Optimization of vectors for effective delivery and expression of genetic content
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Publication Number
US-9382534-B2
Publication Date
2016-07-05
Expiration Date
Abstract
Lentiviral vectors, plates, kits and methods are provided that permit improved efficiency for selection of a promoter sequence for use in a lentiviral application. Through various embodiments of the present invention, a researcher may first evaluate and then choose in a modular fashion, a vector comprising a promoter that is effective for expression in a particular cell line or type.
Core Innovation
A plate is provided for selection of a promoter sequence. The plate comprises a first row of a plurality of loci and a second row of a plurality of loci, and at least two loci in the first row each comprise a first lentiviral vector including a promoter sequence selected from human CMV, mouse CMV, human EF1α, mouse EF1α, CAG, mouse PGK, and UBC, and a reporter sequence. At least two loci in the second row each comprise a second lentiviral vector that is the same as the first lentiviral vector except that it comprises a different promoter sequence selected from the same group.
The plate is arranged so that the first locus in the first row and the first locus in the second row are in a first column, and the second locus in the first row and the second locus in the second row are in a second column. The amount of the first lentiviral vector in the first locus in the first row is from 2 fold to 6 fold the amount of the first lentiviral vector in the second locus in the first row, and correspondingly the amount of the second lentiviral vector in the first locus in the second row is from 2 fold to 6 fold the amount of the second lentiviral vector in the second locus in the second row.
In further embodiments, at least one of the lentiviral vectors comprises an miRNA scaffolding sequence derived from an endogenous pri-miRNA sequence. The lentiviral vectors can further include a mature strand insert sequence and a star strand insert sequence within an miRNA scaffolding sequence, with the mature sequence at least 60% complementary to the star sequence, and neither insert sequence includes a sequence derived from an endogenous miRNA prior-miRNA scaffolding. A control row can also be included in which every locus has no lentiviral vectors, and additional loci can be defined in consecutive positions with lentiviral vectors matched for identity and amount to specific corresponding earlier loci in the same row.
Claims Coverage
The independent claim covers a promoter-selection plate that uses paired rows of loci containing lentiviral vectors that differ by promoter choice from a defined promoter set, with matched column positioning and quantitative fold-differences in vector amounts. Dependent claims further refine the plate layout, include promoter-vector sequence constraints, and add specific control loci/rows and lentiviral-vector component constraints.
Promoter-selection plate with matched lentiviral vectors across columns
A plate for selection of a promoter sequence comprising a first row of loci and a second row of loci, wherein at least two loci in the first row comprise a first lentiviral vector with a promoter sequence selected from human CMV, mouse CMV, human EF1α, mouse EF1α, CAG, mouse PGK, and UBC and a reporter sequence; and at least two loci in the second row comprise a second lentiviral vector the same as the first except having a different promoter sequence from the same group, with corresponding loci positioned in matched columns.
Fold-difference in lentiviral vector amounts between paired loci
The plate where the amount of the first lentiviral vector in a first locus in the first row is from 2 fold to 6 fold the amount in a second locus in the first row, and wherein the amount of the second lentiviral vector in a first locus in the second row is from 2 fold to 6 fold the amount in a second locus in the second row.
Endogenous-pri-miRNA derived miRNA scaffolding in lentiviral vectors
A plate where the lentiviral-vector-containing plate further includes an miRNA scaffolding sequence derived from an endogenous pri-miRNA sequence.
Mature/star insert constraints within miRNA scaffolding
A plate where the lentiviral-vector-containing plate further includes a mature and a star strand insert sequence within an miRNA scaffolding sequence, where the mature sequence is at least 60% complementary to the star sequence and neither insert sequence includes a sequence derived from an endogenous miRNA prior-miRNA scaffolding.
Additional loci with matched identity and amount to earlier loci
A plate where additional consecutive loci include lentiviral vectors matched for identity and amount to specific corresponding earlier loci in the same row.
Control row with absence of lentiviral vectors
A plate where a control row is included and every locus in the control row has no lentiviral vectors.
The claims center on arranging loci in matched rows and columns containing lentiviral vectors that differ by promoter choice from a defined set, while using paired loci with defined 2-fold to 6-fold differences in vector amounts. Dependent claims further specify lentiviral-vector components such as endogenous-pri-miRNA derived miRNA scaffolding, mature/star insert sequence constraints, an absence-of-lentiviral-vectors control row, and additional consecutive loci with matched identity and amount to earlier loci.
Stated Advantages
Improved promoter/transduction/shRNA performance.
Customization of promoter selection using the plate format.
Documented Applications
Empirically choosing promoters for highest signal at the lowest MOI in specific cell types.
Evaluating promoter activity differences across cell type/species and using qualitative fluorescence with quantitative knockdown.
Assessing performance differences in difficult-to-transduce Jurkat cells.
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