A Mosaic Pavement/Late Romanesque/Early Renaissance

Ink, watercolor/laminated/white paper; 34.5" x 52"Part of the Archives' Visual Materials collectio

EcoScale, a semi-quantitative tool to select an organic preparation based on economical and ecological parameters

A novel post-synthesis analysis tool is presented which evaluates quality of the organic preparation based on yield, cost, safety, conditions and ease of workup/purification. The proposed approach is based on assigning a range of penalty points to these parameters. This semi-quantitative analysis can easily be modified by other synthetic chemists who may feel that some parameters should be assigned different relative penalty points. It is a powerful tool to compare several preparations of the same product based on safety, economical and ecological features

A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell’s endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple “Phylomer” CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne’s muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics

RNA therapeutics in the treatment of retinal disease - delivering the potential

Purpose : Treatment options for inherited retinal diseases (IRD), the leading cause of vision loss in persons aged 15 - 45 years have been limited, however, novel gene and molecular therapeutics are now demonstrating significant potential in the treatment of IRDs. RNA therapeutics hold unique promise in these diseases; although achieving safe and efficient delivery of molecular drugs to the retina and the retinal pigmented epithelium in particular, remains a significant obstacle to clinical application. Antisense oligomers (AO) are a well-established class of RNA therapeutic whose potential is yet to be fully realised due to this delivery challenge. We report an AO conjugate that traffics through the vitreous after intravitreal administration, reaching the deepest layers of the retina and localising to the nuclei to modulate gene expression. This class of therapeutic holds substantial promise in the treatment of IRDs. Methods : We exploit peptide libraries derived from 82 microorganism genomes and 118 synthetic viral genes to identify cell penetrating peptides (CPP) to deliver AO cargos to cells in vitro and to tissues and organs in vivo. The CPPs were screened initially against mammalian cells using a cytosolic extraction method, followed by next generation sequencing and selection using a combination of algorithms known to produce a favourable toxicology and efficacy profile in the eye. Results : The CPPs were conjugated to an antisense morpholino oligomer designed to mediate exon selection in a reporter mRNA. Standout performance in the latter assay, when administered via intravitreal injection, and a clean toxicology profile identified a lead peptide for our retinal disease program. CPP conjugation to our candidate AO therapeutic and evaluation in IRD patient-derived retinal pigmented epithelium rescued target gene expression and improved cell function. Conclusions : Notable CPPs in pre-clinical and clinical development include chemical stabilisation or poly-arginine that can limit efficacy or increase toxicity. Our discovery peptides are derived from nature, lack chemical modifications, and yield optimal amino acid sequences with enhanced efficacy and toxicity performance. The lead CPP, HPG_0031, traffics the AO through the vitreous, into the retinal pigment epithelium with no evidence of retinal damage, resulting in enhanced exon skipping and 6-fold lower cytotoxicity than the competitor CPP

β-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 β-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length β-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of β-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split β-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 µM. Together, the β-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells

GFP-complementation assay to detect functional CPP and protein delivery into living cells

Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell's membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease