Protocol to characterize the melanoma tumor immune microenvironment in mice from single cell to flow cytometry analysis.

Here, we present a protocol to study and describe immune cells that surround or infiltrate tumor cells or get through the body of a melanoma syngeneic mice model. We describe steps for creating and establishing the syngeneic mouse model, euthanasia, and tumor or organ harvest. We then detail procedures to rapidly achieve a single-cell suspension from different tissue samples to further quantify and analyze the phenotype of the immune cell population (lymphocytes T and B, tumor-associated macrophages, and myeloid-derived suppressor cells) by flow cytometry.

Ancestral library identifies conserved reprogrammable liver motif on AAV capsid.

Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction-a finding potentially relevant to preventing hepatoxicities seen in clinical studies.