Bone-marrow-homing lipid nanoparticles for genome editing in diseased and malignant haematopoietic stem cells.

Therapeutic genome editing of haematopoietic stem cells (HSCs) would provide long-lasting treatments for multiple diseases. However, the in vivo delivery of genetic medicines to HSCs remains challenging, especially in diseased and malignant settings. Here we report on a series of bone-marrow-homing lipid nanoparticles that deliver mRNA to a broad group of at least 14 unique cell types in the bone marrow, including healthy and diseased HSCs, leukaemic stem cells, B cells, T cells, macrophages and leukaemia cells. CRISPR/Cas and base editing is achieved in a mouse model expressing human sickle cell disease phenotypes for potential foetal haemoglobin reactivation and conversion from sickle to non-sickle alleles. Bone-marrow-homing lipid nanoparticles were also able to achieve Cre-recombinase-mediated genetic deletion in bone-marrow-engrafted leukaemic stem cells and leukaemia cells. We show evidence that diverse cell types in the bone marrow niche can be edited using bone-marrow-homing lipid nanoparticles.

Lung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models.

Approximately 10% of Cystic Fibrosis (CF) patients, particularly those with CF transmembrane conductance regulator (CFTR) gene nonsense mutations, lack effective treatments. The potential of gene correction therapy through delivery of the CRISPR/Cas system to CF-relevant organs/cells is hindered by the lack of efficient genome editor delivery carriers. Herein, we report improved Lung Selective Organ Targeting Lipid Nanoparticles (SORT LNPs) for efficient delivery of Cas9 mRNA, sgRNA, and donor ssDNA templates, enabling precise homology-directed repair-mediated gene correction in CF models. Optimized Lung SORT LNPs deliver mRNA to lung basal cells in Ai9 reporter mice. SORT LNP treatment successfully corrected the CFTR mutations in homozygous G542X mice and in patient-derived human bronchial epithelial cells with homozygous F508del mutations, leading to the restoration of CFTR protein expression and chloride transport function. This proof-of-concept study will contribute to accelerating the clinical development of mRNA LNPs for CF treatment through CRISPR/Cas gene correction.

Lipid Nanoparticle (LNP) Chemistry Can Endow Unique In Vivo RNA Delivery Fates within the Liver That Alter Therapeutic Outcomes in a Cancer Model.

Within the field of lipid nanoparticles (LNPs) for RNA delivery, the focus has been mainly placed on organ level delivery, which can mask cellular level effects consequential to therapeutic applications. Here, we studied a pair of LNPs with similar physical properties and discovered how the chemistry of the ionizable amino lipid can control the endogenous LNP identity, affecting cellular uptake in the liver and altering therapeutic outcomes in a model of liver cancer. Although most LNPs accumulate in the liver after intravenous administration (suggesting that liver delivery is straightforward), we observed an unexpected behavior when comparing two similar LNP formulations (5A2-SC8 and 3A5-SC14 LNPs) that resulted in distinct RNA delivery within the organ. Despite both LNPs possessing similar physical properties, ability to silence gene expression in vitro, strong accumulation within the liver, and a shared pKa of 6.5, only 5A2-SC8 LNPs were able to functionally deliver RNA to hepatocytes. Factor VII (FVII) activity was reduced by 87%, with 5A2-SC8 LNPs carrying FVII siRNA (siFVII), while 3A5-SC14 LNPs carrying siFVII produced baseline FVII activity levels comparable to the nontreatment control at a dosage of 0.5 mg/kg. Protein corona analysis indicated that 5A2-SC8 LNPs bind apolipoprotein E (ApoE), which can drive LDL-R receptor-mediated endocytosis in hepatocytes. In contrast, the surface of 3A5-SC14 LNPs was enriched in albumin but depleted in ApoE, which likely led to Kupffer cell delivery and detargeting of hepatocytes. In an aggressive MYC-driven liver cancer model relevant to hepatocytes, 5A2-SC8 LNPs carrying let-7g miRNA were able to significantly extend survival up to 121 days. Since disease targets exist in an organ- and cell-specific manner, the clinical development of RNA LNP therapeutics will require an improved understanding of LNP cellular tropism within organs. The results from our work illustrate the importance of understanding the cellular localization of RNA delivery and incorporating further checkpoints when choosing nanoparticles beyond biochemical and physical characterization, as small changes in the chemical composition of LNPs can have an impact on both the biofate of LNPs and therapeutic outcomes.

Enhancing CRISPR/Cas gene editing through modulating cellular mechanical properties for cancer therapy.

Genome editing holds great potential for cancer treatment due to the ability to precisely inactivate or repair cancer-related genes. However, delivery of CRISPR/Cas to solid tumours for efficient cancer therapy remains challenging. Here we targeted tumour tissue mechanics via a multiplexed dendrimer lipid nanoparticle (LNP) approach involving co-delivery of focal adhesion kinase (FAK) siRNA, Cas9 mRNA and sgRNA (siFAK + CRISPR-LNPs) to enable tumour delivery and enhance gene-editing efficacy. We show that gene editing was enhanced >10-fold in tumour spheroids due to increased cellular uptake and tumour penetration of nanoparticles mediated by FAK-knockdown. siFAK + CRISPR-PD-L1-LNPs reduced extracellular matrix stiffness and efficiently disrupted PD-L1 expression by CRISPR/Cas gene editing, which significantly inhibited tumour growth and metastasis in four mouse models of cancer. Overall, we provide evidence that modulating the stiffness of tumour tissue can enhance gene editing in tumours, which offers a new strategy for synergistic LNPs and other nanoparticle systems to treat cancer using gene editing.

Optimization of phospholipid chemistry for improved lipid nanoparticle (LNP) delivery of messenger RNA (mRNA).

Lipid nanoparticles (LNPs) have been established as an essential platform for nucleic acid delivery. Efforts have led to the development of vaccines that protect against SARS-CoV-2 infection using LNPs to deliver messenger RNA (mRNA) coding for the viral spike protein. Out of the four essential components that comprise LNPs, phospholipids represent an underappreciated opportunity for fundamental and translational study. We investigated this avenue by systematically modulating the identity of the phospholipid in LNPs with the goal of identifying specific moieties that directly enhance or hinder delivery efficacy. Results indicate that phospholipid chemistry can enhance mRNA delivery by increasing membrane fusion and enhancing endosomal escape. Phospholipids containing phosphoethanolamine (PE) head groups likely increase endosomal escape due to their fusogenic properties. Additionally, it was found that zwitterionic phospholipids mainly aided liver delivery, whereas negatively charged phospholipids changed the tropism of the LNPs from liver to spleen. These results demonstrate that the choice of phospholipid plays a role intracellularly by enhancing endosomal escape, while also driving organ tropism in vivo. These findings were then applied to Selective Organ Targeting (SORT) LNPs to manipulate and control spleen-specific delivery. Overall, selection of the phospholipid in LNPs provides an important handle to design and optimize LNPs for improved mRNA delivery and more effective therapeutics.

Zwitterionic Phospholipidation of Cationic Polymers Facilitates Systemic mRNA Delivery to Spleen and Lymph Nodes.

Polymers represent a promising therapeutic platform for extrahepatic messenger RNA (mRNA) delivery but are hampered by low in vivo efficacy due to polyplex serum instability and inadequate endosomal escape following systemic administration. Here, we report the rational design and combinatorial synthesis of zwitterionic phospholipidated polymers (ZPPs) via cationic polymer postmodification by alkylated dioxaphospholane oxides to deliver mRNA to spleen and lymph nodes in vivo. This modular postmodification approach readily produces tunable zwitterionic species for serum resistance and introduces alkyl chains simultaneously to enhance endosomal escape, thereby transforming deficient cationic polymers to efficacious zwitterionic mRNA carriers without the need to elaborately synthesize functional monomers. ZPPs mediated up to 39 500-fold higher protein expression than their parent cationic counterparts in vitro and enabled efficacious mRNA delivery selectively in spleen and lymph nodes following intravenous administration in vivo. This zwitterionic phospholipidation methodology provides a versatile and generalizable postmodification strategy to introduce zwitterions into the side chains of cationic polymers, extending the utility of cationic polymer families for precise mRNA delivery and demonstrating substantial potential for immunotherapeutic applications.

Hydrophobic Optimization of Functional Poly(TPAE-co-suberoyl chloride) for Extrahepatic mRNA Delivery following Intravenous Administration.

Messenger RNA (mRNA) has generated great attention due to its broad potential therapeutic applications, including vaccines, protein replacement therapy, and immunotherapy. Compared to other nucleic acids (e.g., siRNA and pDNA), there are more opportunities to improve the delivery efficacy of mRNA through systematic optimization. In this report, we studied a high-throughput library of 1200 functional polyesters for systemic mRNA delivery. We focused on the chemical investigation of hydrophobic optimization as a method to adjust mRNA polyplex stability, diameter, pKa, and efficacy. Focusing on a region of the library heatmap (PE4K-A17), we further explored the delivery of luciferase mRNA to IGROV1 ovarian cancer cells in vitro and to C57BL/6 mice in vivo following intravenous administration. PE4K-A17-0.2C8 was identified as an efficacious carrier for delivering mRNA to mouse lungs. The delivery selectivity between organs (lungs versus spleen) was found to be tunable through chemical modification of polyesters (both alkyl chain length and molar ratio in the formulation). Cre recombinase mRNA was delivered to the Lox-stop-lox tdTomato mouse model to study potential application in gene editing. Overall, we identified a series of polymer-mRNA polyplexes stabilized with Pluronic F-127 for safe and effective delivery to mouse lungs and spleens. Structure-activity relationships between alkyl side chains and in vivo delivery were elucidated, which may be informative for the continued development of polymer-based mRNA delivery.

All-In-One Dendrimer-Based Lipid Nanoparticles Enable Precise HDR-Mediated Gene Editing In Vivo.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein gene editing is poised to transform the treatment of genetic diseases. However, limited progress has been made toward precise editing of DNA via homology-directed repair (HDR) that requires careful orchestration of complex steps. Herein, dendrimer-based lipid nanoparticles (dLNPs) are engineered to co-encapsulate and deliver multiple components for in vivo HDR correction. BFP/GFP switchable HEK293 cells with a single Y66H amino acid mutation are employed to assess HDR-mediated gene editing following simultaneous, one-pot delivery of Cas9 mRNA, single-guide RNA, and donor DNA. Molar ratios of individual LNP components and weight ratios of the three nucleic acids are systematically optimized to increase HDR efficiency. Using flow cytometry, fluorescence imaging, and DNA sequencing to quantify editing, optimized 4A3-SC8 dLNPs edit >91% of all cells with 56% HDR efficiency in vitro and >20% HDR efficiency in xenograft tumors in vivo. Due to the all-in-one simplicity and high efficacy, the developed dLNPs offer a promising route toward the gene correction of disease-causing mutations.

Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing.

Endosomal escape remains a fundamental barrier hindering the advancement of nucleic acid therapeutics. Taking inspiration from natural phospholipids that comprise biological membranes, we report the combinatorial synthesis of multi-tailed ionizable phospholipids (iPhos) capable of delivering messenger RNA or mRNA/single-guide RNA for gene editing in vivo. Optimized iPhos lipids are composed of one pH-switchable zwitterion and three hydrophobic tails, which adopt a cone shape in endosomal acidic environments to facilitate membrane hexagonal transformation and subsequent cargo release from endosomes. Structure-activity relationships reveal that iPhos chemical structure can control in vivo efficacy and organ selectivity. iPhos lipids synergistically function with various helper lipids to formulate multi-component lipid nanoparticles (called iPLNPs) for selective organ targeting. Zwitterionic, ionizable cationic and permanently cationic helper lipids enable tissue-selective mRNA delivery and CRISPR-Cas9 gene editing in spleen, liver and lungs (respectively) following intravenous administration. This rational design of functional phospholipids demonstrates substantial value for gene editing research and therapeutic applications.

A Systematic Study of Unsaturation in Lipid Nanoparticles Leads to Improved mRNA Transfection In Vivo.

Lipid nanoparticles (LNPs) represent the leading concept for mRNA delivery. Unsaturated lipids play important roles in nature with potential for mRNA therapeutics, but are difficult to access through chemical synthesis. To systematically study the role of unsaturation, modular reactions were utilized to access a library of 91 amino lipids, enabled by the synthesis of unsaturated thiols. An ionizable lipid series (4A3) emerged from in vitro and in vivo screening, where the 4A3 core with a citronellol-based (Cit) periphery emerged as best. We studied the interaction between LNPs and a model endosomal membrane where 4A3-Cit demonstrated superior lipid fusion over saturated lipids, suggesting its unsaturated tail promotes endosomal escape. Furthermore, 4A3-Cit significantly improved mRNA delivery efficacy in vivo through Selective ORgan Targeting (SORT), resulting in 18-fold increased protein expression over parent LNPs. These findings provide insight into how lipid unsaturation promotes mRNA delivery and demonstrate how lipid mixing can enhance efficacy.

Hydrophobic Domain Structure of Linear-Dendritic Poly(ethylene glycol) Lipids Affects RNA Delivery of Lipid Nanoparticles.

In this work, a series of linear-dendritic poly(ethylene glycol) (PEG) lipids (PEG-GnCm) were synthesized through a strategy using sequential aza- and sulfa-Michael addition reactions. The effect of modulating the hydrophobic domain of linear-dendritic PEG lipids was systematically investigated for in vitro and in vivo small RNA delivery as the surface-stabilizing component of 5A2-SC8 dendrimer lipid-based nanoparticles (DLNPs). The lipid alkyl lengths (C8, C12, and C16) and dendrimer generations (G1, G2, and G3) were altered to create PEG-GnCm with different physical properties and anchoring potential. The tail chemical structure of PEG-GnCm did not affect the formulation of 5A2-SC8 DLNPs, including the nanoparticle size, RNA encapsulation, and stability. However, the tail chemical structure did dramatically affect the RNA delivery efficacy of the formed 5A2-SC8 DLNPs with different PEG-GnCm. First-generation PEG lipids (PEG-G1C8, PEG-G1C12, and PEG-G1C16) and a second-generation PEG lipid (PEG-G2C8) formed 5A2-SC8 DLNPs that could deliver siRNAs effectively in vitro and in vivo. 5A2-SC8 DLNPs formulated with second-generation PEG lipids (PEG-G2C12 and PEG-G2C16) and all three third-generation PEG lipids (PEG-G3C8, PEG-G3C12, and PEG-G3C16) lost the ability to deliver siRNA effectively in vitro and in vivo. Overall, we determined that the hydrophobic domain chemical structure of linear-dendritic poly(ethylene glycol) lipids affected the RNA delivery of DLNPs by impacting the escape of 5A2-SC8 DLNPs from endosomes at early cell incubation times, thereby indicating how PEG lipid anchoring and chemical structure can modulate in vitro and in vivo siRNA delivery efficacies.

Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing.

CRISPR-Cas gene editing and messenger RNA-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse cellular origin. However, it is currently impossible to rationally design nanoparticles that selectively target specific tissues. Here, we report a strategy termed selective organ targeting (SORT) wherein multiple classes of lipid nanoparticles are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen- and liver-targeted SORT lipid nanoparticles were designed to selectively edit therapeutically relevant cell types including epithelial cells, endothelial cells, B cells, T cells and hepatocytes. SORT is compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA/single guide RNA and Cas9 ribonucleoprotein complexes, and is envisioned to aid the development of protein replacement and gene correction therapeutics in targeted tissues.