Non-viral DNA vectors and uses thereof for expressing FVIII therapeutics
Inventors
Kerr, Douglas Anthony • KLATTE, DEBRA • Samayoa, Phillip • Silver, Nathaniel
Assignees
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Publication Number
US-12338275-B2
Publication Date
2025-06-24
Expiration Date
Abstract
The application describes ceDNA vectors having linear and continuous structure for delivery and expression of a transgene. ceDNA vectors comprise an expression cassette flanked by two ITR sequences, where the expression cassette encodes a transgene encoding FVIII protein. Some ceDNA vectors further comprise cis-regulatory elements, including regulatory switches. Further provided herein are methods and cell lines for reliable gene expression of FVIII protein in vitro, ex vivo and in vivo using the ceDNA vectors. Provided herein are method and compositions comprising ceDNA vectors useful for the expression of FVIII protein in a cell, tissue or subject, and methods of treatment of diseases with said ceDNA vectors expressing FVIII protein. Such FVIII protein can be expressed for treating disease, e.g., hemophilia A.
Core Innovation
The invention relates to capsid-free closed-ended DNA (ceDNA) vectors for expressing Factor VIII (FVIII) therapeutics. The ceDNA vector comprises at least one nucleotide sequence positioned between flanking inverted terminal repeats (ITRs), and the FVIII-encoding nucleotide sequence has at least 90% identity to SEQ ID NO: 395. The disclosure also describes flanking AAV ITRs, including wild-type and modified forms, with symmetric, substantially symmetric, and asymmetric configurations.
The flanking ITRs are associated with terminal resolution and Rep binding features, and altered ITR sequences are described as changing overall ITR three-dimensional conformation. The disclosure further includes optional cis-regulatory elements and regulatory switches associated with the expression cassette. The ceDNA vector is presented with regulatory component classes including promoter, enhancer, 5′ UTR and/or intron, 3′ UTR, and/or at least one poly A sequence.
The disclosure also addresses delivery and formulation concepts, including pharmaceutical delivery with lipid nanoparticle (LNP) formulation and exosome/microparticle delivery formats. It describes ceDNA vector delivery for in vivo administration, including liver/circulating expression and hemophilia A treatment.
Claims Coverage
The independent claim set centers on a capsid-free closed-ended DNA (ceDNA) vector having an FVIII-encoding sequence positioned between flanking inverted terminal repeats (ITRs), with sequence identity constrained to SEQ ID NO: 395. Additional inventive features cover regulatory components, engineered ITR structure and origin constraints, a cell-contacting expression method in vitro or in vivo, and LNP-containing compositions.
Capsid-free closed-ended DNA vector with FVIII between flanking ITRs
A capsid-free closed-ended DNA (ceDNA) vector comprising at least one nucleotide sequence positioned between flanking inverted terminal repeats (ITRs), wherein the at least one nucleotide sequence encodes at least one Factor VIII (FVIII) protein, and wherein the at least one nucleotide sequence that encodes the at least one FVIII protein has at least 90% identity to SEQ ID NO: 395.
Variant FVIII-encoding nucleotide sequence identity constraints
The ceDNA vector includes a nucleotide sequence that is at least 85% identical to SEQ ID NO: 214.
Regulatory element combination linked to FVIII encoding sequence
The ceDNA vector includes a promoter linked to an FVIII-encoding nucleotide sequence, an enhancer, optionally a 5′ UTR and/or intron, a 3′ UTR, and/or at least one poly A sequence.
Engineered flanking ITR origin, structure, and conformation constraints
The ceDNA vector has flanking ITRs with defined terminal resolution and Rep binding features, originating from specified virus types/serotypes, with allowed symmetric, substantially symmetric, and asymmetric configurations and altered ITR sequence designs affecting overall three-dimensional conformation.
Cell contacting method for FVIII protein expression in vitro or in vivo
A method for expressing an FVIII protein in a cell by contacting the cell with the ceDNA vector, where the cell is in vitro or in vivo.
Lipid nanoparticle composition with the ceDNA vector
A composition including the ceDNA vector together with a lipid nanoparticle (LNP).
Overall, the claim coverage centers on a capsid-free closed-ended DNA vector carrying an FVIII-encoding cassette between flanking ITRs with specified sequence identity. Additional inventive features cover regulatory component combinations, engineered ITR designs with defined terminal resolution and Rep binding features, a cell-contacting expression method in vitro or in vivo, and LNP-containing compositions.
Stated Advantages
Sustained transgene expression for extended periods in vivo.
Sustained expression is maintained after re-dosing.
Liver-specific delivery and intracellular presence in hepatocytes are demonstrated.
Durable ocular expression is demonstrated over an extended period after sub-retinal injection.
Maintained expression is demonstrated in Rag2 immune-deficient mice, with increased expression after re-dose.
Durable expression is maintained using promoter and CpG modulation approaches compared with a CAG promoter with high unmethylated CpGs.
Serum FVIII expression is demonstrated after ceDNA injection.
Capsid-free non-viral DNA, avoiding viral capsid/packaging size constraint.
Aims for rapid onset and sustained liver/circulating expression.
Repeatable/dosable administration concepts.
Potential LNP formulation.
Documented Applications
In vivo transgene expression applications are documented, including luciferase expression over extended periods after LNP-encapsulated ceDNA delivery and after re-dosing.
Liver-specific delivery and intracellular presence in hepatocytes are documented using RNAscope in situ hybridization.
Ocular expression applications are documented in rats over about 99 days after sub-retinal injection of ceDNA, contrasted with plasmid.
Immune-related evaluation is documented using Rag2 immune-deficient mice to assess maintained expression and increased expression after re-dose.
FVIII expression applications are documented, including serum FVIII expression measurements after ceDNA injection.
Variant screening applications are documented, including screening multiple FVIII ceDNA sequence variants.
Hemophilia A treatment using the disclosed ceDNA vectors expressing FVIII.
In vitro and in vivo expression of an FVIII protein in a cell by contacting the cell with the ceDNA vector.
Delivery of ceDNA-containing vesicles using purified microvesicles/exosomes, including targeting via surface receptors.
Pharmaceutical compositions for in vivo delivery of ceDNA vectors encoding FVIII using exosome/microparticle and lipid nanoparticle (LNP) formulations.
Treatment of hemophilia A with sustained transgene expression via LNP-ceDNA.
Assessment of FVIII expression and efficacy using assays including luciferase reporter studies.
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