on study day 64 were not statistically significantly correlated with peroneal CMAP amplitude on study day 183, p=0.3612, such that higher pNF-H levels were associated with a trend toward lower peroneal CMAP amplitudes. In subjects who received sham controls, log-transformed pNF-H levels at study day 64 were statistically significantly correlated with peroneal CMAP amplitude at study day 183, p = 0.0142, such that higher pNF-H levels were associated with lower peroneal CMAP amplitude. Fig.33 .

[0345] These results suggest that current pNF-H levels may predict future overall motor neuron health in infants with infantile-onset SMA.

[0346] Example 32: Correlation of Ulna CMAP Amplitude on Day 183 with pNF-H Levels on Day 64

[0347] Plasma samples from the clinical study ENDEAR at study day 64 were analyzed for pNF-H levels, and peroneal CMAP amplitude was assessed at study day 183 (ISIS 396443 and sham controls). For this analysis, a linear regression model was constructed to determine if pNF-H levels at study day 64 were associated with ulnar CMAP amplitude at study day 183. pNF-H levels at study day 64 were significantly associated with ulnar CMAP amplitude at study day 183, p=0.0002. See Fig.34 .

[0348] These results suggest that current pNF-H levels may predict future overall motor health in infants with infantile-onset SMA.

[0349] Example 33: Correlation of Ulna CMAP Amplitude on Day 183 with pNF-H Levels on Day 64

[0350] pNF-H levels in plasma samples from the clinical study ENDEAR on study day 64 were analyzed, and ulnar CMAP amplitudes on study day 183 (ISIS 396443 and sham controls) were assessed. For this analysis, a linear regression model was constructed to determine whether pNF-H levels on study day 64 were associated with ulnar CMAP amplitudes on study day 183. A separate analysis was performed in subjects receiving ISIS 396443 and sham controls. In subjects receiving ISIS 396443, log-transformed pNF-H levels on study day 64 were not statistically significantly correlated with ulnar CMAP amplitudes on study day 183, p=0.0952, such that higher pNF-H levels were associated with a trend toward lower ulnar CMAP amplitudes. In subjects receiving sham controls, log-transformed pNF-H levels at study day 64 were statistically significantly correlated with ulnar CMAP amplitude at study day 183, p = 0.0281, such that higher pNF-H levels were associated with lower ulnar CMAP amplitude. Fig.35 .

[0351] These results suggest that current pNF-H levels may predict future overall motor neuron health in infants with infantile-onset SMA.

[0352] Example 34: ENDEAR Baseline Characteristics Dichotomized by Median pNF-H Levels

[0353] The following is a table providing baseline characteristics dichotomized according to median pNF-H levels (<15,400 and ≥15,400 pg / mL) from the ENDEAR trial.

[0354]

[0355] CHOP INTEND = Children's Hospital of Philadelphia Infant Neuromuscular Disorders Examination;

[0356] CMAP = compound muscle action potential;

[0357] HINE-2 = Hammersmith Infant Neurological Examination Section 2.

[0358] a Median baseline pNF-H.

[0359] b Results for continuous variables were from Student's two-sample t test, and results for proportions were from Fisher's exact test.

[0360] c The numbers of participants in the table refer to participants with non-missing baseline pNF-H in either category.

[0361] Example 35: Correlation between Baseline Characteristics and Log(pNF-H) Levels in ENDEAR

[0362] The table below provides the correlation between baseline characteristics and log(pNF-H) levels in the ENDEAR trial.

[0363]

[0364] Example 36: CHERISH Baseline Characteristics Dichotomized According to Median pNF-H Levels

[0365] The following is a table providing baseline characteristics dichotomized according to median pNF-H levels from the CHERISH trial (<1,200 and ≥1,200 pg / mL).

[0366]

[0367] HFMSE = Hammersmith Functional Motor Scale–Extended.

[0368] a Median baseline pNF-H.

[0369] b Results for continuous variables were from Student's two-sample t test, and results for proportions were from Fisher's exact test.

[0370] c The numbers of participants in the table refer to participants with non-missing baseline pNF-H in either category.

[0371] Example 37: Correlation between Baseline Characteristics and Log(pNF-H) Levels in CHERISH

[0372] The table below provides the correlation between baseline characteristics and log(pNF-H) levels in the CHERISH trial.

[0373]

[0374] HFMSE = Hammersmith Functional Motor Scale–Extended.

[0375] WHO = World Health Organization

[0376] Example 38: Predicting future motor function by measuring the percent change in pNF-H levels after treatment

[0377] Receiver operating characteristic (ROC) curves were used to attempt to select the time period for measuring the percent change in neurofilament levels that best predicted future motor function in treated individuals. In ENDEAR, the percent change in pNF-H levels was measured at Day 29, Day 64, and Day 183 and compared to motor function in treated subjects at Day 302. The ROC curves revealed that the percent change in pNF-H levels at Day 64 was a better predictor of motor function at Day 302 than the percent change in pNF-H levels at Day 29 or Day 183. See Figures 36A-36C In ENDEAR, percent change in pNF-H levels at Day 64 predicted CHOP INTEND (≥4-point improvement) and motor milestone (more improvement than worsening in HINE-2 motor milestones) responders at Day 302, even after controlling for age at first dose. Figures 37A-37B In CHERISH, the percent change in pNF-H levels at day 85 predicted HFMSE (Hammersmith Functional Motor Scale–Extended; ≥3-point improvement), RULM (Revised Upper Limb Module; ≥2-point improvement), and WHO (World Health Organization; achievement of ≥1 motor milestone) responders at day 456, even after controlling for disease duration. Figures 38A-38C .

[0378] Example 39: Cerebrospinal fluid pNF-H levels at baseline and after treatment with Nussenelsen

[0379] Baseline cerebrospinal fluid (CSF) pNF-H levels were measured in presymptomatic, infantile-onset, and late-onset patients and in patients with 2 or 3 SMN2 copies. Baseline pNF-H levels in the CSF were highest in presymptomatic infants and in the youngest infants with 2 SMN2 copies. Figures 39A-39B In the NURTURE study, treatment with nussenelsen was associated with a rapid decrease in pNF-H levels in CSF, which then stabilized. Fig.40 .

[0380] Example 40: Phosphorylated neurofilament heavy chain (pNF-H) and pNF-H in different matrices in multiple spinal muscular atrophy populations Comparison of neurofilament light chain (NF-L)

[0381] The concentrations of pNF-H and NF-L in plasma and cerebrospinal fluid (CSF) were compared in individuals with presymptomatic (most likely to develop type I / II), infantile-onset (had or were most likely to develop type I / II), or late-onset SMA (had or were most likely to develop type II / III) from the Knudsen clinical trial. pNF-H concentrations in plasma and CSF were assessed using the ProteinSimple™ SimplePlex ELLA immunoassay. NF-L concentrations were assessed using the SIMOA assay (Quanterix™). Results reported below are in pg / mL.

[0382] For each SMA population, pNF-H and NF-L concentrations were similar in CSF. However, in plasma, NF-L concentrations were lower than pNF-H concentrations. The difference between NF-L and pNF-H concentrations in plasma was more pronounced in presymptomatic infants compared with infants with infantile-onset SMA. In CSF, the percent reduction in pNF-H and NF-L concentrations before symptomatic onset (day 183: Fig.41A ) and infantile-onset (percent reduction at day 302: Fig.41B ) SMA group decreased at a similar rate over time after nussenelsen treatment. In infantile-onset (day 64) percent reduction: Fig.41C ) and late onset (percent reduction at day 169: Fig.41D ) showed comparable results in plasma pNF-H and NF-L concentrations in the SMA group. Fig.41C The absolute values ​​of the data shown in Fig.41E is presented in Fig.41D The absolute values ​​of the data shown in Fig.41F In addition, the rates of change of pNF-H concentrations in plasma and CSF were similar in the presymptomatic and infantile-onset SMA cohorts. For NF-L, plasma and CSF concentrations declined along similar trajectories in the infantile-onset cohort.

[0383] Overall, baseline pNF-H and NF-L geometric mean (95% CI) concentrations were 20139 (10075-40257) and 7272 (3287-16090) pg / mL, respectively, in presymptomatic infants with 2 SMN2 copies, and 952 (367-2470) and 519 (231-1164) pg / mL, respectively, in those with 3 SMN2 copies. In participants with infantile-onset SMA, baseline pNF-H and NF-L geometric mean (95% CI) concentrations were 3791 (2980-4823) and 3718 (2832-4882) pg / mL, respectively. In participants with late-onset SMA, baseline pNF-H and NF-L geometric mean (95% CI) concentrations were 381 (331-438) and 185 (154-222) pg / mL, respectively.

[0384] Thus, like plasma pNF-H levels, CSF pNF-H and NF-L levels appeared to be highest in presymptomatic SMA participants with 2 copies of SMN2 and lowest in those with late-onset SMA.

[0385] Other Implementations

[0386] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. A method of treating spinal muscular atrophy (SMA) in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of an SMA therapy, wherein the human subject has previously been determined in a biological sample obtained from the human subject to have neurofilament levels above a control prior to initiation of the SMA therapy.

2. A method of treating spinal muscular atrophy (SMA) in a human subject in need thereof, the method comprising: measuring neurofilament levels in a biological sample obtained from the human subject prior to initiating SMA therapy; as well as A therapeutically effective amount of a SMA therapy is administered to the human subject.

3. The method of claim 1 or 2, wherein the neurofilament level in the biological sample is greater than 400 pg / mL.

4. A method of treating spinal muscular atrophy (SMA) in a human subject in need thereof, the method comprising: measuring neurofilament levels in a first biological sample obtained from the human subject prior to initiating SMA therapy; administering an SMA therapy to the human subject; as well as After initiating the SMA therapy, measuring neurofilament levels in a second biological sample obtained from the human subject.

5. The method of claim 4, wherein the level of neurofilaments measured in the second biological sample is lower than the level of neurofilaments measured in the first biological sample.

6. The method of claim 5, wherein the level of neurofilaments measured in the second biological sample is between 10% and 95% of the level of neurofilaments measured in the first biological sample.

7. The method of any one of claims 4 to 6, wherein the second biological sample is obtained from the human subject 40 to 90 days after initiation of the SMA therapy.

8. The method of any one of claims 4 to 6, wherein the second biological sample is obtained from the human subject 50 to 80 days after initiation of the SMA therapy.

9. The method of any one of claims 4 to 6, wherein the second biological sample is obtained from the human subject 60 to 70 days after initiation of the SMA therapy.

10. The method of any one of claims 4 to 6, wherein the second biological sample is obtained from the human subject about 64 days after initiation of the SMA therapy.

11. The method of any one of claims 7 to 10, wherein the level of neurofilaments measured in the second biological sample is reduced by at least 50% compared to the level of neurofilaments measured in the first biological sample.

12. The method of any one of claims 7 to 10, wherein the neurofilament level measured in the second biological sample is reduced by at least 60% compared to the neurofilament level measured in the first biological sample.

13. The method of any one of claims 7 to 10, wherein the level of neurofilaments measured in the second biological sample is reduced by at least 70% compared to the level of neurofilaments measured in the first biological sample.

14. The method of any one of claims 7 to 10, wherein the neurofilament level measured in the second biological sample is reduced by less than 50% compared to the neurofilament level measured in the first biological sample.

15. The method of any one of claims 7 to 10, wherein the neurofilament level measured in the second biological sample is reduced by less than 40% compared to the neurofilament level measured in the first biological sample.

16. The method of any one of claims 7 to 15, wherein the dosage of the SMA therapy is altered for subsequent administration to the human subject based on the percentage decrease in the neurofilament levels measured in the second biological sample compared to the neurofilament levels measured in the first biological sample.

17. The method of claim 4, wherein the level of neurofilaments measured in the second biological sample is higher than the level of neurofilaments measured in the first biological sample.

18. The method of any one of claims 4 to 17, wherein administration of the SMA therapy continues.

19. The method of claim 17, wherein administration of the SMA therapy is discontinued.

20. A method of treating spinal muscular atrophy (SMA) in a human subject in need thereof, the method comprising: measuring the level of neurofilaments in a first biological sample obtained from the human subject prior to administering the candidate amount of the SMA therapy; measuring the level of neurofilaments in a second biological sample obtained from the human subject after administering the candidate amount of the SMA therapy, wherein the level of neurofilaments in the second biological sample is lower than the level of neurofilaments in the first biological sample, thereby indicating that the candidate amount of the SMA therapy is a therapeutically effective amount; After measuring decreased neurofilament levels in the second biological sample, administering the therapeutically effective amount of the SMA therapy to the human subject.

21. A method for predicting the prognosis of spinal muscular atrophy (SMA), the method comprising: measuring neurofilament levels in a biological sample obtained from a human subject having a mutation in both copies of the SMN1 gene that results in a lack of functional SMN protein; and comparing the neurofilament level measured in the biological sample to a control, Wherein the level of neurofilaments measured in the biological sample compared to the control predicts the severity or type of SMA that the subject will develop.

22. The method of claim 21, wherein the biological sample is obtained from the human subject prior to initiating SMA therapy, and wherein the level of neurofilaments measured in the biological sample compared to the control predicts the severity or type of SMA that the human subject will develop in the absence of treatment.

23. The method of claim 21, wherein the biological sample is obtained from the human subject after initiation of SMA therapy, and wherein the level of neurofilaments measured in the biological sample compared to the control predicts the severity or type of SMA that the subject will develop while receiving the SMA therapy.

24. The method of claim 23, wherein the biological sample is obtained from the human subject at least two weeks after initiation of the SMA therapy.

25. The method of claim 23, wherein the biological sample is obtained from the human subject at least two months after initiation of the SMA therapy.

26. A method for predicting the prognosis of spinal muscular atrophy (SMA), the method comprising: measuring neurofilament levels in a first biological sample obtained from a human subject having a mutation in both copies of the SMN1 gene that results in a lack of functional SMN protein prior to initiating SMA therapy; measuring neurofilament levels in a second biological sample obtained from the human subject after initiation of the SMA therapy; as well as comparing the level of neurofilaments measured in the second biological sample to the level of neurofilaments measured in the first biological sample, Wherein the level of neurofilaments measured in the second biological sample, compared to the level of neurofilaments measured in the first biological sample, predicts the severity or type of SMA that the subject will develop.

27. The method of claim 26, wherein the second biological sample is obtained from the human subject 40 to 90 days after initiation of the SMA therapy.

28. The method of claim 26, wherein the second biological sample is obtained from the human subject 50 to 80 days after initiation of the SMA therapy.

29. The method of claim 26, wherein the second biological sample is obtained from the human subject 60 to 70 days after initiation of the SMA therapy.

30. The method of claim 26, wherein the second biological sample is obtained from the human subject about 64 days after initiation of the SMA therapy.

31. The method of any one of claims 26 to 30, wherein the level of neurofilaments measured in the second biological sample is reduced by at least 50% compared to the level of neurofilaments measured in the first biological sample.

32. The method of any one of claims 26 to 30, wherein the neurofilament level measured in the second biological sample is reduced by at least 60% compared to the neurofilament level measured in the first biological sample.

33. The method of any one of claims 26 to 30, wherein the level of neurofilaments measured in the second biological sample is reduced by at least 70% compared to the level of neurofilaments measured in the first biological sample.

34. The method of any one of claims 26 to 30, wherein the neurofilament level measured in the second biological sample is reduced by less than 50% compared to the neurofilament level measured in the first biological sample.

35. The method of any one of claims 26 to 30, wherein the neurofilament level measured in the second biological sample is reduced by less than 40% compared to the neurofilament level measured in the first biological sample.

36. The method of any one of the preceding claims, wherein the SMA therapy is Knudsen or a Knudsen salt.

37. The method of any one of claims 1 to 35, wherein the SMA therapy is nussenelson sodium.

38. The method of any one of claims 1 to 35, wherein the SMA therapy is olesoxim, AVX-101, CK-2127107, RG7916, RG7800, RO7034067, LMI070, or SRK-015.

39. The method of any of the preceding claims, wherein the control is a pre-established neurofilament cutoff value.

40. The method of any one of claims 1 to 38, wherein the control is the level of neurofilaments in a biological sample or samples obtained from one or more human subjects not suffering from SMA.

41. A method for measuring neurofilament levels, the method comprising: providing a biological sample obtained from a human subject having a mutation in both copies of the SMN1 gene that results in a lack of functional SMN protein; as well as The level of neurofilaments in the biological sample is measured.

42. The method of any one of the preceding claims, wherein the neurofilament is neurofilament heavy chain.

43. The method of any one of the preceding claims, wherein the neurofilament is a phosphorylated neurofilament heavy chain.

44. The method of any one of claims 1 to 41, wherein the neurofilament is a neurofilament mid-chain / intermediate chain.

45. The method of any one of claims 1 to 41, wherein the neurofilament is a neurofilament light chain.

46. ​​The method of any of the preceding claims, wherein the biological sample is blood, serum, plasma, or cerebrospinal fluid.

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