Compositions and methods for treating ocular pharyngeal muscular dystrophy (OPMD)

Abstract

The present invention relates to a modified adeno-associated virus (AAV) delivery vector comprising a'silent and alternative 'DNA construct, compositions comprising the same, and the use of the modified AAV and compositions for treating ocular pharyngeal dystrophy (OPMD) in a subject suffering from or susceptible to OPMD. In particular, the present invention relates to an AAV having a capsid protein having a modified subunit 1 (VP1) and comprising a'silence and substitution 'DNA construct wherein the'silence and substitution' DNA construct comprises (i) a DNA guided RNAi (ddRNAi) construct encoding a short hairpin microRNA (shmiR) that targets PABPN1 that results in OPMD, and (ii) a PABPN1 construct encoding a functional PABPN1 protein having an mRNA transcript that is not targeted at (i) by the shmiR. The invention also relates to a method of treating OPMD comprising direct injection of the AAV of the invention to the pharyngeal muscle of a subject.

Description

[0001] This application is a divisional application of the invention patent application filed on February 28, 2020, with application number 202080017400.7 and invention title "Composition and method for treating oculopharyngeal muscular dystrophy (OPMD)".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional No. 62 / 812,187, filed February 28, 2019, the entire contents of which are incorporated herein by reference.

[0004] References to sequence lists submitted electronically via EFS-Web

[0005] This application includes a sequence list submitted electronically via EFS-Web (name: "4226_0190001_SeqListing_ST25.txt"; size: 55,600 bytes; created on February 26, 2019), which is incorporated herein by reference in its entirety. Technical Field

[0006] This invention relates to a modified adeno-associated virus (AAV) delivery vector comprising a 'silencing and replacement' DNA construct, compositions comprising the same, and the use of the modified AAV and compositions for treating OPMD in individuals suffering from or susceptible to oculopharyngeal muscular dystrophy (OPMD). Background Technology

[0007] OPMD is an autosomal dominant, slowly progressive, late-onset degenerative muscle disease. The main characteristics are progressive ptosis (drooping eyelid) and dysphagia (dysphagia). The pharyngeal and cricopharyngeal muscles are specific targets of OPMD. Proximal limb weakness tends to follow later in the disease progression. The mutation causing the disease is an aberrant amplification of the (GCN)n trinucleotide repeat in the coding region of poly(A)-binding protein nucleus 1 (PABPN1). This amplification results in the expansion of polyalanine bundles at the N-terminus of the PABPN1 protein: the normal protein contains 10 alanine residues, which are amplified to 11 to 18 mutant alanine residues (expPABPN1). The main pathological marker of the disease is the nuclear aggregates of expPABPN1. The misfolding of the amplified PABPN1 leads to the accumulation of insoluble polymeric fibrous aggregates within the nucleus of affected cells. PABPN1 is a readily aggregated protein, and the alanine-amplified PABPN1 in OPMD exhibits a higher aggregation rate than the wild-type normal protein. However, it remains unclear whether the nuclear aggregates in OPMD have pathological or protective functions due to cellular defense mechanisms.

[0008] Currently, there are no approved treatments, pharmacological or other therapies available for OPMD. Symptomatic surgical interventions can partially correct ptosis and improve swallowing function in individuals with moderate to severe disease. For example, cricopharyngeal myotomy is currently the only possible treatment available to improve swallowing function in these patients. However, this does not correct the progressive degeneration of the pharyngeal muscle system, which often leads to dysphagia and death from asphyxiation.

[0009] Therefore, therapeutic agents are still needed to treat patients who have OPMD and / or are susceptible to OPMD. Summary of the Invention

[0010] This invention is partly based on the inventors' understanding that there are currently no approved therapeutic agents for treating OPMD. Therefore, this invention provides a therapeutic agent for treating OPMD based on a modified adeno-associated virus (AAV) delivery vector comprising a 'silencing and substitution' construct comprising (i) one or more RNAi agents targeting a region of the PABPN1 mRNA transcript that causes OPMD, and (ii) a PABPN1 substitution construct for expressing wild-type (functional) human PABPN1 protein having an mRNA transcript that is not targeted by the RNAi agent of this invention. This invention also provides a method of treating OPMD using the AAV delivery vector and compositions comprising therein.

[0011] According to one example, the present invention provides an adeno-associated virus (AAV) comprising:

[0012] (a) A viral capsid protein from AAV9 comprising a modified subunit 1 (VP1) sequence wherein amino acids at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87; and

[0013] (b) A polynucleotide sequence comprising (i) a DNA-guided RNAi (ddRNAi) construct comprising a nucleic acid including a sequence encoding a short hairpin microRNA (shmiR); and (ii) a PABPN1 construct comprising a nucleic acid including a sequence encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR encoded by the ddRNAi construct.

[0014] In one example, the modified AAV9 VP1 sequence, relative to the AAV9 VP1 sequence shown in SEQ ID NO: 87, includes serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, and serine at position 44. For example, the modified AAV9 VP1 sequence, relative to the sequence shown in SEQ ID NO: 87, may include the following modifications: A1S, A26E, Q40R, K43D, and A44S. In one example, the modified AAV9 VP1 sequence includes the sequence shown in SEQ ID NO: 88.

[0015] In one instance, the viral capsid protein, relative to the full-length wild-type AAV serotype 9 capsid sequence shown in SEQ ID NO: 89, includes mutants A42S, A67E, Q81R, K84D, and A85S. In another instance, the viral capsid protein includes the amino acid sequence shown in SEQ ID NO: 90.

[0016] The present invention also provides an AAV comprising:

[0017] (a) A viral capsid protein from AAV8 comprising a modified subunit 1 (VP1) sequence wherein amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence shown in SEQ ID NO: 91; and

[0018] (b) A polynucleotide sequence comprising (i) a (ddRNAi) construct comprising a nucleic acid including a sequence encoding shmiR; and (ii) a PABPN1 construct comprising a nucleic acid including a sequence encoding a functional PABPN1 protein having an mRNA transcript not targeted by shmiR encoded by the ddRNAi construct.

[0019] In one example, relative to the AAV8 VP1 sequence shown in SEQ ID NO: 91, the modified AAV8 VP1 sequence includes serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, serine at position 44, and lysine at position 64. For example, relative to the sequence shown in SEQ ID NO: 91, the modified AAV8 VP1 sequence may include the following modifications: A1S, A26E, Q40R, K43D, A44S, and Q64K. In one example, the modified AAV8 VP1 sequence includes the sequence shown in SEQ ID NO: 92.

[0020] In one example, the viral capsid protein, relative to the full-length wild-type AAV serotype 8 capsid sequence shown in SEQ ID NO: 93, includes mutants A42S, A67E, Q81R, K84D, A85S, and Q105K. In another example, the viral capsid protein includes the amino acid sequence shown in SEQ ID NO: 94.

[0021] In each of the foregoing examples, the modified viral capsid protein is a delivery vector comprising a polynucleotide construct including both the ddRNAi and PABPN1 constructs. In one example, the polynucleotide sequence comprises both the ddRNAi and PABPN1 constructs in the 5' to 3' direction. In another example, the polynucleotide sequence comprises both the PABPN1 and ddRNAi constructs in the 5' to 3' direction.

[0022] The polynucleotide may also include an inverted terminal repeat (ITR) sequence derived from an AAV serotype. For example, the ITR may be side-joined with sequences including the ddRNAi construct and the PABPN1 construct. In some instances, the ITR is derived from the AAV2 serotype (e.g., SEQ ID NO: 95 and / or SEQ ID NO: 96).

[0023] In one instance, the DNA sequence encoding the functional PABPN1 protein is codon-optimized so that its mRNA transcript is not targeted by the shmiR of the ddRNAi construct. For example, the sequence encoding the functional PABPN1 protein could be the sequence shown in SEQ ID NO: 73.

[0024] In one instance, the ddRNAi construct and the sequence encoding the functional PABPN1 protein are operatively linked to a promoter located upstream of both the ddRNAi construct and the sequence encoding the functional PABPN1 protein. In some instances, the promoter is a muscle-specific promoter.

[0025] In one instance, the shmiR encoded by the ddRNAi construct, or each shmiR, includes:

[0026] Effector sequences with a length of at least 17 nucleotides;

[0027] Effector complementary sequences;

[0028] Stem-loop sequences; and

[0029] Primary microRNA (pri-miRNA) backbone;

[0030] The effector sequence is substantially complementary to the corresponding length of the region in any of the RNA transcripts shown in SEQ ID NO: 1-13.

[0031] In one instance, at least one shmiR encoded by the ddRNAi construct is selected from the group consisting of:

[0032] shmiR includes the effector sequence shown in SEQ ID NO: 15 and the complementary effector sequence shown in SEQ ID NO: 14;

[0033] shmiR includes the effector sequence shown in SEQ ID NO: 17 and the complementary effector sequence shown in SEQ ID NO: 16;

[0034] shmiR includes the effector sequence shown in SEQ ID NO: 19 and the complementary effector sequence shown in SEQ ID NO: 18;

[0035] shmiR includes the effector sequence shown in SEQ ID NO: 21 and the complementary effector sequence shown in SEQ ID NO: 20;

[0036] shmiR includes the effector sequence shown in SEQ ID NO: 23 and the complementary effector sequence shown in SEQ ID NO: 22;

[0037] shmiR includes the effector sequence shown in SEQ ID NO: 25 and the complementary effector sequence shown in SEQ ID NO: 24;

[0038] shmiR includes the effector sequence shown in SEQ ID NO: 27 and the complementary effector sequence shown in SEQ ID NO: 26;

[0039] shmiR includes the effector sequence shown in SEQ ID NO: 29 and the complementary effector sequence shown in SEQ ID NO: 28;

[0040] shmiR includes the effector sequence shown in SEQ ID NO: 31 and the complementary effector sequence shown in SEQ ID NO: 30;

[0041] shmiR includes the effector sequence shown in SEQ ID NO: 33 and the complementary effector sequence shown in SEQ ID NO: 32;

[0042] shmiR includes the effector sequence shown in SEQ ID NO: 35 and the complementary effector sequence shown in SEQ ID NO: 34;

[0043] shmiR including the effector sequence shown in SEQ ID NO: 37 and the complementary effector sequence shown in SEQ ID NO: 36; and

[0044] shmiR includes the effector sequence shown in SEQ ID NO: 39 and the complementary effector sequence shown in SEQ ID NO: 38.

[0045] In one specific instance, the ddRNAi construct encodes an shmiR comprising the effector sequence shown in SEQ ID NO: 31 and the effector complement sequence shown in SEQ ID NO: 30; and an shmiR comprising the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO: 38. For example, the ddRNAi construct may encode an shmiR named shmiR13 as described herein and an shmiR named shmiR17 as described herein.

[0046] In one example, shmiR, or each shmiR, includes the following in the 5' to 3' direction:

[0047] 5' flanking sequence of the pri-miRNA backbone;

[0048] Effector complementary sequences;

[0049] Stem-loop sequence;

[0050] Effect subsequences; and

[0051] The 3' flanking sequence of the pri-miRNA backbone.

[0052] In another instance, shmiR, or each shmiR, includes the following in the 5' to 3' direction:

[0053] 5' flanking sequence of the pri-miRNA backbone;

[0054] Effect subsequence;

[0055] Stem-loop sequence;

[0056] Effector complementary sequences; and

[0057] The 3' flanking sequence of the pri-miRNA backbone.

[0058] In one instance, the stem-loop sequence is the sequence shown in SEQ ID NO: 40.

[0059] In one instance, the pri-miRNA backbone is the pri-miR-30a backbone. For example, the 5' flanking sequence of the pri-miRNA backbone may be the sequence shown in SEQ ID NO: 41, and the 3' flanking sequence of the pri-miRNA backbone may be the sequence shown in SEQ ID NO: 42.

[0060] In one instance, the ddRNAi construct comprises at least two nucleic acids, each encoding a shmiR, wherein each shmiR comprises an effector sequence substantially complementary to the RNA transcript corresponding to the PABPN1 protein that causes OPMD, and wherein each shmiR comprises a distinct effector sequence.

[0061] In one instance, each of at least two nucleic acids in the ddRNAi construct may encode an shmiR comprising an effector sequence substantially complementary to a region of corresponding length in the RNA transcripts shown in one of SEQ ID NOs: 1, 2, 4, 7, 9, 10, and 13. For example, the at least two nucleic acids in the ddRNAi construct are selected from the group consisting of:

[0062] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 15 and the effector complement sequence shown in SEQ ID NO: 14 (shmiR2);

[0063] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 17 and the effector complement sequence shown in SEQ ID NO: 16 (shmiR3);

[0064] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 21 and the effector complement sequence shown in SEQ ID NO: 20 (shmiR5);

[0065] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 27 and the effector complement sequence shown in SEQ ID NO: 26 (shmiR9);

[0066] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 31 and the effector complement sequence shown in SEQ ID NO: 30 (shmiR13);

[0067] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 33 and the effector complement sequence shown in SEQ ID NO: 32 (shmiR14); and

[0068] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO: 38 (shmiR17).

[0069] For example, at least two nucleic acids in the ddRNAi construct can be selected from the group consisting of: a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 56 (shmiR2) or a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 56 (shmiR2); a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 57 (shmiR3) or a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 57 (shmiR3); a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 59 (shmiR5) or a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 59 (shmiR5); a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 62 (shmiR9) or a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 62 (shmiR9); a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 64 (shmiR13); a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 65 (shmiR14) or a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 65 (shmiR14); and a nucleic acid comprising SEQ ID NO: 56 (shmiR2); The DNA sequence shown in SEQ ID NO: 68 (shmiR17) or the nucleic acid consisting of the DNA sequence shown in SEQ ID NO: 68 (shmiR17).

[0070] In one instance, each of at least two nucleic acids in the ddRNAi construct encodes an shmiR comprising an effector sequence substantially complementary to a region of corresponding length in the RNA transcripts shown in one of SEQ ID NO: 2, 9, 10, and 13. For example, the at least two nucleic acids in the ddRNAi construct may be selected from the group consisting of:

[0071] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 17 and the effector complement sequence shown in SEQ ID NO: 16 (shmiR3);

[0072] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 31 and the effector complement sequence shown in SEQ ID NO: 30 (shmiR13);

[0073] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 33 and the effector complement sequence shown in SEQ ID NO: 32 (shmiR14); and

[0074] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO: 38 (shmiR17).

[0075] For example, at least two nucleic acids in the ddRNAi construct may be selected from the group consisting of: a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 57 (shmiR3) or the DNA sequence shown in SEQ ID NO: 57 (shmiR3); a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or the DNA sequence shown in SEQ ID NO: 64 (shmiR13); a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 65 (shmiR14) or the DNA sequence shown in SEQ ID NO: 65 (shmiR14); and a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or the DNA sequence shown in SEQ ID NO: 68 (shmiR17).

[0076] In one specific instance, at least two nucleic acids are selected from nucleic acids comprising a DNA sequence encoding shmiR or composed of a DNA sequence encoding shmiR, where shmiR includes the effector sequence shown in SEQ ID NO: 31 and the effector complement sequence shown in SEQ ID NO: 30 (shmiR13); and nucleic acids comprising a DNA sequence encoding shmiR or composed of a DNA sequence encoding shmiR, where shmiR includes the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO: 38 (shmiR17). For example, at least two nucleic acids may be nucleic acids comprising a DNA sequence shown in SEQ ID NO: 64 (shmiR13) or composed of a DNA sequence shown in SEQ ID NO: 64 (shmiR13); and nucleic acids comprising a DNA sequence shown in SEQ ID NO: 68 (shmiR17) or composed of a DNA sequence shown in SEQ ID NO: 68 (shmiR17).

[0077] In any of the foregoing examples, the ddRNAi construct and the PABPN1 construct are operatively linked to a promoter. In one example, the ddRNAi construct and the PABPN1 construct are operatively linked to the same promoter, such as a muscle-specific promoter. Compositions comprising the AAV of the present invention and one or more pharmaceutically acceptable carriers are also provided.

[0078] The present invention also provides various baculovirus vectors for producing the AAVs of the present invention in insect cells. In one example, the various baculovirus vectors include:

[0079] (a) A first baculovirus vector comprising a nucleic acid molecule encoding an AAV viral capsid protein having a VP1 sequence modified as described herein; and

[0080] (b) A second baculovirus vector comprising a polynucleotide encoding the ddRNAi construct and the PABPN1 construct as described herein, with an AAV inverted terminal repeat (ITR) sequence attached to the side.

[0081] In one example, a first baculovirus vector includes a nucleic acid molecule encoding a viral capsid protein from AAV9, the viral capsid protein including a modified VP1 sequence, wherein amino acids at positions 1, 26, 40, 43, and 44 are sequence-modified relative to the corresponding wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87, and a second baculovirus vector includes a polynucleotide sequence comprising (i) a ddRNAi construct encoding shmiR and (ii) a PABPN1 construct encoding a functional PABPN1 protein having an mRNA transcript not encoded by the ddRNAi construct targeting shmiR.

[0082] In one example, the modified AAV9 VP1 sequence, relative to the AAV9 VP1 sequence shown in SEQ ID NO: 87, includes serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, and serine at position 44. For example, the modified AAV9 VP1 sequence, relative to the sequence shown in SEQ ID NO: 87, may include the following modifications: A1S, A26E, Q40R, K43D, and A44S. In one example, the modified AAV9 VP1 sequence includes the sequence shown in SEQ ID NO: 88.

[0083] In one instance, the viral capsid protein, relative to the full-length wild-type AAV serotype 9 capsid sequence shown in SEQ ID NO: 89, includes mutants A42S, A67E, Q81R, K84D, and A85S. In another instance, the viral capsid protein includes the amino acid sequence shown in SEQ ID NO: 90.

[0084] In another example, a first baculovirus vector includes a nucleic acid molecule encoding a viral capsid protein from AAV8, the viral capsid protein including a modified VP1 sequence, wherein amino acids at positions 1, 26, 40, 43, 44, and 64 are sequence-modified relative to the corresponding wild-type AAV8 VP1 sequence shown in SEQ ID NO: 91, and a second baculovirus vector includes a polynucleotide sequence comprising (i) a ddRNAi construct encoding shmiR and (ii) a PABPN1 construct encoding a functional PABPN1 protein having an mRNA transcript not encoded by the ddRNAi construct that targets shmiR.

[0085] In one example, relative to the AAV8 VP1 sequence shown in SEQ ID NO: 91, the modified AAV8 VP1 sequence includes serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, serine at position 44, and lysine at position 64. For example, relative to the sequence shown in SEQ ID NO: 91, the modified AAV8 VP1 sequence may include the following modifications: A1S, A26E, Q40R, K43D, A44S, and Q64K. In one example, the modified AAV8 VP1 sequence includes the sequence shown in SEQ ID NO: 92.

[0086] In one example, the viral capsid protein, relative to the full-length wild-type AAV serotype 8 capsid sequence shown in SEQ ID NO: 93, includes mutants A42S, A67E, Q81R, K84D, A85S, and Q105K. In another example, the viral capsid protein includes the amino acid sequence shown in SEQ ID NO: 94.

[0087] In each of the foregoing embodiments, the AAV ITR sequence may originate from the same serotype as the viral capsid protein encoded by the nucleic acid molecule within the first baculovirus vector. In another example, the AAV ITR sequence originates from a different AAV serotype, such as AAV2. In some examples, the ITR sequence originates from AAV serotype 2 and includes the sequences shown in SEQ ID NO: 95 and / or SEQ ID NO: 96.

[0088] As described herein, the second baculoviral vector includes a ddRNAi construct encoding one or more shmiRs that target PABPN1. Exemplary ddRNAi constructs encoding shmiRs (including combinations of shmiRs) that target PABPN1 are described herein. In one example, the second baculoviral vector may include ddRNAi constructs encoding shmiR13 and shmiR17, and a polynucleotide construct including a sequence encoding a functional PABPN1 protein that is codon-optimized such that its mRNA transcript is not targeted by the shmiRs of the ddRNAi construct (e.g., the sequence shown in SEQ ID NO: 73). For example, a second baculovirus vector may include a ddRNAi construct comprising a DNA sequence encoding an shmiR including the effector sequence shown in SEQ ID NO: 31 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 31, such as the effector complementary sequence shown in SEQ ID NO: 30 (shmiR13) or a ddRNAi construct thereof, and a nucleic acid comprising a DNA sequence encoding an shmiR, wherein the shmiR includes the effector sequence shown in SEQ ID NO: 39 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 39, such as the effector complementary sequence shown in SEQ ID NO: 38 (shmiR17) or a nucleic acid thereof. For example, the second baculovirus vector may include a ddRNAi construct comprising the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or a nucleic acid composed of the DNA sequence shown in SEQ ID NO: 64 (shmiR13), and a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or a nucleic acid composed of the DNA sequence shown in SEQ ID NO: 68 (shmiR17).

[0089] Based on the example where the first baculovirus vector does not encode the AAV Rep protein, various baculovirus vectors may further include:

[0090] (c) A third baculovirus vector comprising a polynucleotide sequence encoding at least one large AAV Rep protein selected from Rep78 and Rep68 and at least one small AAV Rep protein selected from Rep52 and Rep40.

[0091] At least one of various baculovirus vectors may include a polynucleotide encoding an assembly-activation protein (AAP). In one example, a baculovirus vector encoding a capsid protein includes a polynucleotide encoding AAP. In alternative examples, baculoviruses encoding the Rep protein and / or baculoviruses encoding ddRNAi and PABPN1 constructs include a polynucleotide encoding AAP.

[0092] The present invention also provides a method for producing AAV as described herein in insect cells, the method comprising:

[0093] (i) Insect cells comprising the various baculovirus vectors described herein are cultured in a medium under conditions sufficient to induce AAV production in the cells; and optionally...

[0094] (ii) Recover AAV from the culture medium and / or cells.

[0095] In one instance, the method involves co-infecting insect cells with a baculovirus vector.

[0096] In one example, a method for producing AAV includes recovering AAV from culture medium and / or cells. In another embodiment, a method for producing AAV includes recovering AAV from culture medium and / or cells and then purifying AAV. In one example, AAV is recovered from cells. In one example, AAV is recovered from culture medium. In one example, AAV is recovered from both cells and culture medium.

[0097] The present invention also provides AAV generated by the method described herein.

[0098] The present invention also provides a method for treating subjects suffering from oculopharyngeal muscular dystrophy (OPMD), the method comprising administering the AAV of the present invention or a composition comprising thereof to the subject; wherein the AAV or composition is administered by direct injection into the pharyngeal muscles of the subject. In one example, the AAV of the present invention or a composition comprising thereof is administered by direct injection into the pharyngeal muscles of the subject. For example, the pharyngeal muscles include one or more of the inferior retractor, middle retractor, superior retractor, palatopharyngeal, eustachian tube retractor, stylopharyngeal, or any combination thereof. In one example, the AAV of the present invention or a composition comprising thereof is administered by direct injection into the tongue muscles of the subject. Attached Figure Description

[0099] Figure 1A is a schematic diagram illustrating the construct used to simultaneously silence the endogenous PABPN1 gene and replace it with codon-optimized PABPN1, which is generated by subcloning two shmiRs targeting wtPABPN1 into the 3' untranslated region of the codon-optimized PABPN1 transcript between two pAAV2 ITRs (ITRs not shown in the schematic diagram).

[0100] Figure 1 B is a schematic diagram illustrating the “silencing and substitution” construct (SR-construct), which is designed to simultaneously silence the endogenous PABPN1 gene and substitute it with codon-optimized PABPN1. This codon-optimized PABPN1 is generated by subcloning two shmiRs (shmiR17 and shmiR13) targeting wtPABPN1 into the 3' untranslated region of the codon-optimized PABPN1 transcript in the pAAV2 vector backbone.

[0101] Figure 1 C shows the predicted secondary structure of a representative shmiR construct, including the 5' flanking region, the sense strand of siRNA, the stem / loop linker sequence, the antisense strand of siRNA, and the 3' flanking region.

[0102] Figure 2 This is a schematic diagram illustrating the SR construct. In the SR construct, both the "replacement" and "silence" boxes are inserted into a single vector containing the Spc512 muscle-specific promoter. Two shmiR sequences are inserted into the 3' UTR of the codon-optimized PABPN1 box.

[0103] Figure 3A The expression of shRNA in the tibialis anterior muscle (TA) of A17 mice injected with the SR-construct is shown. RNA was extracted from TA samples 14 weeks after SR-construct administration.

[0104] Figure 3B This study demonstrates the silencing of PABPN1 expression (including expPABPN1) in the TA muscle of A17 mice treated with the SR-construct. RNA was extracted from TA samples 14 weeks after SR-construct administration.

[0105] Figure 3C This demonstrates the restoration of normal PABPN1 levels in the A17 mouse model after treatment with the SR-construct. RNA was extracted from TA muscle samples 14 weeks after SR-construct administration.

[0106] Figure 4AThe SR construct showed a significant reduction in the formation of insoluble aggregates (intranuclear inclusions (INIs)) including PABPN1, demonstrating a dose-response effect. The SR construct was injected into the TA muscle of A17 mice. Muscle tissue was collected and fixed for histological studies 14 weeks after SR construct administration. Immunofluorescence for PABPN1 showed green, while immunofluorescence for laminin showed red.

[0107] Figure 4B The quantification of the percentage of cell nuclei containing INI in muscle sections showed that treatment with the SR-construct significantly reduced the amount of INI compared with untreated A17 TA muscle (one-way ANOVA test performed by Bonferroni post-hoc test, *p<0.001, ns: not significant).

[0108] Figure 5A The study showed that the maximum force generated by the TA muscle in A17 mice was significantly increased in a dose-dependent manner by the SR construct. Maximum force was measured using in situ muscle physiology methods.

[0109] Figure 5B Normalized muscle weight to body weight (BW) of TA muscle treated with the SR construct in A17 mice is shown. Normalized muscle weight was comparable to that of control FvB mice at TA injection doses above 1e10 vg (mean ± SEM, n = 10, one-way ANOVA performed using Bonferroni post-hoc test, *p < 0.05, **p < 0.001, *p < 0.01, ns: not significant).

[0110] Figure 6A This shows the maximum force generated in the TA muscles of A17 mice 14 weeks after administration of the SR-construct. Maximum force values ​​were measured using in situ muscle physiology methods.

[0111] Figure 6B This shows the maximum force generated in the TA muscles of A17 mice 20 weeks after administration of the SR-construct. Maximum force values ​​were measured using in situ muscle physiology methods.

[0112] Figure 7A The SR-construct was shown to be injected directly into the pharyngeal muscle of an sheep.

[0113] Figure 7B The image shows severe dysphagia in OPMD patients at risk of "accidental ingestion" of a radioactive ointment.

[0114] Figure 8This is a diagram of the vector for the DNA construct designed as BacAAV9-Rep-VPmod. The DNA construct is designed to express AAV Rep protein and modified AAV9 capsid in insect cells. The vector backbone is the pOET1 baculovirus vector backbone (Oxford Expression Technologies), used to prepare AAV containing modified AAV9 capsid protein.

[0115] Figure 9 This is a diagram of the vector for the DNA construct designed as AAV9-VPmod. The DNA construct contains a modified version of the AAV9 capsid gene, which is used to prepare BacAAV9-Rep-VPmod. Figure 8 ).

[0116] Figure 10 This is a vector diagram of a DNA construct designed as AAV9-Rep-VPmod. The DNA construct is designed to express AAV Rep protein and modified AAV9 capsid in insect cells.

[0117] Figure 11 This is a diagram of the vector for the DNA construct designed as BacAAV8-Rep-VPmod. The DNA construct is designed to express the AAV Rep protein and the modified AAV8 capsid in insect cells. The vector backbone is the pOET1 baculovirus vector backbone (Oxford Expression Technologies), used to prepare AAV containing the modified AAV8 capsid protein in insect cells.

[0118] Figure 12 This is a diagram of the vector for the DNA construct designed as AAV8-VPmod. The DNA construct contains a modified version of the AAV8 capsid gene, which is used to prepare AAV8-Rep-VPmod ( Figure 13 ) and BacAAV8-Rep-VPmod ( Figure 11 ).

[0119] Figure 13 This is a vector diagram of a DNA construct designed as wtAAV8-Rep / Cap. The DNA construct is designed to express AAV Rep protein and wtAAV8 capsid in insect cells and to be used to prepare AAV containing wtAAV8 capsid protein.

[0120] Figure 14This is a diagram of the vector for the DNA construct designed as AAV2-GOI. The DNA construct is designed to express two shmiRs flanked by AAV ITR and is used to prepare BacAAV2-GOI (…). Figure 15 ).

[0121] Figure 15 This is a vector diagram of the DNA construct designed as BacAAV2-GOI. The DNA construct is designed to express two shmiRs flanking AAV ITRs (AAV2-GOI) within the pOET1 baculovirus vector backbone (Oxford Expression Technologies). This construct is used to prepare AAVs including AAV9 capsid proteins expressing GOIs encoding the two shmiRs.

[0122] Figure 16 A-16C shows the total number of shmiR copies expressed per cell in JHU67 cells infected with (i) AAV8 with unmodified VP1 produced in mammalian cells (VecBio), (ii) AAV8 with modified VP1 produced in insect cells via baculovirus (BacVPmod), and (iii) AAV8 with unmodified VP1 produced in insect cells via baculovirus (Ben10), at 4x10e9, 8x10e9, and 1.6x10e10 AAV vector genomes. AAV with a wild-type capsid produced in mammalian cells expressed high levels of shmiR, with expression being almost undetectable, compared to AAV with a wild-type capsid produced in insect cells. AAV with a capsid with modified VP1 produced in insect cells showed a significant increase in expression and, consequently, a significant increase in function, compared to AAV produced in insects using an unmodified wild-type capsid.

[0123] Figure 17 This figure shows the total number of shmiR copies expressed from C2C12 cells expressing the AAV internalization receptor (AAV-R) and infected with (i) AAV9 with unmodified VP1 produced in mammalian cells and (ii) AAV9 with modified VP1 produced by baculovirus in insect cells, at 4x10e9, 8x10e9, and 1.6x10e10 AAV vector genomes. Both recombinant viruses produced the same levels of shmiR and exhibited the same function.

[0124] Keys of sequence lists

[0125] SEQ ID NO: 1: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 2.

[0126] SEQ ID NO: 2: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 3.

[0127] SEQ ID NO: 3: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 4.

[0128] SEQ ID NO: 4: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 5.

[0129] SEQ ID NO: 5: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 6.

[0130] SEQ ID NO: 6: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 7.

[0131] SEQ ID NO: 7: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 9.

[0132] SEQ ID NO: 8: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 11.

[0133] SEQ ID NO: 9: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 13.

[0134] SEQ ID NO: 10: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 14.

[0135] SEQ ID NO: 11: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 15.

[0136] SEQ ID NO: 12: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 16.

[0137] SEQ ID NO: 13: RNA sequence corresponding to the mRNA transcript region of the PABPN1 protein, known as PABPN1 mRNA region 17.

[0138] SEQ ID NO: 14: RNA effector complementary sequence of shmiR named shmiR2.

[0139] SEQ ID NO: 15: RNA effector sequence of shmiR named shmiR2.

[0140] SEQ ID NO: 16: RNA effector complementary sequence of shmiR named shmiR3.

[0141] SEQ ID NO: 17: RNA effector sequence of shmiR named shmiR3.

[0142] SEQ ID NO: 18: RNA effector complementary sequence of shmiR named shmiR4.

[0143] SEQ ID NO: 19: RNA effector sequence of shmiR named shmiR4.

[0144] SEQ ID NO: 20: RNA effector complementary sequence of shmiR named shmiR5.

[0145] SEQ ID NO: 21: RNA effector sequence of shmiR named shmiR5.

[0146] SEQ ID NO: 22: The complementary RNA effector sequence of shmiR named shmiR6.

[0147] SEQ ID NO: 23: RNA effector sequence of shmiR named shmiR6.

[0148] SEQ ID NO: 24: RNA effector complementary sequence of shmiR named shmiR7.

[0149] SEQ ID NO: 25: RNA effector sequence of shmiR named shmiR7.

[0150] SEQ ID NO: 26: The complementary RNA effector sequence of shmiR named shmiR9.

[0151] SEQ ID NO: 27: RNA effector sequence of shmiR named shmiR9.

[0152] SEQ ID NO: 28: RNA effector complementary sequence of shmiR named shmiR11.

[0153] SEQ ID NO: 29: RNA effector sequence of shmiR named shmiR11.

[0154] SEQ ID NO: 30: RNA effector complementary sequence of shmiR named shmiR13.

[0155] SEQ ID NO: 31: RNA effector sequence of shmiR named shmiR13.

[0156] SEQ ID NO: 32: RNA effector complementary sequence of shmiR named shmiR14.

[0157] SEQ ID NO: 33: RNA effector sequence of shmiR named shmiR14.

[0158] SEQ ID NO: 34: RNA effector complement sequence of shmiR named shmiR15.

[0159] SEQ ID NO: 35: RNA effector sequence of shmiR named shmiR15.

[0160] SEQ ID NO: 36: RNA effector complementary sequence of shmiR named shmiR16.

[0161] SEQ ID NO: 37: RNA effector sequence of shmiR named shmiR16.

[0162] SEQ ID NO: 38: RNA effector complementary sequence of shmiR named shmiR17.

[0163] SEQ ID NO: 39: RNA effector sequence of shmiR named shmiR17.

[0164] SEQ ID NO: 40: RNA stem-loop sequence of shmiR

[0165] SEQ ID NO: 41: 5' flanking sequence of the pri-miRNA backbone.

[0166] SEQ ID NO: 42: 3' flanking sequence of the pri-miRNA backbone

[0167] SEQ ID NO: 43: RNA sequence of shmiR named shmiR2.

[0168] SEQ ID NO: 44: RNA sequence of shmiR named shmiR3.

[0169] SEQ ID NO: 45: RNA sequence of shmiR named shmiR4.

[0170] SEQ ID NO: 46: RNA sequence of shmiR named shmiR5.

[0171] SEQ ID NO: 47: RNA sequence of shmiR named shmiR6.

[0172] SEQ ID NO: 48: RNA sequence of shmiR named shmiR7.

[0173] SEQ ID NO: 49: RNA sequence of shmiR named shmiR9.

[0174] SEQ ID NO: 50: RNA sequence of shmiR named shmiR11.

[0175] SEQ ID NO: 51: RNA sequence of shmiR named shmiR13.

[0176] SEQ ID NO: 52: RNA sequence of shmiR named shmiR14.

[0177] SEQ ID NO: 53: RNA sequence of shmiR named shmiR15.

[0178] SEQ ID NO: 54: RNA sequence of shmiR named shmiR16.

[0179] SEQ ID NO: 55: RNA sequence of shmiR named shmiR17.

[0180] SEQ ID NO: 56: DNA sequence encoding shmiR named shmiR2.

[0181] SEQ ID NO: 57: DNA sequence encoding shmiR named shmiR3.

[0182] SEQ ID NO: 58: DNA sequence encoding shmiR named shmiR4.

[0183] SEQ ID NO: 59: DNA sequence encoding shmiR named shmiR5.

[0184] SEQ ID NO: 60: DNA sequence encoding shmiR named shmiR6.

[0185] SEQ ID NO: 61: DNA sequence encoding shmiR named shmiR7.

[0186] SEQ ID NO: 62: DNA sequence encoding shmiR named shmiR9.

[0187] SEQ ID NO: 63: DNA sequence encoding shmiR named shmiR11.

[0188] SEQ ID NO: 64: DNA sequence encoding shmiR named shmiR13.

[0189] SEQ ID NO: 65: DNA sequence encoding shmiR named shmiR14.

[0190] SEQ ID NO: 66: DNA sequence encoding shmiR named shmiR15.

[0191] SEQ ID NO: 67: DNA sequence encoding shmiR named shmiR16.

[0192] SEQ ID NO: 68: DNA sequence encoding shmiR named shmiR17.

[0193] SEQ ID NO: 69: DNA sequence encoding dual construct version 1 of PABPN1 under the control of the muscle-specific CK8 promoter shmiR3 and shmiR14 and under the control of Spc512 codon-optimized shmiR3 and shmiR14.

[0194] SEQ ID NO: 70: DNA sequence encoding dual construct version 1 of PABPN1 under the control of the muscle-specific CK8 promoter shmiR17 and shmiR13 and under the control of Spc512 codon-optimized.

[0195] SEQ ID NO: 71: DNA sequence encoding coPABPN1 and shmiR dual construct version 2, known as shmiR3 and shmiR14, under the control of Spc512.

[0196] SEQ ID NO: 72: DNA sequence encoding coPABPN1 and shmiR dual construct version 2 named shmiR17 and shmiR13 under the control of Spc512.

[0197] SEQ ID NO: 73: DNA sequence of human codon-optimized PABPN1 cDNA sequence.

[0198] SEQ ID NO: 74: Amino acid sequence of codon-optimized human PABPN1 protein.

[0199] SEQ ID NO: 75: Amino acid sequence of wild-type human PABPN1 protein with FLAG tag.

[0200] SEQ ID NO: 76: Amino acid sequence of codon-optimized human PABPN1 protein with FLAG tag.

[0201] SEQ ID NO: 77: DNA sequence of the primer named wtPABPN1-Fwd.

[0202] SEQ ID NO: 78: DNA sequence of the primer named wtPABPN1-Rev

[0203] SEQ ID NO: 79: DNA sequence of the probe named wtPABPN1- probe

[0204] SEQ ID NO: 80: DNA sequence of the primer named optPABPN1-Fwd

[0205] SEQ ID NO: 81: DNA sequence of the primer named optPABPN1-Rev

[0206] SEQ ID NO: 82: DNA sequence of the probe named optPABPN1- probe

[0207] DNA sequence of primer named shmiR3-FWD (SEQ ID NO: 83)

[0208] DNA sequence of primer named shmiR13-FWD (SEQ ID NO: 84)

[0209] DNA sequence of primer named shmiR14-FWD (SEQ ID NO: 85)

[0210] DNA sequence of primer named shmiR17-FWD (SEQ ID NO: 86)

[0211] SEQ ID NO: 87 is the wild-type VP1 subsequence of AAV serotype 9, including the PLA2 domain and flanking sequences.

[0212] SEQ ID NO: 88 is a modified VP1 subsequence of AAV serotype 9, including the PLA2 domain and flanking sequences.

[0213] SEQ ID NO: 89 Full-length wild-type AAV serotype 9 capsid.

[0214] SEQ ID NO: 90 Full-length modified AAV serotype 9 capsid.

[0215] SEQ ID NO: 91 is the wild-type VP1 subsequence of AAV serotype 8, including the PLA2 domain and flanking sequences.

[0216] SEQ ID NO: 92 is a modified VP1 subsequence of AAV serotype 8, including the PLA2 domain and flanking sequences.

[0217] SEQ ID NO: 93 Full-length wild-type AAV serotype 8 capsid.

[0218] SEQ ID NO: 94 Full-length modified AAV serotype 8 capsid.

[0219] SEQ ID NO: 95AAV2 5'ITR sequence.

[0220] SEQ ID NO: 96AAV2 3'ITR sequence.

[0221] SEQ ID NO: 97 Full-length wild-type AAV serotype 2 capsid.

[0222] SEQ ID NO: 98: RNA sequence encoding wild-type human PABPN1 protein. Detailed Implementation

[0223] summary

[0224] Throughout this specification, unless otherwise specified or required by context, references to a single step, feature, composition of matter, group of steps, group of features, or group of composition of matter shall be considered to include one or more (i.e., one or more) of those steps, features, compositions of matter, groups of steps, groups of features, or groups of composition of matter.

[0225] Those skilled in the art will understand that, in addition to those specifically described, the invention is susceptible to variations and modifications. It should be understood that the invention includes all such variations and modifications. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, and any and all combinations of any two or more of said steps or features.

[0226] This invention is not limited to the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this invention.

[0227] Unless otherwise specifically stated, any other instances of the invention should be adapted to the necessary modifications.

[0228] Unless otherwise expressly defined, all technical and scientific terms used herein should be regarded as having the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0229] Unless otherwise stated, the recombinant DNA, recombinant protein, cell culture, and immunological techniques used in this invention are standard methods well known to those skilled in the art. These techniques are described and explained in the following sources: J. Perbal, *A Practical Guide to Molecular Cloning*, John Wiley and Sons (1984); Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Press (1989); T.A. Brown (ed.), *Essential Molecular Biology: A Practical Approach*, Volumes 1 and 2, IRL Press (1991); DM. Gl. Glover and BD. Hames (eds.), *DNA Cloning: A Practical Approach*, Volumes 1-4, IRL Press (1995 and 1996); and F.M. Usubel et al. (eds.), *Current Protocols in Molecular Biology*, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J. E. Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0230] Throughout this specification, unless the context otherwise requires, the word “comprise” or variations such as “comprises” or “comprising” should be understood to imply inclusion of the stated steps or elements or integers, or groups of steps or elements or integers, but not to exclude any other steps or elements or integers or elements or groups of integers.

[0231] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be understood to provide explicit support for both meanings or either meaning.

[0232] Selected definition

[0233] “RNA” refers to a molecule comprising at least one ribonucleotide residue. “Ribonucleotide” refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose moiety. This term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and RNA altered to differ from naturally occurring RNA through the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may include the addition of non-nucleotide substances, such as to the end or interior of siRNA, for example, at one or more nucleotides of the RNA. The nucleotides in the RNA molecules of this invention may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0234] The term "RNA interference" or "RNAi" generally refers to RNA-dependent silencing of gene expression induced by double-stranded RNA (dsRNA) molecules in the cytoplasm of cells. dsRNA molecules reduce or inhibit the transcription products of target nucleic acid sequences, thereby silencing the gene or reducing its expression.

[0235] As used herein, the term "double-stranded RNA" or "dsRNA" refers to an RNA molecule having a double-stranded structure and comprising an effector sequence and an effector complement sequence of similar length. The effector sequence and effector complement sequence can be a single RNA strand or separate RNA strands. The "effector sequence" (often referred to as the "guide strand") is substantially complementary to the target sequence, which in this invention is a region of the PABPN1 mRNA transcript. The "effector sequence" may also be referred to as an "antisense sequence." The "effector complement sequence" will be sufficiently complementary to the effector sequence such that it can anneal with the effector sequence to form a double strand. At this point, the effector complement sequence will be substantially homologous to the region of the target sequence. It will be apparent to those skilled in the art that the term "effector complement sequence" may also be referred to as "complement of the effector sequence" or a sense sequence.

[0236] As used herein, the term "double-stranded" refers to a region in two complementary or substantially complementary nucleic acids (e.g., RNA) of a single-stranded nucleic acid (e.g., RNA), or a region in two complementary or substantially complementary regions of a single-stranded nucleic acid (e.g., RNA) that form base pairs with each other, by Watson-Crick base pairing or any other manner that allows a stable double-stranded structure between complementary or substantially complementary nucleotide sequences. Those skilled in the art will understand that 100% complementarity is not required within a double-stranded region; substantial complementarity is permitted. Substantial complementarity includes complementarity that may include 79% or greater. For example, a single mismatch (i.e., co-pairing, 18 base pairs, and one mismatch) in a double-stranded region consisting of 19 base pairs results in 94.7% complementarity, making the double-stranded region substantially complementary. In another example, two mismatches (i.e., 17 base pairs and two mismatches) in a double-stranded region consisting of 19 base pairs result in 89.5% complementarity, making the double-stranded region substantially complementary. In another example, three mismatches in a double-stranded region consisting of 19 base pairs (i.e., 16 base pairs and 3 mismatches) result in 84.2% complementarity, making the double-stranded region essentially complementary, and so on.

[0237] dsRNAs can be provided as hairpin or stem-loop structures, having a double-stranded region consisting of an effector sequence and an effector complement sequence linked by at least two nucleotide sequences called a stem-loop. When dsRNAs are provided as hairpin or stem-loop structures, they can be referred to as “hairpin RNA,” “short hairpin RNAi,” or “shRNA.” Other dsRNA molecules provided in or generating hairpin or stem-loop structures include primary miRNA transcripts (pri-miRNAs) and pre-miRNAs. Pre-miRNA shRNAs can be naturally produced from pri-miRNAs through the action of Drosha and Pasha enzymes, which recognize and release the region of the primary miRNA transcript that forms the stem-loop structure. Alternatively, pri-miRNA transcripts can be engineered to replace the natural stem-loop structure with an artificial / recombinant stem-loop structure. That is, an artificial / recombinant stem-loop structure can be inserted into or cloned into the pri-miRNA backbone sequence lacking its natural stem-loop structure. When designed with stem-loop sequences to express pri-miRNA molecules, Drosha and Pasha recognize and release artificial shRNA. The dsRNA molecules produced using this method are called "shmiRNA," "shmiR," or "microRNA framework shRNA."

[0238] As used herein, the term "complementary" in relation to a sequence refers to complementarity with a sequence that pairs via Watson-Crick base pairing, whereby guanine (G) pairs with cytosine (C), and adenine (A) pairs with uracil (U) or thymine (T). A sequence may be complementary to the full length of another sequence, or it may be complementary to a specific portion or length of another sequence. Those skilled in the art will recognize that U can be present in RNA, and T can be present in DNA. Therefore, an A within either an RNA or DNA sequence can pair with a U in an RNA sequence or a T in a DNA sequence. Those skilled in the art will also recognize that G present in RNA can pair with either C or U in RNA.

[0239] As used herein, the term "substantially complementary" is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between nucleic acid sequences, for example, between an effector sequence and an effector complement, or between an effector sequence and a target sequence. It should be understood that a nucleic acid sequence does not necessarily have to be 100% complementary to its target or complementary sequence. The term covers sequences complementary to another sequence except for the overhang. In some cases, the sequence is complementary to another sequence except for 1-2 mismatches. In some cases, the sequence is complementary except for 1 mismatch. In some cases, the sequence is complementary except for 2 mismatches. In other cases, the sequence is complementary except for 3 mismatches. In still other cases, the sequence is complementary except for 4 mismatches.

[0240] In the context of the shRNA or shmiR of this invention, the term "encoded" should be understood to mean an shRNA or shmiR capable of being transcribed from a DNA template. Therefore, the nucleic acid encoding, or encoding the shRNA or shmiR of this invention, will comprise a DNA sequence that serves as a template for transcribing the corresponding shRNA or shmiR.

[0241] The terms "DNA-directed RNAi construct" or "ddRNAi construct" refer to nucleic acids comprising a DNA sequence that, upon transcription, produces shRNA or shmiR molecules (preferably shmiR) that induce RNAi. A ddRNAi construct may comprise a nucleic acid transcribed into a single RNA capable of self-annealing into a hairpin structure having a double-stranded region connected by a stem-loop link of at least two nucleotides, i.e., shRNA or shmiR; or transcribed into a single RNA having multiple shRNAs or shmiRs; or transcribed into multiple RNA transcripts, each capable of folding into a single shRNA or shmiR. ddRNAi constructs may be provided within a larger "DNA construct" comprising one or more additional DNA sequences. For example, a ddRNAi construct may be provided within a DNA construct comprising an additional DNA sequence encoding a functional PABPN1 protein, which has been codon-optimized so that its mRNA transcript is not targeted by the shmiR of the ddRNAi construct. The ddRNAi construct and / or the DNA construct including the construct can be contained in an expression vector (e.g., a plasmid) that is operatively linked to a promoter.

[0242] As used herein, the term "operable ligation" or "operable ligation" (or similar) means the association of a coding nucleic acid sequence with or in a manner that promotes expression of the coding sequence with a regulatory sequence (e.g., a promoter). Regulatory sequences include promoters, enhancers, and other expression control elements that are well known in the art and selected to guide the expression of the coding sequence.

[0243] As used herein, the term "inverted terminal repeat" or "ITR" refers, in either the plural or singular form, to a sequence located at one end of a vector that, when combined with a complementary sequence located at the opposite end of the vector, forms a hairpin structure. This pair of inverted terminal repeats is involved in the rescue of AAV DNA, replication, and packaging within the host genome. ITRs are also used for the efficient capsidation of AAV DNA and the generation of fully assembled AAV particles.

[0244] The term "vector" should be understood as referring to a vehicle that introduces nucleic acids into a cell. Vectors include, but are not limited to, plasmids, phage particles, viruses, bacteria, and other vectors derived from viruses or bacteria. A "plasmid" is a circular double-stranded DNA molecule. The useful type of vector used according to the invention is a viral vector in which a heterologous DNA sequence is inserted into a viral genome that can be modified to delete one or more viral genes or portions thereof. Some vectors are capable of autonomous replication in host cells (e.g., vectors having a replication origin that functions in the host cell). Other vectors can stably integrate into the host cell's genome, thereby replicating along with the host genome. As used herein, the term "expression vector" should be understood to mean a vector capable of expressing the RNA molecules of the invention.

[0245] "Functional PABPN1 protein" should be understood as a PABPN1 protein possessing the functional properties of wild-type PABPN1 protein, such as the ability to control mRNA polyadenylation and / or intron splicing sites in mammalian cells. Therefore, "functional PABPN1 protein" should be understood as a PABPN1 protein that does not induce OPMD when expressed or present in a test subject. In one instance, the "functional PABPN1 protein" mentioned herein refers to the human wild-type PABPN1 protein. The sequence of the human wild-type PABPN1 protein is given in NCBI RefSeq NP_004634. Therefore, a functional human PABPN1 protein may possess the in vivo functional properties of the human PABPN1 protein listed in NCBI RefSeq NP_004634.

[0246] As used herein, the terms “treating,” “treat,” or “treatment,” and variations thereof, refer to a clinical intervention designed to alter the natural processes of the treated individual or cells during a clinicopathological process. Desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. Therefore, treatment of OPMD includes reducing or inhibiting the expression of the PABPN1 protein that causes OPMD in a subject and / or expressing a PABPN1 protein with a normal length of polyalanine residues in the subject. Preferably, treatment of OPMD includes reducing or inhibiting the expression of the PABPN1 protein that causes OPMD in a subject and / or expressing a PABPN1 protein with a normal length of polyalanine residues in the subject. For example, if one or more of the above-described therapeutic outcomes are achieved, the individual is successfully “treated.”

[0247] The "therapeutic effective dose" is at least the minimum concentration or amount required to achieve a significant improvement in OPMD symptoms, such as a significant improvement in one or more symptoms of OPMD (including, but not limited to, ptosis, dysphagia, and muscle weakness in subjects). The therapeutic effect dose described in this article can vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of shmiR, the nucleic acid encoding shmiR, ddRNAi constructs, DNA constructs, expression vectors, or compositions comprising them to elicit a desired response in an individual and / or the ability of the expression vector to express a functional PABPN1 protein in subjects. The therapeutically effective amount is also the amount by which any toxic or adverse effect of shmiR, the nucleic acid encoding shmiR, the ddRNAi construct, the DNA construct, the expression vector, or a composition comprising thereof exceeds the therapeutically beneficial effect of shmiR, the nucleic acid encoding shmiR, the ddRNAi construct, the DNA construct, the expression vector, or a composition comprising thereof, to inhibit, suppress, or reduce the expression of PABPN1 protein causing OPMD in a subject (considered alone or in combination with the expression of functional PABPN1 protein).

[0248] As used herein, "subject" or "patient" can be a human or non-human animal that suffers from OPMD or is genetically predisposed to OPMD, i.e., a human or non-human animal that has a variant of the PABPN1 gene that causes OPMD. "Non-human animal" can be a primate, livestock (e.g., sheep, horses, cattle, pigs, donkeys), companion animals (e.g., pets such as dogs and cats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, *Caenorhabditis elegans*, zebrafish), performance animals (e.g., racehorses, camels, greyhounds), or captive wild animals. In one instance, the subject or patient is a mammal. In another instance, the subject or patient is a human.

[0249] The terms “reduced expression,” “reduced expression,” or similar terms refer to the absence or observable reduction in the level of the protein and / or mRNA product of a target gene (e.g., the PABPN1 gene). The reduction need not be absolute, but can be partial, sufficient to result in a detectable or observable change in RNAi caused by shmiR, the nucleic acid encoding shmiR, the ddRNAi construct, the DNA construct, the expression vector, or compositions including those of the present invention. The reduction can be measured by determining the decrease in the level of mRNA and / or protein product from the target nucleic acid relative to cells lacking shmiR, the nucleic acid encoding shmiR, the ddRNAi construct, the DNA construct, the expression vector, or compositions including therein, and can be as low as 1%, 5%, or 10%, or can be absolute, i.e., 100% inhibition. The reduction effect can be determined by examining the outward characteristics of cells or organisms (i.e., quantitative and / or qualitative phenotypes of cells or organisms), and may also include detecting the presence or change in the amount of expPABPN1 nuclear aggregates in cells or organisms after administration of shmiR of the present invention, nucleic acid encoding shmiR, ddRNAi construct, DNA construct, expression vector, or composition including the thereof.

[0250] As used herein, a "delivery system" refers to a vector used to package foreign genetic material such as DNA or RNA, and which can be introduced into a cell. Delivery systems can include viral vectors, such as adenovirus-associated virus (AAV) vectors, retroviral vectors, adenovirus vectors (AdV), and lentivirus (LV) vectors. As described herein, viral vectors can be used to deliver and express foreign genetic material in cells. Therefore, the viral expression vectors described herein can be used as delivery systems.

[0251] As used herein, the term "adeno-associated virus" or "AAV" refers to a group of viruses within the family Parvoviridae that contain a short (approximately 4.7 kb) single-stranded DNA genome and are dependent on the presence of helper viruses (e.g., adenoviruses used for their replication). This invention also relates to vectors derived from AAV, for example, used as gene transfer vectors.

[0252] As used herein, the term "serotype" in the context of AAV is used to distinguish an AAV having a capsid that is serologically different from other AAV serotypes. Serological specificity is determined on the basis of the lack of cross-reactivity between antibodies against an AAV compared to another AAV. This difference in cross-reactivity is typically due to differences in capsid protein sequence / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0253] As used herein, in the context of AAV, the terms “viral capsid protein,” “capsid protein,” “capsid polypeptide,” or similar terms refer to the polypeptide of AAV that possesses self-assembly activity to produce the protein shell of AAV particles, also known as the outer shell protein or VP protein. It consists of three subunits, VP1, VP2, and VP3, which are typically expressed by a single nucleic acid molecule and interact to form an icosahedral symmetrical capsid. The capsid structure of AAV is described in Bernard N. Fields et al., *Virusology*, Vol. 2, Chapters 69 & 70 (4th ed., Lippincott-Raven).

[0254] As used herein, the term “promoter” generally refers to a DNA sequence that participates in the recognition and binding of DNA-dependent RNA polymerases and other proteins (trans-acting transcription factors) to initiate and control the transcription of one or more coding sequences, and is typically located upstream of the coding sequence relative to the direction of transcription.

[0255] As used in the context of AAVs comprising modified capsid proteins or VP1 sequences in this invention, the term "improved function" or similar terms should be understood to mean that AAVs comprising modified capsid proteins or VP1 sequences have improved endosome escape activity relative to wild-type AAVs of the same serotype, which have not been modified and are produced in insect cells. As used herein, the terms "endosomal escape activity," "endosome escape activity," or similar terms should be understood to mean the ability of an AAV to escape from its endosome compartment after cellular internalization. Regarding AAV function, it should be understood that AAVs that cannot escape from their endosome compartments after cellular internalization are ineffective, particularly in gene therapy.

[0256] As used in this article, "pharyngeal muscles" refers to one or more groups of muscles that form the pharynx. Pharyngeal muscles may include one or more of the inferior retractor muscles, middle retractor muscles, superior retractor muscles, palatopharyngeal muscles, laryngeal muscles, and / or stylopharyngeal muscles.

[0257] Modified AAV delivery vector for the treatment of OPMD

[0258] Adeno-associated virus (AAV) is a parvovirus that typically requires co-infection with another virus (usually adenovirus or herpesvirus) to initiate and maintain a productive infection cycle. Even without this helper virus, AAV can still infect or transduce target cells via receptor-mediated binding and internalization, penetrating the nuclei of both non-dividing and dividing cells. Because progeny viruses are not generated by AAV infection in the absence of a helper virus, the extent of transduction is limited to the initial cells infected with the virus. This is a characteristic that makes AAV an ideal vector for gene therapy. Furthermore, unlike retroviruses, adenoviruses, and herpes simplex viruses, AAV appears to lack human pathogenicity and virulence (Kay et al., Nature. 424:251 (2003)). Since the genome typically encodes only two genes, it is not surprising that AAV as a delivery vehicle is limited by a packaging capacity of 4.5 kb / s. However, while this size limitation may restrict the delivery of genes for alternative gene therapies, it does not adversely affect the packaging and expression of shorter sequences such as shmiR and shRNA. For these reasons, the present invention considers the use of AAV as a vector or system for delivering the PABPN1 'silencing and replacement' construct for the treatment of OPMD. Generally, AAVs for gene therapy applications are preferably selected from those serotypes capable of infecting humans, such as AAVs selected from the group consisting of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 (or variants thereof).

[0259] In one example, the present invention provides an AAV comprising:

[0260] (a) A viral capsid protein comprising a modified VP1 sequence wherein specific amino acids within the phospholipase A2 (PLA2) domain and flanking sequences of subunit 1 (VP1) are modified to be more “AAV2-like” relative to the corresponding wild-type sequence; and

[0261] (b) A polynucleotide sequence comprising (i) a DNA-guided RNAi (ddRNAi) construct comprising a nucleic acid including a sequence encoding a short hairpin microRNA (shmiR); and (ii) a PABPN1 construct comprising a nucleic acid including a sequence encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR encoded by the ddRNAi construct.

[0262] In this regard, the inventors have demonstrated that the endosome escape activity of representative AAVs from serotypes other than serotype 2, produced by a baculovirus expression system in insect cells, can be restored or improved by amino acid substitutions at specific sites within the PLA2 domain and its flanking sequences. For example, the inventors have demonstrated that the endosome escape activity of representative AAVs from serotypes other than serotype 2 can be restored or improved by substituting amino acids at corresponding positions within the PLA2 domain and flanking sequences of AAV serotype 2 with amino acids at corresponding positions within the PLA2 domain and flanking sequences. In this regard, since the current strategy is to improve the function of AAVs produced in insect cells, the inventors have demonstrated that it is not necessary to exchange the entire PLA2 domain and flanking sequences with the AAV2 domain and flanking sequences to produce chimeric AAVs, nor is it necessary to produce AAVs expressing a chimeric capsid including the wild-type VP1 / PLA2 sequence and AAV2, such as AAV2 / WT VP1.

[0263] The AAV sequences that can be used to produce AAVs having the modified VP1 sequence described herein can be derived from the genome of any AAV serotype. Typically, AAV serotypes have genomic sequences that are significantly homologous at the amino acid and nucleic acid levels, provide the same genetic functional set, produce physically and functionally similar virions, and replicate and assemble via virtually the same mechanism (with a specific exemption for activity of the PLA2 domain described herein). Suitable AAV nucleic acid and protein sequences for designing and producing the modified AAVs of the present invention are publicly available. The VP1 sequence of wild-type AAVs known to infect humans (and which is covered herein) is described in Chen et al., (2013) J.Vir.87(11):6391-6405. Human or simian adeno-associated virus (AAV) serotypes are preferred sources of AAV nucleotide sequences for use in the content of this invention, more preferably AAV serotypes that typically infect humans (e.g., serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13). The capsid polypeptide sequences of AAV serotypes 1-13 are known in the art, such as AAV1 (GenBank accession number: AAD27757.1, GI:4689097), AAV2 (GenBank accession number: AAC03780.1, GP.2906023), AAV3 (GenBank accession number: AAC55049.1, GI:1408469), AAV4 (GenBank accession number: AAC58045.1, GL2337940), and AAV5 (GenBank accession number: AAD27757 ...4689097), AAV2 (GenBank accession number: AAC03780.1, GP.2906023), AAV3 (GenBank accession number: AAC55049.1, GI:1408469), AAV4 (GenBank accession number: AAC58045.1, GL2337940), and AAV5 (GenBank accession number: AAD27757.1, GI:4689097), AAV2 (GenBank accession number: AAC03780.1, GP.2906023), AAV3 (GenBank accession number: AAC03780.1, GP.2906023), AAV4 (GenBank accession number: AAC03780.1, GL2337940), AAV5 (GenBank accession number: AAD27757.1, GI:1408469), AAV4 (Gen Accession numbers: AAD13756.1, GI-4249658), AAV10 (Genbank accession number: AAT46337.1, GL48728343), AAV11 (Genbank accession number: AAT46339.1, GI:48728346), AAV12 (Genbank accession number: ABI16639.1, GI:112379656), or AAV13 (Genbank accession number: ABZ10812.1, GI:167047087). The polypeptide sequences of the AAV capsid proteins of serotypes 1-13 are also shown in SEQ ID NO: 27-39 of this paper.Furthermore, complete genomes of AAVs from serotypes 1-13 are known in the art, such as AAV1 (NCBI reference sequence: NC_002077.1), AAV2 (Genbank accession number: J01901.1), AAV3 (Genbank accession number: AF028705.1), AAV4 (NCBI reference sequence: NC_001829.1), AAV5 (NCBI reference sequence: NC_006152.1), and AAV6 (Genbank accession number: J01901.1). The gene banks used are: AAV7 (NCBI reference sequence: NC_006260.1), AAV8 (NCBI reference sequence: NC_006261.1), AAV9 (Gene Bank accession number: AY530579.1), AAV10 (Gene Bank accession number: AY631965.1), AAV11 (Gene Bank accession number: AY631966.1), or AAV12 (Gene Bank accession number: DQ813647.1). In a specific example, this invention provides AAV delivery vectors from serotypes 8 and 9.

[0264] In one example, the AAV of the present invention comprises a viral capsid protein from AAV9, which includes a modified VP1 sequence, wherein one or more amino acids at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87. For example, the AAV of the present invention may comprise a viral capsid protein from AAV9, the viral capsid protein comprising a modified VP1 sequence comprising the sequence of serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, serine at position 44, and lysine at position 64, wherein the amino acid positions are defined relative to the wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87, wherein any one or more amino acids at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence. In some instances, no amino acids other than those at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence.

[0265] In one instance, the AAV described herein may include a viral capsid protein from AAV9 having a modified VP1 sequence, wherein any two, three, four, or five amino acids at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87.

[0266] In one example, the AAV described herein may comprise a viral capsid protein from AAV9 having a modified VP1 sequence, wherein any two or more amino acids at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87. For example, the modified VP1 sequence may comprise two or more modifications selected from A1S, A26E, Q40R, K43D, and A44S relative to the sequence shown in SEQ ID NO: 87.

[0267] In one instance, the AAV described herein may comprise a viral capsid protein from AAV9 having a modified VP1 sequence, wherein amino acids at any three or more of positions 1, 26, 40, 43, and 44 of the corresponding wild-type AAV9 VP1 shown in SEQ ID NO: 87 are modified. For example, the modified VP1 sequence may comprise three or more modifications selected from A1S, A26E, Q40R, K43D, and A44S relative to the sequence shown in SEQ ID NO: 87.

[0268] In one example, the AAV described herein may comprise a viral capsid protein from AAV9 having a modified VP1 sequence, wherein any four or more amino acids at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87. For example, the modified VP1 sequence may comprise four or more modifications selected from A1S, A26E, Q40R, K43D, and A44S relative to the sequence shown in SEQ ID NO: 87.

[0269] In one example, the AAV described herein may comprise a viral capsid protein from AAV9 having a modified VP1 sequence, wherein amino acids at positions 1, 26, 40, 43, and 44 of the corresponding wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87 are modified. For example, the modified VP1 sequence may comprise the following modifications relative to the sequence shown in SEQ ID NO: 87: A1S, A26E, Q40R, K43D, and A44S. For example, the modified AAV9 VP1 sequence may comprise the amino acid sequence shown in SEQ ID NO: 88. For example, residues at positions 42, 67, 81, 84, and 85 of the corresponding full-length wild-type AAV9 capsid VP1 sequence shown in SEQ ID NO: 89 are modified (e.g., A42S, A67E, Q81R, K84D, and A85S are modified relative to the sequence shown in SEQ ID NO: 89). According to this example, the AAV of the present invention may include a viral capsid protein from AAV9, which includes the modified VP1 sequence shown in SEQ ID NO: 90.

[0270] In one example, the AAV of the present invention comprises a viral capsid protein from AAV8, which includes a modified VP1 sequence, wherein one or more amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence shown in SEQ ID NO: 91. For example, the AAV of the present invention may comprise a viral capsid protein from AAV8, the viral capsid protein comprising a modified VP1 sequence comprising the sequence of serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, serine at position 44, and lysine at position 64, wherein the amino acid positions are defined relative to the wild-type AAV8 VP1 sequence listed in SEQ ID NO: 91, wherein any one or more amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence. In some instances, no amino acids other than those at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence.

[0271] In one instance, the AAV described herein may include a viral capsid protein from AAV8 having a modified VP1 sequence, wherein any two, three, four, or five amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence shown in SEQ ID NO: 91.

[0272] In one instance, the AAV described herein may comprise a viral capsid protein from AAV8 having a modified VP1 sequence, wherein any two or more amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence shown in SEQ ID NO: 91. For example, the modified VP1 sequence may comprise two or more modifications selected from A1S, A26E, Q40R, K43D, A44S, and Q64K relative to the sequence shown in SEQ ID NO: 91.

[0273] In one instance, the AAV described herein may comprise a viral capsid protein from AAV8 having a modified VP1 sequence, wherein amino acids at any three or more of positions 1, 26, 40, 43, 44, and 64 of the corresponding wild-type AAV8 VP1 shown in SEQ ID NO: 91 are modified. For example, the modified VP1 sequence may comprise three or more modifications selected from A1S, A26E, Q40R, K43D, A44S, and Q64K relative to the sequence shown in SEQ ID NO: 91.

[0274] In one instance, the AAV described herein may comprise a viral capsid protein from AAV8 having a modified VP1 sequence, wherein amino acids at any four or more positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence shown in SEQ ID NO: 91. For example, the modified VP1 sequence may comprise four or more modifications selected from A1S, A26E, Q40R, K43D, A44S, and Q64K relative to the sequence shown in SEQ ID NO: 91.

[0275] In one instance, the AAV described herein may comprise a viral capsid protein from AAV8 having a modified VP1 sequence, wherein amino acids at any five or more of positions 1, 26, 40, 43, 44, and 64 of the corresponding wild-type AAV8 VP1 shown in SEQ ID NO: 91 are modified. For example, the modified VP1 sequence may comprise five or more modifications selected from A1S, A26E, Q40R, K43D, A44S, and Q64K relative to the sequence shown in SEQ ID NO: 91.

[0276] In one example, the AAV described herein may comprise a viral capsid protein from AAV8 having a modified VP1 sequence, wherein amino acids at positions 1, 26, 40, 43, 44, and 64 of the corresponding wild-type AAV8 VP1 sequence shown in SEQ ID NO: 91 are modified. For example, the modified VP1 sequence may comprise the following modifications relative to the sequence shown in SEQ ID NO: 91: A1S, A26E, Q40R, K43D, A44S, and Q64K. For example, the modified AAV8 VP1 sequence may comprise the amino acid sequence shown in SEQ ID NO: 92. For example, residues at positions 42, 67, 81, 84, 85, and 105 of the corresponding full-length wild-type AAV8 capsid VP1 sequence shown in SEQ ID NO: 93 are modified (e.g., A42S, A67E, Q81R, K84D, A85S, and Q105K are modified relative to the sequence shown in SEQ ID NO: 93). According to this example, the AAV of the present invention may include a viral capsid protein from AAV8, which includes the modified VP1 sequence shown in SEQ ID NO: 94.

[0277] In each of the above embodiments, the viral capsid protein may include subunit 2 (VP2) and subunit 3 (VP3) sequences from the same AAV serotype as the modified VP1. Preferably, VP1 and VP3 are expressed by the same ORF.

[0278] The AAV genome includes a replication (Rep) gene, which is a protein encoded by the virus that functions in the replication of the viral genome. Therefore, in one example, the AAV described herein includes at least one large AAV Rep protein selected from Rep78 and Rep68 and at least one small AAV Rep protein selected from Rep52 and Rep40. In one example, the AAV described herein includes Rep78 and Rep52. In one example, the AAV described herein includes Rep78 and Rep40. In one example, the AAV described herein includes Rep68 and Rep52. In one example, the AAV described herein includes Rep68 and Rep40. In one example, the AAV described herein includes Rep78, Rep68, Rep52, and Rep40. In each of the above examples, the corresponding small and large Rep proteins may originate from the same AAV serotype as the viral capsid protein. Alternatively, the individual small and large Rep proteins may originate from an AAV serotype different from the viral capsid protein; for example, the Rep protein may originate from AAV2.

[0279] As described herein, AAV can be used as a delivery system in gene therapy. For example, AAV may comprise a polynucleotide encoding a target protein or RNA. As described herein, the AAV of the present invention comprises a polynucleotide sequence including a ddRNAi construct and a PABPN1 construct. The polynucleotide encoding the ddRNAi construct and the PABPN1 construct may be side-joined with an AAV inverted terminal repeat (ITR) sequence. In one example, the AAV ITR sequence is from the same serotype as the viral capsid protein. In another example, the AAV ITR sequence is from a serotype different from the viral capsid protein. In one particular example, the ITR sequence is from AAV serotype 2. In another particular example, the ITR sequence is from AAV serotype 2 and includes the sequences shown in SEQ ID NO: 91 and / or SEQ ID NO: 92.

[0280] As described above, polynucleotides encoding target proteins or RNA, including flanking ITRs, are typically 5,000 nucleotides (nt) or shorter. However, it is also possible to encode DNA that is too large, i.e., polynucleotides exceeding 5,000 nt in length. Oversized DNA is understood herein as DNA exceeding the maximum AAV packaging limit of 5 kbp. Therefore, it is also feasible for the AAV of the present invention to express proteins or RNA typically encoded by genomes larger than 5.0 kb.

[0281] As described herein, the AAV of the present invention also includes polynucleotide sequences incorporated in its genome, which include ddRNAi constructs and PABPN1 constructs for expression in mammalian cells. Exemplary ddRNAi constructs and PABPN1 constructs are described herein (e.g., under the subtitle "ddRNAi constructs") and should be considered as examples adapted to describe the AAV of the present invention with necessary modifications, unless otherwise specifically stated. In this regard, the AAV of the present invention may include polynucleotides comprising ddRNAi constructs encoding any one or more of the shmiRs named shmiR2-shmiR7, shmiR9, shmiR11, or shmiR13-shmiR17 as described herein. However, in certain instances, the AAV of the present invention may comprise a polynucleotide comprising a ddRNAi construct encoding shmiR13 and / or shmiR17, and a polynucleotide construct comprising a sequence encoding a functional PABPN1 protein, the functional PABPN1 protein being codon-optimized such that its mRNA transcript is not targeted by the shmiR of the ddRNAi construct (e.g., the sequence shown in SEQ ID NO: 73). Exemplary ddRNAi constructs encoding shmiR13 and shmiR17 are described and contemplated herein.

[0282] In one specific example, AAV comprises: (a) a viral capsid protein from AAV9 comprising a modified VP1 sequence having modifications of A1S, A26E, Q40R, K43D, and A44S relative to the corresponding wild-type sequence shown in SEQ ID NO: 87 (e.g., a modified VP1 sequence comprising the sequence shown in SEQ ID NO: 88); and (b) a polynucleotide sequence comprising (i) a ddRNAi construct comprising nucleic acids including sequences encoding shmiR13 and shmiR17 as described herein; and (ii) a PABPN1 construct comprising nucleic acids including sequences encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR encoded by the ddRNAi construct (e.g., the codon-optimized sequence shown in SEQ ID NO: 73). The polynucleotide in (b) may be side-joined with an AAV inverted terminal repeat (ITR) sequence from AAV2, as shown in SEQ ID NO: 95 and SEQ ID NO: 96. In some embodiments, the ddRNAi construct includes a DNA sequence encoding an shmiR comprising the effector sequence shown in SEQ ID NO: 31 and a nucleic acid comprising an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 31, such as the effector complementary sequence shown in SEQ ID NO: 30 (shmiR13) or a nucleic acid composed thereof, and a nucleic acid comprising a DNA sequence encoding an shmiR, wherein the shmiR comprises the effector sequence shown in SEQ ID NO: 39 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 39, such as the effector complementary sequence shown in SEQ ID NO: 38 (shmiR17) or a nucleic acid composed thereof. For example, the ddRNAi construct according to this example may include a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or a nucleic acid composed of the DNA sequence shown in SEQ ID NO: 64 (shmiR13), and a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or a nucleic acid composed of the DNA sequence shown in SEQ ID NO: 68 (shmiR17).

[0283] In another specific instance, AAV includes: (a) a viral capsid protein from AAV8 comprising a modified VP1 sequence having modifications of A1S, A26E, Q40R, K43D, A44S, and Q64K relative to the corresponding wild-type sequence shown in SEQ ID NO: 91 (e.g., a modified VP1 sequence comprising the sequence shown in SEQ ID NO: 92); and (b) a polynucleotide sequence comprising (i) a ddRNAi construct comprising nucleic acids including sequences encoding shmiR13 and shmiR17 as described herein; and (ii) a PABPN1 construct comprising nucleic acids including sequences encoding a functional PABPN1 protein having an mRNA transcript not targeted by shmiR encoded by the ddRNAi construct (e.g., a codon-optimized sequence shown in SEQ ID NO: 73). (b) The polynucleotide may be side-joined with an AAV inverted terminal repeat (ITR) sequence from AAV2, as shown in SEQ ID NO: 95 and SEQ ID NO: 96. (b) The polynucleotide may be side-joined with an AAV inverted terminal repeat (ITR) sequence from AAV2, as shown in SEQ ID NO: 95 and SEQ ID NO: 96. In some embodiments, the ddRNAi construct includes a DNA sequence encoding a shmiR comprising the effector sequence shown in SEQ ID NO: 31 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 31, such as the effector complementary sequence shown in SEQ ID NO: 30 (shmiR13) or a nucleic acid composed thereof, and a nucleic acid comprising a DNA sequence encoding a shmiR comprising the effector sequence shown in SEQ ID NO: 39 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 39, such as the effector complementary sequence shown in SEQ ID NO: 38 (shmiR17) or a nucleic acid composed thereof. For example, the ddRNAi construct according to this example may include a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or the DNA sequence shown in SEQ ID NO: 64 (shmiR13), and a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or the DNA sequence shown in SEQ ID NO: 68 (shmiR17).

[0284] In each of the foregoing examples, the polynucleotides encoding the ddRNAi construct and the PABPN1 construct are operatively linked to one or more promoters suitable for expressing the shmiR and PABPN1 proteins in mammalian cells. In one example, the promoter may be a muscle-specific promoter. Suitable muscle-specific promoters are described herein.

[0285] The AAV of the present invention may include a large AAV Rep protein selected from Rep78 and Rep68 and at least one small AAV Rep protein selected from Rep52 and Rep40.

[0286] In this respect, the AAV genome includes Rep genes (i.e., Rep78 and Rep52), proteins that function in viral genome replication. Splicing events in the Rep ORF result in the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40). However, it has been shown that unspliced ​​mRNAs encoding Rep78 and Rep52 proteins are sufficient in insect cells to produce an AAV vector. Therefore, in one instance, the AAV includes a large AAV replication Rep protein selected from Rep78 and Rep68 and at least one small AAV Rep protein selected from Rep52 and Rep40. In one instance, the AAV includes Rep78 and Rep52. In one instance, the AAV includes Rep78 and Rep40. In one instance, the AAV includes Rep68 and Rep52. In one instance, the AAV includes Rep68 and Rep40. In one instance, the AAV includes Rep78, Rep68, Rep52, and Rep40. In each of the above examples, the corresponding small and large Rep proteins can originate from the same AAV serotype as the viral capsid protein. Alternatively, the individual small and large Rep proteins can originate from AAV serotypes different from the viral capsid protein; for example, the Rep protein can originate from AAV serotype 2. In this respect, the Rep sequence is particularly conserved in most serotypes, and Rep sequences have been reported to effectively cross-complement in insect cells.

[0287] Any nucleotide sequence can be incorporated for subsequent expression in mammalian cells transfected with the AAV of this invention, as long as the construct remains within the packaging capacity of the AAV virion.

[0288] As described herein, the AAV described herein has improved functionality when produced in insect cells compared to AAVs that include the corresponding wild-type VP1 sequence.

[0289] Methods and reagents for preparing AAV using modified VP1

[0290] Methods for producing AAV are known in the art. As described, the AAV of the present invention, when produced in insect cells, has improved functionality (e.g., improved endosome escape activity) compared to AAV comprising the corresponding wild-type VP1 sequence. Therefore, methods and reagents for producing AAV in insect cells are contemplated. In some instances, insect cell-compatible vectors, i.e., baculovirus vectors, can be used to produce the AAV of the present invention.

[0291] In one example, the present invention provides various baculovirus vectors for generating the AAV of the present invention in insect cells. These various baculovirus vectors may include:

[0292] (i) A first baculovirus vector comprising a nucleic acid molecule encoding an AAV viral capsid protein having a modified VP1 sequence as described herein; and

[0293] (ii) A second baculovirus vector comprising a polynucleotide encoding the ddRNAi construct and the PABPN1 construct as described herein, with an AAV inverted terminal repeat (ITR) sequence attached to the side.

[0294] In one instance, the AAV ITR sequence originates from the same serotype as the viral capsid protein encoded by the nucleic acid molecule within the first baculovirus vector. In another instance, the AAV ITR sequence originates from a different AAV serotype, such as AAV2. In some instances, the ITR sequence originates from AAV serotype 2 and includes the sequences shown in SEQ ID NO: 95 and / or SEQ ID NO: 96.

[0295] In some instances, the AAV includes a capsid protein from AAV9, which includes a modified VP1 as described herein. In other instances, the AAV includes a capsid protein from AAV8, which includes a modified VP1 as described herein. Therefore, a first baculoviral vector may include a nucleic acid molecule encoding a viral capsid protein of AAV8 or AAV9 having a modified VP1 sequence. The modified VP1 sequence of an AAV comprising a capsid protein from AAV9 or AAV8 has been described herein, and unless otherwise specifically stated, it should be understood that it is applicable, with necessary modifications, to the examples of the invention described for producing the AAV of the present invention using baculoviral vectors.

[0296] As described herein, the second baculoviral vector comprises a ddRNAi construct encoding one or more shmiRs targeting PABPN1. Exemplary ddRNAi constructs encoding shmiRs (including combinations of shmiRs) targeting PABPN1 are described herein and, unless specifically stated otherwise, should be considered as examples of the invention described with necessary modifications for generating AAVs of the present invention. In one particular example, the second baculoviral vector may comprise ddRNAi constructs encoding shmiR13 and shmiR17, and a polynucleotide construct comprising a sequence encoding a functional PABPN1 protein, the functional PABPN1 protein being codon-optimized such that its mRNA transcript is not targeted by the shmiRs of the ddRNAi construct (e.g., the sequence shown in SEQ ID NO: 73). For example, a second baculovirus vector may include a ddRNAi construct comprising a DNA sequence encoding an shmiR including the effector sequence shown in SEQ ID NO: 31 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 31, such as the effector complementary sequence shown in SEQ ID NO: 30 (shmiR13) or a ddRNAi construct composed thereof, and a nucleic acid comprising a DNA sequence encoding an shmiR, wherein the shmiR includes the effector sequence shown in SEQ ID NO: 39 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 39, such as the effector complementary sequence shown in SEQ ID NO: 38 (shmiR17) or a nucleic acid composed thereof. For example, the second baculovirus vector may include a ddRNAi construct comprising the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or a nucleic acid composed of the DNA sequence shown in SEQ ID NO: 64 (shmiR13), and a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or a nucleic acid composed of the DNA sequence shown in SEQ ID NO: 68 (shmiR17).

[0297] In each of the foregoing examples, the polynucleotides encoding the ddRNAi construct and the PABPN1 construct can be operatively linked to the promoter. In one example, the promoter can be a muscle-specific promoter.

[0298] Similarly, nucleic acid molecules encoding AAV viral capsid proteins can be operatively linked to promoters suitable for expression of the capsid proteins in insect cells. Suitable promoters for expression in insect cells are known in the art and are considered for use herein.In this regard, methods for molecular engineering and peptide expression in insect cells have been previously described, for example, in Summers and Smith, A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex. (1986); Luckow, InProkop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152 (1991); King, LA and RD Possee, The baculovirus expression system, Chapman and Hall, United Kingdom (1992); O'Reilly, DR, LK Miller, VA Luckow, Baculovirus Expression Vectors: A Laboratory Manual, New York (1992); WH Freeman and Richardson, CD, Baculovirus Expression Protocols, Methods in Molecular Biology, Vol. 39 (1992); US Patent 4,745,051; US2003148506; WO2003 / 074714; Kotin RM (2011), Human Molecular Genetics, 20(R1):R2-R6; Aucoin et al. (2006), Biotechnol. Bioeng. 95(6):1081-1092; and van Oers et al. (2015), Journal of General Virology 96:6-23.Clearly, promoters and other such regulatory elements known in the art are considered for use in the nucleic acids of the present invention. In some embodiments, the promoter is a polyhedron promoter or a p10 promoter.

[0299] Based on the example where the first baculovirus vector does not encode the AAV Rep protein, various baculovirus vectors further include:

[0300] (iii) A third baculovirus vector comprising a polynucleotide sequence encoding at least one large AAV Rep protein selected from Rep78 and Rep68 and at least one small AAV Rep protein selected from Rep52 and Rep40.

[0301] In this respect, the AAV genome includes Rep genes (i.e., Rep78 and Rep52), proteins that function in viral genome replication. Splicing events in the Rep ORF result in the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40). However, it has been shown that unspliced ​​mRNA encoding Rep78 and Rep52 proteins is sufficient in insect cells to generate an AAV vector. Therefore, in one instance, a third baculovirus vector comprises a polynucleotide sequence encoding at least one large AAV replication Rep protein selected from Rep78 and Rep68 and at least one small AAV Rep protein selected from Rep52 and Rep40. In one instance, a third baculovirus vector comprises polynucleotide sequences encoding Rep78 and Rep52. In one instance, a third baculovirus vector comprises polynucleotide sequences encoding Rep78 and Rep40. In one instance, a third baculovirus vector comprises polynucleotide sequences encoding Rep68 and Rep52. In one example, the third baculovirus vector includes polynucleotide sequences encoding Rep68 and Rep40. In another example, the third baculovirus vector includes polynucleotide sequences encoding Rep78, Rep68, Rep52, and Rep40. In each of the above examples, the corresponding small and large Rep proteins may originate from the same AAV serotype as the viral capsid protein. Alternatively, the individual small and large Rep proteins may originate from an AAV serotype different from the viral capsid protein; for example, the Rep protein may originate from AAV serotype 2. In this respect, the Rep sequence is particularly conserved in most serotypes, and Rep sequences have been reported to efficiently cross-complement in insect cells.

[0302] In each of the foregoing embodiments describing various baculovirus vectors, the polynucleotide sequence encoding the Rep protein within the third baculovirus vector can be operatively linked to a promoter for expressing the Rep protein in insect cells. Suitable promoters for expression in insect cells are known in the art and are contemplated herein. In one specific example, the promoter may be, for example, a polyhedral promoter or a p10 promoter. The nucleotide sequences encoding each Rep protein can be operatively linked to the same promoter. Alternatively, each sequence encoding the Rep protein can be operatively linked to its own promoter.

[0303] At least one of the various baculovirus vectors will include a polynucleotide encoding the assembly-activation protein (AAP) required for AAV capsid assembly. In one example, the baculovirus vector encoding a capsid protein includes a polynucleotide encoding AAP. In alternative examples, baculoviruses encoding the Rep protein and / or baculoviruses encoding ddRNAi and PABPN1 constructs include a polynucleotide encoding AAP.

[0304] Methods for producing AAV suitable for gene therapy (e.g., methods that cannot replicate AAV) are well known in the art and are considered herein. For example, AAV can be produced in insect cells using a baculovirus system, such as... US20120028357 A1 WO2007046703 US20030148506A1 ,WO2017184879、 US20040197895The methods described in A1 and WO2007148971 are incorporated herein by reference. Recombinant AAV can also be produced in mammalian cells (adhesive cells and suspension cells) using methods described in WO2015031686, WO2009097129, WO2007127264, WO1997009441 and WO2001049829, the contents of which are incorporated herein by reference. Methods for producing recombinant AAV for gene therapy are also described in the following literature: Berns KI and Giraud C (1996), Biology of adeno-associated virus, Curr Top Microbiol Immunol 218:1-23; Snyder and Flotte (2002), Curr Opin Biotechnol, 13:418–423; and Synder RO and Moullier P, Adeno-associated virus: methods and protocols, New York: Humana Press (2011), the contents of which are incorporated herein by reference.

[0305] ddRNAi construct

[0306] As described herein, the AAV of the present invention includes a DNA-guided RNAi (ddRNAi) construct comprising a DNA sequence encoding a short hairpin microRNA (shmiR). The shmiR encoded by the ddRNAi construct includes:

[0307] Effector sequences with a length of at least 17 nucleotides;

[0308] Effector complementary sequences;

[0309] Stem-loop sequences; and

[0310] Primary microRNA (pri-miRNA) backbone.

[0311] In one example, the effector sequence is substantially complementary to a region of corresponding length in any of the RNA transcripts shown in SEQ ID NO: 1-13. Preferably, the effector sequence is less than 30 nucleotides in length. For example, a suitable effector sequence can be in the range of 17-29 nucleotides in length. In a particularly preferred example, the effector sequence is 21 nucleotides in length. More preferably, the effector sequence is 21 nucleotides in length, and the complementary effector sequence is 20 nucleotides in length.

[0312] In some embodiments, the shmiR encoded by the ddRNAi construct includes an effector sequence substantially complementary to a region of corresponding length in the RNA transcript, which includes or consists of any one of the sequences shown in SEQ ID NO: 1-13 (i.e., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13). For example, the effector sequence may be substantially complementary to a region of corresponding length in the RNA transcript, which includes or consists of any one of the sequences shown in SEQ ID NO: 1-13, and contains 4 mismatched bases relative to the RNA transcript. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of any one of the sequences shown in SEQ ID NO: 1-13, and containing 3 mismatched bases relative to the RNA transcript. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of any one of the sequences shown in SEQ ID NO: 1-13, and containing 2 mismatched bases relative to the RNA transcript. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of any one of the sequences shown in SEQ ID NO: 1-13, and containing 1 mismatched base relative to the RNA transcript. For example, the effector sequence may be 100% complementary to a region of corresponding length in an RNA transcript comprising or consisting of any one of the sequences shown in SEQ ID NO: 1-13.

[0313] In one example, the shmiR encoded by the ddRNAi construct comprises an effector sequence substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 9. The shmiR according to this example is also referred to herein as "shmiR13". For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 9, and contains 4 mismatched bases relative to the RNA transcript. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 9, and contains 3 mismatched bases relative to the RNA transcript. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 9, and contains 2 mismatched bases relative to the RNA transcript. For example, the effector sequence can be substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 9, and containing one mismatched base relative to the RNA transcript. For example, the effector sequence can be 100% complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 9.

[0314] In one example, the shmiR encoded by the ddRNAi construct comprises an effector sequence substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 13. The shmiR according to this example is also referred to herein as "shmiR17". For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 13 and containing 4 mismatched bases relative to the RNA transcript. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 13 and containing 3 mismatched bases relative to the RNA transcript. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 13 and containing 2 mismatched bases relative to the RNA transcript. For example, the effector sequence can be substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 13, and containing one mismatched base relative to the RNA transcript. For example, the effector sequence can be 100% complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 13.

[0315] According to one example, the effector sequence of shmiR is substantially complementary to a region of corresponding length in the PABPN1 miRNA transcript described herein and, relative to its inclusion of 1, 2, 3, or 4 mismatched bases, preferably the mismatch is not located in the region corresponding to the shmiR seed region, i.e., the region of nucleotides 2-8 of the effector sequence.

[0316] In some instances, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 14 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 14; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 15 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 15 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 15 may be the sequence shown in SEQ ID NO: 14. The shmiR according to this example is referred to below as "shmiR2".

[0317] In one example, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 16 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 16; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 17 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 17 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 17 may be the sequence shown in SEQ ID NO: 16. The shmiR according to this example is referred to below as "shmiR3".

[0318] In one example, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 18 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 18; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 19 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 19 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 19 may be the sequence shown in SEQ ID NO: 18. The shmiR according to this example is referred to below as "shmiR4".

[0319] In one example, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 20 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 20; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 21 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 21 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 21 may be the sequence shown in SEQ ID NO: 20. The shmiR according to this example is referred to below as "shmiR5".

[0320] In one example, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 22 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence can form a double strand with the sequence shown in SEQ ID NO: 22; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 23 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 23 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 23 may be the sequence shown in SEQ ID NO: 22. The shmiR according to this example is referred to below as "shmiR6".

[0321] In one example, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 24 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence can form a double strand with the sequence shown in SEQ ID NO: 24; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 25 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 25 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 25 may be the sequence shown in SEQ ID NO: 24. The shmiR according to this example is referred to below as "shmiR7".

[0322] In one example, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 26 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 26; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 27 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 27 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 27 may be the sequence shown in SEQ ID NO: 26. The shmiR according to this example is referred to below as "shmiR9".

[0323] In one example, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 28 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 28; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 29 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 29 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 29 may be the sequence shown in SEQ ID NO: 28. The shmiR according to this example is referred to below as "shmiR11".

[0324] In one example, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 30 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 30; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 31 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 31 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 31 may be the sequence shown in SEQ ID NO: 30. The shmiR according to this example is referred to below as "shmiR13".

[0325] In one example, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 32 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 32; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 33 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 33 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 33 may be the sequence shown in SEQ ID NO: 32. The shmiR according to this example is referred to below as "shmiR14".

[0326] In one example, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 34 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 34; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 35 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 35 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 35 may be the sequence shown in SEQ ID NO: 34. The shmiR according to this example is referred to below as "shmiR15".

[0327] In one example, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 36 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 36; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 37 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 37 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 37 may be the sequence shown in SEQ ID NO: 36. The shmiR according to this example is referred to below as "shmiR16".

[0328] In one example, the ddRNAi construct may include a DNA sequence encoding shmiR, which includes: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 38 except for 1, 2, 3, or 4 base mismatches, provided that the effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 38; and (ii) an effector complementary sequence, which includes a sequence substantially complementary to the effector sequence. For example, the shmiR encoded by the ddRNAi construct may include the effector sequence shown in SEQ ID NO: 39 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 39 and capable of forming a double strand with it. The effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 39 may be the sequence shown in SEQ ID NO: 38. The shmiR according to this example is referred to below as "shmiR17".

[0329] In any of the instances described herein, the shmiR encoded by the ddRNAi construct of the present invention may include, in the 5' to 3' direction:

[0330] 5' flanking sequence of the pri-miRNA backbone;

[0331] Effector complementary sequences;

[0332] Stem-loop sequence;

[0333] Effect subsequences; and

[0334] The 3' flanking sequence of the pri-miRNA backbone.

[0335] In any of the instances described herein, the shmiR encoded by the ddRNAi construct of the present invention may include, in the 5' to 3' direction:

[0336] 5' flanking sequence of the pri-miRNA backbone;

[0337] Effect subsequence;

[0338] Stem-loop sequence;

[0339] Effector complementary sequences; and

[0340] The 3' flanking sequence of the pri-miRNA backbone.

[0341] Suitable loop sequences can be selected from those known in the art. However, an exemplary stem-loop sequence is shown in SEQ ID NO: 40.

[0342] Suitable primary microRNA (pri-miRNA or pri-R) backbones for nucleic acids used in this invention may be selected from those known in the art. For example, the pri-miRNA backbone may be selected from the pri-miR-30a backbone, pri-miR-155 backbone, pri-miR-21 backbone, and pri-miR-136 backbone. However, the pri-miRNA backbone is preferably the pri-miR-30a backbone. According to an example where the pri-miRNA backbone is the pri-miR-30a backbone, the 5' flanking sequence of the pri-miRNA backbone is shown in SEQ ID NO: 41, and the 3' flanking sequence of the pri-miRNA backbone is shown in SEQ ID NO: 42. Therefore, ddRNAi constructs encoding the shmiR of this invention (e.g., one or more of shmiR2-shmiR7, shmiR9, shmiR11, and shmiR13-shmiR17 described herein) may include a DNA sequence encoding the sequence shown in SEQ ID NO: 41 and a DNA sequence encoding the sequence shown in SEQ ID NO: 42.

[0343] In one instance, the ddRNAi construct may include a DNA sequence selected from any of the sequences shown in SEQ ID NO: 56-68.

[0344] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 56, and encodes shmiR (shmiR2) which comprises or consists of the sequence shown in SEQ ID NO: 43.

[0345] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 57 and encodes shmiR (shmiR3) which comprises or consists of the sequence shown in SEQ ID NO: 44.

[0346] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 58, and encodes shmiR (shmiR4) which comprises or consists of the sequence shown in SEQ ID NO: 45.

[0347] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 59, and encodes shmiR (shmiR5) which comprises or consists of the sequence shown in SEQ ID NO: 46.

[0348] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 60, and encodes shmiR (shmiR6) which comprises or consists of the sequence shown in SEQ ID NO: 47.

[0349] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 61 and encodes shmiR (shmiR7) which comprises or consists of the sequence shown in SEQ ID NO: 48.

[0350] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 62, and encodes shmiR (shmiR9) which comprises or consists of the sequence shown in SEQ ID NO: 49.

[0351] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 63, and encodes shmiR (shmiR11) which comprises or consists of the sequence shown in SEQ ID NO: 50.

[0352] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 64, and encodes shmiR (shmiR13) which comprises or consists of the sequence shown in SEQ ID NO: 51.

[0353] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 65 and encodes shmiR (shmiR14) which comprises or consists of the sequence shown in SEQ ID NO: 52.

[0354] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 66 and encodes shmiR (shmiR15) which comprises or consists of the sequence shown in SEQ ID NO: 53.

[0355] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 67 and encodes shmiR (shmiR16) which comprises or consists of the sequence shown in SEQ ID NO: 54.

[0356] In one instance, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 68, and encodes shmiR (shmiR17) which comprises or consists of the sequence shown in SEQ ID NO: 55.

[0357] The exemplary ddRNAi constructs of the present invention encode one or more shmiRs selected from shmiR2, shmiR3, shmiR5, shmiR9, shmiR13, shmiR14, and shmiR17 described herein. Particularly preferred are ddRNAi constructs encoding one or more shmiRs selected from shmiR3, shmiR13, shmiR14, and shmiR17 described herein. For example, the ddRNAi construct may encode shmiR13 as described herein. For example, the ddRNAi construct may encode shmiR17 as described herein.

[0358] Those skilled in the art will understand that the ddRNAi constructs described herein can encode multiple shmiRs that target RNA transcripts corresponding to the PABPN1 protein that causes OPMD.

[0359] Therefore, in one instance, the ddRNAi construct includes two or more nucleic acids encoding the shmiR described herein, such as two, three, four, five, six, seven, eight, nine, or ten nucleic acids encoding the shmiR described herein.

[0360] In one example, the ddRNAi construct comprises a nucleic acid containing a DNA sequence encoding shmiR2 or composed of a DNA sequence encoding shmiR2, and at least one other nucleic acid encoding shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR2 are described herein and should be adapted to this example of the present invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 56, and encoding shmiR comprising or composed of the sequence shown in SEQ ID NO: 43, the at least one other nucleic acid according to the present invention encoding shmiR targeting a region of the PABPN1 mRNA transcript. For example, the ddRNAi construct may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 56 (shmiR2) or the DNA sequence shown in SEQ ID NO: 56 (shmiR2), and (ii) a DNA sequence encoding one of shmiR3-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR17 or any corresponding shRNA thereof, or a nucleic acid comprising the DNA sequence encoding one of shmiR3-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR17.

[0361] In one example, the ddRNAi construct comprises a nucleic acid containing or composed of a DNA sequence encoding shmiR3 and at least one other nucleic acid encoding a shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR3 are described herein and should be adapted to this example of the invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 57, and encoding a shmiR comprising or composed of the sequence shown in SEQ ID NO: 44, the at least one other nucleic acid according to the present invention encoding a region of the PABPN1 mRNA transcript. For example, the ddRNAi construct may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 57 (shmiR3) or the DNA sequence shown in SEQ ID NO: 57 (shmiR3), and (ii) a DNA sequence comprising encoding one of shmiR2, shmiR4-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR17 or any corresponding shRNA thereof, or a nucleic acid comprising the DNA sequence encoding one of shmiR2, shmiR4-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR17.

[0362] In one example, the ddRNAi construct comprises a nucleic acid containing a DNA sequence encoding shmiR4 or composed of a DNA sequence encoding shmiR4 and at least one other nucleic acid encoding shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR4 are described herein and should be adapted to this example of the invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 58, and encoding shmiR comprising or composed of the sequence shown in SEQ ID NO: 45, the at least one other nucleic acid according to the present invention encoding shmiR targeting a region of the PABPN1 mRNA transcript. For example, ddRNAi constructs may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 58 (shmiR4) or the DNA sequence shown in SEQ ID NO: 58 (shmiR4), and (ii) a DNA sequence encoding one of shmiR2, shmiR3, shmiR5-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR17 or any corresponding shRNA thereof, or a nucleic acid comprising the DNA sequence encoding one of shmiR2, shmiR3, shmiR5-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR17.

[0363] In one example, the ddRNAi construct comprises a nucleic acid containing a DNA sequence encoding shmiR5 or composed of a DNA sequence encoding shmiR5 and at least one other nucleic acid encoding shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR5 are described herein and should be adapted to this example of the present invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 59, and encoding shmiR comprising or composed of the sequence shown in SEQ ID NO: 46, the at least one other nucleic acid according to the present invention encoding shmiR targeting a region of the PABPN1 mRNA transcript. For example, the ddRNAi construct may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 59 (shmiR5) or the DNA sequence shown in SEQ ID NO: 59 (shmiR5), and (ii) a DNA sequence encoding one of shmiR2-shmiR4, shmiR6-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR17 or any corresponding shRNA thereof, or a nucleic acid comprising the DNA sequence encoding one of shmiR2-shmiR4, shmiR6-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR17.

[0364] In one example, the ddRNAi construct comprises a nucleic acid containing a DNA sequence encoding shmiR6 or composed of a DNA sequence encoding shmiR6 and at least one other nucleic acid encoding shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR6 are described herein and should be adapted to this example of the present invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 60, and encoding shmiR comprising or composed of the sequence shown in SEQ ID NO: 47, the at least one other nucleic acid according to the present invention encoding shmiR targeting a region of the PABPN1 mRNA transcript. For example, the ddRNAi construct may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 60 (shmiR6) or the DNA sequence shown in SEQ ID NO: 60 (shmiR6), and (ii) a DNA sequence comprising encoding one of shmiR2, shmiR5-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR17 or any corresponding shRNA thereof, or a nucleic acid comprising the DNA sequence encoding one of shmiR2, shmiR5-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR17.

[0365] In one example, the ddRNAi construct comprises a nucleic acid containing a DNA sequence encoding shmiR7 or composed of a DNA sequence encoding shmiR7 and at least one other nucleic acid encoding shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR7 are described herein and should be adapted to this example of the invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 61, and encoding shmiR comprising or composed of the sequence shown in SEQ ID NO: 48, the at least one other nucleic acid according to the present invention encoding a region of the PABPN1 mRNA transcript. For example, the ddRNAi construct may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 61 (shmiR7) or the DNA sequence shown in SEQ ID NO: 61 (shmiR7), and (ii) a DNA sequence encoding one of shmiR2-shmiR6, shmiR9, shmiR11 or shmiR13-shmiR17 or any corresponding shRNA thereof, or a nucleic acid comprising the DNA sequence encoding one of shmiR2-shmiR6, shmiR9, shmiR11 or shmiR13-shmiR17.

[0366] In one example, the ddRNAi construct comprises a nucleic acid containing a DNA sequence encoding shmiR9 or composed of a DNA sequence encoding shmiR9 and at least one other nucleic acid encoding shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR9 are described herein and should be adapted to this example of the invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 62, and encoding shmiR comprising or composed of the sequence shown in SEQ ID NO: 49, the at least one other nucleic acid according to the present invention encoding a region of the PABPN1 mRNA transcript. For example, the ddRNAi construct may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 62 (shmiR9) or the DNA sequence shown in SEQ ID NO: 62 (shmiR9), and (ii) a nucleic acid comprising a DNA sequence encoding one of shmiR2-shmiR7, shmiR11 or shmiR13-shmiR17 or any of their corresponding shRNAs or a nucleic acid comprising a DNA sequence encoding one of shmiR2-shmiR7, shmiR11 or shmiR13-shmiR17.

[0367] In one example, the ddRNAi construct comprises a nucleic acid containing or composed of a DNA sequence encoding shmiR11 and at least one other nucleic acid encoding shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR11 are described herein and should be adapted to this example of the invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 63, and encoding shmiR comprising or composed of the sequence shown in SEQ ID NO: 50, the at least one other nucleic acid according to the present invention encoding a region of the PABPN1 mRNA transcript. For example, ddRNAi constructs may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 63 (shmiR11) or the DNA sequence shown in SEQ ID NO: 63 (shmiR11), and (ii) a DNA sequence encoding one of shmiR2-shmiR7, shmiR9 or shmiR13-shmiR17 or any of their corresponding shRNAs, or a nucleic acid comprising the DNA sequence encoding one of shmiR2-shmiR7, shmiR9 or shmiR13-shmiR17.

[0368] In one example, the ddRNAi construct comprises a nucleic acid containing or composed of a DNA sequence encoding shmiR13 and at least one other nucleic acid encoding shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR13 are described herein and should be adapted to this example of the invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 64, and encoding shmiR comprising or composed of the sequence shown in SEQ ID NO: 51, the at least one other nucleic acid according to the present invention encoding a region of the PABPN1 mRNA transcript. For example, the ddRNAi construct may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or the DNA sequence shown in SEQ ID NO: 64 (shmiR13), and (ii) a DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11 or shmiR14-shmiR17 or any of their corresponding shRNAs, or a nucleic acid comprising the DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11 or shmiR14-shmiR17.

[0369] In one example, the ddRNAi construct comprises a nucleic acid containing or composed of a DNA sequence encoding shmiR14 and at least one other nucleic acid encoding shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR14 are described herein and should be adapted to this example of the invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 65, and encoding shmiR comprising or composed of the sequence shown in SEQ ID NO: 52, the at least one other nucleic acid according to the present invention encoding a region of the PABPN1 mRNA transcript. For example, ddRNAi constructs may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 65 (shmiR14) or composed of the DNA sequence shown in SEQ ID NO: 65 (shmiR14), and (ii) a DNA sequence comprising encoding one of shmiR2-shmiR7, shmiR9, shmiR11 or shmiR13, shmiR15-shmiR17 or any corresponding shRNA thereof, or a nucleic acid comprising the DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11 or shmiR13, shmiR15-shmiR17.

[0370] In one example, the ddRNAi construct comprises a nucleic acid containing or composed of a DNA sequence encoding shmiR15 and at least one other nucleic acid encoding shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR15 are described herein and should be adapted to this example of the invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 66, and encoding shmiR comprising or composed of the sequence shown in SEQ ID NO: 53, the at least one other nucleic acid according to the present invention encoding a region of the PABPN1 mRNA transcript. For example, ddRNAi constructs may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 66 (shmiR15) or the DNA sequence shown in SEQ ID NO: 66 (shmiR15), and (ii) a DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR14 or shmiR16-shmiR17 or any corresponding shRNA thereof, or a nucleic acid comprising the DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR14 or shmiR16-shmiR17.

[0371] In one example, the ddRNAi construct comprises a nucleic acid containing or composed of a DNA sequence encoding shmiR16 and at least one other nucleic acid encoding shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR16 are described herein and should be adapted to this example of the present invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 67, and encoding shmiR comprising or composed of the sequence shown in SEQ ID NO: 54, the at least one other nucleic acid according to the present invention encoding a region of the PABPN1 mRNA transcript. For example, ddRNAi constructs may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 67 (shmiR16) or composed of the DNA sequence shown in SEQ ID NO: 67 (shmiR16), and (ii) a DNA sequence comprising encoding one of shmiR2-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR15, or shmiR17 or any corresponding shRNA thereof, or a nucleic acid comprising the DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR15, or shmiR17.

[0372] In one example, the ddRNAi construct comprises a nucleic acid containing or composed of a DNA sequence encoding shmiR17 and at least one other nucleic acid encoding shmiR targeting a region of the PABPN1 mRNA transcript according to the present invention. Exemplary nucleic acids encoding shmiR17 are described herein and should be adapted to this example of the invention with necessary modifications. In one example, the ddRNAi construct comprises a nucleic acid and at least one other nucleic acid according to the present invention, the nucleic acid comprising or composed of the DNA sequence shown in SEQ ID NO: 68, and encoding shmiR comprising or composed of the sequence shown in SEQ ID NO: 55, the at least one other nucleic acid according to the present invention encoding a region of the PABPN1 mRNA transcript. For example, ddRNAi constructs may include (i) a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or the DNA sequence shown in SEQ ID NO: 68 (shmiR17), and (ii) a DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR16 or any of their corresponding shRNAs, or a nucleic acid comprising the DNA sequence encoding one of shmiR2-shmiR7, shmiR9, shmiR11 or shmiR13-shmiR16.

[0373] Based on one example where the ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 1. This document describes suitable nucleic acids encoding shmiRs with effector sequences substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 1.

[0374] Based on one example where the ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 2. This document describes suitable nucleic acids encoding shmiRs with effector sequences substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 2.

[0375] Based on one example where the ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 4. This paper describes suitable nucleic acids encoding shmiRs with effector sequences for shmiR5, the effector sequences being substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 4.

[0376] Based on one example where the ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 7. This document describes, for shmiR9, a suitable nucleic acid encoding a shmiR having an effector sequence substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 7.

[0377] Based on one example where the ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 9. This document describes suitable nucleic acids encoding shmiRs with effector sequences for shmiR13, the effector sequences being substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 9.

[0378] Based on one example where the ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 10. This document describes suitable nucleic acids encoding shmiRs with effector sequences for shmiR14, the effector sequences being substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 10.

[0379] Based on one example where the ddRNAi construct encodes multiple shmiRs, at least one of the shmiRs includes an effector sequence substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 13. This document describes suitable nucleic acids encoding shmiRs with effector sequences for shmiR17, the effector sequences being substantially complementary to a region of corresponding length in an RNA transcript comprising or consisting of the sequence shown in SEQ ID NO: 13.

[0380] Exemplary ddRNAi constructs of multiple shmiRs of the present invention include at least two nucleic acids, each nucleic acid including a DNA sequence encoding the shmiR of the present invention, wherein each shmiR includes a different effector sequence.

[0381] In one example, at least two nucleic acids encode shmiRs comprising effector sequences substantially complementary to regions of corresponding length in the RNA transcripts shown in one of SEQ ID NOs: 1, 2, 4, 7, 9, 10, and 13. Exemplary nucleic acids of the present invention encoding shmiRs comprising effector sequences substantially complementary to regions of corresponding length in the RNA transcripts shown in SEQ ID NOs: 1, 2, 4, 7, 9, 10, and 13 are described herein and should be adapted to this example of the invention with necessary modifications.

[0382] In one instance, the ddRNAi construct includes at least two nucleic acids selected from the group consisting of:

[0383] Includes a DNA sequence encoding shmiR or a nucleic acid composed thereof, which includes the effector sequence shown in SEQ ID NO: 15 and the effector complementary sequence shown in SEQ ID NO: 14, for example including the DNA sequence shown in SEQ ID NO: 56 (shmiR2) or a nucleic acid composed thereof;

[0384] Includes a DNA sequence encoding shmiR or a nucleic acid composed thereof, which includes the effector sequence shown in SEQ ID NO: 17 and the effector complementary sequence shown in SEQ ID NO: 16, for example including the DNA sequence shown in SEQ ID NO: 57 (shmiR3) or a nucleic acid composed thereof;

[0385] Includes a DNA sequence encoding shmiR or a nucleic acid composed thereof, which includes the effector sequence shown in SEQ ID NO: 21 and the effector complementary sequence shown in SEQ ID NO: 20, for example including the DNA sequence shown in SEQ ID NO: 59 (shmiR5) or a nucleic acid composed thereof;

[0386] Includes a DNA sequence encoding shmiR or a nucleic acid composed thereof, which includes the effector sequence shown in SEQ ID NO: 27 and the effector complementary sequence shown in SEQ ID NO: 26, for example including the DNA sequence shown in SEQ ID NO: 62 (shmiR9) or a nucleic acid composed thereof;

[0387] Includes a DNA sequence encoding shmiR or a nucleic acid composed thereof, which includes the effector sequence shown in SEQ ID NO: 31 and the effector complementary sequence shown in SEQ ID NO: 30, for example including the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or a nucleic acid composed thereof;

[0388] Includes a DNA sequence encoding shmiR or a nucleic acid thereof, comprising the effector sequence shown in SEQ ID NO: 33 and the effector complement sequence shown in SEQ ID NO: 32, such as including the DNA sequence shown in SEQ ID NO: 65 (shmiR14) or a nucleic acid thereof; and

[0389] Includes a DNA sequence encoding shmiR or a nucleic acid composed thereof, comprising the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO: 38, for example, including the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or a nucleic acid composed thereof.

[0390] In one example, each of at least two nucleic acids in the ddRNAi construct encodes an shmiR comprising an effector sequence substantially complementary to a region of corresponding length in the RNA transcripts shown in one of SEQ ID NO: 2, 9, 10, and 13. Exemplary nucleic acids encoding shmiRs comprising effector sequences substantially complementary to regions of corresponding length in the RNA transcripts shown in SEQ ID NO: 2, 9, 10, and 13 are described herein and should be adapted to this example of the invention with necessary modifications.

[0391] In one instance, the ddRNAi construct includes at least two nucleic acids selected from the group consisting of:

[0392] Includes a DNA sequence encoding shmiR or a nucleic acid composed thereof, which includes the effector sequence shown in SEQ ID NO: 17 and the effector complementary sequence shown in SEQ ID NO: 16, for example including the DNA sequence shown in SEQ ID NO: 57 (shmiR3) or a nucleic acid composed thereof;

[0393] Includes a DNA sequence encoding shmiR or a nucleic acid composed thereof, which includes the effector sequence shown in SEQ ID NO: 31 and the effector complementary sequence shown in SEQ ID NO: 30, for example including the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or a nucleic acid composed thereof;

[0394] Includes a DNA sequence encoding shmiR or a nucleic acid thereof, comprising the effector sequence shown in SEQ ID NO: 33 and the effector complement sequence shown in SEQ ID NO: 32, such as including the DNA sequence shown in SEQ ID NO: 65 (shmiR14) or a nucleic acid thereof; and

[0395] Includes a DNA sequence encoding shmiR or a nucleic acid composed thereof, comprising the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO: 38, for example, including the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or a nucleic acid composed thereof.

[0396] In one example, the ddRNAi construct includes a nucleic acid encoding shmiR, which includes an effector sequence substantially complementary to a region of a corresponding length in the RNA transcript shown in SEQ ID NO: 9, and a nucleic acid encoding shmiR, which includes an effector sequence substantially complementary to a region of a corresponding length in the RNA transcript shown in SEQ ID NO: 13. For example, the ddRNAi construct may include:

[0397] (a) comprising a DNA sequence encoding shmiR or a nucleic acid thereof, including the effector sequence shown in SEQ ID NO: 31 and the effector complement sequence shown in SEQ ID NO: 30, for example including the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or a nucleic acid thereof; and

[0398] (b) includes a DNA sequence encoding shmiR or a nucleic acid thereof, comprising the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO: 38, for example, the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or a nucleic acid thereof.

[0399] The exemplary ddRNAi construct of the present invention includes a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or the DNA sequence shown in SEQ ID NO: 64 (shmiR13) and a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or the DNA sequence shown in SEQ ID NO: 68 (shmiR17).

[0400] In one example, the ddRNAi construct includes a nucleic acid encoding shmiR, which includes an effector sequence substantially complementary to a region of a corresponding length in the RNA transcript shown in SEQ ID NO: 2, and a nucleic acid encoding shmiR, which includes an effector sequence substantially complementary to a region of a corresponding length in the RNA transcript shown in SEQ ID NO: 10. For example, the ddRNAi construct may include:

[0401] (a) comprising a DNA sequence encoding shmiR or a nucleic acid thereof, including the effector sequence shown in SEQ ID NO: 17 and the effector complement sequence shown in SEQ ID NO: 16, for example including the DNA sequence shown in SEQ ID NO: 57 (shmiR3) or a nucleic acid thereof; and

[0402] (b) Includes a DNA sequence encoding shmiR or a nucleic acid composed thereof, comprising the effector sequence shown in SEQ ID NO: 33 and the effector complement sequence shown in SEQ ID NO: 32, for example, including the sequence shown in SEQ ID NO: 65 (shmiR14) or a nucleic acid composed thereof.

[0403] The exemplary ddRNAi construct of the present invention includes a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 57 (shmiR3) or the DNA sequence shown in SEQ ID NO: 57 (shmiR3) and a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 65 (shmiR14) or the DNA sequence shown in SEQ ID NO: 65 (shmiR14).

[0404] According to an example of the ddRNAi construct of the present invention encoding two or more shmiRs, two or more of the nucleic acids encoding shmiRs can form separate portions of the same polynucleotide in the ddRNAi construct.

[0405] In some instances, the nucleic acid encoding shmiR, or each nucleic acid encoding shmiR, may include, or be operatively linked to, additional elements, for example, to facilitate transcription of shmiR. For instance, a ddRNAi construct may include one or more promoters operatively linked to a sequence encoding the shmiR described herein. Other elements, such as transcription terminators and initiators, are known in the art and / or described herein.

[0406] In each of the foregoing examples of the ddRNAi constructs describing the content of this invention, the nucleic acid encoding shmiR, or each nucleic acid, is operatively linked to a promoter. For example, the ddRNAi construct as described herein may include a single promoter operatively linked to the nucleic acid encoding shmiR, or each nucleic acid, included therein, for example, to drive the expression of one or more shmiRs from the ddRNAi construct. In another example, each nucleic acid encoding shmiR included in the ddRNAi construct is operatively linked to a separate promoter.

[0407] Depending on the instance where multiple promoters exist, the promoters may be the same or different. For example, a construct may include multiple copies of the same promoter, each operatively linked to a different nucleic acid of the invention. In another instance, each promoter operatively linked to the nucleic acid encoding shmiR of the invention is different. For example, in a ddRNAi construct encoding two shmiRs, the two nucleic acids encoding shmiR are each operatively linked to a different promoter.

[0408] In one instance, the promoter is a constitutive promoter. When referring to promoters, the term "constitutive" means that the promoter is capable of directing the transcription of an operable, linked nucleic acid sequence in the absence of a specific stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters can direct the expression of coding sequences in virtually any cell and tissue. Promoters used for transcription of shmiR include ubiquitin, CMV, β-actin, histone H4, EF-1α, or promoters of pgk genes controlled by RNA polymerase II, or promoter elements controlled by RNA polymerase I.

[0409] In one example, Pol II promoters such as CMV, SV40, U1, β-actin, or hybrid Pol II promoters are used. Other suitable Pol II promoters are known in the art and can be used according to this example of the invention. For example, in the ddRNAi construct of the invention expressing pri-miRNA, the Pol II promoter system may be preferred, wherein the pri-miRNA is processed into one or more shmiRs by the enzymes Drosha and Pasha. In the ddRNAi construct of the invention comprising sequences encoding multiple shmiRs under the control of a single promoter, the Pol II promoter system may also be preferred. A Pol II promoter system may also be desirable where tissue specificity is required.

[0410] In another instance, promoters controlled by RNA polymerase III are used, such as the U6 promoter (U6-1, U6-8, U6-9), the H1 promoter, the 7SL promoter, the human Y promoter (hY1, hY3, hY4 (see Maraia et al., Nucleic Acids Res 22(15): 3045-52(1994)) and hY5 (see Maraia et al., Nucleic Acids Res 24(18): 3552-59(1994)), the human MRP-7-2 promoter, the adenovirus VA1 promoter, the human tRNA promoter, or the 5S ribosomal RNA promoter.

[0411] Suitable promoters for the ddRNAi constructs of the present invention are described in U.S. Patent Nos. 8,008,468 and 8,129,510.

[0412] In one instance, the promoter is the RNA Pol III promoter. For example, the promoter is the U6 promoter (e.g., U6-1, U6-8, or U6-9 promoters). In another instance, the promoter is the H1 promoter.

[0413] In the case of the ddRNAi construct of the present invention encoding multiple shmiRs, each nucleic acid in the ddRNAi construct is operatively linked to a U6 promoter, such as a separate U6 promoter.

[0414] In one example, the promoter in the ddRNAi construct is the U6 promoter. For example, the promoter could be the U6-1 promoter. For example, the promoter could be the U6-8 promoter. For example, the promoter could be the U6-9 promoter.

[0415] In some instances, promoters with variable strength are used. For example, using two or more strong promoters (e.g., Pol III promoters) can induce cell death by, for example, removing the pooling of available nucleotides or other cellular components required for transcription. Furthermore, or alternatively, using several strong promoters can induce toxic levels of shmiR expression in cells. Thus, in some instances, one or more promoters in a multi-promoter ddRNAi construct are weaker than other promoters in the construct, or all promoters in the construct can express shmiR at a rate less than the maximum rate. Promoters can also be modified using various molecular techniques, or by modifying various regulatory elements, to obtain weaker or stronger levels of transcription. One method to achieve reduced transcription is to modify sequence elements within the promoter that are known to control promoter activity. For example, proximal sequence elements (PSEs) are known to affect the activity of the human U6 promoter (see Domitrovich et al., Nucleic Acids Res, 31:2344-2352 (2003)). Replacing the PSE element present in strong promoters such as human U6-1, U6-8, or U6-9 promoters with an element from a weak promoter such as human U6-7 reduces the activity of the hybrid U6-1, U6-8, or U6-9 promoter. This method is used in the example described in this application, but other means of achieving this result are known in the art.

[0416] The promoters used in the ddRNAi constructs of this invention can also be tissue-specific or cell-specific. When applied to promoters, the term "tissue-specific" refers to a promoter capable of guiding the selective transcription of the target nucleic acid in a specific type of tissue (e.g., tissue of the eye or muscle) when expression of the same target nucleotide sequence is relatively lacking in different types of tissues (e.g., liver). When applied to promoters, the term "cell-specific" refers to a promoter capable of guiding the selective transcription of the target nucleic acid in a specific type of cell when expression of the same target nucleotide sequence is relatively lacking in different types of cells within the same tissue. According to one example, muscle-specific promoters, such as Spc512 or CK8, are used. However, other muscle-specific promoters are known in the art and are contemplated for use in conjunction with the ddRNAi constructs of this invention.

[0417] In one instance, the ddRNAi construct of the present invention may additionally include one or more enhancers to increase the expression of shmiR as described herein. Enhancers suitable for examples of the present invention include the Apo E HCR enhancer, the CMV enhancer (Xia et al., Nucleic Acids Res, 31-17 (2003)), and other enhancers known to those skilled in the art. Suitable enhancers for use in the ddRNAi construct of the present invention are described in U.S. Patent No. 8,008,468.

[0418] In another example, the ddRNAi construct of the present invention may include a transcription terminator linked to a nucleic acid encoding the shmiR of the present invention. In the case of ddRNAi constructs comprising multiple nucleic acids described herein (i.e., encoding multiple shmiRs), the terminator linked to each nucleic acid may be the same or different. For example, in the ddRNAi construct of the present invention using the RNA pol III promoter, the terminator may be a continuous fragment of four or more, five or more, or six or more T residues. However, when using different promoters, the terminator may be different and matched to the promoter from the gene that produces the terminator. These terminators include, but are not limited to, terminators for SV40 polyadenylate, the Adv VA1 gene, the 5S ribosomal RNA gene, and human t-RNA. Other combinations of promoters and terminators are known in the art and contemplated for use in the ddRNAi constructs of the present invention.

[0419] Furthermore, promoters and terminators can be mixed and matched, as is typically done using RNA pol II promoters and terminators.

[0420] In one instance, the promoter and terminator combinations used for each nucleic acid in a ddRNAi construct that includes multiple nucleic acids are different to reduce the likelihood of DNA recombination events between components.

[0421] An exemplary ddRNAi construct of the present invention includes a nucleic acid comprising or composed of a DNA sequence encoding shmiR13 as described herein, operably linked to a promoter, and a nucleic acid comprising or composed of a DNA sequence encoding shmiR17 as described herein, operably linked to a promoter. For example, an exemplary ddRNAi construct of the present invention includes a nucleic acid comprising or composed of a DNA sequence shown in SEQ ID NO: 64 operably linked to a promoter, and a nucleic acid comprising or composed of a DNA sequence shown in SEQ ID NO: 68 operably linked to a promoter. In one example, each nucleic acid in the ddRNAi construct encoding shmiR is operably linked to a separate promoter. In another example, each nucleic acid in the ddRNAi construct encoding shmiR is operably linked to the same promoter. For example, the promoter or each promoter may be a U6 promoter, such as U6-1, U6-8, or U6-9 promoters. For example, the promoter or each promoter may be a muscle-specific promoter, such as Spc512 or CK8 promoters.

[0422] According to one example, the nucleic acids in the ddRNAi construct encoding shmiR13 and shmiR17 are operably linked to the same Spc512 promoter, and the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 72. According to another example, the nucleic acids in the ddRNAi construct encoding shmiR13 and shmiR17 are operably linked to the same CK8 promoter, and the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 70.

[0423] Another exemplary ddRNAi construct of the present invention comprises a nucleic acid comprising a DNA sequence encoding shmiR3, operably linked to a promoter, or comprising a nucleic acid comprising a DNA sequence encoding shmiR14, operably linked to a promoter. For example, an exemplary ddRNAi construct of the present invention comprises a nucleic acid comprising a DNA sequence operably linked to a promoter, as shown in SEQ ID NO: 57, or comprising a nucleic acid comprising a DNA sequence operably linked to a promoter, and a nucleic acid comprising a DNA sequence operably linked to a promoter, as shown in SEQ ID NO: 65. In one instance, each nucleic acid in the ddRNAi construct encoding shmiR is operably linked to a separate promoter. In another instance, each nucleic acid in the ddRNAi construct encoding shmiR is operably linked to the same promoter. For example, the promoter or each promoter may be a U6 promoter, such as U6-1, U6-8, or U6-9. For example, the promoter or each promoter may be a muscle-specific promoter, such as Spc512 or CK8.

[0424] According to an example where the nucleic acids in the ddRNAi construct encoding shmiR3 and shmiR14 are operably linked to the same Spc512 promoter, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 71. According to an example where the nucleic acids in the ddRNAi construct encoding shmiR3 and shmiR14 are operably linked to the same CK8 promoter, the ddRNAi construct comprises or consists of the DNA sequence shown in SEQ ID NO: 69.

[0425] Furthermore, the ddRNAi construct may include one or more multiple cloning sites and / or strategically located unique restriction sites, making it easy to remove or replace promoters, nucleic acids encoding shmiR, and / or other regulatory elements. The ddRNAi construct can be assembled from smaller oligonucleotide components using strategically positioned restriction sites and / or complementary sticky ends. The base vector used in one method according to the invention comprises a plasmid with multiple links, wherein all sites are unique (although this is not absolutely required). Sequentially, each promoter is inserted between its designated unique sites to produce a cassette with one or more promoters, all of which may have variable orientations. Sequentially, annealed primer pairs are again inserted downstream of each individual promoter at unique sites to produce single, double, or multiple expression cassette constructs. The insert can be moved into the AAV backbone using two unique restriction enzyme sites (identical or different) flanking the single, double, or multiple expression cassette insert.

[0426] The ddRNAi construct can be generated using any suitable genetic engineering techniques known in the art, including but not limited to standard techniques such as PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing. The ddRNAi construct (or polynucleotides comprising it) may also include sequences necessary for packaging the ddRNAi construct into viral particles and / or sequences that allow the ddRNAi construct to integrate into the genome of a target cell. In some instances, the viral construct, or each viral construct, additionally contains genes that allow viral replication and proliferation; however, such genes will be provided in trans form. Additionally, each viral construct may contain genes or genetic sequences from the genome of any known organism incorporated or modified in its natural form. For example, the viral construct may include sequences for replicating the construct in bacteria.

[0427] Testing the shmiR or ddRNAi constructs of this invention

[0428] Cell culture model

[0429] An example of a cell line used as a cell culture model for OPMD is the HEK293T cell line (HEK293T, ATCC, Manassas, USA), which has been transfected with a vector expressing either normal Ala10-human PAPPN1-FLAG (Ala10) or the mutant Ala17-human PAPPN1-FLAG (Ala17), the latter being a marker of OPMD.

[0430] Other examples of cell lines that can be used as OPMD cell culture models are C2C12 mouse muscle cells and ARPE-19 human retinal cells.

[0431] Another example of a cell line used as a cell culture model for OPMD is a primary mouse myoblast (IM2) cell line stably transfected to express either normal Ala10-human PAPPN1-FLAG (Ala10) or the mutant Ala17-human PAPPN1-FLAG (Ala17). An exemplary IM2-derived cell line stably expressing the mutant Ala17-human PAPPN1-FLAG (Ala17) is the H2kB-D7e cell line. The H2kB-D7e cell line is also described in Raz et al., (2011) American Journal of Pathology, 179(4): 1988-2000.

[0432] Other cell lines suitable for OPMD cell culture models are known in the art, for example, described in Fan et al. (2001) Human Molecular Genetics, 10:2341-2351, Bao et al. (2002) The Journal of Biological Chemistry, 277:12263-12269 and Abu-Baker et al. (2003) Human Molecular Genetics, 12:2609-2623.

[0433] As illustrated herein, the activity of the shmiR of the present invention is determined by administering a nucleic acid encoding shmiR or a ddRNAi construct or expression vector including therein into cells and subsequently measuring the expression level of RNA or protein encoded by the PABPN1 gene. For example, intracellular PABPN1 gene expression can be determined using one or more probes or primers specific to PABPN1 by any one or more of RT-PCR, quantitative PCR, semi-quantitative PCR, or in situ hybridization under stringent conditions. PABPN1 mRNA or DNA can also be detected by PCR, Western blot, or ELISA using one or more probes or primers specific to PABPN1.

[0434] Polynucleotides for detecting PABPN1 expression that can be used with RT-PCR, quantitative PCR, or semi-quantitative PCR techniques are known and commercially available (Thermo Fisher). However, polynucleotides that can be designed for PCR-based detection methods based on available sequence information of PABPN1 can be used with methods and / or software known in the art. In one example, the presence or absence of PABPN1 mRNA can be detected using RT-PCR with standard methods known in the art. In one example, the presence or absence or relative abundance of PABPN1 peptides or proteins can be detected using any one or more of Western blotting, ELISA, or other standard quantitative or semi-quantitative techniques available in the art, or a combination of these techniques. Techniques for antibody recognition dependent on PABPN1 are considered and described. In one example, the presence or absence or relative abundance of PABPN1 peptides can be detected using techniques including antibody capture of PABPN1 peptides and electrophoretic resolution of the captured PABPN1 peptides, such as using Isonostic... TM Assay (Target Discovery, Inc.). Antibodies against PABPN1 protein are available for purchase.

[0435] Various methods for normalizing the difference between transfection or transduction efficiency and sample recovery rate are known in the art.

[0436] The nucleic acids, ddRNAi constructs, or expression vectors of the present invention, relative to the level of expression of the PABPN1-encoded mRNA or protein, or the level of nuclear aggregation of the PABPN1 protein in the absence of the RNA of the present invention, are considered useful for therapeutic applications, for example, treating OPMD by reducing the expression of endogenous PABPN1 and replacing some or all of the endogenous PABPN1 containing the PABPN1 protein described herein that causes OPMD.

[0437] animal models

[0438] Several small animal models can be used to study OPMD, examples of which are described in Uyama et al., (2005) Acta Myologica, 24(2):84-88 and Chartier and Simonelig (2013) Drug Discovery Today: Technologies, 10:e103-107. An exemplary animal model is the A17.1 transgenic mouse model, previously described in Davies et al. (2005) Nature Medicine, 11:672-677 and Trollet et al. (2010) Human Molecular Genetics, 19(11):2191-2207.

[0439] Any of the aforementioned animal models can be used to determine the efficacy of the shmiR or ddRNAi constructs of the present invention in knocking down, reducing, or inhibiting the expression of RNA or protein encoded by the PABPN1 gene.

[0440] This article has described a method for determining PABPN1 gene expression using a cell model, and this method has been adapted to this embodiment of the invention with necessary modifications.

[0441] PABPN1 construct

[0442] As described herein, the AAV of the present invention comprises a polynucleotide sequence including a PABPN1 construct. In this regard, the AAV of the present invention provides a reagent for replacing, for example, a cell or animal protein with the functional PABPN1 protein. The functional PABPN1 protein is not the cause of OPMD, nor is it encoded by an mRNA transcript that is targeted by shmiR encoded by a ddRNAi construct also included in the AAV as described herein.

[0443] In one instance, the PABPN1 construct includes a nucleic acid, such as DNA or cDNA, encoding a functional PABPN1 protein. For example, the nucleic acid encoding the functional PABPN1 protein may be codon-optimized, for instance, containing one or more degenerate or wobble bases relative to the wild-type PABPN1 nucleic acid but encoding the same amino acid, such that the corresponding mRNA sequence encoding the functional PABPN1 protein is not recognized by the shmiR encoded and expressed from the ddRNAi construct. For example, the codon-optimized nucleic acid encoding the functional PABPN1 protein may include one or more degenerate or wobble bases relative to the wild-type PABPN1 nucleic acid within a region targeted by one or more shmiRs encoded and expressed by the ddRNAi construct. In one instance, one or more degenerate or wobble bases are located within the seed region of the effector sequence of the shmiR encoded and expressed by the ddRNAi construct.

[0444] In one example, the nucleic acid of the PABPN1 construct encoding a functional PABPN1 protein is codon-optimized such that its corresponding mRNA sequence is not recognized by the shmiR encoded and expressed from the ddRNAi construct. Preferably, the functional PABPN1 protein encoded by the codon-optimized nucleic acid sequence comprises the amino acid sequence shown in SEQ ID NO: 74, i.e., the amino acid sequence of the wild-type human PABPN1 protein. Those skilled in the art will understand that there are many combinations of nucleotide sequences that can be used to encode a functional PABPN1 protein, and the choice of nucleotide sequence will ultimately depend on the effector sequence of the shmiR encoded and expressed by the ddRNAi construct, i.e., such that the codon-optimized nucleic acid is not recognized by the shmiR. In one example, the PABPN1 construct comprises a nucleic acid containing the sequence shown in SEQ ID NO: 73. In one example, the nucleic acid encoding a functional PABPN1 protein may also include a Kozak sequence.

[0445] In one example, a codon-optimized nucleic acid encoding a functional PABPN1 protein is operatively linked to a promoter suitable for expressing the functional PABPN1 protein. A promoter suitable for expressing the functional PABPN1 protein in muscle may be particularly suitable. An exemplary promoter suitable for nucleic acids encoding a functional PABPN1 protein is the Spc512 promoter. Another exemplary promoter suitable for nucleic acids encoding a functional PABPN1 protein is the CK8 promoter. However, any suitable promoter known in the art may be used. For example, other suitable promoters for use with nucleic acids encoding a functional PABPN1 protein are described in US20110212529A1.

[0446] In one instance, the PABPN1 construct and the ddRNAi construct are operatively linked to the same promoter within the same polynucleotide, for example, both are operatively linked to the Spc512 promoter. According to this instance, a single promoter drives the expression of both functional PABPN1 protein and shmiR.

[0447] As described herein, promoters that can be used in some instances of the present invention may be tissue-specific or cell-specific.

[0448] In one example, the codon-optimized nucleic acid encoding the functional PABPN1 protein of the present invention may additionally include one or more enhancers to increase the expression of the functional PABPN1 protein and its corresponding mRNA transcript. Enhancers suitable for this example of the present invention will be known to those skilled in the art.

[0449] Testing functionality PABPN1

[0450] animal models

[0451] An exemplary animal model for studying OPMD has been described.

[0452] Any of the aforementioned animal models can be used to determine the efficacy of the PABPN1 construct or AAV including it in replacing functional PABPN1 protein in vivo in the presence of one or more shmiRs expressed by the ddRNAi of the present invention.

[0453] This document has described a method for determining PABPN1 expression in a cell model, and this method has been adapted to this embodiment of the invention with necessary modifications.

[0454] In one instance, histological and morphological analyses can be used to determine the efficacy of the reagents of the present invention in replacing functional PABPN1 protein in vivo in the presence of one or more shmiRs expressed by the ddRNAi of the present invention. Other assays that can be used to determine the efficacy of the reagents of the present invention in replacing functional PABPN1 protein in vivo are described in Trollet et al., (2010) Human Molecular Genetics, 19(11): 2191-2207.

[0455] PABPN1 'Silence and Substitution' DNA Construct

[0456] As described herein, the AAV of the present invention comprises a single polynucleotide comprising a ddRNAi construct and a PABPN1 construct as described herein. That is, the ddRNAi construct and the PABPN1 construct can be provided as a combined DNA construct (also referred to herein as a 'silence and replacement' construct or SR construct) packaged in a modified AAV as described herein for delivery to a patient. Example 2 describes an exemplary DNA construct comprising a nucleic acid encoding a functional PABPN1 protein and the ddRNAi construct of the present invention.

[0457] A single DNA construct including a ddRNAi construct and a PABPN1 construct may include, for example, one or more promoters to drive the expression of a functional PABPN1 protein and / or shmiR encoded by the ddRNAi construct. Promoters used in some instances of the invention may be tissue-specific or cell-specific. Exemplary promoters are muscle-specific promoters, such as Spc512 and CK8. However, any suitable promoters known in the art are considered for use in the DNA constructs described herein, such as those described in US20110212529A1.

[0458] DNA constructs, including ddRNAi and PABPN1 constructs, are packaged in modified AAVs as described herein for delivery to patients.

[0459] In one example, the DNA construct includes a muscle-specific promoter, such as the Spc512 promoter, in the 5' to 3' orientation, the PABPN1 construct described herein, and the ddRNAi construct described herein, for example, wherein the ddRNAi construct is located in the 3' untranslated region (UTR) of the nucleic acid encoding the functional PABPN1 protein. The DNA construct according to this embodiment is shown in... Figure 1 A.

[0460] The exemplary DNA construct according to this embodiment includes, in the 5' to 3' direction:

[0461] (a) Muscle-specific promoters, such as Spc512;

[0462] (b) The PABPN1 construct described herein, comprising the DNA sequence encoding a functional PABPN1 protein having an mRNA transcript not targeted by shmiR encoded by the ddRNAi construct; and

[0463] (c) The ddRNAi construct of the present invention comprising a nucleic acid including a DNA sequence encoding shmiR17 as described herein and a nucleic acid including a DNA sequence encoding shmiR13 as described herein.

[0464] According to this embodiment, the DNA construct may include or consist of the DNA sequence shown in SEQ ID NO: 72.

[0465] An exemplary ddRNAi construct including shmiR13 and shmiR17 in the DNA construct of the present invention comprises a DNA sequence encoding shmiR comprising the effector sequence shown in SEQ ID NO: 31 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 31, such as the effector complementary sequence shown in SEQ ID NO: 30 (shmiR13) or a nucleic acid composed thereof, and a nucleic acid comprising a DNA sequence encoding shmiR, which comprises the effector sequence shown in SEQ ID NO: 39 and an effector complementary sequence substantially complementary to the sequence shown in SEQ ID NO: 39, such as the effector complementary sequence shown in SEQ ID NO: 38 (shmiR17) or a nucleic acid composed thereof. For example, the ddRNAi construct according to this example of the DNA construct may include a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or the DNA sequence shown in SEQ ID NO: 64 (shmiR13), and a nucleic acid comprising the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or the DNA sequence shown in SEQ ID NO: 68 (shmiR17).

[0466] An exemplary PABPN1 construct included in the DNA constructs of the present invention comprises a codon-optimized sequence as shown in SEQ ID NO: 73 and encodes the functional PABPN1 protein as shown in SEQ ID NO: 74.

[0467] While certain examples have been described, it should be understood that DNA constructs according to the invention may include any ddRNAi construct encoding one or more shmiRs of RNA transcripts targeting PABPN1 as described herein. However, ddRNAi constructs encoding shmiRs described in Examples 1 to 5 herein may be particularly suitable for inclusion in the DNA constructs of the invention. Similarly, it should be understood that DNA constructs according to the invention may include any PABPN1 construct encoding a functional PABPN1 protein whose transcript is not targeted by shmiRs expressed from the ddRNAi construct.

[0468] Composition and carrier

[0469] In some instances, the AAV of the present invention can be provided in pharmaceutical compositions formulated for delivery to patients, such as human patients.

[0470] The compositions of the present invention may also include one or more pharmaceutically acceptable carriers or diluents. For example, the composition may include a carrier suitable for delivering the AAV of the present invention into the muscle of a test subject after administration. Carriers suitable for the formulation and delivery of AAV are known in the art and are covered herein.

[0471] The composition will desirably include materials that increase the biostability of the AAV of the present invention and / or materials that increase the ability of the AAV to selectively target and / or penetrate myocytes. The therapeutic compositions of the present invention can be administered in a pharmaceutically acceptable carrier (e.g., physiological saline), selected based on the mode and route of administration and standard pharmaceutical practice. Pharmaceutical compositions comprising one or more AAVs of the present invention can be readily formulated by those skilled in the art. In some cases, isotonic formulations are used. Typically, additives for isotonicity may include sodium chloride, dextran, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate-buffered saline are preferred. Stabilizers include gelatin and albumin. In some instances, vasoconstrictors are incorporated into the formulation. Sterile and pyrogen-free compositions according to the present invention are provided. The standard reference book in this field, Remington: The Science and Practice of Pharmacy (formerly Remington's Pharmaceutical Sciences), Mack Publishing, and in USP / NF, describes suitable drug carriers for drug formulations and the drug requirements.

[0472] The volume, concentration, formulation, and administration regimen of a pharmaceutical composition can be specifically tailored to maximize cellular delivery while minimizing toxicities such as inflammatory responses. For example, relatively large volumes (5, 10, 20, 50 ml or more) with correspondingly low concentrations of active ingredients can be used if necessary, as well as to include anti-inflammatory compounds such as corticosteroids.

[0473] The compositions of the present invention can be formulated for administration via any suitable route (e.g., a route suitable for delivery to the pharyngeal muscles of a test subject). For example, routes of administration include, but are not limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral administration, as well as percutaneous or via inhalation or suppository. Exemplary routes of administration include intravenous (IV) injection, intramuscular (IM) injection, oral administration, intraperitoneal, intradermal, intraarterial, and subcutaneous injection. In one example, the compositions of the present invention are formulated for intramuscular injection (e.g., formulated for administration to the pharyngeal muscles). In a preferred embodiment, administration is made directly to the pharyngeal muscles of the test subject. For example, the pharyngeal muscles may include one or more of the inferior retractor, middle retractor, superior retractor, palatopharyngeal, eustachian tube retractor, stylopharyngeal, or any combination thereof. In another preferred embodiment, administration is made directly to the tongue muscles of the test subject. Such compositions can be used for pharmaceutical applications and can be readily formulated in a suitable sterile, pyrogen-free vehicle (e.g., buffered saline for injection) for parenteral administration, such as intramuscular injection (e.g., direct administration to the pharyngeal muscle), intravenous injection (including intravenous infusion), subcutaneous injection, and intraperitoneal administration. In a preferred embodiment, the route of administration, such as intramuscular injection (e.g., direct administration to the pharyngeal muscle), achieves efficient delivery to muscle tissue and transfection of the ddRNAi construct encoding the PABPN1 of the present invention and codon-optimized nucleic acid therein, as well as the expression of shmiR and codon-optimized nucleic acid.

[0474] Treatment

[0475] Certain aspects of the present invention relate to administering an AAV or a composition comprising the same AAV as described herein to human subjects in need of treatment and / or inhibition of the expression of endogenous PABPN1 protein (including PABPN1 protein that causes OPMD), wherein the composition is administered by direct injection into the pharyngeal muscle of the subject.

[0476] In some embodiments, an AAV or a composition including the AAV described herein can be used to treat OPMD in subjects suffering from OPMD. Similarly, an AAV as described herein or a composition including the AAV can be used to prevent the development or progression of one or more symptoms of OPMD in subjects suffering from or susceptible to OPMD.

[0477] In some embodiments, after administration of AAV or a composition comprising AAV by direct injection into the pharyngeal muscles of the subject, the subject exhibits improved swallowing function.

[0478] As described herein, the AAV and / or compositions of the present invention comprise the ddRNAi construct and the PABPN1 construct of the present invention, the PABPN1 construct comprising a codon-optimized nucleic acid encoding the functional PABPN1 protein of the present invention. Therefore, administration of the AAV or the compositions can effectively (i) inhibit, reduce, or knock out the expression of endogenous PABPN1, including PABPN1 protein comprising a polyalanine bundle that induces OPMD amplification, and (ii) provide expression of functional PABPN1 protein that is not targeted by shmiRs that inhibit, reduce, or knock out endogenous PABPN1 expression. Therefore, the AAV or the compositions of the present invention can restore PABPN1 protein function, such as post-transcriptional processing of RNA, in cells or animals to which they are administered.

[0479] In some embodiments, treatment of OPMD may include administration of AAV by direct injection into the pharyngeal muscle of the subject or a composition comprising AAV as described herein.

[0480] In some embodiments, the route of administration is IM (e.g., direct injection into the pharyngeal muscle of the subject) and achieves efficient delivery to muscle tissue and transduction of the ddRNAi construct and PABPN1 construct of the present invention, including the expression of codon-optimized nucleic acid encoding PABPN1 and shmiR targeting wild-type PABPN1 mRNA transcript, and the expression of codon-optimized nucleic acid therein.

[0481] The effective dose level for any particular patient will depend on a number of factors, including: the composition used; the patient's age, weight, general health condition, sex, and diet; the time of administration; the route of administration; the binding rate of the AAV or a composition including the AAV as described herein; the duration of treatment; and other relevant factors.

[0482] The efficacy of AAV or compositions comprising it in reducing or inhibiting the expression of PABPN1 protein that causes OPMD and in expressing an amount of functional PABPN1 protein that does not cause OPMD sufficient to restore PABPN1 function can be determined by assessing the muscle contractile properties and / or dysphagia of the treated subjects. Methods for testing swallowing ability and muscle contractile properties are known in the art. For example, dysphagia can be assessed using fluoroscopy, UGI endoscopy, or esophageal manometry and impedance testing. Other methods for assessing the clinical characteristics of OPMD are described in Rüegg et al., (2005), Swiss Medical Weekly, 135:574-586.

[0483] Table 1 - Target regions of PABPN1

[0484]

[0485] Table 2 - shmiR effectors and their complementary sequences

[0486]

[0487] Table 3 - shmiR sequences

[0488]

[0489] Table 4 - shmiR encoding boxes

[0490]

[0491] Example 1 – Design of shmiR targeting PABPN1

[0492] Sequences representing potential targets for siRNA construct design were identified from the PABPN1 mRNA sequence using publicly available siRNA design algorithms (including Ambion, Promega, Invitrogen, Origene, and MWG). The selected sequences were conserved across human, non-human primate, bovine, and mouse species. The sequences encoding candidate siRNAs were incorporated into the pre-miR30a scaffold to generate sequences encoding short hairpin microRNAs (shmiRs), which include a 5' flanking region (SEQ ID NO: 41), the siRNA sense strand sequence (effector complement sequence), a stem / loop connector sequence (SEQ ID NO: 40), the siRNA antisense strand (effector sequence), and a 3' flanking region (SEQ ID NO: 42). The predicted secondary structures of representative shmiRs are shown below. Figure 1 Table C shows the target regions of the designed shmiR PABPN1 mRNA transcripts in Table 1, and the corresponding shmiR effector sequences (antisense strands) in Table 2.

[0493] Example 2 – Generating a single “silencing and substitution construct” for simultaneous gene silencing of endogenous PABPN1 and substitution with codon-optimized PABPN1.

[0494] Single-stranded adeno-associated virus type 2 (ssAAV2) plasmids expressing shmiR17 and shmiR13 (e.g., as described in Tables 3 and 4) and optPABPN1 sequence were created.

[0495] The silencing and substitution construct (hereinafter referred to as the “SR construct”) is generated by subcloning the DNA sequences encoding shmiR17 and shmiR13 (as shown in Table 4) into the 3' untranslated region of the optPABPN1 transcript in the pAAV2 vector backbone (pAAV-shmiR viral plasmid). Expression of optPABPN1 and both shmiRs in a single transcript is driven by the muscle-specific promoter Spc512. Figure 1 (A) Figure 1 (B) and Figure 2 A schematic diagram of the SR construct is provided.

[0496] Then, recombinant pseudotyped AAV vectors were generated. In short, HEK293T cells were cultured in a cell factory in Dulbecco's modified Eagle's medium, supplemented with 10% FBS, and incubated at 37°C and 5% CO2. The pAAV-shmiR viral plasmid (SR-construct) and pAAV helper virus and pAAVrepcap8 plasmid or pAAVrepcap9 plasmid or pAAV helper virus and pAAVRH74 plasmid were conjugated with calcium phosphate according to the manufacturer's instructions. Triple transfection was then performed using the pAAV-shmiR plasmid (SR-construct) combined with pAAV helper virus and one of the following capsids: pAAVrepcap8, pAAVrepcap9, or pAAVRH74 in HEK293T cells. HEK293T cells were then cultured at 37°C and 5% CO2 for 72 hours. Afterward, the cells were lysed and purified by fractional-gradient ultracentrifugation with iodixanol (Sigma-Aldrich) followed by cesium chloride ultracentrifugation. The vector genome was quantified using quantitative polymerase chain reaction (Q-PCR).

[0497] Example 3 – In vivo study using a single vector “silence and substitution” approach.

[0498] To test the in vivo efficacy of the SR-construct described in Example 2 in an OPMD-related disease model, the SR-construct was administered alone at a specific dose range via intramuscular injection into the tibialis anterior (TA) muscle of 10-12 week old A17 mice. The dosage was 7.5 x 10⁻⁶ per muscle. 11 2.5x10 11 5x10 10 1x10 10 2x10 9 and 4x10 8Each vector genome (vg) was used. Age-matched A17 mice were injected with saline as the untreated group. Mice were sacrificed 14 or 20 weeks after treatment.

[0499] Example 4 – Quantitative measurement of shmiR production, PABPN1 silencing, and codon-optimized PABPN1 expression in SR-construct-treated A17 mice.

[0500] After 14 weeks of SR-construct treatment, TA muscle from A17 mice in Example 3 was harvested and RNA was extracted. The SR-construct-dependent expression of shmiR in TA muscle was quantified. Figure 3A The quantitative expression level of shmiR depends on the SR-construct dose, PABPN1 (including expPABPN1) ( Figure 3B Silence and normal PABPN1 levels ( Figure 3C The same applies to the recovery of ).

[0501] Example 5 – Reduction of intranuclear inclusions (INI) in A17 mice treated with SR-construct.

[0502] The effect of the SR-construct on the persistence of intranuclear inclusions (INIs) was tested in A17 mice at week 14 of Example 3. FvB wild-type mice were also included as healthy controls. Muscle was harvested and placed for histological studies 14 weeks after AAV injection. Sections were pretreated with 1M KCl to preferentially elute all soluble PABPN1 from the tissue. Immunofluorescence of PABPN1 (green) and laminin (a protein abundant in the extracellular matrix of muscle cells) (red) was detected in the treated muscle fractions and showed a significant reduction in the number of PABPN1-positive intranuclear inclusions (INIs) in SR-construct treated muscle, exhibiting a dose-response effect. Figure 4A Quantification of the percentage of cell nuclei containing INIs in muscle sections showed that treatment with the SR-construct significantly reduced the number of INIs compared to untreated A17 muscle (using a one-way ANOVA test with Bonferroni post-hoc test, ***p<0.001, ns: not significant). Figure 4B ).

[0503] Example 6 – Treatment with SR-construct improved the physiological properties and function of the treated muscle.

[0504] In A17 mice at week 14 of Example 3, the physiological characteristics and function of the treated muscles were measured. FvB wild-type mice were also included as healthy controls. The maximum force generated by the TA muscles was measured by in situ muscle physiology. Figure 5AThe SR-construct significantly increased maximum force generated by TA muscle in a dose-dependent manner. Normalized muscle mass relative to body weight (BW) was also measured 14 weeks after SR-construct administration. Figure 5B When the dose of TA injection exceeded 1e10 vg, the muscle weight of the SR-treated group normalized to body weight was comparable to that of the control FvB mice (mean ± SEM, n = 10, one-way ANOVA performed using Bonferroni post-hoc test, *p < 0.05, **p < 0.001, *p < 0.01, ns: not significant).

[0505] Example 7 – Muscle function recovers over time

[0506] The maximum force generated by the TA muscle in A17 mice and FvB wild-type mice treated with the SR-construct was measured 14 weeks after administration of the SR-construct. Figure 6A ) and SR-construct 20 weeks after administration ( Figure 6B In situ muscle physiology was used to measure the effect. For moderate doses (1e10 vg and 6e10 vg per TA), the beneficial effect on muscle strength was more significant at 20 weeks compared with 14 weeks post-injection (mean ± SEM, n = 10, one-way ANOVA performed using Bonferroni post-hoc test, ***p < 0.001, **p < 0.01).

[0507] Example 8 – Direct administration to the pharyngeal muscles of sheep

[0508] When the SR construct was injected directly into the pharyngeal muscle of sheep, PABPN1 was found to be highly conserved between sheep and humans, including all amino acid residues except for one amino acid residue at position 95.

[0509] The SR construct was directly injected into the pharyngeal muscle of sheep. Figure 7A Two animals in the sheep study were injected with 1.5e13 vg of the SR-construct into the cricopharyngeal muscle (CP) and 1.0e13 vg of the SR-construct into the pharyngeal muscle (pharynx). The remaining 10 animals treated with the SR-construct (1.0e10 vg to 1.0e13 vg) received only CP injections. The total CP injection volume was 1.5 ml (3 times, 0.5 ml each time). The total pharyngeal injection volume was 6 ml (2 injections on each side, 1.5 ml each time).

[0510] Radiographic imaging using a radiolabeled cream showed that human patients with OPMD had severe dysphagia and a risk of "accidental ingestion." Figure 7B ).

[0511] Example 9 – Design, Production, and Testing of Modified AAV VP1 Sequences

[0512] In this example, an AAV with viral capsid protein subunit 1 (VP1) was designed and prepared, in which specific sequence modifications, namely amino acid substitutions, were introduced into the phospholipase A2 (PLA2) domain and flanking sequences to restore the phospholipase activity and viral function of AAVs when produced in insect cells. Furthermore, based on multiple sequence alignment of VP1 subsequences including the PLA2 domain and flanking sequences of various representative AAV serotypes, a common VP1 subsequence including the PLA2 domain and flanking sequences was prepared, including sequence modifications designed to restore phospholipase activity. The wild-type AAV9 VP1 subsequence is shown in SEQ ID NO: 87.

[0513] 9.1 Design of Modified AAV9 VP1 Sequence

[0514] The N-terminal 180 amino acids of the VP1 proteins of AAV9 (SEQ ID NO: 89), AAV8 (SEQ ID NO: 93), and AAV2 (SEQ ID NO: 97) were sequenced using the BLASTp alignment tool. Based on these alignments, the PLA2 domain and flanking sequences from AAV8 and AAV9 showed high conservation with the corresponding sequences in AAV2. Based on these sequence alignments, modified AAV9 VP1 and AAV8 VP1 sequences were designed using computer simulations. The modified AAV9 VP1 sequence was designed by replacing amino acids at positions 42, 67, 81, 84, and 85 of the sequence shown in SEQ ID NO: 89 with amino acids present at the corresponding positions in the AAV2 VP1 sequence shown in SEQ ID NO: 97, i.e., A42S, A67E, Q81R, K84D, and A85S within the sequence of SEQ ID NO: 89. One of the substituted positions in the modified AAV9 VP1 sequence is located in a region flanking the PLA2 domain (but is considered to potentially be involved in the folding and / or activity of the PLA2 domain), and four of the modified residue positions are located within the PLA2 domain itself. Similarly, the modified AAV8 VP1 sequence was designed by replacing amino acids at positions 42, 67, 81, 84, 85, and 105 of the sequence shown in SEQ ID NO: 93 with amino acids present at the corresponding positions in the AAV2 VP1 sequence shown in SEQ ID NO: 97, i.e., A42S, A67E, Q81R, K84D, A85S, and Q105K in the sequence of SEQ ID NO: 93.

[0515] 9.2 Production of baculovirus vectors expressing structural and non-structural AAV9 proteins

[0516] Prepare a baculovirus vector (BacAAV9-Rep-VPmod) encoding AAV9 capsid proteins including VP1, VP2, and VP3 subunits and the AAV9 non-structural proteins Rep78, Rep68, Rep52, and Rep40. Figure 8 In short, in GenScript (AAV9-VPmod, Figure 9 A DNA construct encoding the AAV9 capsid protein was synthesized, containing a modified AAV9 VP1 subunit encoded by the sequence shown in SEQ ID NO: 90, and flanking NotI and ApaI restriction sites. The wtAAV9-Rep plasmid (Virovek, Hayward, CA) encoding the non-structural proteins Rep78, Rep68, Rep52, and Rep40, as well as the capsid proteins VP1, VP2, and VP3, and the assembly activation protein (AAP) was used as the backbone for accepting the AAV9-VPmod DNA construct. NotI and ApaI were used... I The AAV9-VPmod DNA construct and the wtAAV9-Rep plasmid were digested with enzymes, and then the AAV9-VPmod DNA construct was ligated into the wtAAV9-Rep plasmid backbone to replace the wt capsid protein coding sequence to generate AAV9-Rep-VPmod. Figure 10 The AAV9-Rep-VPmod intermediate was then cloned into the pOET1 baculovirus transfer vector (Oxford Expression Technologies). To facilitate this, the EcoRV site was inserted into the AAV9-Rep-VPmod intermediate using Quickchange technology to generate the AAV9-Rep-VPmod-EcoRV intermediate. The AAV9-Rep-VPmod-EcoRV intermediate and pOET1 were then digested with NotI and EcoRV (Oxford Expression Technologies), and the insert was then ligated into the pOET1 backbone to generate the final AAV9-Rep-VPmod clone (BacAAV9-Rep-CapPL). Figure 8 ).

[0517] 9.3 Production of baculovirus vectors expressing structural and non-structural AAV8 proteins

[0518] Prepare a baculovirus vector (BacAAV8-Rep-VPmod) encoding AAV8 capsid proteins including VP1, VP2, and VP3 subunits and modified AAV8 non-structural proteins Rep78 and Rep52. Figure 11 In short, in GenScript (AAV8-VPmod), Figure 12DNA constructs encoding AAV8 capsid proteins (VP1, VP2, and Vp3) were synthesized. The AAV8 capsid proteins have a modified VP1 subunit including the sequence shown in SEQ ID NO: 94, and have flanking NotI and Apa. I Restriction sites. The wtAAV8-Rep / Cap plasmid (Virovek, Hayward, CA) encoding non-structural proteins Rep78, Rep68, Rep52, and Rep40, as well as capsid proteins VP1, VP2, and VP3, and assembly activation protein (AAP) was used as the backbone for receiving the AAV8-VPmod DNA construct. NotI and Apa were used. I The AAV8-VPmod DNA construct and the wtAAV8-Rep / Cap plasmid were digested with enzymes, and then the AAV8-VPmod DNA construct was ligated into the wtAAV8-Rep / Cap plasmid backbone to replace the wt capsid protein coding sequence to generate AAV8-Rep-VPmod. Figure 13 The AAV8-Rep-VPmod intermediate was then cloned into the pOET1 baculovirus transfer vector (Oxford Expression Technologies). To facilitate this, the EcoRV site was inserted into the AAV8-Rep-VPmod intermediate using QuickChange technology to generate the AAV8-Rep-VPmod-EcoRV intermediate. The AAV8-Rep-VPmod-EcoRV intermediate and pOET1 were then digested with NotI and EcoRV, and the insert was ligated into the pOET1 backbone (Oxford Expression Technologies) to generate the final AAV8-Rep-VPmod clone (BacAAV8-Rep-VPmod). Figure 11 ).

[0519] 9.4 Production of baculovirus vectors expressing the target gene (GOI)

[0520] Prepare a baculoviral vector encoding a target gene (GOI) with a side-joined AAV2 inverted terminal repeat (ITR). Simply put, in one case, this is achieved by digesting the AAV2-GOI construct with NotI. Figure 14The researchers at Oxford Expression Technologies cloned a DNA construct encoding shmiR, which encodes two human PAPPN1 transcripts targeting AAV2 ITR, into the pOET1 baculovirus transfer vector (Oxford Expression Technologies), and then ligated the AAV2-GOI construct into the pOET1 backbone to produce the final clone (BacAAV2-GOI). Figure 15 The second GOI was also prepared in the same manner as described above, although it encodes three shmiRs that target different regions of the HBV polymerase gene transcript.

[0521] 9.5P0 Baculovirus Original Species Production

[0522] Baculovirus P0 primary strain was generated using the Oxford Expression Technologies baculoCOMPLETE system (according to the manufacturer's instructions). Simply put, 1 million Sf9 cells were seeded into 6-well plates and allowed to adhere to the plate 1 hour before transfection. In 1 ml of TC100 medium, 500 ng of Bac-AAV2-GOI plasmid, BacAAV8-Rep-CapPL, or BacAAV9-Rep-CapPL was mixed with 500 ng of flash BAC DNA and baculoFECTIN transfection reagent (according to the manufacturer's protocol). After incubating at room temperature for 30 minutes, the transfection mixture was added to the seeded Sf9 cells. The 6-well plates were incubated at 28°C. 24 hours after transfection, 1 ml of Sf9 medium was added to the cells. 5 days after transfection, the medium containing the P0 baculovirus primary strain was collected and stored at 4°C. Therefore, P0 baculoviruses were generated for BacAAV8-Rep-CapPL, BacAAV9-Rep-CapPL, and Bac-AAV2-GOI.

[0523] 9.6 Preparation of AAV in mammalian cells

[0524] The function of AAV prepared in mammalian cells was compared with that of AAV prepared in insect cells as described above. To compare the biological activity (function) of recombinant AAV prepared in mammalian and insect cells, mammalian cells were infected in vitro with various titers of virus, and the expression of treated shmiR was quantified using qRT PCR.

[0525] For these experiments, the commercial supplier (Vector Biolabs; https: / / www.vectorbiolabs.com) prepared recombinant AAV8 particles expressing three shmiRs targeting HBV polymerase gene transcripts in mammalian cells. Additionally, recombinant AAV9 particles expressing two shmiRs targeting human PABPN1 were prepared by a second mammalian cell supplier, Nationwide Children's Hospital vector core. https: / / www.nationwidechildrens.org / research / resources-infrastructure / core- facilities / viralvector-core-clinical-manufacturing-facility ).

[0526] Bioactivity was assessed for (i) AAV8 with unmodified VP1 generated in mammalian cells (vector Biolabs), (ii) AAV8 with modified VP1 generated in insect cells via baculovirus (using BacAAV8-Rep-VPmod as described herein), and (iii) AAV8 with unmodified wt VP1 generated in insect cells via baculovirus using wtAAV8-Rep / Cap (Ben10, Virovek, Hayward, CA), each encoding three shmiRs targeting the HBV polymerase gene (HBV shmiRs named all-4_m3, shRNA8v2_p1, and All-9_p1). Briefly, JHU67 cells were infected with the above-described modified or unmodified recombinant viral preparations at MOIs of 4x10e9, 8x10e9, and 1.6x10e10, and shmiR expression of each of the three shmiRs was quantified at 72 hours post-infection. To quantify shmiR expression, RNA was extracted from infected cells using the Qiagen RNA Mini Kit (Qiagen). RNA was reverse transcribed using the Qiagen miScript Kit (Qiagen). The cDNA was then used with specific primers designed to amplify the shmiR target for qPCR to determine the total copy number present in the sample.

[0527] like Figure 16 As shown in A-16C, cells infected with AAV8 prepared in mammalian cells and containing unmodified wt VP1 produced easily detectable levels of shmiR, while AAV8 produced in insect cells from baculoviruses and containing unmodified wt VP1 produced very little shmiR (if any). Conversely, AAV8 with modified VP1 produced by baculoviruses in insect cells produced relatively high levels of shmiR, indicating increased function of these AAVs compared to AAV8 with unmodified wt VP1 produced by baculoviruses in insect cells.

[0528] The bioactivity of (i) AAV9 with unmodified capsid proteins produced in mammalian cells (Nafionwide) and (ii) AAV9 with modified capsid proteins produced in insect cells using BACAAV9-Rep-VPmod (as described herein) produced by baculovirus, was also evaluated, along with two shmiR-targeting transcripts (named sh13 and sh17) each encoding human PABPN1. Briefly, C2C12 cells expressing the AAV internalization receptor were infected with 4x10e9, 8x10e9, and 1.6x10e10 vector genomes. After 72 hours of incubation, cells were harvested, RNA was extracted, and shmiR expression of the two shmiRs was quantified using the qPCR method described above.

[0529] like Figure 17 As shown, the two formulations exhibited very similar shmiR expression levels, indicating very similar viral functions.

[0530] Although demonstrated in the context of AAVs from serotypes 8 and 9, it is expected that when produced from baculovirus expression systems in insect cells, modifying the VP1 subunit sequence of other AAV serotypes (except serotype 2) according to the methods described herein will restore AAV function.

[0531] Those skilled in the art will understand that various changes and / or modifications can be made to the above embodiments without departing from the broad scope of this disclosure. Therefore, this embodiment is to be considered illustrative rather than restrictive in all respects.

[0532] Some embodiments of the present invention are as follows:

[0533] 1. An adeno-associated virus (AAV) comprising:

[0534] (a) A viral capsid protein from AAV9 comprising a modified subunit 1 (VP1) sequence, wherein the amino acids at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87; and

[0535] (b) A polynucleotide sequence comprising (i) a DNA-guided RNAi (ddRNAi) construct comprising a nucleic acid including a sequence encoding a short hairpin microRNA (shmiR); and (ii) a PABPN1 construct comprising a nucleic acid including a sequence encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR encoded by the ddRNAi construct.

[0536] 2. The AAV according to embodiment 1, wherein the modified VP1 sequence comprises, relative to the wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87, serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43 and serine at position 44.

[0537] 3. The AAV according to embodiment 1 or 2, wherein the modified AAV9 VP1 sequence includes the sequence shown in SEQ ID NO: 88.

[0538] 4. The AAV according to any one of embodiments 1 to 3, wherein the polynucleotide sequence comprises the ddRNAi construct and the PABPN1 construct in the 5' to 3' direction.

[0539] 5. The AAV according to any one of embodiments 1 to 3, wherein the polynucleotide sequence comprises the PABPN1 construct and the ddRNAi construct in the 5' to 3' direction.

[0540] 6. The AAV according to any one of embodiments 1 to 5, wherein the polynucleotide sequence at (b) comprises an inverted terminal repeat (ITR) sequence from an AAV serotype, and wherein the ITR is side-joined with the sequence comprising the ddRNAi construct and the PABPN1 construct.

[0541] 7. The AAV according to embodiment 6, wherein the ITR is derived from the AAV2 serotype.

[0542] 8. The AAV according to any one of embodiments 1 to 7, wherein the sequence encoding the functional PABPN1 protein is codon-optimized such that its mRNA transcript is not targeted by the shmiR of the ddRNAi construct.

[0543] 9. The AAV according to any one of embodiments 1 to 8, wherein the sequence encoding the functional PABPN1 protein is as shown in SEQ ID NO: 73.

[0544] 10. The AAV according to any one of embodiments 1 to 9, wherein the ddRNAi construct and the sequence encoding the functional PABPN1 protein are operatively linked to a promoter located upstream of the ddRNAi construct and the sequence encoding the functional PABPN1 protein.

[0545] 11. The AAV according to embodiment 10, wherein the promoter is a muscle-specific promoter.

[0546] 12. The AAV according to any one of embodiments 1 to 11, wherein the shmiR comprises:

[0547] Effector sequences with a length of at least 17 nucleotides;

[0548] Effector complementary sequences;

[0549] Stem-loop sequences; and

[0550] Primary microRNA (pri-miRNA) backbone;

[0551] The effector sequence is substantially complementary to the corresponding length of the region in any of the RNA transcripts shown in SEQ ID NO: 1-13.

[0552] 13. The AAV according to any one of embodiments 1 to 12, wherein the shmiR is selected from the group consisting of:

[0553] shmiR includes the effector sequence shown in SEQ ID NO: 15 and the complementary effector sequence shown in SEQ ID NO: 14;

[0554] shmiR includes the effector sequence shown in SEQ ID NO: 17 and the complementary effector sequence shown in SEQ ID NO: 16;

[0555] shmiR includes the effector sequence shown in SEQ ID NO: 19 and the complementary effector sequence shown in SEQ ID NO: 18;

[0556] shmiR includes the effector sequence shown in SEQ ID NO: 21 and the complementary effector sequence shown in SEQ ID NO: 20;

[0557] shmiR includes the effector sequence shown in SEQ ID NO: 23 and the complementary effector sequence shown in SEQ ID NO: 22;

[0558] shmiR includes the effector sequence shown in SEQ ID NO: 25 and the complementary effector sequence shown in SEQ ID NO: 24;

[0559] shmiR includes the effector sequence shown in SEQ ID NO: 27 and the complementary effector sequence shown in SEQ ID NO: 26;

[0560] shmiR includes the effector sequence shown in SEQ ID NO: 29 and the complementary effector sequence shown in SEQ ID NO: 28;

[0561] shmiR includes the effector sequence shown in SEQ ID NO: 31 and the complementary effector sequence shown in SEQ ID NO: 30;

[0562] shmiR includes the effector sequence shown in SEQ ID NO: 33 and the complementary effector sequence shown in SEQ ID NO: 32;

[0563] shmiR includes the effector sequence shown in SEQ ID NO: 35 and the complementary effector sequence shown in SEQ ID NO: 34;

[0564] shmiR including the effector sequence shown in SEQ ID NO: 37 and the complementary effector sequence shown in SEQ ID NO: 36; and

[0565] shmiR includes the effector sequence shown in SEQ ID NO: 39 and the complementary effector sequence shown in SEQ ID NO: 38.

[0566] 14. The AAV according to any one of embodiments 1 to 13, wherein the shmiR comprises, in the 5' to 3' direction:

[0567] The 5' flanking sequence of the pri-miRNA backbone;

[0568] The complementary sequence of the effectors;

[0569] The stem-loop sequence;

[0570] The effect subsequence; and

[0571] The 3' flanking sequence of the pri-miRNA backbone.

[0572] 15. The AAV according to embodiment 14, wherein the stem-loop sequence is the sequence shown in SEQ ID NO: 40.

[0573] 16. The AAV according to embodiment 14 or 15, wherein the pri-miRNA backbone is the pri-miR-30a backbone.

[0574] 17. The AAV according to any one of embodiments 14 to 16, wherein the 5' flanking sequence of the pri-miRNA backbone is as shown in SEQ ID NO: 41, and the 3' flanking sequence of the pri-miRNA backbone is as shown in SEQ ID NO: 42.

[0575] 18. The AAV according to any one of embodiments 1 to 17, wherein the ddRNAi construct comprises at least two nucleic acids, each nucleic acid encoding a shmiR, wherein each shmiR comprises an effector sequence substantially complementary to an RNA transcript corresponding to the PABPN1 protein that causes OPMD, and wherein each shmiR comprises a different effector sequence.

[0576] 19. The AAV according to embodiments 1 to 18, wherein each of the at least two nucleic acids encodes an effector sequence comprising a region substantially complementary to a corresponding length in the RNA transcripts shown in one of SEQ ID NOs: 1, 2, 4, 7, 9, 10 and 13.

[0577] 20. The AAV according to embodiment 19, wherein the at least two nucleic acids are selected from the group consisting of:

[0578] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 15 and the effector complement sequence shown in SEQ ID NO: 14 (shmiR2);

[0579] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 17 and the effector complement sequence shown in SEQ ID NO: 16 (shmiR3);

[0580] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 21 and the effector complement sequence shown in SEQ ID NO: 20 (shmiR5);

[0581] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 27 and the effector complement sequence shown in SEQ ID NO: 26 (shmiR9);

[0582] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 31 and the effector complement sequence shown in SEQ ID NO: 30 (shmiR13);

[0583] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 33 and the effector complement sequence shown in SEQ ID NO: 32 (shmiR14); and

[0584] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO: 38 (shmiR17).

[0585] 21. The AAV according to embodiment 19, wherein the at least two nucleic acids are selected from the group consisting of:

[0586] Includes the DNA sequence shown in SEQ ID NO: 56 (shmiR2) or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 56 (shmiR2);

[0587] Includes the DNA sequence shown in SEQ ID NO: 57 (shmiR3) or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 57 (shmiR3);

[0588] Includes the DNA sequence shown in SEQ ID NO: 59 (shmiR5) or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 59 (shmiR5);

[0589] Includes the DNA sequence shown in SEQ ID NO: 62 (shmiR9) or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 62 (shmiR9);

[0590] Includes the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 64 (shmiR13);

[0591] Includes the DNA sequence shown in SEQ ID NO: 65 (shmiR14) or a nucleic acid composed of the DNA sequence shown in SEQ ID NO: 65 (shmiR14); and

[0592] Includes the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 68 (shmiR17).

[0593] 22. The AAV according to embodiment 19, wherein each of the at least two nucleic acids encodes an effector sequence comprising a region of substantially complementary length to a region of corresponding length in the RNA transcripts shown in one of SEQ ID NO: 2, 9, 10 and 13.

[0594] 23. The AAV according to embodiment 19, wherein the at least two nucleic acids are selected from the group consisting of:

[0595] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 17 and the effector complement sequence shown in SEQ ID NO: 16 (shmiR3);

[0596] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 31 and the effector complement sequence shown in SEQ ID NO: 30 (shmiR13);

[0597] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 33 and the effector complement sequence shown in SEQ ID NO: 32 (shmiR14); and

[0598] Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO: 38 (shmiR17).

[0599] 24. The AAV according to embodiment 19, wherein the at least two nucleic acids are selected from the group consisting of:

[0600] Includes the DNA sequence shown in SEQ ID NO: 57 (shmiR3) or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 57 (shmiR3);

[0601] Includes the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 64 (shmiR13);

[0602] Includes the DNA sequence shown in SEQ ID NO: 65 (shmiR14) or a nucleic acid composed of the DNA sequence shown in SEQ ID NO: 65 (shmiR14); and

[0603] Includes the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 68 (shmiR17).

[0604] 25. The AAV according to any one of embodiments 1 to 24, wherein the ddRNAi construct comprises:

[0605] (a) comprising a DNA sequence encoding shmiR or a nucleic acid consisting of a DNA sequence encoding shmiR, wherein shmiR comprises the effector sequence shown in SEQ ID NO: 31 and the effector complement sequence shown in SEQ ID NO: 30 (shmiR13); and

[0606] (b) includes a DNA sequence encoding shmiR or a nucleic acid consisting of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO: 38 (shmiR17).

[0607] 26. The AAV according to embodiment 25, wherein the ddRNAi construct comprises:

[0608] (a) comprising the DNA sequence shown in SEQ ID NO: 64 (shmiR13) or a nucleic acid consisting of the DNA sequence shown in SEQ ID NO: 64 (shmiR13); and

[0609] (b) Includes the DNA sequence shown in SEQ ID NO: 68 (shmiR17) or a nucleic acid consisting of the DNA sequence shown in SEQ ID NO: 68 (shmiR17).

[0610] 27. A pharmaceutical composition comprising an AAV according to any one of embodiments 1 to 26 and one or more pharmaceutically acceptable carriers.

[0611] 28. A method of treating a subject suffering from oculopharyngeal muscular dystrophy (OPMD), comprising administering to the subject an AAV according to any one of embodiments 1 to 26 or a pharmaceutical composition according to embodiment 27.

[0612] 29. The method according to embodiment 28, wherein the composition is administered by direct injection into the pharyngeal muscle of the subject.

[0613] 30. The method according to embodiment 29, wherein the pharyngeal muscles include one or more of the inferior retractor muscle, the middle retractor muscle, the superior retractor muscle, the palatopharyngeal muscle, the eustachian tube pharyngeal muscle, the styloid process pharyngeal muscle, or any combination thereof.

Claims

1. An adeno-associated virus (AAV) comprising: (a) A viral capsid protein from AAV9 comprising a modified subunit 1 (VP1) sequence comprising any four of the following: serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, and / or serine at position 44, wherein the amino acids at positions 1, 26, 40, 43, and 44 are numbered relative to the sequence shown in SEQ ID NO: 87, and wherein any four or more of the amino acids at positions 1, 26, 40, 43, and 44 are substituted relative to the corresponding wild-type AAV9 VP1 sequence; as well as (b) A polynucleotide sequence comprising (i) a DNA-guided RNAi (ddRNAi) construct comprising a nucleic acid including a sequence encoding a short hairpin microRNA (shmiR); and (ii) a PABPN1 construct comprising a nucleic acid including a sequence encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR encoded by the ddRNAi construct.

2. The AAV according to claim 1, wherein, The modified VP1 sequence, relative to the wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87, includes glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, and serine at position 44.

3. The AAV according to claim 2, wherein, The modified VP1 sequence comprises the amino acid sequence shown in SEQ ID NO: 87, except for the amino acid substitutions A26E, Q40R, K43D, and A44S relative to the amino acid sequence shown in SEQ ID NO:

87.

4. The AAV according to claim 1, wherein, The modified VP1 sequence, relative to the wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87, includes serine at position 1, glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, and serine at position 44.

5. The AAV according to claim 1, wherein, The modified AAV9 VP1 sequence includes the sequence shown in SEQ ID NO:

88.

6. The AAV according to claim 1, wherein, The polynucleotide sequence at (b) includes the ddRNAi construct and the PABPN1 construct in the 5' to 3' direction.

7. The AAV according to claim 1, wherein, The polynucleotide sequence at (b) includes the PABPN1 construct and the ddRNAi construct in the 5' to 3' direction.

8. The AAV according to claim 1, wherein, The polynucleotide sequence at (b) includes an inverted terminal repeat (ITR) sequence from the AAV serotype, and wherein the ITR is flanked by sequences comprising the ddRNAi construct and the PABPN1 construct.

9. The AAV according to claim 8, wherein, The ITR is derived from the AAV2 serotype.

10. The AAV according to claim 1, wherein, The sequence encoding the functional PABPN1 protein is codon-optimized so that its mRNA transcript is not targeted by the shmiR of the ddRNAi construct.

11. The AAV according to claim 1, wherein, The sequence encoding the functional PABPN1 protein is shown in SEQ ID NO:

73.

12. The AAV according to claim 1, wherein, The ddRNAi construct and the sequence encoding the functional PABPN1 protein are operatively linked to a promoter located upstream of the ddRNAi construct and the sequence encoding the functional PABPN1 protein.

13. The AAV according to claim 12, wherein, The promoter is a muscle-specific promoter.

14. The AAV according to claim 1, wherein, The shmiR includes: Effector sequences with a length of at least 17 nucleotides; Effector complementary sequences; Stem-loop sequences; and Primary microRNA (pri-miRNA) backbone; The effector sequence is substantially complementary to the corresponding length of the region in any of the RNA transcripts shown in SEQ ID NO: 1-13.

15. The AAV according to claim 1, wherein, The shmiR is selected from the following groups: shmiR includes the effector sequence shown in SEQ ID NO: 15 and the complementary effector sequence shown in SEQ ID NO: 14; shmiR includes the effector sequence shown in SEQ ID NO: 17 and the complementary effector sequence shown in SEQ ID NO: 16; shmiR includes the effector sequence shown in SEQ ID NO: 19 and the complementary effector sequence shown in SEQ ID NO: 18; shmiR includes the effector sequence shown in SEQ ID NO: 21 and the complementary effector sequence shown in SEQ ID NO: 20; shmiR includes the effector sequence shown in SEQ ID NO: 23 and the complementary effector sequence shown in SEQ ID NO: 22; shmiR includes the effector sequence shown in SEQ ID NO: 25 and the complementary effector sequence shown in SEQ ID NO: 24; shmiR includes the effector sequence shown in SEQ ID NO: 27 and the complementary effector sequence shown in SEQ ID NO: 26; shmiR includes the effector sequence shown in SEQ ID NO: 29 and the complementary effector sequence shown in SEQ ID NO: 28; shmiR includes the effector sequence shown in SEQ ID NO: 31 and the complementary effector sequence shown in SEQ ID NO: 30; shmiR includes the effector sequence shown in SEQ ID NO: 33 and the complementary effector sequence shown in SEQ ID NO: 32; shmiR includes the effector sequence shown in SEQ ID NO: 35 and the complementary effector sequence shown in SEQ ID NO: 34; shmiR including the effector sequence shown in SEQ ID NO: 37 and the complementary effector sequence shown in SEQ ID NO: 36; and shmiR includes the effector sequence shown in SEQ ID NO: 39 and the complementary effector sequence shown in SEQ ID NO:

38.

16. The AAV according to claim 1, wherein, The shmiR includes, in the 5' to 3' direction: 5' flanking sequence of the pri-miRNA backbone; The complementary sequence of the effectors; The stem-loop sequence; The effect subsequence; and The 3' flanking sequence of the pri-miRNA backbone.

17. The AAV according to claim 16, wherein, The stem-loop sequence is the sequence shown in SEQ ID NO:

40.

18. The AAV according to claim 16, wherein, The pri-miRNA backbone is the pri-miR-30a backbone.

19. The AAV according to claim 16, wherein, The 5' flanking sequence of the pri-miRNA backbone is shown in SEQ ID NO: 41, and the 3' flanking sequence of the pri-miRNA backbone is shown in SEQ ID NO:

42.

20. The AAV according to claim 1, wherein, The ddRNAi construct comprises at least two nucleic acids, each encoding a shmiR, wherein each shmiR comprises an effector sequence substantially complementary to the RNA transcript corresponding to the PABPN1 protein that causes oculopharyngeal dystrophy (OPMD), and wherein each shmiR comprises a distinct effector sequence.

21. The AAV of claim 1, wherein each of the at least two nucleic acids encodes an effector sequence comprising a region of substantially complementary length to a region of corresponding length in the RNA transcripts shown in one of SEQ ID NOs: 1, 2, 4, 7, 9, 10 and 13.

22. The AAV according to claim 21, wherein, The at least two nucleic acids are selected from the group consisting of: Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 15 and the effector complement sequence shown in SEQ ID NO: 14; Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 17 and the effector complement sequence shown in SEQ ID NO: 16; Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 21 and the effector complement sequence shown in SEQ ID NO: 20; Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 27 and the effector complement sequence shown in SEQ ID NO: 26; Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 31 and the effector complement sequence shown in SEQ ID NO: 30; Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 33 and the effector complement sequence shown in SEQ ID NO: 32; and It includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO:

38.

23. The AAV according to claim 21, wherein, The at least two nucleic acids are selected from the group consisting of: Includes the DNA sequence shown in SEQ ID NO: 56 or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 56; Includes the DNA sequence shown in SEQ ID NO: 57 or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 57; Includes the DNA sequence shown in SEQ ID NO: 59 or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 59; Includes the DNA sequence shown in SEQ ID NO: 62 or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 62; Includes the DNA sequence shown in SEQ ID NO: 64 or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 64; Includes the DNA sequence shown in SEQ ID NO: 65 or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 65; and Includes the DNA sequence shown in SEQ ID NO: 68 or nucleic acids composed of the DNA sequence shown in SEQ ID NO:

68.

24. The AAV according to claim 21, wherein, Each of the at least two nucleic acids encodes an effector sequence comprising a region of substantially complementary length to the corresponding length in the RNA transcripts shown in one of SEQ ID NO: 2, 9, 10 and 13.

25. The AAV according to claim 21, wherein, The at least two nucleic acids are selected from the group consisting of: Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 17 and the effector complement sequence shown in SEQ ID NO: 16; Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 31 and the effector complement sequence shown in SEQ ID NO: 30; Includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 33 and the effector complement sequence shown in SEQ ID NO: 32; and It includes a DNA sequence encoding shmiR or a nucleic acid composed of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO:

38.

26. The AAV according to claim 21, wherein, The at least two nucleic acids are selected from the group consisting of: Includes the DNA sequence shown in SEQ ID NO: 57 or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 57; Includes the DNA sequence shown in SEQ ID NO: 64 or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 64; Includes the DNA sequence shown in SEQ ID NO: 65 or nucleic acids composed of the DNA sequence shown in SEQ ID NO: 65; and Includes the DNA sequence shown in SEQ ID NO: 68 or nucleic acids composed of the DNA sequence shown in SEQ ID NO:

68.

27. The AAV according to claim 1, wherein, The ddRNAi construct includes: (a) comprising a DNA sequence encoding shmiR or a nucleic acid consisting of a DNA sequence encoding shmiR, wherein shmiR comprises the effector sequence shown in SEQ ID NO: 31 and the effector complement sequence shown in SEQ ID NO: 30; and (b) includes a DNA sequence encoding shmiR or a nucleic acid consisting of a DNA sequence encoding shmiR, wherein shmiR includes the effector sequence shown in SEQ ID NO: 39 and the effector complement sequence shown in SEQ ID NO:

38.

28. The AAV according to claim 27, wherein, The ddRNAi construct includes: (a) comprising the DNA sequence shown in SEQ ID NO: 64 or a nucleic acid consisting of the DNA sequence shown in SEQ ID NO: 64; and (b) Includes the DNA sequence shown in SEQ ID NO: 68 or a nucleic acid consisting of the DNA sequence shown in SEQ ID NO:

68.

29. The AAV according to claim 1, wherein, (a) The modified VP1 sequence, relative to the wild-type AAV9 VP1 sequence shown in SEQ ID NO: 87, includes glutamic acid at position 26, arginine at position 40, aspartic acid at position 43 and serine at position 44; (b) The ddRNAi construct includes: (i) includes a DNA sequence encoding shmiR or a nucleic acid consisting of a DNA sequence encoding shmiR, wherein shmiR includes SEQ ID NO: The effector sequence shown in SEQ ID NO: 31 and the complementary effector sequence shown in SEQ ID NO: 30; and (ii) Includes a DNA sequence encoding shmiR or a nucleic acid consisting of a DNA sequence encoding shmiR, wherein shmiR includes SEQ ID NO: The effector sequence shown in SEQ ID NO: 39 and the complementary effector sequence shown in SEQ ID NO: 38; and (c) The sequence encoding the functional PABPN1 protein is codon-optimized so that its mRNA transcript is not targeted by shmiR encoded by the ddRNAi construct.

30. The AAV according to claim 29, wherein, The modified VP1 sequence comprises the amino acid sequence shown in SEQ ID NO: 87, except for the amino acid substitutions A26E, Q40R, K43D, and A44S relative to the amino acid sequence shown in SEQ ID NO:

87.

31. The AAV according to claim 30, wherein, The ddRNAi construct includes: (a) comprising the DNA sequence shown in SEQ ID NO: 64 or a nucleic acid consisting of the DNA sequence shown in SEQ ID NO: 64; and (b) Includes the DNA sequence shown in SEQ ID NO: 68 or a nucleic acid consisting of the DNA sequence shown in SEQ ID NO:

68.

32. A pharmaceutical composition comprising AAV according to any one of claims 1 to 31 and one or more pharmaceutically acceptable carriers.

33. Use of AAV in the preparation of a medicament according to any one of claims 1 to 31, wherein the medicament is used to treat subjects suffering from oculopharyngeal muscular dystrophy (OPMD).

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