Methods for selection and generation of genome edited T cells

Inventors

Marson, Alexander • Lavieu, Gregory G. • Mocciaro, Annamaria • Roth, Theodore L. • Soumillon, Magali • Bennett, Hayley M.

Assignees

University of California San Diego UCSD • Bruker Cellular Analysis Inc

Abstract

Methods are described herein for isolating clonal populations of T cells having a defined genetic modification. The methods are performed, at least in part, in a microfluidic device comprising one or more sequestration pens. The methods include the steps of: maintaining individual T cells (or precursors thereof) that have undergone a genomic editing process in corresponding sequestration pens of a microfluidic device; expanding the T cells into respective clonal populations of T cells; detecting, in one or more T cells of each clonal population, the absence of a cell surface marker that was present in the individual T cells (or precursors thereof); and detecting, in one or more T cells of each clonal population, the presence of a first nucleic acid sequence that is indicative of the presence of an on-target genome edit in the clonal population of T cells. Also described are compositions comprising one or more clonal populations of T cells isolated according to the methods disclosed herein.

Core Innovation

The invention provides methods for isolating genome-edited clonal T-cell populations in microfluidic devices using a sequestration pen. A first T cell that has undergone a genome editing process is maintained individually in the sequestration pen and expanded into a clonal population of T cells. The approach links sequestration-based maintenance with subsequent clonal expansion inside the microfluidic device.

Clonal cells are identified by detecting edit-related biological signatures in one or more T cells of the clonal population. Successful edits are identified by detecting an absence of a cell surface marker that was present in the first T cell or precursor thereof. Additionally or alternatively, the approach detects the presence of a first nucleic acid sequence indicating an on-target genome edit in the clonal population of T cells.

The disclosed microfluidic device architecture includes conditioned inner surfaces of the sequestration pen. At least one inner surface, or a portion thereof, comprises covalently-linked molecules with linking groups covalently bound to the inner surface and moieties that provide an organic and/or hydrophilic layer suitable for maintenance and/or expansion of the genome-edited first cell.

Claims Coverage

The partial content identifies three independent claims. Across these claims, the inventive coverage centers on sequestration-pen based maintenance and clonal expansion of genome-edited T cells in a microfluidic device, together with verification of on-target edits by cell-surface marker loss and/or nucleic-acid sequence detection, and, in one claim, a conditioned inner surface comprising covalently-linked molecules providing an organic and/or hydrophilic layer.

The independent claim set covers microfluidic generation of clonal populations from a genome-edited first T cell maintained in a sequestration pen, with on-target edit confirmation via nucleic-acid sequence detection and, in one independent claim, via absence of a prior cell surface marker. One independent claim further requires a conditioned sequestration-pen inner surface with covalently-linked molecules providing an organic and/or hydrophilic layer suitable for maintenance and/or expansion.

Stated Advantages

Documented Applications