Combinatory treatment of sma with sarna and mRNA modulators

Abstract

This article provides methods and compositions relating to combinations of (a) agents that increase the expression of the SMN2 gene or protein and (b) modulators that increase the splicing or stability of SMN2 mRNA produced by functional SMN2 mRNA and SMN protein, and their use in the treatment of SMA and related disorders or diseases. In some embodiments, the method relates to the use of SMN2 saRNA and SMN2 mRNA modulators to alleviate the symptoms of SMA.

Description

Technical Field

[0001] [Cross-reference]

[0002] This application claims the benefit of PCT patent application No. PCT / CN2020 / 106200, filed on July 31, 2020, which is incorporated herein by reference in its entirety.

[0003] [By incorporating the provided sequence list as a text file]

[0004] Accompanying this document is a sequence list in the text file "SMN2-Combo sequence listing_ST25 final", created on July 30, 2021, with a size of 81Kb. The contents of this text file are incorporated herein by reference in their entirety. Prior Technology

[0005] Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder, affecting approximately 1 in 6,000-8,000 newborns, and is a leading genetic cause of infant mortality. SMA is caused by a homozygous deletion or mutation of the telomere copy of the survival motor neuron (SMN1) gene on chromosome 5q13.4, leading to a decrease in the protein levels of the survival motor neuron (SMN).

[0006] The SMN protein is encoded by two SMN genes (SMN1 and SMN2), whose coding sequences differ fundamentally in one nucleotide of exon 7: cytosine (C) is replaced by thymine (T) in the SMN2 gene (Koufert, DD et al., Proteins of Surviving Motor Neurons in Spinal Muscular Atrophy. Human Molecular Genetics (1997)). This crucial difference creates a hidden splicing site and results in approximately 90% skipping of exon 7 in mature SMN mRNA transcribed from the SMN2 gene. SMN2 mRNA lacking exon 7 (SMN2Δ7) produces an unstable and rapidly degraded truncated SMN protein. In SMA patients, the SMN1 gene no longer produces any SMN protein, and the amount of full-length SMN protein produced by SMN2 is insufficient to compensate for the loss of SMN1, leading to apoptotic death of motor neurons in the anterior horn of the spinal cord, skeletal muscle atrophy, and consequent weakness (Monani, UR et al., Human centromere survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(- / -) mice and results in mouse spinal muscular atrophy. Human Molecular Genetics (2000)). The severity of symptoms in SMA patients depends on the copy number of the SMN2 gene in the patient's cells—the higher the copy number, the milder the symptoms (Halada, Y. et al., Correlation between SMN2 copy number and clinical phenotype of spinal muscular atrophy: Three SMN2 copies failed to rescue some patients from disease severity. J Neurol (2002)).

[0007] To develop therapeutics for SMA, one strategy is to use splice modulators (SMs) to stimulate exon 7 inclusion during splicing. In this regard, the antisense oligonucleotide (ASO) drug Spinraza® has been approved by the U.S. Food and Drug Administration (FDA) (Hua, Y. and A. Kleiner. Antisense-mediated exon inclusion. Molecular Biology Approaches (2012) and Stan, C.A. and D. Castanoto. FDA-approved oligonucleotide therapy in 2017. Molecular Diagnostics (2017)). Another drug, Risdiplam (RG7916), an investigational oral small molecule, is in the process of submitting a New Drug Application (NDA) to the FDA (Lamdas, S. and L. Selve. New treatments for spinal muscular atrophy: a review of currently available data. Pharmacological Expert Opinion (2020)). Both drugs have altered the treatment of SMA by significantly prolonging patient survival and improving motor development indicators. Despite these improvements, treated patients, especially children, are far from leading normal lives. Several plausible reasons can explain the inadequacy of SMs. One is the ceiling effect, which limits the maximum achievable level of full-length SMN protein restored through treatment. SM has no effect on SMN2 transcription and therefore does not increase the amount of available SMN2 pre-mRNA. To restore SMN protein to its normal physiological levels, SM would ideally achieve 100% in vivo efficiency in converting SMN2Δ7 mRNA into full-length mRNA, an ideal effect unlikely to occur in reality. Therefore, the maximum therapeutic effect that SM can provide to patients is limited by the availability of SMN2 pre-mRNA.

[0008] Another approach to treating SMA involves stimulating SMN2 transcription to increase levels of full-length SMN protein. Previously, various epigenetic modifiers, such as histone deacetylase (HDAC) inhibitors (e.g., sodium butyrate, valproic acid) and non-HDAC inhibitors (e.g., hydroxyurea, celecoxib, salbutamol, etc.), have been tested in vitro and in mouse SMA models, but these have failed to demonstrate significant clinical efficacy (Lenke, S. and A. Austa. Novel roles of epigenetic modification and chromatin remodeling in spinal muscular atrophy, Journal of Neurochemistry (2009)). One explanation for this failure is the lack of target specificity in epigenetic modifiers. Improved approaches and formulations are needed for treating SMN deficiency-related disorders such as spinal muscular atrophy. Summary of the Invention

[0009] Double-stranded RNAs (dsRNAs) targeting gene regulatory sequences (including promoters) have been shown to upregulate target genes at the transcriptional level in a sequence-specific manner via a mechanism known as RNA initiation (RNAa) (Li, LC et al., Small dsRNA Induces Transcriptional Initiation in Human Cells, Proceedings of the National Academy of Sciences (2006)). These dsRNAs are referred to as small initiator RNAs (saRNAs). A previously disclosed patent application (PCT / CN2019 / 129025) (incorporated herein by reference in its entirety) describes the identification of functional saRNAs targeting the SMN2 promoter and their activity in inducing SMN2 mRNA expression in normal human cells and primary cells derived from SMA patients.

[0010] The embodiments disclosed herein are based in part on the surprising finding that a combination of (a) one or more small promoter RNAs (saRNAs) that initiate or upregulate the expression of the SMN2 gene in cells (also referred to herein as "SMN2 saRNA") and (b) one or more regulators of SMN2 mRNA splicing or stability that increase the production of functional SMN2 mRNA (also referred to herein as "SMN2 mRNA regulators") can significantly increase the levels of full-length SMN2 mRNA and full-length SMN protein. This combination therapy strategy can provide enhanced therapeutic benefits compared to monotherapy, thus maximizing treatment outcomes, for example, in patients with SAM.

[0011] In some respects, pharmaceutical compositions are provided for the treatment or delay of the onset or progression of an SMN deficiency-related disorder (such as SMA) in an individual, the composition comprising (a) one or more agents that increase the expression of the SMN2 gene or protein and (b) one or more regulators that increase the production of functional SMN2 mRNA splicing or stability.

[0012] In some embodiments, the formulation that increases the expression of the SMN2 gene or protein may include any suitable formulation having this activity, including macromolecules and small molecules. Examples of macromolecules are proteins, protein complexes and glycoproteins, and nucleic acids such as DNA, RNA, and PNA (peptide nucleic acids). Examples of small molecules are peptides, peptide analogs (e.g., peptide-like substances), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, and organic or inorganic compounds, such as heteroorganometallic compounds or organometallic compounds. Any of these formulations may be used sequentially or in combination by the methods described herein.

[0013] In some embodiments, the agent for increasing the expression of the SMN2 gene or protein is at least one saRNA (referred to herein as "SMN2 saRNA") or its recombinant vector, or at least one small molecule compound. In specific embodiments, the agent for increasing the expression of the SMN2 gene or protein is at least one SMN2 saRNA. In some embodiments, the SMN2 saRNA comprises a sense strand and an antisense strand, or comprises a single strand, or a mixture thereof.

[0014] In some embodiments disclosed herein, the SMN2 mRNA regulator is an antisense oligonucleotide (ASO) or a small molecule, such as a pyridazine derivative. In some embodiments disclosed herein, the SMN2 mRNA regulator is selected from Nusinersen (Spinraza®, also referred to herein as ASO-10-27), Risdiplam, Rigosertib, and Branaplam.

[0015] In some embodiments disclosed herein, the SMN2 saRNA comprises a first strand having at least 90% identity with the following regions of the SMN2 gene promoter: (a) the -1639 to -1481 region of the SMN2 gene promoter (SEQ ID NO: 472), (b) the -1090 to -1008 region of the SMN2 gene promoter (SEQ ID NO: 473), (c) the -994 to -180 region of the SMN2 gene promoter (SEQ ID NO: 474), or (d) the -144 to -37 region of the SMN2 gene promoter (SEQ ID NO: 475).

[0016] In some embodiments disclosed herein, the first strand of the SMN2 saRNA has at least 75% homology or complementarity with a promoter region segment of the SMN2 gene that is 16-35 nucleotides in length.

[0017] In some embodiments disclosed herein, the first strand of the SMN2 saRNA molecule has at least 75% homology or complementarity with any nucleotide sequence selected from SEQ ID NO:315-471.

[0018] In some embodiments disclosed herein, the sense strand has at least 75% homology with any nucleotide sequence selected from SEQ ID NO:1-157, and the antisense strand has at least 75% homology with any nucleotide sequence selected from SEQ ID NO:158-314.

[0019] In some embodiments disclosed herein, the sense strand comprises a nucleotide sequence selected from any one of SEQ ID NO: 1-157, and the antisense strand comprises a nucleotide sequence selected from any one of SEQ ID NO: 158-314.

[0020] In some embodiments disclosed herein, at least one nucleotide is a chemically modified nucleotide.

[0021] In some embodiments, the compositions provided herein also include one or more pharmaceutically acceptable carriers, such as aqueous carriers, liposomes, polymers, or peptides.

[0022] In some embodiments disclosed herein, the composition comprises 1-150 nM of SMN2 saRNA and 1-50 nM of ASO SMN2 mRNA regulator.

[0023] In some embodiments of this disclosure, the composition comprises 1-150 nM of SMN2 saRNA and 1-3000 nM of a small molecule pyridazine derivative SMN2 mRNA regulator, such as Risdiplam. In some embodiments, the composition comprises 300-2000 nM Risdiplam, which increases the amount of full-length SMN protein in treated cells by at least 10% compared to baseline measurements taken before treatment or compared to untreated cell populations. In other embodiments, the composition of this disclosure reduces the amount of SMN2Δ7 in treated cells compared to baseline measurements taken before treatment.

[0024] In some embodiments disclosed herein, the SMN2 saRNA is DS06-0004 (also known as RAG6-281), DS06-0031 (also known as RAG6-1266), or DS06-0067 (also known as RAG6-293).

[0025] Certain embodiments of this disclosure relate to methods for treating or delaying the onset or progression of SMN deficiency-related disorders in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising (a) one or more agents that increase the expression of the SMN2 gene or protein, and (b) one or more SMN2 mRNA splicing or stability regulators that increase the production of functional SMN2 mRNA. In certain embodiments of the methods provided herein, the agent that increases the expression of the SMN2 gene or protein is saRNA (hereinafter referred to as "SMN2 saRNA") or its recombinant vector, or a small molecule compound. In certain embodiments of the methods provided herein, the SMN2 mRNA regulator is an antisense oligonucleotide (ASO) or a small molecule compound, such as a pyridazine derivative. In certain embodiments of this disclosure, the SMN2 mRNA regulator is selected from Nusinersen (Spinraza®, also referred to herein as ASO-10-27), Risdiplam, Rigosertib, and Branaplam.

[0026] In some embodiments of the method provided herein, the SMN2 saRNA comprises a strand that is at least 90% identical to the following regions of the SMN2 gene promoter: the -1639 to -1481 region of the SMN2 gene promoter (SEQ ID NO: 472), the -1090 to -1008 region of the SMN2 gene promoter (SEQ ID NO: 473), the -994 to -180 region of the SMN2 gene promoter (SEQ ID NO: 474), or the -144 to -37 region of the SMN2 gene promoter (SEQ ID NO: 475).

[0027] In some embodiments of the method provided herein, one strand of the SMN2 saRNA has at least 75% homology or complementarity with a fragment of the SMN2 gene promoter region of 16-35 nucleotides in length.

[0028] In any of the embodiments described herein, the individual suffers from SMA. In yet another embodiment, the individual with SMA has reduced or abnormal expression of the full-length SMN protein. Simple Explanation of the Diagram

[0029] picture [1] shows the structure of the SMN2 gene, the target site of the saRNA, and the location of the PCR primers. Figure [1A] shows the structure of the SMN2 gene and its 2kb promoter region. The target sites of saRNAs DS06-0004, DS06-0067, and DS06-0031, relative to the transcription start site (TSS) of SMN2, are shown at positions -281, -293, and -1266, respectively. Figure [1B] shows the positions of PCR primers used for RT-qPCR (SMN2FL F+SMN2FL R,SMN△7 F+SMN△7 R) and semi-quantitative RT-PCR (SMN-exon6-F+SMN-exon8-R).

[0030] picture [2A] to Figure [2C] shows a schematic diagram of the differences between the SMN1 and SMN2 genes and the semi-quantitative RT-PCR / DdeI digestion assay. The G→A variant in exon 8 of SMN2 creates a recognition site for the DdeI restriction enzyme (Fig. 2A). PCR primer pairs SMN-exon6-F and SMN-exon8-R amplified a 507 bp product (SMN2FL) and a 453 bp product (SMN2△7). To distinguish between the SMN1 and SMN2 products, the SMN2FL product was digested with DdeI into 392 bp and 115 bp fragments (Fig. 2B), and the SMN2△7 product was digested into 338 bp and 115 bp fragments (Fig. 2C).

[0031] picture [3A] to Figure [3G] shows the effects of saRNA (DS06-0004), ASO-10-27, and Risdiplam on the expression of full-length (SMN2FL) and exon-skipped (SMN2Δ7) SMN2 mRNA in GM03813 cells. GM03813 cells were treated with specified concentrations of ASO-10-27, saRNA (DS06-0004), and Risdiplam for 72 h. As a control, analog samples were transfected in the absence of oligonucleotides (control, mock). The mRNA levels of SMN2FL and SMNΔ7 were determined by RT-qPCR using two pairs of primers in separate PCR reactions. Figures 3A to 3C show the mRNA levels of SMN2FL and SMNΔ7 as determined by RT-qPCR. Figure 3D shows the mRNA levels of SMN2FL and SMNΔ7 as determined by semi-quantitative PCR. The PCR products of SMN2 were digested with DdeI enzyme and separated on a 2% agarose gel. TBP gene amplification was also used as a control for RNA loading. [Figures 3E to 3G] show the SMN2FL and SMN2△7 levels derived from the band intensities of the quantitative PCR products in Figure 3D. The values ​​(y-axis) are the band intensities of SMN2 relative to the control treatment after normalization to the TBP band intensity. SMN2FL: Full-length PCR product of SMN2 after digestion (392 bp); SMN△7: PCR product after digestion skipping SMN exon 7 (338 bp); Ladder band: 100 bp DNA marker.

[0032] [Figures 4A to 4E] illustrate the combined effects of saRNA (DS06-0004) and ASO-10-27 on the expression of SMN2FL and SMN2Δ7 SMN2 mRNA in GM00232 cells. GM00232 cells were transfected with ASO-10-27 and DS06-0004 at specified concentrations, alone or in combination, for 72 hours. Analog samples were transfected in the absence of oligonucleotides as a control. Total RNA was extracted from treated cells using the Qiagen RNeasy kit and reverse transcribed to obtain cDNA. The mRNA levels of SMN2FL and SMNΔ7 were then determined by RT-qPCR. [Figures 4A and 4D] show the mRNA levels of SMN2FL and SMN△7 as determined by RT-qPCR. [Figure 4B] shows the mRNA levels of SMN2FL and SMNΔ7 as determined by semi-quantitative PCR. The PCR product of SMN2 was digested with DdeI enzyme and separated on a 2% agarose gel. TBP gene amplification was also used as a control for RNA loading. [Figures 4C and 4E] show the SMN2FL and SMN2△7 levels derived from the band intensities of the quantitative PCR products in 4B. The values ​​(y-axis) are the band intensities of SMN2FL and SMN2△7 normalized to TBP band intensity relative to the control treatment. SMN2FL, full-length PCR product of SMN2 after digestion (392 bp); SMN△7, PCR product of SMN exon 7 skipped after digestion (338 bp), ASO, ASO-10-27.

[0033] [Figures 5A to 5C] illustrate the combined effect of saRNA (DS06-0004) and ASO-10-27 on the expression of full-length SMN protein in type I SMA cells GM00232. ASO-10-27 and DS06-0004 were transfected into GM00232 cells at specified concentrations, alone or in combination, for 72 hours. As a control, analog samples were transfected in the absence of oligonucleotides. Proteins were harvested from the treated cells and immunoblotting was performed using an antibody against human SMN protein for protein analysis. An antibody against α / β-tubulin was used for immunoblotting as a control for protein loading. [Figure 5A] shows SMN protein expression from cells treated with analog and cells treated alone with ASO-10-27 or in combination with DS06-0004. [Figure] 5B shows SMN protein expression from cells treated with analog and cells treated alone with DS06-0004 or in combination with ASO-10-27. [Figure] 5C shows the relative fold change in SMN protein levels derived from the quantitative band intensities in Figures 5A and 5B. The values ​​(y-axis) are the relative band intensities of SMN protein after normalization to the band intensities of α / β-tubulin. ASO, ASO-10-27.

[0034] [Figures 6A to 6E] illustrate the combined effects of saRNA (DS06-0004) and ASO-10-27 on the expression of SMN2FL and SMN2Δ7 SMN2 mRNA in GM03813 cells. GM03813 cells were transfected with ASO-10-27 and DS06-0004 at specified concentrations, alone or in combination, for 72 hours. Analog samples were transfected in the absence of oligonucleotides as a control. Total RNA was extracted from treated cells using the Qiagen RNeasy kit and reverse transcribed to obtain cDNA. The mRNA levels of SMN2FL and SMNΔ7 were then determined by RT-qPCR. [Figures 6A and 6D] show the mRNA levels of SMN2FL and SMN△7 as determined by RT-qPCR. [Figure 6B] shows the mRNA levels of SMN2FL and SMNΔ7 as determined by semi-quantitative PCR. The PCR product of SMN2 was digested with DdeI enzyme and separated on a 2% agarose gel. TBP gene amplification was also used as a control for RNA loading. [Figure 6C and] [Figure 6E] shows the SMN2FL and SMN2△7 levels derived from the band intensities of the quantitative PCR products in Figure 6B. The values ​​(y-axis) are the band intensities of SMN2FL and SMN2△7 normalized to TBP band intensity relative to the control treatment. SMN2FL, full-length PCR product of SMN2 after digestion (392 bp); SMN△7, PCR product of SMN exon 7 skipped after digestion (338 bp), ASO, ASO-10-27.

[0035] [Figures 7A to 7C] illustrate the combined effect of saRNA (DS06-0004) and ASO-10-27 on the expression of full-length SMN protein in type II SMA cells. ASO-10-27 and DS06-0004 were transfected into GM00232 cells at specified concentrations, alone or in combination, for 72 hours. As a control, analog samples were transfected in the absence of oligonucleotides. Proteins were harvested from the treated cells and immunoblotting was performed using an antibody against human SMN protein for protein analysis. An antibody against α / β-tubulin was used for immunoblotting as a control for protein loading. [Figure 7A] shows SMN protein expression from cells treated with analog and cells treated alone with ASO-10-27 or in combination with DS06-0004. [Figure 7B] shows SMN protein expression from cells treated with analog and cells treated alone with DS06-0004 or in combination with ASO-10-27. [Figure 7C] shows the relative fold change in SMN protein levels derived from the quantitative band intensities in Figures 7A and 7B. The values ​​(y-axis) are the relative band intensities of SMN protein after normalization to the band intensities of α / β-tubulin. ASO, ASO-10-27.

[0036] [Figures 8A to 8F] illustrate the combined effect of saRNA (DS06-0004) and Risdiplam on the expression of SMN2FL and SMN2Δ7 SMN2 mRNA in GM00232 cells. Risdiplam and DS06-0004 were transfected into GM00232 cells at specified concentrations, alone or in combination, for 72 hours. DMSO samples served as a Risdiplam medium control, and analogous treatments served as a control for saRNA transfection. Both DMSO samples and control treatments were transfected in the absence of oligonucleotides. Total RNA was extracted from treated cells using the Qiagen RNeasy kit and reverse transcribed to obtain cDNA. The mRNA levels of SMN2FL and SMNΔ7 were then determined by RT-qPCR. [Figure 8A] shows the mRNA levels of SMN2FL and SMN△7 as determined by RT-qPCR. [Figures 8B and 8C] show the relative SMN2FL mRNA levels in cells treated with different concentrations of DS06-0004 and Risdiplam in combination. [Figure 8D] shows the mRNA levels of SMN2FL and SMNΔ7 as determined by semi-quantitative PCR. The PCR product of SMN2 was digested with DdeI enzyme and separated on a 2% agarose gel. TBP gene amplification was also used as a control for RNA loading. [Figures 8E and 8F] show the SMN2FL levels derived from the band intensities of the quantitative PCR products in Figure 8D. The values ​​(y-axis) are the band intensities of SMN2FL and SMN2Δ7 relative to the control or DMSO treatment after normalization to the band intensity of TBP. SMN2FL, full-length PCR product of digested SMN2 (392 bp); SMNΔ7, PCR product of SMN exon 7 after digestion (338 bp).

[0037] [Figures 9A and 9B] show the combined effect of saRNA (DS06-0004) and Risdiplam on the expression of full-length SMN protein in type I SMA cells GM00232. Risdiplam and DS06-0004 were transfected into GM00232 cells at specified concentrations, alone or in combination, for 72 hours. DMSO samples were used as a Risdiplam medium control, and analogous treatments were used as a control for saRNA transfection. DMSO samples and control treatments were transfected in the absence of oligonucleotides. Proteins were harvested from the treated cells and immunoblotting was performed using an antibody against human SMN protein for protein analysis. An antibody against α / β-tubulin was used for immunoblotting as a control for protein loading. [Figure 9A] shows the expression of SMN protein in cells treated with DMSO and in cells treated alone with Risdiplam or in combination with DS06-0004. [Figure 9B] shows the fold change in SMN protein levels derived from the quantitative band intensity in Figure 9A. The values ​​(y-axis) are the relative band intensities of SMN protein after normalization to the band intensities of α / β-tubulin.

[0038] [Figures 10A to 10F] illustrate the combined effects of saRNA (DS06-0004) and Risdiplam on the expression of SMN2FL and SMN2Δ7 SMN2 mRNA in GM03813 cells. Risdiplam and DS06-0004 were transfected into GM03813 cells at specified concentrations, alone or in combination, for 72 hours. DMSO samples served as a Risdiplam transfection control, and analogous treatment served as a control for saRNA transfection. The DMSO samples and control treatments were also transfected in the absence of oligonucleotides. Total RNA was extracted from treated cells using the Qiagen RNeasy kit and reverse transcribed to obtain cDNA. The mRNA levels of SMN2FL and SMNΔ7 were then determined by RT-qPCR. [Figure 10A] shows the mRNA levels of SMN2FL and SMN△7 as determined by RT-qPCR. [Figures 10B and 6C] show the relative SMN2FL mRNA levels in cells treated with different concentrations of DS06-0004 and Risdiplam. [Figure 10D] shows the mRNA levels of SMN2FL and SMNΔ7 as determined by semi-quantitative PCR. The PCR product of SMN2 was digested with DdeI enzyme and separated on a 2% agarose gel. TBP gene amplification was also used as a control for RNA loading. [Figures 10E and 10F] show the SMN2FL levels derived from the band intensities of the quantitative PCR products from 10D. The values ​​(y-axis) are the band intensities of SMN2FL and SMN2Δ7 relative to the control or DMSO treatment after normalization to the band intensity of TBP. SMN2FL, full-length PCR product of digested SMN2 (392 bp); SMNΔ7, PCR product of digested SMN exon 7 skipped (338 bp).

[0039] [Figures 11A-11B] illustrate the combined effect of saRNA (DS06-0004) and Risdiplam on the expression of full-length SMN protein in type II SMA cells GM03813. Risdiplam and DS06-0004 were transfected into GM03813 cells at specified concentrations, alone or in combination, for 72 hours. DMSO samples served as a Risdiplam medium control, analogous treatment served as a control for saRNA transfection, and DMSO samples and control treatments were transfected in the absence of oligonucleotides. Proteins were harvested from the treated cells and immunoblotting was performed using an antibody against human SMN protein for protein analysis. An antibody against α / β-tubulin was used for immunoblotting as a control for protein loading. [Figure] 11A shows SMN protein expression from cells treated with analog and cells treated alone with Risdiplam or in combination with DS06-0004. [Figure] 11B shows the relative fold change in SMN protein levels derived from the quantitative band intensity in 11A. The values ​​(y-axis) are the relative band intensities of SMN protein after normalization to the band intensity of α / β-tubulin.

[0040] [Figures 12A-12E] illustrate the combined effects of saRNAs (DS06-0031 and DS06-0067) and ASO-10-27 on the expression of SMN2FL and SMN2Δ7 SMN2 mRNA and SMN protein in GM03813 cells. GM03813 cells were transfected with DS06-0031 or DS06-0067 alone or in combination with ASO-10-27 at 10 nM for 72 hours. Analog samples were transfected in the absence of oligonucleotides as a control. dsCon2 transfected with unrelated oligonucleotides served as a control. DS06-332i, an SMN2 siRNA, was transfected as a control. Total RNA was extracted from treated cells using the Qiagen RNeasy kit and reverse transcribed to obtain cDNA. The mRNA levels of SMN2FL and SMNΔ7 were then determined by RT-qPCR and semi-quantitative RT-PCR. Proteins were harvested from treated cells and immunoblotting was performed using an antibody against human SMN protein via Western blotting assay. An antibody against α / β-tubulin was also used as a control for protein loading. [Figure 12A] shows the mRNA levels of SMN2FL and SMN△7 as determined by RT-qPCR. [Figure 12B] shows the mRNA levels of SMN2FL and SMNΔ7, determined by semi-quantitative PCR and isolated on a 2% agarose gel. The TBP gene was also amplified as a control for RNA loading. [Figure 12C] shows the SMN2FL and SMN2△7 mRNA levels derived from the band intensities of the quantitative PCR products in Figure 12B. The values ​​(y-axis) are the band intensities of SMN2FL and SMN2△7 relative to the control treatment after normalization to the band intensity of TBP. SMN2FL, full-length PCR product of SMN2 (507 bp); SMN△7, PCR product skipping exon 7 of SMN (453 bp). [Figure 12D] shows the protein blot of SMN protein expression. [Figure 12E] shows the relative fold change in SMN protein levels derived from the quantitative band intensity of 12D. The values ​​(y-axis) are the relative band intensities of SMN proteins normalized to the band intensities of α / β-tubulin.

[0041] [Figures 13A to 13C] show the combined effects of saRNA (LNP-R6-04M1) and LNP-ASO-10-27 or Risdiplam on the expression of full-length (SMN2FL) and skipped-7 (SMN2Δ7) SMN2 mRNA in SMA type III mice (PND7). SMA type III mice were treated with three groups of mice: Group 1 (n=3) – LNP-R6-04M1 administered alone on day 1 (p1, 10 μg) and day 3 (p3, 10 μg) after birth; Group 2 (n=3) – a combination of LNP-R6-04M1 and LNP-ASO-10-27 (p1, 10 μg and p3, 10 μg) administered via intraventricular injection at specified concentrations; and Group 3 (n=3) – LNP-R6-04M1 and Risdiplam (0.3 mg / kg, 1 mg / kg, and 3 mg / kg) administered via intraventricular injection. Mice treated with saline served as untreated controls. Following treatment, RNA was isolated from two tissues (brain and liver) using the Qiagen RNeasy kit, reverse transcribed to obtain cDNA, and then the mRNA levels of SMN2FL and SMNΔ7 were determined by RT-qPCR. [Figure 13A] shows the mRNA levels of SMN2FL and SMNΔ7 in the brains of SMA type III mice as determined by RT-qPCR. [Figure 13B] shows the SMN2FL mRNA levels of SMN2FL and SMNΔ7 in the liver of SMA type III mice as determined by RT-qPCR. [Figure 13C] shows the SMN2FL mRNA levels in the spinal cord of SMA type III mice as determined by RT-qPCR. The SMN2FL and SMN2Δ7 mRNA levels are shown as the mean relative to three animals / groups (n=3 to 7) treated with saline after normalization to a Tbp reference level. Implementation

[0042] This invention is based on research related to methods for improving the therapeutic effect on SMN deficiency-related disorders by initiating / upregulating SMN2 gene expression and increasing full-length SMN2 expression.

[0043] In this disclosure, we also demonstrate that combining SMA patient cells with SMN2 saRNA and SMN2 mRNA modulators (e.g., ASO, such as Nusinersen, or pyridazine derivatives including but not limited to Risdiplam and Branaplam) yields significantly higher levels of full-length SMN2 mRNA and SMN protein than either compound alone. This combination therapy strategy can provide enhanced therapeutic benefits compared to monotherapy, such as by improving clinical symptoms in patients diagnosed with SMN deficiency-related disorders, or by reducing undesirable side effects associated with monotherapy, thereby maximizing patient outcomes (e.g., SMA patients).

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0045] In this application, unless otherwise expressly stated in the context, singular forms such as "a" and "this" include plural objects.

[0046] [definition]

[0047] As used herein, the term "SMN deficiency-related disorders" refers to diseases caused by a deficiency of the full-length SMN protein from any cause. "SMN deficiency-related disorders" include, but are not limited to, spinal muscular atrophy (SMA), neurogenic-type arthrogryposis multiplex congenita (congenital AMC), and amyotrophic lateral sclerosis (ALS). For SMN1 (human), the GenBank genetic reference is gene ID: 6606.

[0048] The term "spinal muscular atrophy" or "SMA" includes, but is not limited to, spinal muscular atrophy (SMA) types 1 through 4; proximal spinal muscular atrophy; childhood-onset SMA type I (Werdnig-Hoffmann disease); type II (intermediate, chronic), type III (Kugelberg-Welander disease, or juvenile spinal muscular atrophy); and the relatively newer adult-onset type IV. (Conference report: International Federation of SMA Conference, Neuromuscular Diseases; 2:423-428.) The term SMA also includes late-onset SMA (also known as SMA types 3 and 4, mild SMA, adult-onset SMA, and Kugelberg-Welander disease). The term SMA also includes other forms of SMA, including X-linked disease, spinal muscular atrophy with respiratory distress (SMARD), spinal and bulbar muscular atrophy (Kennedy disease, or bulbar-spinal muscular atrophy), and distal spinal muscular atrophy. The term SMA includes all forms of SMA described in Arnold, WD, Castle, D. & Kissell, JT New Era of Treatment: Diagnosis and Treatment of Spinal Muscles, Muscles and Nerves (2015); and Butchbach, MER Copy number variations of the gene for surviving motor neurons: Relationship with spinal muscular atrophy and other neurodegenerative diseases. Frontiers in Molecular Biosciences (2016).

[0049] When SMA symptoms appear at birth or 6 months of age, the disease is called SMA type 1 (also known as infantile onset or Werdnig-Hoffmann disease). Infants typically have generalized muscle weakness, a weak cry, and respiratory distress. They often have difficulty swallowing and sucking and do not meet developmental milestones such as being able to sit up without being told. These infants have an increased risk of aspiration and developmental delays. Typically, these infants have two or three copies of the SMN2 gene. (Buchbach, Copy number variations in the MER surviving motor neuron gene: Relationship with spinal muscular atrophy and other neurodegenerative diseases. Frontiers in Molecular Biosciences. (2016), which is incorporated herein by reference in its entirety.)

[0050] When SMA develops between 3 and 15 months of age, before the child is able to stand or walk independently, it is called SMA type 2, or intermediate SMA or Dubowitz's disease. Children with SMA type 2 typically have three copies of the SMA2 gene (Arnold, WD, Castle, D. & Kissell, JT New Era of Treatment: Diagnosis and Treatment of Spinal Muscles, Muscles and Nerves (2015), which is incorporated herein by reference in its entirety). Muscle weakness occurs primarily in the proximal (closer to the center of the body), involving the lower limbs more often than the upper limbs. Typically, the facial and eye muscles are unaffected. (Buchbach, Copy number variations in the MER surviving motor neuron gene: Relationship with spinal muscular atrophy and other neurodegenerative diseases. Frontiers in Molecular Biosciences. (2016), which is incorporated herein by reference in its entirety).

[0051] Late-onset SMA (also known as type 3 and 4 SMA, mild SMA, adult-onset SMA, and Kugelberg-Welander disease) causes varying degrees of weakness. Patients with type 3 SMA have 3 to 4 copies of the SMN2 gene. Type 3 SMA (juvenile onset) accounts for 30% of all SMA cases (Arnold, WD, Castle, D. & Kisser, JT New Era of Treatment: Diagnosis and Treatment of Spinal Muscles, Muscles and Nerves (2015)). Symptoms typically appear between 18 months and adulthood. Affected individuals achieve independent mobility. However, proximal weakness in these patients can lead to falls and difficulty climbing stairs. Over time, many lose the ability to stand and walk, thus requiring wheelchairs for mobility. Many of these patients also develop foot deformities, scoliosis, and respiratory muscle weakness.

[0052] Type 4 SMA is late-onset and accounts for less than 5% of all SMA cases. These patients have 4 to 8 copies of the SMN2 gene (Buchbach, Copy number variation of the MER surviving motor neuron gene: its relationship with spinal muscular atrophy and other neurodegenerative diseases. Frontiers in Molecular Biosciences. (2016)). The age of onset is not well-defined, but it is usually after age 30. Type 4 is a mild form of SMA, therefore lifespan remains normal. Patients can achieve motor development milestones and maintain mobility throughout their lives.

[0053] As used herein, the terms "subject" and "individual" are used interchangeably and refer to any living organism that may be treated with the compounds disclosed herein. The term "patient" refers to a human subject or individual, including infants, children, and adults.

[0054] The "therapeutic effective amount" of a component is an amount sufficient to achieve the desired therapeutic effect, and therefore does not require a cure or complete remission. In the embodiments disclosed herein, therapeutic efficacy is the improvement of any disease marker, and the therapeutic effective amount is sufficient to cause a clinically significant improvement in the treated individual's condition / symptoms. The phrases "therapeutic effective amount" and "effective amount" herein mean an amount sufficient to best prevent a clinically significant deficit in the treated individual's activity, function, and responsiveness by at least about 15%, preferably at least 50%, and more preferably at least 90%.

[0055] The effective amount can vary depending on factors such as the subject's body size and weight, disease type, or the specific compound of the present invention. For example, the choice of the compound of the present invention can affect the composition of the "effective amount." Those skilled in the art will be able to study the factors included herein and determine the effective amount of the compound of the present invention without excessive experimentation.

[0056] The administration regimen affects the composition of the effective dose. The compounds of the present invention can be administered to subjects before or after the onset of SMN deficiency-related disorders. Furthermore, several separate doses and staggered doses can be administered daily or sequentially, or the dose can be administered continuously by infusion or as a single bolus injection. Additionally, the dose of one or more compounds of the present invention can be increased or decreased proportionally depending on the urgency of the treatment or prevention situation. The administerable doses of the components of this application can vary within a wide range and will naturally be suitable for individual requirements in each situation.

[0057] As used herein, the terms "treat," "treated," "treating," or "treatment" have the meaning commonly understood in the medical field and therefore do not require a cure or complete remission, and include any beneficial or desired clinical outcome. Non-limiting examples of such beneficial or desired clinical outcomes include prolonged survival or relief of symptoms, including one or more of the following, compared to expected survival without treatment: proximal skeletal muscle weakness and atrophy, inability to sit or walk independently, dysphagia, dyspnea, etc.

[0058] As used in this article, “preventing” or “delaying” disease means inhibiting the full development of the disease.

[0059] The term "biological sample" refers to any tissue, cell, fluid, or other material derived from a living organism (e.g., a human subject). In some embodiments, the biological sample is serum or blood.

[0060] As used in this article, the term "sequence identity" or "sequence homology" means that an oligonucleotide chain (sense or antisense) of saRNA has at least 80% similarity to a region on the coding or template chain of the promoter sequence of the target gene.

[0061] In the embodiments disclosed herein, the target gene is SMN2. "Target sequence" refers to a sequence fragment in the promoter sequence of the target gene that is homologous to or complementary to a sense oligonucleotide chain or antisense oligonucleotide chain of SMN2 saRNA. "Target gene promoter sequence" refers to the non-coding sequence of the target gene. In this specification, "complementary to the promoter sequence of the target gene" refers to the coding strand of that sequence, also known as the non-model strand, i.e., a nucleic acid sequence identical to the gene's coding sequence.

[0062] As used herein, the terms “sense strand” and “sense oligonucleotide strand” are interchangeable, and the sense oligonucleotide strand of a small activating RNA (saRNA) molecule refers to the first nucleic acid strand of the coding strand of the target gene promoter sequence in the double strand containing the saRNA.

[0063] As used herein, the terms “antisense strand” and “antisense oligonucleotide strand” are interchangeable, and the antisense oligonucleotide strand of a saRNA molecule refers to the second nucleic acid strand in the saRNA duplex that is complementary to the sense oligonucleotide strand.

[0064] As used herein, the term "first oligonucleotide chain" can refer to either a sense chain or an antisense chain. The sense chain of a saRNA is an oligonucleotide chain homologous to the coding strand of the promoter DNA sequence of the target gene in the saRNA duplex. The antisense chain is an oligonucleotide chain complementary to the sense chain in the saRNA duplex.

[0065] As used herein, the term "second oligonucleotide chain" can also be a sense chain or an antisense chain. If the first oligonucleotide chain is a sense chain, then the second oligonucleotide chain is an antisense chain; and if the first oligonucleotide chain is an antisense chain, then the second oligonucleotide chain is a sense chain.

[0066] As used herein, the term "promoter" refers to a nucleic acid sequence that does not encode a protein but regulates its transcription by being spatially associated with a nucleic acid sequence that encodes a protein or RNA. Generally, eukaryotic promoters contain 100 to 5,000 base pairs, although this length range is not intended to limit the use of the term "promoter" herein. While promoter sequences are typically located at the 5' end of sequences that encode proteins or RNA, they are also present in exons and introns.

[0067] As used herein, the term "coding strand" refers to the DNA strand of the target gene that cannot be transcribed, whose nucleotide sequence is identical to the transcribed RNA sequence (in the RNA, the T in the DNA is replaced by U). The coding strand of the double-stranded DNA sequence of the target gene promoter described in this disclosure refers to the promoter sequence on the same DNA strand as the coding strand of the target gene.

[0068] As used herein, the term "template strand" refers to the other double-stranded DNA strand of the target gene, which is complementary to the coding strand and can serve as a template for transcription into RNA complementary to the transcribed RNA (AU, GC). During transcription, RNA polymerase binds to the template strand and moves along the 3'→5' direction of the template strand, catalyzing RNA synthesis in the 5'→3' direction. The template strand of the double-stranded DNA sequence of the target gene promoter described in this disclosure refers to the promoter sequence on the same DNA strand as the target gene's DNA template strand.

[0069] As used herein, the term "transcription start site" or TSS refers to the nucleotide on the template strand of a gene that indicates the initiation of transcription. TSS sites can be located on the template strand of a promoter region. Genes can have multiple transcription start sites.

[0070] As used herein, the term "dangling" refers to the 5' or 3' end of an oligonucleotide chain having one or more non-base-paired nucleotides, which are generated by the other strand extending beyond one strand within the double-stranded oligonucleotide. A single-stranded region extending beyond the 3' and / or 5' ends of the double-stranded oligonucleotide is called a dangling. In some embodiments, the length of the dangling end is 0 to 6 nucleotides. It should be understood that a 0-nucleotide dangling means no dangling.

[0071] As used herein, the terms "gene initiation," "initiation of gene expression," "gene upregulation," and "upregulated gene expression" are used interchangeably and refer to an increase or upregulation of the transcription, translation, expression, or activity of a specific nucleic acid sequence, as determined by measuring the transcriptional level, mRNA level, protein level, enzymatic activity, methylation state, chromatin state or conformation, translational level or activity or state of a gene in a cell or biological system. These activities or states can be determined directly or indirectly. Furthermore, "gene initiation" or "initiation of gene expression" refers to an increase in activity associated with a nucleic acid sequence, regardless of the mechanism of this initiation. For example, gene initiation occurs at the transcriptional level to increase transcription into RNA, and the RNA is translated into protein, thereby increasing protein expression.

[0072] As used herein, the terms "small promoter RNA," "saRNA," and "small promoter ribonucleic acid" are used interchangeably to refer to ribonucleic acid molecules capable of upregulating the expression of target genes. These can be double-stranded nucleic acid molecules, consisting of a first nucleic acid strand containing a ribonucleotide sequence that is sequence homologous to the non-coding nucleic acid sequence of the target gene (such as a promoter and enhancer) and a second nucleic acid strand containing a nucleotide sequence complementary to the first strand. saRNA can also consist of synthetic or vector-expressed single-stranded RNA molecules that can form a hairpin structure with two complementary regions within the molecule, wherein the first region contains a ribonucleotide sequence that is sequence homologous to the target sequence of the gene promoter, and the second region contains a ribonucleotide sequence complementary to the first region. The length of the double-stranded region of a saRNA molecule is typically about 10 to about 50, about 12 to about 48, about 14 to about 46, about 16 to about 44, about 18 to about 42, about 20 to about 40, about 22 to about 38, about 24 to about 36, about 26 to about 34, and about 28 to about 32 base pairs, typically about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 base pairs. Additionally, the terms "small initiator RNA," "saRNA," and "small initiator ribonucleic acid" also include nucleic acids other than ribonucleotides, including but not limited to modified nucleotides or similar substances.

[0073] As used in this article, the term "synthetic" refers to the manner in which oligonucleotides are synthesized, including any means that can synthesize or chemically modify ribonucleic acid, such as chemical synthesis, in vitro transcription, vector expression, etc.

[0074] [The composition of a combination of saRNA and mRNA regulators]

[0075] Certain embodiments disclosed herein provide compositions comprising (a) one or more agents that increase the expression of the SMN2 gene or protein and (b) a combination of one or more regulators that increase the SMN2 mRNA splicing or stability of functional SMN2 mRNA production.

[0076] Administering this composition to a patient can treat or delay the onset of SMN deficiency-related disorders such as spinal muscular atrophy. In some embodiments, the composition increases the amount of full-length SMN protein, for example by increasing the amount of full-length SMN2 mRNA through initiation / upregulation of SMN2 transcriptional binding to the splicing of exon 7 inclusions. In some embodiments, the full-length SMN protein is increased in an amount sufficient to alleviate symptoms associated with SMN deficiency-related disorders. In some embodiments, the full-length SMN protein is increased by at least 10%.

[0077] [Agents that increase the expression of the SMN2 gene or protein]

[0078] In some embodiments, at least one of one or more formulations that increase the expression of the SMN2 gene or protein is a saRNA. SMN2 saRNA initiates or upregulates the expression of the SMN2 gene in cells where the SMN2 gene is normally expressed.

[0079] In a typical implementation, the first strand of the SMN2 saRNA contains a 16-35 nucleotide segment with at least 75% sequence identity or sequence complementarity to the promoter region of the SMN2 gene, thereby enabling the initiation or upregulation of gene expression.

[0080] Specifically, the first strand of the SMN2 saRNA has homology or complementarity with regions of the SMN2 gene derived from: the -1639 to -1481 region of the SMN2 promoter gene (SEQ ID NO: 472), the -1090 to -1008 region of the SMN2 gene promoter (SEQ ID NO: 473), the -994 to -180 region of the SMN2 gene promoter (SEQ ID NO: 474), or the -144 to -37 region of the SMN2 gene promoter (SEQ ID NO: 475), and has at least 75%, such as at least about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%, of homology or complementarity. More specifically, one strand of the SMN2 saRNA has at least 75%, for example, at least about 79%, or about 99%, of homology or complementarity with any nucleotide sequence selected from SEQ ID NO: 315-471.

[0081] In this disclosure, the SMN2 saRNA comprises a sense nucleic acid fragment and an antisense nucleic acid fragment. The sense and antisense nucleic acid fragments contain complementary regions capable of forming a double-stranded nucleic acid structure that promotes the expression of the SMN2 gene in cells via an RNA initiation mechanism. The sense and antisense nucleic acid fragments of the saRNA can reside on two different nucleic acid strands or on the same nucleic acid strand. When the sense and antisense nucleic acid fragments reside on two strands, at least one saRNA strand has a 3' overhang of 0-6 nucleotides in length; preferably, both strands have 3' overhangs of 2 or 3 nucleotides in length, and preferably the nucleotide of the overhang is deoxythymidine (dT). When the sense and antisense nucleic acid fragments of the saRNA reside on the same nucleic acid strand, preferably, the saRNA is a single-stranded hairpin nucleic acid molecule, wherein the complementary regions of the sense and antisense nucleic acid fragments form a double-stranded nucleic acid structure. In such saRNAs, the sense and antisense nucleic acid fragments are 16-35 nucleotides in length, and can be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides.

[0082] In one embodiment, the sense strand of the disclosed SMN2 saRNA has at least 75% homology to any nucleotide sequence selected from SEQ ID NO:1-157, for example, at least about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%, and its antisense strand has at least 75% or about 99% homology to any nucleotide sequence selected from SEQ ID NO:158-314. Specifically, the sense strand of the disclosed SMN2 saRNA comprises any nucleotide sequence selected from SEQ ID NO:1-157 or optionally consists of such nucleotide sequences; the antisense strand of the disclosed SMN2 saRNA comprises any nucleotide sequence selected from SEQ ID NO:158-314 or optionally consists of any nucleotide sequence selected from SEQ ID NO:158-314.

[0083] In some embodiments disclosed herein, the SMN2 saRNA comprises a sense nucleic acid strand and an antisense nucleic acid strand, wherein the sense nucleic acid strand includes at least one region that is complementary to at least one region on the antisense nucleic acid strand to form a double-stranded nucleic acid structure capable of initiating SMN2 gene expression in cells.

[0084] In some embodiments disclosed herein, the sense nucleic acid strand and the antisense nucleic acid strand are located on two different nucleic acid strands.

[0085] In some embodiments disclosed herein, sense and antisense nucleic acid fragments are located on the same nucleic acid strand to form a hairpin single-stranded nucleic acid molecule, wherein complementary regions of sense and antisense nucleic acid fragments form a double-stranded nucleic acid structure.

[0086] In some embodiments disclosed herein, at least one nucleic acid strand has a 3' overhang of 0 to 6 nucleotides in length.

[0087] In some embodiments disclosed herein, both nucleic acid strands have 3' overhangs of 2-3 nucleotides in length.

[0088] In some embodiments disclosed herein, the lengths of the sense and antisense nucleic acid strands are 16 to 35 nucleotides, respectively.

[0089] All nucleotides of the SMN2 saRNA described herein may be natural, i.e., non-chemically modified nucleotides, or at least one nucleotide may be a chemically modified nucleotide, wherein the chemical modification is one or a combination of the following modifications:

[0090] (1) Modification of nucleotide phosphodiester bonds in the nucleotide sequence of SMN2 saRNA;

[0091] (2) Modification of the 2'-hydroxyl group of the ribose in the nucleotide sequence of SMN2 saRNA;

[0092] (3) Modification of the bases in the nucleotide sequence of SMN2 saRNA.

[0093] The chemical modifications of nucleotides or saRNA disclosed herein are well known to those skilled in the art. The modification of the phosphodiester bond refers to the modification of oxygen in the phosphodiester bond, including thiophosphate modification and borophosphate modification. Both modifications stabilize the SMN2 saRNA structure, maintaining high specificity and high affinity for base pairing.

[0094] Ribose modification refers to the modification of the 2'-OH group in the pentose of nucleotides, that is, the introduction of certain substituents at the hydroxyl position of the ribose, such as 2'-fluorine modification, 2'-side oxymethyl modification, 2'-oxyethylene methoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid (LNA), 2'-amino modification, and 2'-deoxy modification.

[0095] Base modification refers to the modification of nucleotide bases, such as 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, and 2,6-diaminopurine modification.

[0096] These modifications can increase the bioavailability of SMN2 saRNA, increase affinity for target sequences, and enhance resistance to nuclease hydrolysis in cells.

[0097] In addition, to promote the entry of SMN2 saRNA into cells, lipophilic groups such as cholesterol can be introduced at the ends of the sense or antisense strands of SMN2 saRNA, based on the above modifications, to promote the action of gene promoter regions in the cell membrane, nuclear membrane, and nucleus, which are composed of lipid bilayers.

[0098] The SMN2 saRNA disclosed herein effectively initiates or upregulates the expression of the SMN2 gene in cells upon contact, with a preferred upregulation of at least 10%.

[0099] One aspect of this disclosure provides a cell comprising the SMN2 saRNA disclosed herein or a nucleic acid encoding the SMN2 saRNA disclosed herein. In one embodiment, the cell is a mammalian cell, preferably a human cell. Such cells may be ex vivo, such as a cell line or cell line, or may be present in a mammal, such as a human (including infants, children, or adults).

[0100] Another aspect of the invention provides a pharmaceutical composition comprising the above-described SMN2 saRNA or nucleic acid encoding the SMN2 saRNA according to the invention, an SMN2 mRNA regulator, and one or more pharmaceutically acceptable carriers. In one embodiment, the pharmaceutically acceptable carrier comprises one or more of an aqueous carrier, liposomes, polymers, and peptides. In one embodiment, the pharmaceutically acceptable carrier may be, for example, RNase-free water or an RNase-free buffer. The composition may contain 1-150 nM, for example, 1-100 nM, for example, 1-50 nM, for example, 1-20 nM, for example, 10-100 nM, 10-50 nM, 20-50 nM, 20-100 nM, for example, 50 nM of the above-described SMN2 saRNA according to the invention or nucleic acid encoding the SMN2 saRNA.

[0101] Another aspect of this disclosure relates to the use of the SMN2 saRNA described herein, the nucleic acid encoding SMN2 saRNA described herein, or a composition containing such SMN2 saRNA or the nucleic acid encoding SMN2 saRNA described herein, in combination with an SMN2 mRNA regulator for the preparation of one or more compositions for increasing the amount of full-length SMN protein expressed in cells.

[0102] On the other hand, the present invention provides an isolated SMN2 gene saRNA targeting site having any consecutive 16-35 nucleotide sequence in the promoter region of the SMN2 gene, preferably selected from any consecutive 16-35 nucleotide sequence of any one of the nucleotide sequences in SEQ ID NO:472-475. In particular, the target site comprises or is selected from the sequence shown in any nucleotide sequence in SEQ ID NO:315-471.

[0103] Another embodiment provides pharmaceutical compositions or medicaments comprising the compounds of the present invention and a therapeutically inert carrier, diluent, or pharmaceutically acceptable excipient, and methods for preparing such compositions and medicaments using the compounds of the present invention. In some embodiments, the SMN2 saRNA and SMN2 mRNA regulator of the present invention are present in separate pharmaceutical compositions. In other embodiments, the SMN2 saRNA and SMN2 mRNA regulator are present in the same pharmaceutical composition.

[0104] The components disclosed herein are formulated, administered, and applied in accordance with good medical practice. Factors to be considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of delivery of the formulation, the method of administration, the timing of administration, and other factors known to the physician.

[0105] Compositions containing any small molecule compounds described herein, such as Risdiplam or Branaplam, may be administered separately from the SMN2 saRNA composition by any suitable method, including oral, topical (including oral and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal, epidural, and intranasal administration. Intralesional administration may also be performed if local treatment is desired. For the SMN2 saRNA composition, delivery may be by parenteral infusion, including intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, administration of the compositions of the present invention may optionally be by parenteral infusion, including intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intraventricular, intravitreal, or subcutaneous administration; or by oral, intranasal, inhalation, vaginal, or rectal administration.

[0106] The small molecule compounds described herein, such as Risdiplam and Branaplam, can be administered in any convenient form, such as tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain components common to pharmaceutical preparations, such as diluents, carriers, pH adjusters, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for altering osmotic pressure, buffers, masking agents, antioxidants, and other active agents. Such compositions may also contain other substances with therapeutic value.

[0107] Typical formulations are prepared by mixing the compounds of the present invention with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail, for example, in Ansel HC et al., Ansel’s Pharmaceutical Dosage Forms and Delivery Systems (2004), Lippincott, Williams and Wilkins, Philadelphia; Gennaro AR et al., Remington: The Science and Practice of Pharmaceuticals (2000), Lippincott, Williams and Wilkins, Philadelphia; and Roe RC, Handbook of Pharmaceutical Excipients (2005), Pharmaceutical Press, Chicago. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, flow aids, processing aids, colorants, sweeteners, flavoring agents, diluents and other known additives to provide an elegant appearance for the drug (i.e., the compounds of the present invention or their pharmaceutical components) or to facilitate the manufacture of the pharmaceutical product (i.e., the drug).

[0108] On the other hand, the present invention provides use of combinations or compositions of any embodiments described herein in the preparation of a medicament for treating an individual with SMN deficiency-related disorders. According to certain embodiments of the use, SMN deficiency-related disorders include inherited neuromuscular diseases, preferably spinal muscular atrophy. Use according to certain embodiments is also provided where the individual is a mammal, preferably a human.

[0109] [SMN2 mRNA regulator]

[0110] As used herein, the term "SMN2 mRNA regulator" refers to regulators of SMN2 mRNA splicing or stability that increase the production of functional SMN2 mRNA and functional SMN protein. The term "SMN2 mRNA regulator" includes formulations that, for example, alter the splicing pattern of SMN2 pre-mRNA by blocking the action of the intron repressive splice region of intron 7 of the SMN2 gene to contain all the information required to produce a functional full-length SMN protein. SMN2 mRNA regulators include those that increase the desired splicing and subsequent protein production by stabilizing the interaction between the spliceosome and SMN2 pre-mRNA (Journal of Medicinal Chemistry, Dec 27, 2018; 61(24):11021-11036), and those that enhance the stability of the transient double-stranded RNA structure formed by the SMN2 pre-mRNA and U1 small nucleoribonucleoprotein (snRNP) complex (Natural Chemical Biology, July 2015; 11(7):511-7). In some instances, SMN2 mRNA regulators modulate the splicing of SMN2 pre-mRNA to include exon 7 in the processed transcript. Alternatively, the SMN2 mRNA regulators disclosed herein include agents capable of increasing the level of functional SMN protein by preventing exon 7 from being spliced ​​out of mature SMN mRNA during splicing. SMN2 mRNA modulators according to this disclosure also include those described in U.S. Patent 10,436,802 and U.S. Patent 10,420,753 (the entire contents of each of these are incorporated herein by reference).

[0111] Examples of SMN2 mRNA regulators disclosed herein include pyridazine derivatives, such as those described in WO2014028459A1 (the entire contents of which are incorporated herein by reference). Specific examples of SMN2 mRNA regulators include Branaplam (also known as LMI070) and Risdiplam (also known as RG7916 or RO7034067).

[0112]

[0113] Other examples of SMN2 mRNA regulators according to this disclosure include antisense oligonucleotides, such as those capable of antisense targeting, substituting, and / or disrupting intron sequences in the SMN2 gene to increase the production of full-length SMN2 (SMN2FL) transcripts (transcripts containing exon 7) during splicing. In some embodiments, Nusinersen, marketed as Spinraza®, is suitable for use in combination according to the disclosure.

[0114] Treatment methods for SMA and related disorders

[0115] Another aspect of the present invention relates to a method for treating or delaying the onset of an individual's SMN deficiency-related disease, the method comprising administering to the individual a therapeutically effective amount of the SMN2 saRNA described herein, a nucleic acid encoding the SMN2 saRNA described herein, or a composition comprising the SMN2 saRNA of the present invention or a nucleic acid encoding the SMN2 saRNA described herein. The subject may be a mammal, such as a human. The subject may be an infant, a child, or an adult. In one embodiment, the disease caused by insufficient expression of the full-length SMN protein or a mutation in the SMN1 gene may include, for example, SMA. In one embodiment, the disease caused by insufficient expression of the full-length SMN protein, a mutation or deletion of the SMN1 gene, and / or insufficient expression of the full-length SMN protein is SMA. In one embodiment, the SMA of the present invention includes SMA type I, SMA type II, SMA type III, and SMA type IV.

[0116] Another aspect of the invention relates to the use of a combination of the disclosed SMN2 saRNA, a nucleic acid encoding the disclosed SMN2 saRNA, or a composition comprising the disclosed SMN2 saRNA or a nucleic acid encoding the disclosed SMN2 saRNA, and a disclosed SMN2 mRNA regulator in the preparation of a drug for treating or delaying the onset of SMN deficiency-related disorders. The subject may be a mammal, such as a human. The subject may be an infant, child, or adult. In one embodiment, the disorder associated with SMN deficiency may include, for example, SMA. In one embodiment, the SMA of the present invention includes SMA type I, SMA type II, SMA type III, and SMA type IV.

[0117] Also provided is the use of any combination of SMN2 saRNA and SMN2 mRNA regulators as described herein, or the use of a composition of any combination of SMN2 saRNA and SMN2 mRNA regulators as described herein in the preparation of a formulation for increasing the amount of full-length SMN protein in cells. In some embodiments, the cells are mammalian cells, preferably human cells. In some embodiments, the cells are present in a human body. In some embodiments, the person is a patient suffering from symptoms caused by an SMN deficiency-related disorder. In some embodiments, the combination or a composition thereof is administered in an amount effective in treating an SMN deficiency-related disorder. In some embodiments, the symptoms caused by an SMN deficiency-related disorder are symptoms associated with a hereditary neuromuscular disease, preferably spinal muscular atrophy.

[0118] In some embodiments, the combination of SMN2 saRNA and an SMN2 mRNA modulator achieves an increase in full-length SMN protein greater than that achieved by administering the same amount of either substance alone, with reduced toxicity or unwanted side effects. In some embodiments, the combination of SMN2 saRNA and an SMN2 mRNA modulator achieves an increase in full-length SMN protein greater than the additive effect of treatment with the same amount of either substance alone. In some embodiments, when used in the embodiments described herein, the amount of SMN2 saRNA or SMN2 mRNA modulator administered is less than that used for general treatment.

[0119] In some implementations, the combination of SMN2 saRNA and the SMN2 mRNA modulator achieved greater clinical improvement than the combined effect of using the same amount of either substance alone.

[0120] This disclosure also relates to a method for increasing the amount of full-length SMN protein in cells, comprising administering to cells a combination of 1) an SMN2 mRNA regulator and 2) at least one of the following: the SMN2 saRNA described herein, a nucleic acid encoding the SMN2 saRNA described herein, or a composition containing the SMN2 saRNA described herein or a nucleic acid encoding the SMN2 saRNA.

[0121] In any of the embodiments provided herein, such SMN2 saRNA, nucleic acid encoding the SMN2 saRNA disclosed herein, or a composition comprising such SMN2 saRNA or nucleic acid encoding the SMN2 saRNA disclosed herein may be directly introduced into cells, or may be generated intracellularly after the nucleotide sequence encoding SMN2 saRNA is introduced into cells, preferably mammalian cells, more preferably human cells. Such cells may be ex vivo, such as cell lines, or may be present in mammals (such as humans). In some embodiments, the person is a patient or individual suffering from an SMN deficiency-related disorder. In some embodiments, a composition comprising nucleic acid encoding SMN2 saRNA or nucleic acid comprising the aforementioned saRNA or SMN2 saRNA encoding the present invention is combined with a composition comprising at least one SMN2 mRNA regulator, in amounts sufficient to treat the SMN deficiency-related disorder. In one embodiment, the SMN deficiency-related disorder is SMA. In one embodiment, the SMA disclosed herein includes SMA type I, SMA type II, SMA type III, and SMA type IV.

[0122] In some embodiments, the combination of SMN2 saRNA and an SMN2 mRNA modulator achieves an increase in full-length SMN protein greater than that achieved by administering the same amount of either substance alone. In some embodiments, the combination of SMN2 saRNA and an SMN2 mRNA modulator has reduced toxicity and / or reduced undesirable side effects compared to treatment with monotherapy. In some embodiments, the combination of SMN2 saRNA and an SMN2 mRNA modulator achieves an increase in full-length SMN protein greater than the additive effect of treatment with the same amount of either substance alone. In some embodiments, either or both of SMN2 saRNA and the SMN2 mRNA modulator are administered in amounts less than those typically used for monotherapy.

[0123] In some implementations, the combination of SMN2 saRNA and SMN2 mRNA modulators achieved greater clinical improvement than the effect of using the same amount of either substance alone. In some implementations, the combination of SMN2 saRNA and SMN2 mRNA modulators achieved greater than the additive clinical improvement compared to the effect of using the same amount of either substance alone.

[0124] In some embodiments, baseline measurements are obtained from biological samples, as defined herein, and are obtained from the individual prior to administration of the therapy described herein. In some embodiments, the biological samples are peripheral blood mononuclear cells, plasma, serum, skin tissue, or cerebrospinal fluid (CSF). In some embodiments, elevated SMN protein levels in peripheral blood mononuclear cells and skin are correlated with SMN protein levels in central nervous system (CNS) neurons, suggesting that changes in these levels in blood or skin can be used as a non-invasive alternative to determine changes in SMN protein levels in the CNS. In yet another embodiment, the combination provided herein increases the amount of full-length SMN protein by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, and at least 195% compared to baseline measurements. At least 200%, at least 210%, at least 215%, at least 220%, at least 225%, at least 230%, at least 235%, at least 240%, at least 245%, at least 250%, at least 255%, at least 260%, at least 265%, at least 270%, at least 275%, at least 280%, at least 285%, at least 290%, at least 295%, at least 300%, at least 310%, at least 315%, at least 320%, at least 325%, at least 330%, at least 335%, at least 340%, at least 345%, at least 350%, at least 355%, at least 360%, at least 365%, at least 370%, at least 375%, at least 380%, at least 385%, at least 390%, at least 395%, at least 400%.

[0125] In this disclosure, the term "co-administration" of one or more SMN2 saRNAs and one or more SMN2 mRNA regulators can be simultaneous (i.e., within 15 minutes, 30 minutes, or 1 hour), nearly simultaneous (i.e., within 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, or 24 hours), or delayed by several days or weeks, for example, for up to 4 or 5 weeks.

[0126] In this disclosure, the term "co-administration" can refer to simultaneous or concurrent administration (i.e., within 15 minutes, 30 minutes, or 1 hour), nearly simultaneous or substantially simultaneous administration (i.e., within 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, or 24 hours), or delayed by several days or weeks, for example, by up to 4 or 5 weeks.

[0127] The dosage of the components disclosed herein can be varied within a wide range of limitations and will certainly be suitable for individual requirements in each case.

[0128] In one particular implementation, the combination of SMN2 saRNA and SMN2 mRNA regulators showed greater than additive effects or synergistic effects in treating, preventing, delaying the progression and / or improving diseases caused by inactivation mutations or deletions of the SMN1 gene and / or associated with loss or defect of SMN1 gene function, as well as in protecting cells associated with the pathophysiology of the disease, particularly in treating, preventing, delaying the progression of spinal muscular atrophy (SMA) and / or improving SMA.

[0129] In some implementations, a first dose of the pharmaceutical composition according to this disclosure is administered when the subject is less than 1 week, less than 1 month, less than 3 months, less than 6 months, less than 1 year, less than 2 years, less than 15 years, or greater than 15 years old.

[0130] In some embodiments, at least one pharmaceutical composition containing SMN2 saRNA and at least one other pharmaceutical composition containing an SMN2 mRNA regulator are administered simultaneously, nearly simultaneously, or co-administered at different times. In some embodiments, the pharmaceutical composition containing an SMN2 mRNA regulator and the pharmaceutical composition containing SMN2 saRNA are co-administered within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, within 11 hours, within 12 hours, within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, or within 5 weeks. A single dose can be SMN2 saRNA, and can be a single dose of 0.1 to 15 mg, a single dose of 1 mg, a single dose of 2 mg, a single dose of 3 mg, a single dose of 4 mg, a single dose of 5 mg, a single dose of 6 mg, a single dose of 7 mg, a single dose of 8 mg, a single dose of 9 mg, a single dose of 10 mg, a single dose of 11 mg, a single dose of 12 mg, a single dose of 13 mg, a single dose of 14 mg, or a single dose of 15 mg. A single dose can be an SMN2 mRNA regulator, and can be a single dose of 0.1 to 15 mg, a single dose of 1 mg, a single dose of 2 mg, a single dose of 3 mg, a single dose of 4 mg, a single dose of 5 mg, a single dose of 6 mg, a single dose of 7 mg, a single dose of 8 mg, a single dose of 9 mg, a single dose of 10 mg, a single dose of 11 mg, a single dose of 12 mg, a single dose of 13 mg, a single dose of 14 mg, or a single dose of 15 mg.

[0131] In some embodiments, a single 4.8 mg dose of the SMN2 mRNA modulator is ASO, administered via lumbar puncture as an intrathecal injection. In some embodiments, the SMN2 mRNA modulator is Nusinersen. In some embodiments, the dose may be a single dose of 5.16 mg, 5.40 mg, 7.2 mg, 7.74 mg, 8.10 mg, 9.6 mg, 10.32 mg, 10.80 mg, 11.30 mg, 12 mg, 12.88 mg, 13.5 mg, 14.13 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg.

[0132] In some embodiments, when administering a dose of SMN2 saRNA and / or SMN2 mRNA via intrathecal injection through lumbar puncture, using a smaller gauge needle may reduce or improve one or more symptoms associated with the lumbar puncture procedure. In some embodiments, symptoms associated with lumbar puncture include, but are not limited to, post-lumbar puncture syndrome, headache, back pain, fever, constipation, nausea, vomiting, and puncture site pain. In some embodiments, using a 24-gauge or 25-gauge needle for lumbar puncture reduces or improves one or more post-lumbar puncture symptoms. In some embodiments, using a 21-gauge, 22-gauge, 23-gauge, 24-gauge, or 25-gauge needle for lumbar puncture may reduce or improve post-lumbar puncture syndrome, headache, back pain, fever, constipation, nausea, vomiting, and / or puncture site pain.

[0133] The recommended dosing frequencies are approximate. For example, in some embodiments, if the recommended dosing frequency is a dose on day 1 and a second dose on day 29, then SMA patients may receive a second dose 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 days after receiving the first dose. In some embodiments, if the recommended dosing frequency is a dose on day 1 and a second dose on day 15, then SMA patients may receive a second dose 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after receiving the first dose. In some embodiments, if the recommended dosing frequency is a dose on day 1 and a second dose on day 85, then SMA patients may receive a second dose 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 days after receiving the first dose.

[0134] In some implementations, the dose and / or volume of the injection will be adjusted based on the patient’s age, the patient’s CSF volume, or the patient’s age and / or estimated CSF volume. (See, for example, Matsuzawa J, Matsui M, Kawai T, Noguchi K, Gur RC, Birk W, Miyazaki T. Age-related volume changes of gray and white matter in healthy infants. Cerebral Cortex 2001 Apr; 11(4):335-342, which is incorporated herein by reference in its entirety.)

[0135] [Example]

[0136] The invention is further illustrated by the following embodiments. These embodiments are provided for illustrative purposes only and should not be construed as limiting the scope or content of the invention in any way.

[0137] [Example 1: Effects of saRNA (DS06-0004), ASO (Nusinersen), and Risdiplam on the expression of full-length and exon-skipping SMN2 mRNA in GM03813 cells.]

[0138] To determine the optimal concentrations of saRNA (DS06-0004), Nusinersen (ASO-10-27), and Risdiplam for cell treatment, saRNA and ASO were transfected into GM03813 cells at different concentrations. Risdiplam was dissolved in DMSO and added to cultured GM03813 cells at different concentrations.

[0139] "Gm03813 cells" refers to fibroblasts provided by Coreell Medical Research Institute. This cell line is described as having spinal muscular atrophy, type II; motor neuron 1 with SMA2 survival, telomeres; SMN1. The associated gene is SMN1; the chromosomal location is 5q12.2-q13.3, and the allelic variant is described as deletion of exons 7 and 8 in exon 1, spinal muscular atrophy, type I; the identified mutation is EX7-8DEL. Phenotypic data from fibroblasts derived from the skin (arm) of the following subject, characterized as: clinically affected; born at full term without complications; rolling over at 6 months of age; babbling at 9 months of age; marked muscle atrophy and weakness by 12 months of age; lack of deep tendon reflexes; constipation; the donor subject had 3 copies of the SMN2 gene; PCR analysis showed that the donor subject's deletion of exons 7 and 8 of the SMN1 gene was homozygous; a similarly affected brother (not in the repository); the mother was GM03814 (fibroblast) / GM24474 (iPSC); the father was GM03815 (fibroblast); see also GM23240 (iPSC-lentivirus) and GM24468 (iPSC-episode); previously classified as SMA I, but data from the proband, such as onset characteristics and SMN2 dosage, support a reclassification to SMA II.

[0140] After 72 hours, total cellular RNA was isolated from the treated cells and reverse transcribed into cDNA. SMN2 mRNA expression was assessed by RT-qPCR using primer pairs specific to SMN2FL or SMN2Δ7. SMN2 mRNA expression was also assessed by semi-quantitative RT-PCR using primers amplifying both SMN2FL and SMN2Δ7, followed by DdeI digestion (PCR / digestion). PCR produced two bands: 507 bp (SMN2FL) and 453 bp (SMN2Δ7). After digestion, both bands decreased by 115 bp, producing two products: 392 bp (SMN2FL) and 338 bp (SMN2Δ7), as shown on the gel in Figure 3D. Figures 3A through 3D show the dose-dependent changes in SMN2FL and SMNΔ7 mRNA assessed by RT-qPCR and PCR / digestion, respectively. Figures 3E through 3G are dot plots of data from the quantitative band intensities in Figure 3D.

[0141] As shown in Figure 3A, treatment with 1 nM ASO-10-27 increased SMN2FL by 1.5-fold, and at 5 nM, it caused a 2.0-fold increase in peak expression, while decreasing SMN2Δ7. Higher doses did not induce further SMN2FL expression or decrease SMN2Δ7 expression. Similarly, PCR / digestion analysis showed that SMN2FL expression reached its peak when cells were treated with 10 nM ASO, while SMNΔ7 expression was almost at its lowest value at 5 nM (Figure 3E). Treatment with 100 nM and 1000 nM Risdiplam increased SMN2FL mRNA levels by 1.2-fold and 1.8-fold, respectively, and decreased SMN2Δ7 by 36% and 98%, respectively (Figures 3C and 3G). Since SMN2 mRNA regulators, including ASO-10-27 and Risdiplam, increase SMN2FL mRNA by regulating SMN2 splicing to include more exon 7, the maximum amount of SMN2FL they can induce depends on the amount of SMN2 pre-mRNA available, which is not altered by SMN2 mRNA regulators. Consistent with this view, data show a ceiling effect of ASO-10-27 and Risdiplam on splice regulator-induced increases in SMN2FL (maximum increase of approximately 2-fold).

[0142] In contrast, saRNA (DS06-0004) induced SMN2FL and SMN2Δ7 expression to levels higher than those of the SMN2 mRNA regulator, and induced expression in a dose-dependent manner across a concentration range of 1 nM to 50 nM, with maximum fold changes of 2.9-fold and 2.7-fold, respectively. 100 nM of DS06-0004 did not further increase SMN2 mRNA expression (Fig. 3B and Fig. 3F). Consistent results were obtained by PCR / digestion analysis (Fig. 3D and Fig. 3F).

[0143] Unlike SMN2 mRNA regulators (ASO-10-27 and Risdiplam) that increase SMN2FL levels by transforming (lowering) SMN2Δ7 levels, the SMN2 saRNA disclosed here increases SMN2 mRNA levels by acting on SMN2 transcription, resulting in a simultaneous increase in both SMN2FL and SMN2Δ7. The data shown in Figure 3 clearly demonstrate the mechanistic differences between the SMN2 mRNA regulators and the SMN2 saRNA.

[0144] [Example 2: Combined effect of saRNA (DS06-0004) and ASO-10-27 on the expression of full-length SMN2 mRNA and SMN2 mRNA skipping exon 7 in GM00232 cells.]

[0145] To determine whether the combination of saRNA (DS06-0004) and ASO-10-27 enhances the induction of SMN2FL in type I SMA cells, GM00232 cells were transfected with DS06-0004 and ASO-10-27 alone or in different concentrations for 72 hours. The expression of SMN2 in the treated cells was assessed by RT-qPCR (Fig. 4A and 4D) and PCR / digestion (Fig. 4B, 4C, and 4E).

[0146] "GM00232 cells" refers to fibroblasts provided by Coreell Medical Research Institute. This cell line is described as spinal muscular atrophy type I (SMA1). The donor subject has two copies of the SMN2 gene (data from multiple sources, including Stabley et al. 2015, PMID 26247043) and is homozygous for the deletion of exons 7 and 8 of the SMN1 gene. The relevant gene is SMN1; the chromosomal location is 5q12.2-q13.3; the allele variant is described as deletion of exons 7 and 8; spinal muscular atrophy, type I; and the identified mutation is EX7-8DEL. Phenotypic data were derived from skin (arm) fibroblasts of the following subjects, characterized by progressive muscle atrophy; lack of deep tendon reflexes; abnormal EMG; and the donor subject had two copies of the SMN2 gene (data from several sources, including Stabley et al. 2015, PMID 26247043) and was homozygous for the deletion of exons 7 and 8 of the SMN1 gene.

[0147] As shown in Figure 4A, 1 nM, 5 nM, and 25 nM ASO-10-27 lead to an increase of SMN2FL by 1.3 times, 1.8 times, and 1.9 times, respectively, while decreasing SMN2Δ7. 1 nM, 5 nM, and 25 nM DS06-0004 increase SMN2FL by 1.7 times, 2.4 times, and 2.4 times, respectively, and increase SMN2Δ7 by 1.5 times, 1.9 times, and 2.1 times, respectively.

[0148] When 1 nM ASO-10-27 was combined with increasing concentrations of DS06-0004 (1 nM, 5 nM, and 25 nM) for cell transfection, SMN2FL was induced by 2.2-fold, 2.6-fold, and 2.9-fold, respectively, while SMN2Δ7 was altered by 1.1-fold, 0.7-fold, and 0.4-fold, respectively. Furthermore, treatment of cells with 5 nM ASO-10-27 combined with increasing concentrations of DS06-0004 (1 nM, 5 nM, and 25 nM) induced an increase in SMN2FL by 2.8-fold, 3.4-fold, and 3.7-fold, respectively, and altered SMN2Δ7 by 0.09-fold, 0.05-fold, and 0.04-fold, respectively. Furthermore, treatment of cells with a combination of 25 nM ASO-10-27 and increasing concentrations of DS06-0004 (1 nM, 5 nM, and 25 nM) induced SMN2FL increases of 3.1-fold, 4.0-fold, and 4.0-fold, respectively, and completely eliminated SMN2Δ7 expression. Compared with 25 nM ASO-10-27 alone (which induced a 1.9-fold increase in SMN2FL), the combined treatment of ASO-10-27 and DS06-0004 increased SMN2FL by 4-fold, twice the effect of ASO-10-27 alone.

[0149] The RT-qPCR results shown in Figure 4A further validated the PCR / DdeI digestion. Consistent with the RT-qPCR results, ASO-10-27 alone at 25 nM induced a 2.3-fold increase in SMN2FL mRNA, while the combination of ASO-10-27 (25 nM) and DS06-0004 (25 nM) resulted in the highest induction of SMN2FL (4.1-fold) and a simultaneous decrease of SMN2Δ7 (0.14-fold) (Figures 4B, 4C, and 4E).

[0150] The data shown in Figure 4 collectively demonstrate that the saRNA DS06-0004 alone has strong activity in inducing SMN2 mRNA expression, especially SMN2FL expression in type I SMA cells with two copies of the SMN2 gene. Maximum SMN2FL induction can be achieved when the SMN2 saRNA is combined with ASO-10-27.

[0151] [Example 3: Combined effect of saRNA (DS06-0004) and ASO-10-27 on SMN protein levels in GM00232 cells.]

[0152] To further verify the effects of ASO-10-27 and DS06-0004, alone or in combination, on SMN2 gene expression, Western blot analysis was performed in GM00232 cells transfected alone or in combination with ASO-10-27 and DS06-0004. As shown in Figures 5A and 5C, 1 nM, 5 nM, and 25 nM of ASO-10-27 resulted in a 1.4-fold, 2.3-fold, and 2.9-fold increase in SMN protein, respectively. 1 nM, 5 nM, and 25 nM of DS06-0004 increased SMN2FL by 1.2-fold, 1.3-fold, and 1.7-fold, respectively (Figures 5B and 5C). The expected protein band of 35 kDa is the full-length SMN protein (Fig. 5A and Fig. 5B), while the SMNΔ7 protein does not appear on the protein blot because it is rapidly degraded (Li, TT et al. SMNΔ7 is the major product of the centromere survival motor neuron (SMN2) gene, which prolongs the survival time of mice with spinal muscular atrophy and is associated with the full-length SMN. Human Molecular Genetics (2005).

[0153] Cells treated with a combination of 1 nM ASO-10-27 and DS06-0004 at increasing concentrations (1 nM, 5 nM, and 25 nM) induced SMN protein levels of 2.4, 2.6, and 2.9 times, respectively (Figs. 5A to 5C, 5A, and 5B contain replicates of the combined treatments).

[0154] Cells were further treated with a combination of 5 nM ASO-10-27 and DS06-0004 at increasing concentrations (1 nM, 5 nM and 25 nM) to induce SMN protein levels of 3.1-fold, 3.0-fold and 3.3-fold, respectively (Figures 5A to 5C, Figures 5A and 5B contain replicates of the combined treatment).

[0155] In addition, treatment of cells with a combination of 25 nM ASO-10-27 and DS06-0004 at increasing concentrations (1 nM, 5 nM and 25 nM) induced SMN protein levels of 3.1-fold, 3.3-fold and 2.6-fold, respectively (Figures 5A to 5C, Figures 5A and 5B contain replicates of the combined treatment).

[0156] These data collectively confirm that using ASO-10-27 in combination with DS06-0004 induces higher levels of SMN protein than using either one alone.

[0157] [Example 4: Combined effect of saRNA (DS06-0004) and ASO-10-27 on the expression of full-length SMN2 mRNA and SMN2 mRNA skipping exon 7 in GM03813 cells.]

[0158] To determine whether the combination of saRNA (DS06-0004) and ASO-10-27 enhanced the induction of SMN2FL in type II SMA cells, GM03813 cells were transfected with DS06-0004 and ASO-10-27 alone or in different concentrations for 72 hours. The expression of SMN2 in the treated cells was assessed by RT-qPCR (Fig. 6A and Fig. 6D) and PCR / digestion (Fig. 6B, Fig. 6C, and Fig. 6E). As shown in Fig. 6A and Fig. 6D, 1 nM, 5 nM, and 25 nM of ASO-10-27 led to a 1.2-fold, 2.1-fold, and 2.1-fold increase in SMN2FL, respectively, while simultaneously reducing SMN2Δ7. DS06-0004 at 1 nM, 5 nM, and 25 nM increased SMN2FL by 2.1, 2.6, and 2.2 times, respectively, and increased SMN2Δ7 by 2.5, 2.5, and 2.1 times, respectively.

[0159] As shown in Figures 6A and 6D, treatment of cells with a combination of 1 nM ASO-10-27 and increasing concentrations of DS06-0004 (1 nM, 5 nM, and 25 nM) induced SMN2FL to increase by 2.6-fold, 2.8-fold, and 3.0-fold, respectively, and SMN2Δ7 to increase by 1.7-fold, 1.4-fold, and 0.8-fold, respectively. Furthermore, treatment of cells with a combination of 5 nM ASO-10-27 and increasing concentrations of DS06-0004 (1 nM, 5 nM, and 25 nM) induced SMN2FL to increase by 3.3-fold, 4.2-fold, and 4.8-fold, respectively, and SMN2Δ7 to increase by 0.2-fold, 0.2-fold, and 0.1-fold, respectively. Furthermore, treatment of cells with a combination of 25 nM ASO-10-27 and increasing concentrations of DS06-0004 (1 nM, 5 nM, and 25 nM) induced SMN2FL increases of 3.8-fold, 4.7-fold, and 4.0-fold, respectively, and completely eliminated SMN2Δ7 expression. Compared with treatment with 25 nM ASO-10-27 alone (which induced a 2.1-fold increase in SMN2FL), the combination treatment with ASO-10-27 and DS06-0004 increased SMN2FL by 4.7-fold, more than twice the effect of ASO-10-27 alone.

[0160] The RT-qPCR results shown in Figure 6A were further validated by semi-quantitative RT-PCR followed by DdeI digestion. Consistent with the RT-qPCR results, ASO-10-27 alone at 25 nM induced a 2.1-fold increase in SMN2FL mRNA, while the combination of ASO-10-27 (25 nM) and DS06-0004 (5 nM) resulted in the highest induction of SMN2FL (2.7-fold) and a simultaneous decrease of SMN2Δ7 (0.18-fold) (Figures 6B, 6C, and 6E).

[0161] The data shown in Figure 6 collectively demonstrate that the SMN2 saRNA DS06-0004 alone is highly active in inducing SMN2 mRNA expression, especially SMN2FL, in type II SMA cells containing three copies of the SMN2 gene. Maximum SMN2FL induction was achieved when the SMN2 saRNA was combined with ASO-10-27. This data confirms that the same type II SMA cells induced by either agent alone (…) Compared to SMN protein levels in GM03813 cells, the combination of ASO-10-27 and DS06-0004 induced higher levels of SMN protein in type II SMA cells (GM03813 cells). Compared to the [GM03813] cell population, this data also determined the levels of SMN protein induced in cells treated with the combination according to this disclosure. As described herein, GM03813 cells have two copies of SMN2 and were used as a model of SMA.

[0162] [Example 5: Combined effect of saRNA (DS06-0004) and ASO-10-27 on SMN protein levels in GM03813 cells.]

[0163] To further verify the effects of ASO-10-27 and DS06-0004 alone or in combination on SMN2 gene expression, Western blot analysis was performed on GM03813 cells transfected alone or in combination with ASO-10-27 and DS06-0004. As shown in Figures 7A and 7C, 1 nM, 5 nM, and 25 nM of ASO-10-27 resulted in a 1.2-fold, 1.5-fold, and 1.9-fold increase in SMN protein, respectively. 1 nM, 5 nM, and 25 nM of DS06-0004 increased SMN2FL by 1.5-fold, 1.5-fold, and 1.6-fold, respectively (Figures 7B and 7C).

[0164] Cells treated with a combination of 1 nM ASO-10-27 and increasing concentrations of DS06-0004 (1 nM, 5 nM, and 25 nM) induced SMN protein levels of 1.4, 1.6, and 1.8 times, respectively (Figures 7A to 7C, Figures 7A and 7B contain replicates of the combined treatments).

[0165] Further treatment of cells with a combination of 5 nM ASO-10-27 and DS06-0004 at increasing concentrations (1 nM, 5 nM and 25 nM) increased SMN protein by 2.0-fold, 2.2-fold and 2.4-fold, respectively (Figures 7A to 7C, Figures 7A and 7B contain replicates of the combined treatment).

[0166] In addition, treatment of cells with a combination of 25 nM ASO-10-27 and DS06-0004 at increasing concentrations (1 nM, 5 nM and 25 nM) increased SMN protein by 2.2-fold, 2.9-fold and 2.8-fold, respectively (Figures 7A to 7C, Figures 7A and 7B contain replicates of the combined treatment).

[0167] These data collectively confirm that the combination of ASO-10-27 and DS06-0004 induced higher levels of SMN protein in type II SMA cells. Specifically, the data demonstrates that the combination of ASO-10-27 and DS06-0004 induced higher levels of SMN protein in type II SMA cells (GM03813 cells) compared to SMN protein levels induced in the same type II SMA cells (GM03813 cells) treated with either agent alone. The data also determine the SMN protein levels induced in cells treated with the combination according to this disclosure compared to the untreated GM03813 cell population. As described herein, GM03813 cells possess two copies of SMN2 and are used as a model of SMA.

[0168] [Example 6: Combined effect of saRNA (DS06-0004) and Risdiplam on the expression of full-length SMN2 mRNA and SMN2 mRNA skipping exon 7 in type I SMA GM00232 cells.]

[0169] To determine whether the combination of SMN2 saRNA and the small SMN2 mRNA regulator Risdiplam enhances the induction of SMN2FL in type I SMA cells, GM00232 cells were treated with DS06-0004 and Risdiplam alone or in combination at different concentrations for 72 hours. SMN2 gene expression in the treated cells was assessed by RT-qPCR (Fig. 8A-8C) and PCR / digestion (Fig. 8D-8F). As shown in Fig. 8A-8C, 50 nM, 250 nM, and 1250 nM Risdiplam resulted in a 1.2-fold, 1.8-fold, and 1.9-fold increase in SMN2FL, respectively, while decreasing SMN2Δ7. 1 nM, 5 nM, and 25 nM DS06-0004 increased SMN2FL by 1.8-fold, 2.1-fold, and 2.0-fold, respectively, and increased SMN2Δ7 by 1.6-fold, 1.6-fold, and 1.7-fold, respectively.

[0170] Cell treatment with a combination of 50 nM Risdiplam and DS06-0004 at increasing concentrations (1 nM, 5 nM, and 25 nM) resulted in a 2.2-fold, 2.6-fold, and 2.5-fold increase in SMN2FL, and a 1.3-fold, 1.3-fold, and 1.2-fold increase in SMN2Δ7, respectively. Furthermore, treatment with a combination of 250 nM Risdiplam and DS06-0004 at increasing concentrations (1 nM, 5 nM, and 25 nM) resulted in a 3.0-fold, 3.4-fold, and 3.4-fold increase in SMN2FL, and a 0.3-fold, 0.3-fold, and 0.3-fold change in SMN2Δ7, respectively. Furthermore, treatment of cells with a combination of 1250 nM Risdiplam and escalating concentrations of DS06-0004 (1 nM, 5 nM, and 25 nM) increased SMN2FL by 3.6-fold, 3.9-fold, and 4.0-fold, respectively, and completely eliminated SMN2Δ7 expression. Compared to 1.9-fold SMN2FL induction by 1250 nM Risdiplam alone, the combination of Risdiplam and DS06-0004 increased SMN2FL by 4-fold, more than doubling the effect of Risdiplam alone.

[0171] The RT-qPCR results shown in Figure 8A were further validated by PCR / DdeI digestion. Consistent with the RT-qPCR results, at 1250 nM, Risdiplam alone led to a 2.3-fold increase in SMN2FL mRNA, while the combination of Risdiplam (1250 nM) and DS06-0004 (1 nM) resulted in the maximum observed induction of SMN2FL (3.2-fold) (Figures 8D to 8F).

[0172] The data shown in Figure 8 together demonstrate that the saRNA DS06-0004 alone has strong activity in inducing SMN2 mRNA expression, especially in inducing SMN2FL expression in type I SMA cells. Maximum SMN2FL induction was achieved when the SMN2 saRNA was combined with Risdiplam.

[0173] [Example 7: Combined effect of saRNA (DS06-0004) and Risdiplam on SMN protein levels in GM00232 cells.]

[0174] To further verify the effects of Risdiplam and DS06-0004, alone or in combination, on SMN2 gene expression in type I SMA cells GM00232, Western blot analysis was performed on GM00232 cells transfected with Risdiplam alone and in combination with saRNA DS06-0004. As shown in Figures 9A and 9B, 50 nM, 250 nM, and 1250 nM of Risdiplam resulted in a 1.7-fold, 1.9-fold, and 2.6-fold increase in SMN protein, respectively.

[0175] Cells were treated with a combination of 50 nM Risdiplam and DS06-0004 at increasing concentrations (1 nM, 5 nM and 25 nM) to induce an increase in SMN protein of 2.3-fold, 2.9-fold and 3.3-fold, respectively (Figures 9A and 9B).

[0176] Cells were further treated with a combination of 250 nM Risdiplam and DS06-0004 at increasing concentrations (1 nM, 5 nM and 25 nM), which induced an increase in SMN protein of 2.6-fold, 2.9-fold and 2.7-fold, respectively (Figures 9A and 9B).

[0177] In addition, treatment of cells with a combination of 1250 nM Risdiplam and DS06-0004 at increasing concentrations (1 nM, 5 nM and 25 nM) induced an increase in SMN protein of 2.4-fold, 2.7-fold and 2.7-fold, respectively (Fig. 9A and Fig. 9B).

[0178] These data together confirm that the combined effect of saRNA and Risdiplam in increasing SMN2 expression can be verified at the protein level.

[0179] [Example 8: Combined effect of saRNA (DS06-0004) and Risdiplam on the expression of full-length SMN2 mRNA and SMN2 mRNA skipping exon 7 in type II SMA GM03813 cells.]

[0180] To determine whether the combination of saRNA and Risdiplam enhances the induction of SMN2FL in type II SMA cells, GM03813 cells were transfected with DS06-0004 and Risdiplam alone or in different concentrations of combination for 72 hours. The expression of SMN2 mRNA in the treated cells was assessed by RT-qPCR (Fig. 10A-10C) and PCR / digestion (Fig. 10D-10F). As shown in Fig. 10A-10C, 50 nM, 250 nM, and 1250 nM of Risdiplam resulted in a 1.0-fold, 1.4-fold, and 2.1-fold increase in SMN2FL, respectively, while decreasing SMN2Δ7. DS06-0004 at 1 nM, 5 nM, and 25 nM increased SMN2FL by 1.7-fold, 2.2-fold, and 2.3-fold, respectively, and increased SMN2Δ7 by 1.8-fold, 2.3-fold, and 2.3-fold, respectively.

[0181] Cells were treated with a combination of 50 nM Risdiplam and DS06-0004 at increasing concentrations (1 nM, 5 nM, and 25 nM), which induced SMN2FL to increase by 1.9-fold, 2.5-fold, and 2.3-fold, respectively, and SMN2Δ7 to increase by 1.2-fold, 1.7-fold, and 1.4-fold, respectively. Further treatment with a combination of 250 nM Risdiplam and DS06-0004 at increasing concentrations (1 nM, 5 nM, and 25 nM) induced SMN2FL to increase by 2.5-fold, 3.1-fold, and 3.4-fold, respectively, and SMN2Δ7 to increase by 0.4-fold, 0.6-fold, and 0.6-fold, respectively. Furthermore, treatment of cells with a combination of 1250 nM Risdiplam and escalating concentrations of DS06-0004 (1 nM, 5 nM, and 25 nM) induced SMN2FL increases of 3.2-fold, 3.6-fold, and 3.3-fold, respectively, and completely eliminated SMN2Δ7 expression. Compared to 1250 nM Risdiplam treatment alone (which induced a 2.1-fold increase in SMN2FL), the combination treatment with Risdiplam and DS06-0004 increased SMN2FL by 3.6-fold, almost twice the effect of Risdiplam alone.

[0182] Figure 10A further validated this result using PCR / DdeI digestion. Consistent with the RT-qPCR results, at 1250 nM, Risdiplam alone led to a 2.1-fold increase in SMN2FL mRNA, while the combination of Risdiplam (1250 nM) and DS06-0004 (25 nM) resulted in the highest SMN2FL induction (3.8-fold) (Figures 10D to 10F).

[0183] Figure 10 shows that the saRNA DS06-0004 alone has strong activity in inducing SMN2 mRNA expression, especially SMN2FL in type II SMA cells. When the SMN2 saRNA is combined with Risdiplam, the maximum observed increase in SMN2FL can be achieved.

[0184] [Example 9: Combined effect of saRNA (DS06-0004) and Risdiplam on SMN protein levels in type II SMA GM03813 cells.]

[0185] To further verify the effects of Risdiplam and DS06-0004, alone or in combination, on SMN2 gene expression in type II SMA cells GM03813, Western blot analysis was performed on GM03813 cells treated with Risdiplam and DS06-0004, alone or in combination. As shown in Figures 11A and 11B, 50 nM and 250 nM Risdiplam resulted in a 1.1-fold and 1.7-fold increase in SMN protein, respectively.

[0186] Cells were treated with a combination of 50 nM Risdiplam and DS06-0004 at increasing concentrations (1 nM, 5 nM and 25 nM) to induce an increase in SMN protein of 1.3-fold, 1.4-fold and 1.8-fold, respectively (Figures 11A and 11B).

[0187] Cells were further treated with a combination of 250 nM Risdiplam and DS06-0004 at increasing concentrations (1 nM, 5 nM and 25 nM), which induced an increase in SMN protein of 2.1-fold, 2.3-fold and 2.0-fold, respectively (Figure 11A and Figure 11B).

[0188] These data collectively confirm that the combined effect of saRNA and Risdiplam in inducing SMN2 expression can be verified at the protein level.

[0189] [Example 10: Combined effect of saRNA (DS06-0031 and DS06-0067) and ASO-10-27 on the expression of full-length SMN2 mRNA and SMN2 mRNA skipping exon 7 in GM03813 cells.]

[0190] To determine the combined effect of ASO-10-27 with two saRNAs (DS06-0031 and DS06-0067), which induced greater SMN2Δ7 expression than SMN2FL mRNA in type II SMA cells, presumably attributable to transcriptional-coupled splicing regulation (Fig. 12A and Fig. 12B), GM03813 cells were transfected with DS06-0031 or DS06-0067 and ASO-10-27 alone or in combination at 10 nM for 72 hours. SMN2 expression in the treated cells was assessed by RT-qPCR (Fig. 12A) and semi-quantitative RT-PCR (Fig. 12B and Fig. 12C). As shown in Figure 12A, DS06-0031 and DS06-0067 at 10nM change SMN2FL by a factor of 0.9 and a factor of 1.3, respectively, and change SMN2△7 by a factor of 1.9 and a factor of 2.4, respectively, while ASO-10-27 changes SMN2FL and SMN2△7 by a factor of 1.4 and a factor of 0.3, respectively.

[0191] When DS06-0031 or DS06-0067 was combined with ASO-10-27 at 10 nM during cell transfection, SMN2FL was induced by 2.0-fold and 3.2-fold, respectively, and SMN2Δ7 was reduced by 0.3-fold and 0.05-fold, respectively.

[0192] The RT-qPCR results shown in Figure 12A were further validated by semi-quantitative RT-qPCR. Consistent with the RT-qPCR results, ASO-10-27 alone at 10 nM led to a 1.4-fold increase in SMN2FL mRNA, and when combined with DS06-0031 and DS06-0067, it led to a 1.8-fold and 2.3-fold increase in SMN2FL, respectively, while reducing SMN2Δ7 by 0.3-fold and 0.02-fold (Figures 12B and 12C).

[0193] In addition, SMN protein levels were assessed by Western blot assay. Consistent with SMN2FL expression, ASO-10-27 increased SMN protein levels by 3.6-fold and 3.3-fold, respectively, in the presence of DS06-0031 and DS06-0067, while increasing them by 2.3-fold when ASO-10-27 was used alone (Figures 12D and 12E).

[0194] Figure 12 demonstrates the strong activity of individual saRNAs (DS06-0031 and DS06-0067) in inducing SMN2 (especially SMNΔ7) expression. When they bind to ASO-10-27, they achieve maximum induction of SMN2FL and a decrease in SMN2Δ7. This data also indicates that although the transcriptional initiation and subsequent increase in pre-mRNA induced by certain saRNAs primarily reflect an increase in SMN2Δ7, this increase in pre-mRNA provides additional substrates for exon 7 inclusions of SMN2 mRNA regulators (e.g., ASO), leading to a significant enhancement in SMN2FL mRNA and protein expression.

[0195] [Example 11: Combined effects of saRNA (LNP-R6-04M1) with LNP-ASO-10-27 or Risdiplam on the expression of SMN2FL and SMN2Δ7 in SMA type III mice.]

[0196] The effects of LNP-R6-04M1 in combination with LNP-ASO-10-27 or with Risdiplam were evaluated in SMA type III mice. Newborn mice were divided into 10 treatment groups:

[0197] Treatment group 1: LNP-R6-04M1 was administered via ICV injection (10ug LNP-R6-04M1 was injected twice, at P1 and P3 respectively).

[0198] Treatment group 2: LNP-ASO-10-27 was administered via ICV injection (10ug LNP-ASO-10-27 was injected twice, at P1 and P3, respectively).

[0199] Treatment group 3: Risdiplam was administered via intraperitoneal injection at a concentration of 0.3 mg / kg at point P1;

[0200] Treatment group 4: Risdiplam was administered via intraperitoneal injection at a concentration of 1 mg / kg at point P1;

[0201] Treatment group 5: Risdiplam was administered via intraperitoneal injection at a concentration of 3 mg / kg at point P1;

[0202] Treatment group 6: Combined treatment with LNP-R6-04M1 and LNP-ASO-10-27. LNP-R6-04M1 was administered via ICV injection at P1 (10ug), and LNP-ASO-10-27 was administered via ICV injection at P3 (10ug).

[0203] Treatment group 7: Combined treatment with LNP-ASO-10-27 and LNP-R6-04M1. LNP-ASO-10-27 was administered via ICV injection at P1 (10ug), and LNP-R6-04M1 was administered via ICV at P3 (10ug).

[0204]

[0205] Treatment group 8: Combination therapy with LNP-R6-04M1 and Risdiplam. LNP-R6-04M1 was administered via ICV injection at P1 (10ug), and Risdiplam was administered via IP injection at a concentration of 0.3 mg / kg at P3.

[0206] Treatment group 9: Combination therapy with LNP-R6-04M1 and Risdiplam. LNP-R6-04M1 was administered via ICV at P1 (10ug), and Risdiplam was administered via IP at a concentration of 1 mg / kg at P3.

[0207] SMA type III mice were treated twice with saline by subcutaneous (SC) injections at P1 (5 μL) and P3 (5 μL). P1 and P3 represent days 1 and 3 after birth, respectively.

[0208] Treatment group 10: Mice were treated with physiological saline, and the levels of SMN2FL and SMN2Δ7 mRNA in brain, liver and spinal cord tissues were quantified by RT-qPCR.

[0209] P1 and P3 refer to the first and third day postpartum, respectively. [result]

[0210] As shown in Figure 13A, LNP-R6-04M1 (treatment group 1) induced a 1.2-fold increase in SMN2Δ7 mRNA expression in the brain compared to the control (treatment group 10), without regulating SMN2FL mRNA expression. LNP-ASO-10-27 (treatment group 2) induced a 1.6-fold increase in SMN2FL mRNA expression in the brain compared to the control group (treatment group 10), and induced a 0.7-fold decrease in SMN2Δ7 mRNA expression. Risdipalm at concentrations of 0.3 mg / kg (treatment group 3), 1 mg / kg (treatment group 4), and 3 mg / kg (treatment group 5) all induced a 1.1-fold, 1.3-fold, and 1.0-fold increase in SMN2FL mRNA expression in the brain compared to the control group, respectively, and induced a 1.0-fold increase in SMN2Δ7 mRNA expression in the brain compared to the control group (treatment group 10).

[0211] The combined treatment of LNP-R6-04M1 (10ug) at P1 and LNP-ASO-10-27 (10ug) at P3 (treatment group 6) induced a 1.8-fold increase in SMN2FL mRNA expression in the brain compared to the control group (treatment group 10), and a 0.8-fold decrease in SMN2Δ7 mRNA expression in the brain compared to the control group (treatment group 10).

[0212] The combined treatment of LNP-ASO-10-27 at P1 (10ug) and LNP-R6-04M1 at P3 (10ug) (treatment group 7) induced a 2.0-fold increase in SMN2FL mRNA expression and a 0.6-fold decrease in SMN2Δ7 mRNA expression in the brain compared to the control group (treatment group 10).

[0213] Compared with the control group (treatment group 10), the combined treatment of LNP-R6-04M1 at P1 (10ug) and Risdiplam at a concentration of 0.3mg / kg (treatment group 8) induced a 1.2-fold increase in SMN2FL mRNA expression and a 1.0-fold increase in SMN2Δ7 mRNA expression in the brain, respectively.

[0214] Compared with the control group (treatment group 10), the combined treatment of LNP-R6-04M1 at P1 (10ug) and Risdiplam at a concentration of 1mg / kg (treatment group 9) induced a 1.3-fold increase in SMN2FL mRNA expression and a 1.0-fold increase in SMN2Δ7 mRNA expression in the brain.

[0215] As shown in Figure 13B, LNP-R6-04M1 (treatment group 1) did not induce an increase in SMN2FL mRNA expression in the liver. LNP-ASO-10-27 (treatment group 2) induced a 1.7-fold increase in SMN2FL mRNA expression in the liver compared to the control group (treatment group 10), and induced a 0.9-fold decrease in SMN2Δ7 mRNA expression in the liver. Compared to the control group (treatment group 10), Risdipalm at concentrations of 0.3 mg / kg (treatment group 3), 1 mg / kg (treatment group 4), and 3 mg / kg (treatment group 5) all induced a 1.1-fold, 1.5-fold, and 1.0-fold increase in SMN2FL mRNA expression in the liver, respectively, and induced a 1.0-fold, 0.9-fold, and 1.0-fold increase in SMN2Δ7 mRNA expression in the liver, respectively.

[0216] The combined treatment of LNP-R6-04M1 at P1 (10ug) and LNP-ASO-10-27 at P3 (10ug) (treatment group 6) induced a 1.0-fold increase in SMN2FL mRNA expression in the liver compared to the control group (treatment group 10), and induced a 0.9-fold decrease in SMN2Δ7 mRNA expression in the liver compared to the control group (treatment group 10).

[0217] The combined treatment of LNP-ASO-10-27 at P1 (10ug) and LNP-R6-04M1 at P3 (10ug) (treatment group 7) induced a 1.6-fold increase in SMN2FL mRNA expression in the liver compared to the control group (treatment group 10), and induced a 1.0-fold increase in SMN2Δ7 mRNA expression in the liver compared to the control group (treatment group 10).

[0218] The combined treatment of LNP-R6-04M1 at P1 (10ug) and Risdiplam at a concentration of 0.3mg / kg (treatment group 8) induced a 1.6-fold increase in SMN2FL mRNA expression and a 1.1-fold increase in SMN2Δ7 mRNA expression in the liver, respectively.

[0219] The combined treatment of LNP-R6-04M1 at P1 (10ug) and Risdiplam at a concentration of 1mg / kg (treatment group 9) induced a 1.5-fold increase in SMN2FL mRNA expression and a 1.1-fold increase in SMN2Δ7 mRNA expression in the liver, respectively.

[0220] As shown in Figure 13C, LNP-R6-04M1 (treatment group 1) did not induce an increase in SMN2FL gene expression in the spinal cord. LNP-ASO-10-27 (treatment group 2) induced a 1.3-fold increase in SMN2FL mRNA expression in the spinal cord compared to the control group, and induced a 0.8-fold decrease in SMN2Δ7 mRNA expression in the spinal cord.

[0221] The combined treatment of LNP-R6-04M1 at P1 (10ug) and LNP-ASO-10-27 at P3 (10ug) (treatment group 6) induced a 1.8-fold increase in SMN2FL mRNA expression in the spinal cord compared to the control group, and a 1.2-fold increase in SMN2Δ7 mRNA expression in the spinal cord compared to the control group.

[0222] The combined treatment of LNP-ASO-10-27 at P1 (10ug) and LNP-R6-04M1 at P3 (10ug) (treatment group 7) induced a 2.2-fold increase in SMN2FL mRNA expression in the spinal cord compared to the control group, and a 1.1-fold increase in SMN2Δ7 mRNA expression in the spinal cord compared to the control group.

[0223] As shown in Figures 13A to 13C, combined treatment with SMN2 saRNA and SMN2 mRNA regulators led to an increase in SMN2FL mRNA expression and SMN2Δ7 mRNA expression.

[0224] [Materials and methods]

[0225] Design and synthesis of oligonucleotides

[0226] SMN2 saRNAs, including DS06-0004 (also known as RAG6-281), DS06-0031 (also known as RAG6-1266), and DS06-0067 (also known as RAG6-293), were designed to target the SMN2 gene promoter at positions -281, -1266, and -293 relative to the transcription start site of SMN2, respectively (Figure 1). The SMN2 saRNAs were synthesized using a solid-phase technique on a K&A DNA synthesizer (K&A Laborgeraete GbR, Schaafheim, Germany). In short, phosphoramidine monomers were sequentially added to a solid vector to generate the desired full-length oligonucleotides. Each base addition loop consisted of four chemical reactions: detrimethylation, coupling, oxidation / thiolization, and capping. After synthesis, the solid vector was then transferred to screw-cap microcentrifuge tubes. For a 1 μM synthesis scale, add a mixture of 33% methylamine in ethanol and 1 mL of ammonium hydroxide. Then, heat the tube containing the solid support in an oven at 60–65 °C for 2 hours, followed by cooling to room temperature. Collect the lysis solution and evaporate to dryness in a speedvac. Dissolve the crude RNA oligonucleotide product, still carrying the 2'-TBDMS group, in 0.1 mL of DMSO. After adding 1 mL of triethylamine 3HF, cap the tube and shake vigorously to ensure complete dissolution. Heat the tube in an oven at 60–65 °C for 3–3.5 hours. Remove the tube from the oven and cool to room temperature. Cool the solution containing the fully demethylated silyl oligonucleotide on dry ice. Carefully add 0.5 mL of 2 mL of ice-cold n-butanol (-20 °C) to precipitate the oligonucleotide. Filter the precipitate, wash with 1 mL of ice-cold n-butanol, and then dissolve the precipitate in 1 M TEAA (triethylammonium acetate). The crude oligonucleotides were then purified by ion-exchange (IEX) HPLC using a Source 15Q column. The purity of the fractions was analyzed by ion-exchange (IEX) HPLC using a Column DNA Pac™ PA100. After producing a desalted purified single-stranded solution, duplexes were prepared by annealing two complementary single-stranded oligonucleotides and then lyophilized into powder.

[0227] ASO-10-27: Antisense Oligonucleotide (ASO) ASO-10-27, also known as Nusinersen (Spinraza), is synthesized using the same technique as described above, except that the final annealing step is omitted. ASO-10-27 is a single-stranded ASO modified with 2'-O-2-methoxyethyl (MOE). It induces exon 7 inclusions by targeting the intron splicing silencer (ISS) at intron 7 of the SMN2 gene (Hua, Y et al., antisense masking of the hnRNP A1 / A2 intron splicing silencer can correct SMN2 splicing in transgenic mice. American Journal of Human Genetics (2008). The sequence of ASO-10-27 is:

[0228] meU*meC*meA*meC*meU*meU*meU*meC*meA*meU*meA*meA*meU*meG*meC*meU*meG*meG, where me, 2'MOE, *, phosphate thioester (PS) backbone modification, and all cytosine (C) are 5'-methylcytosine. The lyophilized oligonucleotides are suspended in RNase-free water for cell transfection or diluted with physiological saline to a suitable concentration for in vivo injection.

[0229] Cell culture and processing

[0230] Fibroblasts derived from SMA patients were obtained from the Corell Medical Research Institute (Camden, New Jersey, USA) and included GM00232 (SMA type I with 2 copies of the SMN2 gene) and GM03813 (SMA type II with 3 copies of the SMN2 gene). These cells were cultured in modified MEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 15% fetal bovine serum (Sigma-Aldrich), 1% NEAA (Gibco), and 1% penicillin / streptomycin (Gibco) at 5% CO2 and 37°C. For transfection with oligonucleotides, including saRNA, siRNA, and ASO, cells were seeded at a density of 1 × 10⁵ cells / well in 6-well plates and transfected with different concentrations of oligonucleotides using RNAiMax (Invitrogen, Carlsbad, CA) for 72 hours according to the manufacturer's reverse transfection protocol (unless otherwise specified). The sequences of saRNA, SMN2 siRNA (DS06-332i), control dsRNA (dsCon2), and ASO are listed in Table 1. For cellular ribdiplam treatment, unless otherwise specified, ribdiplam dissolved in DMSO (HY-109101, MedChem Express Company, Monmouth Junction, NJ, USA) was added directly to the cells at the desired concentration and left for 72 hours.

[0231] [Table 1: Sequence and Double-Strand Composition of Oligonucleotide Chains]

[0232]

[0233] RNA isolation and quantitative reverse transcription polymerase chain reaction (RT-qPCR)

[0234] For RNA isolation from cultured cells, total cellular RNA was isolated from treated cells using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to its manual. To isolate RNA from animal tissues, tissues were collected and stored in RNA later (AM7021, Thermo Fisher). Total RNA was then isolated using the MagPure Total RNA Micro LQ kit (Magen, R6621, China) via an auto-pure96 machine (ALLSHENG, China). The resulting RNA (1 μg) was reverse transcribed into cDNA using a PrimeScript RT kit containing a gDNA Eraser (Takara, Shlga, Japan). The cDNA was amplified using SYBR Premix Ex Taq II reagents (Takara, Shlga, Japan) and primers specifically for amplifying full-length (SMN2FL) or Δ7 SMN2 mRNA (SMN2Δ7) on an ABI 7500 rapid real-time PCR system (Applied Biosystems; Foster City, CA) (Figure 1). The reaction conditions were: 95℃ for 3 seconds (1 cycle) and 60℃ for 30 seconds (40 cycles). TBP gene amplification served as an internal control. All primer sequences are listed in Table 2. RT and RT-qPCR reactions are shown in Tables 3 and 4.

[0235] Table 2: Primer sequences for RT-qPCR detection

[0236] Table 3: RT reaction

[0237]

[0238]

[0239] Semi-quantitative RT-PCR / DdeI digestion assay

[0240] To simultaneously amplify both SMN2FL and SMN2Δ7 in a single reaction, cDNA was amplified by semi-quantitative RT-PCR using primers spanning exon 7 of SMN2 (Table 5) (Figure 2A). The PCR conditions were: 94°C for 2 minutes (one loop), 98°C for 10 seconds, 60°C for 15 seconds, 72°C for 32 seconds, 30 loops, and a final extension at 72°C for 5 minutes. The PCR reactions are listed in Table 6. To further distinguish between SMN1 mRNA and SMN2, the PCR products of SMN were digested with DdeI restriction endonuclease (R0175L, NEB) and then separated by 2% agarose gel electrophoresis. Due to nucleotide variations in exon 8 of SMN2, the DdeI recognition site is located in the PCR product amplified from the SMN2 gene rather than the SMN1 gene. DdeI digestion releases a 115bp fragment from SMN2FL and SMN2Δ7, producing three fragments: 507 (SMN1FL), 338 (SMN2Δ7), 392 (SMN2FL), and 115bp (Figures 2B and 2C). The TBP gene was also amplified as an RNA loading control. The DdeI digestion conditions were: 37°C for 60 minutes, 65°C for 20 minutes, for one cycle. The DdeI digestion reactions are listed in Table 7.

[0241]

[0242]

[0243] Table 7: Digestion reaction of DdeI

[0244] Western blot

[0245] Proteins were harvested from transfected cells using 1×RIPA buffer containing protease inhibitors, and protein concentrations were determined using a BCA protein assay kit (Beyotime, P0010, China). To isolate proteins from animal tissues, tissues were collected and lysed with 1×RIPA buffer. Protein concentrations were measured using a BCA protein assay kit. Protein electrophoresis (10 μg protein / well) was performed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gels, and then transferred to polyvinylidene fluoride (0.45 μm PVDF) membranes. The membranes were blotted overnight at 4°C with either anti-SMN (CST, 19276, USA) or anti-α / β-tubulin (CST, 2148s, USA) antibody. After washing three times with TBST buffer, the membrane was incubated with anti-IgG and a second antibody conjugated with horseradish peroxidase (CST, 7074s and 7076s, USA) at room temperature (RT) for 1 hour. The membrane was then washed three times with TBST buffer for 10 minutes each time, and analyzed using ImageLab (BIO-RAD, Chemistry Doctm MP Imaging System). The band densities of SMN protein and α / β-tubulin were quantified using ImageJ software.

[0246] Animal program

[0247] All animal procedures were performed by certified laboratory personnel using protocols that comply with local and state regulations and were approved by the agency’s Animal Care and Use Committee. As previously described by Shelley et al. (Shelley, HM et al., Mouse Model of Spinal Muscular Atrophy, Nature Genetics (2000)), SMA-like mice were obtained from the Jackson Laboratory by transgenic homozygous knockout mice with human SMN2, specifically Smn exon 7 (Smn- / -SMN2+ / -). Tail tips were collected on day 0 (P0) after birth, and each pup was identified by paw print. Genotyping was performed by PCR analysis using the following set of three specific primers: S1, 5'-ATAACACCACCACTCTTACTC-3' and S2, 5'-GTAGCCGTGATGCCATTGTCA-3' (a 1,150 bp band for the wild-type allele) and S1 and H1, 5'-AGCCTGAAGAACGAGATCAGC-3' (a 950 bp band for the mutant allele). PCR products were detected by 1% agarose gel electrophoresis. Severe SMA mice (Smn- / -, SMN+ / 0) were produced. Litterctes who were heterozygous for mouse Smn (Smn1+ / -, SMN2+ / -) served as controls.

[0248] Lipid nanoparticle (LNP) formulation

[0249] Lipid stock solution containing DLin-KC2-DMA (50 mg / mL), cholesterol (10 mg / mL), DSPC (7.5 mg / mL), and PEG2000-DMPE (20 mg / mL) dissolved in 100% ethanol was rapidly mixed with oligonucleotide stock solution (20 mg / mL, 0.05 mM citrate buffer, pH 4.0) at a volume ratio of 1:3 using a microfluidic chip at a flow rate of 12 mL / min. The molar ratio of DLin-KC2-DMA, cholesterol, DSPC, and PEG2000-DMPE was 50:38.5:10:1.5. The pre-formed vesicles were then dialyzed in 1X PBS (pH 7.4) for 12 hours using dialysis tubes. The fineness of the LNPs was assessed by dynamic light scattering using a Brookhaven NanoBrook 90Plus Zeta. RNA concentration was assessed using an A260 nanometer using a NanoPhotometer N50.

[0250] Intraventricular ventricle (ICV) injection

[0251] Tail tips were collected on day 0 (PND0) for genotyping by PCR, and mice were grouped into type I SMA mice (Smn- / -, SMN2+ / -), type III SMA mice (Smn- / -, SMN2+ / +), and heterozygous (Het) controls (Smn1+ / -, SMN2+ / -). Bilateral intraventricular (ICV) injections with 2% isoflurane were administered to the pups at P1 and P3 using a 29-gauge syringe (2 μL per side, 5 mg / ml) at depths of 1.5 mm or 3.6 mm. Intraperitoneal (IP) injections were administered to the lower abdominal region of the newborn mice. The sequences of saRNA (LNP-R6-04M1) and LNP-ASO-10-27 are listed in Table 1.

[0252] Table 8. Sequences of saRNA targeting the SMN2 promoter and its target DNA.

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[0263] Table 9. DNA sequence of the saRNA hotspot region on the SMN2 promoter

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[0265] [Equivalents and inclusion by reference]

[0266] All references cited herein are incorporated by reference as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent were specifically and individually indicated to be incorporated by reference in their entirety for all purposes. Pursuant to 37 CFR §1.57(B)(1), the applicant wishes to incorporate this statement by reference in connection with each publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent, each of which is expressly identified pursuant to 37 CFR §1.57(b)(2), even if such reference is not directly adjacent to the specific statement incorporated by reference. The inclusion of a specific statement of incorporation by reference (if any) in the specification does not in any way weaken the general statement of incorporation by reference. The references cited herein do not imply an admission that the references are relevant prior art, nor do they constitute any admission of the content or dates of such publications or documents.

[0267] Although the invention has been specifically shown and described with reference to preferred embodiments and various alternative embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made without departing from the spirit and scope of the invention.

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[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

Claims

1. A pharmaceutical composition comprising a saRNA or a recombinant vector encoding the saRNA that increases the expression of the SMN2 gene or protein, and an SMN2 mRNA regulator that increases the production of functional SMN protein by regulating SMN2 mRNA splicing or stability, said combination being selected from any one of the following: (a) a saRNA or a recombinant vector encoding the saRNA comprising the first nucleic acid shown in SEQ ID NO: 476 and the second nucleic acid shown in SEQ ID NO: 477, and Nusinersen; (b) a saRNA or a recombinant vector encoding the saRNA comprising the first nucleic acid shown in SEQ ID NO: 476 and the second nucleic acid shown in SEQ ID NO: 477, and Risdiplam; (c) a saRNA or a recombinant vector encoding the saRNA comprising the first nucleic acid shown in SEQ ID NO: 478 and the second nucleic acid shown in SEQ ID NO: 479, and Nusinersen; (d) a saRNA comprising the first nucleic acid shown in SEQ ID NO: 480 and the second nucleic acid shown in SEQ ID NO:

479. (e) saRNA of the second nucleic acid shown in SEQ ID NO: 481 or a recombinant vector encoding the saRNA, and Nusinersen; and (e) saRNA of the first nucleic acid shown in SEQ ID NO: 488 and the second nucleic acid shown in SEQ ID NO: 496 or a recombinant vector encoding the saRNA, and Nusinersen.

2. The composition as claimed in claim 1, wherein at least one nucleotide of the saRNA is a chemically modified nucleotide.

3. The composition as claimed in claim 1, wherein the sequence of Nusinersen is as shown in SEQ ID NO:

486.

4. The composition as claimed in claim 1, wherein the composition comprises at least one pharmaceutically acceptable carrier.

5. The composition as claimed in claim 4, wherein at least one pharmaceutically acceptable carrier is selected from aqueous carriers, liposomes, polymers, and peptides.

6. Use of a pharmaceutical composition as described in any one of claims 1 to 5 in the preparation of a medicament for treating or delaying the onset or progression of an individual’s SMN deficiency-related disorder.

7. The use as described in claim 6, wherein the individual suffers from SMA.

8. The use as described in claim 7, wherein the individual suffering from SMA has reduced or abnormal expression of the full-length SMN protein.