Devices and methods for administering a therapeutic composition to pharyngeal muscles

Abstract

The present disclosure relates to devices and methods for administering a therapeutic composition to pharyngeal muscles. In particular, the devices and methods of the present disclosure can be used to treat or prevent dysphagia, including dysphagia associated with oculopharyngeal muscular dystrophy (OPMD) in individuals having or susceptible to OPMD. Additionally, the present disclosure relates to devices and methods for delivering a modified adeno-associated virus (AAV) delivery vector comprising a'silencing and replacement' DNA construct, including compositions comprising the DNA construct.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 510,750, filed June 28, 2023, the contents of which are incorporated herein by reference in their entirety. Furthermore, the applicant has recently disclosed therapeutic agents and compositions for treating oculopharyngeal muscular dystrophy (OPMD) in the following applications: U.S. Provisional Patent Application No. 62 / 812,187, filed February 28, 2019; U.S. Provisional Patent Application No. 62 / 747,089, filed October 17, 2018; International Patent Application PCT / AU2019 / 051134, filed October 17, 2019; and International Patent Application PCT / AU2020 / 050182, filed February 28, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to apparatus and methods for administering therapeutic compositions to the pharyngeal muscles. Specifically, the apparatus and methods of this disclosure can be used to treat or prevent dysphagia, including OPMD-related dysphagia in individuals who have or are susceptible to oculopharyngeal dysphagia (OPMD). Additionally, this disclosure relates to apparatus and methods for delivering a modified adeno-associated virus (AAV) delivery vector comprising a 'silencing and replacement' DNA construct, such as a composition comprising said DNA construct. Background Technology

[0004] OPMD is a rare and slowly progressive late-onset muscular dystrophy that initially appears in middle age and develops with age, characterized by progressive ptosis (drooping eyelid), dysphagia (difficulty swallowing), and proximal limb weakness. OPMD is caused by a specific mutation in the gene encoding PABPN1 on chromosome 14 (position q11.2-q13). Therefore, people with OPMD experience increasing difficulty swallowing (dysphagia), putting them at risk of injury or death due to choking, aspiration of food or beverages into the lungs (absorption), aspiration pneumonia, weight loss, and malnutrition.

[0005] The mutation causing OPMD is an aberrant amplification of the (GCN)n trinucleotide repeat sequence in the coding region of the poly(A)-binding protein nucleus 1 (PABPN1) gene. The presence of the trinucleotide repeat leads to the amplification of the polyalanine channel at the N-terminus of the PABPN1 protein: 10 alanine residues are present in the normal protein, but 11 to 18 alanine residues are amplified in the mutant form (expPABPN1). Intranuclear aggregates, designated as “intranuclear inclusions,” are the main histopathological feature of this disease. The misfolding of the amplified PABPN1 protein causes the accumulation of insoluble polymeric fibrous aggregates within the nucleus of affected cells, as PABPN1 is a readily aggregated protein.

[0006] Currently, there are no approved medications for treating OPMD, and none have shown significant changes in the natural history of the disease. Palliative surgical interventions, such as cricopharyngeal myotomy, have been used. Cricopharyngeal myotomy releases tension in the cricopharyngeal (CP) muscle, and this surgery can temporarily alleviate the effects of dysphagia in some patients. Direct injection of botulinum toxin (Botox) into the CP muscle has also been used to temporarily improve swallowing through chemically induced relaxation of the targeted muscle. While both techniques can temporarily alleviate dysphagia experienced by some patients, oropharyngeal symptoms inevitably recur, and disease progression continues. Repeated dilation of the upper esophageal sphincter has shown some efficacy in OPMD patients with moderate dysphagia, but further clinical studies are needed to confirm the results and characterize long-term outcomes.

[0007] As background, under normal circumstances, a food bolus leaves the oral cavity and is propelled across the length of the pharynx by the coordinated contractions of the superior, middle, and inferior pharyngeal constrictors, on its way to the esophagus. As the bolus approaches the opening at the upper end of the esophagus, subsequent relaxation of the cricopharyngeal muscles allows the bolus to enter the esophagus and proceed into the stomach. In the case of OPMD, the pharyngeal constrictors are weakened and atrophied, and therefore unable to sustain the required level of force to support the propulsion of the food bolus that defines the normal swallowing process. Restoring the muscle fiber size and force-generating capacity of the weakened and atrophied pharyngeal constrictors is expected to significantly enhance these muscle groups' ability to support the food bolus through the pharynx and towards the esophagus. Therefore, the pharyngeal constrictors (inferior and middle) are more relevant therapeutic targets, and increased muscle strength can enhance the function of these muscles, thereby reducing swallowing difficulties in patients with OPMD.

[0008] The applicant has recently disclosed therapeutic compositions for treating OPMD in WO2017 / 177277, WO2018 / 107228, WO2019 / 043630, WO2020 / 077412, and WO2020 / 172720. However, there is still a need for improved methods and means of delivering compositions, such as those developed and disclosed by the applicant, to the pharyngeal muscles.

[0009] Any discussion of documents, actions, materials, devices, articles, or the like included in this specification shall not be construed as an admission that any or all of these matters constitute part of the prior art or common knowledge in the field relating to this disclosure prior to the priority date of each appended claim. Summary of the Invention

[0010] In a first aspect of this disclosure, an injection needle is provided, comprising:

[0011] Needle cannula, the needle cannula comprising:

[0012] The proximal portion has a proximal end;

[0013] The distal portion has a distal end;

[0014] The intermediate portion, disposed between the proximal portion and the distal portion; and

[0015] A lumen 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 lumen opening at or near the distal end of the distal portion.

[0016] The proximal and distal portions are each substantially straight, and the intermediate portion is curved such that the distal portion extends relative to the proximal portion at an angle between 75° and 105°; and

[0017] The length of the proximal portion is shorter than the length of the distal portion.

[0018] The distal portion may extend relative to the proximal portion at an angle between approximately 85° and 95°. For example, the distal portion may extend relative to the proximal portion at an angle of approximately 90°.

[0019] The radius of curvature of the intermediate portion can be between approximately 2.5 mm and 3.1 mm. For example, the radius of curvature of the intermediate portion can be approximately 2.8 mm.

[0020] The length of the proximal portion may be, for example, less than 90%, less than 80%, less than 70%, or less than 60% or less than 50% of the length of the distal portion.

[0021] The length of the distal portion may be greater than the sum of the length of the proximal portion and the length of the intermediate portion (e.g., the arc length of the intermediate portion).

[0022] The total length of the needle cannula can be between about 10 mm and about 27 mm. In some instances, the total length of the needle cannula can be between about 16 mm and about 22 mm. For example, the total length of the needle cannula can be between about 18 mm and 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 intermediate 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.

[0023] In one instance:

[0024] The distal portion extends relative to the proximal portion at an angle between approximately 85° and approximately 95° (e.g., approximately 90°); and

[0025] The radius of curvature of the middle section is between approximately 2.5 mm and 3.1 mm.

[0026] In one instance:

[0027] The length of the proximal portion is less than 70% of the length of the distal portion;

[0028] 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 (e.g., the arc length of the intermediate portion); and

[0029] The total length of the needle cannula is between approximately 10 mm and 27 mm.

[0030] In one instance:

[0031] The distal portion extends relative to the proximal portion at an angle between approximately 85° and 95°.

[0032] The length of the distal portion is between approximately 7 mm and 15 mm; and

[0033] The length of the proximal portion is between approximately 2 mm and 6 mm.

[0034] The needle cannula can be approximately 26-28 gauge, for example, a 27 gauge needle. The outer diameter of the needle cannula can be, for example, between approximately 0.3 mm and 0.5 mm. The inner diameter of the needle cannula can be, for example, between approximately 0.1 mm and 0.3 mm. The wall thickness of the needle cannula can be, for example, between approximately 0.03 mm and 0.17 mm.

[0035] The dead volume of the needle cannula can, for example, be between about 0.5 µL / 25.4 mm and 1.5 µL / 25.4 mm.

[0036] The needle cannula may include a bevel formed at the distal end of the needle. The bevel may include a beveled edge facing away from the inner fold 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 beveled at an angle of about 5° to about 15°. The length of the bevel may be about 0.5 mm to about 3.5 mm. The exit of the needle cannula may be located at the edge of the bevel.

[0037] The injection needle may further include a needle hub connected to the needle cannula. The needle cannula may project distally from the needle hub. The proximal end of the proximal portion of the needle cannula may be defined at a boundary where the needle cannula extends from the needle hub. In some embodiments, the needle cannula may further include a support portion extending proximally from the proximal end of the proximal portion of the needle cannula, and the support portion being disposed within the needle hub. The lumen may further extend through the support portion.

[0038] The needle cannula may have an inlet located at or near the proximal end of the proximal portion, or at the proximal end of the support portion (if present). The lumen may extend between the inlet and the outlet.

[0039] The needle hub may have a distal portion adjacent to the proximal end of the proximal portion of the needle sheath. The distal portion of the needle hub may be forged.

[0040] The injection needle can be specifically used as a pharyngeal muscle injection needle, for example. The injection needle can be used to inject therapeutic drugs directly into the pharyngeal muscles. Direct injection can occur, for example, during open surgery. That is, direct injection into the pharyngeal muscles can occur after an incision is made in the subject's neck to obtain direct access to the pharyngeal muscles. The injection needle can be used specifically for direct injection into the pharyngeal constrictor muscles and / or any of the following specific muscles: the hypopharyngeal constrictor, the middle pharyngeal constrictor, the superior pharyngeal constrictor, the palatopharyngeal muscle, the eustachian tube pharyngeal muscle, the stylopharyngeal muscle, or any combination thereof.

[0041] The injection needle can be used to treat or prevent dysphagia. In some instances, dysphagia is associated with oculopharyngeal dystrophy (OPMD); for example, dysphagia can be secondary to a genetically confirmed diagnosis of OPMD.

[0042] On one hand, a pharyngeal muscle injection needle is provided, the pharyngeal muscle injection needle comprising:

[0043] The proximal portion has a proximal end;

[0044] The distal portion has a distal end;

[0045] The intermediate portion, disposed between the proximal portion and the distal portion; and

[0046] A lumen 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 lumen opening at or near the distal end of the distal portion.

[0047] The proximal portion and the distal portion are each substantially straight, and the intermediate portion is curved such that the distal portion extends at an angle relative to the proximal portion.

[0048] The pharyngeal muscle injection needle may include any one or more of the features of the injection needle described above with respect to the first aspect of this disclosure. For example, the intermediate portion may be curved such that the distal portion extends relative to the proximal portion at an angle between 75° and 105° (e.g., at an angle between about 85° and about 95°, or at an angle of about 90°). As another example, the length of the proximal portion is shorter than the length of the distal portion.

[0049] The pharyngeal muscle injection needle can be used to treat or prevent dysphagia. In one instance, dysphagia can be associated with oculopharyngeal dystrophy (OPMD), for example, dysphagia can be secondary to a genetically confirmed diagnosis of OPMD.

[0050] On the other hand, a method for forming an injection needle, such as a pharyngeal muscle injection needle, is provided, wherein the method comprises:

[0051] A needle cannula is provided, the needle cannula comprising a proximal end and a distal end, and a lumen extending through the needle cannula between the proximal end and the distal end, the lumen opening at an outlet at the distal end of the distal portion or adjacent to the distal end; and

[0052] The needle cannula is bent such that it includes a substantially straight proximal portion having the proximal end, a substantially straight distal portion having the distal end, and an intermediate portion disposed between the proximal portion and the distal portion, the intermediate portion being curved such that the distal portion extends at an angle relative to the proximal portion.

[0053] The method allows the injection needle to include any one or more of the features of the injection needle described above with respect to the first aspect of this disclosure. For example, the intermediate portion may be curved such that the distal portion extends relative to the proximal portion at an angle between 75° and 105° (e.g., at an angle between about 85° and about 95°, or at an angle of about 90°). As another example, the length of the proximal portion is shorter than the length of the distal portion.

[0054] In one aspect of this disclosure, a method of administering a therapeutic composition to the pharyngeal muscle of a subject is provided, the method comprising injecting the therapeutic composition directly into the pharyngeal muscle of the subject using a bent or curved injection needle. In some instances, the bent or curved injection needle comprises:

[0055] The proximal portion has a proximal end;

[0056] The distal portion has a distal end;

[0057] The intermediate portion, disposed between the proximal portion and the distal portion; and

[0058] A lumen 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 lumen opening at or near the distal end of the distal portion.

[0059] The proximal portion and the distal portion are each substantially straight, and the intermediate portion is curved such that the distal portion extends at an angle relative to the proximal portion.

[0060] The bent or curved injection needle may include any one or more of the features of the injection needle described above with respect to the first aspect of this disclosure. For example, the intermediate portion may be curved such that the distal portion extends relative to the proximal portion at an angle between 75° and 105° (e.g., at an angle between about 85° and about 95°, or at an angle of about 90°). As another example, the length of the proximal portion is shorter than the length of the distal portion.

[0061] In a preferred embodiment, the method includes injecting the therapeutic composition directly into the pharyngeal muscle of the subject using an injection needle described herein according to the first aspect.

[0062] The pharyngeal muscles may include any of the following specific muscles: the hypopharyngeal constrictor, the middle pharyngeal constrictor, the superior pharyngeal constrictor, the palatopharyngeal muscle, the eustachian tube constrictor, the stylopharyngeal muscle, or any combination thereof. In a particular example, the method includes applying the therapeutic composition to the middle pharyngeal constrictor and the hypopharyngeal constrictor.

[0063] The therapeutic composition can be administered to the pharyngeal muscles via direct injection after making an incision in the subject's neck (i.e., intramuscular injection), the incision being sufficient to provide direct access to at least a portion of the subject's pharyngeal muscles. Therefore, the method may involve making one or more incisions (towards the skin) in the subject's neck to provide direct access to the subject's pharyngeal muscles, and then administering the therapeutic composition to the pharyngeal muscles via direct intramuscular injection. For example, an incision can be made on the right side of a pharyngeal region on the right side of a human or animal body, and the region is adequately dissected to access at least the right side of one or more pharyngeal muscles. A needle cannula can then be directly inserted into the right side of one or more pharyngeal muscles. Alternatively or additionally, an incision can be made on the left side of a pharyngeal region on the left side of a human or animal body, and the region is adequately dissected to access the left side of one or more pharyngeal muscles. A needle cannula can then be directly inserted into the left side of one or more pharyngeal muscles.

[0064] The therapeutic compositions described herein can be injected directly into one or more sites in the pharyngeal muscles, into one or more pharyngeal muscles, into one or more sides of the pharyngeal muscles (e.g., right or left), or any combination thereof. Injection at multiple sites can help improve the delivery of the therapeutic composition. For example, in the case of delivering a viral vector-based therapeutic composition, injection at multiple sites can improve delivery to the pharyngeal muscle tissue. In one example, the therapeutic composition can be injected into the midpharyngeal constrictor muscle and the hypopharyngeal constrictor muscle.

[0065] This disclosure also provides a method of administering a therapeutic composition to the pharyngeal muscles of a subject, the method comprising injecting the therapeutic composition directly into the pharyngeal muscles of the subject using a bent or curved injection needle. In some preferred embodiments, the injection needle is as described herein with reference to the first aspect.

[0066] The therapeutic composition can be applied to one or more pharyngeal muscles selected from the following: hypopharyngeal constrictor, middle pharyngeal constrictor, superior pharyngeal constrictor, palatopharyngeal muscle, eustachian tube constrictor, styloid pharyngeal muscle, and any combination thereof. For example, the method may include applying the therapeutic composition to the middle pharyngeal constrictor and the hypopharyngeal constrictor.

[0067] The therapeutic composition can be administered to the pharyngeal muscles by direct injection after making an incision in the subject's neck, the incision being sufficient to provide direct access to at least a portion of the subject's pharyngeal muscles. According to this example, the method may include making one or more incisions in the subject's neck to provide direct access to at least a portion of the subject's pharyngeal muscles, and then administering the therapeutic composition to the pharyngeal muscles by direct intramuscular injection.

[0068] In some instances, the therapeutic composition is used to treat or prevent dysphagia, such as dysphagia associated with oculopharyngeal dysphagia (OPMD). Therefore, the therapeutic composition may be effective in treating or preventing dysphagia associated with OPMD. The therapeutic composition may also be effective in treating or preventing one or more other symptoms of OPMD.

[0069] A therapeutic composition effective for treating or preventing dysphagia associated with OPMD, used in the methods of this disclosure, may comprise a viral delivery vector comprising a 'silencing and replacement' construct comprising:

[0070] (i) A DNA-guided RNAi (ddRNAi) construct containing nucleic acids, wherein the nucleic acids comprise a sequence encoding a short hairpin microRNA (shmiR); and

[0071] (ii) A PABPN1 construct containing nucleic acid, the nucleic acid containing a sequence encoding a functional PABPN1 protein, the sequence having an mRNA transcript not targeted by the shmiR encoded by the ddRNAi construct.

[0072] In some instances, the viral delivery vector is adeno-associated virus (AAV) comprising a viral capsid protein from AAV9, the viral capsid protein comprising a modified subunit 1 (VP1) sequence, wherein amino acids at positions 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence shown in SEQ ID NO: 17. For example, the AAV may comprise a viral capsid protein from AAV9 comprising a VP1 sequence having a modified phospholipase 2 (PLA2) domain, the domain comprising the sequence shown in SEQ ID NO: 27. For example, the AAV may comprise a viral capsid protein from AAV9 comprising mutants A67E, Q81R, K84D, and A85S relative to the full-length wild-type AAV serotype 9 capsid sequence shown in SEQ ID NO: 19, optionally wherein the viral capsid protein comprises the amino acid sequence shown in SEQ ID NO: 28.

[0073] In some instances, the viral delivery vector is adeno-associated virus (AAV) comprising a viral capsid protein from AAV9, the viral capsid protein comprising a modified subunit 1 (VP1) sequence, wherein amino acids at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9 VP1 sequence shown in SEQ ID NO: 17. For example, the AAV may comprise a viral capsid protein from AAV9 comprising a VP1 sequence having a modified phospholipase 2 (PLA2) domain, the domain comprising the sequence shown in SEQ ID NO: 18. For example, the AAV may comprise a viral capsid protein from AAV9 comprising mutants A42S, A67E, Q81R, K84D, and A85S relative to the full-length wild-type AAV serotype 9 capsid sequence shown in SEQ ID NO: 19, optionally wherein the viral capsid protein comprises the amino acid sequence shown in SEQ ID NO: 20.

[0074] In some instances, the viral delivery vector is adeno-associated virus (AAV) comprising a viral capsid protein from AAV8, the viral capsid protein comprising a modified subunit 1 (VP1) sequence, wherein amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence shown in SEQ ID NO: 21. For example, the AAV may comprise a viral capsid protein from AAV8 comprising a VP1 sequence having a modified PLA2 domain, the domain comprising the sequence shown in SEQ ID NO: 22. For example, the AAV may comprise a viral capsid protein from AAV8 comprising mutations A42S, A67E, Q81R, K84D, A85S, and Q105K relative to the full-length wild-type AAV serotype 8 capsid sequence shown in SEQ ID NO: 23, optionally wherein the viral capsid protein comprises the amino acid sequence shown in SEQ ID NO: 24.

[0075] In some instances, the PABPN1 construct within the 'silencing and replacement' construct comprises a nucleic acid molecule encoding a functional PABPN1 protein, said nucleic acid molecule being codon-optimized such that its corresponding mRNA sequence is not recognized by shmiR encoded and expressed from the ddRNAi construct. For example, the codon-optimized nucleic acid may comprise the sequence shown in SEQ ID NO:15. Preferably, the codon-optimized nucleic acid encodes the amino acid sequence shown in SEQ ID NO:16.

[0076] In some instances, the ddRNAi construct comprises a DNA sequence encoding a shmiR, wherein the shmiR comprises an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 1 and / or substantially complementary to the sequence shown in SEQ ID NO: 2. In some instances, the ddRNAi construct comprises a DNA sequence encoding a shmiR comprising an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 1 and a DNA sequence encoding a shmiR comprising an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 2. For example, the ddRNAi construct may comprise a DNA sequence encoding a shmiR (shmiR13) comprising the effector sequence shown in SEQ ID NO: 4 and the effector complement sequence shown in SEQ ID NO: 3; and a DNA sequence encoding a shmiR (shmiR17) comprising the effector sequence shown in SEQ ID NO: 6 and the effector complement sequence shown in SEQ ID NO: 5. For example, the ddRNAi construct may include the DNA sequence shown in SEQ ID NO: 12 or a nucleic acid composed therefrom (shmiR13) and the DNA sequence shown in SEQ ID NO: 13 or a nucleic acid composed therefrom (shmiR17).

[0077] In one particular instance, a therapeutic composition effective for treating or preventing dysphagia associated with OPMD and administered in the methods of this disclosure is designated as BB-301 and comprises an AAV9 carrier, said carrier comprising:

[0078] (a) A viral capsid protein from AAV9, said viral capsid protein comprising mutations A67E, Q81R, K84D, and A85S identified relative to the full-length wild-type AAV9 capsid sequence shown in SEQ ID NO: 19, optionally said viral capsid protein comprising the amino acid sequence shown in SEQ ID NO: 28; and

[0079] (b) A 'silence and replacement' construct, which includes:

[0080] (i) A muscle-specific promoter (optionally, the promoter is the Spc512 muscle-specific promoter);

[0081] (ii) A DNA-guided RNAi (ddRNAi) construct comprising: a nucleic acid comprising a sequence encoding shmiR13 as described herein (optionally the DNA sequence shown in SEQ ID NO: 12) or composed thereof; and a nucleic acid comprising a sequence encoding shmiR17 as described herein (optionally the DNA sequence shown in SEQ ID NO: 13); and

[0082] (iii) A PABPN1 construct comprising a nucleic acid sequence encoding a functional PABPN1 protein comprising the amino acid sequence shown in SEQ ID NO: 16, wherein the nucleic acid sequence encoding the functional PABPN1 protein is a DNA sequence that is codon-optimized such that the corresponding mRNA sequence of the DNA sequence is not targeted by the shmiR encoded by the ddRNAi construct (optionally wherein the codon-optimized nucleic acid comprises the sequence shown in SEQ ID NO: 15).

[0083] The muscle-specific promoter is operatively linked to the ddRNAi construct and the PABPN1 construct.

[0084] In each of the examples above, the method may comprise administering a therapeutic composition (such as the examples described herein and the designated BB-301) for treating or preventing dysphagia associated with OPMD in a volume ranging from about 25 µl to about 75 µl per injection site. The therapeutic composition may be administered at a concentration between about 1.0+E12 vg / mL and about 1.0+E15 vg / mL. For example, the concentration of the therapeutic composition administered to a subject for treating dysphagia associated with OPMD may be between about 5.0+E12 vg / mL and about 1.0+E14 vg / mL, such as between about 5.0+E12 vg / mL and 5.0+E13 vg / mL.

[0085] The therapeutic composition (e.g., BB-301) can be applied (by injection) to multiple sites within the pharyngeal muscle group. For example, the method may involve injecting the therapeutic composition at approximately 2 to approximately 30 or more sites within the pharyngeal muscle. In some instances, the method may involve injecting the therapeutic composition at one or more locations within the pharyngeal muscle at approximately 2 to approximately 30 sites (e.g., approximately 4 to approximately 8 sites). For example, the method may involve injecting the therapeutic composition at one or more segments or approximately 4 to approximately 8 sites within each of the following segments of the pharyngeal muscle: a segment located on the left lateral side of the pharyngeal suture of the middle pharyngeal constrictor muscle; a segment located on the right lateral side of the pharyngeal suture of the middle pharyngeal constrictor muscle; a segment located on the left lateral side of the pharyngeal suture of the hypopharyngeal constrictor muscle; and a segment located on the right lateral side of the pharyngeal suture of the hypopharyngeal constrictor muscle. For example, the method may include: injecting the therapeutic composition at approximately four sites in a segment located on the left lateral side of the pharyngeal suture of the middle pharyngeal constrictor muscle; injecting the therapeutic composition at approximately four sites in a segment located on the right lateral side of the pharyngeal suture of the middle pharyngeal constrictor muscle; injecting the therapeutic composition at approximately eight sites in a segment located on the left lateral side of the pharyngeal suture of the hypopharyngeal constrictor muscle; and injecting the therapeutic composition at approximately eight sites in a segment located on the right lateral side of the pharyngeal suture of the hypopharyngeal constrictor muscle.

[0086] In each of the foregoing examples of multiple injections of a therapeutic composition (e.g., BB-301) into the pharyngeal muscle (including methods of injection at multiple locations or sites within the pharyngeal muscle), each injection volume may be from about 12.5 μL to about 25 µL to about 75 µL, preferably from 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 at each injection site may be a single injection volume or the cumulative volume of multiple injections administered at the injection site. For example, administering a volume of about 75 µL per injection site may comprise administering three injections of about 25 µL per injection site.

[0087] In each of the foregoing examples of intramuscular injection of the 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 the subject in a single treatment may be from about 1.0e13 vg / subject to about 5.5e13 vg / subject. For example, the total dose of the therapeutic composition administered to the subject in a single treatment may be about 1.2e13 vg / subject ('low dose'). For example, the total dose of the therapeutic composition administered to the subject in a single treatment may be about 3.6e13 vg / subject ('moderate dose'). For example, the total dose of the therapeutic composition administered to the subject in a single treatment may be about 5.4e13 vg / subject ('high dose').

[0088] In each of the foregoing examples, the therapeutic composition described herein (e.g., BB-301) may be provided in a pre-filled syringe.

[0089] This disclosure also provides a pre-filled syringe containing a therapeutic composition (e.g., BB-301) effective for treating or preventing dysphagia associated with OPMD. As described herein, the therapeutic composition (e.g., BB-301) may comprise an AAV9 carrier including:

[0090] (a) A viral capsid protein from AAV9, said viral capsid protein comprising mutations A67E, Q81R, K84D, and A85S identified relative to the full-length wild-type AAV9 capsid sequence shown in SEQ ID NO: 19, optionally said viral capsid protein comprising the amino acid sequence shown in SEQ ID NO: 28; and

[0091] (b) A 'silence and replacement' construct, which includes:

[0092] (i) A muscle-specific promoter (optionally, the promoter is the Spc512 muscle-specific promoter);

[0093] (ii) A DNA-guided RNA interference (ddRNAi) construct comprising: a nucleic acid comprising a sequence encoding shmiR13 as described herein (optionally the DNA sequence shown in SEQ ID NO: 12) or composed thereof; and a nucleic acid comprising a sequence encoding shmiR17 as described herein (optionally the DNA sequence shown in SEQ ID NO: 13); and

[0094] (iii) A PABPN1 construct comprising a nucleic acid sequence encoding a functional PABPN1 protein comprising the amino acid sequence shown in SEQ ID NO: 16, wherein the nucleic acid sequence encoding the functional PABPN1 protein is a DNA sequence that is codon-optimized such that the corresponding mRNA sequence of the DNA sequence is not targeted by the shmiR encoded by the ddRNAi construct (optionally wherein the codon-optimized nucleic acid comprises the sequence shown in SEQ ID NO: 15).

[0095] The muscle-specific promoter is operatively linked to the ddRNAi construct and the PABPN1 construct.

[0096] This disclosure also provides an injection device comprising: a pre-filled syringe containing a therapeutic composition (e.g., BB-301) effective for treating or preventing dysphagia associated with OPMD described herein; and an injection needle of this disclosure coupled to the pre-filled syringe. For example, the injection device may comprise a pre-filled syringe as described above coupled to a curved or bent injection needle comprising any one or more of the features of an injection needle described above with respect to the first aspect of this disclosure (e.g., needle A described herein). For example, the pre-filled syringe can be coupled to an injection needle comprising a needle cannula, the needle cannula comprising: a proximal portion having a proximal end; a distal portion having a distal end; an intermediate portion disposed between the proximal and distal portions; and a lumen extending through the proximal, intermediate, and distal portions between the proximal end of the proximal portion and the distal end of the distal portion, the lumen opening at or near the distal end of the distal portion. The proximal and distal portions can be substantially straight, and the intermediate portion can be curved such that the distal portion extends at an angle relative to the proximal portion. The distal portion can extend relative to the proximal portion at an angle between 75° and 105° (e.g., between about 85° and about 95°, or about 90°). Alternatively or additionally, the length of the proximal portion can be shorter than the length of the distal portion. The needle cannula can be between about 10 mm and about 27 mm, for example, the total length is about 16 mm to about 22 mm, for example, the total length is about 18 mm to about 20 mm, for example, about 19 mm. The needle cannula can be about 26-28 gauge, for example, a 27 gauge needle. The needle cannula can have a beveled needle tip. In a preferred embodiment, the injection needle includes a needle cannula comprising: a proximal portion having a proximal end; a distal portion having a distal end; an intermediate portion disposed between the proximal and distal portions; and a lumen extending through the proximal, intermediate, and distal portions between the proximal end of the proximal portion and the distal end of the distal portion, the lumen opening at or near the distal end of the distal portion, and wherein the proximal and distal portions are substantially straight, and the intermediate portion is curved such that the distal portion extends relative to the proximal portion at an angle between about 85° and about 95° (optionally about 90°).The length of the proximal portion may be shorter than the length of the distal portion, the total length of the needle cannula may be approximately 18 mm to approximately 20 mm, for example, approximately 19 mm, the needle cannula may be approximately 26-28 gauge, for example, 27 gauge needle, and / or the needle cannula may have a beveled needle tip.

[0097] The injection device can deliver a therapeutic composition (e.g., BB-301) in multiple injections. The injection device can be configured to deliver a therapeutic composition (e.g., BB-301 as described herein) from a pre-filled syringe in a series of single or multiple injections. Each injection can have a predetermined injection volume. For example, each injection volume can be from about 12.5 µl to about 75 µl, preferably from about 50 µl to about 75 µl. For example, each injection volume can be 50 µl. For example, each injection volume can be 75 µl.

[0098] The injection device may further include an injection volume setting mechanism operable to set and / or adjust the injection volume dispensed for any injection during one or more injections.

[0099] The injection volume setting mechanism is operable to set the injection volume between about 12.5 µL and about 75 µL, or between about 50 µL and about 75 µL. For example, the injection volume setting mechanism is operable to set the injection volume to 50 µL. For example, the injection volume setting mechanism is operable to set the injection volume to 75 µL. The injection setting mechanism is operable to select from a set of discrete injection volumes. The set of discrete injection volumes may include one or more of the following: about 12.5 µL; about 25 µL; about 50 µL; and about 75 µL. The injection device may be configured to provide auditory and / or tactile feedback to the user to indicate that the injection is complete.

[0100] Therefore, it is contemplated that this disclosure could 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 treating or preventing dysphagia associated with OPMD, and the pre-filled syringe is capable of delivering the therapeutic composition (e.g., BB-301 as described herein) in multiple injections, each injection having a predetermined injection volume. Each injection volume may be from about 12.5 µl to about 75 µl, from about 25 µl to about 75 µl, preferably from 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.

[0101] This disclosure also provides a kit comprising a group of two or more injection devices as described herein, wherein 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 treating a single subject. The single therapeutic dose may comprise: approximately 1.2e13 vg / subject; approximately 3.6e13 vg / subject; or approximately 5.4e13 vg / subject.

[0102] In some instances, the kit may contain four of the aforementioned injection devices. For example, a kit containing four of the aforementioned injection devices may be configured to treat a subject according to the method described herein, wherein: one injection device is used to inject the therapeutic composition at multiple sites (e.g., four or more injection sites) within a segment located on the left lateral side of the pharyngeal suture of the midpharyngeal constrictor muscle; one injection device is used to inject the therapeutic composition at multiple sites (e.g., four or more injection sites) within a segment located on the right lateral side of the pharyngeal suture of the midpharyngeal constrictor muscle; one injection device is used to inject the therapeutic composition at multiple sites (e.g., eight or more injection sites) within a segment located on the left lateral side of the pharyngeal suture of the hypopharyngeal constrictor muscle; and one injection device is used to inject the therapeutic composition at multiple sites (e.g., eight or more injection sites) within a segment located on the right lateral side of the pharyngeal suture of the hypopharyngeal constrictor muscle. 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 in the injection apparatus may contain between about 200 µL and about 2000 µL, or between about 400 µL and about 1000 µL, such as 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 the therapeutic composition (e.g., BB-301).

[0103] In some examples, the kit includes two injection devices containing between about 200 µL and about 1000 µL of the therapeutic composition; and two injection devices containing between about 400 µL and about 2000 µL of the therapeutic composition. In some examples, the kit includes two injection devices containing between about 200 µL and about 500 µL of the therapeutic composition; and two injection devices containing between about 400 µL and about 1000 µL of the therapeutic composition.

[0104] One or more injection devices in the kit can be configured to dispense the therapeutic composition (e.g., BB-301) in a series of injections, with each injection volume being at least 25 μL. One or more injection devices in the kit can be configured to dispense the therapeutic composition (e.g., BB-301) in a series of injections, with each injection volume being approximately 50 μL. One or more injection devices in the kit can be configured to dispense the therapeutic composition (e.g., BB-301) in a series of injections, with each injection volume being approximately 75 μL.

[0105] In some instances, the method comprises applying the therapeutic composition to one or more locations of the pharyngeal muscles, one or more pharyngeal muscles, one or more sides of the pharyngeal muscles, or any combination thereof.

[0106] In any of the above aspects, the therapeutic composition can be administered to the pharyngeal muscles by making an incision in the subject's neck to obtain a direct access to the pharyngeal muscles and then injecting directly.

[0107] Sequence List Symbol Explanation

[0108] SEQ ID NO: 1: RNA sequence of region 13 of the mRNA transcript corresponding to the PABPN1 protein.

[0109] SEQ ID NO: 2: RNA sequence of region 17 of the mRNA transcript corresponding to the PABPN1 protein.

[0110] SEQ ID NO: 3: RNA effector complement sequence of shmiR designated as shmiR13.

[0111] SEQ ID NO: 4: RNA effector sequence of shmiR designated as shmiR13.

[0112] SEQ ID NO: 5: RNA effector complement sequence of shmiR designated as shmiR17.

[0113] SEQ ID NO: 6: RNA effector sequence of shmiR designated as shmiR17.

[0114] SEQ ID NO: 7: RNA stem-loop sequence of shmiR.

[0115] SEQ ID NO: 8: 5' flanking sequence of the primary miRNA backbone.

[0116] SEQ ID NO: 9: 3' flanking sequence of the primary miRNA backbone.

[0117] SEQ ID NO: 10: RNA sequence of shmiR designated as shmiR13.

[0118] SEQ ID NO: 11: RNA sequence of shmiR designated as shmiR17.

[0119] SEQ ID NO: 12: DNA sequence encoding shmiR designated as shmiR13.

[0120] SEQ ID NO: 13: DNA sequence encoding shmiR designated as shmiR17.

[0121] SEQ ID NO: 14: DNA sequence encoding coPABPN1 and shmiR designated as shmiR17 and shmiR13 controlled by Spc512, dual construct version 2.

[0122] SEQ ID NO: 15: DNA sequence of PABPN1 cDNA sequence optimized with human codons.

[0123] SEQ ID NO: 16: Amino acid sequence of human wild-type PABPN1 protein.

[0124] SEQ ID NO: 17: Wild-type VP1 subsequence of AAV serotype 9, containing PLA2 domain and flanking sequence.

[0125] SEQ ID NO: 18: Modified VP1 subsequence variant 1 of AAV serotype 9, containing PLA2 domain and flanking sequences.

[0126] SEQ ID NO: 19: Full-length wild-type AAV serotype 9 capsid.

[0127] SEQ ID NO: 20: Full-length modified AAV serotype 9 capsid variant 1.

[0128] SEQ ID NO: 21: Wild-type VP1 subsequence of AAV serotype 8, containing PLA2 domain and flanking sequence.

[0129] SEQ ID NO: 22: Modified VP1 subsequence of AAV serotype 8, containing PLA2 domain and flanking sequence.

[0130] SEQ ID NO: 23: Full-length wild-type AAV serotype 8 capsid.

[0131] SEQ ID NO: 24: Full-length modified AAV serotype 8 capsid.

[0132] SEQ ID NO: 25: AAV2 5' ITR sequence variant 1.

[0133] SEQ ID NO: 26: AAV2 3' ITR sequence variant 1.

[0134] SEQ ID NO: 27: Modified VP1 subsequence variant 2 of AAV serotype 9, containing PLA2 domain and flanking sequences.

[0135] SEQ ID NO: 28: Full-length modified AAV serotype 9 capsid variant 2.

[0136] SEQ ID NO: 29: AAV2 5' ITR sequence variant 2.

[0137] SEQ ID NO: 30: AAV2 3' ITR sequence variant 2. Attached Figure Description

[0138] The embodiments will now be described by way of example only, with reference to the accompanying drawings, in which:

[0139] Figure 1 This is a side view of an injection needle according to an embodiment of the present disclosure;

[0140] Figure 2 yes Figure 1 Another side view of the injection needle;

[0141] Figure 3 yes Figure 1 An additional side view of the injection needle with example dimensions and angles;

[0142] Figure 4 yes Figure 1 An enlarged schematic diagram of the distal portion and distal tip of the injection needle;

[0143] Figure 5 yes Figure 1 An enlarged frontal elevation view of the distal portion and distal tip of the injection needle;

[0144] Figure 6A yes Figure 1 An enlarged cross-sectional view of the middle section of the injection needle;

[0145] 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;

[0146] Figure 7AThis is a side view of an alternative injection needle design for comparative purposes (injection needle B discussed in this paper).

[0147] Figure 7B yes Figure 7A An enlarged perspective view of the tip of the alternative injection needle shown in the image;

[0148] Figure 8A and 8B This is a schematic diagram of the human neck and the use of an injection needle according to an embodiment of the present disclosure;

[0149] Figure 9 A is a schematic diagram illustrating a construct for simultaneously performing gene silencing of endogenous PAPPN1 and replacing it with codon-optimized PAPPN1. The construct is generated by subcloning two shmiRs targeting wtPABPN1 and mPABPN1 into the 3' untranslated region of a codon-optimized PAPPN1 transcript and placing them between two pAAV2 ITRs (ITRs are not shown in the schematic diagram).

[0150] Figure 9 B is a schematic diagram illustrating a 'silencing and replacement' construct (SR construct) designed to simultaneously perform gene silencing of endogenous PAPPN1 and replacement with codon-optimized PAPPN1. This construct is generated by subcloning two shmiRs (shmiR17 and shmiR13) targeting wtPABPN1 and mPABPN1 into the 3' untranslated region of a codon-optimized PAPPN1 transcript within the pAAV2 vector backbone; and

[0151] Figure 10 This is a schematic diagram of the posterior part of the human pharyngeal muscles. Detailed Implementation

[0152] General definition

[0153] Unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include plural indicators.

[0154] Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” shall be understood to imply inclusion of the stated elements, integers or steps, or groups of elements, groups of integers or groups of steps, but not to exclude any other elements, integers or steps, or groups of elements, groups of integers or groups of steps.

[0155] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be regarded as providing explicit support for both or either of these meanings.

[0156] Those skilled in the art will understand that this disclosure is open to variations and modifications beyond those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, and any and all combinations of any two or more of said steps or features.

[0157] Those skilled in the art will understand that numerous variations and / or modifications can be made to the above embodiments without departing from the broad general scope of this disclosure. Therefore, the embodiments of the present invention should be considered illustrative rather than restrictive in all respects.

[0158] Throughout this specification, unless otherwise expressly stated or required by the context, references to individual steps, features, material compositions, groups of steps, or groups of features or material compositions shall be deemed to cover one or more (i.e., one or more) of such steps, features, material compositions, groups of steps, or groups of features or material compositions.

[0159] This disclosure is not limited to the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are obviously within the scope of this disclosure.

[0160] Unless otherwise expressly stated, any instance of this disclosure herein, with necessary modifications, shall be considered applicable to any other instance of this disclosure.

[0161] Unless otherwise specified, all technical and scientific terms used herein should be regarded as having the same meaning as commonly understood by a person skilled in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0162] Unless otherwise specified, all technical and scientific terms used herein should be regarded as having the same meaning as commonly understood by a person skilled in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0163] Unless otherwise stated, the recombinant DNA, recombinant protein, cell culture, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained in the literature from the following sources: 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 (ed.), *Essential Molecular Biology: A Practical Approach*, Volumes 1 & 2, IRL Press (1991); DM Glover and BD Hames (ed.), *DNA Cloning: A Practical Approach*, Volumes 1–4, IRL Press (1995 and 1996); and FMAusubel et al. (ed.), *Current Protocols in Molecular Biology*. Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988); and JE Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0164] Selected definition

[0165] “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-ribose-furanose moiety. The term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and altered RNA that 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, such as to the end or interior of siRNA, for example at one or more nucleotides of RNA. The nucleotides in the RNA molecules of this disclosure may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0166] The term "RNA interference" or "RNAi" generally refers to RNA-dependent silencing of gene expression induced by double-stranded RNA (dsRNA) molecules in the cytoplasm of cells. dsRNA molecules reduce or inhibit the transcription products of target nucleic acid sequences, thereby silencing or reducing gene expression.

[0167] As used herein, the term "double-stranded RNA" or "dsRNA" refers to an RNA molecule having a double-stranded structure and comprising an effector sequence and an effector complement sequence of similar length. The effector sequence and effector complement sequence may be in a single RNA strand or in separate RNA strands. The "effector sequence" (often referred to as the "guide strand") is substantially complementary to the target sequence, in this example, a region of the PABPN1 mRNA transcript. The "effector sequence" may also be referred to as an "antisense sequence." The "effector complement sequence" will be sufficiently complementary to the effector sequence such that it can anneal to the effector sequence to form a double strand. In this respect, the effector complement sequence will be substantially homologous to a region of the target sequence. As will be apparent to those skilled in the art, the term "effector complement sequence" may also be referred to as the "complement of the effector sequence" or a sense sequence.

[0168] As used herein, the term "double-stranded" refers to a region in two complementary or substantially complementary nucleic acids (e.g., RNA), or two complementary or substantially complementary regions in a single-stranded nucleic acid (e.g., RNA), which form base pairs with each other through Watson-Crick base pairing or any other means that allows for a stable double-stranded structure between complementary or substantially complementary nucleotide sequences. Those skilled in the art will understand that 100% complementarity is not required within a double-stranded region; substantial complementarity is permitted. Substantial complementarity can include 79% or greater complementarity. For example, a single mismatch (i.e., 18 base pairs and one mismatch) in a double-stranded region of 19 base pairs achieves 94.7% complementarity, thus making the double-stranded region substantially complementary. In another example, two mismatches (i.e., 17 base pairs and two mismatches) in a double-stranded region of 19 base pairs achieve 89.5% complementarity, thus making the double-stranded region substantially complementary. In yet another example, three mismatches in a double-stranded region consisting of 19 base pairs (i.e., 16 base pairs and three mismatches) achieve 84.2% complementarity, thus making the double-stranded region essentially complementary, and so on.

[0169] dsRNAs can be provided as hairpin or stem-loop structures, having a double-stranded region comprising an effector sequence and an effector complement sequence linked by at least two nucleotide sequences, referred to as a stem-loop. When dsRNAs are provided as hairpin or stem-loop structures, they can be referred to as “hairpin RNA”, “short hairpin RNAi agents”, or “shRNA”. Other dsRNA molecules provided in or generating hairpin or stem-loop structures include primary miRNA transcripts (primary miRNAs) and precursor microRNAs (precursor miRNAs). Precursor miRNAs (shRNAs) can be naturally generated from primary miRNAs by the action of Drosha and Pasha enzymes, which recognize and release the region of the primary miRNA transcript that forms the stem-loop structure. Alternatively, primary miRNA transcripts can be engineered to replace the natural stem-loop structure with an artificial / recombinant stem-loop structure. That is, an artificial / recombinant stem-loop structure can be inserted into or cloned into a primary miRNA backbone sequence lacking a natural stem-loop structure. With stem-loop sequences engineered to be expressed as part of a primary miRNA molecule, Drosha and Pasha recognize and release the artificial shRNA. dsRNA molecules produced using this method are called “shmiRNA,” “shmiR,” or “microRNA framework shRNA.”

[0170] As used herein, the term "complementary" in relation to a sequence refers to the complementarity of sequences through Watson-Crick base pairing, whereby guanine (G) pairs with cytosine (C), and adenine (A) pairs with uracil (U) or thymine (T). A sequence may be complementary to the entire length of another sequence, or it may be complementary to a specific portion or length of another sequence. Those skilled in the art will recognize that U can be present in RNA, and T can be present in DNA. Thus, A within an RNA or DNA sequence can 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 can pair with C or U in RNA.

[0171] As used herein, the term "substantially complementary" indicates a sufficient degree of complementarity or exact pairing to allow stable and specific binding between nucleic acid sequences (e.g., between an effector sequence and an effector complement sequence, or between an effector sequence and a target sequence). It should be understood that the nucleic acid sequence does not need to be 100% complementary to its target or complement sequence. Except for overhangs, the term encompasses sequences complementary to another sequence. In some cases, the sequence is complementary to other sequences except for 1-2 mismatches. In some cases, the sequence is complementary except for 1 mismatch. In some cases, the sequence is complementary except for 2 mismatches. In other cases, the sequence is complementary except for 3 mismatches. In still other cases, the sequence is complementary except for 4 mismatches.

[0172] As used in the context of the shRNA or shmiR disclosed herein, the term “encode” should be understood to mean an shRNA or shmiR capable of being transcribed from a DNA template. Thus, the nucleic acid encoding or coding the shRNA or shmiR of this disclosure will contain a DNA sequence that serves as a template for transcription of the corresponding shRNA or shmiR.

[0173] The terms "DNA-guided RNAi construct" or "ddRNAi construct" refer to nucleic acids containing a DNA sequence that, upon transcription, produces shRNA or shmiR molecules (preferably shmiR) that initiate RNAi. A ddRNAi construct may contain nucleic acids transcribed into a single RNA capable of self-annealing into a hairpin structure having a double-stranded region connected by a stem-loop of at least two nucleotides, i.e., shRNA or shmiR; or transcribed into a single RNA having multiple shRNAs or shmiRs; or transcribed into multiple RNA transcripts, each capable of folding into a single shRNA or shmiR. A ddRNAi construct may be provided in a larger "DNA construct" containing one or more additional DNA sequences. For example, a ddRNAi construct may be provided in a DNA construct containing additional DNA sequences encoding a functional PABPN1 protein (whose codons have been optimized so that its mRNA transcript is not targeted by the shmiR of the ddRNAi construct). ddRNAi constructs and / or DNA constructs containing ddRNAi constructs can be in expression vectors, for example, operatively linked to a promoter.

[0174] As used herein, the term “operationally ligated” or “operationally linked” (or similar expressions) means that a coding nucleic acid sequence is linked or associated with a regulatory sequence (e.g., a promoter) in a manner that promotes the expression of the coding sequence. Regulatory sequences include promoters, enhancers, and other expression control elements that are recognized and selected for guiding the expression of the coding sequence.

[0175] “Vector” will be understood as a medium used to introduce nucleic acids into cells. Vectors include, but are not limited to, plasmids, phage particles, viruses, bacteria, and media derived from viruses or bacteria. A “plasmid” is a circular double-stranded DNA molecule. A useful type of vector used according to this disclosure is a viral vector in which a heterologous DNA sequence is inserted into a viral genome, which may be modified to delete one or more viral genes or portions thereof. Some vectors are capable of autonomous replication in host cells (e.g., vectors having a replication origin that functions in the host cell). Other vectors can stably integrate into the genome of a host cell and thereby replicate along with the host genome. As used herein, the term “expression vector” will be understood as a vector capable of expressing the RNA molecules of this disclosure.

[0176] "Functional PABPN1 protein" should be understood to mean a PABPN1 protein that possesses the functional properties of the wild-type PABPN1 protein (e.g., the ability to control sites 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. In one instance, "functional PABPN1 protein" as used herein refers to the human wild-type PABPN1 protein. The sequence of the human wild-type PABPN1 protein is shown in NCBI RefSeq NP_004634. Therefore, a functional human PABPN1 protein may possess the functional properties of the human PABPN1 protein shown in NCBI RefSeq NP_004634 in vivo.

[0177] As used herein, the term "dysphagia" describes a swallowing disorder, including inability to swallow, difficulty in swallowing, or discomfort in swallowing. Dysphagia may also affect speech and / or breathing in subjects with swallowing disorders. In some cases, dysphagia is secondary to a diagnosis of other symptoms or conditions (e.g., OPMD). For example, dysphagia may be secondary to a subject's OPMD genetic diagnosis.

[0178] As used herein, the terms “treating,” “treat,” or “treatment,” and variations thereof, refer to a clinical intervention designed to alter the natural processes of the treated individual or cells during a clinicopathological process. Desired therapeutic effects include reduced disease progression rates, improved or alleviated disease states, and remission or improved prognosis. Treatment of dysphagia can improve the strength and / or coordination of pharyngeal muscles, and thereby improve swallowing in the subject. Treatment of dysphagia can also improve speech and / or breathing in the subject. In the case of treatment 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 the PABPN1 protein with 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 the PABPN1 protein with normal length of polyalanine residues in the subject. Treatment of underlying OPMD can treat associated dysphagia. For example, if one or more of the above treatment outcomes are achieved, the individual is considered to have been successfully "treated".

[0179] As used herein, the terms “prevent,” “preventing,” or similar terms should be understood to include preventing, reducing, or eliminating the development of at least one symptom of the condition. As used in the context of dysphagia, the terms “prevent” or “preventing” should be understood to prevent, reduce, or eliminate the development of one or more symptoms associated with dysphagia (as described above). For example, the methods of this disclosure can be used to prevent dysphagia in subjects who have been genetically diagnosed with OPMD (e.g., including PABPN1 allele classifications: (GCN)12, (GCN)13, (GCN)14, (GCN)15, or (GCN)16) but have not yet exhibited physical symptoms of dysphagia. Therefore, the methods of this disclosure can be used to prevent, reduce, or eliminate the development of dysphagia in subjects, despite their genetic predisposition to OPMD and associated dysphagia.

[0180] A “therapeutic effective amount” is at least the minimum concentration or amount required to affect a measurable improvement in the dysphagia to be treated. Based on instances where dysphagia is associated with OPMD, a therapeutic effective amount is an amount sufficient to affect a measurable improvement in one or more symptoms of OPMD (e.g., dysphagia and weight loss in the subject). Measurable improvement can be determined by a variety of clinical assessment methods, such as: assessing pharyngeal constrictor muscle function by measuring the pharyngeal area at maximal contraction; assessing swallowing efficiency by determining total pharyngeal residue; assessing subject-reported oropharyngeal dysphagia using the Sydney Swallow Questionnaire; and assessing swallowing ability using a timed cold water drinking test. An exemplary clinical assessment method for measuring improvement in one or more symptoms of OPMD, as considered herein, is described in Example 3 of this disclosure.

[0181] The therapeutically effective dose described herein can vary depending on factors such as the patient's disease state, age, sex, and weight. The therapeutically effective dose of the compositions described herein for treating OPMD and OPMD-associated dysphagia can further vary depending on various factors, such as the ability of shmiR, the nucleic acid encoding shmiR, the ddRNAi construct, the DNA construct, the expression vector, or the composition comprising thereof to elicit the desired response in an individual and / or the ability of the expression vector to express functional PABPN1 protein in a subject. The therapeutically effective dose is also a dose in which any toxic or adverse effects of shmiR, the nucleic acid encoding shmiR, the ddRNAi construct, the DNA construct, the expression vector, or the composition comprising thereof are outweighed by the therapeutically beneficial effects of shmiR, the nucleic acid encoding shmiR, the ddRNAi construct, the DNA construct, the expression vector, or the composition comprising thereof to inhibit, suppress, or reduce the expression of PABPN1 protein causing OPMD-associated dysphagia, considered alone or in combination with the therapeutically beneficial effects of expressing functional PABPN1 protein in a subject.

[0182] As used herein, "subject" or "patient" can be a human or non-human animal that has or is genetically susceptible to OPMD (i.e., has a variant of the PABPN1 gene that causes OPMD). "Non-human animal" can be a primate, livestock (e.g., sheep, horses, cattle, pigs, donkeys), companion animals (e.g., pets such as dogs and cats), laboratory testing animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, *C. elegans*, zebrafish), performance animals (e.g., racehorses, camels, greyhounds), or captive wild animals. In one instance, the subject or patient is a mammal. In another instance, the subject or patient is a human.

[0183] The terms “reduced expression,” “reduced expression,” or similar terms refer to the absence or observable decrease in the level of protein and / or mRNA products from a target gene (e.g., the PABPN1 gene). The reduction is not necessarily absolute, but can be a partial reduction sufficient to cause a detectable or observable change as a result of the influence of RNAi by the shmiR disclosed herein, the nucleic acid encoding the shmiR, the ddRNAi construct, the DNA construct, the expression vector, or a composition containing the thereof. The reduction can be measured by determining a decrease in the level of mRNA and / or protein products from the target nucleic acid relative to cells lacking the shmiR, the nucleic acid encoding the shmiR, the ddRNAi construct, the DNA construct, the expression vector, or a composition containing the thereof, and can be as low as 1%, 5%, or 10%, or can be absolute, i.e., 100% inhibition. The reduction effect can be determined by examining the external characteristics of cells or organisms (i.e., quantitative and / or qualitative phenotypes), and may include detecting the presence or changes in the number of intranuclear inclusions that partially or completely contain expPABPN1 in cells or organisms after administration of the shmiR of this disclosure, nucleic acids encoding shmiR, ddRNAi constructs, DNA constructs, expression vectors, or compositions containing the thereof.

[0184] As used herein, the terms “delivery system,” “delivery vector,” and similar terms refer to a vector used to package foreign genetic material (such as DNA or RNA) and which can be introduced into cells. Delivery vectors can include viral vectors, such as adeno-associated virus (AAV) vectors, retroviral vectors, adenovirus vectors (AdV), and lentiviral (LV) vectors. As described herein, viral vectors can be used to deliver and express foreign genetic material in cells. Therefore, viral expression vectors as described herein can be used as delivery vectors.

[0185] As used herein, the term "adeno-associated virus" or "AAV" refers to a group of viruses within the family Parvoviridae that contain a short (approximately 4.7 kb) single-stranded DNA genome and whose replication depends on the presence of helper viruses, such as adenoviruses. This disclosure also considers vectors derived from AAVs, for example, as gene transfer agents.

[0186] As used herein, the term “serotype” as used in the context of AAV is used to distinguish AAVs having a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined based on the lack of cross-reactivity between one AAV antibody and another AAV. Such differences in cross-reactivity are usually due to differences in capsid protein sequence / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). The VP1 sequence of known wild-type AAVs infecting humans is described in Chen et al., (2013) Journal of Virology 87(11):6391-6405, and includes, for example, 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 accession number: AAD27757.1, GI: 4689097), AAV2 (Genbank accession number: AAC03780.1, GP.2906023), AAV3 (Genbank accession number: AAC55049.1, GI: 1408469), AAV4 (Genbank accession number: AAC58045.1, GL2337940), AAV5 (Genb ANK login number: AAD13756.1, GI-4249658), AAV10 (Genbank login number: AAT46337.1, GL48728343), AAV11 (Genbank login number: AAT46339.1, GI: 48728346), AAV12 (Genbank login number: ABI16639.1, GI: 112379656) or AAV13 (Genbank login number: ABZ10812.1, GI: 167047087).Furthermore, the complete genomes of AAV serotypes 1-13 are known in the art, for example, AAV1 (NCBI reference sequence NC_002077.1), AAV2 (GenBank accession number: J01901.1), AAV3 (GenBank accession number: AF028705.1), AAV4 (NCBI reference sequence: NC_001829.1), AAV5 (NCBI reference sequence: NC_006152.1), and AAV6 (GenBank: A F028704.1), AAV7 (NCBI reference sequence: NC_006260.1), AAV8 (NCBI reference sequence: NC_006261.1), AAV9 (GenBank accession number: AY530579.1), AAV10 (GenBank accession number: AY631965.1), AAV11 (GenBank accession number: AY631966.1), or AAV12 (GenBank accession number: 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, particularly when different AAVs within the same serotype have separated from different host species and / or different geographical locations.

[0187] As used in the context of AAV herein, the terms “viral capsid protein,” “capsid protein,” “capsid polypeptide,” or similar terms refer to polypeptides of AAV that possess self-assembling activity to produce the protein coat (also known as the capsid protein or VP protein) of AAV particles. It comprises three subunits, VP1, VP2, and VP3, typically expressed from a single nucleic acid molecule and interacting together to form an icosahedral symmetrical capsid. The capsid structure of AAV is described in BERNARD N. FIELDS et al., *Virusology*, Vol. 2, Chapters 69 and 70 (4th ed., Lippincott-Raven Publishers).

[0188] As used herein, the term “promoter” generally refers to a DNA sequence involved in recognizing and binding DNA-dependent RNA polymerases and other proteins (trans-acting transcription factors) to initiate and control transcription of one or more coding sequences, and is typically located upstream of the coding sequence relative to the direction of transcription.

[0189] As used herein, “pharyngeal muscle” refers to one or more of the group of muscles that form the pharynx. Pharyngeal muscles may include one or more of the following: hypopharyngeal constrictor, middle pharyngeal constrictor, superior pharyngeal constrictor, palatopharyngeal muscle, eustachian tube pharyngeal muscle, and / or styloid pharyngeal muscle.

[0190] Injection needle

[0191] In one embodiment according to this disclosure, and as Figures 1 to 3 As shown, the injection needle 10 includes a needle cannula 20 having a proximal portion 21, a distal portion 23, and a middle portion 22. The proximal portion has a proximal end 211, the distal portion has a distal end 231, and an outlet 25 located at or adjacent to the distal end 231. The middle portion is disposed between the proximal portion 21 and the distal portion 23. A lumen 26 extends through the proximal portion 21, the middle portion 22, and the distal portion 23 between the proximal end 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.

[0192] like Figures 1 to 3 As shown, the injection needle 10 also includes a needle hub 30 connected to the 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 include a support portion 28 (in... Figure 1 (Indicated by dashed lines in the image), the support portion extends proximally from the proximal end 211 of the proximal portion 21 of the needle cannula, and the support portion is disposed (e.g., fully disposed) within the needle hub 30. The lumen 26 may further extend through the support portion 28.

[0193] The needle cannula 20 includes an inlet 24 located at or near the proximal end 211 of the proximal portion 21, or at or near the proximal end 281 of the support portion 28 (if present). For example, when fluid is injected into a patient, the fluid can enter the needle cannula 20 via the inlet 24 and proceed to the outlet 25.

[0194] In this embodiment, the needle hub 30 includes a distal portion 31 adapted to connect to the needle cannula 20 (e.g., by securing or embedding a support portion 28 of the needle cannula therein) and an opposing proximal portion 32. The proximal portion 32 includes threads 33 on its outer surface, adapted to engage with complementary threads of another device (such as a syringe barrel), wherein a fluid substance (e.g., a therapeutic composition) can be stored prior to delivery through the needle cannula 20. Figure 1 and 3 As seen in the image, the distal portion 31 can be die-forged (gradually tapering towards the central axis).

[0195] In this embodiment, the proximal portion 21 and the distal portion 23 are each substantially straight. In this embodiment, the intermediate portion 22 is bent, and more specifically, curved, such that the distal portion 23 extends relative to the proximal portion 21 at an angle between approximately 75° and 105°.

[0196] The boundary between the proximal portion 21 and the intermediate portion 22 of the needle cannula 20 can be defined at a location where the needle cannula 20 extends in a straight line from the proximal portion 21 to a bend or curve at the intermediate portion 22. Similarly, the boundary between the intermediate portion 22 and the distal portion 23 of the needle cannula 20 can be defined at a location where the needle cannula 20 extends in a straight direction from the intermediate portion 22 to a location where it bends or curves at the intermediate portion 22.

[0197] Angle of bending

[0198] The middle portion 22 of the needle cannula 20 is bent, and more specifically curved (e.g., arched), to assist in guiding anatomical structures at the injection site. This particular configuration has been found to be particularly suitable for delivering therapeutic compositions directly to one or more pharyngeal muscles, with the needle cannula effectively guiding the anatomical structures surrounding the pharyngeal muscles.

[0199] As shown in the figure, for example in Figure 6A and 6B In the middle portion, the portion may be bent or curved, such that the distal portion 23 extends relative to the proximal portion 21 at an angle β between approximately 75° and 105°. For example, as... Figure 6B As shown in the embodiments, the middle portion 22 may be bent or curved, such that the distal portion 23 extends at an angle of approximately 105° relative to the proximal portion 21.

[0200] In some embodiments, the distal portion 23 extends relative to the proximal portion 21 at an angle β between about 80° and 100°. In other embodiments, the distal portion 23 extends relative to the proximal portion 21 at an angle between about 85° and 95°. In other embodiments, the distal portion 23 extends relative to the proximal portion 21 at an angle between about 87° and 93°. In other embodiments, and as in, for example... Figure 6A As shown, the distal portion 23 extends at an angle β of approximately 90° relative to the proximal portion 21.

[0201] As indicated above, the proximal portion 21 and the distal portion 23 may each be perfectly straight. However, in other embodiments, the proximal portion 21 and / or the distal portion 23 may have one or more bends, angles, or irregularities while still being substantially straight compared to the intermediate portion. In such cases, when determining the angles at which the proximal portion 21 and the distal portion 23 extend, this may be based on a conceptual straight line extending between the proximal end 211 of the proximal portion 21 and the proximal end of the intermediate portion 22, and a conceptual straight line extending between the distal end of the intermediate portion 22 and the distal end 231 of the distal portion 23, respectively.

[0202] In some embodiments, the proximal portion 21, the intermediate portion 22, and the distal portion 23 of the needle cannula 20 extend entirely in a common plane. Alternatively, these portions may not extend entirely in a common plane. For example, the needle cannula may be bent or flexed in three dimensions.

[0203] Injection device

[0204] This disclosure also provides an injection device 200, such as the drug product injection device (DPID) 200 shown in FIG8, which includes a needle 10 coupled to a syringe 100 (e.g., a pre-filled syringe).

[0205] The pre-filled syringe 100 may include a barrel and a plunger housed 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 may slide longitudinally relative to the barrel to expel the therapeutic composition from the syringe 100.

[0206] The injection device 200 may include an injection volume setting mechanism operable to set and / or adjust the injection volume dispensed with any injection during 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 configured to be coupled to one or more components of the pre-filled syringe.

[0207] The injection volume setting mechanism may be operable to set the injection volume between about 12.5 µL and about 75 µL, or between about 50 µL and about 75 µL. For example, the injection setting mechanism may be operable to select from a set of discrete injection volumes. The set of discrete injection volumes may include one or more of the following: about 12.5 µL; about 25 µL; about 50 µL; and about 75 µL.

[0208] The injection device can be configured to provide auditory and / or tactile feedback to the user to indicate that the injection is complete. For example, the plunger of the pre-filled syringe 100 may include at least one set of successive plunger formations that provide auditory and tactile feedback to the user through a series of plunger "clicks" when the treatment composition is expelled.

[0209] Use of injection needles

[0210] 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 can be used to specifically inject directly into the pharyngeal constrictor muscle and / or any of the following specific muscles: the hypopharyngeal constrictor muscle, the middle pharyngeal constrictor muscle, the superior pharyngeal constrictor muscle, the palatopharyngeal muscle, the eustachian tube pharyngeal muscle, the stylopharyngeal muscle, or any combination thereof.

[0211] In one embodiment, and as Figures 8A to 8B As shown, an open surgical procedure is performed to allow direct injection into the pharyngeal muscle using the injection needle 10. (Reference) Figure 8A For example, a first incision 511 is made on the first lateral side of the patient's neck 500 using a scalpel 504 to expose at least a portion 521 of at least one pharyngeal muscle, such as the hypopharyngeal constrictor 501, the middle pharyngeal constrictor 502, the superior pharyngeal constrictor 503, or a portion of another pharyngeal muscle. The first lateral side can be the right or left side of the patient's neck (e.g., the left side...). Figure 8A and 8B (As shown in the image).

[0212] A second incision 512 (not shown) may be made on the second lateral side to expose the other side of the hypopharyngeal constrictor muscle 501, the middle pharyngeal constrictor muscle 502, the superior pharyngeal constrictor muscle 503, or other pharyngeal muscles.

[0213] refer to Figure 8B The distal portion of the needle cannula 20 of the injection needle 10 is forced to penetrate the exposed portions of the pharyngeal muscles 501, 502, and 503. The injection device 200 is operated to expel the therapeutic composition from the syringe 100, through the needle cannula 20, and directly into the pharyngeal muscles 501, 502, and 503.

[0214] This injection procedure can be used to treat or prevent dysphagia associated with oculopharyngeal muscular dystrophy (OPMD). For example, in this case, the injection needle can be considered, alternatively or additionally, as a treatment injection needle for oculopharyngeal muscular dystrophy (OPMD). This document describes a method of administering a therapeutic composition using the injection needle of this disclosure to treat or prevent dysphagia associated with OPMD.

[0215] The injection procedure can be repeated on the relatively lateral side of the patient's neck, for example, after a second incision has been made on the relatively lateral side of the patient's neck. Typically, the injection can be administered directly to one or more sites within the pharyngeal muscles, to one or more pharyngeal muscles, to one or more sides of the pharyngeal muscles (e.g., right or left), or any combination thereof. Injections at multiple sites can help increase the delivery of pharyngeal muscle tissue. Injections can also be repeated over time (e.g., weeks, months, or years) to address the issue of diminishing effective amounts of therapeutic agents over time.

[0216] radius of curvature

[0217] As indicated, the middle section 22 is curved.

[0218] like Figure 6AAs shown, the curved middle portion 22 has an arc length l and a substantially constant radius r. Since the proximal portion 21 extends at an angle β of substantially 90° relative 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, and the radius of curvature r is approximately 2.8 mm, satisfying the following equation: radius = circumference / 2π (radius of curvature r = 4(4.4 mm) / 2π = 2.8 mm).

[0219] However, for example, such as Figure 6B As shown in the figure, this illustration depicts an embodiment where the needle cannula 20 has a distal portion 23 that extends relative to the proximal portion at an alternative angle β of approximately 105°, and 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 can be referred to as a series of values ​​r. v .

[0220] In some embodiments, the radius of curvature (r or r0) of the curved middle portion 22 v The radius of curvature (r or r0) is between approximately 2.5 mm and approximately 3.1 mm. In other embodiments, the radius of curvature of the curved intermediate portion 22 is between approximately 2.5 mm and approximately 3.1 mm. v The radius of curvature (r or r0) is between 2.6 mm and 3.0 mm. In another embodiment, the radius of curvature of the curved middle portion 22 is between 2.6 mm and 3.0 mm. v The radius of curvature (r or r0) is between 2.7 mm and 2.9 mm. Furthermore, in another embodiment, the radius of curvature of the curved intermediate portion 22 is... v It is approximately 2.8 mm.

[0221] Those skilled in the art will understand that the arc length and / or radius of curvature of the needle cannula can be varied to scale the needle cannula relative to the patient’s anatomy or other aspects, while maintaining, for example, a constant bending angle between the proximal and distal portions of the needle cannula.

[0222] Total length

[0223] For example, such as Figure 2 As shown, 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 middle portion 22, and the length C of the distal portion 23. Figure 3 In the embodiment shown, the total length of the needle cannula can be approximately 19 mm.

[0224] However, those skilled in the art will understand that the needle cannula 20 described herein may have different lengths, for example, to be scaled relative to the patient’s anatomy or other aspects. In some embodiments, the total length of the needle cannula is between about 10 mm and about 27 mm, between about 13 mm and about 25 mm, between about 15 mm and about 23 mm, between about 17 mm and about 21 mm, or about 19 mm.

[0225] Needle size

[0226] Needle size (G) describes the size of the lumen or orifice of the needle cannula. A higher needle size corresponds to a smaller lumen, and therefore typically a finer needle. Needle sizes are selected for different purposes by balancing factors such as pain minimization, tissue type, rate of substance introduction, and impact on the integrity of the contents. For example, insulin needles are typically fine needles (29G to 31G) to minimize pain during subcutaneous injection, while thicker needles (16G to 17G) can be used for blood donation because the increased lumen exerts less pressure on fragile red blood cells. In this respect, needle size can also be selected based on its impact on the integrity of the needle's components (e.g., viral expression vectors).

[0227] The injection needle described herein can be used during open surgery under anesthesia, and therefore pain minimization may not be a priority. During trials with different needle designs (including those involving injection into the pharyngeal muscle), the priority was minimizing leakage and maximizing the expected tissue transduction of functional virus. This is particularly important given the small volume of drug product delivered at each injection site. In the context of the applicant's drug product BB-301, it was found that needle size has an impact on the proportion of AAV that remains functional after the drug product flows through the injection needle, likely due to the effects of biomechanical forces (e.g., flow rate, pressure, and shear stress) on viral particles during injection. Therefore, a needle size was selected that balances the need to minimize leakage and maximize functional AAV transduction into tissue on the one hand, and the need for a needle with sufficient strength to maintain its curvature and withstand mechanical deformation during use on the other.

[0228] exist Figure 1 In the illustrated embodiment, the needle cannula shown is 27G. Typically, the needle cannula according to this disclosure can be about 23-31, about 25-29, about 26-28, or about 27G or other sizes.

[0229] For ease of reference, the dimensions of the needle cannula described in this article can be found in the Birmingham needle scale.

[0230] However, in some embodiments, the needle cannula 20 may not conform to any standard.

[0231] outer diameter and inner diameter

[0232] In some embodiments, the outer diameter 202 of the needle cannula 20 is between about 0.3 mm and about 0.5 mm, between about 0.35 mm and about 0.45 mm, between about 0.40 mm and about 0.42 mm, or about 0.41 mm, such as... Figure 4 and 5 As shown in the image.

[0233] 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 gradually). The outer diameter 202 may vary continuously along the cannula 20 or vary at one or more discrete segments of the cannula 20.

[0234] In some embodiments, the inner diameter 201 of the needle cannula is between about 0.1 mm and 0.3 mm, between about 0.15 mm and 0.25 mm, between about 0.20 mm and 0.22 mm, or about 0.21 mm.

[0235] 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 gradually). For example, the inner diameter 201 may vary continuously along the cannula 20 or vary at one or more discrete segments of the cannula 20.

[0236] In some embodiments, the wall thickness of the needle cannula 20 is between about 0.03 mm and about 0.17 mm, between about 0.05 mm and about 0.15 mm, between about 0.07 mm and about 0.13 mm, or about 0.1 mm.

[0237] needle volume

[0238] In some embodiments, the injection needle includes a dead volume between approximately 0.5 µL / 25.4 mm and 1.5 µL / 25.4 mm. In some embodiments, the total dead volume may be between approximately 0.7 mm. 3 With approximately 0.9 mm 3 Between, and approximately 0.76 mm 3 With approximately 0.86 mm 3 Between, between approximately 0.80 mm 3 With approximately 0.82 mm 3Between or approximately 0.81 mm 3 .

[0239] Relative length of the segment

[0240] In some embodiments, different portions of the needle sleeve 20 may have different lengths.

[0241] For example, in Figure 2 and 3 In the embodiment shown, 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.

[0242] In some embodiments, and as Figure 2 and 3 As shown in the embodiments, the length C of the distal portion 23 can be substantially equal to 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 can be about 50% or greater 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.

[0243] needle tip

[0244] Preferably, the distal tip 27 of the needle cannula 20 is beveled, making the distal tip 27 sharp, such as, for example... Figure 1-5 As shown. Without being bound by any theory, the applicant believes that including the beveled distal tip 27 facilitates clean and efficient penetration of tissue during use, thereby minimizing the possibility of leakage of the drug product from the tissue after injection and release.

[0245] like Figures 1 to 3 As shown, the orientation of the bevel of the distal tip 27 is such that the edge of the bevel faces away from the inner bend of the needle sleeve 20. Figure 1 (Refer to the inner bend side of the IB general marking). For example, the edge of the bevel of the distal tip 27 may be opposite to the center of curvature of the middle part of the arch 22 ( Figure 1 (The curvature center point is generally marked by reference to CP).

[0246] like Figure 4 As shown, preferably, the distal tip includes a long bevel. For example, the distal tip is beveled at an angle α between about 5° and about 15°, between about 10° and about 12.5°, between about 11° and about 12°, or about 11.5°.

[0247] For example, such as Figure 2 and 3As shown in Figure -5, the length D of the inclined plane can be between about 0.5 mm and about 3.5 mm, between about 1 mm and about 3 mm, between about 1.5 mm and about 2.5 mm, or about 2 mm.

[0248] The distal tip 27 may include one or more additional features, such as additional bevels, lancets, or heels.

[0249] Needle material

[0250] The needle sheath 20 of the injection needle 10 described herein may comprise stainless steel, which may contain iron and chromium and / or nickel. The needle sheath 20 may comprise nickel-coated stainless steel. Specifically, the needle sheath may comprise AISI (American Iron and Steel Institute) 304 stainless steel.

[0251] The needle sleeve 20 may contain annealed metal, for example, to improve ductility and / or reduce brittleness. The needle sleeve 20 may be annealed before or after bending or forming the intermediate portion 22.

[0252] Other materials may be suitable, which maintain the integrity of the composition required for injection.

[0253] Reagent test kit

[0254] This disclosure also provides a kit comprising a set of injection devices 200. The injection devices 200 may be provided in an assembled or disassembled state (e.g., wherein the needle 10 is coupled to or detached from a pre-filled syringe).

[0255] The pre-filled syringe of the injection device may collectively contain a single therapeutic dose of the therapeutic composition for treating a single subject. For example, a single therapeutic dose may contain: about 1.2e13 vg / subject (low dose); about 3.6e13 vg / subject (medium dose); or about 5.4e13 vg / subject (high dose) of the therapeutic composition designated as BB-301 as described herein.

[0256] Each injection device in the injection apparatus may contain at least 200 µL of therapeutic composition. For example, each injection device may contain between about 200 µL and about 2000 µL, or between about 400 µL and about 1000 µL, such as 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, the kit comprises two injection devices, each containing between about 200 µL and about 1000 µL of therapeutic composition; and two injection devices, each containing between about 400 µL and about 2000 µL of therapeutic composition. In some instances, the kit includes two injection devices, each containing between about 200 µL and about 500 µL of a therapeutic composition; and two injection devices, each containing between about 400 µL and about 1000 µL of a therapeutic composition.

[0257] One or more injection devices in the injection apparatus can be configured to dispense a drug product in a series of injections with an injection volume of 25 μL. One or more injection devices in the injection apparatus can be configured to dispense a drug product in a series of injections with an injection volume of 50 μL. One or more injection devices in the injection apparatus can be configured to dispense a drug product in a series of injections with an injection volume of 75 μL.

[0258] Alternatively, the kit may include a set of pre-filled syringes 100, and the injection needles 10 may be provided separately.

[0259] Application and treatment targeting the pharyngeal muscles

[0260] This disclosure also provides a method of administering a therapeutic composition to the pharyngeal muscles of a subject. The administration method preferably comprises injecting the therapeutic composition directly into the pharyngeal muscles of the subject using a bent or curved injection needle. In a particularly preferred embodiment, the administration method comprises injecting the therapeutic composition directly into the pharyngeal muscles of the subject using an injection needle 10 as described herein.

[0261] The pharyngeal muscles are a group of muscles that form the pharynx, located at the back of the oral cavity, thus determining the shape of its lumen and influencing its acoustic characteristics as a primary resonant cavity. The pharyngeal muscles (involuntary skeletal muscles) assist in pushing food into the esophagus. The pharynx has two muscular layers: the outer circular layer and the inner longitudinal layer. The outer circular layer includes the hypopharyngeal constrictor, middle pharyngeal constrictor, and superior pharyngeal constrictor. During swallowing, these muscles contract to push the bolus downward (an involuntary process). The inner longitudinal layer includes the stylopharyngeal muscle, eustachian tube pharyngeal muscle, and palatopharyngeal muscle. During swallowing, these muscles work to shorten and widen the pharynx.

[0262] The methods disclosed herein may include applying a therapeutic composition to any one or more muscles within the pharyngeal muscle group. For example, the methods disclosed herein may include injecting the therapeutic composition directly into any one or more of the following specific muscles using a bent or curved injection needle: the hypopharyngeal constrictor, the midpharyngeal constrictor, the superior pharyngeal constrictor, the palatopharyngeal muscle, the eustachian tube constrictor, the stylopharyngeal muscle, or any combination thereof. In one example, the method includes applying the therapeutic composition to the midpharyngeal constrictor and the hypopharyngeal constrictor.

[0263] The therapeutic composition can be administered to the pharyngeal muscles via direct injection after making an incision in the subject's neck (i.e., intramuscular injection), the incision being sufficient to provide direct access to at least a portion of the subject's pharyngeal muscles. Therefore, the administration method may involve making one or more incisions in the subject's neck (e.g., to the skin) to provide direct access to the subject's pharyngeal muscles, and then administering the therapeutic composition to the pharyngeal muscles via direct intramuscular injection 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 a pharyngeal region on the right side of a human or animal body, and the region may be adequately dissected to access at least the right side of one or more pharyngeal muscles. A needle cannula may then be directly inserted into the right side of one or more pharyngeal muscles. Alternatively, or additionally, an incision may be made on the left side of a pharyngeal region on the left side of a human or animal body, and the region may be adequately dissected to access the left side of one or more pharyngeal muscles. A needle cannula may then be directly inserted into the left side of one or more pharyngeal muscles.

[0264] The therapeutic composition described herein can be injected directly into one or more sites in the pharyngeal muscles, into one or more pharyngeal muscles, into one or more sides of the pharyngeal muscles (e.g., right or left), or any combination thereof. Injection at multiple sites can help increase transduction of pharyngeal muscle tissue. In one example, the therapeutic composition can be injected into the midpharyngeal constrictor and hypopharyngeal constrictor muscles. Direct injection can be applied to specific portions of the muscles, such as the thyropharynx or cricopharynx.

[0265] In cases where it is desired to inject into multiple sites within the pharyngeal muscle and / or administer multiple doses, a curved or bent injection needle (e.g., the injection needle 10 described herein) may be used in conjunction with a pre-filled syringe configured to deliver multiple doses or volumes to one or more muscles via microinjection.

[0266] In some instances, therapeutic compositions administered to the pharyngeal muscles using a bent or curved needle (e.g., needle 10) according to this disclosure are used to treat or prevent dysphagia. In one instance, dysphagia is associated with oculopharyngeal dystrophy (OPMD). According to this instance, the method may comprise administering a therapeutic composition to treat or prevent OPMD (and thereby treat or prevent the associated symptoms, such as dysphagia). The therapeutic composition for treating or preventing OPMD may be based on a modified adeno-associated virus (AAV) delivery vector comprising a 'silencing and replacement' construct comprising (i) one or more RNAi agents targeting regions of the PABPN1 mRNA transcript that cause OPMD, and (ii) a PABPN1 replacement construct for expression of wild-type (functional) human PABPN1 protein having an mRNA transcript not targeted by the RNAi agents of this disclosure. Therefore, the method may comprise administering a modified AAV delivery carrier containing the 'silencing and replacement' construct to the subject's pharyngeal muscles via direct injection (e.g., intramuscular injection into the pharyngeal muscles after making an incision in the pharyngeal region to provide access to the pharyngeal muscles, as described herein) using a bent or curved injection needle (e.g., injection needle 10). The applicant has previously described therapeutic compositions according to this example in WO2017 / 177277, WO2018 / 107228, WO2019 / 043630, WO2020 / 077412, and WO2020 / 172720, the contents of each of which are incorporated herein by reference in their entirety.

[0267] In one instance, a therapeutic composition administered directly to the pharyngeal muscles of a subject using a bent or curved injection needle (e.g., injection needle 10 described herein) comprises an AAV, said AAV comprising:

[0268] (a) A viral capsid protein from AAV9, the viral capsid protein comprising a modified subunit 1 (VP1) sequence, wherein amino acids at positions 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9VP1 sequence shown in SEQ ID NO: 17; and

[0269] (b) A 'silencing and replacement' construct comprising: (i) a DNA-guided RNAi (ddRNAi) construct containing nucleic acids, the nucleic acids containing a sequence encoding a short hairpin microRNA (shmiR); and (ii) a PABPN1 construct containing nucleic acids, the nucleic acids containing a sequence encoding a functional PABPN1 protein having an mRNA transcript not targeted by the shmiR encoded by the ddRNAi construct. For example, relative to the AAV9 VP1 sequence shown in SEQ ID NO: 17, a modified AAV9 VP1 sequence may contain glutamic acid at position 26, arginine at position 40, aspartic acid at position 43, and serine at position 44. For example, relative to the sequence shown in SEQ ID NO: 17, a modified AAV9 VP1 sequence may contain the following modifications: A26E, Q40R, K43D, and A44S. In one example, the modified AAV9 VP1 sequence comprises the sequence shown in SEQ ID NO: 27. In one example, the viral capsid protein comprises mutants A67E, Q81R, K84D, and A85S relative to the full-length wild-type AAV serotype 9 capsid sequence shown in SEQ ID NO: 19. In one example, the viral capsid protein comprises the amino acid sequence shown in SEQ ID NO: 28.

[0270] In one instance, a therapeutic composition administered directly to the pharyngeal muscles of a subject using a bent or curved injection needle (e.g., injection needle 10 described herein) comprises an AAV, said AAV comprising:

[0271] (a) A viral capsid protein from AAV9, said viral capsid protein comprising a modified subunit 1 (VP1) sequence, wherein amino acids at positions 1, 26, 40, 43, and 44 are modified relative to the corresponding wild-type AAV9VP1 sequence shown in SEQ ID NO: 17; and

[0272] (b) A 'silencing and replacement' construct comprising: (i) a DNA-guided RNAi (ddRNAi) construct containing nucleic acids, the nucleic acids containing a sequence encoding a short hairpin microRNA (shmiR); and (ii) a PABPN1 construct containing nucleic acids, the nucleic acids containing a sequence encoding a functional PABPN1 protein, the sequence protein having an mRNA transcript not targeted by the shmiR encoded by the ddRNAi construct. For example, relative to the AAV9 VP1 sequence shown in SEQ ID NO: 17, a modified AAV9 VP1 sequence may contain a serine at position 1, a glutamate at position 26, an arginine at position 40, an aspartic acid at position 43, and a serine at position 44. For example, relative to the sequence shown in SEQ ID NO: 17, a modified AAV9 VP1 sequence may contain the following modifications: A1S, A26E, Q40R, K43D, and A44S. In one example, the modified AAV9 VP1 sequence comprises the sequence shown in SEQ ID NO: 18. In one example, the viral capsid protein comprises mutants A42S, A67E, Q81R, K84D, and A85S relative to the full-length wild-type AAV serotype 9 capsid sequence shown in SEQ ID NO: 19. In one example, the viral capsid protein comprises the amino acid sequence shown in SEQ ID NO: 20.

[0273] In another instance, a therapeutic composition administered directly to the pharyngeal muscles of a subject using a bent or curved injection needle (e.g., injection needle 10 described herein) comprises an AAV, said AAV comprising:

[0274] (a) A viral capsid protein from AAV8, said viral capsid protein comprising a modified subunit 1 (VP1) sequence, wherein amino acids at positions 1, 26, 40, 43, 44, and 64 are modified relative to the corresponding wild-type AAV8 VP1 sequence shown in SEQ ID NO: 21; and

[0275] (b) A polynucleotide sequence comprising: (i) a ddRNAi construct containing a nucleic acid that includes a sequence encoding shmiR; and (ii) a PABPN1 construct containing a nucleic acid that includes a sequence encoding a functional PABPN1 protein, the sequence having an mRNA transcript not targeted by the shmiR encoded by the ddRNAi construct. For example, relative to the AAV8 VP1 sequence shown in SEQ ID NO: 21, a modified AAV8 VP1 sequence may contain a serine at position 1, a glutamic acid at position 26, an arginine at position 40, an aspartic acid at position 43, a serine at position 44, and a lysine at position 64. For example, relative to the sequence shown in SEQ ID NO: 21, a modified AAV8 VP1 sequence may include the following modifications: A1S, A26E, Q40R, K43D, A44S, and Q64K. In one example, the modified AAV8 VP1 sequence comprises the sequence shown in SEQ ID NO: 22. In one example, the viral capsid protein comprises mutants A42S, A67E, Q81R, K84D, A85S, and Q105K relative to the full-length wild-type AAV serotype 8 capsid sequence shown in SEQ ID NO: 23. In one example, the viral capsid protein comprises the amino acid sequence shown in SEQ ID NO: 24.

[0276] In each of the above examples, the modified viral capsid protein is a delivery vector containing a polynucleotide construct and a 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.

[0277] The polynucleotide may further comprise an inverted terminal repeat (ITR) sequence derived from an AAV serotype. For example, the ITR may be side-joined with sequences comprising the ddRNAi construct and the PABPN1 construct. In some instances, the ITR is derived from the AAV2 serotype. For example, an ITR derived from the AAV2 serotype may comprise the 5' ITR sequence shown in SEQ ID NO: 29 and the 3' ITR sequence shown in SEQ ID NO: 30. For example, an ITR derived from the AAV2 serotype may comprise the 5' ITR sequence shown in SEQ ID NO: 25 and the 3' ITR sequence shown in SEQ ID NO: 26. However, other ITR sequence variants, including those from AAV2, are known in the art and are considered herein.

[0278] A therapeutic composition administered to a subject according to the method of administration of this disclosure to treat or prevent dysphagia associated with OPMD comprises an AAV, the AAV comprising a 'silencing and replacement' construct comprising a PABPN1 construct. In this respect, the AAV provides an agent that replaces a functional PABPN1 protein, for example, for use in cells or animals. The functional PABPN1 protein will not cause OPMD and will not be encoded by an mRNA transcript targeted by shmiR encoded by a ddRNAi construct as described herein, which is also contained within the AAV.

[0279] In one instance, the PABPN1 construct contains a nucleic acid, such as DNA or cDNA, encoding a functional PABPN1 protein. For example, the nucleic acid encoding the functional PABPN1 protein may be codon-optimized, for instance, containing one or more degenerate or wobbly bases relative to wild-type PABPN1 nucleic acid, but encoding the same amino acid, so that the corresponding mRNA sequence encoding the functional PABPN1 protein will not be recognized by the shmiR 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 wobbly bases relative to wild-type PABPN1 nucleic acid within a region targeted by one or more shmiRs encoded and expressed from the ddRNAi construct. In one instance, one or more degenerate or wobbly bases reside within a seed region of the effector sequence of the shmiR encoded and expressed from the ddRNAi construct.

[0280] In one example, the nucleic acid of the PABPN1 construct encoding a functional PABPN1 protein is codon-optimized such that its corresponding mRNA sequence is not recognized by the shmiR encoded and expressed from the ddRNAi construct. Preferably, the functional PABPN1 protein encoded by the codon-optimized nucleic acid sequence comprises the amino acid sequence shown in SEQ ID NO: 16, i.e., the amino acid sequence of the wild-type human PABPN1 protein. Those skilled in the art will understand that many combinations of nucleotide sequences can be used to encode a functional PABPN1 protein, and the choice of nucleotide sequence will ultimately depend on the effector sequence of the shmiR encoded and expressed from the ddRNAi construct, i.e., such that the codon-optimized nucleic acid is not recognized by the shmiR. In one example, the PABPN1 construct comprises a nucleic acid containing the sequence shown in SEQ ID NO: 15. In one example, the nucleic acid encoding the functional PABPN1 protein may also contain a Kozak sequence.

[0281] In one example, a codon-optimized nucleic acid encoding a functional PABPN1 protein is operatively linked 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 suitable. An 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.

[0282] In one instance, the PABPN1 construct and the ddRNAi construct are operatively linked to the same promoter within the same polynucleotide, for example, both are operatively linked to the Spc512 promoter. According to this example, a single promoter drives the expression of both functional PABPN1 protein and shmiR.

[0283] A therapeutic composition administered to a subject according to the method of administration of this disclosure for treating or preventing dysphagia associated with OPMD comprises an AAV, said AAV comprising a 'silencing and replacement' construct, said 'silencing and replacement' construct itself comprising a DNA-guided RNAi (ddRNAi) construct. The ddRNAi construct comprises a DNA sequence encoding one or more short hairpin microRNAs (shmiRs) encoding an mRNA transcript targeting endogenous PABPN1.

[0284] shmiR encoded by the ddRNAi construct or each shmiR contains:

[0285] The effector sequence is at least 17 nucleotides in length;

[0286] Complementary effect sequences;

[0287] Stem-loop sequences; and

[0288] Primary microRNA (primary miRNA) backbone;

[0289] The effector sequence is substantially complementary to the corresponding length region in the RNA transcript shown in SEQ ID NO: 1 or 2. Preferably, the length of the effector sequence, or each effector sequence, will be less than 30 nucleotides. For example, a suitable effector sequence length may be in the range of 17-29 nucleotides. In a particularly preferred example, the effector sequence length is 21 nucleotides. More preferably, the effector sequence length will be 21 nucleotides, and the length of the effect complement sequence will be 20 nucleotides.

[0290] In one example, the shmiR encoded by the ddRNAi construct contains an effector sequence substantially complementary to a region of corresponding length in an RNA transcript comprising the sequence shown in SEQ ID NO: 1 or thereof. The shmiR according to this example is also referred to herein as "shmiR13". For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising the sequence shown in SEQ ID NO: 1 or thereof and contains 4 mismatched bases relative to it. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising the sequence shown in SEQ ID NO: 1 or thereof and contains 3 mismatched bases relative to it. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising the sequence shown in SEQ ID NO: 1 or thereof and contains 2 mismatched bases relative to it. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising the sequence shown in SEQ ID NO: 1 or thereof and contains 1 mismatched base. For example, the effector sequence may be 100% complementary to a region of the corresponding length in an RNA transcript containing the sequence shown in SEQ ID NO: 1 or composed thereof.

[0291] In one example, the shmiR encoded by the ddRNAi construct contains an effector sequence substantially complementary to a region of corresponding length in an RNA transcript comprising the sequence shown in SEQ ID NO: 2 or thereof. The shmiR according to this example is also referred to herein as "shmiR17". For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising the sequence shown in SEQ ID NO: 2 or thereof and contains 4 mismatched bases relative to it. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising the sequence shown in SEQ ID NO: 2 or thereof and contains 3 mismatched bases relative to it. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising the sequence shown in SEQ ID NO: 2 or thereof and contains 2 mismatched bases relative to it. For example, the effector sequence may be substantially complementary to a region of corresponding length in an RNA transcript comprising the sequence shown in SEQ ID NO: 2 or thereof and contains 1 mismatched base. For example, the effector sequence may be 100% complementary to a region of the corresponding length in an RNA transcript containing the sequence shown in SEQ ID NO: 2 or composed thereof.

[0292] Based on the fact that the effector sequence of shmiR is substantially complementary to the corresponding length region in the PABPN1 miRNA transcript described herein and, relative to instances containing 1, 2, 3, or 4 mismatched bases, it is preferred that the mismatch is not located in the region corresponding to the seed region of shmiR (i.e., nucleotides 2-8 of the effector sequence).

[0293] In one example, the ddRNAi construct may comprise a DNA sequence encoding shmiR, said shmiR comprising: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 3, except for one, two, three, or four base mismatches, provided that said effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 3; and (ii) an effector complement sequence comprising a sequence substantially complementary to said effector sequence. For example, the shmiR encoded by the ddRNAi construct may comprise the effector sequence shown in SEQ ID NO: 4 and an effector complement sequence substantially complementary to the sequence shown in SEQ ID NO: 4 and capable of forming a double strand with it. The effector complement sequence substantially complementary to the sequence shown in SEQ ID NO: 4 may be the sequence shown in SEQ ID NO: 3. The shmiR according to this example is designated hereinafter as "shmiR13".

[0294] In one example, the ddRNAi construct may comprise a DNA sequence encoding shmiR, said shmiR comprising: (i) an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 5, except for one, two, three, or four base mismatches, provided that said effector sequence is capable of forming a double strand with the sequence shown in SEQ ID NO: 5; and (ii) an effector complement sequence comprising a sequence substantially complementary to said effector sequence. For example, the shmiR encoded by the ddRNAi construct may comprise the effector sequence shown in SEQ ID NO: 6 and an effector complement sequence substantially complementary to the sequence shown in SEQ ID NO: 6 and capable of forming a double strand with it. The effector complement sequence substantially complementary to the sequence shown in SEQ ID NO: 6 may be the sequence shown in SEQ ID NO: 5. The shmiR according to this example is designated hereinafter as "shmiR17".

[0295] In one instance, at least one shmiR encoded by the ddRNAi construct is:

[0296] shmiR containing the effect sequence shown in SEQ ID NO: 4 and the effect complement sequence shown in SEQ ID NO: 3; or

[0297] shmiR containing the effect sequence shown in SEQ ID NO: 6 and the effect complement sequence shown in SEQ ID NO: 5.

[0298] In one particular instance, the ddRNAi construct encodes an shmiR containing the effector sequence shown in SEQ ID NO: 4 and the effector complement sequence shown in SEQ ID NO: 3; and an shmiR containing the effector sequence shown in SEQ ID NO: 6 and the effector complement sequence shown in SEQ ID NO: 5. For example, the ddRNAi construct may encode an shmiR designated as shmiR13 as described herein and an shmiR designated as shmiR17 as described herein.

[0299] In one instance, shmiR, or each type of shmiR, includes the following in the 5' to 3' direction:

[0300] 5' flanking sequence of the primary miRNA backbone;

[0301] Complementary effect sequences;

[0302] Stem-loop sequence;

[0303] Effect sequence; and

[0304] The 3' flanking sequence of the primary miRNA backbone.

[0305] In another instance, shmiR, or each type of shmiR, includes the following in the 5' to 3' direction:

[0306] 5' flanking sequence of the primary miRNA backbone;

[0307] Effect sequence;

[0308] Stem-loop sequence;

[0309] Complementary effect sequences; and

[0310] The 3' flanking sequence of the primary miRNA backbone.

[0311] Suitable loop sequences can be selected from loop sequences known in the art. However, an exemplary stem-loop sequence is shown in SEQ ID NO: 7.

[0312] In one instance, the primary miRNA backbone is the primary miR-30a backbone. For example, the 5' flanking sequence of the primary miRNA backbone may be the sequence shown in SEQ ID NO: 8, and the 3' flanking sequence of the primary miRNA backbone may be the sequence shown in SEQ ID NO: 9.

[0313] In one instance, the ddRNAi construct contains at least two nucleic acids, each encoding a shmiR, wherein each shmiR contains an effector sequence substantially complementary to the RNA transcript corresponding to the PABPN1 protein that causes OPMD, and wherein each shmiR contains a distinct effector sequence.

[0314] In one example, the ddRNAi construct encodes an shmiR containing an effector sequence substantially complementary to a region of corresponding length in the RNA transcript shown in SEQ ID NO: 1, and an shmiR containing an effector sequence substantially complementary to a region of corresponding length in the RNA transcript shown in SEQ ID NO: 2. For example, the ddRNAi construct may contain:

[0315] A nucleic acid comprising a DNA sequence or thereof, said DNA sequence encoding a shmiR (shmiR13) comprising the effector sequence shown in SEQ ID NO: 4 and the effector complement sequence shown in SEQ ID NO: 3; and

[0316] The nucleic acid comprising a DNA sequence or thereof, the DNA sequence encoding a shmiR (shmiR17) comprising the effector sequence shown in SEQ ID NO: 6 and the effector complement sequence shown in SEQ ID NO: 5.

[0317] The exemplary ddRNAi constructs of this disclosure comprise a DNA sequence or nucleic acid comprising the sequence shown in SEQ ID NO: 12 (e.g., encoding shmiR13, which comprises or is composed of the sequence shown in SEQ ID NO: 10) and a DNA sequence or nucleic acid comprising the sequence shown in SEQ ID NO: 13 (e.g., encoding shmiR17, which comprises or is composed of the sequence shown in SEQ ID NO: 11).

[0318] In any of the foregoing examples, the ddRNAi construct and the PABPN1 construct may be operatively linked to a promoter. In one example, the ddRNAi construct and the PABPN1 construct are operatively linked 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 contemplated for use in conjunction with the ddRNAi construct of this disclosure.

[0319] As described herein, therapeutic compositions administered to subjects to treat or prevent dysphagia associated with OPMD may comprise a single polynucleotide (or 'silence and replacement' construct) containing a ddRNAi construct and a 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 to herein as a 'silence and replacement' 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 exemplary 'silence and replacement' construct is described herein.

[0320] The 'silence and replacement' construct can be packaged in a modified AAV as described in this article for delivery to the patient.

[0321] In one instance, the 'silence and replacement' construct includes a muscle-specific promoter (e.g., the Spc512 promoter), the PABPN1 construct described herein, and the ddRNAi construct described herein, for example, wherein the ddRNAi construct is located in the 3' untranslated region (UTR) of the nucleic acid encoding the functional PABPN1 protein. Figure 9 A shows the 'Silence and Replace' builder based on this instance.

[0322] The exemplary 'silence and replacement' construct based on this example includes, in the 5' to 3' direction:

[0323] (a) Muscle-specific promoters, such as Spc512;

[0324] (b) The PABPN1 construct as 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

[0325] (c) The ddRNAi construct of this disclosure comprises nucleic acids including a DNA sequence encoding shmiR17 as described herein and nucleic acids including a DNA sequence encoding shmiR13 as described herein. According to this example, the 'silence and replacement' construct may comprise or consist of the DNA sequence shown in SEQ ID NO: 14. Figure 9 B shows the 'Silence and Replace' builder based on this instance.

[0326] An exemplary ddRNAi construct for inclusion in the 'silencing and replacement' constructs of this disclosure comprises: a DNA sequence encoding shmiR13 and shmiR17, the shmiR comprising the effector sequence shown in SEQ ID NO: 4 and an effector complement sequence substantially complementary to the sequence shown in SEQ ID NO: 4, for example, the effector complement sequence shown in SEQ ID NO: 3; and a DNA sequence encoding shmiR17, the shmiR comprising the effector sequence shown in SEQ ID NO: 6 and an effector complement sequence substantially complementary to the sequence shown in SEQ ID NO: 6, for example, the effector complement sequence shown in SEQ ID NO: 5. For example, an instance of this 'silence and replacement' construct may contain a DNA sequence or nucleic acid comprising the DNA sequence shown in SEQ ID NO: 12 (e.g., encoding shmiR13, which contains or is composed of the sequence shown in SEQ ID NO: 10) and a DNA sequence or nucleic acid comprising the DNA sequence shown in SEQ ID NO: 13 (e.g., encoding shmiR17, which contains or is composed of the sequence shown in SEQ ID NO: 11).

[0327] An exemplary PABPN1 construct for inclusion in the 'silence and replacement' constructs of this disclosure comprises the codon-optimized sequence shown in SEQ ID NO: 15 and encodes the functional PABPN1 protein shown in SEQ ID NO: 16.

[0328] Those skilled in the art will understand that the therapeutic composition will be formulated for delivery to a patient, e.g., a human patient. Therefore, a therapeutic composition for administration to a subject according to the methods of this disclosure may comprise one or more pharmaceutically acceptable carriers or diluents. For example, the therapeutic composition may comprise a carrier suitable for delivering the AAV of this disclosure to said muscle after injection into the muscle of the subject. Carriers suitable for the formulation and delivery of AAV are known in the art and are contemplated herein. Suitable drug carriers for pharmaceutical formulations and drug necessities are described in the following literature: *Remington: The Science and Practice of Pharmacy* (formerly known as *Remington's Pharmaceutical Sciences*), Mack Publishing Co., which is the standard reference text in this art; and USP / NF.

[0329] The volume, concentration, and formulation of the therapeutic composition administered to a subject according to the methods of this disclosure, as well as the dosing regimen, can be specifically tailored to maximize cellular delivery while minimizing toxicities such as inflammatory responses, and using correspondingly low concentrations of active agents (e.g., AAV carriers containing 'silencing and replacement' constructs for treating OPMD). Anti-inflammatory compounds (such as corticosteroids) can also be administered (e.g., orally) for systemic distribution.

[0330] According to one example, a therapeutic composition for treating dysphagia associated with OPMD is administered in volumes ranging from about 25 µl to about 75 µl per injection site. For example, the therapeutic composition may be administered in volumes of about 25 µl, or about 30 µl, or about 35 µl, or about 40 µl, or about 45 µl, or about 50 µl, or about 55 µl, or about 60 µl, or about 65 µl, or about 70 µl, or about 75 µl per injection site.

[0331] In another example, the therapeutic composition may be administered in volumes of about 50 µl to about 75 µl per injection site. For example, the therapeutic composition may be administered in volumes of about 50 µl, or about 55 µl, or about 60 µl, or about 65 µl, or about 70 µl, or about 75 µl per injection site.

[0332] The volume of the therapeutic composition applied at each injection site can be the volume of a single injection or the cumulative volume of multiple injections applied at one injection site. For example, an injection volume of approximately 75 µL per injection site can be applied as three injections of 25 µL per injection site, or as a single injection of 25 µL and a single injection of 50 µL.

[0333] In each of the foregoing examples describing the application of the therapeutic composition at multiple injection sites, the total dose administered to the subject in a single treatment may be approximately 1.5E. +12 vg to approximately 9.5E +13 vg. For example, the total dose of the therapeutic composition administered to the subject may be about 5.0E. +12 vg to approximately 6.0E +13 vg. For example, the total dose of the therapeutic composition administered to the subject may be about 1.0E. +13 vg to approximately 5.5E +13 vg. In some instances, the total dose of the therapeutic composition administered was approximately 1.0E. +13vg / subject. In some instances, the total dose of the therapeutic composition administered was approximately 1.2E. +12 vg / subject. In some instances, the total dose of the therapeutic composition administered was approximately 3.6E. +13 vg / subject. In some instances, the total dose of the therapeutic composition administered was approximately 5.4E. +13 vg / subject.

[0334] Therefore, when the therapeutic composition is administered in volumes ranging from about 25 µl to about 75 µl per injection site, the therapeutic composition can be administered at concentrations between about 1.0+E12 vg / mL and about 1.0+E15 vg / mL. For example, the concentration of the therapeutic composition administered to the subject can be between about 5.0+E12 vg / mL and about 1.0+E14 vg / mL, such as between about 5.0+E12 vg / mL and 5.0+E13 vg / mL.

[0335] As described herein, it is desirable to inject a therapeutic composition at multiple sites within the pharyngeal muscle group and / or administer multiple doses to one or more muscles within the pharyngeal muscle group, including applying the therapeutic composition to one or more sites within the pharyngeal muscles, one or more pharyngeal muscles, one or more sides of the pharyngeal muscles (e.g., right or left side), or any combination thereof. Injecting at multiple sites can help improve delivery to the pharyngeal muscle tissue. For example, the method may involve injecting the therapeutic composition at about two to about 30 or more sites within the pharyngeal muscles. Based on an example where multiple injection sites are separated between the left and right sides of the midline of the pharyngeal muscles, the method may include injecting the therapeutic composition at about one to about 15 or more sites (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more sites) in the muscle on the left side of the midline of the pharyngeal muscles and at about one to about 15 or more sites (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more sites) in the muscle on the right side of the midline of the pharyngeal muscles. For example, the method may involve injecting the therapeutic composition into about one to about 15 or more sites (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more sites) on the left side of the midline of the thyropharyngeal (TP) muscle and about one to about 15 or more sites (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more sites) within the muscle on the right side of the midline of the thyropharyngeal (TP) muscle. Alternatively or additionally, the method may involve injecting the therapeutic composition at approximately one to approximately 15 or more sites (e.g., approximately 4 sites) on the left side of the midline of the hypopharyngeal (HP) muscle and at approximately one to approximately 15 or more sites (e.g., approximately 4 sites) within the muscle on the right side of the midline of the hypopharyngeal (HP) muscle. According to a specific example in which the therapeutic composition is applied to the HP and TP muscles, the composition may be injected at 4 sites on the left side of the midline of the HP muscle, 4 sites on the right side of the midline of the HP muscle, 8 sites on the left side of the midline of the TP muscle, and 8 sites on the right side of the midline of the TP muscle.

[0336] The method may also involve injecting the therapeutic composition multiple times into more than one pharyngeal muscle. Different pharyngeal muscles are described and considered herein. However, in one example, the therapeutic composition may be injected into the middle pharyngeal constrictor muscle at multiple sites and into the hypopharyngeal constrictor muscle at multiple sites. Direct injection may also be applied to multiple sites of other pharyngeal muscles, such as the thyropharynx or cricopharynx.

[0337] As described herein, a therapeutic composition can be administered to the pharyngeal muscles via direct injection after making an incision in the subject's neck (i.e., intramuscular injection), said incision being sufficient to provide direct access to at least a portion of the subject's pharyngeal muscles. Therefore, a method for treating OPMD-associated dysphagia in a subject with OPMD may involve making one or more incisions in the subject's neck (e.g., to the skin) to provide direct access to the subject's pharyngeal muscles, and then administering the therapeutic composition as described herein to the pharyngeal muscles via direct intramuscular injection 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 a pharyngeal region on the right side of a human or animal body, and said region may be adequately dissected to access at least the right side of one or more pharyngeal muscles. A needle cannula may then be directly inserted into the right side of one or more pharyngeal muscles. Alternatively, or additionally, an incision may be made on the left side of a pharyngeal region on the left side of a human or animal body, and said region may be adequately dissected to access the left side of one or more pharyngeal muscles. Then, the needle cannula can be directly inserted into the left side of one or more pharyngeal muscles.

[0338] Example

[0339] Example 1: Dye Research

[0340] In this study, the inventors (1) evaluated the maximum volume that could be injected into the hypopharyngeal and thyropharyngeal muscles of a beagle dog without observable leakage of the injected material using trypan blue (Gibco No. 15250-061), and (2) evaluated different injection needles to identify a delivery device suitable for delivering the applicant's OPMD 'Silence and Replacement' therapeutic agent "BB-301" without observable leakage of the therapeutic composition. The beagle dog was chosen as the animal model because its muscle mass and size are comparable to those of a human.

[0341] animal

[0342] This study, conducted in June 2020 at the Boisbonne Center (a large animal facility), included two female beagle dogs, Rosette and Rillette, aged 4 years and 10 months and 4 years and 9 months, respectively. Both animals were euthanized at the end of the study.

[0343] Needle

[0344] This study evaluated the ability of two different injection needles, "Needle A" and "Needle B," to administer trypan blue to the hypopharyngeal, thyropharyngeal, and cricopharyngeal muscles of animals, as well as the ability of trypan blue delivery to affect the expected volume without observed leakage of trypan blue from the injected muscle after injection.

[0345] Needle A is typically configured according to the injection needle 10 described herein and includes the 3Dose Next Generation BoNT drug delivery injector (catalog number 3DOSE-GREEN-125) from VlowMedical, which is coupled to a 27G, 19 mm needle having a bent, arc-shaped middle portion such that the proximal and distal portions of the needle extend at 90 degrees relative to each other.

[0346] Needle B also includes the 3Dose next-generation BoNT drug delivery injector from Vlow Medical (catalog number 3DOSE-GREEN-125), but is coupled to the 25G, 19-22 mm nuclear hydrolysis needle from Accuspire (catalog number J075-25G), typically as follows. Figure 7A and 7B As shown in the image.

[0347] Figure 7A An injection needle 400, typically configured according to needle B, is shown, comprising a needle cannula 420 and a seat 430. (See diagram) Figure 7A As shown, the needle cannula 420 has a generally straight portion near the seat (proximal portion 421), while the middle portion 422 and the distal portion 423 are curved. It can be seen that the radius of curvature is large, and the curvature angle θ between the proximal portion 421 and the distal portion 423 is approximately 160°. Figure 7B The distal portion 423 of the sleeve 420 is shown to have a flat, elliptical tip 427.

[0348] Surgical procedures and dye injection

[0349] Rillette

[0350] The surgery was performed under general anesthesia with appropriate analgesia, and all procedures were performed by a veterinarian. A single incision was made at the right level of the pharyngeal region, and this region was dissected to provide access to the segments of the HP and TP muscles to the right of the midline. After exposing the HP and TP muscle segments to the right of the midline, trypan blue dye was administered at 75 µl per injection via multiple direct intramuscular injections (to 4, 5, or 9 discrete injection sites) using a 3-dose unit syringe device (Vlow Medical, Netherlands) to control the volume of each injection. The dye was drawn into a microinjector using a 21G, 2-inch needle (catalog number 301155) from BD and injected using a 27G, 19 mm needle (p / n 10101008, N (27 / 19 / 4x2 / A90)) at a 90-degree angle from Hamilton. A surgical microscope was used for all injections.

[0351] Table 1 below presents a summary of trypan blue dye injections performed on the HP and TP muscle segments to the right of the midline and in the cricopharyngeal (CP) muscle.

[0352] After the dye is applied, the dye leakage in the muscle area is evaluated by visual inspection.

[0353] Approximately 5 minutes after injection, the surgical incision is closed with a staple, and the dog is turned to access the left side of the pharyngeal region. A single incision is then made at the left level of the pharyngeal region, and this area is dissected to provide access to segments of the HP and TP muscles on the left side of the midline. After exposing the HP and TP muscles on the left side of the midline, trypan blue dye is applied using a 3-dose unit syringe device (Vlow Medical, Netherlands) via multiple direct intramuscular injections (25 µl, 50 µl, or 75 µl per injection at 1, 2, or 4 discrete injection sites) to control the volume of each injection. The dye was drawn into a microinjector using a 21G, 2-inch needle from BD (catalog number 301155), and injected using a 27G, 19 mm needle at a 90-degree angle from Hamilton (p / n 10101008, N(27 / 19 / 4x2 / A90)) or a 25G, 22 mm nucleolysis needle from Accuspire (catalog number J075-25G). A surgical microscope was used for all injections.

[0354] Table 1 below presents a summary of trypan blue dye injections performed on the left side of the midline in the HP and TP muscle segments and in the CP muscle.

[0355] After the dye is applied, the dye leakage in the muscle area is evaluated by visual inspection.

[0356] Approximately 5 minutes after injection, the surgical incision was closed with staples, and whole blood samples (total 20 mL, divided into 2 x 10 mL portions and frozen at < -70°C) were obtained on EDTA-coated tubes.

[0357] The dog was then euthanized by intravenous injection of a lethal dose of pentobarbital at a dose of 1 mL / kg (180 mg / kg).

[0358] Table 1 - Dye injections performed on "Rillette"

[0359]

[0360] After the animal is euthanized, the entire pharyngeal region (including the trachea, esophagus, and tongue) is dissected.

[0361] The left and right segments (length, width, depth) of the HP and TP muscles, CP muscles, and lateral extensor toes muscles were meticulously dissected, measured, and evaluated. Each muscle was then placed flat in a histological box, directly frozen in liquid nitrogen, and subsequently stored at < -70°C. It should be noted that it was not possible to label the left and right segments of the muscles. The characteristics of each muscle sample are summarized in Table 3 below.

[0362] Liver samples were also collected, cut into small pieces, and placed in a total of 15 DNase-free, RNase-free, and PCR-clean microtubes, which were then directly frozen in liquid nitrogen and stored at < -70°C.

[0363] The following observations were made:

[0364] Except for the right segment of TP, which was only partially stained, the muscle segments of TP, HP, and CP were completely stained with trypan blue dye.

[0365] After evaluating the muscles of the entire pharyngeal region, some residual dye staining was observed at the cartilage and esophageal wall levels. These observations, in addition to the increased level of dye leakage observed for RILLETTE compared to that recorded for ROSETTE, can be explained by the higher injection volume of trypan blue dye used for RILLETTE relative to the volume used for ROSETTE.

[0366] Rosette

[0367] Surgery is performed under general anesthesia with appropriate analgesia, and all surgeries are performed by veterinarians.

[0368] A single horizontal incision is made on the left side of the pharyngeal region, and this region is dissected to provide access to segments of the hypopharyngeal (HP) and thyropharyngeal (TP) muscles on the left side of the midline.

[0369] After exposing the HP and TP muscle segments to the left of the midline, trypan blue dye was administered using a 3-dose unit syringe device (Vlow Medical, Netherlands) at 50 µl per injection via multiple direct intramuscular injections (to 4 or 8 discrete injection sites) to control the volume of each injection. The dye was drawn into a microinjector using a 21G, 2-inch needle (catalog number 301155) from BD and intramuscularly injected using a 27G, 19 mm needle (p / n10101008, N(27 / 19 / 4x2 / A90)) at a 90-degree angle from Hamilton. A surgical microscope was used for all injections.

[0370] Table 2 below presents a summary of trypan blue dye injections in the HP and TP muscle segments to the left of the midline.

[0371] After the dye is applied, the dye leakage in the muscle area is evaluated by visual inspection.

[0372] Approximately 5 minutes after the injection, the surgical incision is closed with staples, and the dog is turned over to access the right side of the pharyngeal region.

[0373] Then, a single incision is made at the right level in the pharyngeal region, and this region is dissected to provide access to segments of the HP and TP muscles on the right side of the midline.

[0374] After exposing the HP and TP muscle segments to the right of the midline, trypan blue dye was applied via multiple direct intramuscular injections (50 µl or 100 µl per injection at 2, 3, or 4 discrete injection sites) using a 3-dose unit syringe device (Vlow Medical, Netherlands) to control the volume of each injection. The dye was drawn into a microinjector using a 21G, 2-inch needle (catalog number 301155) from BD and injected using a 27G, 19 mm needle (p / n 10101008, N (27 / 19 / 4x2 / A90)) at a 90-degree angle from Hamilton. A surgical microscope was used for all injections.

[0375] Table 2 below presents a summary of trypan blue dye injections performed on the HP and TP muscle segments to the right of the midline and in the cricopharyngeal (CP) muscle.

[0376] After the dye is applied, the dye leakage in the muscle area is evaluated by visual inspection.

[0377] Approximately 5 minutes after injection, the surgical incision was closed with staples, and whole blood samples (total 24 mL, divided into 2 x 10 mL + 1 x 4 mL, and frozen at < -70°C) were obtained on EDTA-coated tubes.

[0378] The dog was then euthanized by intravenous injection of pentobarbital at a dose of 1 mL / kg (180 mg / kg).

[0379] After the animal is euthanized, the entire pharyngeal region (including the trachea, esophagus, and tongue) is dissected.

[0380] The HP and TP muscles, as well as the left and right segments (length, width, depth) of the lateral extensor digitorum muscles, were meticulously dissected, measured, and evaluated. Each muscle was then laid flat in a histological box, directly frozen in liquid nitrogen, and subsequently stored at < -70°C. It should be noted that it was not possible to label the left and right segments of the muscles. The characteristics of each muscle sample are summarized in Table 4 below.

[0381] Liver samples were also collected, cut into small pieces, and placed in a total of 15 DNase-free, RNase-free, and PCR-clean microtubes, which were then directly frozen in liquid nitrogen and stored at < -70°C.

[0382] The following observations were made:

[0383] The entire HP and TP muscle segments were completely stained with trypan blue dye.

[0384] The left CP muscle segment was not stained, indicating that there was no leakage between different muscles in the pharyngeal anatomy and that the diffusion of dye was limited to the injected pharyngeal muscles.

[0385] After evaluating the muscles of the entire pharyngeal region, some residual dye staining was observed only at the cartilage level. No dye staining was observed at the level of other tissues, including the esophagus and trachea.

[0386] Table 2 - Dye injections performed in dogs 2.

[0387]

[0388] Table 3: Measurements of muscle samples taken from “RILLETTE”.

[0389]

[0390] Table 4: Measurements of muscle sampled from “ROSETTE”.

[0391]

[0392]

[0393] in conclusion

[0394] The following information was obtained from this dye study on two beagle dog subjects:

[0395] ○ No problems were encountered with the surgical approach and anatomy of the pharyngeal region. The bilateral surgical approach (left and right sides of the pharyngeal region) was ideally suited for the dosing protocol to facilitate better visualization of discrete intramuscular injections of each segment of the target pharyngeal muscle.

[0396] As expected, the pharyngeal muscle in question was small and very thin (approximately 4 cm). 2 Up to 5 cm 2 (2 mm to 3 mm deep).

[0397] ○VlowMedical microinjector is ideally suited for drug delivery and is very easy to use.

[0398] ○ Needle A was undoubtedly the most ideally suited for the injection procedure. In contrast, needle B was not sufficiently angled or beveled. Therefore, even with only 25 µL of trypan blue dye injected, extensive leakage was observed at the puncture site.

[0399] ○ The maximum fluid volume that can be administered per injection site is in the range of 50 µL to 75 µL without promoting significant leakage. To limit the risk of leakage when removing the injection needle from the target muscle, it is recommended to pause for about 30 seconds at the end of each injection before withdrawing the needle from the muscle.

[0400] ○ Since no dye leakage was observed when injecting 50 µL of trypan blue, the injection volume of 25 µL of dye was not evaluated. Since significant dye leakage was observed when injecting 100 µL of trypan blue, the injection volume of 150 µL of dye was not evaluated. However, a medium volume of 75 µL trypan blue dye was evaluated. No significant leakage was observed with this medium volume.

[0401] The appropriate number of injection sites in the TP muscle segment to the left or right of the midline is 8 (out of a total of 16 injection sites in the entire TP muscle), and the appropriate number of injection sites in the HP muscle segment to the left or right of the midline is 4 (out of a total of 8 injection sites in the entire muscle). Due to the variability in muscle size among dogs, the appropriate number of injection sites may increase in some animals.

[0402] ○The cricopharyngeal muscle can also be accessed through surgical methods.

[0403] Because the animal subjects had to be turned over to access the contralateral pharyngeal muscle for dye injection, it was impossible to observe trypan blue diffusion within 30 minutes post-injection. However, a diffusion pattern was observed after the animal subjects were euthanized and the pharyngeal region was fully dissected. The dye diffused almost completely within the injected muscle, and the diffusion was generally limited to the injected muscle.

[0404] Example 2: Pilot Dosing Study of the 'Silence and Replacement' Construct BB-301

[0405] BB-301 is a first-in-class gene medicine that uses a “silencing and replacement” approach to treat dysphagia associated with OPMD. Specifically, BB-301 relies on the applicant’s proprietary DNA-guided RNA interference (ddRNAi) platform, which combines RNA interference (RNAi) with classic AAV-based gene therapy. The applicant’s ddRNAi platform also allows for the simultaneous delivery of wild-type replacement genes, and these different gene elements work synergistically to silence the pathogenic mutant gene through RNAi expression and simultaneously replace the mutant gene with a normal (wild-type) gene to restore the natural underlying physiology of the diseased tissue.

[0406] The applicant has previously described BB-301 and its components in WO2017 / 177277, WO2018 / 107228, WO2019 / 043630, WO2020 / 077412 and WO2020 / 172720, the contents of each of which are incorporated herein by reference in their entirety. BB-301 is also described herein.

[0407] BB-301 is injected directly into the pharyngeal muscles, which are known to form the basis of morbidity and mortality in the natural history of dysphagia associated with OPMD. This pilot BB-301 dosing study was conducted in large animal subjects (particularly beagle dogs) to demonstrate that direct intramuscular injection of BB-301 via the needle 10 described herein can be safely performed during open surgery.

[0408] ●Biologically significant, highly consistent, dose-dependent levels of BB-301 tissue transduction (i.e., delivery of the multifunctional gene construct to target pharyngeal muscle cells);

[0409] ●Persistent, widespread, dose-dependent expression of three different genes containing the BB-301 gene construct in pharyngeal muscle cells; and

[0410] ●Persistent and biologically significant levels of intracellular knockdown of target genes in pharyngeal muscle cells (i.e., suppression of expression of the genes of interest).

[0411] BB-301

[0412] BB-301 incorporates Benitec's proprietary AAV9 delivery vector, which comprises: (i) a ddRNAi construct containing two nucleic acids (designated shmiR13 and shmiR17 (described herein)) encoding short hairpin microRNA (shmiR), said nucleic acids targeting the mRNA transcript of OPMD-causing mutant PABPN1; and (ii) a PABPN1 construct containing codon-optimized nucleic acids encoding a functional (wild-type) PABPN1 protein, wherein the mRNA corresponding to the codon-optimized nucleic acids is not targeted by shmiR. Two distinct siRNA species (i.e., siRNA13 and siRNA17) were processed from shmiR, each capable of independently suppressing (i.e., “silencing”) the expression of all forms of the endogenous PABPN1 protein. siRNA13 and siRNA17 silenced both wild-type PABPN1 [wtPABPN1] and mutant PABPN1 (which is the cause of OPMD). The mutant form of PABPN1 protein supports the development and progression of OPMD. In contrast, a codon-optimized sequence encoding a functional wild-type version of the PABPN1 protein within BB-301 was unaffected by the repressive activity of siRNA13 and siRNA17. This codon-optimized PABPN1 protein (i.e., coPABPN1) was used to complement the endogenous form of PABPN1 protein and replace the mutant form of PABPN1 that supports the development and progression of OPMD in diseased tissues.

[0413] Pilot dosing study of BB-301

[0414] A pilot dosing study was conducted to evaluate the safety and bioactivity of two concentrations of BB-301 across three different doses, and to optimize the administration method of BB-301 developed and described in Example 1 of this paper.

[0415] In summary, two different concentrations (1.0+E13 vg / mL and 3.0+E13 vg / mL) were tested across three different doses (1.0+E13 vg / mL and 3.0+E13 vg / mL using low injection volumes, and 3.0+E13 vg / mL using a high injection volume) by direct intramuscular injection into the hypopharyngeal (HP) and thyropharyngeal (TP) muscles of beagle dogs using open surgery and the injection needle 10 described herein. As described in Example 1, the HP muscles of beagle dogs correspond to the midpharyngeal constrictor muscles of human subjects, and the TP muscles of beagle dogs correspond to the hypopharyngeal constrictor muscles of human subjects. Beagles were chosen as a large animal model because their muscle size is comparable to that of humans. The surgical procedure for administering BB-301 to beagle dogs was performed using needle A (which is the injection needle 10 described herein) as described in Example 1 above.

[0416] BB-301 was injected only on day 1 of the pilot dosing study, and the corresponding canine pharyngeal muscles were collected for analysis 8 weeks after BB-301 delivery.

[0417] BB-301 administration was performed by a veterinarian and a licensed ENT physician with extensive experience in providing palliative surgical care for patients with OPMD.

[0418] Interim data points were collected from a complete analysis of pharyngeal muscle tissue isolated from 16 beagle subjects to date (in a study cohort of 24 subjects).

[0419] It is crucial to emphasize the key methodological differences between the current BB-301 beagle pilot dosing study conducted by Benedict and previous beagle dosing studies conducted independently by previous BB-301 licensees. Specifically, the BB-301 dosing studies conducted by previous BB-301 licensees employed a non-ideal route and method of administration to the target pharyngeal muscle tissue and used similarly limited analytical methods at the completion of the dosing phase of the study. In the current study, the inventors have focused on optimizing the route and method of BB-301 administration to the pharyngeal muscle (as described in Example 1) and improving the core analytical methods used after the completion of dosing.

[0420] result

[0421] Table 5 below presents the results of the pilot drug administration study.

[0422] Table 5 - Results of the pilot drug administration study

[0423]

[0424] As shown in Table 5, BB-301 was well transduced into the pharyngeal muscles at all evaluated doses and concentrations using open surgery and the injection needle 10 described herein. Of particular note is that, in this dosing study, BB-301 transduction in the HP muscle was improved by 248-fold (+24,650%) compared to the levels observed in previous preliminary studies of BB-301 conducted by a previous licensee of BB-301, and BB-301 transduction in the TP muscle was improved by 111-fold (+11,027%).

[0425] In summary, these data support the conclusion that open surgery and the injection needle described herein are effective for administering BB-301 (or any therapeutic agent) to the pharyngeal muscles, demonstrating biologically significant, highly consistent, and dose-dependent levels of BB-301 tissue transduction (i.e., delivery of the multifunctional gene construct to target pharyngeal muscle cells). This study also demonstrates persistent, widespread, and dose-dependent expression of three distinct nucleic acids contained within the BB-301 gene construct (i.e., shmiR13 / siRNA13, shmiR17 / siRNA17, and codon-optimized PABPN1) within pharyngeal muscle cells, along with persistent and biologically significant levels of intracellular target gene knockdown (i.e., suppression of expression of the gene of interest) within pharyngeal muscle cells.

[0426] Example 3: First Human Efficacy Study

[0427] A first-in-human Phase 1b / 2a clinical trial has been initiated to evaluate the safety and efficacy of BB-301 in subjects diagnosed with oculopharyngeal muscular dystrophy (OPMD). The United States Food and Drug Administration (FDA) approved the Investigational New Drug (IND) application for BB-301 in June 2023, and the first study subject was safely administered in the Phase 1b / 2a clinical trial of BB-301 (NCT06185673) in November 2023. Detailed information on NCT06185673 is available at ClinicalTrials.gov and is incorporated herein by reference.

[0428] This clinical trial (NCT06185673) is a 3+3 dose-escalation study designed to evaluate three discrete doses of BB-301 to identify the maximum tolerated dose (MTD). The Phase 1b component of this study aims to treat 9–18 OPMD subjects in three dose-escalation cohorts. The Phase 2a component of this study aims to treat up to 12 OPMD subjects within the MTD. Study subjects will be monitored for 52 weeks following BB-301 administration to assess the efficacy of the investigational gene therapy, and safety will be evaluated for 15 years after each subject receives BB-301. Table 6 below summarizes the design of this first-in-human clinical trial.

[0429] Table 6: Design of the first human clinical trial of BB-301.

[0430]

[0431]

[0432] To date, two participants have progressed from the natural history study to the Phase 1b / 2a clinical trial. At the time of writing, interim clinical safety and efficacy data are available for the first study participant, starting from the first protocol-specified post-dose assessment at 90 days after BB-301 administration. Please see the “Results” section below.

[0433] OPMD Natural History Research

[0434] Eligibility for the Phase 1b / 2a clinical trial requires confirmation of pathogenic triplet amplification of PABPN1. Before direct intramuscular injection of BB-301 into the pharyngeal constrictor muscle, each study subject must complete a minimum of six months of evaluation in the OPMD Natural History Study, during which baseline levels of dysphagia are assessed at five discrete time points using quantitative, X-ray-based video-fluorescence swallowing studies. Following the 6-month follow-up in the OPMD Natural History Study, subjects are eligible for screening to enter the BB-301 Phase 1b / 2a clinical trial (NCT06185673). The results of each assessment performed during the OPMD Natural History Study provide a baseline, which can be compared to the baseline after BB-301 administration in the Phase 1b / 2a clinical trial, thereby providing an indication of the efficacy and safety of the BB-301 OPMD gene therapy.

[0435] Evaluation criteria

[0436] Using a variety of X-ray-based techniques, clinical techniques, and subject-reported outcome measurements known in the art, study subjects were assessed for dysphagia (i.e., swallowing difficulty) at all time points outlined in Table 7 (i.e., during the OPMD natural history study and in the Phase 1b / 2a clinical trial following BB-301 administration) to monitor:

[0437] 1. Pharyngeal constrictor muscle function (e.g., assessed by the pharyngeal area at maximum contraction)

[0438] 2. Swallowing efficiency (e.g., assessed by total pharyngeal residue)

[0439] 3. Oropharyngeal swallowing difficulties reported by the participants (e.g., as assessed by the Sydney Swallowing Questionnaire).

[0440] 4. Swallowing ability (e.g., assessed through a timed cold water drinking test)

[0441] At each assessment visit, the study subjects consumed barium-containing foods and liquids of various viscosities (solid foods, and very viscous, medium viscous, and thin liquids), and the swallowing process was assessed using the methods described above.

[0442] Table 7 - Summary of Evaluations of Clinical Development Programs for BB-301

[0443]

[0444] Pharyngeal area at maximal contraction (PhAMPC) was measured using the length of the patient's C2-C4 cervical vertebrae as an anatomical scalar. The "pharyngeal area" measurement includes the area of ​​the visible air cavity or mass at maximal contraction. Measurements were taken on a radiographic frame at the point of most compact contraction of the pharyngeal lumen during swallowing. Normal PhAMPC values ​​for characteristic foods and liquids evaluated in the swallowing task in question range from 0% to 2.2%. PhAMPC was calculated as follows:

[0445]

[0446] The C2-C4 length was also used as an anatomical scalar to measure total pharyngeal residue. Total pharyngeal residue measurement includes the volume (v) of material (res) remaining in the pharynx after the first swallowed bolus. Measurements were taken on a first radiographic frame showing the pyriform fossa (PS) at its lowest position. Normal total pharyngeal residue values ​​should be close to zero. Total pharyngeal residue was calculated as follows:

[0447]

[0448] Subject-reported oropharyngeal dysphagia was measured using the Sydney Swallowing Questionnaire (SSQ), a self-report checklist for assessing subjective symptoms of oropharyngeal dysphagia, with strong content, conception, discrimination, and predictive validity and test-retest reliability across a range of patient populations. The SSQ is a 17-item questionnaire developed to measure the severity of oropharyngeal dysphagia symptoms reported by affected subjects. Except for one question, all questions used a 100 mm long visual analog scale. Possible scores ranged from 0 to 1700, with higher scores indicating greater dysphagia. Healthy subjects without dysphagia were likely to present an average score of approximately 59.0.

[0449] Changes in assessment criteria during natural history studies are used to determine the progression (if any) of dysphagia in study subjects. Mean and final outcomes (i.e., before administration) are assessed in study subjects to determine baseline measurements of the assessment criteria. Any changes can be assessed based on said baseline measurements at days 90, 180, 270, and 360 following treatment with BB-301 DP.

[0450] As discussed above, at the time of writing, both subjects had completed the natural history study and progressed to the phase 1b / 2a clinical trial.

[0451] BB-301 Phase 1b / 2a Clinical Trial Methods

[0452] NCT06185673 is a phase 1b / 2a, first-in-human, single-arm, open-label, sequential, dose-escalation cohort study. Subjects who have previously been enrolled in the Benitec Biopharma Inc. OPMD-NH-001 natural history study and completed at least 6 months of follow-up may be eligible to participate in this interventional study.

[0453] To characterize safety properties and determine the maximum tolerated dose (MTD) / recommended phase 2 dose (RP2D) of BB-301, the phase 1b component was designed to involve dose escalation by including subjects in sequential dose cohorts in which BB-301 was administered as a direct intramuscular injection into the middle pharyngeal constrictor (MPC) and hypopharyngeal constrictor (IPC). BB-301 was administered intramuscularly following open surgical dissection of the pharyngeal region under general anesthesia. An independent Data Safety Monitoring Board (DSMB), operating under the bylaws, will review all available safety data from the cohorts (as they become available) and cumulative information from all administered subjects, and will make decisions regarding the appropriateness of dose escalation, other aspects of the safety properties of BB-301, and the conduct of the study.

[0454] The dose of BB-301 administered to the respective subjects (as shown in Figure 8) will be determined by the group allocation (Phase 1b) and the established MTD / RP2D (Phase 2a). On Day 1, following an open surgical dissection of the pharyngeal region of the neck 500 under general anesthesia, the designated dose of BB-301 will be 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).

[0455] To minimize systemic inflammatory responses and / or specific immune responses in study subjects caused by surgical dissection, prednisone will be administered prophylactically (starting from day -1) and gradually reduced over 8 weeks.

[0456] Injection procedure and device operation

[0457] Unless otherwise stated, the injection procedures and device operation used in the Phase 1b / 2a clinical trials are generally consistent with Examples 1 and 2 of this document.

[0458] Select the 3Dose™ unit injector microinjector (catalog number 3DOSEGREEN-125) from Vlow Medical as injector 100 ( Figure 8B In this example, syringe 100 is a pre-filled multi-injection syringe, which enables repeated administration of a predetermined small volume of BB-301 drug product (DP).

[0459] The syringe 100 includes an injection volume selection mechanism that allows selection of an injection volume range covering 12.5 μL to 50 μL per plunger "click". The injection volume selection mechanism includes a plunger rotatable relative to the syringe barrel for selecting one of multiple plunger configurations corresponding to the desired injection volume. The syringe 100 facilitates the delivery of multiple injections of BB-301 DP into the targeted pharyngeal constrictor muscle, ensuring the delivery of the desired total volume of BB-301 DP to the target muscle. The appropriate plunger setting is selected by rotating the plunger about the central axis of the syringe to facilitate the dispensing of 12.5 μL, 25 μL, or 50 μL of BB-301 DP with each plunger "click" of the syringe 100, and to achieve the predetermined total number of injections on each side of the pharyngeal constrictor muscle, thereby setting the required BB-301 DP volume for each injection.

[0460] As discussed above, syringe 100 allows an operator to administer one or more injections of a selected volume of BB-301 DP (12.5 μL, 25 μL, or 50 μL BB-301 DP) at any injection site within the targeted pharyngeal constrictor muscle by using a single or multiple "clicks" of the plunger at each discrete injection site within the target pharyngeal constrictor muscle of the subject. Additionally, syringe 100 allows an operator to administer multiple injections of a selected volume of BB-301 DP over a surgically accessible anatomical region of the pharyngeal constrictor muscle by targeting discrete injection sites evenly distributed across the muscle plane.

[0461] During the surgical procedure, BB-301 DP is intramuscularly injected into the pharyngeal constrictor muscle using the syringe 100 and injection needle 10 described herein (and will be performed on future OPMD subjects). Specifically, needle A described in Examples 1 and 2 herein is used as injection needle 10. Figure 1 The syringe 100 is coupled to the injection needle 10 (i.e., needle A as described herein) to form a drug product injection device (DPID) 200 (Figure 8).

[0462] For intraoperative administration of BB-301 DP to OPMD subjects currently enrolled in the Phase 1b / 2a clinical trial, two DPID 200s are used per pharyngeal constrictor muscle, i.e., two (2) DPID 200s are used to complete the administration of MPC muscle 502, and two (2) DPID 200s are used to complete the administration of IPC muscle 501. This regimen will also be used for intraoperative administration to future OPMD subjects enrolled in the Phase 1b / 2a clinical trial.

[0463] The total dose of BB-301 DP to be administered to each pharyngeal constrictor muscle will be delivered in a two-step process, which distributes the intramuscular injection in a manner that emphasizes the two separate contralateral portions of the corresponding pharyngeal constrictor muscle, as determined by the relative anatomical position of each tissue segment relative to the pharyngeal suture 506. Figure 10 In this regard, one (1) DPID 200 will be used to complete the administration of each contralateral (i.e., left and right) side of MPC muscle 502. Similarly, one (1) DPID 200 will be used to complete the administration of each contralateral (i.e., left and right) side of IPC muscle 501. Therefore, completing the planned injection of the pharyngeal constrictor muscle for each study subject will require the use of at least four (4) DPID 200 per subject.

[0464] Figure 10 The specific anatomical sites where BB-301 DP was injected are shown, including the left and right components of the MPC muscle 502 and the left and right components of the IPC 501 muscle. Specifically, Figure 10The rear view of the pharyngeal muscles in the neck 500 of the patient is shown, including the hypopharyngeal constrictor muscle 501, the middle pharyngeal constrictor muscle 502, and the superior pharyngeal constrictor muscle 503. Figure 10 The pharyngeal suture 506, which separates the left and right components of the pharyngeal constrictor muscles 501, 502 and 503, is also shown.

[0465] The final dose of BB-301DP administered to each study subject was determined based on the subject’s group assignment (as discussed below).

[0466] Application procedure

[0467] In the Phase 1b dose-escalation study, BB-301 DP was administered to 'Subject 1' and 'Subject 2' during the surgical procedure, following the general administration procedure described below. The same procedure will also be used for the remaining subjects who progressed from the natural history study to the Phase 1b / 2b study.

[0468] Amoxicillin (1 g) was administered intravenously immediately before the administration of BB-301 DP during the procedure (i.e., day 1 of the phase 1b study), and the intravenous administration of amoxicillin was repeated twice daily for the first two days following the administration of BB-301 DP during the procedure.

[0469] BB-301 DP was administered under general anesthesia with endotracheal intubation and appropriate analgesia. The patient was first placed in a supine position, and then a medium-sized probe or a plain endotracheal tube was placed in the esophagus under visual guidance to help identify the pharyngeal junction (“esophageal identification tube”) preoperatively.

[0470] A bilateral neck incision of approximately 5 cm is made along the anterior margin 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 are obliquely inclined laterally, and the larynx is rotated. Blunt dissection is then performed to provide 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 restricted. This approach allows for extensive overall exposure of the posterior surface of the pharynx and the constrictor muscles 501, 502, and 503.

[0471] The cricopharyngeal muscles, hypopharyngeal constrictor 501, and middle pharyngeal constrictor 502 of the pharynx are extensively exposed up to the level of the superior angle of the thyroid cartilage. The cricopharyngeal muscles are more or less distinct from the hypopharyngeal constrictor 501 and are identified by their ring fibers, cricoid cartilage rings, and the lower edge of the hypopharyngeal constrictor 501. The superior portion of the hypopharyngeal constrictor 501 originates from the thyroid cartilage (thyroporopharynx). It is identified between the posterior edges of the thyroid cartilage and each half connects inferiorly to the cricoid cartilage at the joint (cricothyroid joint), where the fibrous pharyngeal suture 506 lies in the posterior midline of the pharynx. The higher region for pharyngeal muscle injection occurs at the level of the superior angle of the thyroid cartilage.

[0472] Using pre-filled BB-301 DPID 200, intramuscular injections of BB-301 DP were performed on each contralateral side (left and right of pharyngeal suture 506) of the inferior pharyngeal constrictor muscle 501 (Constrictor pharyngis inferior) and on each contralateral side (left and right of pharyngeal suture 506) of the middle pharyngeal constrictor muscle 502 (Musculus constrictor pharyngis medius). Each DPID 200 was filled with a final volume of the specified dose of BB-301 DP, sufficient for injection on one side of each pharyngeal constrictor muscle, as shown below:

[0473] ● One DPID 200 filler contains enough BB-301 DP to complete the planned four (4) intramuscular injections into the left segment of the pharyngeal constrictor muscle 502 (left lateral to the pharyngeal suture 506), wherein the injections are evenly distributed across the entire tissue plane (i.e., 4 × 50 μL or 4 × 75 μL).

[0474] ● One DPID 200 filler contains enough BB-301 DP to complete the planned four (4) intramuscular injections into the right segment of the pharyngeal constrictor muscle 502 (right lateral to the pharyngeal suture 506), wherein the injections are evenly distributed across the entire tissue plane (i.e., 4 × 50 μL or 4 × 75 μL).

[0475] ● One DPID 200 filler contains enough BB-301 DP to complete the planned eight (8) intramuscular injections into the left segment of the hypopharyngeal constrictor muscle 501 (left lateral to the pharyngeal suture 506), wherein the injections are evenly distributed across the entire tissue plane (i.e., 8 × 50 μL or 8 × 75 μL).

[0476] ● One DPID 200 is filled with enough BB-301 DP to complete the planned eight (8) intramuscular injections into the right segment of the hypopharyngeal constrictor muscle 501 (right lateral to the pharyngeal suture 506), wherein the injections are evenly distributed across the entire tissue plane (i.e., 8 × 50 μL or 8 × 75 μL).

[0477] Therefore, in order to treat each study subject in each group, the planned injection of pharyngeal constrictor muscle will require at least four (4) DPID 200 per subject.

[0478] The final dose of BB-301 DP administered to each study subject was determined based on subject cohort allocation. In this regard, OPMD subjects enrolled in the Phase 1b dose-escalation study were randomly assigned to the following cohorts:

[0479] ●Group 1: Subjects in Group 1 received a fixed amount of BB-301 IM injected into the corresponding pharyngeal constrictor muscle on the day of administration (as discussed above), with a total dose of 1.2e13 vg / subject (low dose).

[0480] ●Group 2: Subjects in Group 2 received a fixed number of BB-301 IM injections into the corresponding pharyngeal constrictor muscles on the day of administration (as discussed above), with a total dose of 3.6e13 vg / subject (moderate dose).

[0481] ●Group 3: Subjects in Group 3 received a fixed number of BB-301 IM injections into the corresponding pharyngeal constrictor muscles on the day of administration (as discussed above), with a total dose of 5.4e13 vg / subject (high dose).

[0482] Therefore, during the Phase 1b dose escalation study, the dose of BB-301 DP delivered to any subject may vary from subject to subject.

[0483] Following administration of BB-301 DP to study subjects, the dissected tissue was closed by suturing (subcutaneous suturing with 3 / 0 absorbable Vichy sutures or equivalents and skin suturing with non-absorbable monofilaments), with or without drainage and dressings (or bandages) as appropriate. An esophageal identification tube was removed from the esophagus and mouth; this allowed for further examination of the integrity of the superior esophageal sphincter mucosa.

[0484] Following administration of BB-301 during surgery, study subjects were provided with postoperative inpatient care, including twice-daily IV injections of amoxicillin 1 g (e.g., Clamoxyl) on days 0–2, daily examination of the surgical site, removal of drainage tubes (if any) as appropriate on days 1–3, and removal of skin sutures on days 10–12, provided adequate healing was observed.

[0485] Study participants were able to resume a soft diet on day 0-1.

[0486] result

[0487] At the time of writing, clinical efficacy data were available for the first study subject (“Subject 1”) starting from the first protocol-specified post-dose assessment at 90 days after BB-301 administration.

[0488] During the OPMD natural history study, which represented the pre-drug observation period for each subject, Subject 1 experienced progressively worsening dysphagia, as evidenced by the results of the Video Fluorescence Surgery Study (VFSS), the Timed Cold Water Drinking Test, and the key subject-reported outcome measure (Sydney Swallowing Questionnaire). The Video Fluorescence Surgery Study represents the gold standard analytical method for the quantitative assessment of dysphagia (swallowing difficulty) in a clinical setting.

[0489] Table 8 summarizes the preliminary clinical, radiographic, and subject-reported assessment results (90 days post-treatment, “Day 90”) of Subject 1 in the BB-301 Phase 1b / 2a clinical trial (NCT06185673). On Day 90, Subject 1 showed improvement in key video fluorescence microscopy assessments, as well as similar improvements in key subject-reported outcome measures, compared to the mean and final values ​​of the corresponding assessments completed during the pre-dose observation period. Notably, the results of many assessments completed on Day 90 confirmed improvements compared to the initial measurements assessed at the subject’s first visit in the OPMD Natural History Study, which occurred more than 12 months prior to the Day 90 assessment.

[0490] The most significant improvements were observed on day 90 in swallowing tasks that assessed pharyngeal constrictor function and swallowing efficiency while consuming thin liquids, solid foods, and thick non-solid foods (e.g., yogurt or pudding) (see Table 8). These improvements correlated with improvements in the key subject-reported outcome measure, the Sydney Swallowing Questionnaire, indicating improved swallowing function as reported by Subject 1 (see Table 8).

[0491] Table 8 - Summary of swallowing study results for Subject 1

[0492]

[0493]

[0494] Table 8 (continued) - Summary of swallowing study results for Subject 1

[0495]

[0496]

[0497] Conclusions of the first human efficacy study

[0498] As expected, Subject 1 experienced disease progression during the natural history study period. However, after administration of BB-301 DP using the device and method described herein, Subject 1's dysphagia significantly improved. The dose of BB-301 was sufficiently bioactive to promote benefit in Subject 1, and these benefits were visible in the initial follow-up assessment performed on day 90 post-administration. BB-301 did not cause any serious adverse events in Subject 1.

[0499] Based on the results of the Phase 1b dose escalation study in all subjects, the maximum tolerated dose (MTD) / recommended Phase 2 dose (RP2D) for the Phase 2a dose expansion study will be determined.

[0500] Those skilled in the art will understand that numerous variations and / or modifications can be made to the above embodiments without departing from the broad general scope of this disclosure. Therefore, the embodiments of the present invention should be considered illustrative rather than restrictive in all respects.

Claims

1. An injection needle comprising: Needle cannula, the needle cannula comprising: The proximal portion has a proximal end; The distal portion has a distal end; The middle portion is disposed between the proximal portion and the distal portion; as well as A lumen 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 lumen opening at or near the distal end of the distal portion. The proximal portion and the distal portion are each substantially straight, and the intermediate portion is curved such that the distal portion extends relative to the proximal portion at an angle between 75° and 105°. and The length of the proximal portion is shorter than the length of the distal portion.

2. The injection needle of claim 1, wherein the distal portion extends relative to the proximal portion at an angle between about 85° and 95°.

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

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

5. The injection needle according to any one of claims 1 to 4, wherein the length of the proximal portion is less than 90%, less than 80%, less than 70%, or 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 between about 10 mm and 27 mm.

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

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

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

11. The injection needle according to any one of claims 1 to 10, wherein: The distal portion extends relative to the proximal portion at an angle between approximately 85° and 95°; and The radius of curvature of the middle section is between approximately 2.5 mm and 3.1 mm.

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

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

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

15. The injection needle according to any one of claims 1 to 14, wherein the outer diameter of the needle cannula is between about 0.3 mm and about 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 between about 0.1 mm and about 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 between about 0.03 mm and about 0.17 mm.

18. The injection needle according to any one of claims 1 to 17, wherein the dead volume of the needle cannula is between about 0.5 µL / 25.4 mm and about 1.5 µL / 25.4 mm.

19. The injection needle according to any one of claims 1 to 18, wherein the needle cannula includes 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 includes a bevel formed at the distal end of the needle, wherein the bevel includes a beveled edge opposite to the inner fold side of the needle cannula.

21. The injection needle according to any one of claims 1 to 20, wherein the distal tip is beveled at an angle between about 5° and about 15°.

22. The injection needle according to any one of claims 1 to 21, wherein the length of the bevel is between about 0.5 mm and about 3.5 mm.

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

24. The injection needle according to any one of claims 1 to 23, wherein the injection needle further comprises a needle hub connected to the needle sheath, wherein the needle sheath projects distally relative to the needle hub.

25. The injection needle of claim 24, wherein the needle cannula further includes a support portion, and wherein the support portion is disposed within the needle hub.

26. The injection needle according to any one of claims 1 to 25, wherein the needle cannula includes an inlet located 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 of the proximal end adjacent to the proximal portion of the needle sheath, and wherein the distal portion of the needle hub is forged.

28. The injection needle according to any one of claims 1 to 27, wherein the injection needle includes an inlet 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 injecting a therapeutic composition directly into the pharyngeal muscle of a subject.

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

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

32. The use according to any one of claims 29 to 31, wherein the pharyngeal muscle comprises one or more of the following pharyngeal muscles: hypopharyngeal constrictor, middle pharyngeal constrictor, superior pharyngeal constrictor, palatopharyngeal muscle, eustachian tube pharyngeal muscle, styloid process pharyngeal muscle, or any combination thereof.

33. The use according to any one of claims 29 to 32, wherein the therapeutic composition is effective for 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 of forming an injection needle, wherein the method comprises: A needle cannula is provided, the needle cannula comprising a proximal end, a distal end, and a lumen extending through the needle cannula between the proximal end and the distal end; and The needle cannula is bent such that it includes a substantially straight proximal portion having the proximal end, a substantially straight distal portion having the distal end, and an intermediate portion disposed between the proximal portion and the distal portion, the intermediate portion being curved such that the distal portion extends at an angle relative to the proximal portion.

35. The method of claim 34, wherein the portion is curved such that the distal portion extends relative to the proximal portion at an angle between 75° and 105°.

36. The method of 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 an injection needle according to any one of claims 1 to 28.

38. A method of administering a therapeutic composition to the pharyngeal muscles of a subject, the method comprising injecting the therapeutic composition directly into the pharyngeal muscles of the subject using a bent or curved injection needle.

39. The method of claim 38, wherein the bent or kinked injection needle comprises an injection needle that: The proximal portion has a proximal end; The distal portion has a distal end; The middle portion is disposed between the proximal portion and the distal portion; as well as A lumen 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 lumen opening at or near the distal end of the distal portion. The proximal and distal portions are each substantially straight, and the intermediate portion is curved such that the distal portion extends at an angle relative to the proximal portion. Optionally, the injection needle is the injection needle according to any one of claims 1 to 28.

40. The method according to claim 38 or 39, wherein the therapeutic composition is applied to one or more pharyngeal muscles selected from the group consisting of: hypopharyngeal constrictor, middle pharyngeal constrictor, superior pharyngeal constrictor, palatopharyngeal muscle, eustachian tube pharyngeal muscle, styloid pharyngeal muscle, and any combination thereof.

41. The method according to any one of claims 38 to 40, comprising applying the therapeutic composition to the middle pharyngeal constrictor muscle and the lower 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 after making an incision in the neck of the subject, the incision being sufficient to provide a pathway for direct access to at least a portion of the pharyngeal muscle of the subject.

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

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

45. The method of any one of claims 38 to 44, wherein the therapeutic composition comprises a viral delivery vector, the viral delivery vector comprising a 'silencing and replacement' construct, the 'silencing and replacement' construct comprising: (i) A DNA-guided RNAi (ddRNAi) construct containing nucleic acids, wherein the nucleic acids comprise a sequence encoding a short hairpin microRNA (shmiR); and (ii) A PABPN1 construct containing nucleic acid, the nucleic acid containing a sequence encoding a functional PABPN1 protein, the sequence having an mRNA transcript not targeted by the shmiR encoded by the ddRNAi construct.

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

21.

47. The method of claim 45, wherein: (i) The AAV comprises a viral capsid protein from AAV9, the viral capsid protein comprising mutations A67E, Q81R, K84D, and A85S relative to the full-length wild-type AAV serotype 9 capsid sequence shown in SEQ ID NO: 19, optionally wherein the viral capsid protein comprises the amino acid sequence shown in SEQ ID NO: 28; or (ii) The AAV comprises a viral capsid protein from AAV8, the viral capsid protein comprising mutations A42S, A67E, Q81R, K84D, A85S and Q105K relative to the full-length wild-type AAV serotype 8 capsid sequence shown in SEQ ID NO: 23, optionally wherein the viral capsid protein comprises the amino acid sequence shown 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 nucleic acid molecule being codon-optimized such that the corresponding mRNA sequence of the nucleic acid molecule is not recognized by the shmiR encoded and expressed from the ddRNAi construct, optionally wherein the codon-optimized nucleic acid comprises the sequence shown in SEQ ID NO: 15 and encodes the amino acid sequence shown 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, wherein the shmiR comprises an effector sequence substantially complementary to the sequence shown in SEQ ID NO: 1 and / or an effector sequence substantially complementary to the sequence shown in SEQ ID NO:

2.

50. The method of claim 49, wherein the ddRNAi construct comprises: (i) a DNA sequence encoding an shmiR (shmiR13) comprising the effector sequence shown in SEQ ID NO: 4 and the effector complement sequence shown in SEQ ID NO: 3; and a DNA sequence encoding an shmiR (shmiR17) comprising the effector sequence shown in SEQ ID NO: 6 and the effector complement sequence shown in SEQ ID NO: 5; and / or (ii) containing the DNA sequence shown in SEQ ID NO: 12 or a nucleic acid composed therefrom (shmiR13) and containing the DNA sequence shown in SEQ ID NO: 13 or a nucleic acid composed therefrom (shmiR17).

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

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

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

54. An injection device comprising: (i) a pre-filled syringe containing a therapeutic composition effective for treating or preventing dysphagia associated with OPMD; and (ii) The injection needle according to any one of claims 1 to 28, wherein the injection needle is coupled to the pre-filled syringe.

55. The injection device of claim 54, wherein the therapeutic composition comprises an AAV9 carrier, the AAV9 carrier comprising: (a) A viral capsid protein from AAV9, said viral capsid protein comprising mutations A67E, Q81R, K84D, and A85S identified relative to the full-length wild-type AAV9 capsid sequence shown in SEQ ID NO: 19, optionally said viral capsid protein comprising the amino acid sequence shown in SEQ ID NO: 28; and (b) A silence and replacement construct, which includes: (i) A muscle-specific promoter (optionally, the promoter is the Spc512 muscle-specific promoter); (ii) A DNA-guided RNAi (ddRNAi) construct comprising: a nucleic acid comprising a sequence encoding shmiR13 as described herein (optionally the DNA sequence shown in SEQ ID NO: 12) or composed thereof; and a nucleic acid comprising a sequence encoding shmiR17 as described herein (optionally the DNA sequence shown in SEQ ID NO: 13); and (iii) A PABPN1 construct comprising a nucleic acid sequence encoding a functional PABPN1 protein comprising the amino acid sequence shown in SEQ ID NO: 16, wherein the nucleic acid sequence encoding the functional PABPN1 protein is a DNA sequence that is codon-optimized such that the corresponding mRNA sequence of the DNA sequence is not targeted by the shmiR encoded by the ddRNAi construct (optionally wherein the codon-optimized nucleic acid comprises the sequence shown in SEQ ID NO: 15). The muscle-specific promoter is operatively linked to the ddRNAi construct and the PABPN1 construct.

56. The injection device according to claim 54 or claim 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 of claim 56, further comprising an injection volume setting mechanism operable to set the injection volume.

58. The injection device of claim 57, wherein the injection volume setting mechanism is operable to set the injection volume between about 12.5 µL and about 75 µL.

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

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

61. A kit comprising a group of two or more injection devices according to any one of claims 54 to 60, wherein the pre-filled syringes of said injection devices collectively contain a single therapeutic dose of said therapeutic composition for treating a single subject.

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

63. The kit of claim 62, wherein the injection device contains at least 200 µL of the therapeutic composition, such as the therapeutic composition between about 200 µL and about 2000 µL.

64. The kit according to claim 63, comprising: (i) Two injection devices containing the therapeutic composition between about 200 µL and about 500 µL; and (ii) Two injection devices containing the therapeutic composition between about 400 µL and about 1000 µL.

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

66. The kit according to any one of claims 61 to 64, wherein the single therapeutic dose of the therapeutic composition is: The therapeutic composition described in vg / subject; The therapeutic composition described in vg / subject; or The therapeutic composition described in vg / subject.

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