In Vivo Delivery of Nucleic Acids via Glycopolymer Vehicles Affords Therapeutic Infarct Size Reduction In Vivo

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original article

In Vivo Delivery of Nucleic Acids via Glycopolymer Vehicles Affords Therapeutic Infarct Size Reduction In Vivo Michael Tranter1, Yemin Liu2, Suiwen He1, James Gulick3, Xiaoping Ren1, Jeffrey Robbins3, W. Keith Jones1 and Theresa M. Reineke4 Department of Pharmacology, University of Cincinnati, Cincinnati, Ohio, USA; 2Department of Chemistry, Macromolecules and Interfaces Institute, Virginia Tech, Blacksburg, Virginia, USA; 3Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA; 4 Department of Chemistry, University of Minnesota, Minneapolis, Minnesota, USA 1

Using a new class of nontoxic and degradable glycopolymer-based vehicles termed poly(glycoamidoamine)s, we demonstrate virus-like delivery efficacy of oligodeoxynucleotide (ODN) decoys to cardiomyoblasts (H9c2), primary cardiomyocytes, and the mouse heart. These glycopolymers bind and compact ODN decoys into nanoparticle complexes that are internalized by the cell membrane and mediate nuclear uptake of DNA in 90+% of cultured primary cardiomyocytes and 87% of the mouse myocardium. Experimental results reveal that decoys delivered via these glycopolymers block the activation of the transcription factor NF-κB, a major contributor to ­ischemia/reperfusion injury. Decoy complexes formed with glycopolymer T4 significantly blocked downstream gene expression of Cox-2 and limited myocardial infarction in  vivo, phenocopying a transgenic mouse model. These promising delivery vehicles may facilitate highthroughput genetic approaches in animal models. Additionally, the low toxicity, biodegradation, and outstanding delivery efficacy suggest that these nanomedicines may be clinically applicable for gene regulatory therapy. Received 9 June 2011; accepted 12 November 2011; published online 20 December 2011. doi:10.1038/mt.2011.267

Introduction The intracellular delivery of nucleic acids has unprecedented promise for unraveling the intricate genetic and epigenetic mecha­ nisms associated with human health and disease. Indeed, ubiq­ uitous tools such as RNA interference, oligodeoxynucleotide (ODN) transcription factor decoys, and plasmid DNA are help­ ing researchers understand biological processes, disease path­ ways, and are undergoing extensive research and development as novel therapeutics. However, a fundamental problem facing the successful application of these technologies is the development of vehicles that carry genetic material into cells in a manner that is efficient, nontoxic, and does not interfere with normal cellular functions.1–3

Without a vehicle, polynucleotides do not effectively enter cells and can be rapidly digested. Thus, vehicles derived from viruses4 and natural or synthetic materials1–3,5–7 are being widely researched for this purpose. While genetically modified viruses were first introduced into the clinic in 1993 and offer high efficacy,4 serious adverse effects in humans have been reported.8,9 Nonviral systems, including lipid carriers,7 metal-based nanoparticles,10 and cationic polymers,5 are being investigated. Unfortunately, each of the aforementioned delivery systems has limitations that must be overcome in order to be useful in vivo.11 We have created a novel series of glycopolymer-based ­delivery vehicles, termed poly(glycoamidoamine)s (PGAAs), that have the ability to complex with various polynucleotide types and form nanoparticles (termed polyplexes). These materials are created by polymerizing the methyl ester or lactone derivatives of various carbo­ hydrates (D-glucarate (D), meso-galactarate (G), D-mannarate (M), and L-tartarate (T)) with a series of oligoethyleneamine monomers (containing between 1-4 ethyleneamines).12 A subset composed of these carbohydrates and four ethyleneamines in the polymer repeat units (named D4, G4, M4, and T4; see Supplementary Figure S1) have yielded exceptional pDNA delivery efficiency, reporter gene expression, and low toxicity with a variety of cell types.12–15 The cur­ rent study represents the first report of utilizing these nanovehicles in vivo and reveals their unparalleled, virus-like efficacy for deliv­ ering oligonucleotides to the nucleus of primary cells in vitro and in vivo. Herein, we demonstrate that these glycopolymers show great promise for delivery of ODN decoys against NF-κB, a transcrip­ tion factor essential to many aspects of human health16 and which plays a critical role in the development of cardiovascular disease, the leading cause of disease and death in the United States.17–19 After activation, NF-κB translocates from the cellular cytoplasm to the nucleus, binds to its cognate binding sites in promoter or enhancer regions, and regulates the expression of cytokines, growth fac­ tors, and genes that regulate cardiac hypertrophy, myocardial cell death, and cardio protection.17,20,21 For this reason, NF-κB block­ ade can reduce cell death and the extent of myocardial infarction (MI) after ischemia/reperfusion injury.17,20,22 One way to achieve

Correspondence: Theresa M. Reineke, Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, USA. E-mail: [email protected] or W. Keith Jones, Department of Pharmacology and Cell Biophysics, University of Cincinnati, Cincinnati, OH, 45267, USA. E-mail: [email protected] Molecular Therapy vol. 20 no. 3, 601–608 mar. 2012

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Results Characterization of the glycopolymer–NF-κB decoy polyplexes The glycopolymers (D4, G4, M4, T4; see Figure 1) were screened via several assays to determine the best candidate to deliver ODN decoys to functionally block NF-κB in vivo. The capacity of each material to bind and complex ODN decoys into stable nanoparticle complexes (polyplexes) was examined via transmission electron microscopy (TEM), gel electrophoresis, and dynamic light scat­ tering. The results indicated that all the tested glycopolymers (D4, G4, M4, and T4) formed nanoscale (~100-nm diameter) poly­ plexes with NF-κB ODN decoys (Figure 1a and Supplementary Figure S2). The resistance of the polyplexes to nuclease degradation, criti­ cal for efficient delivery, was examined via gel electrophoresis after exposure of polyplex solutions to RQ1 DNase. Polyplexes formed with T4 were more resistant to DNase digestion than those in the D4, G4, and M4 polyplexes (Figure  1b). Furthermore, stability tests in the presence of high salt concentrations showed that T4 polyplexes were more stable (reduced salt-induced decomplex­ ation of T4 from the ODN) than polyplexes made with another glycopolymer. In contrast, M4 polyplexes did provide the weak­ est DNase protection and decomplexed at lower sodium chloride concentrations (Figure 1c) than those made with D4, G4, or T4. Thus, the results of these physical studies show that the T4 glyco­ polymer offers the most stable polyplexes.

Glycopolymer–NF-κB decoy efficacy and toxicity in H9c2 cells To assess the in vitro efficacy of the polyplexes, H9c2 cells were transfected with increasing doses of the glycopolymer–ODN poly­ plexes. Lipofectamine 2000–ODN and JetPEI–ODN complexes were used as positive controls in this study and cells only and naked DNA were used as negative controls. Twenty-four hours after trans­ fection, the cells were treated with TNF-α to stimulate the activation of NF-κB. The efficacy of polyplex-mediated NF-κB blockade was assessed via electrophoretic mobility shift assay (EMSA). Relative to naked decoy, the glycopolymer–ODN significantly decreased the half maximal inhibitory concentration (IC50) for inhibition of NF-κB DNA binding in a dose-dependent manner (Figure 2a–c). The T4 polymer yielded the highest efficacy, decreasing the IC50 602

from >15 μg (naked decoy) to 2.8 μg (Figure 2b,c). Importantly, we did not see inhibition or activation of NF-κB DNA binding either with polymer alone (Figure 2b) or with glycopolymer-scrambled decoy control (Supplementary Figure S3). Thus, the NF-κB block­ ade appears to be ODN sequence-specific and the polymers them­ selves do not elicit an innate inflammatory response at the cellular level (they do not activate NF-κB). The relative efficacy of delivery in this experiment was (in order of increasing IC50) Lipofectamine 2000 > T4 > JetPEI > D4, G4, M4 >> naked decoy (Figure 2c). The performance of the T4 polymer was only slightly lower than that of Lipofectamine 2000, but it was much more effective than JetPEI and all other glycopolymers. Microscopy experiments were also performed to assess the efficiency of decoy delivery to the nucleus of H9c2 cells. After 22 hours, 80% of H9c2 nuclei were positive for Fluorescein isothiocyanate (FITC)-labeled decoys when bound to polymer T4 as opposed to naked decoy, where no significant nuclear entry was found (Figure 2d). MTT assays were employed with each glycopolymer and control to assess toxicity. As shown in Figure 2e, the glycopolymers elicited significantly lower toxicity (higher LD50) in H9c2 cells than either Lipofectamine 2000 or JetPEI, and T4 was by far the most benign delivery agent in this group. In contrast, the

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selective NF-κB inhibition is the application of short (~20 base pairs) exogenous ODN decoys that contain the consensus NF-κB binding sequence; this inhibits NF-κB-dependent genomic effects by competing with the endogenous nuclear NF-κB binding sites.23 However, naked ODN decoys are not efficacious due to their lack of cell and tissue penetration and nuclease degradation during biological transport. Consequently, we demonstrate the ability of our glycopolymers to achieve therapeutic potency of ODN decoys directed against NF-κB into primary cardiomyocytes and the in vivo murine heart, thereby attaining therapeutic benefit in animals. Because ODN decoys can be engineered to titrate most transcription factors with a high degree of specificity, this effective delivery technology has extremely broad significance for in vitro genetic approaches and possibly transgenesis in a variety of spe­ cies in vivo.

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Figure 1 Characterization of glycopolymer–ODN NF-κB decoy polyplexes. (a) The sizes of PGAA/decoy polyplexes examined by transmission electron microscopy (TEM). (b) The ability of glycopolymers to protect decoys from DNase digestion assessed by gel electrophoresis (band disappearance indicates DNA degradation). (c) Polyplex stability, as a function of increasing NaCl concentration, represented by the fraction of PicoGreen excluded from the polyplex structure (more dye intercalates into DNA upon polyplex dissociation). ODN, oligodeoxynucleotide; PGAA, poly(glycoamidoamine).

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Figure 2  Glycopolymer–ODN efficacy and toxicity in H9c2 cells. Cells were treated with increasing doses of either (a) decoy only or (b) T4–decoy polyplexes 24 hours prior to TNF-α treatment to activate NF-κB. Activation of NF-κB was assessed by EMSA. (c) IC50 doses for NF-κB decoys alone (naked decoy) and polyplexes formed with T4, M4, G4, and D4 polymers, Lipofectamine 2000, and JetPEI were calculated from sigmoidal doseresponse curves fitted to EMSA results. (d) Nuclear localization of fluorescently labeled decoys in H9c2 cells were transfected alone or via T4 and the fraction of fluorescently positive nuclei were quantified at the indicated times. (e) MTT assay was used to assess cytotoxicity of delivery vectors. Error bars represent 95% confidence intervals. *P ≤ 0.05 versus all other delivery vectors (c–e). H9c2 cells were transfected with CMV-Luciferase reporter pDNA using either T4 or Lipofectamine 2000 and (f) the fraction of cell survival as well as (g) the activity of the luciferase reporter (relative to the initially measured activity 24 hours after treatment) were tracked for ten days following transfection. *P ≤ 0.05 versus baseline control. #P ≤ 0.05 versus Lipofectamine transfected group. EMSA, electrophoretic mobility shift assay; ODN, oligodeoxynucleotide.

commonly used delivery vehicles Lipofectamine 2000 and JetPEI are associated with extreme cytotoxicity. Juxtaposing these results with the aforementioned data, polymer vector T4 simultaneously shows the highest polyplex stability, efficacy of delivery, and the lowest toxicity. Comparison of toxicity over a ten-day time course follow­ ing transfection using either T4 or Lipofectamine 2000 shows that the toxicity elicited by Lipofectamine 2000 is manifest within the first 24 hours and persists throughout, while no significant toxicity from T4 is detectable at any time point (Figure 2f). As expected, the activity of a T4 and Lipofectamine-delivered cytomegalovirus (CMV)-Luciferase reporter is equally maintained for at least 10 days indicating that the stability of the DNA cargo is independent of the T4 delivery vector (Figure 2g). Efficient nuclear localization as observed with T4 transfection is important for the stability and Molecular Therapy vol. 20 no. 3 mar. 2012

effective duration of the delivered DNA, an important consider­ ation for potential clinical application. However, as our data indi­ cate (Figure 2g), the half-life of the delivered DNA is independent of the delivery vector once the DNA has been released. In fact, ­double-labeling experiments (Supplementary Figure S4) show that the DNA and T4 polymer dissociate very quickly after entry into the cell (within hours; Figure 2d) and the DNA becomes ­localized to the nucleus, while the polymer remains in the cytoplasm.

Glycopolymer–NF-κB decoy delivery and toxicity in neonatal rat ventricular myocytes Next, the ability of the glycopolymers to effectively deliver FITClabeled NF-κB decoys (Figure  3a) to primary neonatal rat ven­ tricular cardiomyocytes (NRVMs), which are notoriously difficult 603

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Delivered NF-κB Oligo Reduces Infarct Size In Vivo

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a delivery vector, FITC-labeled NF-κB decoys were not ­detectable in the cardiomyocytes (Figure 3 and Supplementary Figure S5); however, nearly all primary cardiomyocytes were FITC-decoy positive when transfected with the glycopolymer polyplexes. In all experiments using the glycopolymers, the ODN decoys were found in the nuclei of the primary cells, indicating that the glyco­ polymers facilitate nuclear localization of the decoys. It should be noted that a control experiment was also conducted by forming polyplexes with a scrambled ODN decoy sequence and the gly­ copolymer T4. In this experiment, enhanced nuclear localization of the scrambled decoys was also found, which corroborates our hypothesis that the glycopolymer is responsible for this unusual effect (Supplementary Figure S5). Extrapolating from these and the H9c2 studies, this nuclear delivery might play a role in the high efficacy of the glycopolymer–ODN polyplexes against NF-κB activation. Overall, the results suggest that the glycopolymers promote efficient nuclear delivery of NF-κB decoys in primary NRVMs and that the decoys efficiently block NF-κB activation. To examine the cytotoxicity of the glycopolymers in primary NRVMs, the release of adenylate kinase, a cell death biomarker,24 was monitored (Figure 3c). In this assay, high levels of adenylate kinase release were noted for G4, M4, and JetPEI (JetPEI > G4 > M4), while T4 and Lipofectamine 2000 showed only mild, com­ parable toxicity, evident only at the longer incubation times (6 and 8 hours). Though D4 showed negligible cytotoxicity throughout the experiment, it yielded low delivery efficiency, similar to that of JetPEI and Lipofectamine 2000 in primary cardiomyocytes. Thus, the polymer vector T4 shows the highest efficacy of delivery and the lowest toxicity in NRVMs and thus was selected for use in vivo.

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Figure 3  Glycopolymer–ODN polyplex delivery and toxicity in neonatal rat ventricular myocytes. (a) Uptake of fluorescently (FITC) labeled decoy was tracked by flow cytometry. (b) Confocal microscopy of NRVMs 24 hour after transfection with FITC-labeled decoy (green). A Troponin I-specific antibody (red) was used to identify cardiomyocytes, and all cells were labeled with a nucleus-specific stain (TO-PRO-3; blue). (c) Adenylate kinase release into the culture media was assayed as a cell death marker 2, 4, 6, and 8 hours after transfection. NRVMs, neonatal rat ventricular myocytes; ODN, oligodeoxynucleotide.

to transfect, was tested. The glycopolymers promoted high cel­ lular uptake of the decoys that was either comparable with (as in the case of D4) or higher than (as demonstrated by G4, M4, and T4) Lipofectamine 2000 or JetPEI. Glycopolymer T4 was found to mediate the highest cellular uptake of NF-κB decoys in NRVMs (Figure  3a). It is important to note that the efficiency of T4 in delivering decoys to the NRVMs was far superior to the delivery efficiency of the positive controls. This result is in con­ trast with what was observed in the H9c2 cell line, where the effi­ ciency of T4–decoy delivery was comparable with either JetPEI or Lipofectamine 2000 (but these controls were far more toxic). Confocal immunofluorescence microscopy was used to examine the cellular distribution of NF-κB decoys 24 hours after delivery (Figure 3b) in the primary cells. When transfected in the absence of 604

In vivo delivery and efficacy of T4–decoy polyplexes The potential therapeutic applicability of the glycopolymer poly­ plexes was assessed using an established murine model of isch­ emia/reperfusion injury. Polyplexes were delivered to the heart by injection into the pericardial sac in a minimally invasive survival surgery. Tissue distribution and signal intensity of Alexa488labeled NF-κB decoys were examined 24 hours after pericardial delivery. Representative tissue sections show that, compared with the naked decoys at the same dosage, the T4–decoy polyplexes achieved a more homogeneous distribution, a significantly deeper penetration into the myocardium, and gave a higher fluorescent signal relative to controls (Figure 4a,b). The T4–decoy polyplexes were able to transfect 87% of the myocardium, whereas the naked decoy transfected only 40% (Figure 4d). This was accompanied by a 350% increase (P ≤ 0.05) in the intensity of the fluorescent signal compared with naked decoy (Figure 4c). Examination of the liver at the same time point showed a much lower fluorescent signal in the T4–decoy polyplex group relative to naked decoy (Supplementary Figure S6). We postulate that the lower liver distribution of ODN delivered by T4 may reflect more efficient myocardial uptake of the T4–decoy polyplexes, compared with naked decoys; thus a higher percentage of naked decoys entered the systemic circulation. As NF-κB is a transcription factor, its activity must reflect the activation and/or repression of downstream genes. Therefore, we assessed the effect of T4–ODN polyplexes upon NF-κB-dependent www.moleculartherapy.org vol. 20 no. 3 mar. 2012

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Figure 4  Pericardial delivery of glycopolymer–ODN polyplexes in vivo efficiently silences NF-κB-dependent gene expression and reduces infarct size after I/R injury. Cardiac sections from mice 24 hours after pericardial injection of (a) Alexa488-labeled NF-κB decoy alone or (b) with T4 polymer. Quantitation of Alexa488 uptake to the myocardium by (c) mean pixel intensity and (d) percentage of fluorescent signal positive myocardium (*P ≤ 0.01). (e) Blockade of Cox-2 mRNA expression via pericardial delivery of NF-κB decoy/T4 polyplexes 24 hours prior to intraperitoneal injection of cytokine mixture. Cox-2 mRNA levels were assessed after 3 hours by quantitative real-time polymerase chain reaction. *P ≤ 0.001 versus 0 μg NF-κB decoy/T4. (f) Assessment of the impact on infarct size of 10 μg ODN/T4 delivered via pericardial injection 24 hours prior to a 45-minute coronary occlusion. Results obtained from NF-κB dominant-negative (DN) mice shown for comparison. *P ≤ 0.01 vs. sham-injected C57 wild-type mice. All error bars represent SEM. I/R, ischemia/reperfusion; ODN, oligodeoxynucleotide.

levels of Cox-2 mRNA after cytokine administration. Cox-2 is a well-known NF-κB-dependent gene in the heart and a com­ mon drug target.25 We found that Cox-2 mRNA expression was induced 16-fold following cytokine injection22 and was reduced in a dose-dependent manner with pericardial delivery of T4–NF-κB decoy, with reduction being nearly complete at 10 μg (Figure 4e). This result demonstrates the functional repression of NF-κB activ­ ity by glycopolymer–ODN delivery and indicates cellular entry of the polyplexes in vivo. It has been well established that inhibition of NF-κB protects against ischemia/reperfusion-induced myocardial ­infarction.22  To evaluate the functional efficacy of T4–ODN delivery, we mea­ sured infarct size (normalized to risk region) in mice after 45  minutes of coronary occlusion and 24 hours reperfusion, as previously described (Figure 4f).26 NF-κB decoy or T4 delivered alone, 24 hours prior to ischemia/reperfusion, had no significant Molecular Therapy vol. 20 no. 3 mar. 2012

effect upon infarct size relative to vehicle controls. An equivalent amount of T4–NF-κB decoy polyplexes, but not T4-scrambled (nontargeting) decoy polyplexes, reduced infarct size by more than 40% (P ≤ 0.05). This reduction is comparable to results with pharmacologic27 and genetic22,28 blockade of NF-κB in murine ischemia/reperfusion models (Figure  4f, second bar). Together with the observed reduction of NF-κB-dependent gene expres­ sion, this result demonstrates the ability of NF-κB decoy/T4 poly­ plexes to achieve a functional blockade of NF-κB activity with a therapeutic effect against myocardial infarction in vivo.

Discussion Due to the relevance of NF-κB to human health, and the known involvement of this transcription factor in cardiovascular disease, anti-NF-κB ODN decoys were chosen as the payload for our gly­ copolymer vehicles.17,20,22 We demonstrated that our glycopolymers 605

Delivered NF-κB Oligo Reduces Infarct Size In Vivo

can bind and form stable nanoparticle complexes (polyplexes) with ODN decoys (Figure 1) and effectively knock down NF-κB signaling in cardiomyocytes (Figure 2). We have further demon­ strated that the glycopolymers exhibit an unparalleled combina­ tion of delivery efficacy and low toxicity in cultured cells, primary cardiomyocytes, and in the murine heart in vivo. Remarkably, the polyplexes are internalized by the cell membrane and deliver ODNs with high efficiency to the nucleus of 90+% of cultured primary cardiomyocytes with low toxicity (Figures 2 and 3 and Supplementary Figures S4 and S5). This high nuclear localization was also independent of ODN sequence as scrambled FITC-ODNs were found in the nucleus of primary NRVMs when delivered with T4 (Supplementary Figure S5). Notably, there are noncardiomyo­ cytes that also demonstrate nuclear transfection (Figure 3b). This is expected as isolated cardiomyocyte preparations are never com­ pletely pure, and these cells likely are fibroblasts that are known contaminants. Efficient nuclear localization is important for enhancing func­ tion, such as decoy-mediated transcriptional inhibition or plas­ mid-mediated gene expression, as well as prolonging the half-life of transfected DNA.29 Double-labeling studies (Supplementary Figure S4) showed that at 12 hours, the polymer is localized to the cytoplasm while the DNA is primarily localized to the nucleus. While the exact mechanisms of nuclear accumulation of trans­ fected ODN is currently unknown, it is now understood that the mechanisms of cellular entry play a role in nuclear localization and not all delivery vectors facilitate cellular internalization via similar mechanisms. This suggests that the delivery vectors play a cen­ tral role in determining nuclear accumulation of the transfected nucleic acid.29 Recent work by the Reineke lab indicates that cellular uptake of T4/ODN polyplexes and subsequent nuclear trafficking of the ODN is primarily facilitated by caveolae/lipid raft-mediated endocytosis and is dependent on actin and dynamin active trans­ port mechanisms.30,31 Interestingly, while clathrin is necessary for cellular internalization, it appears inhibitory to subsequent nuclear trafficking through means that remain to be elucidated.31 The current set of glycopolymers have not been shown to be as efficient at the functional delivery of siRNA, perhaps due to the need for siRNA to be localized in the cytoplasm in order to com­ plex with protein components of the RNA-induced silencing com­ plex (RISC) and mediate mRNA degradation; work is currently underway on examining different polymer formulations for this purpose. It is, however, important to note that the currently used T4 has shown high efficiency for the delivery of both small linear decoys and large plasmids.13–15 Thus, the poly(glycoamidoamine) s in general and T4 specifically are useful for delivery of several different types of DNA therapeutics, including decoys, antisense oligonucleotides, and plasmids encoding specific genes. The delivery of nucleic acids to in vitro cell culture systems is readily attainable through many available methods. However, it has been historically proven that it is much more difficult to suc­ cessfully transfect in vivo tissues without significantly altering the physiological homeostasis of the living system. In this work, we show that glycopolymer T4 mediated the delivery of NF-κB decoys to 87% of the murine heart in vivo and resulted in deeper penetra­ tion, more homogenous distribution, and higher signal intensity of fluorescently labeled ODN decoys in the myocardium as compared 606

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with delivery of naked FITC-ODNs (Figure 4a–d). T4–ODN poly­ plexes successfully inhibited NF-κB activation in vivo as evidenced by the dose-dependent decrease in expression of Cox-2 (Figure 4e) and the reduction of ischemia/reperfusion injury (Figure  4f). Importantly, these results show that T4–ODN polyplexes are able to functionally inhibit the transcriptional activation of NF-κB and provide a therapeutic benefit against myocardial infarction in vivo (Figure 4f). After pericardial injection, the polyplexes were local­ ized primarily to the heart and the presence of FITC-ODNs was low in the liver, relative to mice receiving naked ODN (Supplementary Figure S6). These data support that the direct injection of T4–ODN polyplexes into the pericardium allows for quick and efficient uptake into the heart. Although longer term toxicological studies using gly­ copolymer–ODN polyplexes in vivo, and assessing localization to additional tissues, remain to be completed, our results show little or no cytotoxic effects at the cellular level (Figure 2e–f) and the mice survived to the 48-hour endpoint (24 hours after delivery, 24 hours after ischemia/reperfusion), with no signs of ill effects. Also, the administration of glycopolymer alone fails to induce NF-κB activa­ tion (Figure 2b), a traditional hallmark of inflammatory cytokine signaling. These results suggest that the glycopolymer–ODN poly­ plexes do not have adverse inflammatory or innate immune reactiv­ ity often observed with viral delivery vectors.32 In conclusion, the nonviral glycopolymer vehicles offer a prom­ ising technology for delivery of numerous polynucleotide types including transcription factor decoys, transgenes, antisense, and other ODNs. The inhibition of NF-κB in myocardial ischemia/­ reperfusion injury is merely one example of how transcription fac­ tor decoys could be utilized to improve clinical outcome. Though acting at a genetic level, glycopolymer-mediated nucleic acid thera­ peutics are completely reversible, as both the glycopolymers and their nucleic acid payloads naturally biodegrade.33 These polyplexes hold several advantages over adenoviruses (the current gold stan­ dard that causes toxicity and experimental artifacts): (i) the effects are reversible, titratable, and can be modulated, much like tradi­ tional drugs, (ii) therapeutic nucleic acids are not limited to those that can be expressed by a viral genome, and (iii) they do not pose a risk of genetic modification due to insertion and inflammatory sig­ naling.32,34 This technology could facilitate high-throughput func­ tional genetics in mice and has the potential to make translational use of gain- and loss-of-function studies in large animals feasible and rapid. We have demonstrated the power of these glycopolymer vehicles paired with nucleic acid decoy therapeutics in a murine heart disease model and this technology should be applicable to a wide range of research and therapeutic applications.

Materials and Methods All animal experimentation was approved by the Institutional Animal Care and Use Committee at the University of Cincinnati. NF-κB decoys and glycopolymers. Double-stranded NF-κB decoy ODNs (5′-CCTTGAAGGGATTTCCCTCC-3′) and scrambled decoy (5′-TTGCCTGCACTATTCGAGCC-3′) were purchased from Integrated DNA Technologies (Coralville, IA). Glycopolymers D4, G4, M4, and T4 (Supplementary Figure S1) were synthesized as previously described.12,13 JetPEI (linear polyethyleneimine, Avanti Polar Lipids, Alabaster, AL) and Lipofectamine 2000 (Invitrogen, Carlsbad, CA) were used as control vectors at the optimized dosages suggested by the manufacturers (JetPEI: N/P = 5

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© The American Society of Gene & Cell Therapy

where N/P denotes the ratio of secondary amines on the polymer (N) to DNA phosphate groups (P) used to formulate the polyplexes; Lipofectamine 2000: Lipofectamine 2000 (µl) to DNA (µg) ratio of 2.5). Polyplex analysis. Polyplex sizes were measured via dynamic light scatter­ ing at 633 nm on a Zetasizer (Nano ZS) dynamic light scattering instrument (Malvern Instruments, Malvern, UK). Oligonucleotide decoy (0.02 μg/µl in 150 μl H2O) was incubated with polymers D4, G4, M4, or T4 at N/P = 30 for 1 hour to allow for polyplex formation. The intensity-averaged particle size and size distribution of each sample was reported as an average of 12 measurements. Transmission electron microscopy analysis on the polyplex samples was performed according to a previously published method.12 Assessment of DNase protection was completed according to a slightly modified published method34 outlined in the Supplementary Materials and Methods. PicoGreen exclusion assay of polyplex stability. Glycopolymer/decoy

polyplexes (N/P = 30) were prepared in water at a concentration of 0.3 μg  DNA/100 μl. PicoGreen (Molecular Probes, Eugene, OR) solution was prepared in 10 mmol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfo­ nic acid (HEPES) (Sigma, St. Louis, MO). Equal volumes of polyplex and PicoGreen solutions were added to each well of a flat-bottom 96-well plate. The fluorescence of PicoGreen (excitation 485 nm, emission 535 nm) for each sample was measured using a TECAN US plate reader (Research Triangle Park, Durham, NC). Fractional dye exclusion was determined by the following relationship: Dye exclusion = 1 − (Fsample − Fblank) / (FDNAonly − Fblank). H9c2 culture and transfection. H9c2 cells (clone 2-1) were obtained from

the American Type Culture Collection (ATCC) and were cultured accord­ ing to ATCC specifications in Dulbecco’s modified Eagle medium (DMEM) (supplemented with 10% fetal bovine serum, 100 units/mg penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin) in 5% CO2 at 37 °C. H9c2 cells were seeded at 7.5 × 105 cells/100 mm dish and incubated for 24 hours prior to transfection. D4, G4, M4, and T4 (N/P = 30/1), JetPEI (N/P = 5/1), or Lipofectamine 2000 (Lipofectamine 2000 (µl) to DNA (µg) ratio of 2.5) were incubated with oligonucleotide decoys at room temperature for 1 hour prior to transfection. Cells were transfected in 5 ml serum-free media (Opti-MEM, pH 7.2). Supplemented DMEM (7 ml) was added 4 hours after transfection. Fraction of positive nuclei were quantified by visual counting and averaging of fluorescent positive nuclei from nine independent and randomly selected fields of view containing ~30–50 cells each. For analysis of NF-κB activation, 24 hours after initial transfection, cells were treated with TNF-α (25 ng/ml, Peprotech, Rocky Hill, NJ) in 3 ml of Opti-MEM for 30 minutes to stimulate NF-κB activation. Nuclear protein extraction and electrophoretic mobility shift assays (EMSA) were performed according to a similar method as previously described22 and detailed in the Supplementary Materials and Methods. Luciferase assays were done in a 96-well format at 3 × 103 cells/well with 30 ng/well CMVLuc reporter/well (pGL4.10; Promega, Madison, WI) using a Luciferase Assay System kit according to manufacturer’s instructions (Promega). Cell viability was measured via MTT/MTS assays according to a conventional procedure detailed in the Supplementary Materials and Methods. NRVMs. Primary cultures of rat neonatal cardiac myocytes were prepared from 1-day-old Wistar rats (Charles River Laboratories, Wilmington, MA). Briefly, cardiac myocytes were dispersed from the ventricles by diges­ tion with collagenase type IV (Sigma), 0.1% trypsin (Life Technologies, Carlsbad, CA), and 15 μg/ml DNase I (Sigma). Cells were applied on a discontinuous Percoll gradient (1.060/1.086 g/ml) prepared in Ads buf­ fer (116 mmol/l NaCl, 20 mmol/l HEPES, 1 mmol/l NaH2PO4, 5.5 mmol/l glucose, 5.4 mmol/l KCl, 0.8 mmol/l MgSO4, pH 7.35) and centrifuged at 250 g for 3 minutes. Cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 units/mg penicillin, 100 μg/ml streptomycin, and Molecular Therapy vol. 20 no. 3 mar. 2012

Delivered NF-κB Oligo Reduces Infarct Size In Vivo

0.25 μg/ml amphotericin. Cell cultures were obtained in which more than 95% of the cells were myocytes as assessed by immunofluorescence stain­ ing with a monoclonal antibody against sarcomeric myosin. Cellular tox­ icity of the decoy delivery vectors on NRVMs was analyzed by measuring the release of adenylate kinase from damaged cells into the culture media by using the ToxiLight assay (Cambrex, East Rutherford, NJ), according to manufacturer’s specifications. Confocal immunofluorescence analyses. NRVMs were grown in two-well chambered glass slides (100,000 cells/chamber) coated with gelatin (Nalge Nunc International, Naperville, IL) for 3 days. The cells were then trans­ fected with 1 μg of 5′-FITC-labeled decoy complexed with D4, G4, M4, T4 (N/P = 30/1), JetPEI, or Lipofectamine 2000 in serum-free medium. Supplemental DMEM was added to the cultures 4 hours after transfec­ tion. Twenty-four hours after transfection, the cells were fixed using 4% paraformaldehyde and then treated with 0.1% Triton X-100 for 10 min­ utes. Blocking was performed for 30 minutes with a solution containing 1% bovine serum albumin, 0.1% cold fish skin gelatin, 0.1% Tween 20, 0.05% sodium azide, and 0.01 mol/l phosphate buffered saline at pH 7.2. The specimens were then incubated with an Anti-Troponin I antibody (1 μg/ml in phosphate buffered saline, Chemicon, Temecula, CA) for 1 hour and subsequently with an AlexaFluor568-conjugated secondary anti­ body (20 μg/ml in phosphate buffered saline, Molecular Probes, Carlsbad, CA) and TO-PRO-3 nuclear stain (1 μmol/l in phosphate buffered saline, Molecular Probes) for 30 minutes before being examined with confocal microscopy (objective ×40). Flow cytometry. H9c2 cells and primary cardiomyocytes were cultured and transfected as described previously. The cellular uptake of Cy5-labeled decoy in H9c2 cells and primary cardiomyocytes were recorded 4 hours after initial transfection on a FACSCanto II (Becton Dickinson, San Jose, CA) equipped with a helium-neon laser to excite Cy5 (633 nm). Ten thousand events were collected in duplicate for each sample. The positive fluorescence level was established by visual inspection of the histogram of negative control cells such that less than 1% appeared in the positive region. Pericardial delivery of NF-κB decoys. Double-stranded NF-κB decoy was polyplexed with polymer T4 at N/P = 30 in 50 μl total volume for 30 minutes at room temperature. After anaesthetization with pentobarbi­ tal (90 mg/kg intraperitoneally), the mice were maintained on oxygen via intubation. Hearts were accessed via a lateral thoracotomy, and injections were made directly into the pericardial sac using a microsyringe (Hamilton Co, Reno, NV). For fluorescent imaging of decoy localization, mice were sacrificed 24 hours after pericardial injection and hearts and livers were removed, rinsed in cold phosphate buffered saline, fixed overnight in 4% para­formaldehyde, and sectioned on ice at a thickness of 20 µm using a microtome (Leica Microsystems, Wetzlar, Germany). The hearts were imaged using a Zeiss Axioplan Imaging 2 microscope and a Hamamatsu ER CCD-camera. Signal intensity for control hearts (no Alexafluor488) was set to zero (to average out background fluorescence) and any signal above zero was treated as positive for transfection for purposes of estimating (using  Image  J) the area of the ventricle positive for fluorescence (Figure 4d). Secondly, an unbiased measure of pixel density was used in Image J to calculate the fluorescent signal intensity of the entire ventricular sections (minus chambers; Figure 4c). Coronary occlusion. Mice were anesthetized with sodium pentobarbital

(90 mg/kg intraperitoneally) and maintained on oxygen via intubation. The heart was accessed via a lateral thoracotomy. Coronary occlusion was established by placing an 8-0 nylon suture around a piece of soft silicon tub­ ing placed on the left anterior descending (LAD) coronary artery 2–4 mm from the tip of the auricle. Ischemia was achieved by tying the suture for

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Delivered NF-κB Oligo Reduces Infarct Size In Vivo

45 minutes, and reperfusion was established by untying the suture allowing the blood flow to be reestablished.26 To evaluate tissue death (infarct size), hearts were perfused through the aortic root with 1% triphenyltetrazolium chloride (TTC), and phthalo blue dye used, after retying the suture (left in place) to delineate the nonrisk region. Hearts were razor-sectioned after being frozen at −20 °C for several hours, photographs and measurements were taken as described by Ren et al.,26 according to the method of Fishbein et al.35 Results are reported as mean infarct size (as percent of risk region) ± SEM (statistical details are found in the Supplementary Materials and Methods). Analysis of Cox-2 expression. RNA was isolated from the left ventricle 3 hours

following intraperitoneal injection of cytomix (0.1 μg/g TNF-α, 0.001 μg/g IL-1β, and 0.2 μg/g IFN-γ) using a Qiagen RNeasy Mini kit. 1.0 μg total RNA was used to synthesize cDNA and quantitative real-time polymerase chain reaction was done using 100 ng total cDNA (SYBR Green; Applied Biosystems, Carlsbad, CA). Cycling parameters were 90 °C for 10 minutes followed by 40 cycles of 90 °C for 15 seconds and 60 °C for 60 seconds (with data collection at the end of the 60 °C step at each cycle). Expression values were calculated using the delta-delta Ct method36 with normalization to 18S RNA. Primer sequences were 18S sense: 5′-AGTCCCTGCCCTTTGTA CACA-3′; 18S antisense: 5′-5′-CCGAGGGCCTCACTAAACC-3′; Cox-2 sense: 5′-CAACACCTGAGCGGTTACCAC-3′; Cox-2 antisense: 5′-CAG AGGCAATGCGGTTCTG-3′.

SUPPLEMENTARY MATERIAL Figure  S1.  Chemical structures of PGAA glycopolymers. Figure  S2.  Size distributions of PGAA/NF-κB decoy nanoparticles. Figure  S3.  Glycopolymer G4 delivery of scrambled decoy has no effect. Figure  S4.  Dual label localization of decoys and T4. Figure  S5.  Confocal microscopy of PGAA/decoy in primary NVRMs. Figure  S6.  Representative liver sections following in vivo delivery of decoy. Materials and Methods

ACKNOWLEDGMENTS This work was supported by NIH grants HL63034 and HL091478 (W.K.J.), R21EB007244 (T.M.R.), and P01HL69779 (J.R.). T.M.R. also thanks the Beckman Young Investigator Award and Alfred P. Sloan Fellowship programs for support of this project. M. Tranter was supported by an Integrative Graduate Education and Research traineeship from the National Science Foundation. We thank the University of Cincinnati College of Medicine Dean’s Discovery Fund and the University of Cincinnati Institute for Nanoscale Science and Technology for early support of this work. We thank Jackie Belew for support in maintaining mouse breeding colonies and Katye Fichter for performing the MTT assay. University of Cincinnati and W.K.J. have interest in patents based upon oligodeoxynucleotide delivery, these patents have not been licensed and W.K.J. receives no royalties. T.M.R. is a consultant to Techulon, Inc. Techulon has recently licensed the poly(glycoamidoamines) polymers (University of Cincinnati) and is currently marketing one formulation as Glycofect Transfection Reagent. The other authors declared no conflict of interest.

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