Optimized ap4m1 polyneucleotides and expression cassettes and their use
AssigneeBoard of Regents, The University of Texas System
InventorsSteven J. GRAY, Xin CHEN
Life of this patent
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Abstract
Provide herein are polynucleotides comprising a codon-optimized AP4M1 open reading frame (ORF) sequence encoding an AP4M1 polypeptide, vectors (viral or non-viral vectors) comprising the same, pharmaceutical compositions comprising the same, and methods of using the same for delivery of the polynucleotides to a cell or a subject and to treat AP4M1-related genetic disorders such as AP4M1 deficiency and spastic paraplegia type 50.
Claims74
A polynucleotide encoding a human AP4M1 polypeptide, wherein the polynucleotide is codon-optimized for expression in a human cell.
A recombinant adeno-associated virus (rAAV) vector comprising in 5′ to 3′ direction:
- a) a first AAV ITR sequence;
- b) a promoter sequence;
- c) a transgene nucleic acid molecule, wherein the transgene nucleic acid molecule comprises a nucleic acid sequence encoding an AP4M1 polypeptide;
- d) a polyA sequence;
- and
- e) a second AAV ITR sequence.
The rAAV vector of any one of the preceding claims, wherein the transgene nucleic acid sequence encoding for an AP4M1 polypeptide exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased expression in a human subject relative to a mutated AP4M1 nucleic acid sequence.
The rAAV vector of any one of the preceding claims, wherein the first AAV ITR sequence comprises the nucleic acid sequence set forth in SEQ ID NO:2.
The rAAV vector of any one of the preceding claims, wherein the second AAV ITR sequence comprises the nucleic acid sequence set forth in SEQ ID NO:7.
The rAAV vector of any one of the preceding claims, wherein the promoter sequence comprises a Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, a U6 promoter, a JetI promoter, an H1 promoter, a CAG promoter, a hybrid chicken beta-actin (CBA) promoter, an MeCP2 promoter, an EF1 promoter, a ubiquitous chicken β-actin hybrid (CBh) promoter, an U1a promoter, an U1b promoter, an MeCP2 promoter, an MeP418 promoter, an MeP426 promoter, a minimal MeCP2 promoter, a VMD2 promoter, an mRho promoter, an EFla promoter, an Ubc promoter, a human β-actin promoter, a synapsin (hSyn) promoter sequence, a TRE promoter, an Ac5 promoter, a Polyhedrin promoter, a CaMKIIa promoter, a Gall promoter, a TEF1 promoter, a GDS promoter, an ADH1 promoter, an Ubi promoter, a Jet promoter, an UsP promoter, or an α-1-antitrypsin (hAAT) promoter.
The rAAV vector of any one of the preceding claims, wherein the promoter sequence comprises the nucleic acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:39.
The rAAV vector of any one of the preceding claims, wherein the polyA sequence comprises the nucleic acid sequence set forth in SEQ ID NO:6.
An rAAV vector of any one of the preceding claims, comprising, in the 5′ to 3′ direction:
- a) a first AAV ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:2;
- b) a promoter sequence comprising the nucleic acid sequence set forth in SEQ ID NO:3;
- c) a transgene nucleic acid molecule, wherein the transgene nucleic acid molecule comprises a nucleic acid sequence encoding for an AP4M1 polypeptide, wherein the nucleic acid sequence encoding for an AP4M1 polypeptide comprises the nucleic acid sequence set forth in SEQ ID NO:5;
- d) a polyA sequence comprising the nucleic acid sequence set forth in SEQ ID NO:6;
- and
- e) a second AAV ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:7.
An rAAV vector of any one of the preceding claims, comprising, in the 5′ to 3′ direction:
- a) a first AAV ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:2;
- b) a promoter sequence comprising the nucleic acid sequence set forth in SEQ ID NO:39;
- c) a transgene nucleic acid molecule, wherein the transgene nucleic acid molecule comprises a nucleic acid sequence encoding for an AP4M1 polypeptide, wherein the nucleic acid sequence encoding for an AP4M1 polypeptide comprises the nucleic acid sequence set forth in SEQ ID NO:40;
- d) a polyA sequence comprising the nucleic acid sequence set forth in SEQ ID NO:6;
- and
- e) a second AAV ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:7.
An rAAV vector of any one of the preceding claims, comprising, in the 5′ to 3′ direction:
- a) a first AAV ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:2;
- b) a promoter sequence comprising the nucleic acid sequence set forth in SEQ ID NO:3;
- c) an intron sequence comprising the nucleic acid sequence set forth in SEQ ID NO:4;
- d) a transgene nucleic acid molecule, wherein the transgene nucleic acid molecule comprises a nucleic acid sequence encoding for an AP4M1 polypeptide, wherein the nucleic acid sequence encoding for an AP4M1 polypeptide comprises the nucleic acid sequence set forth in SEQ ID NO:5;
- e) a polyA sequence comprising the nucleic acid sequence set forth in SEQ ID NO:6;
- and
- f) a second AAV ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:7.
An rAAV viral vector comprising:
- (i) an AAV capsid protein;
- and
- (ii) an rAAV vector of any one of the preceding claims.
Dependencies are inferred from the claim text.