Messenger RNA lipid-nanoparticle-based therapies represent an emerging class of medicines for a variety of applications. However, anti-poly(ethylene glycol) (anti-PEG) antibodies generated by widely used PEGylated medicines and lipid nanoparticles hinder therapeutic efficacy upon repeated dosing. Here we report the chemical design, synthesis and optimization of high-density brush-shaped polymer lipids that reduce anti-PEG antibody binding to improve protein production consistency in repeated dosing. Brush-shaped polymer lipid parameters, including side chain length, degree of polymerization, anchor alkyl length and surface regimes on lipid nanoparticles modulate anti-PEG antibody binding affinity and control their blood circulation pharmacokinetics. Compared to widely used 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, lipid nanoparticles containing brush-shaped polymer lipids generate superior therapeutic outcomes in protein replacement therapy and genome editing models, reformulating structure-activity guidelines for the design of PEG lipid substitutes. Overall, these findings contribute to the general effort in the development of lipid nanoparticles with low immunogenicity to overcome current roadblocks to nucleic acid medicines.

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. The ability to genetically modify haematopoietic stem cells would allow the durable treatment of a diverse range of genetic disorders but gene delivery to the bone marrow has not been achieved. Here lipid nanoparticles that target and deliver mRNA to 14 unique cells within the bone marrow are presented.

In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)–sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.

Lipid nanoparticles (LNPs) are an essential component of messenger RNA (mRNA) vaccines and genome editing therapeutics. Ionizable amino lipids, which play the most crucial role in enabling mRNA to overcome delivery barriers, have, to date, been restricted to two-dimensional (2D) architectures. Inspired by improved physicochemical properties resulting from the incorporation of three-dimensionality (3D) into small-molecule drugs, we report the creation of 3D ionizable lipid designs through the introduction of bicyclo[1.1.1]pentane (BCP) core motifs. BCP-based lipids enabled efficient in vivo mRNA delivery to the liver and spleen with significantly greater performance over 2D benzene- and cyclohexane-based analogues. Notably, lead BCP-NC2-C12 LNPs mediated ∼90% reduction in the PCSK9 serum protein level via CRISPR/Cas9 gene knockout, outperforming 2D controls and clinically used DLin-MC3-DMA LNPs at the same dose. Here, we introduce BCP-based designs with superior in vivo activity, thereby expanding the chemical scope of ionizable amino lipids from 2D to 3D and offering a promising avenue to improve mRNA and gene editing efficiency for the continued development of genetic medicines.

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.

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.

Coronavirus disease 2019 (COVID-19) caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide as a severe pandemic and caused enormous global health and economical damage. Since December 2019, more than 197 million cases have been reported, causing 4.2 million deaths. In the settings of pandemic it is an urgent necessity for the development of an effective COVID-19 treatment. While in-vitro screening of hundreds of antibodies isolated from convalescent patients is challenging due to its high cost, use of computational methods may provide an attractive solution in selecting the top candidates. Here, we developed a computational approach (SARS-AB) for binding prediction of spike protein SARS-CoV-2 with monoclonal antibodies. We validated our approach using existing structures in the protein data bank (PDB), and demonstrated its prediction power in antibody-spike protein binding prediction. We further tested its performance using antibody sequences from the literature where crystal structure is not available, and observed a high prediction accuracy (AUC = 99.6%). Finally, we demonstrated that SARS-AB can be used to design effective antibodies against novel SARS-CoV-2 mutants that might escape the current antibody protections. We believe that SARS-AB can significantly accelerate the discovery of neutralizing antibodies against SARS-CoV-2 and its mutants.

The mutant form of the guanosine triphosphatase (GTPase) KRAS is a key driver in human tumors but remains a challenging therapeutic target, making KRAS MUT cancers a highly unmet clinical need. Here, we report a class of bottlebrush polyethylene glycol (PEG)–conjugated antisense oligonucleotides (ASOs) for potent in vivo KRAS depletion. Owing to their highly branched architecture, these molecular nanoconstructs suppress nearly all side effects associated with DNA–protein interactions and substantially enhance the pharmacological properties of the ASO, such as plasma pharmacokinetics and tumor uptake. Systemic delivery to mice bearing human non–small-cell lung carcinoma xenografts results in a significant reduction in both KRAS levels and tumor growth, and the antitumor performance well exceeds that of current popular ASO paradigms, such as chemically modified oligonucleotides and PEGylation using linear or slightly branched PEG. Importantly, these conjugates relax the requirement on the ASO chemistry, allowing unmodified, natural phosphodiester ASOs to achieve efficacy comparable to that of chemically modified ones. Both the bottlebrush polymer and its ASO conjugates appear to be safe and well tolerated in mice. Together, these data indicate that the molecular brush–ASO conjugate is a promising therapeutic platform for the treatment of KRAS -driven human cancers and warrant further preclinical and clinical development.

Lipid nanoparticles (LNPs) are a clinically mature technology for the delivery of genetic medicines but have limited therapeutic applications due to liver accumulation. Recently, our laboratory developed selective organ targeting (SORT) nanoparticles that expand the therapeutic applications of genetic medicines by enabling delivery of messenger RNA (mRNA) and gene editing systems to non-liver tissues. SORT nanoparticles include a supplemental SORT molecule whose chemical structure determines the LNP's tissue-specific activity. To understand how SORT nanoparticles surpass the delivery barrier of liver hepatocyte accumulation, we studied the mechanistic factors which define their organ-targeting properties. We discovered that the chemical nature of the added SORT molecule controlled biodistribution, global/apparent pKa, and serum protein interactions of SORT nanoparticles. Additionally, we provide evidence for an endogenous targeting mechanism whereby organ targeting occurs via 1) desorption of poly(ethylene glycol) lipids from the LNP surface, 2) binding of distinct proteins to the nanoparticle surface because of recognition of exposed SORT molecules, and 3) subsequent interactions between surface-bound proteins and cognate receptors highly expressed in specific tissues. These findings establish a crucial link between the molecular composition of SORT nanoparticles and their unique and precise organ-targeting properties and suggest that the recruitment of specific proteins to a nanoparticle's surface can enable drug delivery beyond the liver.

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.

Oligonucleotide-based materials such as spherical nucleic acid (SNA) have been reported to exhibit improved penetration through the epidermis and the dermis of the skin upon topical application. Herein, we report a self-assembled, skin-depigmenting SNA structure, which is based upon a bifunctional oligonucleotide amphiphile containing an antisense oligonucleotide and a tyrosinase inhibitor prodrug. The two components work synergistically to increase oligonucleotide cellular uptake, enhance drug solubility, and promote skin penetration. The particles were shown to reduce melanin content in B16F10 melanoma cells and exhibited a potent antimelanogenic effect in an ultraviolet B-induced hyperpigmentation mouse model.

Despite potency against a variety of cancers in preclinical systems, melittin (MEL), a major peptide in bee venom, exhibits non-specific toxicity, severe hemolytic activity, and poor pharmacological properties. Therefore, its advancement in the clinical translation system has been limited to early-stage trials. Herein, we report a biohybrid involving a bottlebrush-architectured poly(ethylene glycol) (PEG) and MEL. Termed pacMEL, the conjugate consists of a high-density PEG arrangement, which provides MEL with steric inhibition against protein access, while the high molecular weight of pacMEL substantially enhances plasma pharmacokinetics with a ∼10-fold increase in the area under the curve (AUC∞) compared to free MEL. pacMEL also significantly reduces hepatic damage and unwanted innate immune response and all but eliminated hemolytic activities of MEL. Importantly, pacMEL passively accumulates at subcutaneously inoculated tumor sites and exhibits stronger tumor-suppressive activity than molecular MEL. Collectively, pacMEL makes MEL a safer and more appealing drug candidate.

Herein, we report the DNA-mediated self-assembly of bivalent bottlebrush polymers, a process akin to the step-growth polymerization of small molecule monomers. In these "condensation reactions", the polymer serves as a steric guide to limit DNA hybridization in a fixed direction, while the DNA serves as a functional group equivalent, connecting complementary brushes to form well-defined, one-dimensional nanostructures. The polymerization was studied using spectroscopy, microscopy, and scattering techniques and was modeled numerically. The model made predictions of the degree of polymerization and size distribution of the assembled products, and suggested the potential for branching at hybridization junctions, all of which were confirmed experimentally. This study serves as a theoretical basis for the polymer-assembly approach which has the potential to open up new possibilities for suprapolymers with controlled architecture, macromonomer sequence, and end-group functionalities.

Activation of the stimulator of interferon gene (STING) pathway within the tumor microenvironment has been shown to generate a strong antitumor response. Although local administration of STING agonists has promise for cancer immunotherapy, the dosing regimen needed to achieve efficacy requires frequent intratumoral injections over months. Frequent dosing for cancer treatment is associated with poor patient adherence, with as high as 48% of patients failing to comply. Multiple intratumoral injections also disrupt the tumor microenvironment and vascular networks and therefore increase the risk of metastasis. Here, we developed microfabricated polylacticco-glycolic acid (PLGA) particles that remain at the site of injection and release encapsulated STING agonist as a programmable sequence of pulses at predetermined time points that mimic multiple injections over days to weeks. A single intratumoral injection of STING agonist-loaded microparticles triggered potent local and systemic antitumor immune responses, inhibited tumor growth, and prolonged survival as effectively as multiple soluble doses, but with reduced metastasis in several mouse tumor models. STING agonist-loaded microparticles improved the response to immune checkpoint blockade therapy and substantially decreased the tumor recurrence rate from 100 to 25% in mouse models of melanoma when administered during surgical resection. In addition, we demonstrated the therapeutic efficacy of STING microparticles on an orthotopic pancreatic cancer model in mice that does not allow multiple intratumoral injections. These findings could directly benefit current STING agonist therapy by decreasing the number of injections, reducing risk of metastasis, and expanding its applicability to hard-to-reach cancers.

Here, we develop a facile route to bring DNA to the organic phase, which greatly expands the types of structures accessible via DNA macromonomers. Phosphotriester- and exocyclic-amine-protected DNA was synthesized and further modified with a norbornene moiety, which enables homopolymerization via ring-opening metathesis to produce brush-type DNA graft polymers in high yields. Subsequent deprotection cleanly reveals the natural phosphodiester DNA. The method not only achieves high molecular weight DNA graft polymers but, when carried out at low monomer-to-catalyst ratios, also yields oligomers that can be further fractionated to molecularly pure, monodisperse entities with one through ten DNA strands per molecule. In addition, we demonstrate substantial simplification in the preparation of traditionally difficult DNA-containing structures, such as DNA-poly(ethylene glycol) diblock graft copolymers and DNA amphiphiles. We envision that the marriage of oligonucleotides with the vast range of organic-phase polymerizations will result in many new classes of materials with yet unknown properties.

Nucleic acids are generally regarded as the payload in gene therapy, often requiring a carrier for intracellular delivery. With the recent discovery that spherical nucleic acids enter cells rapidly, we demonstrate that nucleic acids also have the potential to act as a delivery vehicle. Herein, we report an amphiphilic DNA-paclitaxel conjugate, which forms stable micellar nanoparticles in solution. The nucleic acid component acts as both a therapeutic payload for intracellular gene regulation and the delivery vehicle for the drug component. A bioreductively activated, self-immolative disulfide linker is used to tether the drug, allowing free drug to be released upon cell uptake. We found that the DNA-paclitaxel nanostructures enter cells ∼100 times faster than free DNA, exhibit increased stability against nuclease, and show nearly identical cytotoxicity as free drug. These nanostructures allow one to access a gene target and a drug target using only the payloads themselves, bypassing the need for a cocarrier system.