Inhalable Bottlebrush Polymer Bioconjugates as Vectors for Efficient Pulmonary Delivery of Oligonucleotides.

Antisense oligonucleotides hold therapeutic promise for various lung disorders, but their efficacy is limited by suboptimal delivery. To address this challenge, we explored the use of inhaled bottlebrush polymer-DNA conjugates, named pacDNA, as a delivery strategy. Inhaled pacDNA exhibits superior mucus penetration, achieving a uniform and sustained lung distribution in mice. Targeting the 5' splice site of an aberrant enhanced green fluorescence protein (EGFP) pre-mRNA in EGFP-654 mice, inhaled pacDNA more efficiently corrects splicing than a B-peptide conjugate and restores EGFP expression in the lung. Additionally, in an orthotopic NCI-H358 non-small-cell lung tumor mouse model, inhaled pacDNA targeting wild-type KRAS mRNA effectively suppresses KRAS expression and inhibits lung tumor growth, requiring a substantially lower dosage compared to intravenously injected pacDNA. These findings demonstrate the potential of bottlebrush polymer-DNA conjugates as a promising agent for enhanced oligonucleotide therapy in the lung and advancing the treatment landscape for lung disorders.

A mechanistic study on the cellular uptake, intracellular trafficking, and antisense gene regulation of bottlebrush polymer-conjugated oligonucleotides.

We have developed a non-cationic transfection vector in the form of bottlebrush polymer-antisense oligonucleotide (ASO) conjugates. Termed pacDNA (polymer-assisted compaction of DNA), these agents show improved biopharmaceutical characteristics and antisense potency in vivo while suppressing non-antisense side effects. Nonetheless, there still is a lack of the mechanistic understanding of the cellular uptake, subcellular trafficking, and gene knockdown with pacDNA. Here, we show that the pacDNA enters human non-small cell lung cancer cells (NCI-H358) predominantly by scavenger receptor-mediated endocytosis and macropinocytosis and trafficks via the endolysosomal pathway within the cell. The pacDNA significantly reduces a target gene expression (KRAS) in the protein level but not in the mRNA level, despite that the transfection of certain free ASOs causes ribonuclease H1 (RNase H)-dependent degradation of KRAS mRNA. In addition, the antisense activity of pacDNA is independent of ASO chemical modification, suggesting that the pacDNA always functions as a steric blocker.

Exploring the Structural Diversity of DNA Bottlebrush Polymers Using an Oligonucleotide Macromonomer Approach.

Herein, we demonstrate that macromonomers consisting of organics-soluble, chemically protected oligonucleotides (protDNA) and poly(ethylene glycol) (PEG) chains can be converted into bottlebrush polymers of distinct architectures via ring-opening metathesis polymerization (ROMP). Using a custom norbornene-containing phosphoramidite, two types of macromonomers were obtained: a linear norbornene-protDNA-PEG structure and a Y-shaped structure where the polymerizable norbornene group is situated at the junction where protDNA and PEG meet. With this strategy, the PEG chains can be placed either near the backbone of the bottlebrush or on its periphery, and in principle anywhere between these two extremes by adjusting the norbornene location, which makes this strategy attractive for constructing architecturally sophisticated oligonucleotide-containing copolymers.

Targeting oncogenic KRAS with molecular brush-conjugated antisense oligonucleotides.

The mutant form of the guanosine triphosphatase (GTPase) KRAS is a key driver in human tumors but remains a challenging therapeutic target, making KRASMUT 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.

Maximizing TLR9 Activation in Cancer Immunotherapy with Dual-Adjuvanted Spherical Nucleic Acids.

Nucleic-acid-based immune adjuvants have been extensively investigated for the design of cancer vaccines. However, nucleic acids often require the assistance of a carrier system to improve cellular uptake. Yet, such systems are prone to carrier-associated adaptive immunity, leading to difficulties in a multidose treatment regimen. Here, we demonstrate that a spherical nucleic acid (SNA)-based self-adjuvanting system consisting of phosphodiester oligonucleotides and vitamin E can function as a potent anticancer vaccine without a carrier. The two functional modules work synergistically, serving as each other's delivery vector to enhance toll-like receptor 9 activation. The vaccine rapidly enters cells carrying OVA model antigens, which enables efficient activation of adaptive immunity in vitro and in vivo. In OVA-expressing tumor allograft models, both prophylactic and therapeutic vaccinations significantly retard tumor growth and prolong animal survival. Furthermore, the vaccinations were also able to reduce lung metastasis in a B16F10-OVA model.

Bottlebrush Polymer-Conjugated Melittin Exhibits Enhanced Antitumor Activity and Better Safety Profile.

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.

Spherical Nucleic Acids for Topical Treatment of Hyperpigmentation.

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.

Self-Assembled DNA-PEG Bottlebrushes Enhance Antisense Activity and Pharmacokinetics of Oligonucleotides.

Herein, we report a novel strategy to enhance the antisense activity and the pharmacokinetics of therapeutic oligonucleotides. Through the DNA hybridization chain reaction, DNA hairpins modified with poly(ethylene glycol) (PEG) form a bottlebrush architecture consisting of a double-stranded DNA backbone, PEG side chains, and antisense overhangs. The assembled structure exhibits high PEG density on the surface, which suppresses unwanted interactions between the DNA and proteins (e.g., enzymatic degradation) while allowing the antisense overhangs to hybridize with the mRNA target and thereby deplete target protein expression. We show that these PEGylated bottlebrushes targeting oncogenic KRAS can achieve much higher antisense efficacy compared with unassembled hairpins with or without PEGylation and can inhibit the proliferation of lung cancer cells bearing the G12C mutant KRAS gene. Meanwhile, these structures exhibit elevated blood retention times in vivo due to the biological stealth properties of PEG and the high molecular weight of the overall assembly. Collectively, this self-assembly approach bears the characteristics of a simple, safe, yet highly translatable strategy to improve the biopharmaceutical properties of therapeutic oligonucleotides.