Adeno-associated virus vectors for treatment of glycogen storage disease
Inventors
Chou, Janice J. • Byrne, Barry J.
Assignees
University of Florida Research Foundation Inc • US Department of Health and Human Services
Publication Number
US-9644216-B2
Publication Date
2017-05-09
Expiration Date
2034-11-25
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Abstract
The present disclosure describes improved adeno-associated virus (AAV) vectors for gene therapy applications in the treatment of glycogen storage disease, particularly glycogen storage disease type Ia (GSD-Ia). Described are recombinant nucleic acid molecules, vectors and recombinant AAV that include a G6PC promoter/enhancer, a synthetic intron, a G6PC coding sequence (such as a wild-type or codon-optimized G6PC coding sequence), and stuffer nucleic acid sequence situated between the G6PC promoter/enhancer and the intron, as well as between the intron and the G6PC coding sequence. The recombinant AAVs disclosed herein exhibit highly efficient liver transduction and are capable of correcting metabolic abnormalities in an animal model of GSD-Ia.
Core Innovation
The invention provides recombinant nucleic acid molecules, adeno-associated virus (AAV) vectors, and recombinant AAVs for gene therapy applications targeting glycogen storage disease type Ia (GSD-Ia). These recombinant nucleic acid molecules include a G6PC promoter/enhancer, a synthetic intron, a G6PC coding sequence (which may be wild-type or codon-optimized), and stuffer nucleic acid sequences positioned between the promoter/enhancer and intron, as well as between the intron and the coding sequence. The inclusion of these elements results in highly efficient liver transduction and correction of metabolic abnormalities in animal models of GSD-Ia.
The problem addressed by the invention arises from the deficiency of glucose-6-phosphatase-α (G6Pase-α), encoded by the G6PC gene, which leads to GSD-Ia characterized by fasting hypoglycemia, hepatomegaly, nephromegaly, and metabolic abnormalities such as hyperlipidemia and lactic acidemia. Although dietary therapies can manage hypoglycemia, they do not correct long-term clinical complications including hepatocellular adenoma, which occurs in a majority of patients and can progress to hepatocellular carcinoma. Previous recombinant AAV vectors using other promoters have shown promise but failed to completely correct hepatic G6Pase-α deficiency.
The invention discloses that using the human G6PC promoter/enhancer region (nucleotides −2684 to −1 relative to the G6PC start site) combined with a synthetic intron and stuffer sequences flanking the intron improves transgene expression efficiency over other promoter/enhancer combinations. Moreover, codon optimization of the G6PC coding sequence further increases translational efficiency, resulting in significantly enhanced hepatic G6Pase-α expression. These features together advance gene therapy approaches capable of sustained correction of GSD-Ia pathology in vivo.
Claims Coverage
The claims define 18 inventive features centering on recombinant nucleic acid molecules, vectors, recombinant AAVs, compositions, and methods of treating GSD-Ia using these constructs.
Recombinant nucleic acid molecule consisting of defined nucleotide sequences
The recombinant nucleic acid molecule comprises nucleotides 182-4441 of SEQ ID NO: 3 or nucleotides 182-4441 of SEQ ID NO: 1, incorporating the G6PC promoter/enhancer, synthetic intron, stuffer sequences, and coding sequence.
Extended recombinant nucleic acid molecules with inverted terminal repeats
The recombinant nucleic acid molecule further comprises nucleotides 17-4819 of SEQ ID NO: 3 or SEQ ID NO: 1 including 5′ and 3′ inverted terminal repeat (ITR) sequences.
Complete recombinant nucleic acid sequences
Recombinant nucleic acid molecules encompass the entire nucleotide sequences of SEQ ID NO: 3 or SEQ ID NO: 1.
Vectors comprising recombinant nucleic acid molecules
Vectors incorporate the recombinant nucleic acid molecules as defined, suitable for gene delivery.
AAV vectors for gene therapy
The vectors are adeno-associated virus (AAV) vectors suitable for hepatic transduction.
AAV serotype specificity
The AAV vector is specified as AAV serotype 8 (AAV8), optimizing liver targeting.
Isolated host cells containing recombinant nucleic acid molecules
Host cells are isolated and contain the recombinant nucleic acid molecules, facilitating vector production.
Recombinant AAV particles comprising recombinant nucleic acid molecules
Recombinant AAV (rAAV) particles that package the defined recombinant nucleic acid molecules.
Serotype 8 recombinant AAV
The recombinant AAV is an rAAV8 serotype, suitable for liver transduction.
Pharmaceutical compositions including recombinant AAV
Pharmaceutical compositions include the rAAV along with pharmaceutically acceptable carriers.
Formulation for intravenous administration
Compositions are formulated for intravenous delivery to subjects.
Method of treating GSD-Ia using recombinant AAV
A method involves selecting a subject with glycogen storage disease type Ia and administering a therapeutically effective amount of the recombinant AAV.
Intravenous administration of recombinant AAV
The recombinant AAV is administered intravenously for therapeutic effect.
Dose range for administration
Recombinant AAV is administered at a dose between about 1×1011 to 1×1014 viral particles (vp)/kg.
Preferred dose ranges
More specific dose ranges include about 1×1012 to 8×1013 vp/kg or about 1×1013 to 6×1013 vp/kg.
Single dose administration
Administration of the recombinant AAV can be accomplished in a single dose.
Multiple dose administration
Administration can alternatively involve multiple doses of recombinant AAV.
The claims collectively cover recombinant nucleic acid molecules comprising G6PC promoter/enhancer, synthetic intron, stuffer sequences, and coding sequences, their incorporation into AAV vectors (particularly AAV8), compositions formulated for intravenous delivery, and methods for treating GSD-Ia by administering therapeutically effective amounts of these recombinant AAV vectors in defined dose ranges.
Stated Advantages
The recombinant AAV vectors with the G6PC promoter/enhancer demonstrate more efficient and persistent hepatic transgene expression compared to vectors using the chicken β-actin promoter/CMV enhancer.
Inclusion of stuffer nucleotide sequences flanking the synthetic intron enhances liver transduction and expression of G6Pase-α.
Codon-optimization of the G6PC coding sequence improves translation efficiency by approximately 1.5 to 2.5-fold, resulting in greater hepatic G6Pase-α expression.
Gene therapy mediated by the disclosed recombinant AAV vectors achieves corrected metabolic abnormalities and prevents hepatic complications such as hepatocellular adenoma in animal models.
Documented Applications
Gene therapy treatment of glycogen storage disease type Ia (GSD-Ia) in subjects, including administration of recombinant AAV vectors expressing G6PC.
Use of recombinant nucleic acid molecules and vectors for efficient hepatic expression of G6Pase-α to restore glucose homeostasis.
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