Semi-stable production of lentiviral vectors
Inventors
Bovolenta, Chiara • Stornaiuolo, Anna • Rizzardi, Paolo • Mavilio, Fulvio
Assignees
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Publication Number
US-9133481-B2
Publication Date
2015-09-15
Expiration Date
Abstract
The present invention provides a new semi-stable packaging cell line and a method to produce lentiviral vectors (LV), using the semi-stable packaging cell line. New methods and packaging cell lines of the invention are generated using a baculo-AAV hybrid system for stable expression of structural and regulatory lentiviral proteins, such system comprising a baculoviral backbone containing an integration cassette flanked by AAV ITRs, in combination with a plasmid encoding rep protein. This system allows to obtain a stable integration of the structural and regulatory HIV-1 proteins gag/pol and rev. The system allows to obtain a cell line including the structural and regulatory HIV proteins gag/pol and rev, to be used for a semi-stable LV production.
Core Innovation
The invention provides a system for stable expression of lentiviral structural and regulatory proteins. The system comprises a hybrid vector with a baculoviral backbone containing an integration cassette flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs) and containing two expression cassettes, wherein the first expression cassette encodes lentiviral gag and pol and the second cassette encodes lentiviral rev and a selection marker. The system also includes an expression plasmid containing the AAV Rep Open Reading Frame (ORF) under the control of a promoter.
The hybrid vector uses an AAV ITR-flanked integration cassette within a baculoviral backbone, together with expression of AAV Rep ORF from an expression plasmid. The arrangement supports stable genomic integration of lentiviral structural and regulatory proteins while maintaining functional lentiviral LV production and transfectability. The document further includes a selection marker for maintaining or characterizing the semi-stable system.
The semi-stable packaging cell line is characterized by cells stably expressing lentiviral gag, pol and rev and containing stably integrated into its genome at least one copy of the AAV ITR-flanked integration cassette. The integration cassette contains two expression cassettes, with gag and pol on a first cassette and rev plus a selection marker on a second cassette. The document further characterizes genetic stability over extended culture, reports stable functional LV production, and positions the approach as reducing recombination-driven replication-competent lentivirus risk.
Claims Coverage
The independent claims define two related inventive scopes: a system for stable expression of lentiviral structural and regulatory proteins and a semi-stable lentiviral packaging cell line with stably integrated gag/pol and rev expression cassettes. Across the independent claims, the coverage includes an AAV ITR-flanked baculoviral hybrid integration cassette with two expression cassettes and Rep ORF expression, implemented as a semi-stable packaging cell line with genome integration and selection marker-containing cassette organization.
Hybrid vector with AAV ITR-flanked integration cassette for gag/pol and rev plus selection marker
A hybrid vector comprising baculoviral backbone containing an integration cassette flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs) including two expression cassettes, wherein the first expression cassette encodes lentiviral gag and pol and the second cassette encodes lentiviral rev and a selection marker.
Expression plasmid expressing AAV Rep ORF under promoter control
An expression plasmid containing the AAV Rep Open Reading Frame (ORF) under the control of a promoter.
Semi-stable lentiviral packaging cell line with stably integrated AAV ITR-flanked integration cassette
A semi-stable lentiviral packaging cell line consisting of cells stably expressing lentiviral gag, pol and rev characterized in that such cells contain stably integrated into its genome at least one copy of an integration cassette flanked by AAV inverted terminal repeats (ITR) including two expression cassettes, wherein the first expression cassette encodes lentiviral gag and pol and the second cassette encodes lentiviral rev and a selection marker.
Overall, the independent claims cover an AAV ITR-flanked integration cassette within a baculoviral hybrid vector that carries two expression cassettes (gag/pol and rev plus selection marker), expression of the AAV Rep ORF from an expression plasmid, and a semi-stable lentiviral packaging cell line with stably integrated copies of the ITR-flanked cassette.
Stated Advantages
Maintains functional LV production and transfectability.
Achieves semi-stable LV production with reduced construct/DNA amounts.
Shows reported surprising genetic stability over extended culture.
Positions the system as safer by reducing recombination-driven replication-competent lentivirus risk.
Documented Applications
Semi-stable lentiviral packaging using a semi-stable lentiviral packaging cell line containing stably integrated lentiviral gag, pol and rev expression cassettes.
Production of lentiviral vectors in which an env gene and a transfer vector are added to semi-stable packaging cells, with env gene choices including RD114-TR and VSV-G.
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