Compositions and methods for controlling production of polypeptides in cells

Inventors

Deisseroth, Karl A. • Ramakrishnan, Charu • Kim, Yoon Seok • Fenno, Lief E.

Assignees

Leland Stanford Junior University

Abstract

The present disclosure provides recombinant expression vectors for modulating production of polypeptides of interest in a target cell or target cell population. Aspects of the disclosure include recombinant expression vectors having coding sequences encoding portions of a polypeptide of interest, where the coding sequences are flanked by recombinase recognition sites. Also provided are methods for using the recombinant expression vectors as well as a device for monitoring expression of the polypeptide of interest.

Core Innovation

The disclosure relates to recombinant expression vectors and methods for modulating production of a polypeptide of interest in a target cell or a target cell population. The vectors include multiple coding sequences encoding portions of the polypeptide of interest, arranged with specific recombinase recognition sites positioned relative to the coding segments in intervening non-coding sequences. The arrangement uses defined 5′ and 3′ positioning of recombinase recognition sites with respect to the coding sequences.

The disclosed vector architecture comprises a first coding sequence and a second coding sequence encoding different portions of the polypeptide of interest, with a first non-coding sequence positioned between the coding sequences that comprises a first recombinase recognition site and a second recombinase recognition site. A third coding sequence is positioned 3′ to the second coding sequence and encodes a further portion of the polypeptide of interest, with a third recombinase recognition site positioned 3′ to the third coding sequence. A second non-coding sequence positioned between the second coding sequence and the third coding sequence comprises a second recombinase recognition site and a third recombinase recognition site.

The disclosure further specifies that recombinase recognition sites can include Cre/lox, Flp/FRT, vCre, and variants of these sites, including loxP and lox variants and FRT-family variants. In some configurations, the coding sequences are arranged in reverse complement orientation, and the polypeptide of interest can be selected from fluorescent polypeptides, calcium indicators, excitatory opsins, and inhibitory opsins. The disclosed system is validated as an INTRSECT optical-neuroscience toolbox with improved fluorescent protein and GECI variants, splicing fidelity assessment, and characterization of off-target expression and mitigation.

Claims Coverage

The partial content provides two independent claims: one directed to a recombinant expression vector and one directed to a method for modulating production of a polypeptide of interest by introducing that vector into target cells. Across the independent claims, the inventive features center on a multi-part coding arrangement, recombinase recognition site placement in intervening non-coding sequences with specified 5′/3′ positioning, and optional constraints relating to recombinase types, coding orientation, and selectable polypeptide categories.

The independent claims are grounded in a recombinant expression vector architecture with three coding sequence portions encoding a polypeptide of interest, arranged relative to recombinase recognition sites located in intervening non-coding sequences with defined 5′/3′ positions. The corresponding method claim covers producing the polypeptide in target cells by introducing the same vector into those cells. Dependent refinements referenced in the partial content further specify recombinase types, recognition-site variant sequences, possible reverse complement coding orientation, and polypeptide-category selection.

Stated Advantages

Documented Applications