Codon-optimized human NPC1 genes for the treatment of Niemann-Pick type C1 deficiency and related conditions
Inventors
Venditti, Charles P. • Pavan, William J. • CHANDLER, Randy J.
Assignees
US Department of Health and Human Services
Publication Number
US-12116577-B2
Publication Date
2024-10-15
Expiration Date
2038-06-20
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Abstract
The present disclosure provides compositions for viral gene therapy, e.g. Adeno-Associated virus-directed gene therapy, and methods of using the same for the treatment and/or prevention of cholesterol storage diseases or disorders, such as Niemann-Pick disease, Type C.
Core Innovation
The present disclosure provides gene therapy vectors, such as Adeno-Associated Virus (AAV) gene therapy vectors, comprising a therapeutic human nucleic acid molecule to ameliorate, treat, and/or correct the cellular defects characteristic of cholesterol storage diseases or disorders, specifically Niemann-Pick disease, type C (NPC). The vectors include codon-optimized NPC1 alleles to maximize human expression and may comprise fusion proteins with tags (e.g., FLAG® tags) for detection and protein transduction domain peptides (PTDs) such as Tat, TP2, or TP10 to allow cross-correction by transferring the therapeutic protein to neighboring cells not directly transduced.
The problem addressed is the lack of effective treatment for NPC, a rare, fatal, autosomal recessive neurodegenerative disease caused primarily by mutations in the NPC1 gene, which leads to intracellular accumulation of unesterified cholesterol and glycosphingolipids. Current absence of curative therapies for NPC, and the early onset and severe progression especially in children, create an urgent need for effective therapeutic approaches.
The invention overcomes limitations of prior approaches by providing codon optimized NPC1 sequences operably linked to promoters (e.g., truncated EF1α promoters) to enhance expression in the CNS, incorporation of multiple stop codons to maximize protein production and avoid aberrant read-through, and fusion to PTD and/or tag moieties to enable cross-correction and facile detection. Furthermore, new generations of AAV vectors with synthetic introns and improved inverted terminal repeats reduce genotoxicity and improve expression, enabling targeted or systemic gene delivery to treat NPC disease effectively.
Claims Coverage
The patent claims include one independent claim focusing on a nucleic acid construct encoding a fusion protein for treatment of NPC1 deficiency, and related dependent claims and compositions.
Human codon-optimized NPC1 fused to a protein transduction domain
A nucleic acid construct encoding a human codon-optimized NPC1 gene translationally fused to a protein transduction domain (PTD) selected from HIV-Tat, TP10, or TP2 to form a NPC1-PTD fusion protein.
Promoter control of the NPC1 gene
The NPC1 gene is operably linked to a promoter selected from a truncated EF1α promoter (EF1t), a mini EF1α promoter, or a short EF1α promoter (EF1α S) to enable expression.
Incorporation of synthetic intron and expression cassettes
The construct optionally includes synthetic intron S positioned between the promoter and NPC1 gene (SEQ ID NO: 25), and comprises expression cassettes selected from SEQ ID NOs: 26-31 and 33-38.
AAV inverted terminal repeats and antibiotic resistance markers
The nucleic acid construct includes first and second AAV inverted terminal repeats (ITRs) flanking the transgene, and may contain a nucleotide sequence encoding an antibiotic resistance marker.
Expression vector and cell embodiments
The construct is included in an expression vector capable of expressing the NPC1 fusion protein, and isolated or purified cells containing the expression vector.
The claims collectively cover codon-optimized NPC1 fusion constructs with PTDs and promoters enabling CNS expression, incorporation of synthetic introns and regulatory sequences, packaged into viral vectors, and methods for treatment of NPC disease via expression of these fusion proteins capable of cross-correcting neighboring cells.
Stated Advantages
The codon-optimized NPC1 alleles enable maximized human expression and unique detectability in preclinical models and patients.
Fusion to protein transduction domains permits cross-correction, improving potency of gene therapy by enabling correction of cells not directly transduced.
Use of truncated or mini EF1α promoters and synthetic introns enhances expression and splicing of the NPC1 transgene.
Improved AAV vectors reduce genotoxicity and facilitate systemic or targeted CNS gene delivery.
Documented Applications
Treatment and/or prevention of cholesterol storage diseases or disorders, such as Niemann-Pick disease, type C.
Gene therapy methods for ameliorating, treating, or preventing symptoms of NPC disease via administration of gene therapy vectors encoding codon optimized NPC1 fusion proteins to CNS tissues.
Use in engineered cell lines expressing NPC1 protein for enzyme replacement therapy or production of therapeutic exosomes containing NPC1.
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