Carolline Rodrigues Menezes

Delivering ribonucleoproteins (RNPs) for in vivo genome editing is safer than using viruses encoding for Cas9 and its respective guide RNA. However, transient RNP activity does not typically lead to optimal editing outcomes. Here we show that the efficiency of delivering RNPs can be enhanced by cell-penetrating peptides (covalently fused to the protein or as excipients) and that lipid nanoparticles (LNPs) encapsulating RNPs can be optimized for enhanced RNP stability, delivery efficiency and editing potency. Specifically, after screening for suitable ionizable cationic lipids and by optimizing the concentration of the synthetic lipid DMG-PEG 2000, we show that the encapsulation, via microfluidic mixing, of adenine base editor and prime editor RNPs within LNPs using the ionizable lipid SM102 can result in in vivo editing-efficiency enhancements larger than 300-fold (with respect to the delivery of the naked RNP) without detectable off-target edits. We believe that chemically defined LNP formulations optimized for RNP-encapsulation stability and delivery efficiency will lead to safer genome editing.

Advanced gene editing methods have accelerated biomedical discovery and hold great therapeutic promise, but safe and efficient delivery of gene editors remains challenging. In this study, we present a virus-like particle (VLP) system featuring nucleocytosolic shuttling vehicles that retrieve pre-assembled Cas-effectors via aptamer-tagged guide RNAs. This approach ensures preferential loading of fully assembled editor ribonucleoproteins (RNPs) and enhances the efficacy of prime editing, base editing, trans-activators, and nuclease activity coupled to homology-directed repair in multiple immortalized, primary, stem cell, and stem-cell-derived cell types. We also achieve additional protection of inherently unstable prime editing guide RNAs (pegRNAs) by shielding the 3'-exposed end with Csy4/Cas6f, further enhancing editing performance. Furthermore, we identify a minimal set of packaging and budding modules that can serve as a platform for bottom-up engineering of enveloped delivery vehicles. Notably, our system demonstrates superior per-VLP editing efficiency in primary T lymphocytes and two mouse models of inherited retinal disease, highlighting its therapeutic potential.

Objetivos: identificar as evidências científicas existentes até o presente momento sobre a efetividade do uso da cloroquina, da hidroxicloroquina associada (ou não) à azitromicina para tratamento da afecção pelo coronavírus e seus possíveis efeitos adversos e tóxicos aos seres humanos. Métodos: a revisão narrativa utilizou-se das bases de dados PubMed, LILACS, SciElo e Google Acadêmico. Nessas, buscaram-se estudos, utilizando-se dos descritores “covid”, “coronavirus”, “SARS-CoV-2”, “chloroquine”, “hydroxychloroquine”, “azithromycin” e “adverse effects” junto com os operadores booleanos “AND” e “OR”. Resultados: sete artigos, das trinta publicações encontradas, atenderam aos critérios de inclusão, sendo utilizados para compor a presente revisão. Dos sete ensaios clínicos analisados, cinco apresentaram resultados de cura e/ou remissão dos sintomas e/ou redução da carga viral dos pacientes, no entanto apresentaram muitas limitações. Conclusão: a literatura científica é escassa e divergente quanto à efetividade dos medicamentos cloroquina e hidroxicloroquina associada (ou não) à azitromicina no tratamento da COVID-19, pela rápida disseminação e instalação da pandemia na esfera global. É necessário a realização de ensaios clínicos pragmáticos, envolvendo um número maior de pacientes, para que seja possível analisar a efetividade no combate ao coronavírus, bem como a segurança do uso desses fármacos.

Rhodopsin, the prototypical class-A G-protein coupled receptor, is a highly sensitive receptor for light that enables phototransduction in rod photoreceptors. Rhodopsin plays not only a sensory role but also a structural role as a major component of the rod outer segment disc, comprising over 90% of the protein content of the disc membrane. Mutations in RHO which lead to structural or functional abnormalities, including the autosomal recessive E150K mutation, result in rod dysfunction and death. Therefore, correction of deleterious rhodopsin mutations could rescue inherited retinal degeneration, as demonstrated for other visual genes such as RPE65 and PDE6B. In this study, we describe a CRISPR/Cas9 adenine base editing strategy to correct the E150K mutation and demonstrate precise in vivo editing in a Rho -E150K mouse model of autosomal recessive retinitis pigmentosa (RP). Using ultraviolet-visible spectroscopy, mass spectrometry, and the G-protein activation assay, we characterized wild-type rhodopsin and rhodopsin variants containing bystander base edits. Subretinal injection of dual-adeno-associated viruses delivering our base editing strategy yielded up to 44% Rho correction in homozygous Rho -E150K mice. Injection at postnatal day 15, but not later time points, restored rhodopsin expression, partially rescued retinal function, and partially preserved retinal structure. These findings demonstrate that in vivo base editing can restore the function of mutated structural and functional proteins in animal models of disease, including rhodopsin-associated RP and suggest that the timing of gene-editing is a crucial determinant of successful treatment outcomes for degenerative genetic diseases.

Membrane frizzled-related protein (MFRP), present in the retinal pigment epithelium (RPE), is an integral membrane protein essential for ocular development and the normal physiology of the retina. Mutations in MFRP are associated with autosomal recessive nonsyndromic nanophthalmos, leading to severe hyperopia and early-onset retinitis pigmentosa. While several preclinical gene-augmentation and gene-editing trials hold promise for future therapies aimed at stopping degeneration and restoring retinal function, the molecular mechanisms involved in MFRP biology are still not well understood. Here, we studied the biochemical properties of MFRP and the molecular consequences of its loss of function in the retinal degeneration 6 (rd6) mouse model. Using transcriptomic and lipidomic approaches, we observed that accumulation of docosahexaenoic acid (DHA) constitutes a primary defect in the MFRP-deficient RPE. In biochemical assays, we showed that MFRP undergoes extensive glycosylation, and it preferentially binds lipids of several classes, including phosphatidylserine and phosphatidylinositol-4-phosphate; as well as binding to several transmembrane proteins, notably adiponectin receptor 1 (ADIPOR1) and inward rectifier potassium channel 13 (KCNJ13). Moreover, MFRP determines the subcellular localization of ADIPOR1 and KCNJ13 in the RPE in vivo. This feature is altered by MFRP deficiency and can be restored by gene-therapy approaches. Overall, our observations suggest that MFRP constitutes an important interaction hub within the apical membrane of RPE cells, coordinating protein trafficking and subcellular localization within the RPE, and lipid homeostasis within the entire retina.