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Effective Antisense Gene Regulation via Non-cationic, Polyethylene Glycol Brushes Xueguang Lu, Fei Jia, Xuyu Tan, Dali Wang, Xueyan Cao, Jiamin Zheng, and Ke Zhang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b05787 • Publication Date (Web): 15 Jul 2016 Downloaded from http://pubs.acs.org on July 16, 2016
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Journal of the American Chemical Society
Effective Antisense Gene Regulation via Non-cationic, Polyethylene Glycol Brushes Xueguang Lu, Fei Jia, Xuyu Tan, Dali Wang, Xueyan Cao, Jiamin Zheng, and Ke Zhang* Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States
Supporting Information Placeholder ABSTRACT: Negatively charged nucleic acids are often complexed with polycationic transfection agents before delivery. Herein, we demonstrate that a non-cationic, biocompatible polymer, polyethylene glycol, can be used as a transfection vector by forming a brush polymer-DNA conjugate. The brush architecture provides embedded DNA strands with enhanced nuclease stability and improved cell uptake. Because of the biologically benign nature of the polymer component, no cytotoxicity was observed. This approach has the potential to address several long-lasting challenges in oligonucleotide therapeutics.
possible that enhanced nuclease stability is an important factor for SNAs to survive the endocytotic processing and 11 eventually enter the cytosol to serve their intended purpose. In other words, enhanced nucleic acid stability may be a crucial if not the bottleneck junction in oligonucleotide therapy for non-cationic systems.
Scheme 1. Structures of pacDNA and Y-shaped PEGDNA conjugate
Oligonucleotide-based gene therapy holds tremendous promise for treating a variety of disorders with a genetic basis, including cancers, neurological diseases, and metabolic 1 conditions. However, since its conceptualization in the 2 1970s, there have only been a relatively small number of commercial successes (e.g. Vitravene, Macugen, and Kynam3 ra), despite powerful advancement in the understanding of 4 the underlying biology. This contrast exemplifies the difficulties in transforming nucleic acids to drugs: poor accumulation at target sites, unwanted innate and adaptive immune responses, nuclease degradation, coagulopathy, poor cellular 5 uptake, and overall low biochemical efficacy. Cationic polymers’ ability to complex with nucleic acids and deliver them to cells has been extensively explored as a 6 route to therapeutic intervention. Despite significant progress, however, these materials are still prone to various degrees of cytotoxic and immunogenic reactions, which limit 7 their clinical application. Recently, a new type of nucleic acid nanostructure, termed spherical nucleic acids (SNAs), has emerged as a non-cationic, single-entity transfection 8 agent. Consisting of tens to hundreds of oligonucleotide strands densely arranged onto a spherical core, SNAs are capable of entering cells in large quantities despite their negative charge and knocking down target genes without significant cytotoxicity and stimulation of the innate immune sys9 tem. Due to the dense arrangement, the SNA oligonucleotides are more stable to nuclease degradation than their free, 10 linear counterparts. These observations led us to suspect that the unusual ability of the SNA to act as an antisense agent and its enzyme stability may not be a coincidence. It is
Inspired by the SNAs, we have developed a novel form of polymer-DNA conjugate, termed polymer-assistedcompaction of DNA (pacDNA), which consists of oligonucleotide (1-3 strands) covalently attached to the backbone of a sterically congested brush polymer with polyethylene glycol 12 (PEG) side chains. By carefully designing the relative lengths of the DNA strands and the PEG side chains, we have shown that the pacDNAs can achieve >20-fold increase in half-life for DNase I, while hybridization with complementary strands remains kinetically unaffected. Therefore, we contemplate that it is possible for the pacDNA to endure the endosome/lysosome environment, and thus enter the cytosol 13 through normal endosomal processing pathways and regulate gene expression with minimal perturbation to the cell. In contrast, cationic species often cause cell mem14 brane/endosome perforation, leading to toxicity. The use of PEG for oligonucleotide delivery can also improve the biopharmaceutical properties of the oligonucleotide by sup-
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pressing unwanted, non-antisense interactions with various 15 proteins. Furthermore, factors previously recognized as important for co-carrier systems such as nucleic acid dissociation from complex and proton buffering capacity do not 11 apply to the pacDNA, thereby simplifying carrier design.
Table 1. GPC analyses for the brush polymers used. Polymer
Composition
Mn (kDa)
Mw (kDa)
PDI
Brush5k
pN-azide5-b-pN-PEG(5k)35
178.8
197.2
1.10
Brush10k
pN-azide5-b-pN-PEG(10k)28
285.5
329.1
1.15
To test our hypothesis, we have designed an antisense pacDNA having 10 kDa PEG side chains that targets the human epidermal growth factor receptor 2 (Her2) mRNA (pacDNA10k, Scheme 1). Her2 is an important biomarker for many cancers including several types of breast and ovarian can16 cers, and antisense control of the Her2 gene has been previ17 ously demonstrated. For controls, we use an improper pacDNA with overly short side chains (5 kDa), and a Y-shaped PEG-DNA conjugate (YPEG-DNA). The pacDNA5k is incapable of effectively protecting the embedded DNA against enzymatic degradation (vide infra). The YPEG is routinely used to form bioconjugates and is found in commercial oligonu18 cleotide drug formulations (Macugen). However, because of the low density of the PEG chains (2 chains), adequate enzymatic protection to the DNA is not anticipated.
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polymerization (ROMP) of norbornenyl bromide (N-Br) and norbornenyl PEG (N-PEG, Mn=5 or 10 kDa, PDI
