Device and method for administering a therapeutic composition to the pharyngeal muscles

Abstract

This disclosure relates to devices and methods for administering therapeutic compositions to the pharyngeal muscles. Specifically, the devices and methods of this disclosure may be used to treat or prevent dysphagia, including dysphagia associated with oculopharyngeal dystrophy (OPMD), in individuals suffering from or predisposed to dysphagia. In addition, this disclosure relates to devices and methods used to deliver modified adeno-associated virus (AAV) delivery vectors containing a "silence and substitution" DNA construct, including compositions containing a "silence and substitution" DNA construct.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 510,750, filed on 28 June 2023, the entire contents of which are incorporated herein by reference. Furthermore, the applicant has recently disclosed therapeutic agents and compositions for the treatment of oculopharyngeal muscular dystrophy (OPMD) through the following applications: U.S. Provisional Patent Application No. 62 / 812,187, filed on 28 February 2019; U.S. Provisional Patent Application No. 62 / 747,089, filed on 17 October 2018; International Patent Application No. PCT / AU2019 / 051134, filed on 17 October 2019; and International Patent Application No. PCT / AU2020 / 050182, filed on 28 February 2020, the entire contents of which are incorporated herein by reference.

[0002] This disclosure relates to devices and methods for administering therapeutic compositions to the pharyngeal muscles. Specifically, the devices and methods of this disclosure may be used in the treatment or prevention of dysphagia, including dysphagia associated with oculopharyngeal dystrophy (OPMD), in individuals suffering from or predisposed to OPMD. In addition, this disclosure relates to devices and methods used to deliver modified adeno-associated virus (AAV) delivery vectors containing a "silence and replacement" DNA construct, including, for example, a composition containing a "silence and replacement" DNA construct. [Background technology]

[0003] OPMD is a rare, slowly progressing tardive muscular dystrophy that first appears in middle age and progresses with age, characterized by progressive ptosis, dysphagia, and proximal limb weakness. OPMD is caused by specific mutations in the gene encoding PABPN1 located on chromosome 14 (positions q11.2-q13). As a result, people with OPMD may experience progressive difficulty swallowing (dysphagia) and may be at risk of choking, aspiration of food or drink into the lungs (aspiration), aspiration pneumonia, weight loss, malnutrition, and death.

[0004] The mutation that causes OPMD is the abnormal elongation of a (GCN)n triplet repeat in the coding region of the poly(A)-binding protein nucleus 1 (PABPN1) gene. The presence of the triplet repeat leads to an elongated polyalanine tract at the N-terminus of the PABPN1 protein; that is, while there are 10 alanine molecules in the normal protein, the mutant form (expPABPN1) has 11-18 alanine molecules. Nuclear aggregates, designated as "nuclear inclusions," constitute a major histopathological feature of the disease. The misfolding of the elongated PABPN1 protein leads to the accumulation of insoluble high-molecular-weight fibril aggregates in the nucleus of affected cells, as PABPN1 is a protein prone to aggregation. [Overview of the project] [Problems that the invention aims to solve]

[0005] There are no approved medications for the treatment of OPMD, nor have any medications demonstrated a significant change in the natural history of the disease. Palliative surgical interventions, such as cricopharyngeal myotomy, have been employed. Cricopharyngeal myotomy works by releasing tension in the cricopharyngeal (CP) muscle, and this surgical procedure can temporarily reduce the effects of dysphagia in some patients. Direct injection of botulinum toxin (Botox) into the CP muscle has also been used to influence temporary improvement of swallowing through chemically induced relaxation of the targeted muscle. While both techniques can provide a temporary reduction in dysphagia experienced by some patients, oropharyngeal symptoms inevitably recur, and disease progression continues without remission. Repeated dilation of the upper esophageal sphincter has shown some efficacy in OPMD patients with moderate dysphagia, but additional clinical studies are needed to support the results and characterize long-term outcomes.

[0006] Under normal conditions, a food bolus leaves the oral cavity and travels across the length of the pharynx to the esophagus via the propulsive activity of coordinated contractions of the superior, middle, and inferior pharyngeal constrictors. As the food bolus approaches the opening of the upper esophagus, subsequent relaxation of the cricopharyngeal muscle allows the bolus to enter the esophagus and proceed to the stomach. In OPMD, the pharyngeal constrictors are weakened and atrophied, and as a result, they cannot consistently exert the level of force required to support the propulsion of the food bolus, which defines the normal swallowing process. Restoration of muscle fiber size and force generation capacity in the weakened and atrophied pharyngeal constrictors is expected to significantly enhance the ability of these muscle groups to support the propulsion of the food bolus through the pharynx to the esophagus. The inferior and middle pharyngeal constrictors are more relevant therapeutic targets, and increased muscle strength may enhance the function of these muscles, thereby reducing dysphagia in OPMD patients.

[0007] The applicant has recently disclosed therapeutic compositions for the treatment of OPMD in WO2017 / 177277, WO2018 / 107228, WO2019 / 043630, WO2020 / 077412, and WO2020 / 172720. However, there remains a need to improve the methods and means of delivering compositions to the pharyngeal muscles, including those therapeutic compositions developed and disclosed by the applicant.

[0008] Nothing included herein with respect to any documents, actions, materials, devices, articles, etc., should be construed as an admission that any or all of these matters constitute part of the basis of the prior art or were common knowledge in the art relating to this disclosure, because they existed prior to the priority date of each of the attached claims. [Means for solving the problem]

[0009] In a first aspect of this disclosure, an injection needle is provided, and the injection needle is, Equipped with a needle cannula, the needle cannula is A proximal portion having a proximal end, A distal portion having a distal end, An intermediate portion located between the proximal and distal portions, A pipe extending between the proximal end of the proximal portion and the distal end of the distal portion, through the proximal portion, the intermediate portion, and the distal portion, the pipe having an outlet at or adjacent to the distal end of the distal portion, The proximal and distal portions are substantially straight, while the intermediate portion is curved such that the distal portion extends from the proximal portion at an angle of 75° to 105°. The proximal portion is shorter than the distal portion.

[0010] The distal portion may extend at an angle of approximately 85° to 95° relative to the proximal portion. For example, the distal portion may extend at an angle of approximately 90° relative to the proximal portion.

[0011] The radius of curvature of the middle portion can be about 2.5 mm to 3.1 mm. For example, the radius of curvature of the middle portion can be, for example, about 2.8 mm.

[0012] The proximal portion can have a length that is, for example, less than 90% of the length of the distal portion, less than 80% of the length of the distal portion, less than 70% of the length of the distal portion, or less than 60% of the length of the distal portion, or less than 50% of the length of the distal portion.

[0013] The length of the distal portion can be greater than the sum of the length of the proximal portion and the length of the middle portion (e.g., the arc length of the middle portion).

[0014] The total length of the needle cannula can be from about 10 mm to about 27 mm. In some examples, the needle cannula can have a total length from about 16 mm to about 22 mm. For example, the needle cannula can have a total length from about 18 mm to about 20 mm, for example, about 19 mm. For example, the length of the distal portion can be between about 7 mm and about 15 mm, the length of the middle portion (e.g., arc length) can be between about 3 mm and about 6 mm, and the length of the proximal portion can be between about 2 mm and about 6 mm.

[0015] In one example, the distal portion extends at an angle of about 85° to about 95° (e.g., about 90°) with respect to the proximal portion, the radius of curvature of the middle portion is from about 2.5 mm to 3.1 mm.

[0016] In one example, the proximal portion has a length that is less than 70% of the length of the distal portion, the length of the distal portion is greater than the sum of the length of the proximal portion and the length of the middle portion (e.g., the arc length of the middle portion), the total length of the needle cannula is from about 10 mm to 27 mm.

[0017] In one example, the distal portion extends at an angle of about 85° to 95° with respect to the proximal portion, the length of the distal portion is from about 7 mm to 15 mm, the length of the proximal portion is from about 2 mm to 6 mm.

[0018] The needle cannula may be approximately 26-28 gauge, for example, a 27 gauge needle. The outer diameter of the needle cannula may be, for example, between approximately 0.3 mm and 0.5 mm. The inner diameter of the needle cannula may be, for example, between approximately 0.1 mm and 0.3 mm. The wall thickness of the needle cannula may be, for example, between approximately 0.03 mm and 0.17 mm.

[0019] The needle cannula may contain, for example, a dead volume of approximately 0.5 μL / 25.4 mm to 1.5 μL / 25.4 mm.

[0020] The needle cannula may have a sloping portion formed at the distal end of the needle. The sloping portion may include a sloping edge that points away from the inward bending side of the needle cannula, for example, away from the center of curvature of the middle portion of the needle cannula. The distal tip may be sloped at an angle between approximately 5° and approximately 15°. The length of the sloping portion may be between approximately 0.5 mm and approximately 3.5 mm. The exit of the needle cannula may be located at the sloping edge.

[0021] The injection needle may further comprise a needle hub connected to a needle cannula. The needle cannula may protrude distally from the needle hub. The proximal end of the proximal portion of the needle cannula may be defined at the boundary where the needle cannula extends from the needle hub. In some embodiments, the needle cannula may further comprise a support portion that extends proximal to the proximal end of the proximal portion of the needle cannula and is disposed inside the needle hub. The tube may further extend through the support portion.

[0022] The needle cannula may have an inlet located at the proximal end of the proximal portion, adjacent to it, or, if present, at the proximal end of the support portion. The tube may extend between the inlet and the outlet.

[0023] The needle hub may have a distal portion adjacent to the proximal end of the proximal portion of the needle cannula. The distal portion of the needle hub may be swaged.

[0024] The injection needle may be used in particular as, for example, a pharyngeal muscle injection needle. The injection needle may be used to directly inject a therapeutic agent into the pharyngeal muscles. Direct injection may occur, for example, during open surgical procedures. That is, a direct injection into the pharyngeal muscles may occur following an incision in the neck of the subject to obtain direct access to the pharyngeal muscles. The injection needle may be used in particular for direct injection into any one of the pharyngeal constrictor muscles and / or the inferior pharyngeal constrictor muscle, middle pharyngeal constrictor muscle, superior pharyngeal constrictor muscle, palatinopharynx muscle, tubopharynx muscle, stylopharynx muscle, or any combination thereof.

[0025] Injection needles may be used to treat or prevent dysphagia. In some cases, dysphagia is associated with oculopharyngeal muscular dystrophy (OPMD), and for example, dysphagia may be secondary to a genetically confirmed diagnosis of OPMD.

[0026] In one embodiment, a pharyngeal muscle injection needle is provided, and the pharyngeal muscle injection needle is, A proximal portion having a proximal end, A distal portion having a distal end, An intermediate portion located between the proximal and distal portions, A pipe extending between the proximal end of the proximal portion and the distal end of the distal portion, through the proximal portion, the intermediate portion, and the distal portion, the pipe having an outlet at or adjacent to the distal end of the distal portion, The injection needle has a proximal and distal portion that are substantially straight, and a middle portion that is curved such that the distal portion extends at a certain angle to the proximal portion.

[0027] A pharyngeal injection needle may include one or more of the features of injection needles described above in relation to a first aspect of the present disclosure. For example, the middle portion may be curved such that the distal portion extends at an angle of 75° to 105° relative to the proximal portion (e.g., an angle of about 85° to about 95°, or an angle of about 90°). As another example, the length of the proximal portion may be shorter than the length of the distal portion.

[0028] Pharyngeal muscle injection needles may be used for the treatment or prevention of dysphagia. In one example, dysphagia may be associated with oculopharyngeal dystrophy (OPMD), for instance, dysphagia may be secondary to a genetically confirmed diagnosis of OPMD.

[0029] In a further embodiment, a method for forming an injection needle, such as a pharyngeal muscle injection needle, is provided, and the method is To provide a needle cannula comprising a proximal end, a distal end, and a tube extending through the needle cannula between the proximal and distal ends, wherein the tube opens at the distal end of the distal portion or at an outlet adjacent thereto. The needle cannula comprises a substantially straight proximal portion having a proximal end, a substantially straight distal portion having a distal end, and an intermediate portion disposed between the proximal and distal portions, wherein the intermediate portion is curved such that the distal portion extends at a certain angle with respect to the proximal portion, and the needle cannula is bent accordingly.

[0030] The method may be such that the injection needle includes one or more of the features of injection needles described above in relation to a first aspect of the present disclosure. For example, the middle portion may be curved such that the distal portion extends at an angle of 75° to 105° relative to the proximal portion (e.g., an angle of about 85° to about 95°, or an angle of about 90°). As another example, the length of the proximal portion may be shorter than the length of the distal portion.

[0031] One aspect of the present disclosure provides a method for administering a therapeutic composition to a target pharyngeal muscle, the method comprising directly injecting the therapeutic composition into the target pharyngeal muscle using a bent or curved needle. In some examples, the bent or curved needle is A proximal portion having a proximal end, A distal portion having a distal end, An intermediate portion located between the proximal and distal portions, A pipe extending between the proximal end of the proximal portion and the distal end of the distal portion, through the proximal portion, the intermediate portion, and the distal portion, the pipe having an outlet at or adjacent to the distal end of the distal portion, The proximal and distal portions are substantially straight, while the intermediate portion is curved such that the distal portion extends at a certain angle relative to the proximal portion.

[0032] A bent or curved injection needle may include one or more of the features of injection needles described above in relation to a first aspect of the present disclosure. For example, the middle portion may be curved such that the distal portion extends at an angle of 75° to 105° relative to the proximal portion (e.g., an angle of about 85° to about 95°, or an angle of about 90°). As another example, the length of the proximal portion may be shorter than the length of the distal portion.

[0033] In one preferred example, the method involves directly injecting the therapeutic composition into the target pharyngeal muscle using a needle described herein in a first aspect.

[0034] The pharyngeal muscles may include any one of the following specific muscles: the inferior pharyngeal constrictor, the oropharynx constrictor, the superior pharyngeal constrictor, the palatinopharynx, the tubal pharyngeal muscle, the stylopharynx, or any combination thereof. In one particular example, the method includes administering a therapeutic composition to the oropharynx and inferior pharyngeal constrictor muscles.

[0035] The therapeutic composition may be administered to the pharyngeal muscles by direct injection (i.e., intramuscular injection) following an incision in the neck of the subject that is sufficient to obtain direct access to at least a portion of the pharyngeal muscles of the subject. Thus, the method may include making one or more incisions (in the skin) in the neck of the subject to provide direct access to the pharyngeal muscles of the subject, and then administering the therapeutic composition by direct intramuscular injection into the pharyngeal muscles. For example, an incision may be made on the right side of the pharyngeal area on the right side of the body of a human or animal, and the area may be sufficiently dissected to access at least one or more pharyngeal muscles on the right side. A needle cannula may then be inserted directly into one or more pharyngeal muscles on the right side. Alternatively or additionally, an incision may be made on the left side of the pharyngeal area on the left side of the body of a human or animal, and the area may be sufficiently dissected to access one or more pharyngeal muscles on the left side. A needle cannula may then be inserted directly into one or more pharyngeal muscles on the left side.

[0036] The therapeutic compositions described herein may be injected directly into one or more sites of the pharyngeal muscles, one or more pharyngeal muscles, one or more sides of the pharyngeal muscles (e.g., the right or left side), or any combination thereof. Injection at multiple sites may aid in improved delivery of the therapeutic composition. For example, in the case of delivery of a viral vector-based therapeutic composition, injection at multiple sites may improve delivery to the pharyngeal muscle tissue. In one example, the therapeutic composition may be injected into the oropharyngeal constrictor muscle and the inferior pharyngeal constrictor muscle.

[0037] This disclosure also provides a method for administering a therapeutic composition to a target pharyngeal muscle, the method comprising directly injecting the therapeutic composition into the target pharyngeal muscle using a bent or curved injection needle. In some preferred examples, the injection needle is one of the injection needles described herein with reference to a first aspect.

[0038] The therapeutic composition may be administered to one or more pharyngeal muscles selected from the inferior pharyngeal constrictor muscle, middle pharyngeal constrictor muscle, superior pharyngeal constrictor muscle, palatinopharynx muscle, tubalopharynx muscle, stylopharynx muscle, and any combination thereof. For example, the method may include administering the therapeutic composition to the middle and inferior pharyngeal constrictor muscles.

[0039] The therapeutic composition may be administered to the pharyngeal muscles by direct injection, following an incision in the neck of the subject, which is sufficient to provide direct access to at least a portion of the pharyngeal muscles of the subject. In this example, the method may include making one or more incisions in the neck of the subject to provide direct access to at least a portion of the pharyngeal muscles of the subject, and then administering the therapeutic composition by direct intramuscular injection into the pharyngeal muscles.

[0040] In some cases, therapeutic compositions are for the treatment or prevention of dysphagia, such as dysphagia associated with oculopharyngeal muscular dystrophy (OPMD). Therefore, therapeutic compositions may be effective in treating or preventing dysphagia associated with OPMD. Therapeutic compositions may also be effective in treating or preventing one or more other symptoms of OPMD.

[0041] A therapeutic composition effective for the treatment or prevention of dysphagia associated with OPMD for use in the method disclosed herein is: (i) A DNA-directed RNAi (ddRNAi) construct comprising nucleic acids containing a sequence encoding a short hairpin microRNA (shmiR), (ii) A viral delivery vector may include a PABPN1 construct comprising a "silence and substitution" construct comprising a PABPN1 construct comprising a nucleic acid comprising a sequence encoding a functional PABPN1 protein having an mRNA transcript that is not targeted by shmiR(s) encoded by the ddRNAi construct.

[0042] In some examples, the viral delivery vector is an adeno-associated virus (AAV) containing a viral capsid protein derived from AAV9 containing a modified subunit 1 (VP1) sequence, wherein the amino acids at positions 26, 40, 43, and 44 are modified compared to the corresponding wild-type AAV9 VP1 sequence described in SEQ ID NO: 17. For example, the AAV may contain a viral capsid protein derived from AAV9 containing a VP1 sequence having a modified phospholipase 2 (PLA2) domain containing the sequence described in SEQ ID NO: 27. For example, the AAV may contain a viral capsid protein derived from AAV9 containing mutations A67E, Q81R, K84D, and A85S with respect to the full-length wild-type AAV serotype 9 capsid sequence described in SEQ ID NO: 19, and optionally, the viral capsid protein may contain the amino acid sequence described in SEQ ID NO: 28.

[0043] In some examples, the viral delivery vector is an adeno-associated virus (AAV) containing a viral capsid protein derived from AAV9 containing a modified subunit 1 (VP1) sequence, wherein the amino acids at positions 1, 26, 40, 43, and 44 are modified compared to the corresponding wild-type AAV9 VP1 sequence described in SEQ ID NO: 17. For example, the AAV may contain a viral capsid protein derived from AAV9 containing a VP1 sequence having a modified phospholipase 2 (PLA2) domain containing the sequence described in SEQ ID NO: 18. For example, the AAV may contain a viral capsid protein derived from AAV9 containing mutations A42S, A67E, Q81R, K84D, and A85S with respect to the full-length wild-type AAV serotype 9 capsid sequence described in SEQ ID NO: 19, and optionally, the viral capsid protein may contain the amino acid sequence described in SEQ ID NO: 20.

[0044] In some examples, the viral delivery vector is an adeno-associated virus (AAV) containing a viral capsid protein derived from AAV8 containing a modified subunit 1 (VP1) sequence, wherein the amino acids at positions 1, 26, 40, 43, 44, and 64 are modified compared to the corresponding wild-type AAV8 VP1 sequence described in SEQ ID NO: 21. For example, AAV may contain a viral capsid protein derived from AAV8 containing a VP1 sequence having a modified PLA2 domain containing the sequence described in SEQ ID NO: 22. For example, AAV may contain a viral capsid protein derived from AAV8 containing mutations A42S, A67E, Q81R, K84D, A85S, and Q105K with respect to the full-length wild-type AAV serotype 8 capsid sequence described in SEQ ID NO: 23, and optionally, the viral capsid protein may contain the amino acid sequence described in SEQ ID NO: 24.

[0045] In some examples, the PABPN1 construct within the “silence and substitution” construct contains a nucleic acid molecule encoding a functional PABPN1 protein, which is codon-optimized so that its corresponding mRNA sequence is not recognized by shmiR(s) encoded and expressed from the ddRNAi construct. For example, the codon-optimized nucleic acid may contain the sequence described in SEQ ID NO: 15. The codon-optimized nucleic acid preferably encodes the amino acid sequence described in SEQ ID NO: 16.

[0046] In some examples, a ddRNAi construct includes a DNA sequence encoding a shmiR that contains an effector sequence substantially complementary to the sequence described in SEQ ID NO: 1, and / or an effector sequence substantially complementary to the sequence described in SEQ ID NO: 2. In one example, a ddRNAi construct includes a DNA sequence encoding a shmiR that contains an effector sequence substantially complementary to the sequence described in SEQ ID NO: 1, and a DNA sequence encoding a shmiR that contains an effector sequence substantially complementary to the sequence described in SEQ ID NO: 2. For example, a ddRNAi construct may include a DNA sequence encoding a shmiR that contains the effector sequence described in SEQ ID NO: 4 and the effector complementary sequence (shmiR13) described in SEQ ID NO: 3, and a DNA sequence encoding a shmiR that contains the effector sequence described in SEQ ID NO: 6 and the effector complementary sequence (shmiR17) described in SEQ ID NO: 5. For example, a ddRNAi construct may include a nucleic acid containing or comprising the DNA sequence (shmiR13) described in SEQ ID NO: 12, and a nucleic acid containing or comprising the DNA sequence (shmiR17) described in SEQ ID NO: 13.

[0047] In one particular example, effective for the treatment or prevention of dysphagia associated with OPMD, the therapeutic composition administered by the method of this disclosure is referred to as BB-301 and comprises an AAV9 vector, the AAV9 vector is (a) A viral capsid protein derived from AAV9, which includes mutations A67E, Q81R, K84D, and A85S, determined in relation to the full-length wild-type AAV9 capsid sequence described in SEQ ID NO: 19, and optionally, the viral capsid protein includes the amino acid sequence described in SEQ ID NO: 28, (b) Silence and replacement structures, including, (i) A muscle-specific promoter (optionally selected, the promoter is the Spc512 muscle-specific promoter) (ii) A DNA-directed RNAi (ddRNAi) construct comprising a nucleic acid comprising or consisting of a sequence encoding shmiR13 as described herein (optionally, the DNA sequence described in Sequence ID No. 12), and a nucleic acid comprising or consisting of a sequence encoding shmiR17 as described herein (optionally, the DNA sequence described in Sequence ID No. 13), (iii) A PABPN1 construct comprising a nucleic acid sequence encoding a functional PABPN1 protein comprising the amino acid sequence described in SEQ ID NO: 16, wherein the nucleic acid sequence encoding the functional PABPN1 protein is a codon-optimized DNA sequence such that its corresponding mRNA sequence is not targeted by shmiR(s) encoded by the ddRNAi construct (optionally, the codon-optimized nucleic acid comprises the sequence described in SEQ ID NO: 15), and the PABPN1 construct comprising: The muscle-specific promoter is operably linked to the ddRNAi construct and the PABPN1 construct.

[0048] In each of the above-described examples, the method may include administering a therapeutic composition for the treatment or prevention of dysphagia associated with OPMD (such as the example described herein and designated as BB-301) in a volume ranging from about 25 μL to about 75 μL per injection site. The therapeutic composition may be administered at a concentration of about 1.0 + E12 vg / mL to about 1.0 + E15 vg / mL. For example, the concentration of the therapeutic composition for the treatment of dysphagia associated with OPMD administered to a subject may be about 5.0 + E12 vg / mL to about 1.0 + E14 vg / mL, for example, about 5.0 + E12 vg / mL to 5.0 + E13 vg / mL.

[0049] The therapeutic composition (e.g., as BB-301) may be administered (by injection) to multiple sites within the pharyngeal muscles. For example, the method may involve injecting the therapeutic composition into about 2 to about 30 or more sites within the pharyngeal muscles. In some examples, the method may involve injecting the therapeutic composition into about 2 to about 30 sites (e.g., about 4 to about 8 sites) in one or more locations within the pharyngeal muscles. For example, the method may involve injecting the therapeutic composition into about 4 to about 8 sites within one or more of the pharyngeal muscles, or within each of the following sections of the pharyngeal muscles: namely, the left section of the oropharyngopharyngeal constrictor muscle lateral to the pharyngeal raphe, the right section of the oropharyngopharyngeal constrictor muscle lateral to the pharyngeal raphe, the left section of the inferior pharyngeal constrictor muscle lateral to the pharyngeal raphe, and the right section of the inferior pharyngeal constrictor muscle lateral to the pharyngeal raphe. For example, the method may include injecting the therapeutic composition into approximately four sites within the left section of the oropharyngeal constrictor muscle located lateral to the pharyngeal raphe; injecting the therapeutic composition into approximately four sites within the right section of the oropharyngeal constrictor muscle located lateral to the pharyngeal raphe; injecting the therapeutic composition into approximately eight sites within the left section of the inferior pharyngeal constrictor muscle located lateral to the pharyngeal raphe; and injecting the therapeutic composition into approximately eight sites within the right section of the inferior pharyngeal constrictor muscle located lateral to the pharyngeal raphe.

[0050] In each of the preceding examples describing the administration of multiple injections of a therapeutic composition (e.g., BB-301) into the pharyngeal muscle, including a method for describing injections at multiple locations or sites within the pharyngeal muscle, each injection volume may be about 12.5 μL to about 25 μL to about 75 μL, preferably about 50 μL to about 75 μL. In one example, each injection volume may be about 50 μL. In another example, each injection volume may be about 75 μL. The therapeutic composition may be administered to one or more injection sites as a single injection per injection site or as multiple injections per injection site. Thus, the volume of the therapeutic composition administered per injection site may be a single injection volume or a cumulative volume of multiple injections administered at the injection site. For example, administering a volume of about 75 μL per injection site may include administering three injections, each having an injection volume of about 25 μL per injection site.

[0051] In each of the above examples describing intramuscular injection of a therapeutic composition (e.g., BB-301) into the pharyngeal muscle, including multiple injections of the therapeutic composition at one or more sites within the pharyngeal muscle, the total dose of the therapeutic composition administered to a subject in a single treatment may range from about 1.0 e13 vg / subject to about 5.5 e13 vg / subject. For example, the total dose of the therapeutic composition administered to a subject in a single treatment may be about 1.2 e13 vg / subject ("low dose"). For example, the total dose of the therapeutic composition administered to a subject in a single treatment may be about 3.6 e13 vg / subject ("medium dose"). For example, the total dose of the therapeutic composition administered to a subject in a single treatment may be about 5.4 e13 vg / subject ("high dose").

[0052] In each of the aforementioned examples, the therapeutic composition described herein (e.g., BB-301) may be provided in a pre-filled syringe.

[0053] This disclosure also provides pre-filled syringes containing a therapeutic composition (e.g., BB-301) effective for the treatment or prevention of dysphagia associated with OPMD. As described herein, the therapeutic composition (e.g., BB-301) may comprise AAV9, which is (a) A viral capsid protein derived from AAV9, which includes mutations A67E, Q81R, K84D, and A85S, determined in relation to the full-length wild-type AAV9 capsid sequence described in SEQ ID NO: 19, and optionally, the viral capsid protein includes the amino acid sequence described in SEQ ID NO: 28, (b) Silence and replacement structures, including, (i) A muscle-specific promoter (optionally selected, the promoter is the Spc512 muscle-specific promoter) (ii) A DNA-directed RNA interference (ddRNAi) construct comprising a nucleic acid comprising or consisting of a sequence encoding shmiR13 as described herein (optionally, the DNA sequence described in Sequence ID No. 12), and a nucleic acid comprising or consisting of a sequence encoding shmiR17 as described herein (optionally, the DNA sequence described in Sequence ID No. 13), (iii) A PABPN1 construct comprising a nucleic acid sequence encoding a functional PABPN1 protein comprising the amino acid sequence described in SEQ ID NO: 16, wherein the nucleic acid sequence encoding the functional PABPN1 protein is a codon-optimized DNA sequence such that its corresponding mRNA sequence is not targeted by shmiR(s) encoded by the ddRNAi construct (optionally, the codon-optimized nucleic acid comprises the sequence described in SEQ ID NO: 15), and the PABPN1 construct comprising: The muscle-specific promoter is operably linked to the ddRNAi construct and the PABPN1 construct.

[0054] The Disclosure also provides an injection device comprising a pre-filled syringe containing a therapeutic composition (e.g., BB-301) effective for the treatment or prevention of dysphagia associated with OPMD as described herein, and an injection needle of the Disclosure coupled to the pre-filled syringe. For example, the injection device may comprise the pre-filled syringe coupled to a curved or bent injection needle having one or more of the above-described features of the injection needle with respect to a first aspect of the Disclosure (e.g., needle A as described herein). For example, the pre-filled syringe may be coupled to an injection needle comprising a needle cannula having a proximal portion having a proximal end, a distal portion having a distal end, an intermediate portion disposed between the proximal portion and the distal portion, and a tube extending through the proximal portion, the intermediate portion, and the distal portion between the proximal end of the proximal portion and the distal end of the distal portion, opening at or adjacent to the distal end of the distal portion. The proximal and distal portions may be substantially straight, and the intermediate portion may be curved such that the distal portion extends at a certain angle to the proximal portion. The distal portion may extend at an angle of 75° to 105° to the proximal portion (for example, at an angle of about 85° to about 95°, or at an angle of about 90°). Alternatively, or in addition, the length of the proximal portion may be shorter than the length of the distal portion. The needle cannula may be, for example, about 10 mm to about 27 mm, and may have an overall length of, for example, about 16 mm to about 22 mm, and may have an overall length of, for example, about 18 mm to about 20 mm, or for example, about 19 mm. The needle cannula may be about 26 to 28 gauge, for example, a 27 gauge needle. The needle cannula may have an inclined needle tip. In one preferred example, the injection needle comprises a needle cannula having a proximal portion having a proximal end, a distal portion having a distal end, an intermediate portion disposed between the proximal and distal portions, a tube extending between the proximal end of the proximal portion and the distal end of the distal portion, and a tube extending between the intermediate portion and the distal portion of the distal portion, the tube opening at an outlet at or adjacent to the distal end of the distal portion, the proximal and distal portions being substantially straight, and the intermediate portion being curved such that the distal portion extends at an angle of about 85 to about 95° (optionally about 90°) with respect to the proximal portion.The length of the proximal portion may be shorter than the length of the distal portion, the needle cannula may have an overall length of about 18 mm to about 20 mm, for example, about 19 mm, the needle cannula may be of about 26 to 28 gauge, for example, a 27 gauge needle, and / or the needle cannula may have an inclined needle tip.

[0055] The injection device may be capable of delivering the therapeutic composition (e.g., BB-301) in multiple injections. The injection device may be configured to deliver the therapeutic composition (e.g., BB-301 as described herein) in one or more series of injections from a pre-filled syringe. Each injection may have a predetermined injection volume. For example, each injection volume may be about 12.5 μL to about 75 μL, preferably about 50 μL to about 75 μL. For example, each injection volume may be 50 μL. For example, each injection volume may be 75 μL.

[0056] The injection device may further include a volume setting mechanism that can be operated to set and / or adjust the amount of injection dispensed in one or more injections.

[0057] The injection volume setting mechanism may be operable to set the injection volume to approximately 12.5 μL to approximately 75 μL, or approximately 50 μL to approximately 75 μL. For example, the injection volume setting mechanism may be operable to set the injection volume to 50 μL. For example, the injection volume setting mechanism may be operable to set the injection volume to 75 μL. The injection volume setting mechanism may be operable to select from a separate set of injection volumes. The separate set of injection volumes may include one or more or each of approximately 12.5 μL, approximately 25 μL, approximately 50 μL, and approximately 75 μL. The injection device may be configured to provide the user with audible and / or tactile feedback to indicate the completion of the injection.

[0058] Accordingly, the present disclosure may provide an injection device comprising a pre-filled syringe coupled to a curved or bent injection needle as described herein, wherein the pre-filled syringe is filled with a therapeutic composition (e.g., BB-301 as described herein) for the treatment or prevention of dysphagia associated with OPMD, and the therapeutic composition (e.g., BB-301 as described herein) can be delivered in multiple injections, each injection having a predetermined injection volume. Each injection volume may be about 12.5 μL to about 75 μL, about 25 μL to about 75 μL, preferably about 50 μL to about 75 μL. For example, each predetermined injection volume may be 50 μL. For example, each predetermined injection volume may be 75 μL.

[0059] This disclosure also provides a kit comprising a set of two or more injection devices as described herein, the pre-filled syringes of the injection devices collectively contain a single therapeutic dose of a therapeutic composition (e.g., BB-301 as described herein) for the treatment of a single subject. A single therapeutic dose may comprise about 1.2 e13 vg / subject, about 3.6 e13 vg / subject, or about 5.4 e13 vg / subject.

[0060] In some examples, the kit may include four injection devices. For example, a kit including four injection devices may be configured to treat a subject according to a method described herein, in which one of the injection devices is used to inject the therapeutic composition at multiple sites (e.g., four or more injection sites) in the left section of the oropharyngopharyngeal constrictor muscle lateral to the pharyngeal raphe; one of the injection devices is used to inject the therapeutic composition at multiple sites (e.g., four or more injection sites) in the right section of the oropharyngopharyngeal constrictor muscle lateral to the pharyngeal raphe; one of the injection devices is used to inject the therapeutic composition at multiple sites (e.g., eight or more injection sites) in the left section of the inferior pharyngeal constrictor muscle lateral to the pharyngeal raphe; and one of the injection devices is used to inject the therapeutic composition at multiple sites (e.g., eight or more injection sites) in the right section of the inferior pharyngeal constrictor muscle lateral to the pharyngeal raphe. Each injection device in the kit may contain at least 200 μL of the therapeutic composition (e.g., BB-301). For example, each injection device may contain approximately 200 μL to 2000 μL, or approximately 400 μL to 1000 μL, for example, approximately 200 μL, approximately 300 μL, approximately 400 μL, approximately 500 μL, approximately 600 μL, approximately 800 μL, approximately 1000 μL, approximately 1500 μL, or approximately 2000 μL of a therapeutic composition (e.g., BB-301).

[0061] In some examples, the kit includes two injection devices containing approximately 200 μL to approximately 1000 μL of therapeutic composition, and two injection devices containing approximately 400 μL to approximately 2000 μL of therapeutic composition. In some examples, the kit includes two injection devices containing approximately 200 μL to approximately 500 μL of therapeutic composition, and two injection devices containing approximately 400 μL to approximately 1000 μL of therapeutic composition.

[0062] One or more injection devices in the kit may be configured to dispense the therapeutic composition (e.g., BB-301) in a series of injections, each having an injection volume of at least 25 μL. One or more injection devices may be configured to dispense the therapeutic composition (e.g., BB-301) in a series of injections, each having an injection volume of about 50 μL. One or more injection devices may be configured to dispense the therapeutic composition (e.g., BB-301) in a series of injections, each having an injection volume of about 75 μL.

[0063] In some examples, the method involves administering a therapeutic composition to one or more locations on the pharyngeal muscles, one or more pharyngeal muscles, one or more sides of the pharyngeal muscles, or any combination thereof.

[0064] In any of the above embodiments, the therapeutic composition may be administered by direct injection into the pharyngeal muscles, following an incision in the neck of the subject to obtain direct access to the pharyngeal muscles.

[0065] Sequence list key Sequence ID 1: RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, designated as PABPN1 mRNA region 13. Sequence ID 2: RNA sequence of the region within the mRNA transcript corresponding to the PABPN1 protein, designated as PABPN1 mRNA region 17. Sequence ID 3: The RNA effector complementary sequence of shmiR designated as shmiR13. Sequence ID 4: RNA effector sequence of shmiR designated as shmiR13. Sequence ID 5: The RNA effector complementary sequence of shmiR designated as shmiR17. Sequence ID 6: RNA effector sequence of shmiR designated as shmiR17. Sequence ID 7: RNA stem-loop sequence of shmiR Sequence ID 8: 5' flanking sequence of the pri-miRNA backbone. Sequence ID 9: 3' flanking sequence of the pri-miRNA backbone Sequence ID 10: The RNA sequence of shmiR designated as shmiR13. Sequence ID 11: The RNA sequence of shmiR designated as shmiR17. Sequence ID 12: The DNA sequence encoding the shmiR designated as shmiR13. Sequence ID 13: The DNA sequence encoding the shmiR designated as shmiR17. Sequence ID 14: DNA sequence of dual construct version 2 encoding coPABPN1 and shmiR designated as shmiR17 and shmiR13 under the control of Spc512. Sequence ID 15: DNA sequence of the human codon-optimized PABPN1 cDNA sequence. Sequence ID 16: Amino acid sequence of human wild-type PABPN1 protein. Sequence ID 17: A wild-type VP1 offspring sequence of AAV serotype 9, containing the PLA2 domain and flanking sequence. Sequence ID 18: A modified VP1 sub-sequence variant 1 of AAV serotype 9, containing a PLA2 domain and a flanking sequence. Sequence ID 19: Full-length wild-type capsid of AAV serotype 9. Sequence ID 20: Full-length modified capsid variant 1 of AAV serotype 9. Sequence ID 21: A wild-type VP1 offspring sequence of AAV serotype 8, containing the PLA2 domain and flanking sequence. Sequence ID 22: A modified VP1 offspring sequence of AAV serotype 8, containing a PLA2 domain and a flanking sequence. Sequence ID 23: Full-length wild-type capsid of AAV serotype 8. Sequence ID 24: Full-length modified capsid for AAV serotype 8. Sequence ID 25: AAV2 5'ITR sequence variant 1. Sequence ID 26: AAV2 3'ITR sequence variant 1. Sequence ID 27: A modified VP1 sub-sequence variant 2 of AAV serotype 9, containing a PLA2 domain and a flanking sequence. Sequence ID 28: Full-length modified capsid variant 2 of AAV serotype 9. Sequence ID 29: AAV2 5'ITR sequence variant 2. Sequence ID 30: AAV2 3'ITR sequence variant 2.

[0066] Next, as a mere example, an embodiment will be described with reference to the attached drawings. [Brief explanation of the drawing]

[0067] [Figure 1] This is a side view of an injection needle according to one embodiment of the present disclosure. [Figure 2] Figure 1 is a further side view of the injection needle. [Figure 3] A further side view of the hypodermic needle of Figure 1, with exemplary dimensions and angles. [Figure 4] Figure 1 is a magnified schematic view of the distal portion and distal tip of the injection needle. [Figure 5] Figure 1 is an enlarged elevation view of the distal portion and distal tip of the injection needle. [Figure 6A] Figure 1 is an enlarged cross-sectional view of the middle portion of the injection needle. [Figure 6B] This is an enlarged cross-sectional view of the middle portion of an injection needle according to another embodiment of the present disclosure. [Figure 7A] This is a side view of an alternative injection needle design tested for comparative purposes (injection needle B discussed herein). [Figure 7B] Figure 7A is a magnified perspective view of the tip of the alternative injection needle shown. [Figure 8A] This is an illustrative diagram of the use of a human neck and a hypodermic needle according to embodiments of the present disclosure. [Figure 8B] This is an illustrative diagram of the use of a human neck and a hypodermic needle according to embodiments of the present disclosure. [Figure 9A]This schematic diagram shows a construct for simultaneously performing gene silencing of endogenous PABPN1 and substitution with codon-optimized PABPN1, generated by subcloning two shmiRs targeting wtPABPN1 and mPABPN1 between two pAAV2 ITRs (ITRs not shown in the schematic diagram) within the 3' untranslated region of a codon-optimized PABPN1 transcript. [Figure 9B] This is a schematic diagram showing a “silence and substitution” construct (SR construct) designed to simultaneously perform gene silencing of endogenous PABPN1 and substitution with codon-optimized PABPN1. This construct is generated by subcloning two shmiRs (shmiR17 and shmiR13) targeting wtPABPN1 and mPABPN1 into the 3' untranslated region of a codon-optimized PABPN1 transcript within the pAAV2 vector backbone. [Figure 10] This is an illustrative diagram of the posterior aspect of the human pharyngeal muscles. [Modes for carrying out the invention]

[0068] explanation General definition Unless otherwise explicitly indicated by the context, the singular forms "a," "an," and "the" include plural demonstratives.

[0069] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” shall be understood to imply that they include the specified elements, components, or steps, or groups of elements, components, or steps, but not to exclude any other elements, components, or steps, or groups of elements, components, or steps.

[0070] The terms "and / or," for example, "X and / or Y," are understood to mean "X and Y" or "X or Y," and are considered to provide explicit support for both meanings or either of them.

[0071] Those skilled in the art will understand that this disclosure is subject to variations and modifications other than those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes, individually or collectively, all of the steps, features, compositions, and compounds referred to or indicated herein, as well as any and all combinations or any two or more of such steps or features.

[0072] Those skilled in the art will understand that numerous variations and / or modifications can be made to the embodiments described above without departing from the broad general scope of this disclosure. Therefore, these embodiments should be considered in all respects as illustrative and not restrictive.

[0073] Throughout this specification, unless otherwise specifically stated or the context requires, any reference to a single step, feature, composition, group of steps, or group of features or compositions shall be construed as encompassing one or more (i.e., one or more) of those steps, features, compositions, group of steps, or group of features or compositions.

[0074] This disclosure is intended for illustrative purposes only and should not be limited in scope by the specific examples described herein. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0075] Any example in this disclosure shall apply mutatis mutandis to any other example in this disclosure unless otherwise specified.

[0076] Unless specifically defined otherwise, all technical and scientific terms used herein shall be construed to have the same meaning as those commonly understood by those skilled in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0077] Unless specifically defined otherwise, all technical and scientific terms used herein shall be construed to have the same meaning as those commonly understood by those skilled in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0078] Unless otherwise indicated, the recombinant DNA, recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. For such techniques, please refer to J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE. Coligan et al. (editors), *Current Protocols in Immunology*, John Wiley & Sons (including all updates to date).

[0079] Selected definition "RNA" means a molecule containing at least one ribonucleotide residue. "Ribonucleotide" means a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranose moiety. The term includes isolated RNA such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinant RNA, and modified RNA which differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material to the terminus(s) of an siRNA, or internally, for example, the addition of one or more nucleotides to RNA. Nucleotides in RNA molecules in this disclosure may also include non-standard nucleotides such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be referred to as analogs, or analogs of naturally occurring RNA.

[0080] The term "RNA interference" or "RNAi" generally refers to RNA-dependent gene expression silencing initiated by double-stranded RNA (dsRNA) molecules in the cytoplasm of a cell. dsRNA molecules reduce or inhibit the transcript of a target nucleic acid sequence, thereby silencing the gene or reducing its expression.

[0081] As used herein, the term “double-stranded RNA” or “dsRNA” refers to an RNA molecule having a duplex structure and containing an effector sequence and an effector complementary sequence of similar lengths. The effector sequence and effector complementary sequence may be located within a single RNA strand or on separate RNA strands. The “effector sequence” (often referred to as the “guide strand”) is substantially complementary to the target sequence, which in this case is the region of the PABPN1 mRNA transcript. The “effector sequence” may also be referred to as the “antisense sequence.” The “effector complementary sequence” is sufficiently complementary to the effector sequence so that it can anneal to the effector sequence to form a duplex. In this regard, the effector complementary sequence is substantially homologous to the region of the target sequence. As will be apparent to those skilled in the art, the term “effector complementary sequence” may also be referred to as the “complement of the effector sequence” or the “sense sequence.”

[0082] As used herein, the term “duplex” refers to regions within two complementary or substantially complementary nucleic acids (e.g., RNA), or regions within two complementary or substantially complementary regions of a single-stranded nucleic acid (e.g., RNA), which base-pair each other by Watson-Crick base pairing or by any other means that enables a stabilized duplex between complementary or substantially complementary nucleotide sequences. Those skilled in the art will understand that within a duplex region, 100% complementarity is not required, and substantial complementarity is acceptable. Substantial complementarity may include 79% or more. For example, a single mismatch in a 19-base-pair duplex region (i.e., 18 base pairs and one mismatch) results in 94.7% complementarity, making the duplex region substantially complementary. In another example, two mismatches in a 19-base pair duplex region (i.e., 17 base pairs and two mismatches) result in 89.5% complementarity, making the duplex region substantially complementary. In yet another example, three mismatches in a 19-base pair duplex region (i.e., 16 base pairs and three mismatches) result in 84.2% complementarity, making the duplex region substantially complementary.

[0083] dsRNA can be provided as a hairpin or stem-loop structure having a duplex region consisting of an effector sequence and an effector complementary sequence linked by at least two nucleotide sequences, called a stem-loop. When dsRNA is provided as a hairpin or stem-loop structure, it may be referred to as “hairpin RNA,” “short hairpin RNAi agent,” or “shRNA.” Other dsRNA molecules that are provided as, or give rise to, a hairpin or stem-loop structure include primary miRNA transcripts (pri-miRNA) and precursor microRNAs (pre-miRNA). Pre-miRNA and shRNA can be spontaneously produced from pri-miRNA by the action of the enzymes Drosha and Pasha, which recognize and release the region of the primary miRNA transcript that forms the stem-loop structure. Alternatively, pri-miRNA transcripts can be manipulated to replace the native stem-loop structure with an artificial / recombinant stem-loop structure. That is, the artificial / recombinant stem-loop structure can be inserted into or cloned into a pri-miRNA backbone sequence that lacks its native stem-loop structure. In the case of stem-loop sequences engineered to be expressed as part of a pri-miRNA molecule, Drosha and Pasha recognize and release the artificial shRNA. The dsRNA molecules produced using this technique are known as "shmiRNA," "shmiR," or "microRNA framework shRNA."

[0084] As used herein, the term “complementary” with respect to sequences refers to the complement of a sequence by Watson-Crick base pairing, in which guanine (G) pairs with cytosine (C), and adenine (A) pairs with either uracil (U) or thymine (T). A sequence may be complementary to the full length of another sequence, or to a particular portion or length of another sequence. Those skilled in the art will recognize that U may be present in RNA and T may be present in DNA. Thus, A in either an RNA or DNA sequence may pair with U in an RNA sequence or T in a DNA sequence. Those skilled in the art will also recognize that G present in RNA may pair with C or U in RNA.

[0085] As used herein, the term “substantially complementary” is used to indicate a sufficient degree of complementarity or exact pairing, for example, between an effector sequence and an effector complementary sequence, or between an effector sequence and a target sequence, so that a stable, specific binding occurs. It is understood that a nucleic acid sequence does not need to be 100% complementary to its target or complementary sequence. The term encompasses sequences that are complementary to another sequence, except for overhangs. In some cases, a sequence is complementary to another sequence with one or two mismatches. In some cases, a sequence is complementary with one mismatch. In some cases, a sequence is complementary with two mismatches. In other cases, a sequence is complementary with three mismatches. In yet another case, a sequence is complementary with four mismatches.

[0086] The term “encoded” as used in the context of shRNA or shmiR in this disclosure should be understood to mean shRNA or shmiR that can be transcribed from a DNA template. Thus, the nucleic acids encoding or encoding shRNA or shmiR in this disclosure will contain a DNA sequence that serves as a template for transcription of the respective shRNA or shmiR.

[0087] The terms “DNA-directed RNAi construct” or “ddRNAi construct” refer to a nucleic acid comprising a DNA sequence that, upon transcription, produces an RNAi-inducing shRNA or shmiR molecule (preferably shmiR). A ddRNAi construct may be provided as a nucleic acid that can be transcribed as a single RNA capable of self-annealing to a hairpin structure having a duplex region linked by a stem-loop of at least two nucleotides, i.e., an shRNA or shmiR, or as a single RNA having multiple shRNAs or shmiRs, or as multiple RNA transcripts, each capable of folding as a single shRNA or shmiR. A ddRNAi construct may be provided within a larger “DNA construct” containing one or more additional DNA sequences. For example, a ddRNAi construct may be provided within a DNA construct containing a further DNA sequence encoding a functional PABPN1 protein, codon-optimized so that its mRNA transcript is not targeted by the shmiR of the ddRNAi construct. A ddRNAi construct and / or a DNA construct containing it may, for example, be in an expression vector operably linked to a promoter.

[0088] As used herein, the terms “operatably linked” or “operatably linked” (or similar) mean that a coding nucleic acid sequence is linked to or associated with a regulatory sequence, such as a promoter, in a manner that promotes the expression of the coding sequence. Regulatory sequences include promoters, enhancers, and other expression regulatory elements recognized in the art and selected to direct the expression of a coding sequence.

[0089] The term "vector" will be understood to mean a carrier for introducing nucleic acids into cells. Examples of vectors include, but are not limited to, plasmids, phagemids, viruses, bacteria, and carriers derived from viral or bacterial sources. A "plasmid" is a circular, double-stranded DNA molecule. A useful type of vector for use in accordance with this disclosure is a viral vector, in which an exogenous DNA sequence is inserted into a viral genome, which can be modified to delete one or more viral genes or portions thereof. Certain vectors are capable of autonomous replication within a host cell (e.g., vectors with an origin of replication that functions within a host cell). Other vectors can be stably integrated into the genome of a host cell, thereby replicating together with the host genome. As used herein, the term "expression vector" will be understood to mean a vector capable of expressing the RNA molecules of this disclosure.

[0090] It should be understood that "functional PABPN1 protein" refers to a PABPN1 protein that possesses the functional characteristics of the wild-type PABPN1 protein, such as the ability to control the site of mRNA polyadenylation and / or intron splicing in mammalian cells. Therefore, "functional PABPN1 protein" will be understood as a PABPN1 protein that does not cause OPMD when expressed or present in a subject. For example, references to "functional PABPN1 protein" in this specification refer to the human wild-type PABPN1 protein. The sequence of the human wild-type PABPN1 protein is listed in NCBI RefSeq NP_004634. Therefore, a functional human PABPN1 protein may possess the in vivo functional characteristics of the human PABPN1 protein listed in NCBI RefSeq NP_004634.

[0091] As used herein, the term “dysphagia” describes a swallowing disorder, including inability to swallow, difficulty swallowing, or discomfort during swallowing. A swallowing disorder may also affect speech and / or respiration in the affected subject. In some cases, dysphagia is secondary to another condition or disorder, such as a diagnosis of OPMD. For example, dysphagia may be secondary to a genetic diagnosis of OPMD in a subject.

[0092] As used herein, the terms “treating,” “treat,” and “treatment,” and their variations, refer to a clinical intervention designed to alter the natural course of an individual or cell being treated during the course of clinicopathology. Desired effects of treatment include a reduction in the rate of disease progression, improvement or alleviation of the disease state, and remission or improved prognosis. Treating dysphagia may result in improved pharyngeal muscle strength and / or coordination, as well as a consequent improvement in swallowing in the subject. Treatment of dysphagia may also improve the subject's speech and / or respiration. In the case of dysphagia associated with OPMD, treatment may include reducing or inhibiting the expression of the PABPN1 protein that causes OPMD in the subject, and / or expressing a PABPN1 protein with the normal length of polyalanine residues in the subject. Preferably, treatment of dysphagia associated with OPMD includes reducing or inhibiting the expression of the PABPN1 protein that causes OPMD in the subject, and expressing a PABPN1 protein with the normal length of polyalanine residues in the subject. Treatment of the underlying OPMD may treat the associated dysphagia. For example, if one or more of the above treatment outcomes are achieved, the individual is said to have been successfully "treated".

[0093] Where used herein, “prevent,” “preventing,” or similar terms should be understood to include preventing, reducing, or eliminating the development of at least one symptom of a condition. Where used in the context of dysphagia, “prevent” or “preventing” should be understood to mean preventing, reducing, or eliminating the development of one or more symptoms associated with dysphagia (as described above). For example, the methods of the present disclosure may be used to prevent dysphagia in subjects who have been genetically diagnosed with OPMD (e.g., PABPN1 allele classification: (GCN)12, (GCN)13, (GCN)14, (GCN)15, or (GCN)16) but who have not yet shown any physical symptoms of dysphagia. Thus, the methods of the present disclosure may be used to prevent, reduce, or eliminate the development of dysphagia in subjects, regardless of their genetic predisposition to OPMD and consequent dysphagia.

[0094] A “therapeutic dose” is defined as the minimum concentration or amount necessary to influence a measurable improvement in the dysphagia being treated. In the example where dysphagia is associated with OPMD, a therapeutic dose is a sufficient amount to influence a measurable improvement in one or more symptoms of OPMD in the subject, e.g., dysphagia and weight loss. Measurable improvement may be determined through several clinical assessment methods, such as assessing pharyngeal constrictor muscle function via pharyngeal area at maximum contraction, assessing swallowing efficiency via determination of total pharyngeal residue, assessing oropharyngeal dysphagia reported by the subject via the Sydney Swallowing Questionnaire, and assessing swallowing ability via the cold water time-measured drinking test. Exemplary clinical assessment methods contemplated herein for measuring improvement in one or more symptoms of OPMD are described in Example 3 of this disclosure.

[0095] The therapeutically effective doses described herein may vary depending on factors such as the patient's disease state, age, sex, and weight. The therapeutically effective doses of the compositions described herein for treating OPMD and OPMD-related dysphagia may further vary depending on factors such as the ability of shmiR, the nucleic acid encoding it, ddRNAi constructs, DNA constructs, expression vectors, or compositions containing them to induce a desired response in an individual, and / or the ability of the expression vector to express functional PABPN1 protein in the subject. The therapeutically effective dose is also such that, either alone or in combination with the therapeutically beneficial effect of the expression of functional PABPN1 protein in the subject, the therapeutically beneficial effect of shmiR, the nucleic acid encoding it, ddRNAi constructs, DNA constructs, expression vectors, or compositions containing them outweighs any toxic or adverse effects of shmiR, the nucleic acid encoding it, ddRNAi constructs, DNA constructs, expression vectors, or compositions containing them in order to inhibit, suppress, or reduce the expression of PABPN1 protein that causes possible OPMD-related dysphagia, either alone or in combination with the therapeutically beneficial effect of the expression of functional PABPN1 protein in the subject.

[0096] As used herein, “subject” or “patient” may be a human or non-human animal that has OPMD or a genetic predisposition to it, i.e., has a PABPN1 gene variant that causes OPMD. “Non-human animal” may 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, C. elegans, zebrafish), competition animals (e.g., racehorses, camels, greyhounds), or wild animals in captivity. In one example, the subject or patient is a mammal. In another example, the subject or patient is a human.

[0097] "Reduced expression," "reduced expression," or similar terms refer to the absence or observable decrease in levels of mRNA product from a protein and / or target gene, e.g., the PABPN1 gene. The decrease does not have to be absolute, but may be partial enough to result in a detectable or observable change as a result of RNAi induced by the shmiR, the nucleic acid encoding it, a ddRNAi construct, a DNA construct, an expression vector, or a composition containing them. The decrease can be measured by determining the decrease in levels of mRNA and / or protein product from the target nucleic acid compared to cells lacking the shmiR, the nucleic acid encoding it, a ddRNAi construct, a DNA construct, an expression vector, or a composition containing them, and may be as small as 1%, 5%, or 10%, or it may be absolute, i.e., 100% inhibition. The effect of the reduction can be determined by examining the external characteristics of cells or organisms, i.e., quantitative and / or qualitative phenotypic tests, and may also include detecting the presence or change in the amount of nuclear inclusions partially or completely containing expPABPN1 in cells or organisms after administration of shmiR, nucleic acids encoding it, ddRNAi constructs, DNA constructs, expression vectors, or compositions containing them.

[0098] As used herein, “delivery system,” “delivery vector,” and similar terms refer to a vector capable of packaging and introducing foreign genetic material, such as DNA or RNA, into a cell. Examples of delivery vectors include viral vectors, such as adeno-associated virus (AAV) vectors, retroviral vectors, adenovirus vectors (AdV), and lentiviral vectors (LV). As described herein, viral vectors can be used to deliver and express foreign genetic material into cells. Therefore, viral expression vectors disclosed herein can be used as delivery vectors.

[0099] As used herein, the terms “adeno-associated virus” or “AAV” refer to a group of viruses within the Parvoviridae family that contain a short (approximately 4.7 kb) single-stranded DNA genome and depend on the presence of a helper virus, such as an adenovirus, for replication. Furthermore, the present disclosure intends, for example, a vector derived from AAV used as a gene transfer vehicle.

[0100] As used herein, the term “serotype,” when used in the context of AAV, is a distinction used to refer to an AAV having a serologically distinct capsid from other AAV serotypes. Serological specificity is determined based on the absence of cross-reactivity between antibodies against one AAV compared to another. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). The VP1 sequences of wild-type AAV known to infect humans are described in Chen et al., (2013) J.Vir. 87(11):6391-6405 (e.g., including 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, for example, AAV1 (Genbank Acc.No: AAD27757.1, GI: 4689097), AAV2 (Genbank Acc.No: AAC03780.1, GP.2906023), AAV3 (Genbank Acc.No: AAC55049.1, GI: 1408469), AAV4 (Genbank Acc.No: AAC58045.1, GL2337940), AAV5 (Genbank Acc.No: AAD13756.1, GI-4249658), AAV10 (Genbank Acc.No: AAT46337.1, GL48728343), AAV11 (Genbank Acc.No:AAT46339.1, GI:48728346), AAV12 (Genbank Acc.No:ABI16639.1, GI:112379656), or AAV13 (Genbank Acc.No:ABZ10812.1, GI:167047087).Furthermore, complete genomes for AAVs from serotypes 1-13 are known in the art, for example, AAV1 (NCBI reference sequence: NC_002077.1), AAV2 (GenBank Acc.No: J01901.1), AAV3 (Genbank Acc.No: AF028705.1), AAV4 (NCBI reference sequence: NC_001829.1), AAV5 (NCBI reference sequence: NC_006152.1), AAV6 (GenBank: AF028704.1), AAV7 (NCBI reference sequence: NC_006260.1), AAV8 (NCBI reference sequence: NC_006261.1), AAV9 (GenBank Acc.No: AY530579.1), AAV10 (Genbank Acc.No: AY631965.1), AAV11 (Genbank Acc.No:AY631966.1) or AAV12 (Genbank Acc.No:DQ813647.1). Those skilled in the art will also understand that sequence variations may exist between the genomes of different AAV strains within the same serotype, especially when different AAVs from the same serotype are isolated from different host species and / or different geographical locations.

[0101] When used herein in the context of AAV, “viral capsid protein,” “capsid protein,” “capsid polypeptide,” or similar terms refer to a polypeptide of AAV that has self-assembling activity to produce the proteinaceous shell (also referred to as coat protein or VP protein) of AAV particles. It is typically composed of three subunits, VP1, VP2, and VP3, which are expressed from a single nucleic acid molecule and interact together to form an icosahedral capsid. The capsid structure of AAV is described in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).

[0102] As used herein, the term “promoter” generally refers to a DNA sequence involved 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 generally located upstream of the coding sequence with respect to the direction of transcription.

[0103] As used herein, “pharyngeal muscles” refers to one or more of the muscle groups that make up the pharynx. The pharyngeal muscles may include one or more of the inferior pharyngeal constrictor, middle pharyngeal constrictor, superior pharyngeal constrictor, palatinopharynx, tubopharynx, and / or stylopharynx.

[0104] syringe needle In one embodiment of the present disclosure, as shown in Figures 1-3, the injection needle 10 includes a needle cannula 20 having a proximal portion 21 with a proximal end 211, a distal portion 23 with a distal end 231 and an outlet 25 located at or adjacent to the distal end 231, and an intermediate portion 22 disposed between the proximal portion 21 and the distal portion 23. The tube 26 extends through the proximal portion 21, the intermediate portion 22, and the distal portion 23 between the proximal portion 211 and the distal end 231, and opens at the outlet 25. In this embodiment, the length of the proximal portion 21 is shorter than the length of the distal portion 23.

[0105] As shown in Figures 1-3, the injection needle 10 also includes a needle hub 30 connected to a needle cannula 20, the needle cannula 20 protruding distally from the needle hub 30. The proximal end 211 of the proximal portion 21 of the needle cannula 20 is located at the boundary where the needle cannula 20 contacts and / or enters the needle hub 30. In some embodiments, the needle cannula 20 may further comprise a support portion 28, shown by the dotted line in Figure 1, which extends proximal to the proximal end 211 of the proximal portion 21 of the needle cannula and is entirely disposed, for example, inside the needle hub 30. A tube 26 may further extend through the support portion 28.

[0106] The needle cannula 20 includes an inlet 24 located at or adjacent to the proximal end 211 of the proximal portion 21, or, if present, at or adjacent to the proximal end 281 of the support portion 28. Fluid can enter the needle cannula 20 through the inlet 24 and proceed to the outlet 25, for example, when injected into a patient.

[0107] In this embodiment, the needle hub 30 comprises a distal end portion 31 adapted to connect to the needle cannula 20 (for example, by fixing or embedding the support portion 28 of the needle cannula therein) and a proximal end portion 32 on the opposite side. The proximal end portion 32 includes threads 33 on its outer surface, which are adapted to engage with complementary threads of an additional device such as a syringe barrel, and a fluid substance (e.g., a therapeutic composition) can be stored before being delivered via the needle cannula 20. As seen in Figures 1 and 3, the distal end portion 31 may be swaged (tapered toward the central axis).

[0108] In this embodiment, the proximal portion 21 and the distal portion 23 are each substantially linear. The intermediate portion 22 in this embodiment is bent, or more specifically curved, such that the distal portion 23 extends at an angle of about 75° to 105° with respect to the angle of the proximal portion 21.

[0109] The boundary between the proximal portion 21 and the intermediate portion 22 of the needle cannula 20 may be defined at the point where the needle cannula 20 transitions from a state in which it extends linearly in the proximal portion 21 to a state in which it is bent or curved in the intermediate portion 22. Similarly, the boundary between the intermediate portion 22 and the distal portion 23 of the needle cannula 20 may be defined at the point where the needle cannula 20 transitions from a state in which it is bent or curved in the intermediate portion 22 to a state in which it extends linearly in the distal portion 23.

[0110] curvature angle The middle portion 22 of the needle cannula 20 is curved, and more specifically (e.g., arc-shaped), to assist in navigating anatomical structures at the injection site. Certain configurations are particularly suitable for direct delivery of a therapeutic composition to one or more pharyngeal muscles, and the needle cannula has been found to be able to effectively guide the anatomical structures surrounding the pharyngeal muscles.

[0111] For example, as shown in Figures 6A and 6B, the intermediate portion may be bent or curved so that the distal portion 23 extends at an angle β of approximately 75° to 105° relative to the proximal portion 21. For example, as shown in the embodiment of Figure 6B, the intermediate portion 22 may be bent or curved so that the distal portion 23 extends at an angle of approximately 105° relative to the proximal portion 21.

[0112] In some embodiments, the distal portion 23 extends from the proximal portion 21 at an angle β of approximately 80° to 100°. In other embodiments, the distal portion 23 extends from the proximal portion 21 at an angle of approximately 85° to 95°. In other embodiments, the distal portion 23 extends from the proximal portion 21 at an angle of approximately 87° to 93°. In other embodiments, as shown in Figure 6A, for example, the distal portion 23 extends from the proximal portion 21 at an angle β of substantially 90°.

[0113] As shown above, the proximal portion 21 and the distal portion 23 may each be perfectly linear. However, in other embodiments, the proximal portion 21 and / or the distal portion 23 may have one or more curves, angles, or irregularities, while being substantially linear compared to the intermediate portion. In such situations, when determining the angles over which the proximal portion 21 and the distal portion 23 each extend, this may be based on a hypothetical straight line extending between the proximal end 211 of the proximal portion 21 and the proximal end of the intermediate portion 22, and a hypothetical straight line extending between the distal end of the intermediate portion 22 and the distal end 231 of the distal portion 23, respectively.

[0114] In some embodiments, the proximal portion 21, intermediate portion 22, and distal portion 23 of the needle cannula 20 extend as a whole within a common plane. Alternatively, the portions may not extend as a whole within a common plane. For example, the needle cannula may bend or curve in three dimensions.

[0115] Injection device The disclosure also provides an injection device 200, such as a drug product injection device (DPID) 200 as shown in Figure 8, which comprises a needle 10 coupled to a syringe 100 (e.g., a pre-filled syringe).

[0116] A pre-filled syringe 100 may comprise a barrel and a plunger received within the barrel. The syringe 100 may be pre-filled with a therapeutic composition (e.g., BB-301 as described herein) stored in the barrel. The plunger is longitudinally slidable relative to the barrel to discharge the therapeutic composition from the syringe 100.

[0117] The injection device 200 may include an injection volume setting mechanism that can be operated to set and / or adjust the amount of injection dispensed in one or more injections. In some examples, a pre-filled syringe includes the injection volume setting mechanism. In other examples, the injection volume setting mechanism may be separate from the pre-filled syringe and may be configured to be coupled to one or more components of the pre-filled syringe.

[0118] The injection volume setting mechanism may be operable to set the injection volume to approximately 12.5 μL to approximately 75 μL, or approximately 50 μL to approximately 75 μL. For example, the injection volume setting mechanism may be operable to select from separate sets of injection volumes. These separate sets of injection volumes may include one or more of approximately 12.5 μL, approximately 25 μL, approximately 50 μL, and approximately 75 μL.

[0119] The injection device may be configured to provide the user with audible and / or tactile feedback to indicate the completion of the injection. For example, the plunger of a pre-filled syringe 100 may include at least one set of consecutive plunger formations, which provide the user with audible and tactile feedback via a series of plunger "clicks" as the therapeutic composition is dispensed.

[0120] Use of injection needles The injection needle 10 in this embodiment is a pharyngeal muscle injection needle. The injection needle 10 is configured to inject the therapeutic composition directly into the pharyngeal muscle. The injection needle may be particularly used for direct injection into any one of the pharyngeal constrictor muscles and / or the inferior pharyngeal constrictor muscle, middle pharyngeal constrictor muscle, superior pharyngeal constrictor muscle, palatinopharynx muscle, tubopharynx muscle, stylopharynx muscle, or any combination thereof.

[0121] In one embodiment, as illustrated in Figures 8A-8B, an open surgical procedure is performed to allow direct injection into the pharyngeal muscle using a needle 10. Referring to Figure 8A, for example, a scalpel 504 is used to make a first incision 511 on a first side of the patient's neck 500, exposing at least a portion 521 of at least one pharyngeal muscle, such as the inferior pharyngeal constrictor muscle 501, the middle pharyngeal constrictor muscle 502, the superior pharyngeal constrictor muscle 503, or a portion of another pharyngeal muscle. The first lateral side may be the right or left side of the patient's neck (the left side is shown in Figures 8A and 8B).

[0122] A second incision 512 (not shown) may occur on the second lateral side and may expose the inferior pharyngeal constrictor muscle 501, the middle pharyngeal constrictor muscle 502, the superior pharyngeal constrictor muscle 503, or the other side of another pharyngeal muscle.

[0123] Referring to Figure 8B, the distal portion of the needle cannula 20 of the injection needle 10 is pushed in so as to penetrate the exposed portions of the pharyngeal muscles 501, 501, and 502. The injection device 200 is operated to push the therapeutic composition out of the syringe 100 and into the pharyngeal muscles 501, 502, and 503 directly through the needle cannula 20.

[0124] This injection process may be used, for example, to treat or prevent dysphagia associated with oculopharyngeal muscular dystrophy (OPMD), in which case the injection needle may be considered additionally or alternatively as an oculopharyngeal muscular dystrophy (OPMD) treatment injection needle. A method for administering a therapeutic composition using the injection needle of the present disclosure to treat or prevent OPMD associated with dysphagia is described herein.

[0125] The injection process may be repeated on the opposite side of the patient's neck, for example, following the creation of a second incision on the opposite side of the patient's neck. Generally, injections may be administered directly into one or more locations within the pharyngeal muscles, one or more pharyngeal muscles, one or more sides of the pharyngeal muscles (e.g., right or left), or any combination thereof. Injections at multiple locations may help increase delivery to the pharyngeal muscle tissue. Injections may also be repeated over time (e.g., weeks, months, or years) to address any decrease in the effective amount of the therapeutic agent over time.

[0126] radius of curvature As shown, the middle section 22 is curved.

[0127] As shown in Figure 6A, the curved intermediate portion 22 has an arc length l and a substantially constant radius r. Since the proximal portion 21 extends at a substantially 90° angle β with respect to the distal portion, the arc length l in this embodiment represents one-quarter of the circumference of the circle. In this embodiment, the arc length l is approximately 4.4 mm, the radius of curvature r is approximately 2.8 mm, and the equation radius = circumference / 2π (radius of curvature r = 4 (4.4 mm) / 2π = 2.8 mm) is satisfied.

[0128] However, as shown in Figure 6B, for example, in one embodiment where the needle cannula 20 has a distal portion 23 extending at an alternative angle β to a proximal portion of about 105°, the radius of curvature r does not need to be substantially constant. Due to this potential difference in the radius of curvature along the intermediate portion, the radius of curvature is within the range of values ​​r. v It can be called that.

[0129] In some embodiments, the radius of curvature of the curved intermediate portion 22 is (r or r v The radius of curvature of the curved intermediate portion 22 is approximately 2.5 mm to approximately 3.1 mm. In other embodiments, the radius of curvature of the curved intermediate portion 22 is (r or r v The radius of curvature of the curved intermediate portion 22 is 2.6 mm to 3.0 mm. In a further embodiment, the radius of curvature of the curved intermediate portion 22 is r or r v The radius of curvature of the curved intermediate portion 22 is 2.7 mm to 2.9 mm. In further embodiments, the radius of curvature of the curved intermediate portion 22 is r or r v ) is approximately 2.8 mm.

[0130] Those skilled in the art will understand that the arc length and / or radius of curvature of a needle cannula may be varied, for example, to scale the needle cannula to the patient's anatomical structure while maintaining a constant bending angle between the proximal and distal portions of the needle cannula, or for other purposes.

[0131] full length For example, as shown in Figure 2, the total length of the needle cannula 20 can be measured as the sum of the length A of the proximal portion 21, the arc length B of the intermediate portion 22, and the length C of the distal portion 23. In the embodiment shown in Figure 3, the total length of the needle cannula may be approximately 19 mm.

[0132] However, it will be understood by those skilled in the art that the needle cannula 20 described herein may have different lengths, for example, to be scaled to the anatomical structure of a patient, or for other purposes. In some embodiments, the total length of the needle cannula is about 10 mm to about 27 mm, about 13 mm to about 25 mm, about 15 mm to about 23 mm, about 17 mm to about 21 mm, or about 19 mm.

[0133] Needle gauge Needle gauge (G) is used to describe the size of the tube or bore of a needle cannula. The higher the gauge value, the smaller the tube and therefore usually the thinner the needle. Needle gauges are selected for different purposes by balancing considerations such as minimizing pain, tissue type, rate of substance penetration, and impact on the integrity of the contents. For example, insulin needles are generally thin (29G-31G) to minimize the pain of subcutaneous injection, while thicker needles (16G-17G) may be used for blood donation because a larger tube creates lower pressure on fragile red blood cells. In this regard, needle gauges may be selected based on the effect of the gauge on the integrity of the needle components for injection, such as viral expression vectors.

[0134] The injection needles described herein may be used during open surgical procedures under anesthesia, and therefore, minimizing pain may not be a priority consideration. During testing with different needle designs, including testing of injections into the pharyngeal muscle, minimizing leakage and maximizing the desired tissue transduction of functional viruses were given priority considerations. This was particularly important given that the amount of drug product delivered at each injection site was small. In the context of the applicant's drug product, BB-301, it was found that the needle gauge affected the percentage of AAV that remained functional following the flow of the drug product through the injection needle, which is likely due to the influence of bioforces on the viral particles during injection (e.g., flow rate, pressure, and shear stress). As a result, a needle gauge was selected that balanced, on the one hand, the need to minimize leakage and maximize the transduction of functional AAV into tissue, and on the other hand, the need for a needle strong enough to maintain its curvature and withstand mechanical deformation during use.

[0135] In the embodiment shown in Figure 1, the needle cannula shown is 27G. Generally, the needle cannulas according to this disclosure may be about 23–31 gauge, about 25–29 gauge, about 26–28, or about 27G, or other.

[0136] For ease of reference, the dimensions of needle cannulas described herein may be referred to in relation to the Birmingham Needle Scale.

[0137] Nevertheless, in some embodiments, the needle cannula 20 may not conform to any gauge standard.

[0138] Outer diameter and inner diameter In some embodiments, the outer diameter 202 of the needle cannula 20 is approximately 0.3 mm to approximately 0.5 mm, approximately 0.35 mm to approximately 0.45 mm, approximately 0.40 mm to approximately 0.42 mm, or approximately 0.41 mm, as shown in Figures 4 and 5.

[0139] The outer diameter 202 of the needle cannula 20 may be substantially constant between the proximal end 211 and the distal end 231 of the cannula 20. Alternatively, although not shown, in some embodiments, the outer diameter 202 of the needle cannula 20 may vary along the length of the cannula 20 (e.g., taper). The outer diameter 202 may vary continuously along the cannula 20, or it may vary in one or more distinct sections of the cannula 20.

[0140] In some embodiments, the inner diameter 201 of the needle cannula is approximately 0.1 mm to 0.3 mm, approximately 0.15 mm to 0.25 mm, approximately 0.20 mm to 0.22 mm, or approximately 0.21 mm.

[0141] The inner diameter 201 of the needle cannula 20 may be substantially constant between the proximal end 211 and the distal end 231 of the cannula 20. Alternatively, although not shown, in some embodiments, the inner diameter 201 of the needle cannula 20 may vary along the length of the cannula 20 (e.g., taper). The inner diameter 201 may vary continuously along the cannula 20, or it may vary in one or more distinct sections of the cannula 20.

[0142] In some embodiments, the wall thickness of the needle cannula 20 is from about 0.03 mm to about 0.17 mm, from about 0.05 mm to about 0.15 mm, from about 0.07 mm to about 0.13 mm, or about 0.1 mm.

[0143] Needle volume In some embodiments, the injection needle includes a dead volume of from about 0.5 μL / 25.4 mm to 1.5 μL / 25.4 mm. In some embodiments, the total dead volume is from about 0.7 mm 3 to about 0.9 mm 3 from about 0.76 mm 3 to about 0.86 mm 3 from about 0.80 mm 3 to about 0.82 mm 3 or about 0.81 mm 3 and may be.

[0144] Relative length of section <B In some embodiments, different portions of the needle cannula 20 may have different lengths.

[0145] For example, in the embodiments shown in FIGS. 2 and 3, the length A of the proximal portion 21 is shorter than the length C of the distal portion 23. In some embodiments, the length A of the proximal portion 21 may be less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of the length C of the distal portion 23.

[0146] In some embodiments, as shown in the embodiments of FIGS. 2 and 3, the length C of the distal portion 23 may be substantially the same as or greater than the sum of the length A of the proximal portion 21 and the arc length B of the intermediate portion 22. That is, the length C of the distal portion 23 may be about 50% or more than 50% of the total length of the needle cannula 20 from the proximal end 211 of the proximal portion to the distal end 231 of the distal portion 231.

[0147] Needle tip Preferably, the distal tip 27 of the needle cannula 20 is angled so that the distal tip 27 is sharp, for example, as shown in Figures 1-5. Although not bound by any one theory, the applicant believes that the inclusion of an angled distal tip 27 facilitates clean puncture of the tissue in use, thereby minimizing potential leakage of the drug product from the tissue after injection and release of the drug product.

[0148] As shown in Figures 1-3, the orientation of the inclined portion of the distal tip 27 may be such that the edge of the inclined portion faces away from the medial curvature side of the needle cannula 20 (the medial curvature side generally marked by reference IB in Figure 1). For example, the edge of the inclined portion of the distal tip 27 may face away from the center of curvature of the arcuate intermediate portion 22 (the center of curvature point generally marked by reference CP in Figure 1).

[0149] As shown in Figure 4, preferably the distal tip has a long inclined section. The distal tip is inclined at angles of, for example, about 5° to about 15°, about 10° to about 12.5°, about 11° to about 12°, or about 11.5°.

[0150] As shown in Figures 2 and 3-5, the inclined section length D can be, for example, about 0.5 mm to about 3.5 mm, about 1 mm to about 3 mm, about 1.5 mm to about 2.5 mm, or about 2 mm.

[0151] The distal tip 27 may have one or more additional features such as an additional bevel, lancet, or heel.

[0152] needle material The needle cannula 20 of the injection needle 10 described herein may include stainless steel that may contain iron and chromium and / or nickel. The needle cannula 20 may include nickel-plated stainless steel. Specifically, the needle cannula may include AISI (American Iron and Steel Institute) 304 stainless steel.

[0153] The needle cannula 20 may contain annealed metal, for example, to improve ductility and / or reduce brittleness. The needle cannula 20 may be annealed before or after bending, or before or after the formation of the intermediate portion 22.

[0154] Other materials may be preferable for maintaining the integrity of the desired component for injection.

[0155] kit The disclosure also provides a kit comprising a set of injection devices 200. The injection devices 200 may be supplied assembled or disassembled (for example, with the needle 10 attached to or detached from a pre-filled syringe).

[0156] A pre-filled syringe of an injection device may collectively contain a single therapeutic dose of a therapeutic composition for the treatment of a single target. For example, a single therapeutic dose may include about 1.2 e13 vg / target (low dose), about 3.6 e13 vg / target (medium dose), or about 5.4 e13 vg / target (high dose) of the therapeutic composition designated BB-301 as described herein.

[0157] Each injection device may contain at least 200 μL of therapeutic composition. For example, each injection device may contain about 200 μL to about 2000 μL, or about 400 μL to about 1000 μL, for example, about 200 μL, about 300 μL, about 400 μL, about 500 μL, about 600 μL, about 800 μL, about 1000 μL, about 1500 μL, or about 2000 μL of therapeutic composition. In some examples, a kit includes two injection devices each containing about 200 μL to about 1000 μL of therapeutic composition, and two injection devices each containing about 400 μL to about 2000 μL of therapeutic composition. In some examples, the kit includes two injection devices, each containing approximately 200 μL to 500 μL of the therapeutic composition, and two more injection devices, each containing approximately 400 μL to 1000 μL of the therapeutic composition.

[0158] One or more injection devices may be configured to dispense a drug product in a series of injections with an injection volume of 25 μL. One or more injection devices may be configured to dispense a drug product in a series of injections with an injection volume of 50 μL. One or more injection devices may be configured to dispense a drug product in a series of injections with an injection volume of 75 μL.

[0159] Alternatively, the kit may include a set of pre-filled syringes 100, and the injection needles 10 may be provided separately.

[0160] Administration to the pharyngeal muscles and treatment This disclosure also provides a method for administering a therapeutic composition to a target pharyngeal muscle. The administration method preferably involves directly injecting the therapeutic composition into the target pharyngeal muscle using a bent or curved injection needle. In a particularly preferred embodiment, the administration method includes directly injecting the therapeutic composition into the target pharyngeal muscle using the injection needle 10 described herein.

[0161] The pharyngeal muscles are a group of muscles that form the pharynx, located at the back of the oral cavity. They determine the shape of the pharynx tube and influence its acoustic properties as the primary resonating cavity. The pharyngeal muscles (involuntary skeleton) assist in pushing food into the esophagus. The pharynx has two muscle layers: the outer cricoid layer and the inner longitudinal layer. The outer cricoid layer includes the inferior pharyngeal constrictor, middle pharyngeal constrictor, and superior pharyngeal constrictor. During swallowing, these muscles contract to propel the bolus downward (an involuntary process). The inner longitudinal layer includes the stylopharyngeal, laryngopharyngeal, and palatinopharyngeal muscles. During swallowing, these muscles act to shorten and expand the pharynx.

[0162] The methods of the present disclosure may include administering a therapeutic composition to one or more muscles within the pharyngeal muscle group. For example, the methods of the present disclosure may include directly injecting a therapeutic composition into one or more specific muscles, such as the inferior pharyngeal constrictor, oropharynx constrictor, superior pharyngeal constrictor, palatinopharynx, tubopharynx, stylopharynx, or any combination thereof, using a bent or curved injection needle. In one example, the method includes administering a therapeutic composition to the oropharynx constrictor and inferior pharyngeal constrictor.

[0163] The therapeutic composition may be administered to the pharyngeal muscles by direct injection (i.e., intramuscular injection) following an incision in the neck of the subject that is sufficient to obtain direct access to at least a portion of the pharyngeal muscles of the subject. Therefore, the method of administration may include making one or more incisions (e.g., in the skin) in the neck of the subject to provide direct access to the pharyngeal muscles (or more) of the subject, and then administering the therapeutic composition by direct intramuscular injection into the pharyngeal muscles using a bent or curved injection needle (e.g., injection needle 10 described herein). For example, an incision may be made on the right side of the pharyngeal area on the right side of the body of a human or animal, and the area may be sufficiently dissected to access at least one or more of the pharyngeal muscles on the right side. The needle cannula may then be inserted directly into one or more of the pharyngeal muscles on the right side. Alternatively or additionally, an incision may be made on the left side of the pharyngeal area on the left side of the body of a human or animal, and the area may be sufficiently dissected to access one or more of the pharyngeal muscles on the left side. The needle cannula may then be inserted directly into one or more of the pharyngeal muscles on the left side.

[0164] The therapeutic compositions described herein may be injected directly into one or more locations of the pharyngeal muscles, one or more pharyngeal muscles, one or more sides of the pharyngeal muscles (e.g., the right or left side), or any combination thereof. Injections at multiple locations may help increase the transduction of pharyngeal muscle tissue. In one example, the therapeutic composition may be injected into the oropharyngial constrictor muscle and the inferior pharyngeal constrictor muscle. Direct injection may be applied to specific parts of muscles, such as the thyropharyngeal muscle or the cricopharyngeal muscle.

[0165] When it is desirable to inject into multiple sites within the pharyngeal muscle and / or to administer multiple doses, a curved or bent injection needle (e.g., injection needle 10 described herein) may be used with a pre-filled syringe configured to deliver multiple doses or volumes to one or more muscles via microinjection.

[0166] In some examples, therapeutic compositions administered to the pharyngeal muscles using a bent or curved injection needle (e.g., injection needle 10) according to the present disclosure are for the treatment or prevention of dysphagia. In one example, the dysphagia is associated with oculopharyngeal dystrophy (OPMD). According to this example, the method may involve administering a therapeutic composition for the treatment or prevention of OPMD (thereby treating or preventing a condition associated therewith, e.g., dysphagia). A therapeutic composition for the treatment or prevention of OPMD may be based on a modified adeno-associated virus (AAV) delivery vector comprising a “silence and substitution” construct, which includes (i) one or more RNAi agents targeting a region of the PABPN1 mRNA transcript that causes OPMD, and (ii) a PABPN1 substitution construct for the expression of wild-type (functional) human PABPN1 protein having an mRNA transcript that is not targeted by the RNAi agents of the present disclosure. Accordingly, the method may involve administering a modified AAV delivery vector containing the “silence and replacement” construct to the target pharyngeal muscle by direct injection (e.g., direct intramuscular injection into the pharyngeal muscle, as described herein, following an incision into the pharyngeal area to provide access to the pharyngeal muscle) using a bent or curved injection needle (e.g., injection needle 10). Therapeutic compositions according to this example have been previously described by the applicant in WO2017 / 177277, WO2018 / 107228, WO2019 / 043630, WO2020 / 077412, and WO2020 / 172720, the contents of each of them being incorporated herein by reference as a whole.

[0167] In one example, a therapeutic composition administered directly to the target pharyngeal muscle using a curved or bent injection needle (e.g., injection needle 10 described herein) includes AAV, which is, (a) A viral capsid protein derived from AAV9 containing a modified subunit 1 (VP1) sequence, wherein the amino acids at positions 26, 40, 43, and 44 are modified compared to the corresponding wild-type AAV9 VP1 sequence described in Sequence ID No. 17, and (b) A “silence and substitution” construct comprising (i) a DNA-directed RNAi (ddRNAi) construct comprising a nucleic acid comprising a sequence encoding a short hairpin microRNA (shmiR), and (ii) a PABPN1 construct comprising a nucleic acid comprising a sequence encoding a functional PABPN1 protein having an mRNA transcript that is not targeted by the shmiR(s) encoded by the ddRNAi construct. For example, a modified AAV9 VP1 sequence may include glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, and serine at position 44, relative to the AAV9 VP1 sequence described in SEQ ID NO: 17. For example, a modified AAV9 VP1 sequence may include the following modified A26E, Q40R, K43D, and A44S relative to the sequence described in SEQ ID NO: 17. In one example, a modified AAV9 VP1 sequence includes the sequence described in SEQ ID NO: 27. In one example, the viral capsid protein contains mutations A67E, Q81R, K84D, and A85S from the full-length wild-type AAV serotype 9 capsid sequence described in SEQ ID NO: 19. In another example, the viral capsid protein contains the amino acid sequence described in SEQ ID NO: 28.

[0168] In one example, a therapeutic composition administered directly to the target pharyngeal muscle using a curved or bent injection needle (e.g., injection needle 10 described herein) includes AAV, which is, (a) A viral capsid protein derived from AAV9 containing a modified subunit 1 (VP1) sequence, wherein the amino acids at positions 1, 26, 40, 43, and 44 are modified compared to the corresponding wild-type AAV9 VP1 sequence described in Sequence ID No. 17, and (b) A “silence and substitution” construct comprising (i) a DNA-directed RNAi (ddRNAi) construct comprising a nucleic acid comprising a sequence encoding a short hairpin microRNA (shmiR), and (ii) a PABPN1 construct comprising a nucleic acid comprising a sequence encoding a functional PABPN1 protein having an mRNA transcript that is not targeted by the shmiR(s) encoded by the ddRNAi construct. For example, a modified AAV9 VP1 sequence may, relative to the AAV9 VP1 sequence described in SEQ ID NO: 17, include 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, a modified AAV9 VP1 sequence may, relative to the sequence described in SEQ ID NO: 17, include the following modified A1S, A26E, Q40R, K43D, and A44S. In one example, a modified AAV9 VP1 sequence includes the sequence described in SEQ ID NO: 18. In one example, the viral capsid protein contains mutations A42S, A67E, Q81R, K84D, and A85S from the full-length wild-type AAV serotype 9 capsid sequence described in SEQ ID NO: 19. In another example, the viral capsid protein contains the amino acid sequence described in SEQ ID NO: 20.

[0169] In another example, a therapeutic composition administered directly to the target pharyngeal muscle using a curved or bent injection needle (e.g., injection needle 10 described herein) includes AAV, which is, (a) A viral capsid protein derived from AAV8 containing a modified subunit 1 (VP1) sequence, wherein the amino acids at positions 1, 26, 40, 43, 44, and 64 are modified compared to the corresponding wild-type AAV8 VP1 sequence described in Sequence ID No. 21, and (b) A polynucleotide sequence comprising (i) a ddRNAi construct comprising a nucleic acid comprising a sequence encoding a shmiR, and (ii) a PABPN1 construct comprising a nucleic acid comprising a sequence encoding a functional PABPN1 protein having an mRNA transcript that is not targeted by the shmiR(s) encoded by the ddRNAi construct. For example, a modified AAV8 VP1 sequence may, with respect to the AAV8 VP1 sequence described in SEQ ID NO: 21, include 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, a modified AAV8 VP1 sequence may, with respect to the sequence described in SEQ ID NO: 21, include the following modified A1S, A26E, Q40R, K43D, A44S, and Q64K. In one example, a modified AAV8 VP1 sequence includes the sequence described in SEQ ID NO: 22. In one example, the viral capsid protein contains mutations A42S, A67E, Q81R, K84D, A85S, and Q105K from the full-length wild-type AAV serotype 8 capsid sequence described in SEQ ID NO: 23. In another example, the viral capsid protein contains the amino acid sequence described in SEQ ID NO: 24.

[0170] In each of the examples described above, the modified viral capsid protein is a polynucleotide delivery vector containing the ddRNAi construct and the PABPN1 construct. In one example, the polynucleotide sequence contains the ddRNAi construct and the PABPN1 construct in the 5' to 3' direction. In another example, the polynucleotide sequence contains the PABPN1 construct and the ddRNAi construct in the 5' to 3' direction.

[0171] The polynucleotide may further contain inverted end repeats (ITRs) derived from AAV serotypes. For example, an ITR may flank sequences containing ddRNAi and PABPN1 constructs on both sides. In some examples, the ITR is derived from the AAV2 serotype. For example, the ITR may be derived from the AAV2 serotype containing the 5'ITR sequence described in SEQ ID NO: 29 and the 3'ITR sequence described in SEQ ID NO: 30. For example, the ITR may be derived from the AAV2 serotype containing the 5'ITR sequence described in SEQ ID NO: 25 and the 3'ITR sequence described in SEQ ID NO: 26. However, other ITR sequence variants, including those derived from AAV2, are known in the art and are contemplated herein.

[0172] A therapeutic composition administered to a subject to treat or prevent dysphagia associated with OPMD according to the administration method of this disclosure comprises an AAV containing a “silence and replacement” construct comprising a PABPN1 construct. In this regard, the AAV provides an agent for replacing, for example, a functional PABPN1 protein in cells or animals. The functional PABPN1 protein is not the cause of OPMD, nor is it encoded by an mRNA transcript targeted by shmiR(plural) encoded by the ddRNAi construct described herein, which is also contained within the AAV.

[0173] For example, a PABPN1 construct may contain a nucleic acid, such as DNA or cDNA, that encodes a functional PABPN1 protein. For example, the nucleic acid encoding the functional PABPN1 protein may be codon-optimized and contain, for example, one or more degenerate or fluctuating bases relative to the wild-type PABPN1 nucleic acid, but encoding the same amino acids, thereby preventing the corresponding mRNA sequence encoding the functional PABPN1 protein from being recognized by the shmiR(s) encoded and expressed from the ddRNAi construct. For example, the codon-optimized nucleic acid encoding the functional PABPN1 protein may contain one or more degenerate or fluctuating bases relative to the wild-type PABPN1 nucleic acid in a region targeted by one or more shmiR(s) encoded and expressed from the ddRNAi construct. For example, one or more degenerate or fluctuating bases may be located within the seed region of the effector sequence of the shmiR(s) encoded and expressed from the ddRNAi construct.

[0174] In one example, a nucleic acid having a PABPN1 construct encoding a functional PABPN1 protein is codon-optimized such that its corresponding mRNA sequence is not recognized by shmiR(s) encoded and expressed from a ddRNAi construct. Preferably, the functional PABPN1 protein encoded by the codon-optimized nucleic acid sequence contains the amino acid sequence described in SEQ ID NO: 16, i.e., the amino acid sequence of the wild-type human PABPN1 protein. Those skilled in the art will recognize that there are several combinations of nucleotide sequences that can be used to encode a functional PABPN1 protein, and that the selection of the nucleotide sequence ultimately depends on the effector sequence of shmiR(s) encoded and expressed from a ddRNAi construct, i.e., on the codon-optimized nucleic acid not being recognized by shmiR(s). In one example, the PABPN1 construct contains a nucleic acid containing the sequence described in SEQ ID NO: 15. In one example, the nucleic acid encoding the functional PABPN1 protein may also contain a Kozak sequence.

[0175] In one example, a codon-optimized nucleic acid encoding a functional PABPN1 protein is operably ligated to a promoter suitable for the expression of the functional PABPN1 protein. A promoter suitable for the expression of the functional PABPN1 protein in muscle may be particularly preferred. One exemplary promoter suitable for use with a nucleic acid encoding a functional PABPN1 protein is the Spc512 promoter. Another exemplary promoter suitable for use with a nucleic acid encoding a functional PABPN1 protein is the CK8 promoter. However, any suitable promoter known in the art may be used.

[0176] In one example, the PABPN1 construct and the ddRNAi construct are operably ligated to the same promoter within the same polynucleotide, for example, both being operably ligated to the Spc512 promoter. In this example, a single promoter drives the expression of both the functional PABPN1 protein and shmiR.

[0177] A therapeutic composition administered to a subject to treat or prevent dysphagia associated with OPMD according to the administration method of this disclosure comprises an AAV containing a “silence and substitution” construct which itself contains a DNA-directed RNAi (ddRNAi) construct. The ddRNAi construct contains a DNA sequence encoding one or more short hairpin microRNAs (shmiRs) that target the mRNA transcript of endogenous PABPN1.

[0178] The shmiR encoded by the ddRNAi construct, or each shmiR, An effector sequence with a length of at least 17 nucleotides, Complementary arrangement of effectors, Stem-loop array, Primary microRNA (pri-miRNA) backbone, Here, the effector sequence is substantially complementary to a region of the corresponding length in the RNA transcript described in SEQ ID NO: 1 or 2. Preferably, the effector sequence or each effector sequence is less than 30 nucleotides in length. For example, a preferred effector sequence may be in the range of 17 to 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 effector complementary sequence is 20 nucleotides in length.

[0179] For example, the shmiR encoded by the ddRNAi construct contains the sequence described in SEQ ID NO: 1, or an effector sequence substantially complementary to a region of the corresponding length in an RNA transcript consisting of the sequence described in SEQ ID NO: 1. In this example, the shmiR is also referred to herein as "shmiR13". For example, the effector sequence may contain the sequence described in SEQ ID NO: 1, or be substantially complementary to a region of the corresponding length in an RNA transcript consisting of the sequence described in SEQ ID NO: 1, and contain four mismatched bases. For example, the effector sequence may contain the sequence described in SEQ ID NO: 1, or be substantially complementary to a region of the corresponding length in an RNA transcript consisting of the sequence described in SEQ ID NO: 1, and contain three mismatched bases. For example, the effector sequence may contain the sequence described in SEQ ID NO: 1, or be substantially complementary to a region of the corresponding length in an RNA transcript consisting of the sequence described in SEQ ID NO: 1, and contain two mismatched bases. For example, the effector sequence may contain the sequence described in SEQ ID NO: 1, or be substantially complementary to a region of the corresponding length in an RNA transcript consisting of the sequence described in SEQ ID NO: 1, and contain one mismatched base. For example, the effector sequence may be 100% complementary to a region of the corresponding length in an RNA transcript that contains or consists of the sequence described in Sequence ID No. 1.

[0180] For example, the shmiR encoded by the ddRNAi construct contains the sequence described in SEQ ID NO: 2, or an effector sequence substantially complementary to a region of the corresponding length in an RNA transcript consisting of the sequence described in SEQ ID NO: 2. In this example, the shmiR is also referred to herein as "shmiR17". For example, the effector sequence may contain the sequence described in SEQ ID NO: 2, or be substantially complementary to a region of the corresponding length in an RNA transcript consisting of the sequence described in SEQ ID NO: 2, and contain four mismatched bases. For example, the effector sequence may contain the sequence described in SEQ ID NO: 2, or be substantially complementary to a region of the corresponding length in an RNA transcript consisting of the sequence described in SEQ ID NO: 2, and contain three mismatched bases. For example, the effector sequence may contain the sequence described in SEQ ID NO: 2, or be substantially complementary to a region of the corresponding length in an RNA transcript consisting of the sequence described in SEQ ID NO: 2, and contain two mismatched bases. For example, the effector sequence may contain the sequence described in SEQ ID NO: 2, or be substantially complementary to a region of the corresponding length in an RNA transcript consisting of the sequence described in SEQ ID NO: 2, and contain one mismatched base. For example, the effector sequence may be 100% complementary to a region of the corresponding length in an RNA transcript that contains or consists of the sequence described in Sequence ID No. 2.

[0181] In cases where the effector sequence of shmiR is substantially complementary to a region of corresponding length in the PABPN1 miRNA transcript described herein and contains one, two, three, or four mismatched bases, it is preferable that the mismatch(s) are not located within the seed region of shmiR, i.e., the region corresponding to nucleotides 2-8 of the effector sequence.

[0182] In one example, a ddRNAi construct may include a DNA sequence encoding a shmiR, which includes (i) an effector sequence substantially complementary to the sequence described in SEQ ID NO: 3, except for one, two, three, or four base mismatches, provided that the effector sequence can form a duplex with the sequence described in SEQ ID NO: 3, and (ii) an effector complementary sequence containing a sequence substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 4, and an effector complementary sequence that is substantially complementary to the sequence described in SEQ ID NO: 4 and can form a duplex with it. The effector complementary sequence substantially complementary to the sequence described in SEQ ID NO: 4 may be the sequence described in SEQ ID NO: 3. The shmiR in this embodiment is hereafter designated as "shmiR13".

[0183] In one example, a ddRNAi construct may include a DNA sequence encoding a shmiR, which includes (i) an effector sequence substantially complementary to the sequence described in SEQ ID NO: 5, except for one, two, three, or four base mismatches, provided that the effector sequence can form a duplex with the sequence described in SEQ ID NO: 5, and (ii) an effector complementary sequence containing a sequence substantially complementary to the effector sequence. For example, a shmiR encoded by a ddRNAi construct may include an effector sequence described in SEQ ID NO: 6, and an effector complementary sequence that is substantially complementary to the sequence described in SEQ ID NO: 6 and can form a duplex with it. The effector complementary sequence substantially complementary to the sequence described in SEQ ID NO: 6 may be the sequence described in SEQ ID NO: 5. The shmiR in this embodiment is hereafter designated as "shmiR17".

[0184] For example, at least one shmiR encoded by the ddRNAi construct is shmiR including the effector sequence described in Sequence ID 4 and the complementary effector sequence described in Sequence ID 3, or shmiR includes the effector sequence described in sequence number 6 and the complementary effector sequence described in sequence number 5.

[0185] In one particular example, a ddRNAi construct encodes a shmiR containing the effector sequence described in SEQ ID NO: 4 and the effector complementary sequence described in SEQ ID NO: 3, and a shmiR containing the effector sequence described in SEQ ID NO: 6 and the effector complementary sequence described in SEQ ID NO: 5. For example, a ddRNAi construct may encode a shmiR designated as shmiR13 as described herein, and a shmiR designated as shmiR17 as described herein.

[0186] For example, shmiR or each shmiR includes the following in the 5' to 3' direction: 5' flanking sequence of the pri-miRNA backbone, Complementary arrangement of effectors, Stem-loop array, Effector arrangement, and 3' flanking sequence of the pri-miRNA backbone.

[0187] In another embodiment, shmiR or each shmiR includes the following in the 5' to 3' direction: 5' flanking sequence of the pri-miRNA backbone, Effector arrangement, Stem-loop array, Complementary arrangement of effectors, and 3' flanking sequence of the pri-miRNA backbone.

[0188] A suitable loop sequence may be selected from those known in the art. However, an exemplary stem-loop sequence is described in Sequence ID No. 7.

[0189] For example, the pri-miRNA backbone is the pri-miR-30a backbone. For instance, the 5' flanking sequence of the pri-miRNA backbone could be the sequence described in SEQ ID NO: 8, and the 3' flanking sequence of the pri-miRNA backbone could be the sequence described in SEQ ID NO: 9.

[0190] For example, a ddRNAi construct contains at least two nucleic acids, each encoding a shmiR, and each shmiR contains an effector sequence substantially complementary to the RNA transcript corresponding to the PABPN1 protein that causes OPMD, and each shmiR contains a different effector sequence.

[0191] For example, a ddRNAi construct encodes a shmiR containing an effector sequence substantially complementary to a region of the corresponding length in the RNA transcript described in SEQ ID NO: 1, and a shmiR containing an effector sequence substantially complementary to a region of the corresponding length in the RNA transcript described in SEQ ID NO: 2. For example, a ddRNAi construct may include: A nucleic acid comprising or consisting of a DNA sequence encoding shmiR(shmiR13) including the effector sequence described in SEQ ID NO: 4 and the effector complementary sequence described in SEQ ID NO: 3, and A nucleic acid comprising, or consisting thereof, a DNA sequence encoding shmiR (shmiR17) which includes the effector sequence described in SEQ ID NO: 6 and the effector complementary sequence described in SEQ ID NO: 5.

[0192] An exemplary ddRNAi construct of this disclosure includes a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 12 (for example, encoding shmiR13 comprising or consisting of the sequence described in SEQ ID NO: 10), and a nucleic acid comprising or consisting of the DNA sequence described in SEQ ID NO: 13 (for example, encoding shmiR17 comprising or consisting of the sequence described in SEQ ID NO: 11).

[0193] In any of the embodiments described above, the ddRNAi construct and the PABPN1 construct may be operably ligated to a promoter. In one example, the ddRNAi construct and the PABPN1 construct are operably ligated to the same promoter, for example, a muscle-specific promoter. The muscle-specific promoter may be the Spc512 or CK8 promoter. However, other muscle-specific promoters are known in the art and are intended to be used in conjunction with the ddRNAi construct of this disclosure.

[0194] As described herein, therapeutic compositions administered to subjects to treat or prevent dysphagia associated with OPMD may comprise a single polynucleotide (or “silence and substitution” construct) containing the ddRNAi construct and the PABPN1 construct as described herein, packaged within an AAV vector for delivery. That is, the ddRNAi construct and the PABPN1 construct may be provided as a combined DNA construct (also referred herein as the “silence and substitution” construct or SR construct), packaged in a modified AAV as described herein for delivery to a patient according to the administration method described herein. An example of a “silence and substitution” construct is described herein.

[0195] The “silence and replacement” construct may be packaged in a modified AAV as described herein for delivery to a patient.

[0196] In one example, the “silence and substitution” construct comprises, oriented from 5' to 3', a muscle-specific promoter, e.g., the Spc512 promoter described herein, a PABPN1 construct, and a ddRNAi construct described herein, the ddRNAi construct positioned in the 3' untranslated region (UTR) of the nucleic acid encoding the functional PABPN1 protein. The “silence and substitution” construct according to this example is illustrated in Figure 9A.

[0197] The exemplary “silence and replacement” construct in this example includes, in the 5' to 3' direction: (a) Muscle-specific promoter, e.g., Spc512, (b) PABPN1 constructs described herein, comprising a DNA sequence encoding a functional PABPN1 protein having an mRNA transcript that is not targeted by shmiR encoded by the ddRNAi construct, and (c) A ddRNAi construct of the present disclosure comprising a nucleic acid comprising a DNA sequence encoding shmiR17 as described herein, and a nucleic acid comprising a DNA sequence encoding shmiR13 as described herein. In this example, the “silence and substitution” construct comprises or comprises the DNA sequence described in SEQ ID NO: 14. The “silence and substitution” construct according to this example is illustrated in Figure 9B.

[0198] Exemplary ddRNAi constructs encoding shmiR13 and shmiR17 for inclusion in the “Silence and Substitution” constructs of this disclosure include a nucleic acid comprising or comprising a DNA sequence encoding an effector complementary sequence substantially complementary to the effector complementary sequence described in SEQ ID NO: 4, and a DNA sequence encoding an effector complementary sequence substantially complementary to the sequence described in SEQ ID NO: 3, and a nucleic acid comprising a DNA sequence encoding an effector complementary sequence substantially complementary to the sequence described in SEQ ID NO: 6, such as a nucleic acid comprising or comprising the effector complementary sequence described in SEQ ID NO: 5. For example, a ddRNAi construct according to this example of the “silence and substitution” construct may include a nucleic acid containing or comprising the DNA sequence described in SEQ ID NO: 12 (e.g., encoding shmiR13 containing or comprising the sequence described in SEQ ID NO: 10), and a nucleic acid containing or comprising the DNA sequence described in SEQ ID NO: 13 (e.g., encoding shmiR17 containing or comprising the sequence described in SEQ ID NO: 11).

[0199] An exemplary PABPN1 construct for inclusion in the “silence and substitution” constructs of this disclosure comprises the codon-optimized sequence described in SEQ ID NO: 15 and encodes the functional PABPN1 protein described in SEQ ID NO: 16.

[0200] Those skilled in the art will recognize that therapeutic compositions are formulated for delivery to patients, for example, human patients. Therefore, a therapeutic composition for administration to a subject according to the method of this disclosure may comprise one or more pharmaceutically acceptable carriers or diluents. For example, a therapeutic composition may comprise a carrier suitable for delivering the AAV of this disclosure to the muscle of a subject following injection. Suitable carriers for the formulation and delivery of AAV are known in the art and are contemplated herein. Suitable pharmaceutical carriers, as well as pharmaceutical essentials for use in pharmaceutical formulations, are described in Remington: The Science and Practice of Pharmacy (formerly Remington's Pharmaceutical Sciences), Mack Publishing Co., standard reference text in the art, and USP / NF).

[0201] The volume, concentration, and formulation of therapeutic compositions for administration to a subject according to the methods of this disclosure, as well as the administration regimens, may be specifically adjusted to maximize cell delivery while minimizing toxicity, such as inflammatory responses, using corresponding low concentrations of active agents (e.g., AAV vectors containing “silence and substitution” constructs for the treatment of OPMD). Anti-inflammatory compounds, such as corticosteroids, may also be administered (e.g., orally) for systemic distribution.

[0202] For example, a therapeutic composition for the treatment of dysphagia associated with OPMD is administered in a volume ranging from approximately 25 μL to approximately 75 μL per injection site. For instance, the therapeutic composition may be administered in a volume of approximately 25 μL, or approximately 30 μL, or approximately 35 μL, or approximately 40 μL, or approximately 45 μL, or approximately 50 μL, or approximately 55 μL, or approximately 60 μL, or approximately 65 μL, or approximately 70 μL, or approximately 75 μL per injection site.

[0203] In another embodiment, the therapeutic composition may be administered in a volume ranging from approximately 50 μL to approximately 75 μL per injection site. For example, the therapeutic composition may be administered in a volume of approximately 50 μL, or approximately 55 μL, or approximately 60 μL, or approximately 65 μL, or approximately 70 μL, or approximately 75 μL per injection site.

[0204] The volume of the therapeutic composition administered per injection site may be the volume of a single injection or the cumulative volume of multiple injections administered at one injection site. For example, an injection volume of approximately 75 μL per injection site may be administered as three injections of 25 μL each, or as one injection of 25 μL and one injection of 50 μL.

[0205] In each of the above examples describing the administration of a therapeutic composition at multiple injection sites, the total dose administered to the subject in a single treatment is approximately 1.5E. +12 vg~about 9.5E +13 It may be vg. For example, the total dose of the therapeutic composition administered to the subject is approximately 5.0E. +12 vg~about 6.0E +13 It may be vg. For example, the total dose of the therapeutic composition administered to the subject is approximately 1.0E. +13 vg~approx.5.5E +13 It may be vg. In some examples, the total dose of the therapeutic composition administered is about 1.0E. +13vg / target. In some examples, the total dose of the therapeutic composition administered is approximately 1.2E. +12 The value is vg / target. In some examples, the total dose of the therapeutic composition is approximately 3.6E. +13 The target was administered vg / target. In some cases, the total dose of the therapeutic composition administered was approximately 5.4E. +13 vg / is the target.

[0206] Therefore, when the therapeutic composition is administered in a volume ranging from about 25 μL to about 75 μL per injection site, the therapeutic composition may be administered at a concentration of about 1.0 + E12 vg / mL to about 1.0 + E15 vg / mL. For example, the concentration of the therapeutic composition administered to the subject may be about 5.0 + E12 vg / mL to about 1.0 + E14 vg / mL, for example, about 5.0 + E12 vg / mL to 5.0 + E13 vg / mL.

[0207] As described herein, it is preferable to administer the therapeutic composition multiple times to one or more muscles within the pharyngeal muscle group, which includes injecting the therapeutic composition into multiple sites within the pharyngeal muscle group and / or administering the therapeutic composition to one or more locations in the pharyngeal muscles, one or more pharyngeal muscles, one or more sides of the pharyngeal muscles (e.g., right or left), or any combination thereof. Injecting at multiple sites may help improve delivery to the pharyngeal muscle tissue. For example, the method may include injecting the therapeutic composition into about 2 to about 30 or more sites within the pharyngeal muscles. In an example where multiple injection sites are divided between the left and right sides of the median raphe of the pharyngeal muscles, the method may involve injecting a therapeutic composition into approximately 1 to approximately 15 or more sites within the muscles to the left of the median raphe of the pharyngeal muscles (e.g., 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 or more sites), and approximately 1 to approximately 15 or more sites within the muscles to the right of the median raphe of the pharyngeal muscles (e.g., 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 or more sites). For example, the method may involve injecting a therapeutic composition into approximately 1 to 15 or more sites on the left side of the median raphe of the thyropharyngeal (TP) muscle (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more sites) and approximately 1 to 15 or more sites within the muscle on the right side of the median raphe of the thyropharyngeal (TP) muscle (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more sites).Alternatively, or in addition, this method may involve injecting the therapeutic composition into approximately 1 to approximately 15 or more sites (e.g., approximately 4 sites) on the left side of the median raphe of the hypopharyngeal (HP) muscle, and approximately 1 to approximately 15 or more sites (e.g., approximately 4 sites) within the muscle on the right side of the median raphe of the hypopharyngeal (HP) muscle. According to one particular example in which the therapeutic composition is administered to the HP and TP muscles, the composition may be injected into 4 sites on the left side of the median raphe of the HP muscle, 4 sites on the right side of the median raphe of the HP muscle, 8 sites on the left side of the intermediate groove of the TP muscle, and 8 sites on the right side of the median raphe of the TP muscle.

[0208] The method may also involve multiple injections of the therapeutic composition into more than one pharyngeal muscle. Different pharyngeal muscles are described and intended herein. However, in one example, the therapeutic composition may be injected into the oropharyngopharyngeal constrictor muscle at multiple sites, and into the inferior pharyngeal constrictor muscle at multiple sites. Direct injection may also be applied to multiple sites of other pharyngeal muscles, such as the thyropharyngeal or cricopharyngeal muscles.

[0209] As described herein, the therapeutic composition may be administered to the pharyngeal muscles by direct injection (i.e., intramuscular injection) following an incision in the neck of the subject sufficient to obtain direct access to at least a portion of the pharyngeal muscles of the subject. Thus, a method for treating dysphagia associated with OPMD in a subject suffering from OPMD may include making one or more incisions (e.g., skin) in the neck of the subject to provide direct access to the pharyngeal muscles (or more) of the subject, and then administering the therapeutic composition described herein by direct intramuscular injection into the pharyngeal muscles using a bent or curved needle (e.g., needle 10 described herein). For example, an incision may be made on the right side of the pharyngeal area on the right side of the body of a human or animal, and the area may be sufficiently dissected to access at least one or more of the pharyngeal muscles on the right side. The needle cannula may then be inserted directly into one or more of the pharyngeal muscles on the right side. Alternatively or additionally, an incision may be made on the left side of the pharyngeal area on the left side of the body of a human or animal, and the area may be sufficiently dissected to access one or more of the pharyngeal muscles on the left side. Next, the needle cannula can be directly inserted into one or more of the pharyngeal muscles on the left side. [Examples]

[0210] Example 1: Staining study In this study, the inventors evaluated suitable delivery devices for the delivery of the applicant's OPMD “Silence and Replacement” therapeutic agent “BB-301” without observable leakage of the therapeutic composition by (1) the maximum volume that could be injected into the hypopharynx and thyropharyngeal muscles of a beagle dog using trypan blue (Gibco #15250-061) without observable leakage of the injected material, and (2) different injection needles. Beagle dogs were selected as an animal model because of their muscle and muscle size being similar to that of humans.

[0211] animal This study, conducted in June 2020 at the Boisbonne Centre (large animal facility), included two female beagles, Rosette and Rillette, aged 4 years and 10 months and 4 years and 9 months, respectively. Both animals were slaughtered upon completion of the study.

[0212] needle This study evaluated two different injection needles, "Needle A" and "Needle B," for their ability to administer trypan blue to the hypopharynx, thyropharynx, and cricopharyngeal muscles of animals, and for their ability to influence the delivery of the intended volume of trypan blue without observing post-injection leakage of trypan blue from the injected muscle.

[0213] Needle A is constructed in general accordance with the injection needle 10 described herein and includes a 3-Dose Next Generation BoNT administration syringe (Cat No. 3DOSE-GREEN-125) from VlowMedical, to which a 27G, 19mm needle is attached, which has a curved, arched middle section, so that the proximal and distal portions of the needle extend at a 90-degree angle to each other.

[0214] Needle B also contained a 3-Dose Next Generation BoNT administration syringe from VlowMedical (Cat No. 3DOSE-GREEN-125), but was generally coupled with a 25G, 19-22mm nuclear hydrolysis needle from Accuspire (Cat No. J075-25G), as shown in Figures 7A and 7B.

[0215] Figure 7A shows an injection needle 400 configured in general terms to needle B, which includes a needle cannula 420 and a hub 430. As shown in Figure 7A, the needle cannula 420 has a substantially straight portion proximal to the hub (proximal portion 421), while the intermediate portion 422 and distal portion 423 are curved. It can be seen that the radius of curvature is large and the angle of curvature θ between the proximal portion 421 and the distal portion 423 is approximately 160°. Figure 7B shows that the distal portion 423 of the cannula 420 has a flat, elliptical tip 427.

[0216] Surgery and dye injections "Rillette" Surgical procedures were performed under general anesthesia with appropriate analgesics, and all surgical procedures were carried out by a veterinarian. A single incision was made at the right level of the pharyngeal area, and the area was dissected to provide access to the HP and TP muscle sections to the right of the median raphe. Once the HP and TP muscle sections to the right of the median raphe were exposed, trypan blue dye was administered at a rate of 75 μL per injection via multiple direct intramuscular injections (4, 5, or 9 separate injection sites) using a 3-Dose Unit Injector device (VlowMedical, Netherlands), with the amount of each injection controlled. The dye was aspirated into a microinjection syringe using a BD 21G, 2-inch needle (catalog no. 301155), and injected using a Hamilton 90-degree angled 27G, 19 mm needle (p / n 10101008, N(27 / 19 / 4x2 / A90)). All injections were performed using a surgical microscope.

[0217] Table 1 below outlines the injections of trypan blue dye into the HP and TP muscle sections on the right side of the median raphe, as well as within the cricopharyngeal (CP) muscle.

[0218] After dye administration, muscle sections were visually inspected for dye leakage.

[0219] Approximately 5 minutes after the injection, the surgical incision was closed with staples, and the dog was inverted to access the left side of the pharyngeal area. A single incision was then made at the left level of the pharyngeal area, and the area was dissected to provide access to the HP and TP muscle sections to the left of the median raphe. Once the HP and TP muscles to the left of the median raphe were exposed, trypan blue dye was administered by multiple direct intramuscular injections (25 μL, 50 μL, or 75 μL per injection for 1, 2, or 4 separate injection sites) using a 3-Dose Unit Injector device (VlowMedical, Netherlands), with the amount of each injection controlled. The dye was drawn into a microinjection syringe (catalog no. 301155) using a 21G, 2-inch needle from BD, and injected using a 90-degree angled 27G, 19mm needle (p / n 10101008, N(27 / 19 / 4x2 / A90)) from Hamilton or a 25G, 22mm nuclear hydrolysis needle (catalog no. J075-25G) from Accuspire. All injections were performed using a surgical microscope.

[0220] A summary of trypan blue dye injections performed in the median raphe and the left HP and TP muscle sections of the CP muscle is presented in Table 1 below.

[0221] After dye administration, muscle sections were visually inspected for dye leakage.

[0222] Approximately 5 minutes after the injection, the surgical incision was closed with staples, and whole blood samples were obtained on EDTA-coated tubes (20 mL total divided into 2 × 10 mL portions and frozen at below -70°C).

[0223] Subsequently, the dogs were euthanized by lethal intravenous injection of pentobarbital at a dose of 1 mL / kg (180 mg / kg). [Table 1]

[0224] After the animals were slaughtered, the entire pharyngeal area (including the trachea, esophagus, and tongue) was dissected.

[0225] The HP and TP muscles, CP muscle, and the left and right sections of the lateral extensor digitorum muscle were precisely dissected, measured, and weighed (length, width, and depth). Each muscle was then placed flat in a histological cassette, directly frozen in liquid nitrogen, and stored at below -70°C. It was not possible to mark the left and right sections of the muscles. The characteristics of each muscle sample are summarized in Table 3 below.

[0226] Additionally, liver samples were collected, cut into small pieces, and placed in a total of 15 DNAse-free, RNAse-free, and PCR-clean microtubes. These were then frozen directly in liquid nitrogen and stored at temperatures below -70°C.

[0227] The following observations were made.

[0228] Except for the right-hand section of the TP, which was only partially stained, the muscle sections of the TP, HP, and CP were fully stained with trypan blue dye.

[0229] After evaluating the entire pharyngeal muscle area, residual dye staining was also observed at the cartilage and esophageal wall levels. These observations can be explained by the larger volume of trypan blue dye injected into the rillette compared to the volume administered to the rosette, as well as the increased level of dye leakage observed in the rillette compared to that recorded in the rosette.

[0230] "rosette" The surgical procedure was performed under general anesthesia with appropriate analgesics, and all surgical procedures were carried out by a veterinarian.

[0231] A single incision was made at the left level of the pharyngeal area, and the area was dissected to provide access to the sections of the hypopharyngeal (HP) and thyropharyngeal (TP) muscles to the left of the median raphe.

[0232] When the HP and TP muscle sections on the left side of the midline suture were exposed, trypan blue dye was administered at 50 μL per injection via multiple direct intramuscular injections (4 or 8 separate injection sites) using a 3 Dose Unit Injector device (Vlow Medical, Netherlands) to control the amount of each injection. A 21G, 2-inch needle (catalog number 301155) from BD was used to aspirate the dye into a micro syringe, and an intramuscular injection was performed using a 27G, 19 mm needle angled at 90 degrees from Hamilton (p / n 10101008, N(27 / 19 / 4x2 / A90)). All injections were performed using a surgical microscope.

[0233] A summary of the trypan blue dye injections performed within the HP and TP muscle sections on the left side of the midline suture is presented in Table 2 below.

[0234] After dye administration, the muscle sections were evaluated by visual inspection for dye leakage.

[0235] Approximately 5 minutes after the injection, the surgical incision was closed with staples, and the dog was reversed to access the right side of the pharyngeal area.

[0236] Next, a single incision was made at the level on the right side of the pharyngeal area, and the area was dissected to provide access to the HP muscle and TP muscle sections on the right side of the midline suture.

[0237] When the HP and TP muscle sections on the right side of the midline suture were exposed, trypan blue dye was administered by multiple direct intramuscular injections (50 μL or 100 μL per injection for 2, 3, or 4 separate injection sites) using a 3 Dose Unit Injector device (VlowMedical, Netherlands) to control the volume of each injection. A BD 21G, 2-inch needle (catalog number 301155) was used to aspirate the dye into a micro-injection syringe, and an injection was performed using a 27G, 19 mm needle angled at 90 degrees from Hamilton (p / n 10101008, N(27 / 19 / 4x2 / A90)). All injections were performed using a surgical microscope.

[0238] A summary of the trypan blue dye injections performed in the HP and TP muscle sections on the right side of the midline suture, as well as in the circular pharyngeal (CP) muscle, is presented in Table 2 below.

[0239] After dye administration, the muscle sections were evaluated by visual inspection for dye leakage.

[0240] Approximately 5 minutes after injection, the surgical incision was closed with staples and whole blood samples were obtained on EDTA-coated tubes (a total of 24 mL was aliquoted into 2 × 10 mL + 1 × 4 mL and frozen at less than -70 °C).

[0241] Subsequently, the dogs were euthanized by a lethal intravenous injection of pentobarbital at a dose of 1 mL / kg (180 mg / kg).

[0242] After animal euthanasia, the entire pharyngeal area (including the trachea and esophagus + tongue) was dissected.

[0243] The left and right sections of the HP and TP muscles, as well as the extensor digitorum longus muscle, were carefully dissected, measured, and weighed (length, width, depth). Then, each muscle was placed flat in a histological cassette and directly frozen in liquid nitrogen and then stored at less than -70 °C. Note that the left and right sections of the muscle could not be marked. The characteristics of each muscle sample are summarized in Table 4 below.

[0244] Additionally, liver samples were collected, cut into small pieces, and placed in a total of 15 DNAse-free, RNAse-free, and PCR-clean microtubes. These were then frozen directly in liquid nitrogen and stored at temperatures below -70°C.

[0245] The following observations were made.

[0246] The entire HP and TP muscle sections were completely stained with trypan blue dye.

[0247] The left CP muscle section was not stained, indicating no leakage between different muscles in the pharyngeal anatomical region, and that the diffusion of the dye was limited to the injected pharyngeal muscle.

[0248] After evaluating the muscles of the pharyngeal area as a whole, residual dye staining was observed only at the cartilage level. No dye staining was observed at the levels of other tissues, including the esophagus and trachea. [Table 2] [Table 3] [Table 4]

[0249] conclusion The following information was obtained from this dye study conducted on two beagle dogs. No problems arose during the surgical approach to and dissection of the pharyngeal region. The bilateral surgical approach (left and right sides of the pharyngeal region) is ideally suited for use in the administration protocol to facilitate better visualization of separate intramuscular injections into each section of the target pharyngeal muscle. ○As expected, the pharyngeal muscle of interest is small and very thin (approximately 4-5 cm). 2 (2-3 mm deep). ○VlowMedical microinjection syringes are ideally suited to the administration process and are very easy to use. ○It is clear that needle A is the most ideally suited for the injection process. In contrast, needle B is not sufficiently angled or inclined. As a result, significant leakage from the puncture site was observed even when only 25 μL of trypan blue dye was injected. ○The maximum fluid volume that can be administered per injection site without causing significant leakage is in the range of 50 μL to 75 μL. To limit the risk of leakage while removing the injection needle from the target muscle, it is recommended to pause for approximately 30 seconds at the end of each injection before withdrawing the needle from the muscle. ○ No dye leakage was observed when 50 μL of trypan blue was injected, so the injection volume of 25 μL of dye was not evaluated. Significant dye leakage was observed when 100 μL of trypan blue was injected, so the injection volume of 150 μL of dye was not evaluated. However, an intermediate dose of 75 μL of trypan blue dye was evaluated. No significant leakage was observed with this intermediate dose. ○The appropriate number of total injection sites in the TP muscle section located to the left or right of the median raphe is 8 injection sites (16 injection sites in total for the entire TP muscle), and the appropriate number of total injection sites in the HP muscle section located to the left or right of the median raphe is 4 injections (8 injection sites in total for the entire muscle). Due to variability in muscle size among dogs, the appropriate number of total injection sites may potentially increase for some animals. ○The cricopharyngeal muscle was also accessible via a surgical approach. ○Observation of trypan blue dye diffusion for 30 minutes post-injection was impossible because the animal subjects had to be inverted to access the contralateral pharyngeal muscles for dye injection. However, after the sacrifice of the animal subjects and complete dissection of the pharyngeal area, the dye diffusion pattern was observed. Dye diffusion in the injected muscle was almost complete, and the dye diffusion was essentially confined to the injected muscle.

[0250] Example 2: Pilot Administration Study of the "Silence and Replace" Construct BB-301 BB-301 is the first gene medicine of its class to adopt a "Silence and Replace" approach for the treatment of dysphagia associated with OPMD. Specifically, BB-301 relies on the applicant's proprietary DNA-directed RNA interference (ddRNAi) platform, which combines RNA interference (RNAi) with classical AAV-based gene therapy. The applicant's ddRNAi platform also enables the co-delivery of wild-type replacement genes, and these distinct gene elements cooperate to silence the expression of mutant genes that cause disease via RNAi and simultaneously replace the mutant genes with normal (wild-type) genes to restore the original underlying physiological functions of diseased tissues.

[0251] BB-301 and its components have been previously described by the applicant in WO2017 / 177277, WO2018 / 107228, WO2019 / 043630, WO2020 / 077412, and WO2020 / 172720, and the content of each of them is incorporated herein in its entirety. BB-301 is also described herein.

[0252] BB-301 is injected directly into the pharyngeal muscles, which are known to underlie the morbidity and mortality rates that characterize the natural course of dysphagia associated with OPMD. This pilot administration study of BB-301 was conducted in large animal subjects, specifically beagle dogs, and aimed to show that direct intramuscular injection of BB-301 using the injection needle 10 described herein could safely achieve the following in an open surgical procedure. ● A biologically significant, highly consistent, dose-dependent level of BB-301 tissue transduction (i.e., delivery of a multifunctional gene construct to target pharyngeal muscle cells), ● Durable, broad, dose-dependent expression of three different genes within pharyngeal muscle cells containing the BB-301 gene construct, and ● A sustained and biologically significant level of target gene knockdown (i.e., inhibition of the expression of the gene of interest) within pharyngeal muscle cells.

[0253] BB-301 BB-301 incorporates Benitec's proprietary AAV9 delivery vector, which includes (i) a ddRNAi construct containing two nucleic acids encoding short hairpin microRNAs (shmiRs) designated shmiR13 and shmiR17 (as described herein) that target the mRNA transcript of mutant PABPN1, which is the cause of OPMD, and (ii) a PABPN1 construct containing a codon-optimized nucleic acid encoding a functional (wild-type) PABPN1 protein, wherein the mRNA corresponding to the codon-optimized nucleic acid is not targeted by shmiR. Two different siRNA species (i.e., siRNA13 and siRNA17) are treated from shmiR, and each of them can independently inhibit (i.e., "silence") the expression of all forms of the endogenous PABPN1 protein (siRNA13 and siRNA17 silence the expression of both wild-type PABPN1 [wtPABPN1] and mutant PABPN1, which is the cause of OPMD). Mutant forms of the PABPN1 protein underlie the development and progression of OPMD. In contrast, the codon-optimized sequence within BB-301 encoding the functional wild-type version of the PABPN1 protein is unaffected by the inhibitory activity of siRNA13 and siRNA17. This codon-optimized PABPN1 protein (i.e., coPABPN1) plays a role in supplementing the endogenous form of the PABPN1 protein and replacing the mutant form of PABPN1 that underlies the development and progression of OPMD in diseased tissue.

[0254] Pilot administration study of BB-301 A pilot dose study was conducted to evaluate the safety and biological activity of two concentrations of BB-301 across three different doses, and to optimize the administration method of BB-301 developed and described herein.

[0255] In short, two different concentrations (1.0+E13vg / mL and 3.0+E13vg / mL) across three different doses (1.0+E13vg / mL, 3.0+E13vg / mL at a low injection volume, and 3.0+E13vg / mL at a high injection volume) were tested via direct intramuscular injection into the hypopharyngeal (HP) and thyropharyngeal (TP) muscles of beagle dogs using the injection needle 10 described herein via an open surgical procedure. As described in Example 1, the HP muscle in beagle dogs corresponds to the oropharyngeal constrictor muscle in human subjects, and the TP muscle in beagle dogs corresponds to the hypopharyngeal constrictor muscle in human subjects. Beagle dogs were also selected as a large animal model due to their muscle size being similar to that of humans. The surgical procedure used to administer BB-301 to the beagle was as described above in Example 1, using needle A (which is the injection needle 10 described herein).

[0256] BB-301 was injected only on day 1 of the pilot administration study, and the corresponding pharyngeal muscles of dogs were collected for analysis 8 weeks after delivery of BB-301.

[0257] BB-301 administration was performed by both veterinarians and otolaryngologists with extensive experience in providing palliative surgical care to OPMD patients.

[0258] Intermediate data points were collected from the completed analysis of pharyngeal muscle tissue isolated from 16 beagle dogs (out of a study group of 24 dogs).

[0259] It is important to highlight the key methodological differences between the current BB-301 pilot administration study in beagle dogs conducted by Benitec and previous beagle dog administration studies independently performed by previous BB-301 licensees. Specifically, the BB-301 administration studies conducted by previous BB-301 licensees employed non-ideal administration routes and methods to target pharyngeal muscle tissue and similarly limited analytical methods at the completion of the administration phase of the studies. In this study, we worked to optimize the route and method of administration of BB-301 to the pharyngeal muscle (as described in Example 1) and to refine the core analytical methods used after the completion of administration.

[0260] result The results of the pilot study are shown in Table 5 below. [Table 5]

[0261] As shown in Table 5, administration of BB-301 to the pharyngeal muscles using open surgical procedures and injection needle 10 as described herein achieved good transduction to the pharyngeal muscles across all evaluated doses and concentrations. Of particular note is the 248-fold improvement (+24,650%) in BB-301 transduction to HP muscles and the 111-fold improvement (+11,027%) in BB-301 transduction to TP muscles compared to the levels of BB-301 transduction observed in previous BB-301 preliminary studies conducted by previous licensees of BB-301.

[0262] Overall, these data support the conclusion that the open surgical procedure and injection needle 10 described herein are effective for the delivery of BB-301 (or any therapeutic agent) to the pharyngeal muscle and demonstrate a biologically significant, highly consistent, and dose-dependent level of BB-301 tissue transduction (i.e., delivery of the multifunctional gene construct to target pharyngeal muscle cells). The study also demonstrated durable, broad-based, and dose-dependent expression of three different nucleic acids contained within the BB-301 gene construct (i.e., shmiR13 / siRNA13, shmiR17 / siRNA17, and codon-optimized PABPN1) within pharyngeal muscle cells, as well as durable and biologically significant levels of target gene knockdown (i.e., inhibition of the expression of the gene of interest) within pharyngeal muscle cells.

[0263] Example 3: Initial Human Efficacy Study An initial human Phase 1b / 2a clinical trial was initiated to evaluate the safety and efficacy of BB-301 in subjects diagnosed with oculopharyngeal muscular dystrophy (OPMD). The Investigational Drug (IND) application for BB-301 was approved by the U.S. Food and Drug Administration (FDA) in June 2023, and the first subjects were safely administered in the Phase 1b / 2a clinical trial of BB-301 (NCT06185673) in November 2023. Details of NCT06185673 are available on ClinicalTrials.gov, which are incorporated herein by reference.

[0264] Clinical trial (NCT06185673) is a 3+3 dose escalation study designed to evaluate three distinct doses of BB-301 to identify the maximum tolerated dose (MTD). The Phase 1b portion of this trial is designed to treat 9 to 18 OPMD subjects in three dose escalation cohorts. The Phase 2a portion of this trial is designed to treat up to 12 OPMD subjects at the MTD. Subjects will be monitored for 52 weeks after administration of BB-301 to assess the efficacy of the investigational gene therapy, and safety assessments will be conducted for 15 years after each subject received BB-301. The design of this initial human clinical trial is summarized in Table 6 below. [Table 6]

[0265] To date, both subjects have progressed from the natural history study to the Phase 1b / 2a clinical trial. As of this writing, interim clinical safety and efficacy data are available for the first subject from the first post-administration assessment time, which was performed 90 days after administration of BB-301 as mandated by the protocol. See the results section below.

[0266] OPMD Natural History Research Eligibility requirements for the Phase 1b / 2a clinical trial require confirmation of pathogenic triplet expansion of PABPN1. Prior to direct intramuscular injection of BB-301 into the pharyngeal constrictor muscle, each subject must complete a minimum of six months of evaluation in the OPMD Natural History Study, assessing baseline levels of dysphagia in a quantitative X-ray-based videofluorescence swallowing study at five separate time points. Following the six-month visit in the OPMD Natural History Study, subjects will then be eligible to undergo screening for entry into the BB-301 Phase 1b / 2a clinical trial (NCT06185673). The results of each assessment performed during the OPMD Natural History Study will provide a baseline that can be compared to the results of the corresponding assessment after administration of BB-301 in the Phase 1b / 2a clinical trial, thereby providing indicators of the efficacy and safety of BB-301 OPMD gene therapy.

[0267] Assessment Criteria Dysphagia (i.e., difficulty swallowing) will be assessed at all time points in the study (i.e., during the OPMD natural history study and during the Phase 1b / 2a clinical trial after BB-301 administration) using various X-ray-based techniques, clinical techniques, and subject-reported outcome measures, each known in the relevant art, as outlined in Table 7, and will be monitored as follows: 1. Pharyngeal constrictor muscle function (assessed by the pharyngeal area at maximum contraction) 2. Swallowing efficiency (assessed by total pharyngeal residue) 3. Oropharyngeal dysphagia reported by the subject (assessed using the Sydney Swallowing Questionnaire) 4. Swallowing ability (assessed by a cold water drinking test)

[0268] At each assessment visit, study participants will ingest a variety of consistent barium-containing foods and liquids (solid foods, as well as very concentrated, moderately concentrated, and dilute liquids), and the swallowing process will be assessed using the methods described above. [Table 7]

[0269] The pharyngeal area at maximum contraction (PhAMPC) is measured using the length of the patient's C2-C4 cervical vertebrae as the anatomical scalar. The "pharyngeal area" measurement includes the area of ​​the air space or bolus visualized at maximum contraction. The measurement occurs on the radiographic frame where the pharyngeal cavity contracts most strongly during swallowing. Normal PhAMPC values ​​range from 0% to 2.2% for characteristic foods and liquids evaluated in the swallowing tasks considered. PhAMPC is calculated as follows:

number

[0270] Total pharyngeal residue is also measured using the C2–C4 length to function as an anatomical scalar. Measurement of total pharyngeal residue includes the volume (v) of material remaining in the pharynx (res) after the initial swallowing of the bolus. The measurement occurs on the first X-ray frame showing the piriform sinus (PS) at its lowest point. Normal total pharyngeal residue should be close to zero. Total pharyngeal residue is calculated as follows:

number

[0271] Subject-reported oropharyngeal dysphagia was measured using the Sydney Swallowing Questionnaire (SSQ), a self-report inventory for assessing subjective symptoms of oropharyngeal dysphagia with strong content validity, construct validity, discrimination validity, predictive validity, and test-retest reliability across a broad patient population. The SSQ is a 17-item questionnaire developed to measure the severity of symptoms of oropharyngeal dysphagia reported by affected subjects. The questionnaire uses a 100mm long visual analog scale for all but one question. Possible scores range from 0 to 1700, with higher scores indicating greater difficulty in swallowing. Healthy subjects without dysphagia may present with an average score of approximately 59.0.

[0272] Changes in assessment criteria throughout the natural history study are used to determine the progression of dysphagia in subjects (if present). Mean and final outcomes (i.e., pre-treatment) are assessed in subjects to determine baseline values ​​of assessment criteria that can assess any changes at 90, 180, 270, and 360 days after BB-301DP treatment.

[0273] As discussed above, at the time of writing, two subjects had completed natural history studies and progressed to Phase 1b / 2a clinical trials.

[0274] BB-301 Phase 1b / 2a clinical trial methodology NCT06185673 is a Phase 1b / 2a, first-time, single-arm, open-label, sequential, dose-escalation cohort study. Participants who were previously enrolled in Benitec Biopharma Inc.'s OPMD-NH-001 Natural History Study and have completed at least six months of follow-up may be eligible to participate in this intervention trial.

[0275] To characterize the safety profile and determine the maximum tolerated dose (MTD) / recommended phase 2 dose (RP2D) of BB-301, the Phase 1b portion is designed to include dose escalation via enrollment of subjects into a continuous dose cohort in which BB-301 is administered as direct intramuscular injection into the oropharyngeal constrictor muscle (MPC) and inferior pharyngeal constrictor muscle (IPC). Intramuscular injection of BB-301 follows open surgical dissection of the pharyngeal region under general anesthesia. An independent Data Safety Monitoring Committee (DSMB), operating under the Charter, will review all safety data available from the cohort, along with cumulative information available from all administered subjects, to determine the appropriateness of dose escalation, other aspects of the safety profile of BB-301, and decisions regarding the conduct of the study.

[0276] The dose of BB-301 administered to each subject (as shown in Figure 8) is defined by cohort assignment (Phase 1b) and the MTD / RP2D to be determined (Phase 2a). On Day 1, the specified dose of BB-301 is injected directly into each contralateral side of the MPC muscle 502 (4 × 50 μL or 4 × 75 μL) and IPC muscle 501 (8 × 50 μL or 8 × 75 μL) after open surgical dissection of the pharyngeal region of the subject's neck 500 under general anesthesia.

[0277] To minimize systemic inflammatory and / or specific immunological responses within the study subject caused by surgical dissection, prednisone is administered prophylactically (starting on day 1) and tapered over 8 weeks.

[0278] Injection procedures and device operation Unless otherwise specified, the injection procedures and device handling used in the Phase 1b / 2a clinical trials are generally consistent with Examples 1 and 2 of this specification.

[0279] The 3Dose® Unit Injector microinjection syringe from Vlow Medical (Cat No. 3DOSE GREEN-125) was selected as injection syringe 100 (Figure 8B). In this example, injection syringe 100 is a pre-filled multi-dose syringe that allows for repeated administration of a predetermined small amount of BB-301 drug (DP).

[0280] Syringe 100 includes an injection volume selection mechanism, allowing selection of an injection volume range of 12.5 μL to 50 μL per plunger "click". The injection volume selection mechanism features a plunger rotatable relative to the barrel of the syringe to select one of several sets of plunger forming sections corresponding to the desired injection volume. Injection syringe 100 facilitates the completion of multiple injections of BB-301DP into the target pharyngeal constrictor muscle and ensures that the desired total volume of BB-301DP is delivered to the target muscle. Setting the desired volume of BB301DP with each injection was achieved by facilitating the dispensing of 12.5 μL, 25 μL, or 50 μL of BB-301DP with each plunger "click" of injection syringe 100 and selecting the appropriate plunger setting via rotation of the plunger around the central axis of the injection syringe to complete a predetermined total number of injections per contralateral pharyngeal constrictor muscle.

[0281] As described above, the injection syringe 100 enabled the operator to complete one or more injections of selected volumes of BB-301DP (12.5 μL, 25 μL, or 50 μL of BB-301DP) at any injection site within the target pharyngeal constrictor muscle through the use of a single or multiple clicks of the plunger at each distinct injection site within the target pharyngeal constrictor muscle. Additionally, the injection syringe 100 enabled the operator to complete multiple injections of selected volumes of BB-301DP across a surgically accessible anatomical region of the pharyngeal constrictor muscle through the targeting of distinct injection sites evenly distributed on the plane of the muscle.

[0282] Intramuscular injection of BB-301DP into the pharyngeal constrictor muscle was performed intraoperatively (and will be performed for future OPMD subjects) via the injection syringe 100 and needle 10 described herein. Specifically, needle A as described in Examples 1 and 2 herein was used as needle 10 (Figure 1). The injection syringe 100 was coupled with needle 10 (i.e., needle A as described herein) to form a drug injection device (DPID) 200 (Figure 8).

[0283] For intraoperative administration of BB-301DP to OPMD subjects currently enrolled in the Phase 1b / 2a clinical trial, two DPID200s are used per pharyngeal constrictor muscle, i.e., two DPID200s to complete administration of MPC muscle 502 and two DPID200s to complete administration of IPC muscle 501. This protocol will also be used for intraoperative administration to future OPMD subjects enrolled in the Phase 1b / 2a clinical trial.

[0284] The total dose of BB-301DP administered to each pharyngeal constrictor muscle is delivered in a two-step process, fractionating the intramuscular injection to focus on two separate contralateral portions of each pharyngeal constrictor muscle, which is determined by the relative anatomical position of each tissue section to the pharyngeal raphe 506 (Figure 10). In this regard, one DPID200 is used to complete the administration of each contralateral (i.e., left and right) side of the MPC muscle 502. Similarly, one DPID200 is used to complete the administration of each contralateral (i.e., left and right) side of the IPC muscle 501. Thus, completion of the planned injections of the pharyngeal constrictor muscles of each subject requires the use of at least four DPID200 per subject.

[0285] Figure 10 shows the specific anatomical sites to be injected with BB-301DP, including the left and right components of the MPC muscle 502 and the left and right components of the IPC muscle 501. Specifically, Figure 10 shows a posterior view of the pharyngeal muscles of the patient's neck 500, including the inferior pharyngeal constrictor muscle 501, the middle pharyngeal constrictor muscle 502, and the superior pharyngeal constrictor muscle 503. Figure 10 also shows the pharyngeal rupture 506 separating the left and right components of the pharyngeal constrictor muscles 501, 502, and 503.

[0286] The final dose of BB-301DP administered to each study participant will be determined according to the participant's cohort assignment (as discussed below).

[0287] Administration Procedure Subjects 1 and 2 received intraoperative administration of BB-301DP in the Phase 1b dose-escalation study according to the following general administration procedure. The same procedure will be used for the remaining subjects progressing from the natural history study to the Phase 1b / 2b study.

[0288] Amoxicillin (1 g) was delivered intravenously immediately before the intraoperative administration of BB-301DP (i.e., on day 1 of the Phase 1b trial), and again twice daily for the first two days after the intraoperative administration of BB-301DP.

[0289] BB-301DP was administered under general anesthesia with endotracheal intubation and appropriate analgesia. The patient was initially positioned supine, and then a medium-sized bougie or standard tracheal tube was visually guided and placed in the esophagus to facilitate preoperative identification of the pharyngeal-esophageal junction ("esophageal identification tube").

[0290] A bilateral cervical incision of approximately 5 cm was made along the anterior edge of the lower third of the sternocleidomastoid muscle (first incision 511 and second incision 512). After separating the omohyoid muscle from the middle thyroid vein, the internal jugular vein and carotid artery were tilted laterally, and the larynx was rotated. Blunt dissection then provided access to the posterior midline of the pharynx. Dissection of the middle thyroid vein and / or inferior thyroid artery is only necessary if access is limited. This approach allows for broad overall exposure of the posterior surface of the pharynx using the constrictor muscles 501, 502, and 503.

[0291] The cricopharyngeal muscles, inferior pharyngeal constrictor 501, and middle pharyngeal constrictor 502 of the pharynx are broadly exposed up to the level of the superior angle of the thyroid cartilage. The cricopharyngeal muscle differs more or less from the inferior pharyngeal constrictor 501 and is distinguished by its annular fibers, annular cartilage ring, and inferior border of the inferior pharyngeal constrictor 501. The upper part of the inferior pharyngeal constrictor 501 arises from the thyroid cartilage (thyropharyngeal portion). This is distinguished between the posterior edges of the thyroid cartilage, with each half articulating inferiorly with the cricoid cartilage (cricothyroid joint) and connecting with the fibrous pharyngeal raphe 506 located on the posterior midline of the pharynx. The higher zone of the pharyngeal muscle arises at the level of the superior angle of the thyroid cartilage.

[0292] Intramuscular injection of BB-301DP was performed using pre-filled BB-301DPID200 units on each contralateral side of the inferior pharyngeal constrictor muscle 501 (left and right of the pharyngeal raphe 506) and on each contralateral side of the middle pharyngeal constrictor muscle 502 (left and right of the pharyngeal raphe 506). Each DPID200 unit was filled with a final volume of BB-301DP sufficient to inject one side of each pharyngeal constrictor muscle, as follows: ● Fill one DPID200 with sufficient BB-301DP and complete four planned intramuscular injections into the left section of the oropharyngeal constrictor muscle 502 (lateral to the pharyngeal raphe 506, left side), spreading the injection evenly across the entire tissue plane (i.e., 4 × 50 μL or 4 × 75 μL). ● Fill one DPID200 with sufficient BB-301DP and complete four planned intramuscular injections into the right section of the oropharyngeal constrictor muscle 502 (lateral to the pharyngeal raphe 506, right side), spreading the injection evenly across the entire tissue plane (i.e., 4 × 50 μL or 4 × 75 μL). ● Fill one DPID200 with sufficient BB-301DP and complete eight planned intramuscular injections into the left section of the inferior pharyngeal constrictor muscle 501 (lateral to the pharyngeal raphe 506, left side), spreading the injections evenly across the entire tissue plane (i.e., 8 × 50 μL or 8 × 75 μL). ● Fill one DPID200 with sufficient BB-301DP and complete eight planned intramuscular injections into the right section of the inferior pharyngeal constrictor muscle 501 (lateral to the pharyngeal raphe 506, right side), spreading the injections evenly across the entire tissue plane (i.e., 8 × 50 μL or 8 × 75 μL).

[0293] Therefore, to treat each study subject in each cohort, the completion of planned injections into the pharyngeal constrictor muscle requires the use of at least four (4)DPID200 per subject.

[0294] The final dose of BB-301DP administered to each study participant was determined / determined according to the participant's cohort assignment. In this regard, OPMD participants enrolled in the Phase 1b dose escalation study were randomized into the following cohorts: ●Cohort 1: In Cohort 1, subjects received a fixed number of BB-301 IM injections into each pharyngeal constrictor muscle on the day of administration (as described above), with a total dose of 1.2e13vg / subject (low dose). ●Cohort 2: In Cohort 2, subjects received a fixed number of BB-301 IM injections into each pharyngeal constrictor muscle on the day of administration (as described above), with a total dose of 3.6e13vg / subject (medium dose). ●Cohort 3: In Cohort 3, subjects received a fixed number of BB-301 IM injections into each pharyngeal constrictor muscle on the day of administration (as described above), with a total dose of 5.4e13vg / subject (high dose).

[0295] Therefore, the dose of BB-301DP delivered to any one subject during the Phase 1b dose escalation study may vary depending on the subject.

[0296] After administering BB-301DP to the study subjects, the detached tissue was closed by suturing (subcutaneous sutures using 3 / 0 absorbable bicryl or equivalent, and subcutaneous sutures using non-absorbable monofiber), with or without drainage and dressings (or bandages) as needed. The esophageal identification tube was removed from the esophagus and oral cavity. This allowed for an additional check of the integrity of the upper esophageal sphincter mucosa.

[0297] Postoperative hospital care for the study subjects is provided after intraoperative administration of BB-301, which includes intravenous administration of 1 g amoxicillin (e.g., clamoxil) twice daily on days 0–2, daily examination of the surgical site, removal of drains (if present) on days 1–3, and removal of skin sutures on days 10–12, subject to the observation of sufficient healing.

[0298] The subjects in the study were able to resume eating soft foods on days 0-1.

[0299] result As of this writing, clinical efficacy data for the first subject ("Subject 1") is available from the first post-administration assessment time, as mandated by the protocol, which was performed 90 days after administration of BB-301.

[0300] During the OPMD Natural History Study, which represents the pre-treatment observation period for each subject, Subject 1 experienced a progressive worsening of dysphagia, as indicated by the results of the videofluoroscopy swallowing study (VFSS), the cold water time drinking test, and the subject's reported primary outcome measure (Sydney Swallowing Questionnaire). The videofluoroscopy swallowing study represents the gold standard analytical method for the quantitative assessment of dysphagia in a clinical setting.

[0301] Table 8 summarizes the preliminary clinical, radiological, and subject-reported assessment results (90 days post-treatment, "Day 90") for Subject 1 of the BB-301 Phase 1b / 2a clinical trial (NCT06185673). At Day 90, Subject 1 showed improvement in major videofluoroscopy assessments, correlated with similar observed improvements in the primary subject-reported outcome measure, compared to both the mean and final values ​​of each assessment completed during the pre-treatment observation period. In particular, the results of many assessments completed at Day 90 showed improvement compared to the initial measurements assessed at the subject's first visit for the OPMD natural history study, which occurred more than 12 months prior to the Day 90 assessment.

[0302] On day 90, the most significant improvements were observed in swallowing tasks, focusing on the assessment of pharyngeal constrictor muscle function and swallowing efficiency in the context of consuming thin liquids, solid foods, and thick non-solid foods (e.g., yogurt or pudding) (see Table 8). The improvements correlated with improvements in the primary subject-reported outcome measure of the Sydney Swallowing Questionnaire, which indicated improved swallowing function as reported by Subject 1 (see Table 8). [Table 8-1] [Table 8-2]

[0303] Conclusions from the initial human efficacy trial As expected, Subject 1 experienced disease progression during the enrollment period in the natural history study. However, after administration of BB-301DP using the device and method described herein, Subject 1 experienced a significant improvement in dysphagia. The dose of BB-301 was biologically active enough to promote the benefits in Subject 1, and these benefits were evident at the initial follow-up assessment conducted 90 days after administration. BB-301 did not cause any serious adverse events in Subject 1.

[0304] Based on the results of the Phase 1b dose escalation trials for all subjects, the maximum tolerated dose (MTD) / recommended Phase 2 dose (RP2D) for the Phase 2a dose expansion trial will be determined.

[0305] Those skilled in the art will understand that numerous variations and / or modifications can be made to the embodiments described above without departing from the broad general scope of this disclosure. Therefore, these embodiments should be considered in all respects as illustrative and not restrictive.

Claims

1. An injection needle, wherein the injection needle is It is equipped with a needle cannula, and the needle cannula is A proximal portion having a proximal end, A distal portion having a distal end, An intermediate portion disposed between the proximal portion and the distal portion, A pipe extending between the proximal end of the proximal portion and the distal end of the distal portion, through the proximal portion, the intermediate portion, and the distal portion, the pipe having an outlet at or adjacent to the distal end of the distal portion, The proximal portion and the distal portion are each substantially linear, and the intermediate portion is curved such that the distal portion extends from the proximal portion at an angle of 75° to 105°. An injection needle in which the length of the proximal portion is shorter than the length of the distal portion.

2. The injection needle according to claim 1, wherein the distal portion extends at an angle of approximately 85° to 95° with respect to the proximal portion.

3. The injection needle according to claim 1, wherein the distal portion extends at an angle of approximately 90° with respect to the proximal portion.

4. The injection needle according to claim 1 or 2, wherein the radius of curvature of the intermediate portion is approximately 2.5 mm to 3.1 mm.

5. The injection needle according to any one of claims 1 to 4, wherein the proximal portion has a length less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of the length of the distal portion.

6. The injection needle according to any one of claims 1 to 5, wherein the length of the distal portion is greater than the sum of the length of the proximal portion and the length of the intermediate portion.

7. The injection needle according to any one of claims 1 to 6, wherein the total length of the needle cannula is approximately 10 mm to 27 mm.

8. The injection needle according to any one of claims 1 to 7, wherein the length of the distal portion is approximately 7 mm to approximately 15 mm.

9. The injection needle according to any one of claims 1 to 8, wherein the length of the intermediate portion is approximately 3 mm to approximately 6 mm.

10. The injection needle according to any one of claims 1 to 9, wherein the length of the proximal portion is approximately 2 mm to approximately 6 mm.

11. The distal portion extends at an angle of approximately 85° to 95° with respect to the proximal portion. The injection needle according to any one of claims 1 to 10, wherein the radius of curvature of the intermediate portion is approximately 2.5 mm to 3.1 mm.

12. The proximal portion has a length less than 70% of the length of the distal portion. The length of the distal portion is greater than the sum of the length of the proximal portion and the length of the intermediate portion, and optionally the arc length of the intermediate portion. The injection needle according to any one of claims 1 to 11, wherein the total length of the needle cannula is approximately 10 mm to 27 mm.

13. The distal portion extends at an angle of approximately 85° to 95° with respect to the proximal portion. The length of the distal portion is approximately 7 mm to 15 mm. The injection needle according to any one of claims 1 to 12, wherein the length of the proximal portion is approximately 2 mm to 6 mm.

14. The injection needle according to any one of claims 1 to 13, wherein the needle cannula is 26 to 28 gauge.

15. The injection needle according to any one of claims 1 to 14, wherein the outer diameter of the needle cannula is approximately 0.3 mm to approximately 0.5 mm.

16. The injection needle according to any one of claims 1 to 15, wherein the inner diameter of the needle cannula is approximately 0.1 mm to approximately 0.3 mm.

17. The injection needle according to any one of claims 1 to 16, wherein the wall thickness of the needle cannula is approximately 0.03 mm to approximately 0.17 mm.

18. The injection needle according to any one of claims 1 to 17, wherein the needle cannula includes a dead volume of approximately 0.5 μL / 25.4 mm to approximately 1.5 μL / 25.4 mm.

19. The injection needle according to any one of claims 1 to 18, wherein the needle cannula has an outlet located at or adjacent to the distal end of the distal portion.

20. The injection needle according to any one of claims 1 to 19, wherein the needle cannula comprises an inclined portion formed at the distal end of the needle, and the inclined portion includes an inclined edge portion that faces away from the inner bending side of the needle cannula.

21. The distal tip is inclined at an angle of approximately 5° to approximately 15°, as described in any one of claims 1 to 20.

22. The inclined portion length is approximately 0.5 mm to approximately 3.5 mm, as described in any one of claims 1 to 21.

23. The injection needle according to any one of claims 1 to 22, wherein the outlet of the needle cannula is located at the inclined edge.

24. The injection needle according to any one of claims 1 to 23, further comprising a needle hub connected to the needle cannula, wherein the needle cannula protrudes distally from the needle hub.

25. The injection needle according to claim 24, wherein the needle cannula further comprises a support portion, the support portion being disposed inside the needle hub.

26. The injection needle according to any one of claims 1 to 25, wherein the needle cannula has an entrance at the proximal end of the support portion.

27. The injection needle according to any one of claims 24 to 26, wherein the needle hub has a distal portion adjacent to the proximal end of the proximal portion of the needle cannula, and the distal portion of the needle hub is swaged.

28. The injection needle according to any one of claims 1 to 27, wherein the injection needle has an entrance located at or adjacent to the proximal end of the proximal portion.

29. Use of an injection needle according to any one of claims 1 to 28 for directly injecting a therapeutic composition into the target pharyngeal muscle.

30. The use according to claim 29, wherein the injection needle is used to directly inject the therapeutic composition into the pharyngeal muscle, following an incision in the neck of the subject to obtain direct access to the pharyngeal muscle.

31. The use according to claim 29 or 30, wherein the injection needle is used to directly inject the therapeutic composition into the target pharyngeal constrictor muscle.

32. The use according to any one of claims 29 to 31, wherein the pharyngeal muscles include one or more of the inferior pharyngeal constrictor, middle pharyngeal constrictor, superior pharyngeal constrictor, palatinaryngeal muscle, tubal pharyngeal muscle, stylopharyngeal muscle, or any combination thereof.

33. The use according to any one of claims 29 to 32, wherein the therapeutic composition is effective in treating oculopharyngeal muscular dystrophy (OPMD), and the injection needle is used to treat or prevent dysphagia associated with oculopharyngeal muscular dystrophy (OPMD) in a subject.

34. A method for forming an injection needle, wherein the method is To provide a needle cannula, the needle cannula comprising a proximal end, a distal end, and a tube extending between the proximal and distal ends through the needle cannula, A method comprising bending the needle cannula such that the intermediate portion curves such that the distal portion extends at a certain angle to the proximal portion.

35. The method according to claim 34, wherein the distal portion is curved so as to extend at an angle of 75° to 105° with respect to the proximal portion.

36. The method according to claim 34 or 35, wherein the length of the proximal portion is shorter than the length of the distal portion.

37. The method according to any one of claims 34 to 36, wherein the injection needle formed is the injection needle according to any one of claims 1 to 28.

38. A method for administering a therapeutic composition to a target pharyngeal muscle, comprising directly injecting the therapeutic composition into the target pharyngeal muscle using a bent or curved injection needle.

39. The aforementioned bent or curved injection needle is A proximal portion having a proximal end, A distal portion having a distal end, An intermediate portion disposed between the proximal portion and the distal portion, An injection needle comprising: a tube extending through the proximal portion, the intermediate portion, and the distal portion between the proximal end of the proximal portion and the distal end of the distal portion, the tube opening at an outlet located at or adjacent to the distal end of the distal portion; The proximal portion and the distal portion are each substantially linear, and the intermediate portion is curved such that the distal portion extends at a certain angle to the proximal portion. The method according to claim 38, wherein the injection needle is optionally the injection needle described in any one of claims 1 to 28.

40. The method according to claim 38 or 39, wherein the therapeutic composition is administered to one or more pharyngeal muscles selected from the inferior pharyngeal constrictor muscle, the middle pharyngeal constrictor muscle, the superior pharyngeal constrictor muscle, the palatinaryngeal muscle, the tubal pharyngeal muscle, the stylopharyngeal muscle, and any combination thereof.

41. The method according to any one of claims 38 to 40, comprising administering the therapeutic composition to the oropharyngeal constrictor muscle and the inferior pharyngeal constrictor muscle.

42. The method according to any one of claims 38 to 41, wherein the therapeutic composition is administered to the pharyngeal muscle by direct injection following an incision in the neck of the subject, which is sufficient to provide direct access to at least a portion of the pharyngeal muscle of the subject.

43. The method according to claim 42, comprising making one or more incisions in the neck of the target to provide direct access to at least a portion of the pharyngeal muscles of the target, and then administering the therapeutic composition by direct intramuscular injection into the pharyngeal muscles.

44. The method according to any one of claims 38 to 43, wherein the therapeutic composition is effective for the treatment or prevention of dysphagia associated with oculopharyngeal muscular dystrophy (OPMD).

45. The therapeutic composition, (i) A DNA-directed RNAi (ddRNAi) construct comprising a nucleic acid containing a sequence encoding a short hairpin microRNA (shmiR), The method according to any one of claims 38 to 44, comprising a viral delivery vector comprising a “silence and substitution” construct, the PABPN1 construct comprising a nucleic acid comprising a sequence encoding a functional PABPN1 protein having an mRNA transcript that is not targeted by the shmiR(plural) encoded by the ddRNAi construct.

46. The aforementioned virus delivery vector is adeno-associated virus (AAV), and the AAV is (i) A viral capsid protein derived from AAV9 containing a modified subunit 1 (VP1), wherein the amino acids at positions 26, 40, 43, and 44 are modified from the corresponding wild-type AAV9 VP1 sequence described in SEQ ID NO: 17, and optionally, the viral capsid protein contains the amino acid sequence described in SEQ ID NO: 27, or (ii) The method according to claim 45, comprising a viral capsid protein derived from AAV8 containing a modified subunit 1 (VP1) sequence, wherein the amino acids at positions 1, 26, 40, 43, 44, and 64 are modified from the corresponding wild-type AAV8 VP1 sequence described in Sequence ID No.

21.

47. (i) The AAV comprises a viral capsid protein derived from AAV9, which includes mutations A67E, Q81R, K84D, and A85S with respect to the full-length wild-type AAV serotype 9 capsid sequence described in SEQ ID NO: 19, and optionally, the viral capsid protein comprises the amino acid sequence described in SEQ ID NO: 28, or (ii) The method of claim 45, wherein the AAV comprises a viral capsid protein derived from AAV8, which includes mutations A42S, A67E, Q81R, K84D, A85S, and Q105K with respect to the full-length wild-type AAV serotype 8 capsid sequence described in SEQ ID NO: 23, and optionally the viral capsid protein comprises the amino acid sequence described in SEQ ID NO:

24.

48. The method according to any one of claims 45 to 47, wherein the PABPN1 construct comprises a nucleic acid molecule encoding a functional PABPN1 protein, the thereof codon-optimized such that its corresponding mRNA sequence is not recognized by the shmiR(s) encoded and expressed from the ddRNAi construct, and optionally the codon-optimized nucleic acid comprises the sequence described in SEQ ID NO: 15 and encodes the amino acid sequence described in SEQ ID NO:

16.

49. The method according to any one of claims 45 to 48, wherein the ddRNAi construct comprises a DNA sequence encoding shmiR, which includes an effector sequence substantially complementary to the sequence described in SEQ ID NO: 1 and / or an effector sequence substantially complementary to the sequence described in SEQ ID NO:

2.

50. The aforementioned ddRNAi construct is (i) a DNA sequence encoding shmiR including the effector sequence described in SEQ ID NO: 4 and the effector complementary sequence (shmiR13) described in SEQ ID NO: 3, and a DNA sequence encoding shmiR including the effector sequence described in SEQ ID NO: 6 and the effector complementary sequence (shmiR17) described in SEQ ID NO: 5, and / or (ii) The method according to claim 49, comprising a nucleic acid containing or consisting of the DNA sequence (shmiR13) described in SEQ ID NO: 12, and a nucleic acid containing or consisting of the DNA sequence (shmiR17) described in SEQ ID NO:

13.

51. The therapeutic composition for the treatment of OPMD is administered in a volume ranging from about 25 μL to about 100 μL per injection site, according to any one of claims 45 to 50.

52. The method according to any one of claims 45 to 51, wherein the therapeutic composition for the treatment of OPMD is administered to the subject at a concentration of about 1.0 + E12 vg / mL to about 1.0 + E14 vg / mL, and optionally, the concentration of the therapeutic composition is about 5.0 + E12 vg / mL to about 5.0 + E13 vg / mL, and optionally, the concentration of the therapeutic composition is about 1.0 + E13 vg / mL to 3.0 + E13 vg / mL.

53. The method according to any one of claims 44 to 52, comprising administering the therapeutic composition to one or more locations on the pharyngeal muscles, one or more pharyngeal muscles, one or more sides of the pharyngeal muscles, or any combination thereof.

54. (i) A pre-filled syringe containing a therapeutic composition effective for the treatment or prevention of dysphagia associated with OPMD, (ii) An injection device comprising an injection needle according to any one of claims 1 to 28, coupled to the pre-filled syringe.

55. The therapeutic composition comprises an AAV9 vector, and the AAV9 vector is (a) A viral capsid protein derived from AAV9, comprising mutations A67E, Q81R, K84D, and A85S, determined in relation to the full-length wild-type AAV9 capsid sequence described in SEQ ID NO: 19, and optionally comprising the viral capsid protein comprising the amino acid sequence described in SEQ ID NO: 28, (b) Silence and replacement structure, wherein the silence and replacement structure are (i) A muscle-specific promoter (optionally, the promoter is the Spc512 muscle-specific promoter) (ii) A DNA-directed RNAi (ddRNAi) construct comprising a nucleic acid comprising or comprising a sequence encoding shmiR13 as described herein (optionally, the DNA sequence described in SEQ ID NO: 12), and a nucleic acid comprising or comprising a sequence encoding shmiR17 as described herein (optionally, the DNA sequence described in SEQ ID NO: 13), (iii) A PABPN1 construct comprising a nucleic acid sequence encoding a functional PABPN1 protein comprising the amino acid sequence described in Sequence ID No. 16, wherein the nucleic acid sequence encoding the functional PABPN1 protein is a codon-optimized DNA sequence such that its corresponding mRNA sequence is not targeted by the shmiR(s) encoded by the ddRNAi construct (optionally, the codon-optimized nucleic acid comprises the sequence described in Sequence ID No. 15), and The injection device according to claim 54, wherein the muscle-specific promoter is operably connected to the ddRNAi construct and the PABPN1 construct.

56. The injection device according to claim 54 or 55, wherein the injection device is configured to deliver the therapeutic composition from the pre-filled syringe in a series of one or more injections, each injection having a predetermined injection volume.

57. The injection device according to claim 56, further comprising an injection volume setting mechanism that can be operated to set the injection volume.

58. The injection device according to claim 57, wherein the injection volume setting mechanism is operable to set the injection volume to approximately 12.5 μL to approximately 75 μL.

59. The injection device according to claim 58, wherein the injection setting mechanism is operable to select from a separate set of injection volumes.

60. The injection device according to claim 59, wherein the set of separate injection volumes comprises at least 25 μL and 50 μL.

61. A kit comprising a set of two or more injection devices according to any one of claims 54 to 60, wherein the pre-filled syringe of the injection device collectively contains a single therapeutic dose of the therapeutic composition for the treatment of a single subject.

62. The kit according to claim 61, comprising four of the aforementioned injection devices.

63. The kit according to claim 62, wherein the injection device contains at least 200 μL of the therapeutic composition, for example, about 200 μL to about 2000 μL of the therapeutic composition.

63. (i) Two injection devices for containing approximately 200 μL to approximately 500 μL of the therapeutic composition, (ii) The kit according to claim 63, comprising two injection devices each containing about 400 μL to about 1000 μL of the therapeutic composition.

64. The kit according to claim 62, wherein one or more of the injection devices are configured to dispense the therapeutic composition in a series of injections each having a 50 μL injection volume, and / or one or more of the injection devices are configured to dispense the therapeutic composition in a series of injections each having a 75 μL injection volume.

65. The single therapeutic dose of the therapeutic composition is The therapeutic composition is 1.2 e13 vg / target, 3.6 e13 vg / target of the therapeutic composition, or The kit according to any one of claims 61 to 64, wherein the therapeutic composition is 5.4e13vg / target.

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