Methods for Treating Oculopharyngeal Muscular Dystrophy (OPMD)

1 . A method for treating a subject suffering from oculopharyngeal muscular dystrophy (OPMD) comprising administering to said subject a composition comprising:

(a) a ddRNAi construct comprising a nucleic acid comprising a DNA sequence which encodes a short hairpin micro-RNA (shmiR); and

(b) a PABPN1 construct comprising a DNA sequence encoding a functional PABPN1 protein having a mRNA transcript which is not targeted by the shmiR(s) encoded by the nucleic acid; wherein the composition is administered by direct injection to a pharyngeal muscle of the subject.

2 . (canceled)

3 . The method of claim 1 , wherein the subject has improved swallowing following administering the composition by direct injection to a pharyngeal muscle of the subject.

4 . The method of claim 1 , wherein the composition comprises an expression vector comprising the ddRNAi construct, the PABPN1 construct, or a combination thereof.

5 . The method of claim 4 , wherein the expression vector comprises, in a 5′ to 3′ direction, the ddRNAi construct and the PABPN1 construct.

6 . The method of claim 4 , wherein the expression vector comprises, in a 5′ to 3′ direction, the PABPN1 construct and the ddRNAi construct.

7 . The method of claim 4 , wherein the expression vector is a plasmid or minicircle.

8 . The method of claim 4 , wherein the expression vector is a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, a retroviral vector, an adenoviral (AdV) vector and a lentiviral (LV) vector.

9 . The method of claim 4 , wherein the ddRNAi construct and/or PABPN1 construct is/are comprised within an expression construct and the expression construct comprises inverted terminal repeats (ITRs) from an AAV serotype, optionally wherein the AAV serotype is AAV2, AAV8 or AAV9.

10 . (canceled)

11 . The method of claim 1 , wherein the DNA sequence encoding the functional PABPN1 protein is codon optimised such that its mRNA transcript is not targeted by the shmiRs of the ddRNAi construct, optionally wherein the DNA sequence encoding the functional PABPN1 protein is set forth in SEQ ID NO: 73.

12 . (canceled)

13 . The method of claim 1 , wherein the DNA sequence encoding the functional PABPN1 protein is operably-linked to a promoter comprised within the PABPN1 construct and positioned upstream of the DNA sequence encoding the functional PABPN1 protein, optionally wherein the promoter comprised within the PABPN1 construct is a muscle-specific promoter.

14 . (canceled)

15 . The method of claim 1 , wherein the shmiR comprises:

an effector sequence of at least 17 nucleotides in length;

an effector complement sequence;

a stemloop sequence; and

a primary micro RNA (pri-miRNA) backbone;

wherein the effector sequence is substantially complementary to a region of corresponding length in an RNA transcript of human PABPN1.

16 . The method of claim 15 , wherein:

(i) the shmiR comprises an effector sequence which is substantially complementary to a region of corresponding length within the RNA sequence set forth in SEQ ID NO: 87;

(ii) the shmiR comprises an effector sequence which is substantially complementary to a region of corresponding length in an RNA transcript set forth in any one of SEQ ID NOs: 1-13; and/or

(iii) the shmiR is selected from the group consisting of:

a shmiR comprising an effector sequence set forth in SEQ ID NO: 15 and an effector complement sequence set forth in SEQ ID NO: 14;

a shmiR comprising an effector sequence set forth in SEQ ID NO: 17 and an effector complement sequence set forth in SEQ ID NO: 16;

a shmiR comprising an effector sequence set forth in SEQ ID NO: 19 and an effector complement sequence set forth in SEQ ID NO: 18;

a shmiR comprising an effector sequence set forth in SEQ ID NO: 21 and an effector complement sequence set forth in SEQ ID NO: 20;

a shmiR comprising an effector sequence set forth in SEQ ID NO: 23 and an effector complement sequence set forth in SEQ ID NO: 22;

a shmiR comprising an effector sequence set forth in SEQ ID NO: 25 and an effector complement sequence set forth in SEQ ID NO: 24;

a shmiR comprising an effector sequence set forth in SEQ ID NO: 27 and an effector complement sequence set forth in SEQ ID NO: 26;

a shmiR comprising an effector sequence set forth in SEQ ID NO: 29 and an effector complement sequence set forth in SEQ ID NO: 28;

a shmiR comprising an effector sequence set forth in SEQ ID NO: 31 and an effector complement sequence set forth in SEQ ID NO: 30;

a shmiR comprising an effector sequence set forth in SEQ ID NO: 33 and an effector complement sequence set forth in SEQ ID NO: 32;

a shmiR comprising an effector sequence set forth in SEQ ID NO: 35 and an effector complement sequence set forth in SEQ ID NO: 34;

a shmiR comprising an effector sequence set forth in SEQ ID NO: 37 and an effector complement sequence set forth in SEQ ID NO: 36; and

a shmiR comprising an effector sequence set forth in SEQ ID NO: 39 and an effector complement sequence set forth in SEQ ID NO: 38.

17 . (canceled)

18 . (canceled)

19 . The method of claim 1 , wherein the shmiR comprises, in a 5′ to 3′ direction:

a 5′ flanking sequence of the pri-miRNA backbone;

the effector complement sequence;

the stemloop sequence;

the effector sequence; and

a 3′ flanking sequence of the pri-miRNA backbone;

optionally wherein:

the stemloop sequence is the sequence set forth in SEQ ID NO: 40;

the pri-miRNA backbone is a pri-miR-30a backbone; and/or

the 5′ flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 41 and

the 3′ flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 42.

20 .- 22 . (canceled)

23 . The method of claim 1 , wherein:

the shmiR comprises a sequence set forth in any one of SEQ ID NOs: 43-55; and/or

the DNA sequence which encodes the shmiR is set forth in any one of SEQ ID NO: 56-68.

24 . (canceled)

25 . The method of claim 1 , comprising administering at least two nucleic acids encoding shmiRs, or administering a ddRNAi construct comprising the at least two nucleic acids, wherein each shmiR comprises an effector sequence which is substantially complementary to a RNA transcript corresponding to a PABPN1 protein which is causative of OPMD, and wherein each shmiR comprises a different effector sequence.

26 . The method of claim 25 , wherein:

(i) each of the at least two nucleic acids encode a shmiR comprising an effector sequence which is substantially complementary to a region of corresponding length in an RNA transcript set forth in one of SEQ ID NOs: 1, 2, 4, 7, 9, 10 and 13;

(ii) the at least two nucleic acids are selected from the group consisting of:

a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 15 and an effector complement sequence set forth in SEQ ID NO: 14 (shmiR2);

a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 17 and an effector complement sequence set forth in SEQ ID NO: 16 (shmiR3);

a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 21 and an effector complement sequence set forth in SEQ ID NO: 20 (shmiR5);

a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 27 and an effector complement sequence set forth in SEQ ID NO: 26 (shmiR9);

a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 31 and an effector complement sequence set forth in SEQ ID NO: 30 (shmiR13);

a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 33 and an effector complement sequence set forth in SEQ ID NO: 32 (shmiR14); and

a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 39 and an effector complement sequence set forth in SEQ ID NO: 38 (shmiR17); and/or

(iii) the at least two nucleic acids are selected from the group consisting of:

a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 56 (shmiR2);

a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 57 (shmiR3);

a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 59 (shmiR5);

a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 62 (shmiR9);

a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 64 (shmiR13);

a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 65 (shmiR14); and

a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 68 (shmiR17).

27 . (canceled)

28 . (canceled)

29 . The method of claim 25 , wherein:

(i) each of the at least two nucleic acids encode a shmiR comprising an effector sequence which is substantially complementary to a region of corresponding length in an RNA transcript set forth in one of SEQ ID NOs: 2, 9, 10 and 13;

(ii) the at least two nucleic acids are selected from the group consisting of:

a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 17 and an effector complement sequence set forth in SEQ ID NO: 16 (shmiR3);

a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 31 and an effector complement sequence set forth in SEQ ID NO: 30 (shmiR13);

a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 33 and an effector complement sequence set forth in SEQ ID NO: 32 (shmiR14); and

a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 39 and an effector complement sequence set forth in SEQ ID NO: 38 (shmiR17); and/or

(iii) the at least two nucleic acids are selected from the group consisting of:

a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 57 (shmiR3);

a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 64 (shmiR13);

a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 65 (shmiR14); and

a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 68 (shmiR17).

30 . (canceled)

31 . (canceled)

32 . The method of claim 1 , wherein said ddRNAi construct comprises:

(i) a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 31 and an effector complement sequence set forth in SEQ ID NO: 30 (shmiR13); and

a nucleic acid comprising or consisting of a DNA sequence encoding a shmiR comprising an effector sequence set forth in SEQ ID NO: 39 and an effector complement sequence set forth in SEQ ID NO: 38 (shmiR17); and/or

(ii) a nucleic acid comprising or consisting of the DNA sequence set forth in SEQ ID NO: 64 (shmiR13); and

a nucleic acid comprising or consisting of the DNA sequence set forth in SEQ ID NO: 68 (shmiR17).

33 . (canceled)

34 . The method of claim 1 , wherein the composition further comprises one or more pharmaceutically acceptable carriers.

35 . The method of claim 1 , wherein the pharyngeal muscle comprises one or more of an inferior constrictor muscle, a middle constrictor muscle, a superior constrictor muscle, a palatopharyngeus muscle, a salpingopharyngeus muscle, a stylopharyngeus muscle, or any combination thereof.