Spatial patterning of proteins is a valuable technique for many biological applications and is the prevailing tool for defining microenvironments for cells in culture, a required procedure in developmental biology and tissue engineering research. However, it is still challenging to achieve protein patterns that closely mimic native microenvironments, such as gradient protein distributions with desirable mechanical properties. By combining projection dynamic mask lithography and protein engineering with non-canonical photosensitive amino acids, we demonstrate a simple, scalable strategy to fabricate any user-defined 2D or 3D stable gradient pattern with complex geometries from an artificial extracellular matrix (aECM) protein. We show that the elastic modulus and chemical nature of the gradient profile are biocompatible and allow useful applications in cell biological research.

Zinc-finger nuclease (ZFN)-based in vivo genome editing is a novel treatment that can potentially provide lifelong protein replacement with single intravenous administration. Three first-in-human open-label ascending single-dose phase 1/2 studies were performed in parallel (starting November 2017) primarily to assess safety and tolerability of ZFN in vivo editing therapy in mucopolysaccharidosis I (MPS I) (n = 3), MPS II (n = 9), and hemophilia B (n = 1). Treatment was well tolerated with no serious treatment-related adverse events. At the 1e13 vg/kg dose, evidence of genome editing was detected through albumin-transgene fusion transcripts in liver for MPS II (n = 2) and MPS I (n = 1) subjects. The MPS I subject also had a transient increase in leukocyte iduronidase activity to the lower normal range. At the 5e13 vg/kg dose, one MPS II subject had a transient increase in plasma iduronate-2-sulfatase approaching normal levels and one MPS I subject approached mid-normal levels of leukocyte iduronidase activity with no evidence of genome editing. The hemophilia B subject was not able to decrease use of factor IX concentrate; genome editing could not be assessed. Overall, ZFN in vivo editing therapy had a favorable safety profile with evidence of targeted genome editing in liver, but no long-term enzyme expression in blood.