Nucleic acid constructs and their use in the treatment of spinal muscular atrophy

Abstract

This invention provides an improved gene therapy for SMA with optimized SMN expression and reduced off-target toxicity. [Solution] A synthetic promoter comprising an enhancer and a core promoter is provided, wherein the synthetic promoter comprises a CMV enhancer and an hSyn promoter.

Description

[Technical Field]

[0001] (Cross-reference with related applications) This application claims the benefit of PCT application number PCT / CN2020 / 107173 filed on 5 August 2020 and PCT application number PCT / CN2020 / 138056 filed on 21 December 2020, each of which is incorporated herein by reference in its entirety. (Reference to electronically submitted sequence listings) This application includes a sequence listing submitted electronically via EFS-Web as an ASCII formatted sequence listing file named "14652-017 228_SEQ_LISTING.txt", created on July 21, 2021, and with a size of 108,587 bytes. This sequence listing submitted via EFS-Web constitutes part of this specification, and the entirety of it is incorporated herein by reference. (1. Technical field) This disclosure relates to nucleic acid constructs, gene therapies based on such constructs, and methods of using them. [Background technology]

[0002] (2.Background) Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder characterized by progressive muscle weakness and hypotonia resulting from the loss of lower motor neurons. Based on the age of onset and the severity of neuromuscular symptoms, four clinical phenotypes have been reported (Lunn and Wang, Lancet, 371(9630):2120-33(2008)). The motor neuron survival (SMN) gene, which is responsible for most cases of SMA, has been identified at chromosomal locus 5q13 (Lefebvre et al., Cell, 80(1):155-65(1995)). This human gene is replicated in telomere copies and centromere copies, SMN1 and SMN2, respectively. SMN-mediated malformation (SMA) is caused by mutations or deletions in the SMN1 gene, and because SMN2 cannot produce a sufficient amount of full-length protein, it leads to SMN protein depletion.

[0003] Treatment strategies, including gene therapy, have been developed based on increased SMN gene expression. However, currently available treatments face various challenges, including insufficient SMN gene expression levels and off-target toxicity. Therefore, there is a need in this field for improved SMA gene therapy with optimized SMN expression and reduced off-target toxicity. [Overview of the project]

[0004] (3. Overview) In one aspect, the herein provides a nucleic acid comprising (i) a first nucleic acid region comprising a nucleic acid sequence encoding an SMN protein or a variant thereof, and (ii) a second nucleic acid region comprising one or more target segments(optional) of one or more endogenous microRNAs (miRNAs), wherein the second nucleic acid region is located at the 3' position of the first nucleic acid region.

[0005] In some embodiments, the SMN protein or its variant includes the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 33.

[0006] In some embodiments, the first nucleic acid region includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34 and 35.

[0007] In some embodiments, the second nucleic acid region includes at least one target segment of cardiac endogenous miRNA. In some embodiments, the cardiac endogenous miRNA is selected from the group consisting of hsa-mir-1-5p, hsa-mir-208a-5p, hsa-mir-208b-5p, hsa-mir-133a-1, and hsa-mir-488-5p. In some embodiments, the cardiac endogenous miRNA includes a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6.

[0008] In some embodiments, the second nucleic acid region includes at least one target segment of an endogenous miRNA of the liver. In some embodiments, the endogenous miRNA of the liver is hsa-mir-122. In some embodiments, the endogenous miRNA of the liver includes a nucleic acid sequence in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO: 4.

[0009] In some embodiments, the second nucleic acid region includes two or more target segments of hsa-mir-133a-1. In some embodiments, the second nucleic acid region includes at least three target segments of hsa-mir-133a-1. In some embodiments, the second nucleic acid region includes at least one target segment of hsa-mir-208a-5p, at least one target segment of hsa-mir-208b-5p, at least one target segment of hsa-mir-122, and at least one target segment of hsa-mir-133a-1. In some embodiments, the second nucleic acid region includes two target segments of hsa-mir-208a-5p, two target segments of hsa-mir-208b-5p, three target segments of hsa-mir-122, and three target segments of hsa-mir-133a-1.

[0010] In some embodiments, the target segment of hsa-mir-1-5p includes a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 7; the target segment of hsa-mir-208a-5p includes at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, The target segment of hsa-mir-208b-5p contains nucleic acid sequences in which 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 8; the target segment of hsa-mir-208b-5p contains nucleic acid sequences in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 9 The target segment of hsa-mir-122 includes a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 10; the target segment of hsa-mir-133a-1 includes a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 9. The target segment of hsa-mir-488-5p includes a nucleic acid sequence in which %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 11; and / or the target segment of hsa-mir-488-5p includes a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 12.

[0011] In some embodiments, the second nucleic acid region comprises at least three repeats of the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the second nucleic acid region further comprises one or more target segments of endogenous hepatic miRNA.

[0012] In some embodiments, the second nucleic acid region includes (i) two repeats of the target segment having the nucleic acid sequence of SEQ ID NO: 8, (ii) two repeats of the target segment having the nucleic acid sequence of SEQ ID NO: 9, (iii) three repeats of the target segment having the nucleic acid sequence of SEQ ID NO: 10, and (iv) three repeats of the target segment having the nucleic acid sequence of SEQ ID NO: 11.

[0013] In some embodiments, the second nucleic acid region further includes one or more linkers between target segments. In some embodiments, the linker includes 1 to 10 nucleotides. In some embodiments, the linker includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16, and SEQ ID NOs: 17.

[0014] In some embodiments, the second nucleic acid region is (i) a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 18, and (ii) a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 19. A nucleic acid sequence, (iii) a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 20, or (iv) a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 21.

[0015] In some embodiments, the first nucleic acid region further includes a promoter. In some embodiments, the promoter includes the nucleic acid sequence of SEQ ID NO: 36 or SEQ ID NO: 37. In some embodiments, the promoter includes a CMV enhancer and an hSyn promoter. In some embodiments, the promoter includes a proC3 enhancer and an hSyn promoter. In some embodiments, the promoter includes a proA5 enhancer and an hSyn promoter. In other embodiments, the promoter includes a proB15 enhancer and an hSyn promoter. In some embodiments, the promoter includes the region of SEQ ID NO: 38 and the region of SEQ ID NO: 39. In some embodiments, the promoter includes the region of SEQ ID NO: 38 and the region of SEQ ID NO: 40. In some embodiments, the promoter includes the region of SEQ ID NO: 38 and the region of SEQ ID NO: 41. In some embodiments, the promoter includes the region of SEQ ID NO: 38 and the region of SEQ ID NO: 42.

[0016] In another embodiment, provided herein are vectors comprising nucleic acids provided herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, AAV44-9, or combinations or variants thereof. In a particular embodiment, the vector is a recombinant AAV9 (rAAV9) vector or a variant thereof.

[0017] In yet another embodiment, provided herein is a recombinant AAV (rAAV) vector comprising (i) a first nucleic acid region comprising a transgene, and (ii) a second nucleic acid region comprising one or more target segments of one or more endogenous miRNAs, wherein at least one target segment is a target segment of cardiac endogenous miRNA, and at least one target segment is a target segment of hepatic endogenous miRNA, and the second nucleic acid region The region is located at the 3' position of the first nucleic acid region, and the rAAV vector is a recombinant AAV vector containing a reverse terminal repeat (ITR) derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, or AAV44-9.

[0018] In some embodiments, the first nucleic acid region includes a nucleic acid sequence encoding an SMA protein or a variant thereof, which comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% are identical to SEQ ID NO: 33.

[0019] In some embodiments, the first nucleic acid region includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34 and 35.

[0020] In some embodiments, the endogenous miRNA of the heart is selected from the group consisting of hsa-mir-1-5p, hsa-mir-208a-5p, hsa-mir-208b-5p, hsa-mir-133a-1, and hsa-mir-488-5p, and / or the endogenous miRNA of the liver is hsa-mir-122.

[0021] In some embodiments, (i) the endogenous miRNA of the heart comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6, and / or (ii) the endogenous miRNA of the liver comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to SEQ ID NO: 4.

[0022] In some embodiments, the second nucleic acid region includes two or more target segments of hsa-mir-133a-1. In some embodiments, the second nucleic acid region includes at least three target segments of hsa-mir-133a-1. In some embodiments, the second nucleic acid region includes at least one target segment of hsa-mir-208a-5p, at least one target segment of hsa-mir-208b-5p, at least one target segment of hsa-mir-122, and at least one target segment of hsa-mir-133a-1. In some embodiments, the second nucleic acid region includes two target segments of hsa-mir-208a-5p, two target segments of hsa-mir-208b-5p, three target segments of hsa-mir-122, and three target segments of hsa-mir-133a-1.

[0023] In some embodiments, (i) the target segment of hsa-mir-1-5p includes a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 7; (ii) the target segment of hsa-mir-208a-5p includes at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% (iii) The target segment of hsa-mir-208b-5p contains nucleic acid sequences in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 8. (iv) The target segment of hsa-mir-122 includes a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 10, (v) The target segment of hsa-mir-133a-1 includes at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, (vi) The target segment of hsa-mir-488-5p contains a nucleic acid sequence in which 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 11, and / or (vi) the target segment of hsa-mir-488-5p contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 12.

[0024] In some embodiments, the second nucleic acid region includes at least three repeats of the nucleic acid sequence of SEQ ID NO: 11.

[0025] In some embodiments, the second nucleic acid region includes (i) two repeats of the target segment having the nucleic acid sequence of SEQ ID NO: 8, (ii) two repeats of the target segment having the nucleic acid sequence of SEQ ID NO: 9, (iii) three repeats of the target segment having the nucleic acid sequence of SEQ ID NO: 10, and (iv) three repeats of the target segment having the nucleic acid sequence of SEQ ID NO: 11.

[0026] In some embodiments, the second nucleic acid region further includes one or more linkers between target segments, and optionally the linkers include 1 to 1500 nucleotides, 1 to 500 nucleotides, 1 to 100 nucleotides, 1 to 50 nucleotides, or 1 to 10 nucleotides.

[0027] In some embodiments, the linker includes a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

[0028] In some embodiments, the second nucleic acid region is (i) a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 18, and (ii) a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 19. A nucleic acid sequence, (iii) a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 20, or (ii) a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 21.

[0029] In some embodiments, the first nucleic acid region further includes a promoter. In some embodiments, the promoter includes the nucleic acid sequence of SEQ ID NO: 36 or SEQ ID NO: 37. In some embodiments, the promoter includes a CMV enhancer and an hSyn promoter. In some embodiments, the promoter includes a proC3 enhancer and an hSyn promoter. In some embodiments, the promoter includes a proA5 enhancer and an hSyn promoter. In other embodiments, the promoter includes a proB15 enhancer and an hSyn promoter. In some embodiments, the promoter includes the region of SEQ ID NO: 38 and the region of SEQ ID NO: 39. In some embodiments, the promoter includes the region of SEQ ID NO: 38 and the region of SEQ ID NO: 40. In some embodiments, the promoter includes the region of SEQ ID NO: 38 and the region of SEQ ID NO: 41. In some embodiments, the promoter includes the region of SEQ ID NO: 38 and the region of SEQ ID NO: 42.

[0030] In some embodiments, the rAAV vectors provided herein include ITRs derived from AAV9.

[0031] In yet another embodiment, provided herein are nucleic acids comprising a nucleic acid sequence encoding an SMN protein or a variant thereof, and a nucleic acid region comprising a synthetic promoter comprising an enhancer and a core promoter, wherein optionally, (i) the synthetic promoter comprises a CMV enhancer and an hSyn promoter, (ii) the synthetic promoter comprises a proC3 enhancer and an hSyn promoter, (iii) the synthetic promoter comprises a proA5 enhancer and an hSyn promoter, or (iv) the synthetic promoter comprises a proB15 enhancer and an hSyn promoter.

[0032] In some embodiments, the hSyn promoter includes a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 38.

[0033] In some embodiments, the CMV enhancer includes a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 39.

[0034] In some embodiments, the proC3 enhancer includes a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 40.

[0035] In some embodiments, the proA5 enhancer includes a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 41.

[0036] In some embodiments, the proB15 enhancer includes a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 42.

[0037] In some embodiments, the SMN protein or its variants include the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% are identical to SEQ ID NO: 33.

[0038] In some embodiments, the nucleic acid sequence encoding the SMN protein or a variant thereof includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 34 and 35.

[0039] In another aspect, provided herein are rAAV vectors comprising the nucleic acid sequence of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.

[0040] In another embodiment, provided herein are rAAV vectors comprising the nucleic acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.

[0041] In yet another embodiment, provided herein are recombinant AAV (rAAV) particles comprising (a) nucleic acids or rAAV vectors provided herein, and (b) the capsid proteins of AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, AAV44-9, or variants thereof. In some embodiments, the capsid protein is the AAV9 capsid protein or a variant thereof.

[0042] In yet another embodiment, provided herein are pharmaceutical compositions comprising nucleic acids, vectors or rAAV vectors or rAAV particles provided herein, and pharmaceutically acceptable excipients.

[0043] In yet another embodiment, the foregoing provides a method for enhancing intracellular SMN protein expression, comprising contacting the cells with a nucleic acid, vector, or rAAV vector, rAAV particles, or pharmaceutical composition provided herein.

[0044] In yet another embodiment, provided herein are methods for treating a disease or disorder of interest, comprising administering to the subject a nucleic acid, vector or rAAV vector, rAAV particles, or pharmaceutical composition provided herein. In some embodiments, the disease or disorder is an SMN-related disease or disorder. In some embodiments, the SMN-related disease or disorder is a disease or disorder associated with insufficient expression of the SMN protein. In some embodiments, the disease or disorder is associated with a defective SMN protein. In other embodiments, the disease or disorder is associated with smn1 gene deletion and / or mutation. In some embodiments, the disease or disorder is spinal muscular atrophy (SMA). In some embodiments, the disease or disorder is SMA-I, SMA-II, SMA-III, or SMA-IV. In some embodiments, the subject is under 2 years of age. [Brief explanation of the drawing]

[0045] (4. Brief explanation of the drawing) [Figure 1] Figure 1A shows an example of the gene design of the rAAV vector provided herein. Figure 1B shows an example of the nucleic acid region of the nucleic acid construct or rAAV vector containing multiple target segments.

[0046] [Figure 2A] Figure 2A shows the SMN protein expression levels of various rAAV vectors provided herein. [Figure 2B] Figure 2B shows the results of transcriptome analysis for the codon-optimized constructs provided herein.

[0047] [Figure 3] Figure 3 shows the survival results of the in vivo efficacy assay for these rAAV particles.

[0048] [Figure 4] Figure 4 shows the open-field activity results of the in vivo efficacy assay of these rAAV particles.

[0049] [Figure 5] Figure 5 shows the body weight results of the in vivo efficacy assay for these rAAV particles.

[0050] [Figure 6] Figure 6 shows a comparison of survival outcomes among rAAVs containing different miRNA target segments.

[0051] [Figure 7A] Figures 7A and 7B show examples of constructs provided herein, including a synthetic protomer. [Figure 7B] Same as above

[0052] [Figure 8A] Figure 8A shows the results of an in vivo efficacy assay of a construct containing the synthetic promoter provided herein, compared to constructs containing other promoters.

[0053] [Figure 8B] Figure 8B shows the in vivo efficacy at low doses of constructs containing various synthetic promoters with different enhancers. [Modes for carrying out the invention]

[0054] (5. Detailed explanation) Part of this disclosure is based on novel nucleic acid constructs (e.g., nucleic acid constructs encoding SMN proteins and containing tissue-specific microRNA target sequences), AAV vectors containing them, and improved properties thereof.

[0055] (5.1.Definition) The techniques and procedures described or referenced herein include those generally well understood by those skilled in the art and / or commonly employed using conventional methods, including, for example, the widely used methods described in Sambrook et al.'s "Molecular Cloning: A Laboratory Manual" (3rd edition, 2001), "Current Protocols in Molecular Biology" (Ausubel et al., eds., 2003), "Therapeutic Monoclonal Antibodies: From Bench to Clinic" (An, ed., 2009), "Monoclonal Antibodies: Methods and Protocols" (Albitar, ed., 2010), and "Antibody Engineering" Volumes 1 and 2 (Kontermann and Dubel, eds., 2nd edition, 2010).

[0056] Unless otherwise defined herein, technical and scientific terms used herein have the meanings that are ordinarily understood by those skilled in the art. For the purpose of interpreting this specification, the following definitions of terms apply, and wherever appropriate, a singular term is also included in its plural form, and vice versa. In the event of any conflict between the definitions of terms given and any document incorporated herein by reference, the definitions given below shall prevail.

[0057] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to amino acid polymers of any length. These polymers may be linear or branched, may contain modified amino acids, and may include non-amino acid groups. The term also includes naturally occurring or indirectly modified amino acid polymers, such as those resulting from disulfide bond formation, glycosylation, lipid modification, acetylation, phosphorylation, or other operations or modifications. Similarly, this definition includes, but is not limited to, amino acid analogs containing, for example, non-natural amino acids, and polypeptides containing one or more other modifications known in the art.

[0058] "Polynucleotide" or "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length, including DNA and RNA. These nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by synthetic reactions. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to short, generally single-stranded, synthetic polynucleotides, but not necessarily, generally less than approximately 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and entirely applicable to oligonucleotides. Cells producing the binding molecules of this disclosure may include parental hybridoma cells, as well as bacterial and eukaryotic host cells into which the nucleic acid encoding the polypeptide has been introduced. Unless otherwise stated, the left end of all single-stranded polynucleotide sequences disclosed herein is the 5' end, and the left-handed direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction in which an RNA transcript is added from 5' to 3' during development is called the transcription direction, the sequence region on a DNA strand having the same sequence as the RNA transcript that is located at the 5' position relative to the 5' end of the RNA transcript is called the "upstream sequence", and the sequence region on a DNA strand having the same sequence as the RNA transcript that is located at the 3' position relative to the 3' end of the RNA transcript is called the "downstream sequence".

[0059] As used herein, “nucleic acid base” is intended to refer to a heterocyclic component capable of forming base pairs with another nucleic acid.

[0060] As used herein, “nucleotide” is intended to mean a nucleoside having a phosphate group, wherein the phosphate group is covalently bonded to the sugar portion of the nucleoside.

[0061] As used herein, "nucleoside" is intended to refer to a nucleic acid base bound to a sugar.

[0062] The asymmetric ends of DNA and RNA strands are called the 5' (5-dash) and 3' (3-dash) ends, with the 5' end having a terminal phosphate group and the 3' end having a terminal hydroxyl group. The 5' (5-dash) end has a fifth carbon in the ring structure of a deoxyribose or ribose sugar at its end. Nucleic acids are synthesized in vivo in the 5'- to 3'- direction because the polymerase used to assemble the new strands attaches each new nucleotide to a 3'-hydroxyl (-OH) group via a phosphate diester bond.

[0063] "Isolated nucleic acids" are nucleic acids, such as RNA and DNA, or mixtures of nucleic acids, which are typically substantially separated from other genomic DNA sequences and proteins, or complexes such as ribosomes and polymerases, that are inherently associated with the native sequence. "Isolated" nucleic acid molecules are those isolated from other nucleic acid molecules present in the natural source of that nucleic acid molecule. Furthermore, "isolated" nucleic acid molecules, such as cDNA molecules, may not substantially contain other cellular materials or culture media if they are produced by recombinant technology, or may not substantially contain chemical precursors or other chemical substances if they are chemically synthesized. This term encompasses nucleic acid sequences extracted from their natural environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule may include an isolated form of that molecule. Specifically, "isolated" nucleic acid molecules encoding polypeptides as described herein are nucleic acid molecules identified and isolated from at least one contaminating nucleic acid molecule that is typically associated in the environment in which they were produced.

[0064] As used herein, the term “homology” refers to the percentage of identity between two polynucleotides or two polypeptide components. Two DNA or polypeptide sequences are “substantially homologous” to each other if their sequences exhibit at least about 50%, at least about 75%, at least about 80%–85%, at least about 90%, at least about 95%–98%, at least about 99%, or any percentage in between, over a defined length of their molecules. As used herein, “substantially homologous” also refers to sequences that exhibit complete identity with a particular DNA or polypeptide sequence.

[0065] As used herein, the term "identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid match of two polynucleotide or polypeptide sequences. Methods for determining the identity percentage are well known in the art. For example, the identity percentage can be determined by a direct comparison of the sequence information of two molecules, which is done by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing that number by the length of the shorter sequence, and multiplying the result by 100. Easily available computer programs can be used to assist in this analysis, for example, in the ALIGN, Dayhoff, MO publication "Atlas of Protein Sequence and Structure," edited by MO Dayhoff, 5th Supplement, 3:353-358, Biomedical Research Foundation, Washington, D.C., the local homology algorithm of Smith and Waterman, Advances in Appl. Math. 2:482-489, 1981, can be applied for peptide analysis. Programs for determining nucleotide sequence identity are available, such as the Wisconsin Sequence Analysis Package, version 8 (available from Genetics Computer Group, Madison, Wisconsin), which also relies on the Smith and Waterman algorithms, including programs like BESTFIT, FASTA, and GAP. These programs are recommended by the manufacturers and readily available with the default parameters described in the Wisconsin Sequence Analysis Package mentioned above. For example, the percentage of identity between a particular nucleotide sequence and a reference sequence can be determined using the Smith and Waterman homology algorithms, along with a default scoring table and a gap penalty for six nucleotide positions. Another method for establishing the percentage of identity in the context of this invention is to use the MPSRCH package of the program developed by John F. Collins and Shane S. Sturrok, copyrighted by the University of Edinburgh, and distributed by Intelligentics, Inc. (Mountain View, California).The Smith-Waterman algorithm can be used from this set of packages when using default parameters in the scoring table (e.g., gap start penalty of 12, gap extension penalty of 1, gap of 6). The "match" values ​​from the generated data reflect "sequence identity". Other suitable programs for calculating the percentage of identity or similarity between sequences are generally known in this field; for example, another alignment program is BLAST, which is used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code = standard; filter = none; strand = both; cutoff value = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sort method = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss-protein+Spupdate+PIR. Details of these programs are well known in this field. Alternatively, homology can be determined by hybridizing polynucleotides under conditions that form stable double helixes between homologous regions, then digesting them with a single-strand specific nuclease(s), and determining the size of the digested fragments. Substantially homologous DNA sequences can be identified, for example, by Southern hybridization experiments under stringent conditions defined for that particular system. Defining appropriate hybridization conditions is within the scope of the art of this field. See, for example, Sambrook et al. (cited above), DNA cloning (cited above), and nucleic acid hybridization (cited above).

[0066] As used herein, the term "vector" refers to a substance used to transport or contain a nucleic acid sequence, for example, to introduce the nucleic acid sequence into a host cell. Applicable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selective sequences or markers that enable stable integration into the chromosomes of host cells. Furthermore, a vector may contain one or more selectable marker genes and appropriate expression regulatory sequences. Selectable marker genes may, for example, provide resistance to antibiotics or toxins, compensate for nutritional deficiencies, or supply essential nutrients absent in the culture medium. Expression regulatory sequences may include constitutive and inducible promoters, transcriptional enhancers, transcriptional terminators, etc., which are well known in the art. When co-expressing two or more nucleic acid molecules, both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. The introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as northern blotting of mRNA or amplification of mRNA by polymerase chain reaction (PCR), immunoblotting of gene product expression, or other appropriate analytical methods for testing the expression of a introduced nucleic acid sequence or its corresponding gene product. The term “vector” includes cloning vehicles and expression vehicles, as well as viral vectors. In some embodiments, the vectors provided herein are recombinant AAV vectors.

[0067] As used herein, the term “recombinant AAV vector (rAAV vector)” means a polynucleotide vector comprising a nucleic acid sequence derived from AAV and one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin). In some embodiments, the one or more heterologous sequences are flanked by at least one, and in some embodiments two, AAV reverse-terminal repeat sequences (ITRs). In some embodiments, such an rAAV vector may be replicated and packaged within an infectious viral capsid particle if it is present in a host cell that is, for example, infected with a suitable helper virus (or expressing suitable helper function) and expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). The rAAV vector may be incorporated into a larger polynucleotide (e.g., within a chromosome or within another vector such as a plasmid used for cloning or transfection) and can be “rescued” by replication and capsid formation if AAV packaging function and suitable helper function are present. rAAV vectors can take on one of many forms, including, but are not limited to, plasmids, linear artificial chromosomes, complexes with lipids, encapsulated within liposomes, and capsid-encapsulated within viral capsid particles, particularly AAV particles. rAAV vectors can be packaged within AAV capsids to produce "recombinant adeno-associated virus capsid particles (rAAV particles)."

[0068] As used herein, the term “heterogeneous” refers, in the context of nucleic acid sequences such as coding and regulatory sequences, to sequences that are not typically bound together and / or sequences that are not typically associated with a particular cell. Therefore, a “heterogeneous” region of a nucleic acid construct or vector is a segment of nucleic acid bound to or within another nucleic acid molecule that is not found in nature in association with other molecules. For example, a heterogeneous region of a nucleic acid construct may include a coding sequence adjacent to a coding sequence that is not found in nature in association with its own coding sequence. Another example of a heterogeneous coding sequence is a construct whose coding sequence itself is not found in nature (e.g., a synthetic sequence with different codons than those in a natural gene).

[0069] As used herein, the term “adjacent” means that, when other elements relate to adjacent sequences, there are one or more adjacent elements upstream and / or downstream, i.e., at the 5' and / or 3' positions, with respect to that sequence. The term “adjacent” is not intended to indicate that the sequences are necessarily consecutive. For example, there may be intervening sequences between the nucleic acid encoding the transgene and the adjacent elements. In sequences where two other elements (e.g., TRs) are “adjacent” (e.g., a transgene), it means that one element is at the 5' position of the sequence and the remaining elements are at the 3' position, but there may be intervening sequences between them.

[0070] As used herein, the term “reverse terminal repeat” or “ITR” sequence refers to a relatively short sequence found at the ends of a viral genome that is oriented in the reverse direction. “AAV reverse terminal repeats (ITRs)” sequences are well known in this art and are typically sequences of approximately 145 nucleotides present at both ends of a natural single-stranded AAV genome. The outermost 125 nucleotides of an ITR can exist in either of two alternative directions, resulting in heterogeneity between different AAV genomes and between the ends of a single AAV genome. The outermost 125 nucleotides also include several self-complementary, shorter regions (referred to as regions A, A', B, B', C, C', and D) that enable the generation of intra-chain base pairs within this portion of the ITR.

[0071] The "coding sequence," or sequence that "codes" a selected polypeptide, is the nucleic acid molecule to be transcribed (in the case of DNA) and the nucleic acid molecule to be translated into a polypeptide (in the case of mRNA), when under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by the 5' (amino) terminal start codon and the 3' (carboxy) terminal translation termination codon. The transcription termination sequence may be located on the 3' side of the coding sequence.

[0072] The term "regulatory sequence" refers to a DNA sequence necessary for the expression of a coding sequence that is operablely linked within a specific host organism. Suitable regulatory sequences for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0073] As used herein, the terms “operatably linked” and similar phrases (e.g., genetically fused) refer to operatably linked nucleic acid sequences or amino acid sequences that are arranged in a functional relationship with one another, when used in relation to nucleic acids or amino acids. For example, operatably linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the precise production of a nucleic acid molecule (e.g., RNA). In some embodiments, operatably linked nucleic acid elements result in the transcription of an open reading frame, ultimately leading to the production of a polypeptide (i.e., expression of the open reading frame). In another example, an operatably linked peptide is a peptide in which multiple functional domains are arranged at appropriate distances from one another to confer the intended function of each domain.

[0074] As used herein, the term “promoter” in the ordinary sense refers to a nucleotide region containing a DNA regulatory sequence, which originates from a gene capable of binding to RNA polymerase to initiate transcription of a downstream (3' direction) coding sequence. Transcription promoters may include “inducible promoters” (where the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressive promoters” (where the expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters.”

[0075] As used broadly in this specification, the term “transgene” means any heterologous nucleotide sequence incorporated into a viral vector, which may, for example, be associated with an expression regulatory sequence, such as a promoter, for expression in a target cell. Those skilled in the art will understand that the expression regulatory sequence is selected based on its ability to promote transgene expression in the target cell. Examples of transgenes are nucleic acids encoding therapeutic polypeptides or detectable markers.

[0076] As used herein, the terms “AAV capsid,” “AAV capsid protein,” or “AAV cap” refer to the protein encoded by the AAV capsid (cap) gene (e.g., VPI, VP2, and VP3) or its variants. For example, this term includes, but is not limited to, capsid proteins derived from any AAV serotype, such as AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAV-2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAVLK03, AAVrh10, AAVrh74, AAV44-9, or their variants. This term also includes capsid proteins expressed by or derived from recombinant AAVs, such as chimeric AAVs.

[0077] As used herein, the terms “AAV capsid particle” or “AAV particle” comprise at least one AAV capsid protein (e.g., VP1 protein, VP2 protein, VP3 protein, or a variant thereof) and optionally encapsulate nucleic acids of the AAV genome or nucleic acids derived from the AAV genome.

[0078] The term "serotype," used in relation to vectors or viral capsids, is defined by a distinct immunological profile based on the capsid protein sequence and capsid structure.

[0079] As used herein, the term “chimeric” means, with respect to a viral capsid or particle, that the capsid or particle contains sequences derived from different parvoviruses, preferably sequences derived from different AAV serotypes, which are described in the literature of Rabinowitz et al., U.S. Patent No. 6,491,907, the disclosure of which is incorporated herein by reference in whole.

[0080] The term "recombinant" refers to a genetic entity that is clearly distinguishable from those commonly found in nature. When applied to polynucleotides or genes, the term means that the polynucleotide is the product of various combinations of cloning, restriction steps and / or ligation steps, and other procedures, resulting in the production of a construct that is clearly distinguishable from polynucleotides found in nature.

[0081] As used herein, the term “recombinant virus” means a virus that has been genetically modified, for example, by the addition or insertion of a heterologous nucleic acid construct into a particle. For example, as used herein, the terms “recombinant AAV particle” or “rAAV” mean an AAV that has been genetically modified, for example, by the deletion or other mutation of the endogenous AAV gene, and / or by the addition or insertion of a heterologous nucleic acid construct into the polynucleotide of an AAV particle.

[0082] As used herein, “detargeting activity” means any detectable or measurable activity resulting from the hybridization of an antisense compound to its target nucleic acid. In some embodiments, detargeting activity is a reduction in the amount or expression of the target nucleic acid or the protein product encoded by that target nucleic acid.

[0083] As used herein, “endogenous miRNA” means known or unknown microRNA expressed by any mammalian cell that, after a processing step, can be hybridized to a target nucleic acid via hydrogen bonding or complexed with a protein / RNA. Non-limiting examples of endogenous miRNA include single-stranded and double-stranded DNA or RNA, or nucleic acid compounds such as antisense oligonucleotides, siRNA, shRNA, ssRNA, miRNA, and lncRNA, as well as occupancy-based compounds.

[0084] As used herein, “detargeting inhibition” means a reduction in the target nucleic acid level in the presence of endogenous miRNA complementary to the target nucleic acid, or in the absence of endogenous miRNA, compared to the target nucleic acid level.

[0085] As used herein, “antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleic acid base sequence capable of hybridizing to a corresponding segment of a target nucleic acid. In some embodiments, the antisense oligonucleotides of this disclosure have sequences that are complementary to the target region in the target nucleic acid by at least 80%, at least about 85%, at least about 90%, or at least about 95%. For example, an endogenous miRNA in which 18 of the 20 nucleic acid bases of the antisense oligonucleotide are complementary to the target region and therefore specifically hybridize to the target region is expressed as 90 percent complementarity. The percentage of complementarity of an endogenous miRNA to a region of the target nucleic acid can be routinely determined using a basic local alignment search tool (BLAST program) (see Altschul et al., J. Mol. Biol., 215, 403-410 (1990); Zhang and Madden, Genome Res., 7, 649-656 (1997)). In some embodiments, the antisense oligonucleotide includes endogenous miRNAs and other miRNAs that hybridize to an rAAV vector expressing SMN1 mRNA, representative sequences of these endogenous miRNAs are described herein.

[0086] As used herein, “wild-type SMN1 transcript” means a transcript produced from an AAV vector containing wild-type SMN1 mRNA with or without an array of tissue-specific miRNA target segments. Therefore, a wild-type SMN1 transcript with or without an array of tissue-specific miRNA target segments is a canonically transcribed mammalian cell “SMN1 sense transcript” and is different from one produced from the coding strand (also called the sense strand) of the host cell smn1 gene.

[0087] As used herein, "specifically hybridizable" means an antisense compound that, under conditions where specific binding is desired, i.e., under physiological conditions in in vivo assays and therapeutic treatments, exhibits sufficient complementarity between the antisense oligonucleotide and the target nucleic acid to induce the desired effect, while exhibiting minimal or no effect on non-target nucleic acids.

[0088] As used herein, "stringent hybridization conditions" or "stringent conditions" refer to conditions under which an oligomer compound hybridizes to its target sequence while hybridizing to other sequences in a minimal number.

[0089] As used herein, “target segment” refers to the nucleotide sequence of a target nucleic acid targeted by an antisense compound (e.g., miRNA). “5' target site” refers to the 5' end nucleotide of the target segment. “3' target site” is intended to refer to the 3' end nucleotide of the target segment. The target segment of a miRNA is a nucleic acid sequence, and its mRNA transcript is specifically hybridizable by the miRNA.

[0090] As used herein, the terms “transfect,” “transform,” or “transform” refer to the process by which exogenous nucleic acids are introduced into or transferred into host cells. A “transfected,” “transformed,” or “transformed” cell is one that has been transfected, transformed, or transformed with exogenous nucleic acids. For example, the term “transfection” is used to refer to the uptake of foreign DNA by a cell, and a cell is “transfected” when exogenous DNA is introduced into the cell membrane. Many transfection techniques are generally known in this field. For example, see Graham et al., (1973) Virology, 52:456; Sambrook et al., (1989) "Molecular Cloning, a laboratory manual," Cold Spring Harbor Laboratories, New York; Davis et al., (1986) "Basic Methods in Molecular Biology," Elsevier; and Chu et al., (1981) Gene, 13:197. Using such techniques, one or more exogenous molecules can be introduced into suitable host cells. Viral "transduction" of cells means the transfer of nucleic acids such as DNA or RNA from viral particles into the cell.

[0091] As used herein, the term “host cell” means a particular cell that can be transfected with nucleic acid molecules, and the offspring or potential offspring of such a cell. A host cell may be a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

[0092] The term "purified" refers to the isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that it constitutes the majority of the sample in which it is present. Typically, substantially purified components in a sample constitute 50%, 80-85%, 90-99%, or, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sample. Techniques for purifying target polynucleotides and polypeptides are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and density gradient precipitation.

[0093] As used herein, the term “medically acceptable” means that it is approved by a federal or state regulatory authority for use in animals, more specifically in humans, or that it is listed in the United States Pharmacopeia, the European Pharmacopoeia, or any other generally accepted pharmacopoeia.

[0094] In Embodiment 1, each component is “pharmaceutically acceptable” to mean that it is compatible with other components of the pharmaceutical formulation and is suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications, and that it is balanced by a reasonable benefit / risk ratio. See, for example, Lippincott Williams and Wilkins, Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th edition; Rowe et al., The Pharmaceutical Press and the American Pharmaceutical Association, 2009; Handbook of Pharmaceutical Additives, 3rd edition; Ash and Ash, Gower Publishing Company, 2007; Pharmaceutical Preformulation and Formulation, 2nd edition; Gibson, CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, the pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed to it at the dose and concentration used. In some embodiments, the pharmaceutically acceptable excipient is a pH-buffered aqueous solution.

[0095] As used herein, the terms “to treat,” “treatment,” and “to treat” refer to the reduction or improvement of the progression, severity, and / or duration of a disease or symptom resulting from the administration of one or more therapies. Treatment may be determined by assessing whether there is a reduction, reduction, and / or relief of one or more symptoms associated with the disease, to the extent that improvement is observed in the patient, even if the patient is still suffering from the disease. The term “treatment” includes both disease control and remission. The terms “to control,” “to control,” and “control” refer to the beneficial effect that a subject receives from a therapy that does not necessarily result in a cure for the disease. "Treatment" or "treating" includes (1) preventing a disease, i.e., preventing the symptoms of a disease or making the disease milder in a person who is potentially exposed to or susceptible to a disease but has not yet experienced the disease or shown symptoms of the disease; (2) blocking a disease, i.e., inhibiting its onset, preventing or delaying its progression, or reversing the disease state; (3) alleviating disease symptoms, i.e., reducing the number of symptoms experienced by the person; and (4) reducing, preventing, or delaying the progression of a disease or its symptoms. The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, symptom, or associated symptom(s).

[0096] As used herein, “administer,” “dosage,” or “administer” means the act of injecting or otherwise physically delivering a substance (e.g., a conjugate or pharmaceutical composition provided herein) to a subject or patient (e.g., a human), for example, by oral, mucosal, topical, intradermal, parenteral, intravenous, intravitreous, intra-articular, subretinal, intramuscular, intrathecal delivery, and / or any other physical delivery method described herein or known in the art. In special embodiments, administration is by intravenous infusion. The conjugates or compositions provided herein may be delivered systemically or to specific tissues.

[0097] As used herein, the terms “effective dose” or “therapeutic effective dose” refer to an amount of therapeutic agent (e.g., a conjugate or pharmaceutical composition provided herein) sufficient to treat, diagnose, prevent, delay or reduce the onset of, and / or improve the severity and / or duration of, a given symptom, disorder or disease and / or related signs. These terms also include the amount necessary to reduce, delay or improve the enhancement or progression of a given disease, to reduce, delay or improve the recurrence, onset or onset of a given disease, and / or to improve or enhance the preventive or therapeutic effect of another therapy, or to act as a bridge to another therapy. In some embodiments, “effective dose” as used herein also refers to the amount of conjugate described herein to achieve the specified result. As used herein, the terms “subject” and “patient” are used interchangeably.

[0098] As used herein, the subject is a mammal, such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkeys and humans), such as humans. In some embodiments, the subject is a mammal diagnosed with a disease or disorder provided herein, such as a human. In other embodiments, the subject is a mammal at risk of developing a disease or disorder provided herein, such as a human. In special embodiments, the subject is a human.

[0099] As used herein, the terms “multiple therapies” and “therapy” may refer to any protocol(s), methods(s), compositions, formulations, and / or agents(s) that can be used to prevent, treat, manage, or improve a disease or disorder or its symptoms (for example, a disease or disorder provided herein, or one or more symptoms or conditions associated therewith). In some embodiments, the terms “multiple therapies” and “therapy” may refer to pharmacotherapy, adjuvant therapy, radiotherapy, surgery, biological therapy, supportive care, and / or other therapies useful for treating, managing, preventing, or improving a disease or disorder or one or more symptoms thereof. In some embodiments, the term “therapy” may refer to therapies other than the conjugates or pharmaceutical compositions described herein.

[0100] As used herein, the term “SMN-related disease or disorder” means a disease or disorder in which SMN is involved, for example, as a symptom or as a direct or indirect factor (including abnormal levels of SMN protein expression). SMN-related disease or disorder includes, but is not limited to, diseases or disorders associated with reduced expression of the smn1 gene or with a mutant smn1 gene.

[0101] The terms "about" and "approximately" mean within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of a given value or range.

[0102] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural form unless otherwise explicitly specified by the context.

[0103] Wherever an embodiment is described herein using the term "includes," it is understood that other similar embodiments described using the phrases "consist of" and / or "essentially consist of" are also provided. Similarly, wherever an embodiment is described herein using the phrase "essentially consist of," it is understood that other similar embodiments described using the phrase "consist of" are also provided.

[0104] The term "between" in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0105] In this specification, the term "and / or" as used in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0106] (5.2. Nucleic acid construct) In one aspect, the herein provides a novel nucleic acid comprising a first nucleic acid region encoding a target protein, and a second nucleic acid region containing one or more target segments (also referred to as miRNA target sequences) of one or more endogenous RNAs.

[0107] In some embodiments, the first nucleic acid region encodes a therapeutic molecule (e.g., SMN protein). In some embodiments, the second nucleic acid region containing the one or more target segments is located at the 3' position of the first nucleic acid region encoding the target protein. In some embodiments, the second nucleic acid region containing the one or more target segments is located immediately after or adjacent to the 3' position of the first nucleic acid region encoding the target protein. In other embodiments, the second nucleic acid region containing the one or more target segments is located one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides) away from the 3' position of the first nucleic acid region encoding the target protein.

[0108] (5.2.1. Nucleic acids encoding the target protein or its variants) In some embodiments, the nucleic acid constructs provided herein include nucleic acid sequences encoding an SMN protein or a variant thereof.

[0109] In some embodiments, the nucleic acid constructs provided herein include a nucleic acid sequence encoding an SMN protein having the amino acid sequence of SEQ ID NO: 33. In some embodiments, the nucleic acid sequence encoding the SMN protein is selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 35.

[0110] In some embodiments, the nucleic acids provided herein have a specific percentage of identity with the nucleic acid sequence of SEQ ID NO: 34 or SEQ ID NO: 35. In some embodiments, the nucleic acids encoding the target protein provided herein have sequence identity with SEQ ID NO: 34 of at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the nucleic acids encoding the target protein provided herein have sequence identity with SEQ ID NO: 35 of at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0111] The determination of the percentage of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using mathematical algorithms. One preferred, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990), modified in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993). Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). A BLAST nucleotide search can be performed using the NBLAST nucleotide program parameter set, e.g., score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search can be performed using the XBLAST program parameter set, for example, with a score of 50 and a word length of 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gap alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997). Alternatively, PSI BLAST may be used to perform iterative searches to detect distant relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI Blast programs, the default parameters for each program (e.g., those for XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) website: http: / / www.ncbi.nlm.nih.gov). Another non-restrictive example of a mathematical algorithm used to compare sequences is the algorithm in Myers and Miller, CABIOS 4:11-17 (1998).This algorithm will be incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program for amino acid sequence comparison, the PAM120 weighted residual table, gap length penalty = 12, and gap penalty = 4 can be used.

[0112] The percentage of identity between two sequences can be determined using techniques similar to those described above, with or without gap tolerance. In calculating the percentage of identity, typically only exact matches are counted.

[0113] Some embodiments, including those of the sequences described herein, may further include one or more modifications to the sugar moiety, internucleoside bonds, or nucleic acid bases.

[0114] In one embodiment, the nucleic acid is human nucleic acid (i.e., nucleic acid derived from the human smn1 gene). In another embodiment, the nucleic acid is non-human nucleic acid (i.e., nucleic acid derived from a non-human smn1 gene).

[0115] In some embodiments, the nucleic acids provided herein include one or more insertions, deletions, inversions, and / or substitutions. In some embodiments, the nucleic acid constructs provided herein include codon-optimized nucleic acid regions. In one embodiment, the nucleic acid encoding smn1 is codon-optimized. In one embodiment, the nucleic acid encoding smn1 is codon-optimized for expression within a eukaryote, for example, in a human. In some embodiments, the coding sequence encoding smn1 is codon-optimized in particular for expression within a cell, for example, in a eukaryotic cell. A eukaryotic cell is a specific organism, which may be, for example, a mammal or of which it may be derived, and such organisms include, for example, humans, or non-human eukaryotes or animals, or mammals discussed herein, such as mice, rats, rabbits, dogs, livestock for food, etc., or non-human mammals or primates. Generally, codon optimization refers to the process of modifying nucleic acid sequences by replacing at least one codon in the native sequence (e.g., approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with a codon that is more or most frequently used in the host cell's gene, while maintaining the native amino acid sequence, in order to enhance expression in the target host cell. Different species exhibit specific biases for certain codons of particular amino acids. Codon bias (differences in codon usage frequency between organisms) often correlates with messenger RNA (mRNA) translation efficiency, which is thought to depend, among other things, on the characteristics of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of selected tRNAs within a cell is generally a reflection of the codons most frequently used in peptide synthesis. Therefore, genes can be tuned based on codon optimization techniques for optimal gene expression within a given organism. Codon frequency tables are readily available, for example, in the "Codon Frequency Database" available at www.kazusa.orjp / codon / , and these tables can be adapted in many ways. See also Nakamura, Y. et al., Nucl. Acids Res. 28:292 (2000).Computer algorithms for codon-optimizing specific sequences for expression within specific host cells are also available; for example, Gene Forge (Aptagen, Jacobus, PA) is similarly available.

[0116] The nucleic acid molecules of this disclosure (including, for example, the smn1 nucleic acid) can be isolated using standard molecular biology techniques. By using all or part of the nucleic acid sequence of interest as a hybridization probe, the nucleic acid molecule can be isolated using standard hybridization and cloning techniques (such as those described in, for example, Sambrook, J., Fritsh, EF., and Maniatis, T., "Molecular Cloning: A Laboratory Manual," 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0117] Nucleic acid molecules for use in the methods of this disclosure can also be isolated by polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based on the sequence of the nucleic acid molecule of interest. Nucleic acid molecules for use in the methods of this disclosure can be amplified according to standard PCR amplification techniques, using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers.

[0118] Furthermore, oligonucleotides corresponding to the target nucleotide sequence can also be chemically synthesized using standard techniques. Numerous methods for the chemical synthesis of polydeoxynucleotides are known, including solid-phase synthesis automated using commercially available DNA synthesizers (see, for example, Itakura et al., U.S. Patent No. 4,598,049; Caruthers et al., U.S. Patent No. 4,458,066; and Itakura, U.S. Patents No. 4,401,796 and 4,373,071, which are incorporated herein by reference). Automated methods for designing synthetic oligonucleotides are available. See, for example, Hoover, DM and Lubowski, J. Nucleic Acids Research, 30(10):e43(2002).

[0119] In other embodiments, the nucleic acid constructs provided herein include nucleic acid sequences encoding SMN protein variants. In some embodiments, the SMN protein variants include amino acid sequences that are identical to SEQ ID NO: 33 by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%.

[0120] It is intended that variants of the proteins described herein can be prepared. For example, peptide variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by the synthesis of the desired polypeptide. Those skilled in the art who recognize the value of such amino acid changes may alter the post-translational process of the peptide.

[0121] A mutation may be a substitution, deletion, or insertion of one or more codons encoding a polypeptide, resulting in a change in its amino acid sequence compared to the original polypeptide.

[0122] Amino acid substitutions may also result from the substitution of one amino acid with another amino acid having similar structural and / or chemical properties, such as a conserved amino acid substitution, like replacing leucine with serine. Mutations can be introduced into the nucleotide sequences encoding the molecules provided herein using standard techniques known to those skilled in the art, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In some embodiments, substitutions, deletions, or insertions include substitutions of fewer than 25 amino acids, fewer than 20 amino acids, fewer than 15 amino acids, fewer than 10 amino acids, fewer than 5 amino acids, fewer than 4 amino acids, fewer than 3 amino acids, or fewer than 2 amino acids compared to the original molecule. In special embodiments, substitutions are conserved amino acid substitutions made at one or more predicted non-essential amino acid residues. Acceptable mutations can be determined by systematically performing amino acid insertions, deletions, or substitutions in the sequence and testing the resulting variants for the activity exhibited by the parent peptide.

[0123] Amino acid insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequences of single or multiple amino acid residues. Examples of terminal insertions include polypeptides with an N-terminal methionyl residue.

[0124] Proteins produced by conservative amino acid substitutions are included in this disclosure. In conservative amino acid substitutions, an amino acid residue is substituted with an amino acid residue having a side chain with a similar charge. As described above, families of amino acid residues having side chains with similar charges are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations may be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and its protein activity can be determined. Conservative substitutions (e.g., within amino acid groups having similar properties and / or side chains) can be performed to maintain or not significantly alter the properties. Examples of substitutions are shown in the table below. [Table 1]

[0125] Amino acids can be grouped according to the similarity of their side-chain properties (see, for example, Lehninger's "Biochemistry" 73-75 (2nd edition, 1975)): (1) Nonpolar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M), (2) Non-charged: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q), (3) Acidic: Asp(D), Glu(E), and (4) Basic: Lys(K), Arg(R), His(H). Separately, native residues may be grouped based on common side-chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro, and (6) Aromatic: Trp, Tyr, Phe.

[0126] For example, any cysteine ​​residues that do not participate in maintaining the proper three-dimensional structure of the polypeptides provided herein can also be substituted with other amino acids, such as alanine or serine, to improve the oxidative stability of the molecule and prevent abnormal crosslinking.

[0127] Non-conservative substitution involves swapping one member of one of these classes with one of another.

[0128] Amino acid insertions include fusions of amino and / or carboxyl terms, as well as intrasequence insertions of single or multiple amino acid residues, ranging in length from one residue to polypeptides containing 100 or more residues. Examples of terminal insertions include polypeptides with an N-terminal methionyl residue.

[0129] Mutations can be induced using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, Biochem J. 237:1-7 (1986), and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques can be applied to cloned DNA to generate polypeptide mutant DNA.

[0130] In another embodiment, provided herein are optimized nucleic acids encoding an SMN protein, comprising the nucleic acid sequence of SEQ ID NO: 35, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 35. In some embodiments, the optimized nucleic acids provided herein further comprise a promoter region comprising SEQ ID NO: 36 or SEQ ID NO: 37. In some special embodiments, provided herein are nucleic acids comprising the nucleic acid of SEQ ID NO: 36 and the nucleic acid of SEQ ID NO: 35. In other special embodiments, provided herein are nucleic acids comprising the nucleic acid of SEQ ID NO: 37 and the nucleic acid of SEQ ID NO: 35.

[0131] (5.2.2. Target segments of endogenous miRNAs) In one embodiment, the nucleic acid construct provided herein comprises, in addition to the nucleic acid encoding the target protein (e.g., SMN protein), a nucleic acid (or a target sequence of miRNA) containing one or more target segments(or segments) of one or more endogenous miRNAs(or segments). As shown in Section 6 below, the inclusion of specific target segments(or segments) of one or more endogenous miRNAs(or segments) has shown advantageous properties in reducing off-target toxicity to gene therapy (e.g., AAV-based gene therapy).

[0132] In some embodiments, the nucleic acid comprises two or more target segments of endogenous miRNAs, the two or more endogenous miRNAs being tissue-specific miRNAs. As used herein, tissue-specific miRNAs are those that are present in greater quantities in one or more specific tissues compared to other parts, such as liver-specific miRNAs and heart-specific miRNAs. In some embodiments, the two or more endogenous miRNAs are specific to the same tissue. In other embodiments, the two or more endogenous miRNAs are specific to different tissues. In some special embodiments, the nucleic acid comprises one or more target segments of one or more liver-specific miRNAs and one or more target segments of one or more heart-specific miRNAs.

[0133] In some embodiments, the nucleic acid includes 1 to 50 target segments. In some embodiments, the nucleic acid includes 1 to 40 target segments. In some embodiments, the nucleic acid includes 1 to 30 target segments. In some embodiments, the nucleic acid includes 1 to 20 target segments. In some embodiments, the nucleic acid includes 1 to 15 target segments. In some embodiments, the nucleic acid includes 5 to 15 target segments. In some embodiments, the nucleic acid includes 7 to 11 target segments.

[0134] A miRNA target segment is a nucleic acid segment that is specifically hybridizable or complementary to a miRNA (e.g., tissue-specific endogenous miRNA). The most common mechanism of hybridization involves hydrogen bonding between complementary nucleic acid bases of nucleic acid molecules (e.g., Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding). Stringent conditions are sequence-dependent and determined by the characteristics and composition of the nucleic acid molecules to be hybridized. Methods for determining whether one sequence is specifically hybridizable with another are well known in this field.

[0135] Endogenous miRNAs and target nucleic acids are complementary if a sufficient number of nucleic acid bases of the endogenous miRNA can form hydrogen bonds with the corresponding nucleic acid bases of the target nucleic acid to produce the desired effect (e.g., antisense inhibition of the target nucleic acid, such as smn1 having the target segment provided herein).

[0136] Even if there are nucleic acid bases that are non-complementary between the miRNA and the smn1 having the target segment, this can be tolerated as long as the miRNA is maintained capable of specifically hybridizing to the target nucleic acid. Furthermore, the miRNA can hybridize to one or more segments of the smn1 having the target segment, such that the intervening or adjacent segments do not participate in hybridization events (e.g., loop structures, mismatches, or hairpin structures).

[0137] In some embodiments, the miRNA or a particular portion thereof provided herein is complementary to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target sequence or portion thereof of the miRNA provided in the nucleic acid construct. The percentage complementarity of a miRNA having a target nucleic acid can be determined using routine methods. For example, a miRNA in which 18 of its 20 nucleic acid bases are complementary to the target region and specifically hybridizes is expressed as having 90 percent complementarity. In this example, the remaining non-complementary nucleic acid bases may be clustered with the complementary nucleic acid bases or scattered, and do not need to be adjacent to each other or to the complementary nucleic acid bases. Therefore, a miRNA with 18 nucleic acid base lengths that has 4 (4) non-complementary nucleic acid bases adjacent to two regions that are perfectly complementary to the target nucleic acid has an overall complementarity of 77.8% with the target nucleic acid and is therefore within the scope of this disclosure. The complementarity percentage of miRNAs containing the target nucleic acid region can be routinely determined using the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656), which are known in this field. Percentage homology, sequence identity, or sequence complementarity can be determined, for example, using the Gap program (Wisconsin Sequence Analysis Package, Unix version 8, Genetics Computer Group, University Research Park, Madison, Wisconsin) with default settings and the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482 489).

[0138] In some embodiments, the miRNA or a particular portion thereof provided herein is perfectly complementary (i.e., 100% complementary) to the target nucleic acid or a particular portion thereof. For example, in some embodiments, the miRNA may be perfectly complementary to the target segment provided herein. As used herein, “perfectly complementary” means that each nucleic acid base of the miRNA can precisely base-pair with the corresponding nucleic acid base of the target nucleic acid. For example, a 20-nucleotide miRNA is perfectly complementary to a 400-nucleotide-length target sequence, provided that there are 20 corresponding nucleic acid base portions of the target nucleic acid that are perfectly complementary to the miRNA. “Perfectly complementary” can also be used with respect to specific portions of a first and / or second nucleic acid. For example, a 20-nucleotide portion of a 30-nucleotide miRNA may be “perfectly complementary” to a 400-nucleotide-length target sequence. The 20 nucleic acid base portions of a 30-nucleotide oligonucleotide are perfectly complementary to the target sequence if the target sequence has a corresponding 20 nucleic acid base portion and each nucleic acid base is complementary to the 20 nucleic acid base portions of the miRNA. In that case, the entire 30-nucleotide miRNA may or may not be perfectly complementary to the target sequence, depending on whether the remaining 10 nucleic acid bases of the miRNA are also complementary to the target sequence.

[0139] The location of the non-complementary nucleic acid base may be at the 5' or 3' end of the miRNA, or at any location between the 5' and 3' ends. Alternatively, one or more non-complementary nucleic acid bases may be located inside the miRNA. If two or more non-complementary nucleic acid bases are present, they may be contiguous (i.e., linked) or discontinuous. In one embodiment, the non-complementary nucleic acid bases are located within the wing segment of a gapmer-type antisense oligonucleotide.

[0140] In some embodiments, a miRNA having a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleic acid bases, or being up to these lengths, contains non-complementary nucleic acid bases (may be more than 8, 7, 6, 5, 4, 3, 2, or 1) relative to the target nucleic acid. In some embodiments, a miRNA having a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleic acid bases, or being up to these lengths, contains non-complementary nucleic acid bases (may be more than 8, 7, 6, 5, 4, 3, 2, or 1) relative to the target nucleic acid.

[0141] The miRNAs provided herein also include those complementary to a portion of the target nucleic acid. As used herein, “portion” means a specific number of consecutive (i.e., linked) nucleic acid bases within a region or segment of the target nucleic acid. “Portion” may also refer to a specific number of consecutive nucleic acid bases of the miRNA. In some embodiments, the miRNA is complementary to at least 8 nucleic acid base portions of the target segment. In some embodiments, the miRNA is complementary to at least 9 nucleic acid base portions of the target segment. In some embodiments, the miRNA is complementary to at least 10 nucleic acid base portions of the target segment. In some embodiments, the miRNA is complementary to at least 11 nucleic acid base portions of the target segment. In some embodiments, the miRNA is complementary to at least 12 nucleic acid base portions of the target segment. In some embodiments, the miRNA is complementary to at least 13 nucleic acid base portions of the target segment. In some embodiments, the miRNA is complementary to at least 14 nucleic acid base portions of the target segment. In some embodiments, the miRNA is complementary to at least 15 nucleic acid base portions of the target segment. Similarly intended are miRNAs that are complementary to at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid base portions of the target segment, or to any two of these ranges defined by these values.

[0142] Table 2 below shows non-exclusive examples of endogenous miRNAs and non-exclusive examples of target segments for those miRNAs. [Table 2]

[0143] In some embodiments, the endogenous miRNA sequence includes SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is at least 80% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is about 80% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is about 81% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is about 82% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is about 83% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is about 84% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is about 85% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is about 86% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is about 87% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is approximately 88% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is approximately 89% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is approximately 90% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is approximately 91% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is approximately 92% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is approximately 93% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is approximately 94% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is approximately 95% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is approximately 96% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is approximately 97% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is approximately 98% identical to SEQ ID NO: 1. In some embodiments, the endogenous miRNA sequence is approximately 99% identical to SEQ ID NO: 1. In some embodiments, the nucleic acid comprises one or more target segments of any miRNA.

[0144] In some embodiments, the endogenous miRNA sequence includes SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is at least 80% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is about 80% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is about 81% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is about 82% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is about 83% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is about 84% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is about 85% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is about 86% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is about 87% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is approximately 88% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is approximately 89% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is approximately 90% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is approximately 91% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is approximately 92% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is approximately 93% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is approximately 94% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is approximately 95% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is approximately 96% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is approximately 97% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is approximately 98% identical to SEQ ID NO: 2. In some embodiments, the endogenous miRNA sequence is approximately 99% identical to SEQ ID NO: 2. In some embodiments, the nucleic acid comprises one or more target segments of any miRNA.

[0145] In some embodiments, the endogenous miRNA sequence includes SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is at least 80% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is about 80% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is about 81% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is about 82% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is about 83% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is about 84% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is about 85% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is about 86% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is about 87% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is approximately 88% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is approximately 89% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is approximately 90% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is approximately 91% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is approximately 92% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is approximately 93% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is approximately 94% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is approximately 95% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is approximately 96% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is approximately 97% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is approximately 98% identical to SEQ ID NO: 3. In some embodiments, the endogenous miRNA sequence is approximately 99% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid comprises one or more target segments of any miRNA.

[0146] In some embodiments, the endogenous miRNA sequence includes SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is at least 80% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is about 80% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is about 81% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is about 82% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is about 83% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is about 84% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is about 85% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is about 86% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is about 87% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is approximately 88% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is approximately 89% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is approximately 90% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is approximately 91% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is approximately 92% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is approximately 93% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is approximately 94% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is approximately 95% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is approximately 96% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is approximately 97% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is approximately 98% identical to SEQ ID NO: 4. In some embodiments, the endogenous miRNA sequence is approximately 99% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid comprises one or more target segments of any miRNA.

[0147] In some embodiments, the endogenous miRNA sequence includes SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is at least 80% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is about 80% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is about 81% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is about 82% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is about 83% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is about 84% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is about 85% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is about 86% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is about 87% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is approximately 88% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is approximately 89% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is approximately 90% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is approximately 91% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is approximately 92% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is approximately 93% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is approximately 94% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is approximately 95% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is approximately 96% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is approximately 97% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is approximately 98% identical to SEQ ID NO: 5. In some embodiments, the endogenous miRNA sequence is approximately 99% identical to SEQ ID NO: 5. In some embodiments, the nucleic acid comprises one or more target segments of any miRNA.

[0148] In some embodiments, the endogenous miRNA sequence includes SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is at least 80% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is about 80% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is about 81% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is about 82% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is about 83% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is about 84% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is about 85% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is about 86% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is about 87% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is approximately 88% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is approximately 89% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is approximately 90% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is approximately 91% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is approximately 92% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is approximately 93% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is approximately 94% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is approximately 95% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is approximately 96% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is approximately 97% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is approximately 98% identical to SEQ ID NO: 6. In some embodiments, the endogenous miRNA sequence is approximately 99% identical to SEQ ID NO: 6. In some embodiments, the nucleic acid comprises one or more target segments of any miRNA.

[0149] In some embodiments, the nucleic acid comprises one or more repeats of a target segment in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleic acid sequence is identical to sequence number 7. In some special embodiments, the nucleic acid comprises one or more repeats of a target segment having sequence number 7. In some embodiments, the nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, ten, or more repeats of any of the above target segments, for example, the target segment having the nucleic acid sequence of sequence number 7.

[0150] In some embodiments, the nucleic acid comprises one or more repeats of a target segment in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleic acid sequence is identical to sequence number 8. In some special embodiments, the nucleic acid comprises one or more repeats of a target segment having sequence number 8. In some embodiments, the nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, ten, or more repeats of any of the above target segments, for example, the target segment having the nucleic acid sequence of sequence number 8.

[0151] In some embodiments, the nucleic acid comprises one or more repeats of a target segment, wherein at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleic acid sequence is identical to sequence number 9. In some special embodiments, the nucleic acid comprises one or more repeats of a target segment having sequence number 9. In some embodiments, the nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, ten, or more repeats of any of the above target segments, for example, the target segment having the nucleic acid sequence of sequence number 9.

[0152] In some embodiments, the nucleic acid comprises one or more repeats of a target segment in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleic acid sequence is identical to sequence number 10. In some special embodiments, the nucleic acid comprises one or more repeats of a target segment having sequence number 10. In some embodiments, the nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, ten, or more repeats of any of the above target segments, for example, the target segment having the nucleic acid sequence of sequence number 10.

[0153] In some embodiments, the nucleic acid comprises one or more repeats of a target segment in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleic acid sequence is identical to sequence number 11. In some special embodiments, the nucleic acid comprises one or more repeats of a target segment having sequence number 11. In some embodiments, the nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, ten, or more repeats of any of the above target segments, for example, the target segment having the nucleic acid sequence of sequence number 11.

[0154] In some embodiments, the nucleic acid comprises one or more repeats of a target segment in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleic acid sequence is identical to sequence number 12. In some special embodiments, the nucleic acid comprises one or more repeats of a target segment having sequence number 12. In some embodiments, the nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, ten, or more repeats of any of the above target segments, for example, the target segment having the nucleic acid sequence of sequence number 12.

[0155] In some embodiments, the nucleic acid comprises at least one target segment of hsa-mir-133a (or hsa-mir-133a-1). In some embodiments, the nucleic acid comprises at least two repeats of the target segment of hsa-mir-133a (or hsa-mir-133a-1). In some embodiments, the nucleic acid comprises at least three repeats of the target segment of hsa-mir-133a (or hsa-mir-133a-1).

[0156] In some embodiments, the nucleic acid comprises at least one target segment of hsa-mir-133a (or hsa-mir-133a-1) and at least one target segment of liver-specific miRNA. In some embodiments, the nucleic acid comprises at least two repeats of the target segment of hsa-mir-133a (or hsa-mir-133a-1) and at least one target segment of liver-specific miRNA. In some embodiments, the nucleic acid comprises at least three repeats of the target segment of hsa-mir-133a (or hsa-mir-133a-1) and at least one target segment of liver-specific miRNA. In some embodiments, the liver-specific miRNA is hsa-mir-122. In some more specific embodiments, the target segment of hsa-mir-133a comprises SEQ ID NO: 11, and the target segment of hsa-mir-122 comprises SEQ ID NO: 10. [Table 3]

[0157] In some embodiments, multiple target segments in the target sequence of the nucleic acid construct may overlap, or they may not overlap. In some embodiments, the target segments within the target region are separated by a number of nucleotides, i.e., about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides, not exceeding that number, not exceeding that number, or within a range defined by any two of the above values. In some embodiments, the target segments within the target sequence are separated by not more than 5 nucleotides, or not exceeding about 5 nucleotides. In some embodiments, the target segments are contiguous.

[0158] In some embodiments, one or more linkers are present between two target segments. In some embodiments, all target segments are linked by linkers. In other embodiments, only portions of the target segments are linked by linkers. In some embodiments, the linkers in the nucleic acid are identical. In other embodiments, different linkers are present in the nucleic acid.

[0159] Examples of linkers are shown in Table 3. In some embodiments, the nucleic acid includes one or more linkers having SEQ ID NO: 13. In some embodiments, the nucleic acid includes one or more linkers having SEQ ID NO: 14. In some embodiments, the nucleic acid includes one or more linkers having SEQ ID NO: 15. In some embodiments, the nucleic acid includes one or more linkers having SEQ ID NO: 16. In some embodiments, the nucleic acid includes one or more linkers having SEQ ID NO: 17. In some embodiments, the nucleic acid includes two or more linkers, each independently selected from SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

[0160] Examples of nucleic acids (or target sequences of miRNAs) containing multiple target segments of multiple endogenous miRNAs are shown in Table 6 of Section 6 below.

[0161] In some embodiments, the nucleic acid (or miRNA target sequence) comprising multiple target segments includes at least one target segment of hsa-mir-208a, at least one target segment of hsa-mir-208b, at least one target segment of hsa-mir-122, and at least one target segment of hsa-mir-133a. In some more specific embodiments, the nucleic acid (or miRNA target sequence) comprising multiple target segments includes two target segments of hsa-mir-208a, two target segments of hsa-mir-208b, three target segments of hsa-mir-122, and three target segments of hsa-mir-133a. In a particular embodiment, the nucleic acid (or miRNA target sequence) comprising multiple target segments includes two repeats of a target segment having the nucleic acid sequence of SEQ ID NO: 8, two repeats of a target segment having the nucleic acid sequence of SEQ ID NO: 9, three repeats of a target segment having the nucleic acid sequence of SEQ ID NO: 10, and three repeats of a target segment having the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, a nucleic acid comprising multiple target segments (or target sequences of miRNA) includes a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 19.

[0162] In some embodiments, a nucleic acid comprising multiple target segments (or target sequences of miRNA) includes a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO: 18.

[0163] In some embodiments, a nucleic acid comprising multiple target segments (or target sequences of miRNA) includes a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO: 20.

[0164] In some embodiments, a nucleic acid comprising multiple target segments (or target sequences of miRNA) includes a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO: 21.

[0165] In some embodiments, the component target segments are in the same order as those of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In other embodiments, the component target segments are in a different order (from 5' to 3') than those of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

[0166] (5.2.3. Synthetic Promoter) As demonstrated in Section 6 below, the specific synthetic promoters provided herein offer remarkable efficacy, particularly in use in AAV-based gene therapies delivering SMN.

[0167] Accordingly, in yet another embodiment, provided herein is a nucleic acid comprising a nucleic acid sequence encoding an SMN protein or a variant thereof, and a nucleic acid region comprising a synthetic promoter including an enhancer and a core promoter.

[0168] In some embodiments, the synthetic promoter includes a CMV enhancer and an hSyn promoter. In some embodiments, the synthetic promoter includes a proC3 enhancer and an hSyn promoter. In some embodiments, the synthetic promoter includes a proA5 enhancer and an hSyn promoter. In other embodiments, the synthetic promoter includes a proB15 enhancer and an hSyn promoter.

[0169] In some embodiments, the synthetic promoter comprises a CMV enhancer and an hSyn promoter, the hSyn promoter comprising a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO: 38, and the CMV enhancer comprising a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO: 39.

[0170] In some embodiments, the synthetic promoter comprises a proC3 enhancer and an hSyn promoter, wherein the hSyn promoter comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO: 38, and the proC3 enhancer comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO: 40.

[0171] In some embodiments, the synthetic promoter comprises a proA5 enhancer and an hSyn promoter, wherein the hSyn promoter comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO: 38, and the proA5 enhancer comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO: 41.

[0172] In some embodiments, the synthetic promoter comprises a proB15 enhancer and an hSyn promoter, wherein the hSyn promoter comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO: 38, and the proB15 enhancer comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO: 42.

[0173] In some embodiments, the SMN protein or its variant includes the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 33.

[0174] In some embodiments, the nucleic acid sequence encoding the SMN protein or a variant thereof includes a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 35.

[0175] Examples of rAAV vectors containing this synthetic promoter are described in Section 6 below.

[0176] In some embodiments, provided herein are rAAV vectors comprising the nucleic acid sequence of SEQ ID NO: 45, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 45.

[0177] In some embodiments, provided herein are rAAV vectors comprising the nucleic acid sequence of SEQ ID NO: 46, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 46.

[0178] In some embodiments, provided herein are rAAV vectors comprising the nucleic acid sequence of SEQ ID NO: 47, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 47.

[0179] In some embodiments, provided herein are rAAV vectors comprising the nucleic acid sequence of Sequence ID No. 48, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with Sequence ID No. 48.

[0180] In some embodiments, provided herein are rAAV vectors comprising the nucleic acid sequence of SEQ ID NO: 49, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 49.

[0181] In some embodiments, provided herein are rAAV vectors comprising the nucleic acid sequence of SEQ ID NO: 50, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 50.

[0182] In some embodiments, provided herein are rAAV vectors comprising the nucleic acid sequence of SEQ ID NO: 51, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 51.

[0183] In some embodiments, provided herein are rAAV vectors comprising the nucleic acid sequence of SEQ ID NO: 52, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 52.

[0184] In some embodiments, provided herein are rAAV vectors comprising the nucleic acid sequence of SEQ ID NO: 53, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with SEQ ID NO: 53.

[0185] (5.3. Recombinant viral vectors and viral particles for gene therapy) Similarly, provided herein are virus-based gene therapies for the delivery of nucleic acids provided herein. Thus, in another embodiment, provided herein are viral vectors (e.g., rAAV vectors) comprising nucleic acids provided herein (including a nucleic acid region encoding a target protein and a nucleic acid region comprising a target segment of miRNA). In yet another embodiment, provided herein are viral particles (e.g., rAAV or rAAV particles) comprising nucleic acids provided herein.

[0186] (5.3.1. Virus-based gene delivery systems) Numerous virus-based systems have been developed for gene transfer into mammalian cells. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and their derivatives. Viral vector technology is well-known in this field and is described, for example, in Sambrook et al.'s work (2001, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory, New York), as well as in other virology and molecular biology manuals.

[0187] In certain embodiments, the viral vectors or viral particles provided herein are derived from adenoviruses. Examples of vectors include those based on or derived from HAd5, ChAd3, HAd26, HAd6, AdCH3NSmut, HAd35, ChAd63, HAd4, and rcAd26. Recombinant adenovirus vectors can be constructed according to methods known in the art. See, for example, O'Connor et al., Virology, 217(1):11-22 (1996); Hardy et al., Journal of Virology, 73(9):7835-7841 (1999); and Hardy et al., Journal of Virology, 71(3):1842-1849 (1997). In some embodiments, longer sequences can be delivered using third-generation adenovirus vectors (also known as “high-capacity adenovirus vectors” (HCAd), helper-dependent, or “gutless” adenovirus vectors) as described herein. In some embodiments, the polynucleotide of interest, e.g., a transgene, is cloned into an adenovirus vector containing only the ITR and packaging signal. The helper adenovirus vector can be co-transfected into HEK cells to produce adenovirus particles. See Lee et al., Genes and Diseases, 4(2):43-63(2007).

[0188] In certain embodiments, the viral vectors or viral particles provided herein are derived from lentiviruses. Examples of vectors include those based on or derived from HIV-1, HIV-2, SIVSM, SIVAGM, EIAV, FIV, VNV, CAEV, or BIV. Lentiviral vectors can be produced based on methods known in the art, e.g., Cribbs et al., BMC Biotechnology, 13:98 (2003); Merten et al., Mol Ther Methods Clin Dev., 13(3):16017 (2016); Durand and Cimarelli, Viruses, 3:132-159 (2011). In some embodiments, third-generation self-inactivating lentiviral vectors are used herein.

[0189] In some embodiments, the viral vectors or viral particles provided herein are derived from herpes simplex virus (HSV). In some embodiments, the herpes simplex virus is herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), or any derivative thereof. Examples of vectors include those based on or derived from HSV-1, HSV-2, CMV, VZV, EBV, and KSHV. HSV-based vectors can be constructed based on methods known in the art, for example, U.S. Patent Nos. 7,078,029, 6,261,552, 5,998,174, 5,879,934, 5,849,572, 5,849,571, 5,837,532, 5,804,413, and 5,658,724, as well as International Patent Application Publications WO91 / 02788, WO96 / 04394, WO98 / 15637, and WO99 / 06583, which are incorporated herein by reference in their entirety.

[0190] In some embodiments, the HSV-based vectors provided herein are amplicon vectors. In other embodiments, the HSV-based vectors provided herein are replication-deficient vectors. In yet another embodiment, the HSV-based vectors provided herein are replication-capable vectors.

[0191] Amplicons are plasmid-derived vectors engineered to contain both an HSV DNA origin (ori) and an HSV cleavage-packaging recognition sequence (pac). When amplicons are transfected into mammalian cells with HSV helper function, they replicate, form head-to-tail concatemers, and are then packaged within viral particles. Two main methods are currently used to generate amplicon particles: one based on infection with a deficient helper HSV, and the other based on transfection of the HSV-1 gene, e.g., a set of pac-deleted duplicate cosmids, or BAC-HSV-1 with pac deletion and ICP27 deletion. In some embodiments, the amplicons used herein can contain large fragments of exogenous DNA (e.g., up to 152 kb) containing multiple copies of the transgene (e.g., up to 15 copies) and are non-toxic.

[0192] In some embodiments, the HSV-based vectors used herein are defective in at least one essential HSV gene, and these HSV-based vectors may also contain deletions of one or more non-essential genes. In some embodiments, the HSV-based vectors are replication-deficient. Most replication-deficient HSV-based vectors contain deletions in which one or more intermediate-early, early, or late HSV genes are removed, thereby inhibiting replication. In other embodiments, the HSV-based vectors are defective in pre-early genes selected from the group consisting of ICP0, ICP4, ICP22, ICP27, ICP47, and combinations thereof. In a particular embodiment, the HSV-based vectors are defective in all of ICP0, ICP4, ICP22, ICP27, and ICP47. Examples of replication-capable vectors include NV-1020 (HSV-1), RAV9395 (HSV-2), AD-472 (HSV-2), NS-gEnull (HSV-1), and ImmunoVEX (HSV-2). Examples of replication-deficient vectors include dl5-29 (HSV-2), dl5-29-41L (HSV-1), DISC-dH (HSV-1 and HSV-2), CJ9gD (HSV-1), TOH-OVA (HSV-1), d106 (HSV-1), d81 (HSV-1), HSV-SIV d106 (HSV-1), and d106 (HSV-1).

[0193] Replication-deficient HSV-based vectors are typically produced in complementary cell lines that provide gene functions necessary for viral replication, which are usually absent in the replication-deficient HSV-based vector itself, at appropriate levels to generate high-titer viral vector stocks. One example of a cell line complements at least one replication-essential gene function, and in some embodiments, all replication-essential gene functions, that are absent in the replication-deficient HSV-based vector. For example, an HSV-based vector lacking ICP0, ICP4, ICP22, ICP27, and ICP47 can be complemented by the human osteosarcoma cell line U2OS. This cell line can also complement non-essential genes (such as UL55) whose absence reduces proliferation or replication efficiency. Complementary cell lines can complement defects in at least one replication-essential gene function encoded by an early region, pre-early region, late region, viral packaging region, virus-associated region, or a combination thereof, including all HSV functions (e.g., functions that enable the proliferation of HSV amplicons containing minimal HSV sequences, such as only the reverse terminal repeat and packaging signal, or only the ITR and HSV promoter). In some embodiments, the cell line further includes complementary genes in a manner that does not overlap with the HSV-based vector, minimizing and substantially eliminating the possibility of the HSV-based vector genome being recombined in the cell's DNA. Thus, because the presence of replicable HSV is minimized, even if unavoidable within the vector stock, it is suitable for certain therapeutic purposes, particularly gene therapy. The construction of complementary cell lines requires standard molecular biology and cell culture techniques well known in this field.

[0194] In certain embodiments, the viral vectors or viral particles provided herein are derived from adeno-associated viruses (AAVs). Further details regarding AAVs are provided in sections 5.3.2 to 5.3.4 below.

[0195] The nucleic acid of interest can be cloned into a vector using any molecular cloning method known in the art, which may include, for example, the use of a restriction endonuclease site and one or more selectability markers. In some embodiments, the nucleic acid is operably ligated to a promoter. Various promoters have been explored for gene expression in mammalian cells, and any promoter known in the art may be used in this disclosure. Promoters can be broadly classified as constitutive promoters or regulatory promoters, such as inducible promoters.

[0196] In some embodiments, the nucleic acids provided herein are operably linked to a constitutive promoter. A constitutive promoter constitutively expresses a heterologous gene (also referred to as a transgene) in a host cell. Examples of constitutive promoters intended herein, but not limited to, include the cytomegalovirus (CMV) promoter, human elongation factor-1 alpha (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), Simian virus 40 early promoter (SV40), and the chicken β-actin promoter (CAGG) in combination with the CMV early enhancer. The efficiency of these constitutive promoters in driving transgene expression has been widely compared in a vast number of studies.

[0197] In some embodiments, the nucleic acids provided herein are operably linked to an inducible promoter. Inducible promoters belong to the category of regulatory promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of genetically engineered immunoeffector cells, the physiological state of genetically engineered immunoeffector cells, inducing factors (i.e., inducers), or combinations thereof.

[0198] In some embodiments, the induction conditions do not induce the expression of endogenous genes in genetically engineered mammalian cells and / or in subjects to whom the pharmaceutical composition is administered. In some embodiments, the induction conditions are selected from the group consisting of induction factors, radiation (ionizing radiation, light, etc.), temperature (heat, etc.), redox state, tumor environment, and activation state of genetically engineered mammalian cells.

[0199] Those skilled in the art will recognize that target cells may require a specific promoter, and such specific promoters may include, but are not limited to, species-specific, induceable, tissue-specific, or cell cycle-specific promoters (Parr et al., Nat. Med. 3:1145-9 (1997), the entire contents of which are incorporated herein by reference). In one embodiment, the promoter is a promoter that is considered efficient in driving the expression of the polynucleotide described herein. Promoter enhances expression in most tissues and includes, but are not limited to, human elongation factor 1α-subunit (EF1α), early cytomegalovirus (CMV), RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, Simian virus 40 (SV40) promoter and CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline-responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, liver-specific chimeric promoter (LSP), telomerase (hTERT) promoter, chicken β-actin (CBA) and its derivatives CAG and mini-CBA, β-glucuronidase (GUSB), or ubiquitin C (UBC). Expression in specific cell types can be restricted using tissue-specific expression elements, such as neural promoters that can be used to restrict expression in neurons, astrocytes, or oligodendrocytes. Non-limiting examples of tissue-specific expression elements for nerve cells include nerve cell-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synapsin (Syn), methyl-CpG binding protein 2 (MeCP2), CaMKII, mGluR2, NFL, NFH, nβ2, PPE, Enk, and the EAAT2 promoter. These promoters can be combined with other short synthetic regulatory elements to generate novel synthetic promoters with center homology in the DNA sequence.

[0200] In some embodiments, the promoter can express heterologous nucleic acids within nerve cells. In some embodiments, the promoter can express heterologous nucleic acids within motor neurons. In some embodiments, the promoter can express heterologous nucleic acids within astrocytes. In some embodiments, the promoter is a human synapsin 1 (hSyn) promoter, or hSyn combined with a neuronal-specific synthetic regulatory element. In some embodiments, the promoter is a glial fibrillary acidic protein (GFAP) or EAAT2 promoter, or GFAP or EAAT2 combined with an astrocyte-specific synthetic regulatory element.

[0201] In one embodiment, the nucleic acid construct includes, but is not limited to, a promoter such as CMV or U6, or CMV or U6 combined with a synthetic regulatory element. As an unspecified example, the promoter in the rAAV vector is a CBA or mini-CBA promoter. As another unspecified example, the promoter in the rAAV vector is a modified mini-CBA promoter. In one embodiment, the rAAV vector has a genetically engineered promoter. In one embodiment, the rAAV vector further includes a synthetic enhancer element.

[0202] In one embodiment, the vector genome includes at least one element that enhances the target specificity and expression of the transgene, such as an intron or a synthetic intron having a modified sequence derived from a mammalian genome (see, for example, Powell et al., “Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy” 2015 (the entire content of which is incorporated herein by reference)). Non-limiting examples of introns include MVM (67-97 bps), factor IX (F.IX) truncated intron 1 (300 bps), β-globin SD / immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor / immunoglobin splice acceptor (500 bps), SV40 late splice donor / splice acceptor (19S / 16S) (180 bps), and hybrid adenovirus splice donor / IgG splice acceptor (230 bps). In one embodiment, the intron may also be a nucleotide of 100-500 length. The introns may also be 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 in length. The promoter may be of a length between 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or between 400 and 500.

[0203] In some embodiments, the vector also includes a selectability marker gene or a reporter gene for selecting cells that express a protein from a population of host cells transfected via the vector. Both the selectability marker and reporter genes may be flanked by appropriate regulatory sequences to enable expression in host cells. For example, the vector may include transcription and translation terminators, start sequences, and promoters useful for regulating the expression of nucleic acid sequences.

[0204] (5.3.2. Recombinant AAV vectors) Furthermore, in certain specific embodiments, the nucleic acids provided herein are delivered by an AAV-based system and are therefore contained within a recombinant AAV vector.

[0205] Any AAV serotype or its variant may be used in this disclosure. Examples of AAV serotypes include, but are not limited to, AAV1 (Genbank accession number NC_002077.1; HC000057.1), AAV2 (Genbank accession number NC_001401.2, JC527779.1), and AAV2i8 (Asokan, A., Literature, 2010, Discov. Med.). 9:399), AAV3 (Genbank accession number NC_001729.1), AAV3-B (Genbank accession number AF028705.1), AAV4 (Genbank accession number NC_001829.1), AAV5 (Genbank accession number NC_006152.1;JC527780.1), AAV6 (Genbank accession number AF028704.1;JC527781.1), AAV7 (Genbank accession number NC_006260.1;JC527782.1), AAV8 (Genbank accession number NC _006261.1;JC527783.1), AAV9 (Genbank accession number AX753250.1;JC527784.1), AAV10 (Genbank accession number AY631965.1), AAVrh10 (Genbank accession number AY243015.1), AAV11 (Genbank accession number AY631966.1), AAV12 (Genbank accession number DQ813647.1), AAV13 (Genbank accession number EU285562.1), AAVLK03, AAVrh74, AAVDJ (Wu This may include Z et al.'s work, J Virol. 80:11393-7 (2006), AAVanc81, Anc82, Anc83, Anc84, Anc110, Anc113, Anc126, or Anc127 (Zin, E. et al.'s work, Cell. Rep. 12:1056 (2016)), AAV_go.1 (Arbetum, AE et al.'s work, J. Virol. 79:15238 (2005)), AAVhu.37, AAVrh8, AAVrh8R, and AAV rh.8 (Wang et al.'s work, Mol. Ther. 18:119-125 (2010)), or their variants.

[0206] AAV variants include, but are not limited to, AAV1 variants (e.g., AAVs containing AAV1 variant capsid protein), AAV2 variants (e.g., AAVs containing AAV2 variant capsid protein), AAV3 variants (e.g., AAVs containing AAV3 variant capsid protein), AAV3-B variants (e.g., AAVs containing AAV3-B variant capsid protein), AAV4 variants (e.g., AAVs containing AAV4 variant capsid protein), AAV5 variants (e.g., AAVs containing AAV5 variant capsid protein), AAV6 variants (e.g., AAVs containing AAV6 variant capsid protein), AAV7 variants (e.g., AAVs containing AAV7 variant capsid protein), AAV8 variants (e.g., AAVs containing AAV8 variant capsid protein), AAVrh8, and AAVrh. This includes 8R (e.g., AAVs containing AAVrh8 or AAVrh8R mutant capsid proteins), AAV9 mutants (e.g., AAVs containing AAV9 mutant capsid proteins), AAV10 mutants (e.g., AAVs containing AAV10 mutant capsid proteins), AAVrh10 mutants (e.g., AAVs containing AAVrh10 mutant capsid proteins), AAV11 mutants (e.g., AAVs containing AAV11 mutant capsid proteins), AAV12 mutants (e.g., AAVs containing AAV12 mutant capsid proteins), AAV13 mutants (e.g., AAVs containing AAV13 mutant capsid proteins), AAVLK03 mutants (e.g., AAVs containing AAVLK03 mutant capsid proteins), or AAVrh74 mutants (e.g., AAVs containing AAVrh74 mutant capsid proteins).

[0207] The recombinant AAV (rAAV) vectors used in this disclosure can be constructed based on known techniques. In some embodiments, the rAAV vector is constructed to include a transcriptionally operably linked component, a regulatory element including a transcription start region, a polynucleotide provided herein, and a transcription termination region. The regulatory element can be selected based on the target cell. In some embodiments, the resulting rAAV vector construct includes a transcriptionally linked component with functional AAV ITR sequences adjacent (5' and 3').

[0208] In some embodiments, a polypeptide provided herein (e.g., encoding an SMN protein) is operably ligated to at least one regulatory sequence. In some embodiments, this regulatory sequence may include, for example, a promoter sequence, an enhancer sequence such as an upstream enhancer sequence (USE), an RNA processing signal such as a splicing signal, a polyadenylation signal sequence, a sequence that stabilizes cytoplasmic mRNA, a post-transcriptional regulatory element (PRE), and / or a microRNA (miRNA) target sequence. In some embodiments, the regulatory sequence may include a sequence that enhances translation efficiency (e.g., a Kozak sequence), a sequence that enhances protein stability, and / or a sequence that enhances protein processing and / or secretion. In some embodiments, a polynucleotide may encode a regulatory miRNA.

[0209] In some embodiments, the control sequence includes a constitutive promoter and / or a regulatory element. In some embodiments, the control sequence includes a tunable promoter and / or a regulatory element. In some embodiments, the control sequence includes a ubiquitous promoter and / or a regulatory element. In some embodiments, the control sequence includes a cell-specific or tissue-specific promoter and / or a regulatory element. In some embodiments, the regulatory element is located at 5' of the protein's coding sequence (i.e., in the 5' untranslated region (5'UTR)). In other embodiments, the regulatory element is located at 3' of the protein's coding sequence (i.e., in the 3' untranslated region (3'UTR)). In some embodiments, the polynucleotide includes more than one regulatory element, for example, two, three, four, or five regulatory elements. If a polynucleotide contains more than one regulatory element, each regulatory element may be located independently at the 5' position, the 3' position, adjacent to the protein coding sequence, or within the protein coding sequence.

[0210] In some embodiments, the regulatory element is an enhancer. In some embodiments, the included regulatory element directs the transcription or expression of the polynucleotide of the protein in vivo. The regulatory element may include a normal regulatory sequence associated with the selected polynucleotide of interest, or a heterogeneous regulatory sequence instead.

[0211] Examples of regulatory sequences include those derived from sequences encoding mammalian or viral genes, such as the neuron-specific enolase promoter, GFAP promoter, SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoters such as the CMV pre-early promoter region (CMVIE), Roussarcoma virus (RSV) promoter, synthetic promoters, and hybrid promoters.

[0212] In some embodiments, the promoter is not cell-specific or tissue-specific. For example, the promoter is considered an ubiquitous promoter. Examples of promoter sequences that can promote expression within multiple intracellular or tissue types include, for example, human elongation factor 1α-subunit (EFla), cytomegalovirus (CMV) pre-initial enhancer and / or promoter, chicken β-actin (CBA) and its derivatives, CAG such as the CBA promoter combined with the S40 intron, β-glucuronidase (GUSB), or ubiquitin C (UBC).

[0213] In some embodiments, the promoter sequence can promote expression within a specific cell type or tissue. For example, in one embodiment, the promoter may be a muscle-specific promoter, such as the mammalian muscle creatine kinase (MCK) promoter, the mammalian desmin (DES) promoter, the mammalian troponin I (TNNI2) promoter, or the mammalian skeletal α-actin (ASKA) promoter. In other embodiments, the promoter sequence may be capable of promoting expression within a neuronal cell or cell type, such as the neuron-specific enolase (NSE), synapsin (Syn), methyl-CpG-binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or heavy chain (NFH), β-globin minigene hb2, preproenkephalin (PPE), enkephalin (Enk), or excitatory amino acid transporter 2 (EAAT2) promoter. In other embodiments, the promoter sequence may promote expression in the liver, for example, an α-1-antitrypsin (hAAT) or thyroxine-binding globulin (TBG) promoter. In yet another embodiment, the promoter sequence may promote expression in cardiac tissue, for example, a cardiomyocyte-specific promoter such as an MHC, cTnT, or CMV-MUC2k promoter.

[0214] In some embodiments, the polynucleotide may include at least one polyadenylation (poly-A) signal sequence known in the art. If a polyadenylation sequence is present, it is generally located between the 3' end of the transgene coding sequence and the 5' end of the 3'ITR. In some embodiments, the polynucleotide further includes a poly-A upstream enhancer sequence on the 5' side of the poly-A signal sequence. In certain examples, the regulatory sequence is a sequence that enhances translation efficiency, such as a Kozak sequence.

[0215] In some embodiments, the polynucleotide includes an intron. In some embodiments, the intron is located within the coding sequence of a protein provided herein. In some embodiments, the intron is located at the 5' or 3' position of the protein coding sequence. In some embodiments, the intron is adjacent to the 5' or 3' end of the protein coding sequence. In some embodiments, the polynucleotide includes two introns. In some embodiments, one intron is located at the 5' position of the protein coding sequence, and another intron is located at the 3' position of the protein coding sequence. In some embodiments, one intron is adjacent to the 5' end of the protein coding sequence, and the second intron is adjacent to the 3' end of the protein coding sequence. In some embodiments, the intron is an SV40 intron, for example, a 5'UTR SV40 intron.

[0216] AAV ITR sequences known in the art can be used in the rAAV vector. In some embodiments, the AAV ITR used in the vector has a wild-type nucleotide sequence. In other embodiments, the AAV ITR sequence used in the vector is not a wild-type sequence, but instead includes, for example, nucleotide insertions, deletions, or substitutions. The AAV ITRs provided herein may be derived from any AAV serotype, including, but not limited to, AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, AAV44-9, or their variants.

[0217] In some embodiments, the 5' and 3' ITRs adjacent to the nucleotide sequence in the rAAV vector provided herein are identical and originate from the same AAV serotype. In other embodiments, the 5' and 3' ITRs adjacent to the nucleotide sequence in the rAAV vector provided herein are different and / or originate from different AAV serotypes.

[0218] In some embodiments, an rAAV vector containing the polynucleotides of adjacent proteins in an AAV ITR can be constructed by directly inserting the polynucleotide of the desired type into the AAV genome, for example, into a cut-out AAV open reading frame, and a specific portion of the AAV genome can be arbitrarily deleted. These methods are described, for example, in the literature by WO1993 / 003769; Kotin (1994), Human Gene Therapy 5:793-801; Shelling and Smith (1994), Gene Therapy 1:165-169; and Zhou et al. (1994), J. Exp. Med. 179:1867-1875.

[0219] In other embodiments, AAV ITRs are excised from the AAV genome or from an AAV vector containing such ITRs, and then fused to the 5' and 3' ends of the polynucleotide sequence of a protein present in another vector using standard ligation techniques.

[0220] In some embodiments, the rAAV vector provided herein comprises a recombinant self-complementary genome. The rAAV comprising the self-complementary genome can typically rapidly form a double-stranded DNA molecule through its partial complementation sequences (e.g., complementing the coding and non-coding strands of the transgene). More specifically, in some embodiments, the rAAV vector provided herein comprises an rAAV genome comprising a first heterologous polynucleotide sequence (e.g., the coding strand of a therapeutic transgene) and a second heterologous polynucleotide sequence (e.g., the non-coding or antisense strand of a therapeutic transgene), wherein the first heterologous polynucleotide sequence can form intra-chain base pairs with the second polynucleotide sequence. In some embodiments, the first and second heterologous polynucleotide sequences are linked by sequences that promote intra-chain base pairing, such as hairpin DNA structures. In some embodiments, the first and second heterologous polynucleotide sequences are linked by a mutant ITR, and the rep protein does not cleave the viral genome at the mutant ITR. rAAV vectors containing self-complementary genomes can be prepared using methods known in the art, such as those described in U.S. Patent Nos. 7,125,717, 7,785,888, 7,790,154, 7,846,729, 8,093,054, and 8,361,457.

[0221] In some embodiments, the polynucleotide molecules in the rAAV vectors provided herein are less than approximately 5 kilobases (kb). In some embodiments, the polynucleotide molecules in the rAAV vectors provided herein are less than approximately 4.5 kb. In some embodiments, the polynucleotide molecules in the rAAV vectors provided herein are less than approximately 4.0 kb. In some embodiments, the polynucleotide molecules in the rAAV vectors provided herein are less than approximately 3.5 kb. In some embodiments, the polynucleotide molecules in the rAAV vectors provided herein are less than approximately 3.0 kb. In some embodiments, the polynucleotide molecules in the rAAV vectors provided herein are less than approximately 2.5 kb.

[0222] In one embodiment (apsect), provided herein is a recombinant AAV (rAAV) vector comprising (i) a first nucleic acid region comprising a transgene, and (ii) a second nucleic acid region comprising one or more target segments of one or more endogenous miRNAs, wherein at least one target segment is a target segment of cardiac endogenous miRNA, and at least one target segment is a target segment of hepatic endogenous miRNA, and the second The nucleic acid region is located at the 3' position of the first nucleic acid region, and the rAAV vector is a recombinant AAV vector comprising a reverse terminal repeat (ITR) derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, or AAV44-9. In some embodiments, the first nucleic acid encodes an SMN or a variant thereof.

[0223] In some specific embodiments, the second nucleic acid region within the rAAV vector contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleic acid sequence identical to SEQ ID NO: 18. In some embodiments, the rAAV vector contains an ITR derived from AAV1. In some embodiments, the rAAV vector contains an ITR derived from AAV2. In some embodiments, the rAAV vector contains an ITR derived from AAV2i8. In some embodiments, the rAAV vector contains an ITR derived from AAV3. In some embodiments, the rAAV vector contains an ITR derived from AAV3-B. In some embodiments, the rAAV vector contains an ITR derived from AAV4. In some embodiments, the rAAV vector includes an ITR derived from AAV5. In some embodiments, the rAAV vector includes an ITR derived from AAV6. In some embodiments, the rAAV vector includes an ITR derived from AAV7. In some embodiments, the rAAV vector includes an ITR derived from AAV8. In some embodiments, the rAAV vector includes an ITR derived from AAVrh8. In some embodiments, the rAAV vector includes an ITR derived from AAVrh8R. In some embodiments, the rAAV vector includes an ITR derived from AAV9. In some embodiments, the rAAV vector includes an ITR derived from AAV10. In some embodiments, the rAAV vector includes an ITR derived from AAVrh10. In some embodiments, the rAAV vector includes an ITR derived from AAV11. In some embodiments, the rAAV vector includes an ITR derived from AAV12. In some embodiments, the rAAV vector includes an ITR derived from AAV13. In some embodiments, the rAAV vector includes an ITR derived from AAV-DJ. In some embodiments, the rAAV vector includes an ITR derived from AAVLK03. In some embodiments, the rAAV vector includes an ITR derived from AAVrh74.

[0224] In some specific embodiments, the second nucleic acid region within the rAAV vector contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleic acid sequence identical to SEQ ID NO: 19. In some embodiments, the rAAV vector contains an ITR derived from AAV1. In some embodiments, the rAAV vector contains an ITR derived from AAV2. In some embodiments, the rAAV vector contains an ITR derived from AAV2i8. In some embodiments, the rAAV vector contains an ITR derived from AAV3. In some embodiments, the rAAV vector contains an ITR derived from AAV3-B. In some embodiments, the rAAV vector contains an ITR derived from AAV4. In some embodiments, the rAAV vector includes an ITR derived from AAV5. In some embodiments, the rAAV vector includes an ITR derived from AAV6. In some embodiments, the rAAV vector includes an ITR derived from AAV7. In some embodiments, the rAAV vector includes an ITR derived from AAV8. In some embodiments, the rAAV vector includes an ITR derived from AAVrh8. In some embodiments, the rAAV vector includes an ITR derived from AAVrh8R. In some embodiments, the rAAV vector includes an ITR derived from AAV9. In some embodiments, the rAAV vector includes an ITR derived from AAV10. In some embodiments, the rAAV vector includes an ITR derived from AAVrh10. In some embodiments, the rAAV vector includes an ITR derived from AAV11. In some embodiments, the rAAV vector includes an ITR derived from AAV12. In some embodiments, the rAAV vector includes an ITR derived from AAV13. In some embodiments, the rAAV vector includes an ITR derived from AAV-DJ. In some embodiments, the rAAV vector includes an ITR derived from AAVLK03. In some embodiments, the rAAV vector includes an ITR derived from AAVrh74.

[0225] In some specific embodiments, the second nucleic acid region within the rAAV vector contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleic acid sequence identical to SEQ ID NO: 20. In some embodiments, the rAAV vector contains an ITR derived from AAV1. In some embodiments, the rAAV vector contains an ITR derived from AAV2. In some embodiments, the rAAV vector contains an ITR derived from AAV2i8. In some embodiments, the rAAV vector contains an ITR derived from AAV3. In some embodiments, the rAAV vector contains an ITR derived from AAV3-B. In some embodiments, the rAAV vector contains an ITR derived from AAV4. In some embodiments, the rAAV vector includes an ITR derived from AAV5. In some embodiments, the rAAV vector includes an ITR derived from AAV6. In some embodiments, the rAAV vector includes an ITR derived from AAV7. In some embodiments, the rAAV vector includes an ITR derived from AAV8. In some embodiments, the rAAV vector includes an ITR derived from AAVrh8. In some embodiments, the rAAV vector includes an ITR derived from AAVrh8R. In some embodiments, the rAAV vector includes an ITR derived from AAV9. In some embodiments, the rAAV vector includes an ITR derived from AAV10. In some embodiments, the rAAV vector includes an ITR derived from AAVrh10. In some embodiments, the rAAV vector includes an ITR derived from AAV11. In some embodiments, the rAAV vector includes an ITR derived from AAV12. In some embodiments, the rAAV vector includes an ITR derived from AAV13. In some embodiments, the rAAV vector includes an ITR derived from AAV-DJ. In some embodiments, the rAAV vector includes an ITR derived from AAVLK03. In some embodiments, the rAAV vector includes an ITR derived from AAVrh74.

[0226] In some specific embodiments, the second nucleic acid region within the rAAV vector contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleic acid sequence identical to SEQ ID NO: 21. In some embodiments, the rAAV vector contains an ITR derived from AAV1. In some embodiments, the rAAV vector contains an ITR derived from AAV2. In some embodiments, the rAAV vector contains an ITR derived from AAV2i8. In some embodiments, the rAAV vector contains an ITR derived from AAV3. In some embodiments, the rAAV vector contains an ITR derived from AAV3-B. In some embodiments, the rAAV vector contains an ITR derived from AAV4. In some embodiments, the rAAV vector includes an ITR derived from AAV5. In some embodiments, the rAAV vector includes an ITR derived from AAV6. In some embodiments, the rAAV vector includes an ITR derived from AAV7. In some embodiments, the rAAV vector includes an ITR derived from AAV8. In some embodiments, the rAAV vector includes an ITR derived from AAVrh8. In some embodiments, the rAAV vector includes an ITR derived from AAVrh8R. In some embodiments, the rAAV vector includes an ITR derived from AAV9. In some embodiments, the rAAV vector includes an ITR derived from AAV10. In some embodiments, the rAAV vector includes an ITR derived from AAVrh10. In some embodiments, the rAAV vector includes an ITR derived from AAV11. In some embodiments, the rAAV vector includes an ITR derived from AAV12. In some embodiments, the rAAV vector includes an ITR derived from AAV13. In some embodiments, the rAAV vector includes an ITR derived from AAV-DJ. In some embodiments, the rAAV vector includes an ITR derived from AAVLK03. In some embodiments, the rAAV vector includes an ITR derived from AAVrh74.

[0227] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO: 22, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 22.

[0228] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO: 23, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 23.

[0229] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO: 24, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 24.

[0230] In some more specific embodiments, provided herein is a vector comprising the nucleic acid sequence of SEQ ID NO: 25, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 25.

[0231] (5.3.3. Recombinant AAV Particles) In another aspect, provided herein is a recombinant AAV (rAAV) or rAAV particle comprising a nucleic acid provided herein and at least one AAV capsid protein. This nucleic acid includes any nucleic acid and rAAV vector described in Sections 5.2 and 5.3.2 above.

[0232] The capsid protein may be derived from the same serotype as the ITR or its derivative. The capsid may also be of a serotype different from the ITR. For example, in certain embodiments, the AAV particles include AAV2 ITR and AAV6 capsid (AAV2 / 6), AAV2 ITR and AAV7 capsid (AAV2 / 7), AAV2 ITR and AAV8 capsid (AAV2 / 8), or AAV2 ITR and AAV9 capsid (AAV2 / 9).

[0233] The native AAV capsid contains AAV VP1, VP2, and VP3 capsid proteins, which are encoded by splicing variants of the AAV cap gene, respectively. Generally, an AAV particle contains three proteins, VP1, VP2, and VP3, where VP2 and VP3 have sequences also included in VP1 because they are truncated versions of VP1. Generally, the amino acid sequence of VP1 defines the serotype of the capsid. Thus, for example, when the VP1 capsid protein encodes the AAV2 VP1 protein, the AAV is of the AAV2 serotype, while when the VP1 capsid protein encodes the AAV8 VP1 protein, the AAV is of the AAV8 serotype.

[0234] In some embodiments, the AAV capsid protein (e.g., VP1, VP2, and / or VP3) in the present rAAV particles is not a native capsid protein. In some embodiments, the AAV capsid protein (e.g., VP1, VP2, and / or VP3) is derived from a native capsid protein.

[0235] In some embodiments, the AAV capsid protein is the VP1 capsid protein. In other embodiments, the AAV capsid protein is the VP2 capsid protein. In other embodiments, the AAV capsid protein is the VP3 capsid protein. In some embodiments, the rAAV particles contain the VP1 capsid protein, the VP2 capsid protein, and / or the VP3 capsid protein. In other embodiments, the rAAV particles contain the VP1 capsid protein, the VP2 capsid protein, and the VP3 capsid protein. In some embodiments, the rAAV particles contain the VP1 capsid protein, the VP2 capsid protein, and / or the VP3 capsid protein, and the capsid proteins of the rAAV particles are of the same serotype. In other embodiments, the rAAV particles comprise VP1 capsid protein, VP2 capsid protein, and VP3 capsid protein, wherein the capsid proteins of the AAV particles are of the same serotype.

[0236] In some embodiments, the capsid protein is a mutant capsid protein. The mutant capsid protein may contain one or more mutations compared to the corresponding reference capsid protein, such as the native parent capsid protein (i.e., the capsid protein from which it is derived), including, for example, amino acid substitutions, amino acid deletions, and heterologous peptide insertions. In some embodiments, the amino acid sequence of the AAV capsid protein is identical to that of the wild-type, reference, or parent AAV capsid protein, except for the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues. In some embodiments, the capsid proteins or AAV particles described herein may be chimeric capsid proteins or AAV particles, each containing two or more protein sequences of AAV serotype capsid proteins or particles.

[0237] In some embodiments, the capsid proteins in the rAAV particles provided herein are derived from the AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, and AAV44-9 capsid proteins. In certain embodiments, the capsid proteins in the rAAV particles provided herein are AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK 03, AAVrh74, AAV44-9 capsid proteins have the same amino acid sequence as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100%.

[0238] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having VP1, VP2 and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV1.

[0239] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having a VP1, VP2 and / or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV2.

[0240] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having VP1, VP2 and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV2i8.

[0241] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having a VP1, VP2 and / or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV3.

[0242] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having a VP1, VP2 and / or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV3-B.

[0243] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having a VP1, VP2 and / or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV4.

[0244] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having a VP1, VP2 and / or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV5.

[0245] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having a VP1, VP2 and / or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV6.

[0246] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having VP1, VP2 and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV7.

[0247] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having a VP1, VP2 and / or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV8.

[0248] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having VP1, VP2 and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAVrh8.

[0249] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having VP1, VP2 and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAVrh8R.

[0250] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having VP1, VP2 and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV9.

[0251] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having VP1, VP2 and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV10.

[0252] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having VP1, VP2 and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAVrh10.

[0253] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having VP1, VP2 and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV11.

[0254] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins having a VP1, VP2, and / or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 100% sequence identity to any amino acid sequence of VP1, VP2, or VP3 of AAV12.

[0255] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins having a VP1, VP2, and / or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 100% sequence identity to any amino acid sequence of VP1, VP2, or VP3 of AAV13.

[0256] In certain embodiments, the AAV particles provided herein comprise VP1, VP2, and / or VP3 capsid proteins having a VP1, VP2, and / or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 100% sequence identity to any amino acid sequence of VP1, VP2, or VP3 of AAV-DJ.

[0257] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having VP1, VP2 and / or VP3 capsid protein sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAVLK03.

[0258] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having a VP1, VP2 and / or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAVrh74.

[0259] In one embodiment, the AAV particles provided herein include VP1, VP2 and / or VP3 capsid proteins having a VP1, VP2 and / or VP3 capsid protein sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 100% sequence identity with respect to any amino acid sequence of VP1, VP2, or VP3 of AAV44-9.

[0260] In some special embodiments, the rAAV particles provided herein include a nucleic acid encoding the target protein provided herein and VP1 of AAV having the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32. In some special embodiments, VP1 has the amino acid sequence of SEQ ID NO: 29. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0261] The rAAV particles described herein may be produced using any suitable method known in the art. For example, host cells (e.g., mammalian cells) can be manipulated to stably express the components necessary for the production of AAV particles. This can be achieved by incorporating a plasmid (or multiple plasmids) containing the AAV rep gene and the AAV cap gene, as well as selectivity markers such as antibiotic resistance genes (e.g., neomycin or ampicillin), into the cell's genome. The cells may be insect cells or mammalian cells, which can then be co-infected with a helper virus (e.g., an adenovirus or baculovirus providing helper function) and an rAAV vector containing 5' and 3' AAV ITRs. The use of selectivity markers enables large-scale production of rAAV. As another non-limiting example, the rep gene and cap gene can be introduced into packaging cells using an adenovirus or baculovirus instead of a plasmid. As yet another non-limiting example, both viral vectors containing the 5' and 3' AAV ITR, as well as the rep and cap genes, can be stably incorporated into the DNA of producer cells, and helper functions can be provided by wild-type adenovirus to produce rAAV.

[0262] AAV helper viruses are viruses that enable AAV to replicate and package by host cells. Helper viruses provide helper functions that facilitate AAV replication. Many helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses such as vaccinia. Adenovirus type 5 (Ad5) of subgroup C is the most commonly used, but adenoviruses encompass numerous different subgroups. Numerous adenoviruses of human, non-human mammalian, and avian origin are publicly known and available from depositaries such as ATCC. Herpesviridae are also available from depositaries such as ATCC and include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Examples of adenovirus helper functions for AAV replication include E1A, E1B, E2A, VA, and E4 or E6 functions.

[0263] A preparation of AAV is described as "substantially free" of helper virus if the ratio of infectious AAV particles to infectious helper virus particles is at least about 10²:1, at least about 10⁴:1, at least about 10⁶:1, or at least about 10⁸:1. The preparation also does not contain equal amounts of helper virus proteins (i.e., proteins that would be present as a result of such levels of helper virus if the aforementioned helper virus particle impurities were present in a disrupted form). Contamination of viral and / or cellular proteins can generally be observed as the presence of Coomassie staining bands on the SDS gel (e.g., the appearance of bands other than those corresponding to AAV capsid proteins VP1, VP2, and VP3).

[0264] In one embodiment, a host cell containing the rAAV vector encodes the target protein provided herein, and an AAV helper function is provided for replicating and encapsulating the polynucleotides adjacent to the AAV ITR, thereby enabling the generation of rAAV particles. The AAV helper function is generally an AAV-derived coding sequence that can be expressed to provide the AAV gene product and then functions in trans for productive AAV replication. The AAV helper function is used herein to complement essential AAV functions that are missing from the rAAV vector. In some embodiments, the AAV helper function includes a major AAV ORF, i.e., the rep and cap coding regions, or one or both of their functional homologs.

[0265] AAV helper function can be introduced into host cells by transfecting the host cells with an AAV helper construct, either before or in parallel with the transfection of the rAAV vector. For example, an AAV helper construct can be used to provide at least transient expression of AAV rep and / or cap genes, complementing AAV functions that are essential for productive AAV infection but are missing. Typically, AAV helper constructs lack an AAV ITR and cannot replicate or package on their own. AAV helper constructs may be in the form of plasmids, phages, transposons, cosmids, viruses, or virions.

[0266] In some embodiments, the host cell may also provide, or is provided with, a non-AAV-derived function or “accessory function” for producing rAAV particles. The accessory function is a non-AAV-derived viral and / or cellular function on which AAV depends for replication, such as non-AAV proteins and RNA necessary for AAV replication, and includes those involved in the activation of AAV gene transcription, step-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis, and AAV capsid assembly. In some embodiments, the virus-based accessory function may be derived from a known helper virus.

[0267] In some embodiments, recombinant AAV particles are produced as a result of infection of host cells with a helper virus and / or accessory functional vector, the produced rAAV particles being infectious replication-deficient viruses and comprising an AAV protein shell in which AAV ITRs encapsulate desired heterologous nucleotide sequences on both sides.

[0268] rAAV particles can be purified from host cells using purification methods known in the art, such as chromatography, CsCl gradients, and other methods described in, for example, U.S. Patent Nos. 6,989,264 and 8,137,948, and International Publication WO2010 / 148143. In some embodiments, residual helper viruses can be inactivated using known methods, such as by heating.

[0269] (5.3.4.Cell) Various host cells can be used to produce the rAAV particles described herein. Suitable host cells for producing AAV particles from the polynucleotides and AAV vectors provided herein include microorganisms, yeast cells, insect cells, and mammalian cells. Typically, such cells can or have been used as recipients of heterologous nucleic acid molecules and can be cultured, for example, in suspension cultures and bioreactors.

[0270] In some embodiments, the cells are mammalian host cells, such as HEK293, HEK293-T, A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, W138, HeLa, 293, Jurkat, 2V6.11, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, and myoblasts.

[0271] In other embodiments, the cells are insect cells, such as Sf9, SF21, SF900+, or mosquito cell lines such as Drosophila cell lines, Aedes albopictus cell lines, silkworm cell lines such as Bombicus mori cell lines, Trichoprusia ní cell lines such as High Five cells, or Lepidopteran cell lines such as Ascalapha odorata cell lines. In some embodiments, the insect cells are cells derived from insect species susceptible to baculovirus infection and include High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, and Ao38. For example, the large-scale production of recombinant AAV within cells such as Sf9 insect cells is described in the literature by Kotin RM. in Hum Mol Genet. 20(R1):R2-R6(2011)doi:10.1093 / hmg / ddr141.Methods for molecular manipulation and expression of polypeptides in insect cells are described, for example, in the literature: Summers and Smith, "A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures," Texas Agricultural Experimental Station Bull. No. 7555, College Station, Texas (1986); King, LA and RD Possee, "The baculovirus expression system," Chapman and Hall, UK (1992); O'Reilly, DR, LK Miller, and VALuckow, "Baculovirus Expression Vectors: A Laboratory Manual," New York (1992); and WH Freeman and Richardson, CD, "Baculovirus Expression Protocols," Methods in Molecular Biology, volume 39 (1995).

[0272] (5.4. Pharmaceutical Compositions) In one aspect, the Disclosure further provides a pharmaceutical composition comprising the vector or viral particle of the Disclosure. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the vector or viral particle provided herein and a pharmaceutically acceptable excipient.

[0273] In some embodiments, provided herein are pharmaceutical compositions comprising a therapeutically effective amount of the rAAV vector provided herein and pharmaceutically acceptable excipients.

[0274] In other embodiments, provided herein are pharmaceutical compositions comprising a therapeutically effective amount of rAAV particles provided herein and pharmaceutically acceptable excipients.

[0275] In specific embodiments, the term “excipient” also means diluent, adjuvant (e.g., (complete or incomplete) Freund’s adjuvant), carrier, or vehicle. Pharmaceutical excipients may be sterile liquids such as water and oils, and may be of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous glucose and glycerol solutions may also be used as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, and malt. These include rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. The compositions may optionally contain small amounts of wetting agents or emulsifiers, or pH buffers. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Examples of suitable pharmaceutical excipients are described in "Remington's Pharmaceutical Sciences" (1990), Mack Publishing Co., Easton, PA. Such compositions would contain, for example, a prophylactic or therapeutically effective amount of the active ingredient provided herein, in a purified form, along with appropriate amounts of excipients to provide a form for appropriate administration to a patient. The formulations need to be adapted to the mode of administration.

[0276] In some embodiments, the choice of excipients is determined to some extent by the specific cells, virus particles, and / or method of administration. Therefore, a variety of suitable formulations exist.

[0277] Typically, acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers, antioxidants such as ascorbic acid, methionine, vitamin E, and sodium metabisulfite; preservatives, isotonic agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents such as EDTA and / or nonionic surfactants.

[0278] Especially when stability is pH-dependent, buffers can be used to adjust the pH to a range that optimizes therapeutic efficacy. Suitable buffers for use in this disclosure include both organic and inorganic acids, as well as their salts. For example, citrates, phosphates, succinates, tartrates, fumarates, glucons, oxalates, lactates, and acetates. Furthermore, buffers may include histidine and trimethylamine salts such as Tris.

[0279] Preservatives can be added to slow the growth of microorganisms. Suitable preservatives for use in this disclosure include octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., benzalkonium chloride, benzalkonium bromide, benzalkonium iodide), benzethonium chloride; thimerosal, phenol, butyl alcohol or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0280] Isotonic agents, sometimes known as "stabilizers," may be present in a composition to adjust or maintain the tonicity of the liquid. When used with large, charged biomolecules such as proteins and antibodies, isotonic agents are often referred to as "stabilizers" because they interact with the charged groups of amino acid side chains, thereby reducing the possibility of intermolecular and intramolecular interactions. Examples of isotonic agents include polyhydric sugar alcohols and trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0281] Examples of additional excipients include (1) bulking agents, (2) dissolution accelerators, (3) stabilizers, and (4) denaturation inhibitors or anti-adhesion agents to container walls. Such excipients include polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitol (e.g., inositol), polyethylene Organic sugars or sugar alcohols such as lycolic acid; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin and dextran.

[0282] Nonionic surfactants or detergents (also known as "wetting agents") may be present to assist in the solubilization of the therapeutic agent and to protect the therapeutic protein from aggregation induced by agitation, and they also allow the formulation to be exposed to shear surface stress without causing denaturation of the therapeutically active protein. Suitable nonionic surfactants include, for example, polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), Pluronic® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. The anionic surfactants that can be used include sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. The cationic detergents include benzalkonium chloride or benzethonium chloride.

[0283] For use in vivo, the pharmaceutical compositions are preferably sterilized. The pharmaceutical compositions may be sterilized by filtration through a sterile filtration membrane. The pharmaceutical compositions described herein can generally be placed in containers having a sterile access port, such as intravenous solution bags or vials with a stopper that can be punctured with a subcutaneous needle.

[0284] The route of administration shall be in accordance with known and accepted methods, for example, by injection or infusion via subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intrafocal, or intra-articular routes, by intravitreous, subretinal injection, local administration, or inhalation, by a single or multiple bolus or infusion over a long period, or by a sustained-release or sustained-release mechanism, in an appropriate manner.

[0285] In another embodiment, the pharmaceutical composition may be provided as a controlled-release or sustained-release system. In one embodiment, a pump can be used to achieve controlled or sustained-release (see, for example, Sefton, Crit. Ref. Biomed. Eng. 14:201-40 (1987); Buchwald et al., Surgery, 88:507-16 (1980); and Saudek et al., N. Engl. J. Med. 321:569-74 (1989)). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the prophylactic or therapeutic agents or compositions provided herein (e.g., "Medical Applications of Controlled Release" (Langer and Wise, eds., 1974); "Controlled Drug Bioavailability, Drug Product Design and Performance" (Smolen and Ball, eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126 (1983); Levy et al., Science 228:190-92 (1985); During et al., Ann. Neurol. 25:351-56 (1989); Howard et al., J. Neurosurg. 71:105-12(1989); see U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326, and International Patent Publications WO99 / 15154 and WO99 / 20253). Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyacrylamide, poly(ethylene glycol), polylactic acid (PLA), poly(lactic acid-co-glycolide) (PLGA), and polyorthoesters.In one embodiment, the polymer used in the sustained-release formulation is inert, free of eluting impurities, has storage stability, is sterilized, and is biodegradable.

[0286] In yet another embodiment, a controlled or sustained-release system can be positioned close to a specific target tissue, such as the nasal passages or lungs, and therefore requires only a portion of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled-release systems are discussed, for example, in Langer, Science 249:1527-33 (1990). Sustained-release formulations comprising one or more of the agents described herein can be prepared using any technique known to those skilled in the art (see, for example, U.S. Patent No. 4,526,938, International Patent Publications WO91 / 05548 and WO96 / 20698, Ning et al., Radiotherapy & Oncology 39:179-89 (1996); Song et al., PDA J. of Pharma. Sci. & Tech. 50:372-97 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54 (1997), and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60 (1997)).

[0287] The pharmaceutical compositions described herein may also contain one or more active compounds or agents as necessary to treat specific conditions. Alternatively, the compositions may further contain cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, or growth inhibitors. Such molecules are preferably present in combinations of amounts effective for the intended purpose.

[0288] The active ingredient may also be encapsulated in microcapsules, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, which may be prepared, for example, by coacervation or interfacial polymerization, to form colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. Such techniques are disclosed in the 18th edition of Remington's Pharmaceutical Sciences.

[0289] Various compositions and delivery systems are known and can be used in the therapeutics provided herein, and these include, but are not limited to, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing the therapeutic molecules provided herein, viral vectors, or nucleic acid constructs as part of other vectors.

[0290] In some embodiments, the pharmaceutical compositions provided herein contain binding molecules and / or viral particles in an effective amount, e.g., a therapeutically effective amount or a prophylactic effective amount, to treat or prevent a disease or disorder. The therapeutic or prophylactic efficacy in some embodiments is monitored by periodically evaluating the treated subject. Repeated administration over several days or more is repeated, depending on the conditions and treatment, until the desired suppression of disease symptoms occurs. However, other dosage regimens may be used and determined.

[0291] (5.5. Method and Use) In another aspect, what is provided herein is a method or use for using a vector or viral particle (rAAV) provided herein.

[0292] Such methods and uses include, for example, therapeutics and uses involving the administration of molecules, rAAV containing them, or compositions to subjects having a disease or disorder. In some embodiments, the molecules, virus particles, and / or compositions are administered in amounts effective for treating the disease or disorder. Uses include the use of virus particles in such methods and therapeutics, and their use in the preparation of pharmaceuticals for carrying out such therapeutics. In some embodiments, the method is carried out by administering virus particles, or compositions containing them, to subjects having or suspected of having a disease or symptoms. In some embodiments, the disease or disorder of the subject is treated by the method.

[0293] In some embodiments, the treatments provided herein cause complete or partial improvement or reduction of a disease or disorder, or symptoms, adverse effects or outcomes, or associated phenotypes. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. These terms include, but are not necessarily, efficacy against, all symptoms or outcomes, or complete cure of the disease, or complete elimination of any symptom, or all symptoms or outcomes.

[0294] When used herein, in some embodiments, the treatments provided herein delay the onset of a disease or disorder, for example, preventing, inhibiting, delaying, blocking, stabilizing, suppressing, and / or postponing the onset of a disease (such as SMA). This delay may be of varying duration depending on the disease history and / or the individual receiving treatment. As will be apparent to those skilled in the art, a sufficient or significant delay is effective in that the individual does not develop the disease or disorder, and can encompass prevention.

[0295] In other embodiments, the methods or uses provided herein prevent disease or disability.

[0296] In some embodiments, the disease or disorder is related to SMN. In some embodiments, the disease or disorder is related to insufficient expression of the SMN protein. In some embodiments, the disease or disorder is related to a defective SMN protein (e.g., a mutant SMN protein). In some embodiments, the vector or viral particle described herein is used to treat a subject with SMA having smn1 deletion and / or mutation. In some embodiments, the subject has one or more smn1 mutations or microdeletions. In some embodiments, the subject has one or more smn1 nonsense mutations. In some embodiments, the subject has one or more smn1 frameshift mutations.

[0297] In some specific embodiments, the disease or disorder is SMA. In some embodiments, the disclosure provides gene therapy for SMN (e.g., SMN1)-related diseases or disorders, such as neuromuscular degenerative diseases, e.g., SMA-I, SMA-II, SMA-III, and SMA-IV. In some embodiments, the disease or disorder is a neuromuscular degenerative disease such as SMA-I, SMA-II, SMA-III, and SMA-IV. In some specific embodiments, the disease or disorder is SMA-I. In some embodiments, the disease or disorder is SMA-II. In other embodiments, the disease or disorder is SMA-III. In yet another embodiment, the disease or disorder is SMA-IV.

[0298] Microdeletions and nonsense mutations in the smn1 gene are the most common genetic factors for SMA-I type. In the vast majority of neurologically healthy individuals, sufficient levels of SMN1 protein expression are detected in the blood. Lack of SMN protein (e.g., SMN1) expression has also been identified as a pathogenesis for other neurodegenerative diseases, including, for example, Parkinson's disease, progressive supranuclear palsy, ataxia, corticobasal syndrome, Huntington's disease-like syndrome, Creutzfeldt-Jakob disease, and Alzheimer's disease. In some embodiments, SMN-related disorders or impairments are SMN protein expression deficiency-related disorders (e.g., SMN1 expression deficiency-related disorders).

[0299] Spinal muscular atrophy (SMA), a premature neuromuscular degenerative disease, is a progressive and incurable disease characterized, for example, by the selective death of motor neurons in the motor cortex, brainstem, and spinal cord. Patients diagnosed with SMA-I develop a progressive muscle phenotype characterized, for example, spasticity, hyperreflexia or hyporeflexia, fasciculations, muscle atrophy, and paralysis. These motor impairments are caused by denervation of the muscles due to the loss of motor neurons. The main pathological features of SMA-I include degeneration of the corticospinal tract and extensive loss of lower motor neurons (LMNs) or anterior horn cells, degeneration and loss of Betz cells and other pyramidal cells in the primary motor cortex, and reactive gliosis in the motor cortex and spinal cord. SMA-I usually leads to death from respiratory failure and / or inflammation within 0.9 to 2 years after diagnosis.

[0300] In some embodiments, symptoms of SMA include, but are not limited to, motor neuron degeneration, muscle weakness, muscle atrophy, muscle rigidity, dyspnea, slurred speech, onset of fasciculations, frontotemporal dementia, and / or premature death, which are improved in the treated subject. In other embodiments, the compositions of this disclosure are applied to one or both of the brain and / or spinal cord. In some embodiments, one or both of muscle coordination and / or muscle function are improved. In some embodiments, the survival time of the subject is extended.

[0301] In some embodiments, overall enhancement of AAV-derived wild-type smn1 or codon-optimized smn1 expression reduces the effect of SMA in the subject.

[0302] In some embodiments, administration of the disclosed composition to a subject may enhance smn1 (e.g., wild-type smn1 or codon-optimized smn1) mRNA transcription in the transduced cells of the subject. In some embodiments, the transcription of wild-type smn1 and / or codon-optimized smn1 is approximately 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, or 500%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40 Enhancement may be possible in transduced cells, including those within the target CNS region or containing specific cells of the target CNS, at levels of ~70%, 40~80%, 40~90%, 40~95%, 40~100%, 50~60%, 50~70%, 50~80%, 50~90%, 50~95%, 50~100%, 60~70%, 60~80%, 60~90%, 60~95%, 60~100%, 70~80%, 70~90%, 70~95%, 70~100%, 80~90%, 80~95%, 80~100%, 90~95%, 90~100%, or 95~100%, 100~500%.

[0303] In some embodiments, the vectors or viral particles described herein may be administered to subjects with early-stage SMA. Early-stage symptoms include, but are not limited to, weak and soft or rigid and spasmodic muscles, muscle spasms and fasciculations, decreased muscle mass (atrophy), fatigue, poor balance, slurred speech, weak grip strength, and / or stumbling while walking. Symptoms may be limited to one area of ​​the body, or mild symptoms may extend to multiple areas. As a non-limiting example, administration of the vectors or particles described herein may reduce the severity and / or onset of symptoms of early-stage SMA.

[0304] In other embodiments, the vectors or viral particles described herein may be administered to subjects in the intermediate or late stages of SMA-I, or the early stages of SMA-II to IV. The intermediate or late stages of SMA-I, or the early stages of SMA-II to IV, include, but are not limited to, more extensive muscular symptoms compared to the early stages, such as some muscles being paralyzed while others are weakened or unaffected, continuous muscle contractions (fasciculations), unused muscles becoming contractured, joints becoming stiff, painful, and deformed, weakness of swallowing muscles potentially leading to choking and further difficulty in feeding and saliva regulation, weakness of masticatory muscles potentially leading to respiratory failure that is more pronounced in a supine position, and / or subjects may exhibit uncontrollable and inappropriate crying-laughing fits (emotional dysregulation). As a non-limiting example, administration of the vectors or viral particles described herein may reduce the severity and / or onset of symptoms of intermediate-stage SMA-I or late-stage SMA-I, or early-stage SMA-II to IV.

[0305] The compositions described herein may be administered to an individual by any route, for example, intravascularly (e.g., intravenous (IV) or intraarterial), directly intraarterial, systemically (e.g., by intravenous injection), or topically (e.g., by intraarterial or intraocular injection). Examples of non-limiting methods of administration include injection into: intravenous (e.g., by infusion pump), intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, percutaneous, transpleural, intraarterial, topical, inhaled (e.g., as a spray mist), mucosal (e.g., via nasal mucosa), subcutaneous, percutaneous, gastrointestinal, intra-articular, intracisional, intraventricular, intracranial, intraurethral, ​​intrahepatic, intratumoral, intravitreous, and subretinal. In some embodiments, the compositions of the present disclosure for the treatment of SMA are administered intravenously, intramuscularly, subcutaneously, intraperitoneally, intrathecally, and / or intraventricularly to a subject in need thereof, enabling the nucleic acids of the present invention to cross one or both of the blood-brain barrier and the blood-spinal cord barrier. In some embodiments, the method includes administering a therapeutically effective amount of the composition of the present disclosure directly to the central nervous system (CNS) of the subject (e.g., using an infusion pump and / or delivery scaffold) (e.g., intraventricular and / or intrathecal). Vectors or viral particles may be used to enhance smn1 gene expression in the subject and / or reduce one or more symptoms of SMA in order to therapeutically treat SMA.

[0306] In some embodiments, a composition of AAV vectors or AAV particles containing the nucleic acid sequences described herein may be administered in a manner that facilitates the composition's entry into the central nervous system and its entry into motor neurons.

[0307] In some embodiments, the AAV vector or AAV particles of the present disclosure may be administered by intramuscular injection.

[0308] In some embodiments, the AAV vector or AAV particles containing the nucleic acids of the present disclosure are administered to a subject by circumferential injection and / or intranasal delivery.

[0309] In some embodiments, the AAV vector or AAV particles comprising the nucleic acids of the foregoing disclosure are administered to a subject by intracranial delivery (e.g., intrathecal or intraventricular administration, e.g., the contents thereof are incorporated herein by reference in their entirety).

[0310] In some embodiments, compositions comprising the AAV vector or particles of the Disclosure are administered intravenously or intracranially to the target central nervous system (CNS). In other embodiments, compositions comprising the AAV vector or particles of the Disclosure are administered to CNS such as nerve cells, motor neurons, microglia, and astrocytes. In other embodiments, compositions comprising the AAV vector or particles of the Disclosure are administered to astrocytes.

[0311] In some embodiments, compositions comprising the AAV vector or particles of the present disclosure may be delivered into specific types of target cells, which include nerve cells, motor neurons; glial cells such as oligodendrocytes, astrocytes, and microglia; and / or other cells surrounding nerve cells, such as T cells.

[0312] In some embodiments, the AAV vector or AAV particles of the present disclosure may be administered in a therapeutically effective dose, for example, a dose sufficient to alleviate and / or prevent at least one symptom associated with the disease, or to provide improvement of the symptom in question.

[0313] In some embodiments, the AAV vector or AAV particles of the present disclosure may be administered to the CNS in therapeutically effective doses to improve the function and / or survival of subjects having SMA. In non-limiting examples, the composition may be administered intravenously and / or intrathecally.

[0314] In some embodiments, the AAV vector or AAV particles of the Disclosure may be administered to a subject (e.g., to the subject's CNS) in a therapeutically effective dose to delay the decline of the subject's function (e.g., determined using known assessment methods such as the SMA Function Rating Scale (SMAFRS)) and / or to extend the subject's ventilator-independent survival (e.g., by reducing mortality or the need for respiratory support). In non-limiting examples, the composition may be administered intravenously and / or intrathecally.

[0315] In some embodiments, the AAV vector or AAV particles of the present disclosure may be administered to the cisterna magna in a therapeutically effective dose to transduce spinal motor neurons and / or astrocytes. In a non-limiting example, the composition may be administered intrathecally.

[0316] In some embodiments, the AAV vector or AAV particles of the present disclosure may be administered by intrathecal injection in a therapeutically effective dose to transduce spinal motor neurons and / or astrocytes. In non-limiting examples, the composition may be administered intrathecally.

[0317] In some embodiments, the AAV vector or AAV particles of the present disclosure may be administered using a bolus injection method.

[0318] In some embodiments, the AAV vector or AAV particles of the present disclosure may be administered using a sustained delivery method lasting several minutes, several hours, or several days. The infusion rate may vary depending on the target, distribution, formulation, or other delivery parameters.

[0319] In some embodiments, the catheter may be positioned at multiple locations on the spine for multi-site delivery. In some embodiments, the AAV vector or AAV particles of the present disclosure may be delivered by continuous infusion and / or bolus infusion. Each delivery site may have a different drug regimen, or the same drug regimen may be used at each delivery site. In an unspecified example, the delivery sites may be the cervical and lumbar regions. In another unspecified example, the delivery site may be the cervical region. In yet another unspecified example, the delivery site may be the lumbar region.

[0320] In some embodiments, subjects may be subjected to spinal anatomical and pathological analysis prior to delivery of the AAV vector or AAV particles described herein. As a non-limiting example, subjects with scoliosis may be given different drug regimens and / or catheter locations compared to subjects without scoliosis.

[0321] In some embodiments, the orientation of the target's spine during delivery of the AAV vector or particles may be perpendicular to the ground. In other embodiments, the orientation of the target's spine during delivery of the AAV vector or AAV particles may be horizontal to the ground.

[0322] In some embodiments, the target vertebra may be angled with respect to the ground during delivery of the AAV vector or AAV particles. The angle of the target vertebra with respect to the ground may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 180 degrees.

[0323] In some embodiments, the delivery method and duration of delivery are selected to provide broad transduction to the spinal cord. As a non-limiting example, intrathecal delivery is used to provide broad transduction along the rostral-caudal length of the spinal cord. As another non-limiting example, multi-site injection provides more uniform transduction along the rostral-caudal length of the spinal cord. As yet another non-limiting example, prolonged injection provides more uniform transduction along the rostral-caudal length of the spinal cord.

[0324] The pharmaceutical compositions of this disclosure may be administered to subjects in any amount effective in reducing, preventing, and / or treating SMN-related disorders (e.g., SMA). The exact required amount may vary from subject to subject, depending on the subject's race, age, and general condition, the severity of the disease, the specific composition, its mode of administration, its mode of activity, etc.

[0325] In some embodiments, the AAV vector or AAV particles of the Disclosure may be administered in any suitable form, such as a liquid solution or suspension, or as a solid suitable for a liquid solution or suspension in a liquid solution, and may be formulated with any suitable and pharmaceutically acceptable excipients. In some embodiments, the AAV vector or particles are formulated. In non-limiting examples, the basicity and / or osmotic pressure of the formulation can be optimized to ensure optimal drug distribution within the central nervous system or a region or component of the central nervous system.

[0326] In some embodiments, the pharmaceutical composition provided herein is a suspension, for example, a refrigerated suspension. In some embodiments, the method further includes stirring the suspension before the administration step to ensure uniform distribution of the suspension. In some embodiments, the method further includes warming the pharmaceutical composition to room temperature before the administration step. The composition may also be administered in a sustained-release formulation. The sustained-release device (e.g., pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.) may be administered by injection or surgically implanted at various locations.

[0327] The compositions of this disclosure are typically formulated in unit dosage forms for ease of administration and uniformity of dosage. However, it will be understood that the total daily dose of the compositions of this disclosure may be determined by the attending physician within the bounds of sound medical judgment. The specific therapeutic efficacy for any particular patient will depend on a variety of factors, including the disorder being treated and its severity; the activity of the specific compound used; the specific composition used; the patient's age, weight, overall health, sex, and diet; the time of administration; the route of administration; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field.

[0328] In some embodiments, the age and sex of the subject can be used to determine the dose of the composition of the Disclosure. As a non-limiting example, older subjects may be administered a higher dose of the composition compared to younger subjects (e.g., 5–10%, 10–20%, 15–30%, 20–50%, 25–50%, or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90%). As another non-limiting example, younger subjects may be administered a higher dose of the composition (e.g., 5-10%, 10-20%, 15-30%, 20-50%, 25-50%, or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90%) compared to older subjects. As yet another non-limiting example, female subjects may be administered a higher dose of the composition (e.g., 5-10%, 10-20%, 15-30%, 20-50%, 25-50%, or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90%) compared to male subjects. As yet another non-limiting example, male subjects may be administered higher doses of the composition compared to female subjects (e.g., 5-10%, 10-20%, 15-30%, 20-50%, 25-50%, or at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90%).

[0329] In some embodiments, the dose of the AAV vector or AAV particles for delivering the nucleic acids of this disclosure can be adapted according to the disease symptom, target, and therapeutic strategy.

[0330] In some embodiments, the concentration of the administered vector or viral particles may vary depending on the manufacturing method and may be selected or optimized based on a concentration determined to be therapeutically effective for a particular route of administration.

[0331] In some embodiments, the vector genome concentration (vg / ml) per milliliter is about 10 8 vg / ml, about 10 9 vg / ml, about 10 10 vg / ml, about 10 11 vg / ml, about 10 12 vg / ml, about 10 13 vg / ml, about 10 14 vg / ml, and about 10 15 vg / ml is selected from the group consisting of. In some embodiments, the concentration is 10 10 vg / ml to 10 14 vg / ml, for example, 10 10 vg / ml to 10 15 vg / ml, 10 10 vg / ml to 10 14 vg / ml, 0 10 vg / ml to 10 13 vg / ml, 10 10 vg / ml to 10 12 vg / ml, 10 10 vg / ml to 10 11 vg / ml, 10 11 vg / ml to 10 14 vg / ml, 10 11 vg / ml to 10 13 vg / ml, 10 11 vg / ml to 10 12 vg / ml, 10 12 vg / ml to 10 14 vg / ml, 10 12 vg / ml to 10 13 vg / ml, 10 13 vg / ml to 10 14 vg / ml, or 10 14 vg / ml to 10 15The concentration is in the range of vg / ml. In some embodiments, the vectors or viral particles provided herein are delivered intravenously, by intracranial injection, or intracisional injection, or intrathecal injection, or intramuscular injection, or intravitreous injection. In some embodiments, the vectors or virus particles provided herein are in volumes between about 0.1 ml and about 20 ml, for example, about 0.1 ml to about 20 ml, about 0.5 ml to about 20 ml, about 1 ml to about 20 ml, about 5 ml to about 20 ml, about 0.1 ml to about 5.0 ml, about 0.1 ml to about 2.0 ml, about 0.1 ml to about 1.0 ml, about 0.1 ml to about 0.8 ml, about 0.1 ml to about 0.6 ml, about 0.1 ml to about 0.4 ml, about 0.1 ml to about 0.2 ml, about 0.2 ml to about 1.0 ml, about 0.2 ml to about 0.8 ml, about 0.2 ml to about 0.6 ml, about 0.2 ml to about 0.6 ml, about 0.2 ml to about 0.4 ml, about 0.4 ml to about 1.0 ml, about 0.4 ml to about 0.8 ml, about 0.4 ml to about 0.6 ml, and about 0.6 It is injected in volumes between ml and approximately 1.0 ml, approximately 0.6 ml and approximately 0.8 ml, approximately 0.8 ml and approximately 1.0 ml, or in volumes of approximately 0.1 ml, approximately 0.2 ml, approximately 0.4 ml, approximately 0.6 ml, approximately 0.8 ml, and approximately 1.0 ml.

[0332] In some embodiments, rAAV containing the nucleic acids described herein is applied to a target at a dose of 1 × 10⁻⁶ 8 ~1 × 10 17 For example, with a dose of vector genome (vg), 1 × 10 9 ~1 × 10 17 Vector genome (vg) or 1 × 10⁻⁶ 14 ~1 × 10 15 For example, with a dose of vector genome (vg), 1 × 10 10 , 2×10 10 , 3 x 10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1 x 10 11 , 2×10 11 , 3 x 10 11、4×10 11 、5×10 11 、6×10 11 、7×10 11 、8×10 11 、9×10 11 、1×10 12 、2×10 12 、3×10 12 、4×10 12 、5×10 12 、6×10 12 、7×10 12 、8×10 12 、9×10 12 、1×10 13 、2×10 13 、3×10 13 、4×10 13 、5×10 13 、6×10 13 、7×10 13 、8×10 13 、9×10 13 、1×10 14 、2×10 14 、3×10 14 、4×10 14 、5×10 14 、6×10 14 、7×10 14 、8×10 14 、9×10 14 、1×10 15、 2×10 15 、3×10 15 、4×10 15 、5×10 15 、6×10 15 、7×10 15 、8×10 15 、9×10 15 、1×10 16 、2×10 16 、3×10 16 、4×10 16 、5×10 16 、6×10 16 、7×10 16 、8×10 16 、9×10 16 、1×10 17、 2×10 17 、3×10 17 、4×10 17 、5×1017 , 6×10 17 , 7×10 17 , 8×10 17 , or 9×10 17 It can be administered in doses containing the vector genome (vg).

[0333] In some embodiments, rAAV containing the nucleic acids described herein is applied to a target at a dose of 1 × 10⁻⁶ 8 ~1 × 10 17 At a dose of vector genome / kg (vg / kg), for example, 1 × 10⁻¹⁶ 13 ~1 × 10 16 For example, a dose of vg / kg, such as 1 × 10 13 , 2×10 13 , 3 x 10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , 9×10 13 , 1 x 10 14 , 2×10 14 , 3 x 10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , or 9×10 14 It can be administered in doses containing vg / kg.

[0334] In some embodiments, one or more additional therapeutic agents may be administered to the subject.

[0335] The efficacy of the compositions described herein can be monitored by several criteria. For example, after treatment using the methods disclosed herein, subjects may be evaluated by one or more clinical parameters, including those described herein, for improvement and / or stabilization and / or delay of the progression of one or more signs or symptoms of a disease state. Examples of such tests are known in the art and include objective and subjective (e.g., subject-reported) methods.

[0336] In some embodiments, the AAV vector or AAV particles of the Disclosure may be delivered to a subject via a single route of administration. In other embodiments, the AAV vector or AAV particles of the Disclosure may be delivered to a subject via multiple administration routes, for example, two, three, four, five, or more than five sites.

[0337] The pharmaceutical composition comprising the AAV vector or AAV particles may be administered as a single daily dose, or the daily dose may be administered in two, three, or four divided doses per day. The compositions provided herein may also be administered multiple times (e.g., two, three, four, or five times) over a period of time (e.g., one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, two years, or three years).

[0338] In some embodiments, compositions comprising the AAV vector or particles of the present disclosure are administered as monotherapy or combination therapy for the treatment of SMA.

[0339] In some embodiments, compositions comprising the AAV vector or particles of the present disclosure may be used in combination with one or more other therapeutic agents. “In combination with” is not intended to imply that the agents must be administered simultaneously and / or formulated for joint delivery, even if these delivery methods are within the scope of the present disclosure. The compositions may be administered simultaneously with, before, or after one or more other desired therapeutic or medical procedures. Generally, each agent will be administered in a dose and / or time schedule determined for that agent.

[0340] In some embodiments, therapeutic agents that can be used in combination with the AAV vectors or particles of this disclosure may be small molecule compounds, including, for example, immunosuppressants, antioxidants, anti-inflammatory agents, anti-apoptotic agents, calcium regulators, anti-glutamates, structural protein inhibitors, and compounds involved in metal ion regulation.

[0341] In some embodiments, compounds for the treatment of SMA that may be used in combination with the vectors or viral particles described herein include, but are not limited to, anti-glutamate agents such as riluzole, topiramate, talampanel, lamotrigine, dextromethorphan, gabapentin, and AMPA antagonists; anti-apoptotic agents such as minocycline, sodium phenylbutyrate, and alimocromol; anti-inflammatory agents such as gangliosides, celecoxib, cyclosporine, azathioprine, cyclophosphamide, plasmaphoresis, glatiramer acetate, and thalidomide; ceftriaxone; beat-lactam antibiotics; pramipexole (dopamine agonist); nimeslide; diazoxide; pyrazolone derivatives; free radical scavengers that inhibit oxidative stress-induced cell death, such as bromocriptine; phenylcarbamate compounds; neuroprotective compounds; and glycopeptides.

[0342] In some embodiments, the therapeutic agents that can be used in combination therapy with the vectors or viral particles described herein may be hormones or variants that can protect against neuronal loss, such as adrenocorticotropic hormone (ACTH) or fragments thereof (e.g., U.S. Patent Publication No. 20130259875); estrogens (e.g., U.S. Patents 6,334,998 and 6,592,845), the contents of which are incorporated herein by reference in their entirety.

[0343] In some embodiments, neurotrophic factors may be used for the treatment of SMA in combination therapy with the AAV vector or AAV particles of this disclosure. Generally, neurotrophic factors are defined as substances that promote the survival, growth, differentiation, proliferation and / or maturation of nerve cells, or that stimulate increased nerve cell activity. In some embodiments, the method further includes the delivery of one or more trophic factors to a subject requiring treatment. The trophic factors may include, but are not limited to, IGF-I, GDNF, BDNF, CTNF, VEGF, coliberine, xaliprodene, thyroid-stimulating hormone-releasing hormone, and ADNF, as well as their variants.

[0344] (5.6. Assay) (5.6.1. mRNA-level analysis) The enhancement of gene levels or expression (e.g., smn1 nucleic acid) can be assayed using various methods known in this field.

[0345] For example, several methods for detecting or quantifying mRNA levels are known in this field. Examples of methods include, but are not limited to, Northern blotting, ribonuclease-protected assays, and PCR-based methods. Using the mRNA sequence of a gene, a probe that is at least partially complementary to the mRNA sequence can be prepared. This probe can then be used to detect mRNA in a sample using any suitable assay, such as a PCR-based method, Northern blotting, or dipstick assay.

[0346] The assay method can be modified depending on the type of mRNA information desired. Examples of methods, though not limited to them, include Northern blotting and PCR-based methods (e.g., qRT-PCR). Methods such as qRT-PCR can also accurately quantify the amount of mRNA in a sample.

[0347] The presence of mRNA in a sample can be determined using any suitable assay platform. For example, the assay may be in the form of a dipstick, membrane, tip, disk, test strip, filter, microsphere, slide, multiwell plate, or optical fiber. The assay system may have a solid support on which nucleic acid corresponding to mRNA is attached. The solid support may include, for example, plastic, silicon, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate, or slide. The components of the assay can be prepared and packaged together as an mRNA detection kit.

[0348] Nucleic acids can be labeled as needed to produce labeled mRNA populations. Generally, samples can be labeled using methods well known in this field (e.g., by using DNA ligase, terminal transferase, or by labeling the RNA backbone). See, for example, Ausubel et al., "Short Protocols in Molecular Biology" (Wiley & Sons, 3rd edition, 1995); and Sambrook et al., "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor, NY, 3rd edition, 2001). In some embodiments, samples are labeled with fluorescent labels. Examples of fluorescent dyes, though not limited to them, include xanthene dyes, fluorescein dyes (e.g., fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE)), rhodamine dyes (e.g., rhodamine 110 (R110), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine 6G (R6G5 or G5), 6-carboxyrhodamine This includes 6G (R6G6 or G6), cyanine dyes (e.g., Cy3, Cy5, and Cy7), Alexa dyes (e.g., Alexa-fluor-555), coumarin, diethylaminocoumarin, umbelliferone, benzimide dyes (e.g., Hoechst 33258), phenanthoridine dyes (e.g., Texas Red), ethidium dyes, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, BODIPY dyes, quinoline dyes, pyrene, fluorescein chlorotriazinyl, eosin dyes, tetramethylrhodamine, lisamin, naphofluorescein, etc.

[0349] A typical mRNA assay method may include the steps of (1) obtaining a target probe bound to a surface, (2) hybridizing a population of mRNA to the surface-bound probe under conditions sufficient to provide specific binding, (3) washing after hybridization to remove nucleic acids that are not specifically bound to the surface-bound probe, and (4) detecting the hybridized mRNA. The reagents used in each of these steps and their operating conditions can vary depending on the specific application.

[0350] Hybridization can be performed under appropriate hybridization conditions, and stringency can be varied as desired. Even under normal conditions, it is sufficient to generate probe / target complexes on a solid surface between complementary binding members, i.e., between the surface-bound target probe and complementary mRNA in the sample. In some embodiments, stringent hybridization conditions may be employed.

[0351] Hybridization is typically performed under stringent hybridization conditions. Standard hybridization techniques (e.g., under conditions sufficient for specific binding of target mRNA in the sample to the probe) are described in Kallioniemi et al., Science, 258:818-821 (1992) and international publication WO93 / 18186. Several guides to general techniques are available, for example, in Tijssen's "Hybridization with Nucleic Acid Probes, Part I and II" (Elsevier, Amsterdam, 1993). For a description of suitable techniques for in situ hybridization, see Gall et al., Meth. Enzymol. 1981, 21:470-480; and Angerer et al., Genetic Engineering: Principles and Methods, Vol 7, pgs 43-65 (Plenum Press, New York, edited by Setlow and Hollaender, 1985). The selection of appropriate conditions, including temperature, salt concentration, polynucleotide concentration, hybridization time, and stringency of washing conditions, will depend on the experimental design, including the sample source, identity of the capture agent, and the expected degree of complementarity, which can be determined as routine experimental matters for those skilled in the art.

[0352] Those skilled in the art will readily recognize that alternative but equivalent hybridization and washing conditions can be used to provide similar stringency conditions.

[0353] After the mRNA hybridization procedure, the surface-bound polynucleotides are typically washed away to remove unbound nucleic acids. Washing can be performed using any convenient washing protocol as described above, provided the washing conditions are those of a standard stringent. Next, the hybridization of the target mRNA to the probe is detected using standard techniques.

[0354] Other methods, such as PCR-based methods, can also be used to detect gene expression. An example of the PCR method can be found in U.S. Patent No. 6,927,024, which is incorporated herein by reference in its entirety. An example of the RT-PCR method can be found in U.S. Patent No. 7,122,799, which is incorporated herein by reference in its entirety. The fluorescent in situ PCR method is described in U.S. Patent No. 7,186,507, which is incorporated herein by reference in its entirety.

[0355] In some embodiments, quantitative reverse transcription PCR (qRT-PCR) can be used for both the detection and quantification of RNA targets (Bustin et al., Clin. Sci. 2005, 109:365-379). Quantitative results obtained by qRT-PCR are generally more informative than qualitative data. Therefore, in some embodiments, qRT-PCR-based assays can be used to measure mRNA levels during cell-based assays. The qRT-PCR method is also useful for monitoring patient treatment. An example of a qRT-PCR-based method can be found, for example, in U.S. Patent No. 7,101,663, which is incorporated herein by reference in its entirety.

[0356] In contrast to analysis using standard reverse transcriptase PCR and agarose gel, qRT-PCR provides quantitative results. An additional advantage of qRT-PCR is its relatively simple and convenient use. Instruments for qRT-PCR, such as the Applied Biosystems 7500, are commercially available, as are reagents, such as TaqMan® "Sequence Detection Chemistry." For example, TaqMan® gene expression assays can be used according to the manufacturer's instructions. These kits are pre-prepared gene expression assays for rapid and reliable detection and quantification of human, mouse, and rat mRNA transcripts. To determine the number of cycles at which the fluorescence signal associated with a specific amplicon accumulation exceeds a threshold (referred to as CT), this data can be analyzed, for example, using the 7500 Real-Time PCR System Sequence Detection Software and a comparative CT relative quantification method. Using this method, the output is expressed as a multiplier change in expression level. In some embodiments, the threshold level can be selected to be automatically determined by the software. In some embodiments, the threshold level is set to be above the baseline but low enough to be within the exponential amplification region of the amplification curve.

[0357] In other embodiments, the target RNA can be detected or quantified by next-generation sequencing (NGS).

[0358] (5.6.2. Analysis of Protein Levels) The amplification of smn1 nucleic acid protein expression can be evaluated by measuring SMN protein levels. Protein levels can be evaluated or quantified by various methods known in the art, such as immunoprecipitation, Western blotting, enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (e.g., caspase activity assay), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS), LC-MS (liquid chromatography-MassSpec), and other methods. Antibodies against the target are specific and available from various suppliers, for example, from the MSRS antibody catalog (Aerie Corporation, Birmingham, Michigan), or can be prepared via conventional monoclonal or polyclonal antibody production methods known in the art. Antibodies useful for detecting mouse, rat, monkey, and human smn1 are commercially available. In the case of MassSpec, protein levels can be measured using labeled or unlabeled methods.

[0359] (5.6.3. In vivo analysis) In vivo assays can be used to evaluate enhanced SMN1 expression, such as improving motor function and respiration while reducing off-target toxicity in the liver and / or heart.

[0360] In some embodiments, the motor function of animals is measured by the ability to recover from immobility in open-field activity. In some embodiments, the respiration of animals is evaluated by whole-body plethysmography, invasive resistance, and compliance measurements.

[0361] In some embodiments, overall survival (OS) and disease-free survival (DFS) are measured by observing the weight and health status of living animals twice a day.

[0362] The tests may be conducted in normal animals or experimental disease models. For administration to animals, oligonucleotides in pharmaceutically acceptable diluents, such as phosphate-buffered saline, can be formulated. Administration includes parenteral routes such as intraperitoneal, intravenous, and subcutaneous. The calculation of oligonucleotide dosage and frequency is within the scope of the skills of those skilled in the art and depends on factors such as the route of administration and animal body weight. After the oligonucleotide treatment period, RNA can be isolated from the target tissue, including liver, heart, spleen, CNS tissue, or CSF, and changes in smn1 nucleic acid expression can be measured, for example, using NGS.

[0363] (5.7. Kits and manufactured goods) Further provided are kits, unit dose products, and manufactured articles containing any of the compositions described herein. In some embodiments, a kit is provided which contains any of the pharmaceutical compositions described herein and preferably provides instructions for use thereof.

[0364] The kit of this application is housed in appropriate packaging. Appropriate packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar bags or plastic bags), etc. The kit may optionally provide additional components such as cushioning material and explanatory information. Accordingly, this application also provides manufactured products including vials (sealed vials, etc.), bottles, jars, flexible packaging, etc.

[0365] The manufactured product may include a container and labels or accompanying documents on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Generally, the container holds a composition effective for treating a disease or disorder described herein (e.g., SMA) and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured with a subcutaneous needle). The label or accompanying document indicates that the composition is used to treat a specific condition in an individual. The label or accompanying document may further include instructions for use for administering the composition to an individual. The label may also indicate instructions for reconstitution and / or use. The container holding the pharmaceutical composition may be a multi-purpose vial that allows for repeated administration (e.g., 2 to 6 doses) of the reconstituted formulation. An accompanying document refers to instructions for use that are customarily included in the market packaging of a therapeutic product and include information regarding indications, usage, dosage, administration method, contraindications, and / or warnings for the use of the therapeutic product. The manufactured article may further include a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0366] The kit or manufactured article may include multiple unit doses of pharmaceutical compositions and instructions for use, packaged in quantities sufficient for storage and use in a pharmacy, such as a hospital pharmacy and a compounding pharmacy.

[0367] For the sake of brevity, certain abbreviations are used herein. One example is the one-letter abbreviations representing amino acid residues. The amino acids, along with their corresponding three-letter and one-letter abbreviations, are as follows: JPEG2026086420000010.jpg141162

[0368] This disclosure is generally disclosed herein using positive language to describe a number of embodiments. This disclosure also specifically includes embodiments in which certain subjects, such as substances or materials, steps and conditions of a method, protocols, procedures, assays or analyses, are all or partially excluded. Accordingly, aspects not expressly included in this disclosure are disclosed herein, even if they are not generally indicated herein in relation to what is not included in this disclosure.

[0369] Numerous embodiments of the present disclosure have been described. Notwithstanding these numerous embodiments, it will be understood that various modifications can be made without departing from the spirit and scope of the present disclosure. Accordingly, the following embodiments are intended to illustrate, and not limit, the scope of the disclosure as described in the claims. [Examples]

[0370] (6. Examples) The following is a description of the various methods and materials used in the research, provided to those skilled in the art to provide a complete disclosure and explanation of how to carry out and use this disclosure. It is not intended to limit the scope of what the inventors consider to be a disclosure, nor is it intended to represent all experiments that have been or may be carried out. Illustrative descriptions written in the present tense are not necessarily performed; rather, they should be understood as demonstrable for producing data relating to the teachings of this disclosure. While efforts have been made to ensure accuracy with respect to numerical values ​​(e.g., quantities, percentages, etc.) used, some experimental errors and deviations should be accounted for.

[0371] (6.1. Example 1: Nucleic acid construct encoding SMN) This example illustrates a representative nucleic acid construct provided herein, comprising a first nucleic acid region encoding an SMN and a second nucleic acid region containing multiple target segments of multiple endogenous microRNAs (miRNAs) (see Figure 1A).

[0372] The coding sequence of human SMN1 (hSMN1) was optimized based on the wild-type SMN1 protein sequence (SEQ ID NO: 33) using algorithms and platforms including GPS technology (ATUM, USA), OptimWiz (GeneWiz, USA), Optimum Gene (GenScript, USA), and PyCUB (J. Kalfon, 2018). Next, the wild-type hSMN1 coding sequence (SEQ ID NO: 34) and the codon-optimized hSMN1 coding sequence (e.g., SEQ ID NO: 35) were cloned to create expression vectors containing the optimized promoter sequence (SEQ ID NO: 36 or SEQ ID NO: 37) provided herein. Codon optimization increased in vitro hSMN1 mRNA expression as measured by transcriptome analysis (Figure 2B), a result consistent with protein expression analysis using Western blotting, which is discussed in more detail below (Figure 2A). In addition, an increase in median survival rate of injected animals was observed, as indicated by in vivo efficacy assays, which will be discussed in more detail below.

[0373] Each mRNA transcript of a target segment used in this nucleic acid construct can be specifically hybridized with liver-specific miRNAs such as hsa-mir-122, and tissue-specific endogenous miRNAs such as heart-specific endogenous miRNAs such as hsa-mir-1-5p, hsa-mir-208a, hsa-mir-208b, hsa-mir-133a, and hsa-mir-448-5p. Examples of target segments for each of the above miRNAs are shown in Table 2 above. Figure 1B shows a specific nucleic acid region containing multiple target segments. Examples of sequences of a second nucleic acid region containing multiple target segments of multiple endogenous miRNAs are also shown in Table 6 below, along with the constituent target segments for each nucleic acid sequence. In Figures 1A, 1B, and Table 6, miR-1 represents the target segment hsa-mir-1-5p, miR-208a represents the target segment hsa-mir-208a-5p, miR-208b represents the target segment hsa-mir-208b-5p, miR-122 represents the target segment hsa-mir-122, miR-133 represents the target segment hsa-mir-133a-1, and miR-488 represents the target segment hsa-mir-488-5p. The 5' (left side) of these example constructs corresponds to the SMN code sequence and its buffer sequence, and the 3' (right side) of these example constructs corresponds to the poly-A sequence and its buffer sequence. Linkers exist between specific target segments. As illustrated, a construct can have multiple repeat sequences. The sequences are provided as examples of constructs, and the order of these target segments is modifiable and is included in this disclosure. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 6-1] [Table 6-2]

[0374] (6.2. Example 2: Construction of an AAV vector containing nucleic acid) In this example, nucleic acid sequences, including those described in Section 6.1, were introduced into an example of an AAV9-derived rAAV vector to create rAAV vectors containing, for example, EXG202, EXG204, EXG205, EXG206, EXG207, EXG209, and EXG211, as shown in Figure 1A. Examples of nucleic acid sequences of the rAAV vectors (i.e., EXG204, EXG207, EXG209, and EXG211) are shown in the table below. The target segments in these rAAV vectors are shown in Tables 6 and 7. Examples of components of the rAAV vectors provided herein are similarly provided in Table 8. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10] [Table 8-1] [Table 8-2]

[0375] (6.3. Example 3: Protein Expression Assay) HEK 293 cells were cultured in 12-well plates containing DMEM supplemented with 10% fetal bovine serum and incubated for 24 hours at 37°C in an incubator with 5% CO2 before the infection procedure. 24 hours before the infection procedure, HEK 293 cells were cultured in 12-well plates at a rate of 4 × 10⁶ cells per well. 5 HEK 293 cells were seeded in 1 mL of DMEM and 10% FBS and incubated in an incubator at 37°C and 5% CO2. The HEK 293 cells were then treated with rAAV-SMN(10) as provided herein. 5 Cells were co-infected with GC / cell and human adenovirus 5 (Ad5) helper virus (10 iu / cell) and incubated at 37°C in a 5% CO2 incubator for 72 hours. Normal 293FT cells were used as a blank control. Cells were harvested after 72 hours, washed with PBS, pelletized by centrifugation, and lysed in cell lysis buffer. Based on the total protein concentration calculated from the BCA assay, samples containing equal amounts of protein (1.25 ug / sample) were individually separated by 4%–12% Bis-Tris gel electrophoresis according to the manufacturer's (Invitrogen) recommendations, then transferred to a nitrocellulose membrane using a power blotter station at 25 V for 7 minutes, and probed with SMN antibody and tubulin antibody.

[0376] The rAAV vectors tested in this embodiment are shown in Table 8 above. For example, EXG101-01-03 contains an hSMN1 sequence (sequence number 36) driven by a promoter sequence (sequence number 35); EXG101-05M contains an hSMN1 sequence (sequence number 37) driven by a promoter sequence (sequence number 35); EXG101-02M contains an hSMN1 sequence (sequence number 37) driven by a promoter sequence (sequence number 34); and EXG101-01M2 contains an hSMN1 sequence (sequence number 36) driven by a promoter sequence (sequence number 34).

[0377] Expression levels were measured by the SMN / tubulin ratio, and the results are shown in Figure 2A.

[0378] (6.4. Example 4: In vivo efficacy assay) The constructs provided herein were tested and analyzed in vivo efficacy assays by treating P0-2 year old mice with spinal muscular atrophy (SMA) carrying two-allele mutations in the motor neuron survival 1 (smn1) gene. In vivo efficacy assays refer to rescue experiments in smn1- / - defective mouse models, measurement of median survival time, and other relevant in vivo parameters such as postural righting and body weight. SMA-I mouse models are defined by the development of progressive muscle weakness associated with the loss of lower motor neurons before the spontaneous acquisition of postural righting or hind leg standing ability.

[0379] As shown in Figures 3-6, mice treated with this construct, e.g., EXG204, demonstrated superior survival rates, open-field activity comparable to untreated wild-type mouse controls, and a very uniform body weight distribution.

[0380] The results also demonstrate that the target segment hsa-mir-133a (e.g., in EXG204 and EXG206) is more effective than the target segments of other miRNAs, such as hsa-mir-1 and hsa-mir-488a (e.g., in EXG202 and EXG205) (see Figure 6). Furthermore, multiple repeats of the target segment hsa-mir-133a, e.g., three repeats, showed better results compared to a single target segment hsa-mir-133a, as exemplified in the comparison between EXG204 and EXG206.

[0381] In summary, this rAAV9 viral vector for smn1 gene delivery expressed human SMN1 protein driven by an optimized promoter sequence, while simultaneously achieving in vivo tolerable hepatotoxicity and cardiotoxicity (>60% better) and maximizing tissue-specific downregulation of SMN1 in the liver and heart through post-transcriptional regulation by a 3' targeting sequence of endogenous microRNA.

[0382] (6.5. Example 5: Structure containing a synthetic promoter) In this study, various nucleic acid constructs containing synthetic promoters were created and tested. Specifically, as shown in Figures 7A and 7B, various enhancers were combined with the core promoter (hSyn) in specific constructs. For example, EXG304 contains the proC3 enhancer and the hSyn promoter; EXG305 contains the proB15 enhancer and the hSyn promoter; EXG306 contains the proA5 enhancer and the hSyn promoter; EXG307 contains the CMV enhancer and the hSyn promoter; and EXG340 is designed by replacing the coding sequence of the SMN1 protein with the codon-optimized sequence of human SMN1, but the other components are the same as EXG307. Various enhancers and promoters are shown in Table 9. The sequences of the rAAV vectors shown in Figure 7B (i.e., EXG301, EXG302, EXG303, EXG304, EXG305, EXG306, and EXG307), EXG340, and EXG341 are shown in Table 10 below. [Table 9-1] [Table 9-2] [Table 9-3] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8] [Table 10-9] [Table 10-10] [Table 10-11] [Table 10-12] [Table 10-13]

[0383] Using the in vivo efficacy assay described in Example 4, 1.98 × 10⁶ 14 Or 3.96 × 10 14 Various constructs with the different promoters described above were examined by single intravenous (IV) administration at dose levels of vg / kg. As shown in Figures 8A and 8B, EXG303, EXG307, and EXG340 showed dose-dependent improvements in survival. All EXG candidates demonstrated consistent therapeutic efficacy compared to GFP-controlled EXG100-07, but EXG307 showed significantly superior efficacy compared to EXG301, EXG204, and EXG101-01. Surprisingly, constructs with different synthetic promoters were administered via single IV at dose levels of 3.96 × 10⁶. 14When administered at vg / kg, the synthetic promoter-containing constructs EXG307 and EXG340 demonstrated a significant improvement in survival rate compared to other promoters, such as EXG304, EXG305, and EXG306. These results demonstrate the superior efficacy of specific combinations of enhancers and specific core promoters, such as CMV enhancers with hSyn promoters, and particularly their superior efficacy when used with SMN1 in AAV vector-mediated gene therapy.

[0384] All patents, published applications, and references cited herein are incorporated as a whole by reference.

[0385] Although exemplary embodiments are specifically shown and described, those skilled in the art will understand that various modifications in form and detail can be made without departing from the scope of embodiments included in the appended claims.

[0386] As stated above, specific embodiments are described herein for illustrative purposes, but it will be understood that various modifications can be made without departing from the concepts and scope provided herein. All references mentioned above are incorporated herein by reference in their entirety.

Claims

1. (i) A first nucleic acid region comprising a nucleic acid sequence encoding the SMN protein or a variant thereof, (ii) A second nucleic acid region comprising one or more target segments of one or more endogenous microRNAs (miRNAs), nucleic acids containing, The second nucleic acid region is located at the 3' position of the first nucleic acid region, and is the nucleic acid.

2. The nucleic acid according to claim 1, wherein the SMN protein or its variant comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence having identity with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of SEQ ID NO:

33.

3. The nucleic acid according to claim 1 or 2, wherein the first nucleic acid region includes a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO:

35.

4. The nucleic acid according to any one of claims 1 to 3, wherein the second nucleic acid region comprises at least one target segment of an endogenous cardiac miRNA.

5. The nucleic acid according to claim 4, wherein the endogenous miRNA of the heart is selected from the group consisting of hsa-mir-1-5p, hsa-mir-208a-5p, hsa-mir-208b-5p, hsa-mir-133a-1, and hsa-mir-488-5p.

6. The nucleic acid according to claim 4, wherein the endogenous miRNA of the heart comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:

6.

7. The nucleic acid according to any one of claims 1 to 6, wherein the second nucleic acid region comprises at least one target segment of an endogenous miRNA of the liver.

8. The nucleic acid according to claim 7, wherein the endogenous miRNA of the liver is hsa-mir-122.

9. The nucleic acid according to claim 7, wherein the endogenous miRNA of the liver comprises a nucleic acid sequence in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to SEQ ID NO:

4.

10. The nucleic acid according to any one of claims 1 to 3, wherein the second nucleic acid region comprises two or more target segments of hsa-mir-133a-1.

11. The nucleic acid according to claim 10, wherein the second nucleic acid region comprises at least three target segments of hsa-mir-133a-1.

12. The nucleic acid according to any one of claims 1 to 3, wherein the second nucleic acid region comprises at least one target segment of hsa-mir-208a-5p, at least one target segment of hsa-mir-208b-5p, at least one target segment of hsa-mir-122, and at least one target segment of hsa-mir-133a-1.

13. The nucleic acid according to claim 12, wherein the second nucleic acid region comprises two target segments of hsa-mir-208a-5p, two target segments of hsa-mir-208b-5p, three target segments of hsa-mir-122, and three target segments of hsa-mir-133a-1.

14. (i) The target segment of hsa-mir-1-5p contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 7. (ii) The target segment of hsa-mir-208a-5p contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 8. (iii) The target segment of hsa-mir-208b-5p contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 9. (iv) The target segment of hsa-mir-122 contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 10. (v) The target segment of hsa-mir-133a-1 contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 11, and / or (vi) The target segment of hsa-mir-488-5p contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 12. The nucleic acid according to any one of claims 5, 8, and 10 to 13.

15. The nucleic acid according to any one of claims 1 to 3, wherein the second nucleic acid region comprises at least three repeats of the nucleic acid sequence of sequence number 11.

16. The nucleic acid according to claim 15, wherein the second nucleic acid region further comprises one or more target segments of endogenous miRNAs of the liver.

17. The aforementioned second nucleic acid region is, (i) Two repeats of the target segment having the nucleic acid sequence of sequence number 8, (ii) Two repeats of the target segment having the nucleic acid sequence of sequence number 9, (iii) Three repeats of the target segment having the nucleic acid sequence of sequence number 10, and (iv) Three repeats of the target segment having the nucleic acid sequence of sequence number 11, A nucleic acid according to any one of claims 1 to 3, comprising:

18. The nucleic acid according to any one of claims 1 to 17, wherein the second nucleic acid region further comprises one or more linkers between target segments, and optionally the linkers comprise 1 to 10 nucleotides.

19. The nucleic acid according to claim 18, wherein the linker comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO:

17.

20. The aforementioned second nucleic acid region is, (i) Nucleic acid sequences in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 18, (ii) Nucleic acid sequences in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 19, (iii) Nucleic acid sequences in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 20, or (iv) Nucleic acid sequences in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 21, A nucleic acid according to any one of claims 1 to 3, comprising:

21. The aforementioned first nucleic acid region is further, (i) A promoter having the nucleic acid sequence of SEQ ID NO: 36 or SEQ ID NO: 37, (ii) A promoter including a CMV enhancer and an hSyn promoter, (iii) A promoter including a proC3 enhancer and an hSyn promoter, (iv) A promoter including a proA5 enhancer and an hSyn promoter, or (v) Promoter including proB15 enhancer and hSyn promoter, A nucleic acid according to any one of claims 1 to 20, comprising: The nucleic acid wherein the hSyn promoter comprises the nucleic acid sequence of SEQ ID NO: 38, the CMV enhancer comprises the nucleic acid sequence of SEQ ID NO: 39, the proC3 enhancer comprises the nucleic acid sequence of SEQ ID NO: 40, the proA5 enhancer comprises the nucleic acid sequence of SEQ ID NO: 41, and / or the proB15 enhancer comprises the nucleic acid sequence of SEQ ID NO:

42.

22. A vector comprising the nucleic acid according to any one of claims 1 to 21.

23. The vector according to claim 22, wherein the vector is a viral vector.

24. The vector according to claim 23, wherein the viral vector is an adeno-associated virus (AAV) vector.

25. The vector according to claim 24, wherein the AAV vector is derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, AAV44-9, or a combination or variant thereof.

26. The vector according to claim 25, wherein the vector is a recombinant AAV9 (rAAV9) vector or a variant thereof.

27. (i) the first nucleic acid region containing the introduced gene, and (ii) A second nucleic acid region comprising one or more target segments of one or more endogenous miRNAs, A recombinant AAV (rAAV) vector containing, At least one target segment is a target segment of endogenous miRNA in the heart, and at least one target segment is a target segment of endogenous miRNA in the liver. The second nucleic acid region is located at the 3' position of the first nucleic acid region, and The rAAV vector is the recombinant AAV vector comprising a reverse terminal repeat (ITR) derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, or AAV44-9.

28. The rAAV vector according to claim 27, wherein the first nucleic acid region comprises a nucleic acid sequence encoding an SMA protein or a variant thereof, which comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% are identical to SEQ ID NO:

33.

29. The nucleic acid according to claim 27 or claim 28, wherein the first nucleic acid region comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO:

35.

30. The rAAV vector according to any one of claims 27 to 29, wherein the endogenous miRNA of the heart is selected from the group consisting of hsa-mir-1-5p, hsa-mir-208a-5p, hsa-mir-208b-5p, hsa-mir-133a-1, and hsa-mir-488-5p, and / or the endogenous miRNA of the liver is hsa-mir-122.

31. (i) The endogenous miRNA of the heart contains a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6, and / or (ii) The endogenous miRNA of the liver contains a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 4. The rAAV vector according to any one of claims 27 to 29.

32. The rAAV vector according to any one of claims 27 to 29, wherein the second nucleic acid region comprises two or more target segments of hsa-mir-133a-1.

33. The rAAV vector according to claim 32, wherein the second nucleic acid region comprises at least three target segments of hsa-mir-133a-1.

34. The rAAV vector according to any one of claims 27 to 29, wherein the second nucleic acid region comprises at least one target segment of hsa-mir-208a-5p, at least one target segment of hsa-mir-208b-5p, at least one target segment of hsa-mir-122, and at least one target segment of hsa-mir-133a-1.

35. The rAAV vector according to any one of claims 27 to 29, wherein the second nucleic acid region comprises two target segments of hsa-mir-208a-5p, two target segments of hsa-mir-208b-5p, three target segments of hsa-mir-122, and three target segments of hsa-mir-133a-1.

36. (i) The target segment of hsa-mir-1-5p contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 7. (ii) The target segment of hsa-mir-208a-5p contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 8. (iii) The target segment of hsa-mir-208b-5p contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 9. (iv) The target segment of hsa-mir-122 contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 10. (v) The target segment of hsa-mir-133a-1 contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 11, and / or (vi) The target segment of hsa-mir-488-5p contains a nucleic acid sequence in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% is identical to sequence number 12. The rAAV vector according to any one of claims 30 and 32-35.

37. The rAAV vector according to any one of claims 27 to 29, wherein the second nucleic acid region comprises at least three repeats of the nucleic acid sequence of sequence number 11.

38. The aforementioned second nucleic acid region is, (i) Two repeats of the target segment having the nucleic acid sequence of sequence number 8, (ii) Two repeats of the target segment having the nucleic acid sequence of sequence number 9, (iii) Three repeats of the target segment having the nucleic acid sequence of sequence number 10, and (iv) Three repeats of the target segment having the nucleic acid sequence of sequence number 11, An rAAV vector according to any one of claims 27 to 29, comprising:

39. The rAAV vector according to any one of claims 27 to 38, wherein the second nucleic acid region further comprises one or more linkers between target segments, and optionally the linkers comprise 1 to 10 nucleotides.

40. The rAAV vector according to claim 39, wherein the linker comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO:

17.

41. The aforementioned second nucleic acid region is, (i) Nucleic acid sequences in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 18, (ii) Nucleic acid sequences in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 19, (iii) Nucleic acid sequences in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 20, or (iv) Nucleic acid sequences in which at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 21, An rAAV vector according to any one of claims 27 to 29, comprising:

42. The aforementioned first nucleic acid region is further, (i) A promoter having the nucleic acid sequence of SEQ ID NO: 36 or SEQ ID NO: 37, (ii) A promoter including a CMV enhancer and an hSyn promoter, (iii) A promoter including a proC3 enhancer and an hSyn promoter, (iv) A promoter including a proA5 enhancer and an hSyn promoter, or (v) Promoter including proB15 enhancer and hSyn promoter, An rAAV vector according to any one of claims 27 to 41, comprising: The rAAV vector wherein, optionally, the hSyn promoter comprises the nucleic acid sequence of SEQ ID NO: 38, the CMV enhancer comprises the nucleic acid sequence of SEQ ID NO: 39, the proC3 enhancer comprises the nucleic acid sequence of SEQ ID NO: 40, the proA5 enhancer comprises the nucleic acid sequence of SEQ ID NO: 41, and / or the proB15 enhancer comprises the nucleic acid sequence of SEQ ID NO:

42.

43. The rAAV vector according to any one of claims 27 to 42, wherein the ITR is derived from AAV9.

44. An rAAV vector comprising the nucleic acid sequence of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO:

25.

45. A nucleic acid comprising a nucleic acid sequence encoding an SMN protein or a variant thereof, and a nucleic acid region comprising a synthetic promoter including an enhancer and a core promoter, optionally, (i) The synthetic promoter comprises a CMV enhancer and an hSyn promoter, (ii) The synthetic promoter comprises a proC3 enhancer and an hSyn promoter, (iii) The synthetic promoter comprises a proA5 enhancer and an hSyn promoter, or (iv) The synthetic promoter comprises a proB15 enhancer and an hSyn promoter, The nucleic acid.

46. The nucleic acid according to claim 45, wherein the hSyn promoter comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 38.

47. The nucleic acid according to claim 45, wherein the CMV enhancer comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 39.

48. The nucleic acid according to claim 45, wherein the proC3 enhancer comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 40.

49. The nucleic acid according to claim 45, wherein the proA5 enhancer comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 41.

50. The nucleic acid according to claim 45, wherein the proB15 enhancer comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical to sequence number 42.

51. The nucleic acid according to any one of claims 45 to 50, wherein the SMN protein or its variant comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence in which at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% are identical to SEQ ID NO:

33.

52. The nucleic acid according to any one of claims 45 to 50, wherein the nucleic acid sequence encoding the SMN protein or a variant thereof includes a nucleic acid sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO:

35.

53. A vector comprising the nucleic acid according to any one of claims 45 to 52.

54. The vector according to claim 53, wherein the vector is a viral vector.

55. The vector according to claim 54, wherein the viral vector is an adeno-associated virus (AAV) vector.

56. The vector according to claim 55, wherein the AAV vector is derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, AAV44-9, or a combination or variant thereof.

57. The vector according to claim 56, wherein the vector is a recombinant AAV9 (rAAV9) vector or a variant thereof.

58. An rAAV vector comprising the nucleic acid sequence of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53, or a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO:

53.

59. (a) a nucleic acid according to any one of claims 1 to 21 and 45 to 52, or an rAAV vector according to any one of claims 27 to 44 and 58, (b) Capsid proteins of AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAV-DJ, AAVLK03, AAVrh74, AAV44-9, or their variants. Recombinant AAV (rAAV) particles containing these particles.

60. The rAAV particle according to claim 59, wherein the capsid protein is the AAV9 capsid protein or a variant thereof.

61. A pharmaceutical composition comprising a nucleic acid according to any one of claims 1 to 21 and 45 to 52, a vector or rAAV vector according to any one of claims 22 to 44 and 53 to 58, or rAAV particles according to any one of claims 45 to 46, 59 and 60, and a pharmaceutically acceptable excipient.

62. A method for enhancing intracellular SMN protein expression, comprising contacting the cells with a nucleic acid according to any one of claims 1 to 21 and 45 to 52, a vector or rAAV vector according to any one of claims 22 to 44 and 53 to 58, rAAV particles according to any one of claims 59 to 60, or a pharmaceutical composition according to claim 61.

63. A method for treating a target disease or disorder, comprising administering to the target a nucleic acid according to any one of claims 1 to 21 and 45 to 52, a vector or rAAV vector according to any one of claims 22 to 44 and 53 to 58, rAAV particles according to any one of claims 59 to 60, or a pharmaceutical composition according to claim 61.

64. The method according to claim 63, wherein the disease or disorder is an SMN-related disease or disorder.

65. The method according to claim 64, wherein the SMN-related disease or disorder is a disease or disorder related to insufficient expression of the SMN protein.

66. The method according to claim 63, wherein the disease or disorder is related to a defective SMN protein.

67. The method according to claim 63, wherein the disease or disorder is related to a deletion and / or mutation of the smn1 gene.

68. The method according to claim 63, wherein the disease or disorder is spinal muscular atrophy (SMA).

69. The method according to claim 63, wherein the disease or disorder is SMA-I type, SMA-II type, SMA-III type, or SMA-IV type.

70. The method according to any one of claims 63 to 69, wherein the subject is under 2 years of age.

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