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.
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.
mRNA‐mediated protein replacement represents a promising concept for the treatment of liver disorders. Children born with fumarylacetoacetate hydrolase (FAH) mutations suffer from Hepatorenal ...Tyrosinemia Type 1 (HT‐1) resulting in renal dysfunction, liver failure, neurological impairments, and cancer. Protein replacement therapy using FAH mRNA offers tremendous potential to cure HT‐1, but is currently hindered by the development of effective mRNA carriers that can function in diseased livers. Structure‐guided, rational optimization of 5A2‐SC8 mRNA‐loaded dendrimer lipid nanoparticles (mDLNPs) increases delivery potency of FAH mRNA, resulting in functional FAH protein and sustained normalization of body weight and liver function in FAH−/− knockout mice. Optimization using luciferase mRNA produces DLNP carriers that are efficacious at mRNA doses as low as 0.05 mg kg−1 in vivo. mDLNPs transfect > 44% of all hepatocytes in the liver, yield high FAH protein levels (0.5 mg kg−1 mRNA), and are well tolerated in a knockout mouse model with compromised liver function. Genetically engineered FAH−/− mice treated with FAH mRNA mDLNPs have statistically equivalent levels of TBIL, ALT, and AST compared to wild type C57BL/6 mice and maintain normal weight throughout the month‐long course of treatment. This study provides a framework for the rational optimization of LNPs to improve delivery of mRNA broadly and introduces a specific and viable DLNP carrier with translational potential to treat genetic diseases of the liver.
Structure‐guided, rational optimization of mRNA‐loaded dendrimer lipid nanoparticles (mDLNPs) is conducted to improve mRNA delivery efficacy for therapeutic application. Delivery of luciferase mRNA by mDLNPs in vivo produces 107 photons s−1 luminescence at 0.1 mg kg−1. In a difficult‐to‐treat Hepatorenal Tyrosinemia Type I (HT‐1) mouse model, mDLNPs effectively deliver fumarylacetoacetate hydrolase (FAH) mRNA that normalizes liver function and significantly extends survival.
The c-fos gene (also known as Fos) is induced by a broad range of stimuli and is a reliable marker for neural activity. Its induction mechanism and available reporter mouse lines are based ...exclusively on c-fos promoter activity. Here we demonstrate that multiple enhancers surrounding the c-fos gene are crucial for ensuring robust c-fos response to various stimuli. Membrane depolarization, brain-derived neurotrophic factor (BDNF) and forskolin activate distinct subsets of the enhancers to induce c-fos transcription in neurons, suggesting that stimulus-specific combinatorial activation of multiple enhancers underlies the broad inducibility of the c-fos gene. Accordingly, the functional requirement of key transcription factors varies depending on the type of stimulation. Combinatorial enhancer activation also occurs in the brain. Providing a comprehensive picture of the c-fos induction mechanism beyond the minimal promoter, our study should help in understanding the physiological nature of c-fos induction in relation to neural activity and plasticity.
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.
A non‐viral, lipid nanoparticle (dLNP) delivery platform capable of co‐encapsulating and delivering Cas9 mRNA, sgRNA, and an ssDNA template is developed for homology‐directed repair (HDR)‐mediated gene correction of DNA mutations in vivo. The relative ratios of lipids and nucleic acids are systematically optimized to induce high HDR efficiency, thereby guiding potential treatment opportunities for correction of mutations that cause disease.
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
. 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.
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.
CRISPR/Cas9-based genome editing has quickly emerged as a powerful breakthrough technology for use in diverse settings across biomedical research and therapeutic development. Recent efforts toward ...understanding gene modification methods
have led to substantial improvements in
genome editing efficiency. Because disease targets for genomic correction are often localized in specific organs, realization of the full potential of genomic medicines will require delivery of CRISPR/Cas9 systems targeting specific tissues and cells directly
. In this Perspective, we focus on progress toward
delivery of CRISPR/Cas components. Viral and nonviral delivery systems are both promising for gene editing in diverse tissues
local injection and systemic injection. We describe the various viral vectors and synthetic nonviral materials used for
gene editing and applications to research and therapeutic models, and summarize opportunities and progress to date for both methods. We also discuss challenges for viral delivery, including overcoming limited packaging capacity, immunogenicity associated with multiple dosing, and the potential for off-target effects, and nonviral delivery, including efforts to increase efficacy and to expand utility of nonviral carriers for use in extrahepatic tissues and cancer. Looking ahead, additional advances in the safety and efficiency of viral and nonviral delivery systems for tissue- and cell-type-specific gene editing will be required to enable broad clinical translation. We provide a summary of current delivery systems used for
genome editing, organized with respect to route of administration, and highlight immediate opportunities for biomedical research and applications. Furthermore, we discuss current challenges for
delivery of CRISPR/Cas9 systems to guide the development of future therapies.
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.
Messenger RNA (mRNA) can treat genetic disease using protein replacement or genome editing approaches but requires a suitable carrier to circumnavigate biological barriers and access the desired cell ...type within the target organ. Lipid nanoparticles (LNPs) are widely used in the clinic for mRNA delivery yet are limited in their applications due to significant hepatic accumulation because of the formation of a protein corona enriched in apolipoprotein E (ApoE). Our lab developed selective organ targeting (SORT) LNPs that incorporate a supplementary component, termed a SORT molecule, for tissue-specific mRNA delivery to the liver, spleen, and lungs of mice. Mechanistic work revealed that the biophysical class of SORT molecule added to the LNP forms a distinct protein corona that helps determine where in the body mRNA is delivered. To better understand which plasma proteins could drive tissue-specific mRNA delivery, we characterized a panel of quaternary ammonium lipids as SORT molecules to assess how chemical structure affects the organ-targeting outcomes and protein corona of lung-targeting SORT LNPs. We discovered that variations in the chemical structure of both the lipid alkyl tail and headgroup impact the potency and specificity of mRNA delivery to the lungs. Furthermore, changes to the chemical structure alter the quantities and identities of protein corona constituents in a manner that correlates with organ-targeting outcomes, with certain proteins appearing to promote lung targeting whereas others reduce delivery to off-target organs. These findings unveil a nuanced relationship between LNP chemistry and endogenous targeting, where the ensemble of proteins associated with an LNP can play various roles in determining the tissue-specificity of mRNA delivery, providing further design criteria for optimization of clinically-relevant nanoparticles for extrahepatic delivery of genetic payloads.
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