Compositions and methods for enhancing the stability of transgenes in poxviruses
Inventors
Kalla, Markus • Rountree, Ryan • Dirmeier, Ulrike
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11052139-B2
Publication Date
2021-07-06
Expiration Date
Abstract
Provided herein are recombinant poxviruses that are stable through successive passaging of the recombinant poxviruses. More particularly, the recombinant poxviruses comprise one or more modified nucleic acids encoding MUC1, CEA, and/or TRICOM antigens, wherein the recombinant poxviruses are stable through successive passaging. Also, provided herein are compositions and method related thereto.
Core Innovation
The invention relates to a recombinant poxvirus vaccine platform in which inserted transgenes are improved for genetic stability during successive passaging. The problem addressed is that transgenes in a prior construct show instability during passaging, including loss and/or decreased expression of MUC1 and CEA and fragment deletions. The document describes modifications designed to maintain transgene integrity.
To address the instability, the recombinant poxvirus encodes a MUC1 peptide having three Variable N-Terminal Repeat (VNTR) domains, and the order of the nucleic acids encoding at least two VNTR domains is different from their order in PANVAC. The VNTR domains are also codon optimized.
The document further describes modified constructs beyond MUC1, including modified CEA with nucleotide substitutions in repetitive nucleotide regions defined by runs of consecutive G/C or T/C nucleotides, and modified TRICOM costimulatory molecules with repetitive-region substitutions. Constructed exemplars are described as maintaining transgene integrity through successive passaging, with the stability supported by PCR and sequencing.
Claims Coverage
The independent claim provides a stable recombinant poxvirus defined by transgene design features affecting VNTR order and codon optimization, while maintaining stability through successive passaging. The provided claims content identifies seven inventive features, with dependent claims further defining VNTR ordering, specific sequence constraints, CEA repetitive-region substitution criteria, and encoding of co-stimulatory molecules.
Stable recombinant poxvirus through successive passaging
A recombinant poxvirus stable through successive passaging, comprising a nucleic acid encoding a MUC1 peptide having three VNTR domains, with stability maintained through successive passaging.
Altered VNTR order relative to PANVAC
The order of the nucleic acids encoding at least two VNTR domains of MUC1 is different from their order in PANVAC.
Codon optimized VNTR domains
The VNTR domains are codon optimized.
Defined VNTR ordering
The order of VNTR #2 and VNTR #1 is reversed.
Specific nucleic acid sequence constraint (SEQ ID NO:2)
The nucleic acid sequence comprises SEQ ID NO:2.
CEA repetitive-region substitution criteria
The nucleic acid encoding a carcinoembryonic antigen (CEA) includes at least one nucleotide substitution within repetitive nucleotide regions defined as three or more consecutive G/C nucleotides and/or three or more consecutive T nucleotides.
Co-stimulatory molecule encoding (B7-1, ICAM-1, LFA-3)
The recombinant poxvirus further comprises a nucleic acid encoding one or more co-stimulatory molecules selected from B7-1, ICAM-1, LFA-3, and combinations thereof.
Overall, the claims coverage centers on maintaining stability through successive passaging by encoding a MUC1 peptide with three VNTR domains whose coding order differs from PANVAC and whose VNTR domains are codon optimized, with dependent claims further narrowing by specific VNTR ordering and sequence features. Additional dependent features extend the platform to CEA repetitive-region substitutions and to recombinant poxviruses that include nucleic acids encoding specified co-stimulatory molecules.
Stated Advantages
Improved genetic stability of inserted transgenes during successive passaging.
Maintenance of transgene integrity through at least passage 4 for a described construct and into passages 7 for additional constructs.
Stability supported by PCR and sequencing, with rationale tied to genetic stability for large-scale manufacturing and regulatory stability.
Documented Applications
Recombinant poxvirus vaccine constructs using modified MUC1, modified CEA repetitive-region substitutions, and modified TRICOM co-stimulatory molecule repetitive-region substitutions.
Heterologous prime-boost regimen concepts described as an MVA prime and a fowlpox boost.
Use of poxvirus hosts including MVA and fowlpox.
Interested in licensing this patent?