Virus-based expression vectors and uses thereof
Inventors
Moss, Bernard • Wyatt, Linda S.
Assignees
US Department of Health and Human Services
Publication Number
US-10894966-B2
Publication Date
2021-01-19
Expiration Date
2035-09-25
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Expression vectors ideal for use in vaccinating individuals against disease based on vaccinia virus and other chordopoxviruses having high expression of recombinant genes and low expression of vector genes in target animals, and low expression of recombinant genes and high expression of vector genes in cells used for propagation.
Core Innovation
The invention provides recombinant chordopoxvirus-based expression vectors, including vaccinia virus vectors, that are engineered to have high expression of heterologous genes in target animals, while exhibiting low expression of vector genes to minimize anti-vector immune responses and competition. These vectors include a heterologous DNA-dependent RNA polymerase gene under control of a pre-replicative promoter and a heterologous repressor protein gene under control of a post-replicative promoter. Additionally, the vector contains at least one inactivating mutation in an open reading frame (ORF) required for expression of post-replicative viral genes, rendering the vector unable to express such genes without complementation.
The problem solved addresses limitations of current poxvirus vectors, which typically do not combine all desired features: non-replicating in humans and animals, high expression of recombinant genes yielding strong immunogenicity, low expression of vector proteins reducing anti-vector immune responses, and stable propagation in continuous cell lines. The disclosed vectors replicate their genome in complementing cells but cannot complete the replication cycle in non-complementing cells, allowing high-level expression of heterologous proteins only in target cells while allowing stable production in complementing cultures.
The system utilizes temporal regulation of viral gene expression, with heterologous elements such as bacteriophage RNA polymerases (e.g., T7 RNA polymerase) and prokaryotic repressors (e.g., LacI) functionally linked to pre- and post-replicative promoters respectively. This design ensures that in complementing cells, repressor expression under post-replicative promoters suppresses recombinant protein expression to favor viral replication and stable vector propagation. Conversely, in non-complementing cells lacking the required transcription factor due to the inactivating mutation, repressor is not expressed, enabling robust recombinant protein production without vector replication.
Claims Coverage
The patent includes one main independent claim focusing on a recombinant chordopoxvirus vector with multiple novel features related to gene expression control and replication.
Recombinant vector architecture combining heterologous expression and inactivating mutation
A recombinant chordopoxvirus comprising: (a) a first nucleic acid sequence encoding a heterologous DNA-dependent RNA polymerase functionally linked to a chordopoxvirus pre-replicative promoter; (b) a second nucleic acid sequence encoding a heterologous repressor protein functionally linked to a chordopoxvirus post-replicative promoter; and (c) at least one inactivating mutation in an ORF required for expression of post-replicative genes, whereby the vector can replicate its genome but not proceed through full replication without complementation.
Use of specific chordopoxvirus pre- and post-replicative promoters
Employment of chordopoxvirus early promoters as pre-replicative promoters (including vaccinia virus early promoters such as thymidine kinase promoter VACVWR094), and chordopoxvirus intermediate or late promoters as post-replicative promoters (such as vaccinia virus I1L promoter VACWR070).
Inactivating mutation targeting transcription factors required for post-replicative gene expression
Introduction of inactivating mutations in ORFs encoding chordopoxvirus transcription factors necessary for post-replicative gene expression, specifically ORFs corresponding to vaccinia virus A8R (VACWR127) or A23R (VACWR143).
Use of heterologous bacteriophage RNA polymerases and prokaryotic repressor proteins
Incorporation of heterologous bacteriophage-derived single subunit DNA-dependent RNA polymerases, such as T7 RNA polymerase, and heterologous prokaryotic repressor proteins, including LacI, to control transcription through recognition of respective promoters and operator sequences.
Strategic genomic positioning of heterologous sequences
Insertion of the first and second nucleic acid sequences between chordopoxvirus ORFs corresponding to vaccinia virus F12 and F13, and inclusion of a third nucleic acid sequence comprising heterologous polynucleotide(s) encoding therapeutic or immunogenic proteins or RNAs under control of a promoter recognized by the heterologous polymerase and an operator recognized by the heterologous repressor, positioned to impede transcription when repressor binds.
The claims collectively cover a recombinant chordopoxvirus vector architecture integrating heterologous RNA polymerase and repressor elements under temporal viral promoters, combined with targeted mutations disabling post-replicative gene expression to confer replication control, enabling high-level recombinant gene expression in non-complementing cells and stable vector propagation in complementing cells.
Stated Advantages
Allows production of high titer recombinant virus vector stocks with stable maintenance of heterologous genetic inserts by reducing growth advantage of mutants in complementing cells.
Enables high expression of heterologous proteins in non-complementing target cells due to absence of repressor protein expression.
Minimizes anti-vector immune responses and resource competition by suppressing vector and recombinant gene expression during propagation in complementing cells.
Provides controlled replication capacity, permitting propagation only in complementing cell lines expressing the necessary transcription factors.
Documented Applications
Vaccination against infectious agents including adenoviruses, herpesviruses, papilloma viruses, polyomaviruses, hepadnaviruses, parvoviruses, astroviruses, caliciviruses, picornaviruses, coronaviruses, flaviviruses, togaviruses, hepeviruses, retroviruses, orthomyxoviruses, arenaviruses, bunyaviruses, filoviruses, paramyxoviruses, rhabdoviruses, reoviruses, and poxviruses in humans and animals.
Production of therapeutic proteins or therapeutic RNA molecules for treatment of illnesses in subjects.
System for manufacturing high-titer recombinant viral vector preparations by culturing recombinant virus vectors in complementing cell lines expressing necessary viral transcription factors.
Interested in licensing this patent?