JPWO2020223356A5

JPWO2020223356A5 -

Info

Publication number: JPWO2020223356A5
Application number: JP2021565001A
Authority: JP
Publication date: 2023-05-08

Claims

A recombinant adeno-associated virus (rAAV), 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 comprising a nucleic acid sequence encoding a chimeric fusion protein comprising a signal peptide and a vIGF2 peptide fused to hGAA comprising at least the active site of hGAA780I; , under the control of regulatory sequences that direct its expression, position 780 being based on the amino acid sequence position numbering in SEQ ID NO:3;
including
Optionally, said rAAV, wherein said vector genome comprises SEQ ID NO:30 or a sequence at least 95% identical thereto.

an expression cassette comprising at least a nucleic acid sequence encoding a chimeric fusion protein comprising a signal peptide and a vIGF2 peptide fused to human acid α-glucosidase (hGAA) comprising the active site of hGAA780I, under the control of regulatory sequences directing its expression; wherein position 780 is based on the amino acid sequence position numbering in SEQ ID NO:3.

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, optionally said miR target sequence is 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 being independently selected from (i) GGAT, (ii) CACGTG, and (iii) GCATGC; 3. The expression cassette of claim 2, wherein the expression cassette is

The expression cassette is
(a) a viral vector selected from a recombinant parvovirus, a recombinant lentivirus, a recombinant retrovirus, and a recombinant adenovirus, optionally wherein said recombinant parvovirus is a clade F adeno-associated virus; optionally, said clade F adeno-associated virus is AAVhu68, or
(b) a non-viral vector selected from naked DNA, naked RNA, inorganic particles, lipid particles, polymer-based vectors, or chitosan-based formulations;
4. The expression cassette of claim 2 or claim 3 carried by.

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;
The rAAV of claim 1 or the expression cassette of claim 2, comprising:

The hGAA780I is
(a) SEQ ID NO: 4 or a sequence at least 95% identical thereto, or
(b) SEQ ID NO: 5 or a sequence at least 95% identical thereto
6. The rAAV of claim 1 or claim 5, or the expression cassette of claim 2 or claim 5, encoded by:

2. The fusion protein of claim 1, wherein said fusion protein comprises SEQ ID NO: 6 or a sequence at least 95% identical thereto, optionally wherein said fusion protein is encoded by SEQ ID NO: 7 or a sequence at least 95% identical thereto. or the expression cassette of claim 2.

said vector genome further comprising at least two tandem repeats of a dorsal root ganglion (DRG)-specific miR-183 target sequence, said at least two tandem repeats being the same or different at least a first miRNA target sequence and at least a second miRNA target sequence, operably linked 3' to said sequence encoding said fusion protein, optionally wherein said miR-183 target sequence is SEQ ID NO:26; and/or two or more of said miRNA target sequences are separated by a spacer, wherein one or more spacers are independently selected from (i) GGAT, (ii) CACGTG, and (iii) GCATGC The rAAV of any one of claims 1 or 5-7, wherein the rAAV is

The vIGF2 peptide is
(a) at least 90% identical to SEQ ID NO:32 and selected from positions 6, 26, 27, 43, 48, 49, 50, 54, 55, and 65 of SEQ ID NO:32; optionally, said at least one substitution is E6R, F26S, Y27L, V43L, F48T, R49S, S50I, A54R of SEQ ID NO: 32 , L55R, and K65R,
(b) at least two at two or more positions selected from positions 6, 26, 27, 43, 48, 49, 50, 54, 55, and 65 of SEQ ID NO:32; a substitution, optionally wherein said at least two substitutions are selected from E6R, F26S, Y27L, V43L, F48T, R49S, S50I, A54R, L55R, and K65R of SEQ ID NO:32 , and/or
(c) an N-terminal deletion at position 1 of SEQ ID NO:32, wherein optionally said vIGF2 peptide comprises an N-terminal deletion at positions 1-4 of SEQ ID NO:32;
The rAAV of any one of claims 1 or 5-8, or the expression cassette of any one of claims 2, 3, or 5-7, comprising:

(a) said vIGF2 peptide has reduced or no affinity for the insulin receptor and IGFR1 compared to the native IGF2 peptide, and/or
(b) the rAAV of any one of claims 1 or 5-9, or claims 2, 3, 5-, wherein said vIGF2 peptide is capable of promoting uptake of hGAA780I into lysosomes within cells; 9. The expression cassette according to any one of 7 or 9.

said nucleic acid sequence further comprises a linker sequence encoding a linker peptide between said vIGF2 nucleotide sequence and said nucleic acid sequence encoding hGAA780I, optionally wherein said linker peptide is any of SEQ ID NOS: 55-60 The rAAV of any one of claims 1 or 5-10, or the expression cassette of any one of claims 2, 3, 5-7, 9, or 10, comprising one.

The signal peptide is
(a) a binding immunoglobulin protein (BiP) signal peptide, optionally wherein said BiP signal peptide comprises an amino acid sequence at least 90% identical to any one of SEQ ID NOS: 49-53; the BiP signal peptide comprises the amino acid sequence of any one of SEQ ID NOS: 49-53, or
(b) is or comprises a Gaussian signal peptide, optionally wherein said Gaussian signal peptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 54; The rAAV of any one of claims 1 or 5-11, or any one of claims 2, 3, 5-7, or 9-11, wherein the Gaussia signal peptide comprises SEQ ID NO:54 expression cassette.

The rAAV of any one of claims 1 or 5-12, wherein said capsid is a clade F capsid, optionally said capsid is an AAVhu68 capsid.

(a) a sequence encoding an expression cassette according to any one of claims 2-7 or 9-12, or
(b) SEQ ID NO: 30 or a sequence at least 95% identical thereto
optionally, wherein said molecule is a plasmid.

15. A host cell comprising the nucleic acid molecule of claim 14.

(a) an expression cassette according to any one of claims 2-7 or 9-12, or
(b) the rAAV of any one of claims 1 or 5-13;
at least one of a pharmaceutically acceptable carrier, excipient, and/or suspending agent;
Optionally, said 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.

rAAV according to any one of claims 1 or 5-10, according to any one of claims 2-7 or 9-12, for use in a method for treating a patient with Pompe disease or the composition of claim 13, wherein the method comprises delivering the expression cassette, the rAAV, or the composition to the patient. said composition.

The expression cassette, the rAAV, or the composition are co-delivered via separate routes and/or the rAAV, the expression cassette, or the composition is via intravenous and/or intrathecal delivery. 18. The rAAV, expression cassette, or composition for use according to claim 17, administered to said patient via a.

18. The rAAV, expression cassette, or composition of claim 17, wherein the rAAV, expression cassette, or composition is delivered in combination with an immunomodulatory agent, and optionally the patient has late-onset Pompe disease or infantile-onset Pompe disease. rAAV, expression cassettes or compositions for use.

rAAV for use according to claim 19, wherein the patient receives combination therapy with bronchodilators, acetylcholinesterase inhibitors, respiratory muscle training (RMST), enzyme replacement therapy, and/or diaphragmatic pacing therapy, expression cassette, or composition.

14. A method according to any one of claims 1 or 5-13 for use in a method for improving myocardial, respiratory and/or skeletal muscle function in patients with a deficiency in alpha-glucosidase (GAA). rAAV, the expression cassette of any one of claims 2-7 or 9-12, or the composition of claim 16, wherein the method comprises: to said patient.