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

Quantitative analysis in outcome assessment of instrumented lumbosacral arthrodesis

The outcome assessment in instrumented lumbosacral fusion mostly focuses on clinical criteria, complications and scores, with a high variability of imaging means, methods of fusion grading and parameters describing degenerative changes, making comparisons between studies difficult. The aim of this retrospective evaluation was to evaluate the interest of quantified radiographic analysis of lumbar spine in global outcome assessment and to highlight the key biomechanical factors involved. Clinical data and Beaujon–Lassale scores were collected for 49 patients who underwent lumbosacral arthrodesis after prior lumbar discectomy (mean follow-up: 5 years). Sagittal standing and lumbar flexion-extension X-ray films allowed quantifying vertebral, lumbar, pelvic and kinematic parameters of the lumbar spine, which were compared to reference values. Statistics were performed to assess evolution for all variables. At long-term follow-up, 90% of patients presented satisfactory clinical outcomes, associated to normal sagittal alignment; vertebral parameters objectified adjacent level degeneration in four cases (8%). Clinical outcome was correlated (r = 0.8) with fusion that was confirmed in 80% of cases, doubtful in 16% and pseudarthrosis seemed to occur in 4% (2) of cases. In addition to clinical data (outcomes comparable to the literature), quantitative analysis accurately described lumbar spine geometry and kinematics, highlighting parameters related to adjacent level’s degeneration and a significant correlation between clinical outcome and fusion. Furthermore, criteria proposed to quantitatively evaluate fusion from lumbar dynamic radiographs seem to be appropriate and in agreement with surgeon’s qualitative grading in 87% of cases