Alphaviral vectors and cell lines for producing recombinant proteins
Inventors
ARANDA, Alejandro • BEZUNARTEA BEZUNARTEA, Jaione • Smerdou Picazo, Cristian • MOLERO SÁNCHEZ, Damaso • MISTIENE, Edita • SOLDEVILA FÀBREGA, Andreu • SERRANO PÉREZ-NIEVAS, Paula
Assignees
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Publication Number
US-10011847-B2
Publication Date
2018-07-03
Expiration Date
Abstract
The present invention relates to polynucleotides and alphaviral vectors for the expression of genes of interest in mammalian cells. Additionally, the invention relates to cells which comprise said polynucleotides and alphaviral vectors and are capable of stably expressing one or more genes of interest. The invention also relates to methods for obtaining said cells, to methods for expressing a gene of interest in said cells, and to methods for replacing the gene of interest stably expressed by said cells with another gene of interest.
Core Innovation
The invention provides alphavirus-replicon polynucleotides designed for integration into mammalian genomes using site-specific recombinase recognition sites. The polynucleotide includes a transcription regulatory sequence and a DNA sequence complementary to an alphavirus replicon, where the complementary DNA sequence is operatively bound to the transcription regulatory sequence. The complementary DNA sequence comprises a replication-directed 5 sequence, a polyprotein coding region for non-structural proteins nsp1 through nsp4, an alphavirus subgenomic promoter, and an alphavirus 3 untranslated sequence.
The complementary replicon region further includes two recombinase recognition sequences positioned for cassette exchange. A first recognition sequence for a site-specific recombinase is located between the sequence complementary to the alphavirus replicon subgenomic promoter and the sequence complementary to the alphavirus replicon 3 untranslated sequence, and a second recognition sequence for a site-specific recombinase is positioned 3 with respect to a transcription termination sequence positioned 3 with respect to the 3 end of the sequence complementary to the alphavirus replicon.
Vectors and gene cassettes are configured to enable recombinase-mediated cassette exchange of stably integrated sequences. The system uses a Cre/LoxP-compatible shuttle vector with a selection gene, allowing swapping of an integrated gene of interest at the recombinase recognition sites. The reported design includes inducible non-cytopathic Semliki Forest Virus replicons and transcription regulatory sequence arrangements that support stable expression without antibiotic maintenance, with observed genetic and phenotypic stability over many passages and induction kinetics upon stimulation.
Claims Coverage
The partial content shows two independent claims, covering a recombinase-flanked alphavirus-replicon complementary polynucleotide with transcription regulation and termination, and a recombinase-flanked gene-of-interest vector containing an alphavirus replication 3 sequence, transcription termination, and a selection gene operatively bound to a promoter. Each independent claim contributes multiple inventive features centered on the ordered arrangement of replicon elements and the placement of recombinase recognition sequences relative to the subgenomic promoter and termination.
Transcription-regulated alphavirus-replicon complementary polynucleotide with ordered replicon elements
A polynucleotide comprising a transcription regulatory sequence and a DNA sequence complementary to an alphavirus replicon, wherein the alphavirus replicon comprises, ordered from the 5 to 3 end: a 5 sequence capable of directing replication of the alphavirus, a nucleotide sequence encoding a polyprotein comprising the sequences of non-structural proteins nsp1, nsp2, nsp3 and nsp4 of an alphavirus, an alphavirus subgenomic promoter, and an alphavirus 3 untranslated sequence, and wherein the DNA sequence complementary to an alphavirus replicon is operatively bound to said transcription regulatory sequence.
Two recombinase recognition sequences positioned for cassette exchange
The DNA sequence complementary to an alphavirus replicon comprises a first recognition sequence for a site-specific recombinase located between the sequence complementary to the alphavirus replicon subgenomic promoter and the sequence complementary to the alphavirus replicon 3 untranslated sequence, and a transcription termination sequence positioned 3 with respect to the 3 end of the sequence complementary to said alphavirus replicon, and a second recognition sequence for a site-specific recombinase positioned 3 with respect to the transcription termination sequence.
Recombinase-flanked gene-of-interest vector with alphavirus replication 3 sequence and selection cassette
A vector comprising a DNA sequence comprising, ordered in the 5 to 3 direction, (i) a first recognition sequence for a site-specific recombinase, (ii) a sequence of a gene of interest, (iii) a 3 sequence necessary for replication of an alphavirus, (iv) a transcription termination sequence, (v) a sequence of a selection gene operatively bound to a promoter, and (vi) a second recognition sequence for a site-specific recombinase.
Overall, claim coverage is directed to a transcription-regulated alphavirus replicon-complementary polynucleotide integrated with two site-specific recombinase recognition sequences strategically positioned relative to the subgenomic promoter and transcription termination. The complementary vector claim adds a recombinase-flanked shuttle DNA arrangement carrying a gene of interest, an alphavirus replication 3 sequence, a transcription termination sequence, and a selection gene operatively bound to a promoter.
Stated Advantages
Stable, inducible high expression without antibiotic maintenance.
High genetic and phenotypic stability over many passages.
Demonstrated ability to perform Cre-driven transgene exchange at the integrated sites.
Documented Applications
Generation of stable mammalian cell lines producing secreted proteins, including CT-1, OSM, and A20 monoclonal antibody chains with functional STAT3 activation.
Use of a master cell line with inducible expression, such as GFP, to characterize induction kinetics.
Adaptation to suspension growth, with handling by delaying induction until high cell density.
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