Shuttle peptide delivers base editor RNPs to rhesus monkey airway epithelial cells in vivo.

Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, we derive the S315 peptide as an improvement over S10 in delivering base editor RNP. Following intratracheal aerosol delivery of Cy5-labeled peptide in rhesus macaques, we confirm delivery throughout the respiratory tract. Subsequently, we target CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieve editing efficiencies of up-to 5.3% in rhesus airway epithelia. Moreover, we document persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restores anion channel function in cultured human airway epithelia. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.

Shuttle Peptide Delivers Base Editor RNPs to Rhesus Monkey Airway Epithelial Cells In Vivo.

Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, to improve base editor RNP delivery, we optimized S10 to derive the S315 peptide. Following intratracheal aerosol of Cy5-labeled peptide cargo in rhesus macaques, we confirmed delivery throughout the respiratory tract. Subsequently, we targeted CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieved editing efficiencies of up to 5.3% in rhesus airway epithelia. Moreover, we documented persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restored anion channel function in cultured human airway epithelial cells. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.

A Phosphoinositide-Binding Protein Acts in the Trafficking Pathway of Hemoglobin in the Malaria Parasite Plasmodium falciparum.

Phosphoinositide lipids play key roles in a variety of processes in eukaryotic cells, but our understanding of their functions in the malaria parasite Plasmodium falciparum is still very much limited. To gain a deeper comprehension of the roles of phosphoinositides in this important pathogen, we attempted gene inactivation for 24 putative effectors of phosphoinositide metabolism. Our results reveal that 79% of the candidates are refractory to genetic deletion and are therefore potentially essential for parasite growth. Inactivation of the gene coding for a Plasmodium-specific putative phosphoinositide-binding protein, which we named PfPX1, results in a severe growth defect. We show that PfPX1 likely binds phosphatidylinositol-3-phosphate and that it localizes to the membrane of the digestive vacuole of the parasite and to vesicles filled with host cell cytosol and labeled with endocytic markers. Critically, we provide evidence that it is important in the trafficking pathway of hemoglobin from the host erythrocyte to the digestive vacuole. Finally, inactivation of PfPX1 renders parasites resistant to artemisinin, the frontline antimalarial drug. Globally, the minimal redundancy in the putative phosphoinositide proteins uncovered in our work supports that targeting this pathway has potential for antimalarial drug development. Moreover, our identification of a phosphoinositide-binding protein critical for the trafficking of hemoglobin provides key insight into this essential process. IMPORTANCE Malaria represents an enormous burden for a significant proportion of humanity, and the lack of vaccines and problems with drug resistance to all antimalarials demonstrate the need to develop new therapeutics. Inhibitors of phosphoinositide metabolism are currently being developed as antimalarials but our understanding of this biological pathway is incomplete. The malaria parasite lives inside human red blood cells where it imports hemoglobin to cover some of its nutritional needs. In this work, we have identified a phosphoinositide-binding protein that is important for the transport of hemoglobin in the parasite. Inactivation of this protein decreases the ability of the parasite to proliferate. Our results have therefore identified a potential new target for antimalarial development.

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia.

The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSAmT/mG mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.

The malaria parasite Plasmodium falciparum Sortilin is essential for merozoite formation and apical complex biogenesis.

The inner membrane complex and the apical secretory organelles are defining features of apicomplexan parasites. Despite their critical roles, the mechanisms behind the biogenesis of these structures in the malaria parasite Plasmodium falciparum are still poorly defined. We here show that decreasing expression of the P. falciparum homologue of the conserved endolysomal escorter Sortilin-VPS10 prevents the formation of the inner membrane complex and abrogates the generation of new merozoites. Moreover, protein trafficking to the rhoptries, the micronemes, and the dense granules is disrupted, which leads to the accumulation of apical complex proteins in the endoplasmic reticulum and the parasitophorous vacuole. We further show that protein export to the erythrocyte and transport through the constitutive secretory pathway are functional. Taken together, our results suggest that the malaria parasite P. falciparum Sortilin has potentially broader functions than most of its other eukaryotic counterparts.

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum.

Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans-cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis-Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages.

Evidence that the Plasmodium falciparum Protein Sortilin Potentially Acts as an Escorter for the Trafficking of the Rhoptry-Associated Membrane Antigen to the Rhoptries.

The rhoptry organelle is critical for the invasion of an erythrocyte by the malaria parasite Plasmodium falciparum. Despite their critical roles, the mechanisms behind their biogenesis are still poorly defined. Our earlier work had suggested that the interaction between the glycosylphosphatidylinositol (GPI)-anchored rhoptry-associated membrane antigen (RAMA) and the soluble rhoptry-associated protein 1 was involved in the transport of the latter from the Golgi apparatus to the rhoptry. However, how this protein complex could interact with the intracellular trafficking machinery was unknown at this stage. Here we show that the P. falciparum homologue of the transmembrane protein sortilin-VPS10 interacts with regions of RAMA that are sufficient to target a fluorescent reporter to the rhoptries. These results suggest that P. falciparum sortilin (PfSortilin) could potentially act as the escorter for the transport of rhoptry-destined cargo. IMPORTANCE The malaria parasite is a massive burden in several parts of the world. Worryingly, the parasite has become resistant to several of the drugs commonly used to treat the disease, and at this time, there is no commercial vaccine. It is therefore critical to identify new targets for the development of antimalarials. To survive in the human body, the malaria parasite needs to invade red blood cells. For this, it uses a variety of effectors stored in organelles forming a structure called the apical complex. The mechanisms behind how the parasite generates the apical complex are poorly understood. In this study, we present evidence that a transmembrane protein called sortilin potentially acts as an escorter to transport proteins from the Golgi apparatus to the rhoptries, a component of the apical complex. Our study provides new insight into the biogenesis of a critical structure of the malaria parasite.

Evidence that the Malaria Parasite Plasmodium falciparum Putative Rhoptry Protein 2 Localizes to the Golgi Apparatus throughout the Erythrocytic Cycle.

Invasion of a red blood cell by Plasmodium falciparum merozoites is an essential step in the malaria lifecycle. Several of the proteins involved in this process are stored in the apical complex of the merozoite, a structure containing secretory organelles that are released at specific times during invasion. The molecular players involved in erythrocyte invasion thus represent potential key targets for both therapeutic and vaccine-based strategies to block parasite development. In our quest to identify and characterize new effectors of invasion, we investigated the P. falciparum homologue of a P. berghei protein putatively localized to the rhoptries, the Putative rhoptry protein 2 (PbPRP2). We show that in P. falciparum, the protein colocalizes extensively with the Golgi apparatus across the asexual erythrocytic cycle. Furthermore, imaging of merozoites caught at different times during invasion show that PfPRP2 is not secreted during the process instead staying associated with the Golgi apparatus. Our evidence therefore suggests that PfPRP2 is a Golgi protein and that it is likely not a direct effector in the process of merozoite invasion.