Lentiviral vectors for delivery of PKLR to treat Pyruvate Kinase Deficiency
Inventors
SEGOVIA, JOSE CARLOS • GOMEZ, Maria G. • NAVARRO, Susana • MEZA, Nestor • BUEREN, JUAN ANTONIO • BRAVO, Maria G.
Assignees
Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas • Centro de Investigación Biomédica en Red • Hospital Universitario Fundación Jiménez Díaz
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Publication Number
US-12496359-B2
Publication Date
2025-12-16
Expiration Date
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 disclosure describes lentiviral gene therapy for pyruvate kinase deficiency (PKD) using an expression cassette arranged 5′ to 3′ in which a promoter is operably linked to a codon-optimized sequence encoding a human PKLR gene product. The expression cassette includes a mutated post-transcriptional regulatory element of woodchuck hepatitis virus (wPRE) positioned downstream of the promoter and PKLR coding sequence, and the mutated wPRE is defined as a chimeric wPRE having at least 90% identity to SEQ ID NO:24.
The document further defines the lentiviral gene delivery configuration in which the expression cassette uses a codon-optimized human PKLR transgene together with the chimeric mutated wPRE. The promoter sequence can be a human phosphoglycerate kinase (PGK) promoter, and the viral vector can include additional nucleotide sequence elements arranged from 5′ to 3′ such as LTRs, an HIV-1 w sequence, a Rev responsive element (RRE), cPPT/CTS, an SV40 poly(A) signal, an SV40 origin of replication, and CMV enhancer and CMV promoter sequences, together with a modified 3′ LTR.
The intent is for PKLR to be expressed at detectable levels in erythroid cells after contacting with an effective amount of the recombinant viral vector. In the disclosed study context, the therapy is evaluated in PKD and is reported to yield stable long-term correction of anemia/erythroid parameters and normalization of erythroid differentiation, together with reduced splenomegaly and liver pathology.
The document reports restoration of RBC glycolysis/pyruvate kinase activity without detectable leukocyte metabolic disturbance, and it summarizes genome-wide LV integration analysis showing polyclonal reconstitution and no evidence of genotoxicity based on integration-site distributions and safety-related assessments.
Claims Coverage
The independent claims are clm-00001 and clm-00015. The claim set centers on the defined expression cassette architecture and erythroid expression of PKLR after contacting with a recombinant viral vector, with two inventive features.
Promoter–codon-optimized human PKLR–chimeric mutated wPRE expression cassette with identity constraints
A polynucleotide comprising an expression cassette comprising, in 5′ to 3′ order: a promoter sequence; a codon-optimized sequence encoding a human PKLR gene product; and a mutated post-transcriptional regulatory element of woodchuck hepatitis virus (wPRE), wherein the mutated wPRE is a chimeric wPRE comprising a sequence having at least 90% identity to SEQ ID NO:24; wherein the promoter sequence is operably linked to the codon-optimized sequence encoding the human PKLR gene product; and wherein the codon-optimized sequence encoding a human PKLR gene product shares at least 90% identity to SEQ ID NO:8.
Erythroid PKLR expression after contacting with a recombinant viral vector
A method for expressing a human PKLR gene in erythroid cells, comprising contacting one or more erythroid cells with an effective amount of a recombinant viral vector, wherein the vector comprises a human phosphoglycerate kinase promoter, a codon-optimized PKLR transgene, and a mutated post-transcriptional regulatory element of woodchuck hepatitis virus, wherein the codon-optimized PKLR transgene shares at least 90% identity with SEQ ID NO:8 and the mutated post-transcriptional regulatory element of the woodchuck hepatitis virus shares at least 90% identity with SEQ ID NO:24, and wherein following said contacting, PKLR is expressed at detectable levels in the one or more erythroid cells.
Across the independent claims, the main inventive elements are the defined 5′ to 3′ expression cassette architecture combining a promoter operably linked to a codon-optimized human PKLR coding sequence and a chimeric mutated wPRE with at least 90% identity to SEQ ID NO:24, together with the requirement that a recombinant viral vector containing these elements results in detectable PKLR expression in erythroid cells.
Stated Advantages
Stable long-term correction of anemia/erythroid parameters and normalization of erythroid differentiation.
Reduced splenomegaly and liver pathology.
Restoration of RBC glycolysis/pyruvate kinase activity without detectable leukocyte metabolic disturbance.
Polyclonal reconstitution and no evidence of genotoxicity based on integration-site distributions and safety-related assessments.
Achieves detectable PKLR expression in erythroid cells following contacting with a recombinant viral vector.
In the disclosed context, supports safety and genotoxicity/insertional mutagenesis assessment with monitoring of vector copy number and integration pattern.
Documented Applications
Gene therapy for pyruvate kinase deficiency (PKD).
Lentiviral vector administration in PKD using an orphan-drug SIN LV vector EU/3/14/1130 (PGK-coRPK.wpre) in autologous HSCT with clinical endpoints focused on safety, engraftment, anemia/reticulocytosis improvements, and vector copy number/integration pattern monitoring.
Expression of a human PKLR gene in erythroid cells after contacting with a recombinant viral vector.
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