C60 in olive oil causes light-dependent toxicity and does not extend lifespan in mice.

C60 is a potent antioxidant that has been reported to substantially extend the lifespan of rodents when formulated in olive oil (C60-OO) or extra virgin olive oil (C60-EVOO). Despite there being no regulated form of C60-OO, people have begun obtaining it from online sources and dosing it to themselves or their pets, presumably with the assumption of safety and efficacy. In this study, we obtain C60-OO from a sample of online vendors, and find marked discrepancies in appearance, impurity profile, concentration, and activity relative to pristine C60-OO formulated in-house. We additionally find that pristine C60-OO causes no acute toxicity in a rodent model but does form toxic species that can cause significant morbidity and mortality in mice in under 2 weeks when exposed to light levels consistent with ambient light. Intraperitoneal injections of C60-OO did not affect the lifespan of CB6F1 female mice. Finally, we conduct a lifespan and health span study in males and females C57BL/6 J mice comparing oral treatment with pristine C60-EVOO and EVOO alone versus untreated controls. We failed to observe significant lifespan and health span benefits of C60-EVOO or EVOO supplementation compared to untreated controls, both starting the treatment in adult or old age. Our results call into question the biological benefit of C60-OO in aging.

Alginate microparticles loaded with basic fibroblast growth factor induce tissue coverage in a rat model of myelomeningocele.

BACKGROUND/PURPOSE: We sought to develop a minimally invasive intra-amniotic therapy for prenatal treatment of myelomeningocele (MMC) in an established rat model. METHODS: Time-dated pregnant rats were gavage-fed retinoic acid to induce MMC. Groups received intraamniotic injections at E17.5 with alginate particles loaded with fluorescent dye, basic fibroblast growth factor (Alg-HSA-bFGF), fluorescently tagged albumin (Alginate-BSA-TR), free bFGF, blank alginate particles (Alg-Blank), or PBS. Groups were analyzed at 3 h for specific particle binding or at term (E21) to determine MMC coverage. RESULTS: Alginate microparticles demonstrated robust binding to the MMC defect 3 h after injection. Of those specimens analyzed at E21, 150 of 239 fetuses (62.8%) were viable. Moreover, 18 of 61 (30%) treated with Alg-HSA-bFGF showed evidence of soft tissue coverage compared to 0 of 24 noninjected (P = 0.0021), 0 of 13 PBS (P = 0.0297), and 0 of 42 free bFGF (P = P < 0.0001). Scaffolds of aggregated particles associated with disordered keratinized tissue were observed covering the defect in 2 of 18 (11%) Alg-BSA-TR and 3 of 19 (16%) Alg-Blank specimens. CONCLUSIONS: Injection of microparticles loaded with bFGF resulted in significant soft tissue coverage of the MMC defect compared to controls. Alginate microparticles without growth factors might result in scaffold development over the fetal MMC. TYPE OF STUDY: Basic science. LEVEL OF EVIDENCE: N/A.

Recombinant Manganese Peroxidase Reduces A2E Burden in Age-Related and Stargardt's Macular Degeneration Models.

Macular degeneration is hallmarked by retinal accumulation of toxic retinoid species (e.g., A2E) for which there is no endogenous mechanism to eliminate it. This ultimately results in progressive dysfunction and loss of vision either in advanced age for genetically normal patients (age-related macular degeneration) or in adolescence for those with inherited genetic mutations (Stargardt's disease). In this article, we present a proof-of-concept study for an enzyme-based therapy to remove these retinoids, modeled on traditional enzyme replacement therapy. Recombinant manganese peroxidase (rMnP) is produced in Pichia pastoris. In vitro, we demonstrate that rMnP breaks down A2E and other lipofuscin fluorophores with limited cellular toxicity, and as this enzyme is mannosylated, it can be taken up into cells through mannose receptor-dependent endocytosis. In vivo, we demonstrate that rMnP can significantly reduce the A2E burden when administered by intravitreal injections. Together, these data provide encouraging results toward the development of an enzyme-based therapy for macular degeneration and indicate the need for additional work to characterize the molecular mechanism of A2E breakdown and to improve the pharmacological parameters of the enzyme.

In utero nanoparticle delivery for site-specific genome editing.

Genetic diseases can be diagnosed early during pregnancy, but many monogenic disorders continue to cause considerable neonatal and pediatric morbidity and mortality. Early intervention through intrauterine gene editing, however, could correct the genetic defect, potentially allowing for normal organ development, functional disease improvement, or cure. Here we demonstrate safe intravenous and intra-amniotic administration of polymeric nanoparticles to fetal mouse tissues at selected gestational ages with no effect on survival or postnatal growth. In utero introduction of nanoparticles containing peptide nucleic acids (PNAs) and donor DNAs corrects a disease-causing mutation in the ß-globin gene in a mouse model of human ß-thalassemia, yielding sustained postnatal elevation of blood hemoglobin levels into the normal range, reduced reticulocyte counts, reversal of splenomegaly, and improved survival, with no detected off-target mutations in partially homologous loci. This work may provide the basis for a safe and versatile method of fetal gene editing for human monogenic disorders.

RPtag as an Orally Bioavailable, Hyperstable Epitope Tag and Generalizable Protein Binding Scaffold.

Antibodies are the most prolific biologics in research and clinical environments because of their ability to bind targets with high affinity and specificity. However, antibodies also carry liabilities. A significant portion of the life-science reproducibility crisis is driven by inconsistent performance of research-grade antibodies, and clinical antibodies are often unstable and require costly cold-chain management to reach their destinations in active form. In biotechnology, antibodies are also limited by difficulty integrating them in many recombinant systems due to their size and structural complexity. A switch to small, stable, sequence-verified binding scaffolds may overcome these barriers. Here we present such a scaffold, RPtag, based on a ribose-binding protein (RBP) from extremophile Caldanaerobacter subterraneus. RPtag binds an optimized peptide with pM affinity, is stable to extreme temperature, pH, and protease treatment, readily refolds after denaturation, is effective in common laboratory applications, was rationally engineered to bind bioactive PDGF-ß, and was formulated as a gut-stable orally bioavailable preparation.