Abstract: Disclosed herein are methods of performing continuous directed evolution in complex biological systems, including metazoan cells. These methods include the infection of engineered, non-naturally occurring metazoan cells with engineered, non-naturally occurring DNA viruses. The generation of infectious viruses that can infect new cells depends on the evolution of a gene of interest which is driven by an error-prone adenoviral polymerase. Also disclosed herein, are the compositions of engineered, non-naturally occurring metazoan cells and engineered, non-naturally occurring DNA viruses that function as components in the continuous directed evolution methodologies.

Abstract: The present disclosure relates to engineered erythroid cells and enucleated cells that include one or more of exogenous HLA-G polypeptides, exogenous immunogenic polypeptides, and exogenous coinhibitory polypeptides wherein the cells are capable of inducing immune tolerance and/or reducing immune response to the exogenous immunogenic polypeptides when administered to a subject.

Abstract: The present disclosure provides customizable enucleated erythroid cells or enucleated cells that can be engineered to include, on their surface, a loadable exogenous antigen-presenting polypeptide, wherein the loadable exogenous antigen-presenting polypeptide comprises one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions stabilize the loadable exogenous antigen-presenting polypeptide on the cell surface. In some embodiments, the loadable exogenous antigen-presenting polypeptide is stabilized on the cell surface in the absence of a polypeptide bound to the loadable exogenous antigen-presenting polypeptide. In some embodiments, the loadable exogenous antigen-presenting polypeptide comprises an exogenous displaceable polypeptide bound to the loadable exogenous antigen-presenting polypeptide. In some embodiments, the loadable exogenous antigen-presenting polypeptide is stabilized on the cell surface upon release of the displaceable polypeptide.

Abstract: Disclosed herein are methodologies and kits for dynamic targeted hypermutation that harness the enzymatic activity of a polynucleic acid-binding protein fused to a nucleobase-editing enzyme to specifically target mutations across a region of interest. These methodologies and kits facilitate the rapid creation of diverse DNA libraries in vivo or in vitro.

Abstract: Disclosed herein are methods of performing continuous directed evolution in complex biological systems, including metazoan cells. These methods include the infection of engineered, non-naturally occurring metazoan cells with engineered, non-naturally occurring DNA viruses. The generation of infectious viruses that can infect new cells depends on the evolution of a gene of interest which is driven by an error-prone adenoviral polymerase. Also disclosed herein, are the compositions of engineered, non-naturally occurring metazoan cells and engineered, non-naturally occurring DNA viruses that function as components in the continuous directed evolution methodologies.