Methods of treating or preventing pyruvate kinase deficiency

Inventors

SEGOVIA, Jose C. • GOMEZ, Maria G. • NAVARRO, Susana • MEZA, Nestor • BUEREN, Juan • BRAVO, Maria G.

Assignees

Centro De Investigaciones Energeticas Medioambientales • Centro de Investigaciones Energeticas Medioambientales y Tecnologicas CIEMAT • Centro de Investigacion Biomedica en Red CIBER • Instituto de Investigacion Sanitaria Fundacion Jimenez Diaz

Abstract

The present disclosure provides polynucleotide cassettes, expression vectors and methods for the expression of a gene in mammalian cells to provide gene therapy for pyruvate kinase deficiency.

Core Innovation

The invention provides an expression cassette comprising, in a 5′ to 3′ order, a promoter sequence comprising a phosphoglycerate kinase (PGK) promoter sequence having a sequence at least 90% identical to SEQ ID NO: 8, a sequence encoding a pyruvate kinase, liver and red blood cell (PKLR) polypeptide, and a mutated woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) comprising a sequence at least 90% identical to SEQ ID NO: 1, with the promoter sequence operably linked to the sequence encoding the PKLR polypeptide. In certain embodiments, the cassette uses exact sequence identities and a codon-optimized PKLR coding sequence.

The described strategy is directed to pyruvate kinase deficiency (PKD) and uses expression from the PKLR transgene in an erythroid expression context. In the vector embodiments, the cassette is incorporated into a self-inactivating lentiviral vector (SIN-LV), including 5′ and 3′ LTRs and other lentiviral-associated regulatory and viral element sequences.

The document further describes recombinant vectors and transduced hematopoietic cell populations for producing therapeutic outcomes in PKD, including pharmaceutical compositions and methods for treating or prophylaxis. Extensive preclinical results are described, including hematologic correction in primary and secondary recipient mice, normalized erythroid differentiation and reduced Epo/splenomegaly/pathology, restoration of RBC glycolysis metabolism while preserving WBC metabolic balance, and vector integration-site mapping with clonal abundance analysis showing no evidence of genotoxicity/insertional oncogenesis based on the reported criteria and pathway enrichment results.

Claims Coverage

The partial content provided identifies one independent claim and describes its core inventive features as an expression cassette architecture based on a PGK promoter and a mutated woodchuck hepatitis virus Wpre linked to a PKLR coding sequence. Dependent claims refine the cassette and further specify associated vector and downstream use features.

Across the independent claim and the refinements described in the dependent claims, the main claim coverage centers on a 5′ to 3′ expression cassette using a PGK promoter (with defined identity to SEQ ID NO: 8), a PKLR coding sequence, and a mutated woodchuck hepatitis virus Wpre (with defined identity to SEQ ID NO: 1), optionally further constrained to exact sequence identities, codon optimization, flanking LTRs, and additional lentiviral packaging and processing elements, with embodiments specifying lentivirus (LV) as the viral vector.

Stated Advantages

Documented Applications