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Publication Number
US-10544397-B2
Publication Date
2020-01-28
Expiration Date
Abstract
This present invention relates to a modified mammalian cell in which the genome of the cell is modified to comprise a sequence encoding CP77 under the control of a promoter such that the modified cell line sustains propagation of a poxvirus that is less able or unable to propagate in the unmodified cell.
Core Innovation
The invention relates to engineered modified mammalian cells, specifically Chinese Hamster Ovary (CHO) cells, for sustaining propagation of a vaccinia virus in vitro. The genome of the CHO cell is modified to comprise a sequence encoding CP77 under the control of a constitutive promoter, addressing CHO non-permissiveness for poxvirus propagation, including vaccinia and vaccinia derivatives such as MVA.
The engineered CHO genome further comprises constitutively expressed poxvirus host range factor components, including a sequence encoding D13L under a constitutive promoter and/or a sequence encoding K1L under a constitutive promoter. A proviso is applied that CP77, D13L and K1L do not include functional orthologs and functional variants, thereby defining an engineered CHO expression set configured to sustain propagation of vaccinia virus in vitro.
The disclosed system additionally contemplates CP77-mediated conversion of CHO to a permissive state for vaccinia and MVA, with constitutive promoter control. Embodiments include CP77 expressed from codon-optimized contiguous nucleotide sequences for expression in CHO cells, and stable continuous CHO cell line embodiments are described for sustaining such propagation.
Claims Coverage
The partial content includes two independent claims: one directed to a modified CHO cell and one directed to a process for propagating vaccinia virus in CHO cells. Across these independent claims, there are two core inventive features: genomic insertion of CP77 under a constitutive promoter to enable sustained vaccinia propagation in CHO, and constitutive encoding and expression of D13L and/or K1L with restrictions on functional orthologs and functional variants.
Constitutive CP77 genomic modification enabling sustained vaccinia propagation in CHO
A modified Chinese Hamster Ovary (CHO) cell in which the genome is modified to comprise a sequence encoding CP77 under the control of a constitutive promoter such that the modified CHO cell line sustains propagation of a vaccinia virus.
Constitutive D13L and/or K1L expression together with CP77, excluding functional orthologs and functional variants
The modified CHO cell further comprises a sequence encoding D13L under the control of a constitutive promoter and/or a sequence encoding K1L under the control of a constitutive promoter, with the proviso that CP77, D13L and K1L do not include functional orthologs and functional variants.
In vitro vaccinia propagation in modified CHO cells expressing CP77 and D13L under constitutive promoters
A process for propagating a vaccinia virus in CHO cells comprising propagating the vaccinia virus in vitro in a CHO cell modified to encode and express CP77 under a constitutive promoter and to encode and express D13L under a constitutive promoter, with the proviso that CP77 and D13L do not include functional orthologs and functional variants.
The independent claim set defines engineered CHO cells for sustained in vitro vaccinia propagation by constitutively encoding CP77 in the CHO genome, optionally combined with constitutive expression of D13L and/or K1L, while restricting CP77/D13L/K1L to exclude functional orthologs and functional variants. The corresponding process claim propagates vaccinia virus in vitro in such modified CHO cells using CP77 and D13L constitutive expression under the same restriction.
Stated Advantages
Enables sustained propagation of a vaccinia virus in non-permissive CHO cells in vitro.
Allows engineered CHO cells to support propagation of vaccinia and vaccinia derivatives such as MVA, as documented by CP77-mediated conversion to permissiveness.
Achieves high virus replication amplification ratios when using CP77-expressing embodiments.
Documented Applications
In vitro propagation of vaccinia virus in engineered modified CHO cells expressing CP77 under a constitutive promoter.
In vitro propagation of vaccinia derivatives including MVA using CP77-containing engineered CHO cells.
Use of D13L- and/or K1L-expressing engineered CHO cells as host cell substrates for vaccinia-based vectors, including embodiments described for D13L-deleted vaccinia vector contexts.
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