Technologies for the Directed Evolution of Cell Therapies

Abstract

The next generation of therapies is moving beyond the use of small molecules and proteins to using whole cells. Compared with the interactions of small-molecule drugs with biomolecules, which can largely be understood through chemistry, cell therapies act in a chemical and physical world and can actively adapt to that world, amplifying complexity but also the potential for truly breakthrough impact. Although there has been success in introducing targeting proteins into cells to achieve a therapeutic effect, for example, chimeric antigen receptor (CAR) T cells, our ability to engineer cells is generally limited to introducing proteins, but not modulating large-scale traits or structures of cellular “machines,” which play critical roles in disease. Example traits include the ability to secrete compounds, deform through tissue, adhere to surrounding cells, apply force to phagocytose targets, or move through extracellular matrix. There is an opportunity to increase the efficacy of cell therapies through the use of quantitative automation tools, to analyze, sort, and select rare cells with beneficial traits. Combined with methods of genetic or epigenetic mutagenesis to create diversity, such approaches can enable the directed cellular evolution of new therapeutically optimal populations of cells and uncover genetic underpinnings of these optimal traits.

High-throughput selection of cells based on accumulated growth and division using PicoShell particles

“…Despite these solvable limitations, the experimental evidence we have presented shows that PicoShell technology has significant advantages. The workflow can be potentially used for directed evolution of cell populations ( 49 ) where mutagenized cells are placed under selection pressures to generate strains based on unique selection criteria that are time dependent (e.g., growth and production of pigments), at the colony level (multicellular construct formation), or require solution exchange steps (lipid staining, enzyme-linked immunosorbent assays [ELISAs]). For example, the technology may be used to produce microalgae strains that overperform in lipid accumulation rates without significantly reducing their rate of growth for biofuel applications.…”

High-throughput selection of microalgae based on biomass accumulation rates in production environments using PicoShell Particles

“…Despite these solvable limitations, the experimental evidence we have presented shows that PicoShell technology has significant advantages. The workflow can be potentially used for directed evolution of cell populations 38 where mutagenized cells are placed under selection pressures to generate novel strains, based on unique selection criteria that are time-dependent (e.g. growth / biomass accumulation), at the colony level (multi-cellular construct formation), or require solution exchange steps (lipid staining, ELISA-based assays).…”

Massively parallel encapsulation of single cells with structured microparticles and secretion-based flow sorting

“…36,37 Finally, processes of directed evolution of cell products and cells themselves can benefit from larger numbers of clones being screened, mutagenized, and expanded across multiple cycles using an efficient process relying on standard equipment and expertise. 8 More broadly, our new approach to encapsulate single entities into uniform compartments with a solid phase can enable a centralized "lab on a particle" platform for a number of single-cell and single-molecule assays. Cell secretion assay.…”

“…7 Finally, the ability to sort rare populations of cells for outlier clones with ultra-high-performing secretion profiles promises to enable cycles of selection and mutagenesis important for directed cellular evolution of the next generation of cell therapies and also can enable performance surveillance of the clones themselves over time. 8 Although secretion of proteins or other biological products is a key function of many cells, approaches to sort cells at high rates based on this function have significant tradeoffs and are not widespread. Intracellular staining of secreted proteins prior to secretion by cells can be performed following fixation and permeabilization.…”