Summary Efficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for neural circuit dissection. Such an approach would also be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological spread through functionally connected and highly distributed networks. Viral vectors, in particular, are powerful gene delivery vehicles for the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, we applied in vivo directed evolution to engineer potent retrograde functionality into the capsid of adeno-associated virus (AAV) — a vector that has shown promise in neuroscience research and the clinic. A newly evolved variant, rAAV2-retro, permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde tracers, and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations.

A family of engineered endopeptidases has been created that is capable of cleaving a diverse array of peptide sequences with high selectivity and catalytic efficiency (k cat /K M > 10 4 M −1 s −1 ). By screening libraries with a selection-counterselection substrate method, protease variants were programmed to recognize amino acids having altered charge, size and hydrophobicity properties adjacent to the scissile bond of the substrate, including Glu↓Arg, a specificity that to our knowledge has not been observed among natural proteases. Members of this artificial protease family resulted from a relatively small number of amino acid substitutions that (at least in one case) proved to be epistatic.Around 2% of the mammalian genome encodes for enzymes involved in protein degradation, underscoring the fundamental role of proteolysis in living organisms 1 . Unregulated proteolysis in vivo is lethal, and hence there is a critical requirement for precise sequence specificity as well as temporal and spatial control over protease activity 2,3 . The generalizable ability to engineer a protease to cleave any desired peptide sequence in an exquisitely selective manner and with high catalytic efficiency is of substantial interest for analytical, biotechnological and even therapeutic applications [4][5][6][7] . Although the utility of structure-guided mutagenesis to swap substrate preferences between homologous proteases has been previously demonstrated8 , 9, the systematic engineering of protease specificity to accommodate a diverse set of substrate sequences while maintaining high catalytic activity has remained elusive. Furthermore, typical directed evolution efforts to modify substrate preferences have given rise to enzymes exhibiting either relaxed selectivity or lower turnover for the new substrate10 ,11 . Herein we report the surprisingly general ability to program a family of highly selective and active proteases with new substrate specificities at both the P1 and P1′ positions, including sequences not recognized by the wild-type (WT) enzyme. Using a dual-substrate selection-counterselection flow cytometric assay to screen libraries derived from various diversification methods, protease variants were engineered to be specific for scissile bonds comprised of amino acids having altered charge, size, and NIH Public Access RESULTS Selection and counterselection strategiesThe Escherichia coli surface endopeptidase OmpT likely plays multiple roles in virulence 12 and exhibits a strong preference for cleavage between pairs of the basic amino acids lysine and arginine (especially the latter), cleaving these pairs with high catalytic efficiency 13 . Importantly, OmpT is not active until incorporated into the E. coli outer membrane, minimizing host lethality 14 . A quantitative, single cell-based assay optimized for dynamic range and sensitivity was developed to isolate only those OmpT variants capable of cleaving a desired selection peptide substrate (SelSub), but not a counterselection substrate (CtsSub) (Fig. 1a, ref...

The human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction, and vascular cell growth. KLK6 is overexpressed in breast and ovarian cancer tissues and has been shown to cleave peptide derived from human myelin protein and Ab amyloid peptide in vitro. Here we analyzed the substrate specificity of KLK1 and KLK6, by substrate phage display using a random octapeptide library. Consistent with earlier biochemical data, KLK1 was shown to exhibit both trypsin-and chymotrypsin-like selectivities with Tyr/Arg preferred at site P1, Ser/Arg strongly preferred at P19, and Phe/Leu at P2. KLK6 displayed trypsin-like activity, with the P1 position occupied only by Arg and a strong preference for Ser in P19. Docking simulations of consensus peptide provide information on the identity of the enzyme residues that are responsible for substrate binding. Bioinformatic analysis suggested several putative KLK6 protein substrates, such as ionotropic glutamate receptor (GluR) and synphilin.