JPWO2020223362A5

JPWO2020223362A5 -

Info

Publication number: JPWO2020223362A5
Application number: JP2021564730A
Authority: JP
Publication date: 2023-05-08

Claims

An expression cassette comprising a nucleic acid sequence encoding human acid α-glucosidase (hGAA), including at least the active site of hGAA780I, under the control of regulatory sequences directing its expression, wherein position 780 is the amino acid in SEQ ID NO:3. Said expression cassette based on position numbering.

wherein said regulatory element directs expression in multiple target tissues, optionally wherein said multiple target tissues comprise two or more of liver cells, skeletal muscle cells, cardiac cells, and central nervous system cells; The expression cassette according to claim 1.

said nucleic acid sequence further comprising at least two tandem repeats of miR target sequences, said at least two tandem repeats comprising at least a first miRNA target sequence and at least a second miRNA target sequence, which may be the same or different; is operably linked 3' to said sequence encoding said fusion protein.

The expression cassette is
(a) a viral vector selected from a recombinant parvovirus, a recombinant lentivirus, a recombinant retrovirus, a recombinant adenovirus, optionally wherein said recombinant parvovirus is an adeno-associated virus; and the adeno-associated virus is AAVhu. 68 or variants thereof, AAV9 or variants thereof, or AAVrh. 10 or variants thereof, or
(b) a non-viral vector selected from naked DNA, naked RNA, inorganic particles, lipid particles, polymer-based vectors, or chitosan-based formulations;
The expression cassette according to any one of claims 1 to 3, carried by.

II A recombinant adeno-associated virus (rAAV) useful for the treatment of type I glycogen storage disease (Pompe) disease, said rAAV comprising
(a) an AAV capsid that targets at least one cell of muscle, heart, and central nervous system;
(b) a vector genome packaged in said AAV capsid, said vector genome directing its expression a nucleic acid sequence encoding human acid alpha-glucosidase (hGAA) comprising at least the active site of hGAA780I; said vector genome comprising under the control of regulatory sequences, position 780 being based on the numbering of the amino acid positions in SEQ ID NO:3;
Said rAAV, comprising

The hGAA is
(a) at least amino acid 204 to amino acid 890 of SEQ ID NO: 3 (hGAA780I), or a sequence at least 95% identical thereto and having Ile at position 780;
(b) at least amino acid 204 to amino acid 952 of SEQ ID NO: 3, or a sequence at least 95% identical thereto and having Ile at position 780;
(c) at least amino acid 123 to amino acid 890 of SEQ ID NO:3, or a sequence that is at least 95% identical thereto and has Ile at position 780;
(d) at least amino acid 70 to amino acid 952 of SEQ ID NO: 3, or a sequence that is at least 95% identical thereto and has Ile at position 780, or
(e) at least amino acid 70 to amino acid 890 of SEQ ID NO:3, or a sequence that is at least 95% identical thereto and has Ile at position 780;
6. The expression cassette of claim 1 or the rAAV of claim 5, comprising:

7. The expression cassette of claim 1 or claim 6 or the rAAV of claim 5 or claim 6, wherein said hGAA780I is a chimeric fusion protein further comprising an exogenous signal sequence.

The AAV capsid is
(a) targeting two or more cells of the liver, muscle, heart, and central nervous system, optionally wherein said regulatory element directs expression in said target cell, and/or
(b) AAVhu. 68 or variants thereof, AAV9 or variants thereof, or AAVrh. 10 or variants thereof.

The hGAA780I is
(a) expressed as full-length SEQ ID NO:3, and/or
(b) encoded by SEQ ID NO:4 or a sequence at least 95% identical thereto, or encoded by SEQ ID NO:5 or a sequence at least 95% identical thereto; An expression cassette according to claim 1, or a rAAV according to any one of claims 5-8.

wherein said vector genome further comprises at least two tandem repeats of miRNA target sequences, said at least two tandem repeats comprising at least a first miRNA target sequence and at least a second miRNA target sequence which may be the same or different; The rAAV of any one of paragraphs 5-9.

said miR target sequences are independently selected from SEQ ID NO:26 and SEQ ID NO:27 and/or two or more of said miRNA target sequences are separated by a spacer, one or more of said spacers is independently selected from (i) GGAT, (ii) CACGTG, and (iii) GCATGC.

A nucleic acid molecule comprising a sequence encoding an expression cassette according to any one of claims 1-4, 6, 7, 9 or 11, optionally wherein said molecule is a plasmid. .

A host cell comprising the nucleic acid molecule of claim 12 .

( a) an expression cassette according to any one of claims 1-4, 6, 7, 9, or 11, or
(b) the rAAV of any one of claims 5 to 11;
at least one of a pharmaceutically acceptable carrier, excipient, and/or suspending agent;
Optionally, the composition is a suspension formulated for intravenous delivery or a suspension formulated for intrathecal, intracisternal, or intracerebroventricular administration The composition, which is a turbid liquid.

An expression cassette according to any one of claims 1-4, 6, 7, 9 or 11, any one of claims 5-11, for use in a method for treating a patient with Pompe disease. 15. The rAAV of claim 14, or the composition of claim 14, wherein said method comprises delivering said expression cassette, said rAAV, or said composition to said patient. rAAV, or said composition.

The expression cassette, the rAAV, or the composition are co-delivered via separate routes and/or the expression cassette, the rAAV, or the composition is via intravenous and/or intrathecal delivery. 16. The expression cassette, rAAV, or composition for use according to claim 15, administered to said patient via a.

16. The expression cassette, AAV, or composition of claim 15, wherein the expression cassette, AAV, or composition is delivered in combination with an immunomodulatory agent, and optionally the patient has late-onset Pompe disease or infantile-onset Pompe disease. Expression cassettes, rAAV, or compositions for use.

18. An expression cassette for use according to claim 17, wherein said patient receives combination therapy with bronchodilators, acetylcholinesterase inhibitors, respiratory muscle training (RMST), enzyme replacement therapy, and/or diaphragmatic pacing therapy; rAAV, or compositions.

12. The composition of claims 1-4, 6, 7, 9 or 11 for use in a method for improving myocardial, respiratory and/or skeletal muscle function in a patient having a deficiency in alpha-glucosidase (GAA). The expression cassette of any one, the rAAV of any one of claims 5-11, or the composition of claim 14, wherein said method comprises: said expression cassette, said rAAV, or said expression cassette, said rAAV, or said composition, comprising delivering said composition to said patient.