Internal Oligoguanidinium-Based Cellular Transporter Enhances

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Internal Oligoguanidinium-based Cellular Transporter Enhances Antisense Efficacy of Morpholinos in in vitro and Zebrafish model Jhuma Bhadra, Sankha Pattanayak, Pragya Paramita Khan, Jayanta Kundu, and Surajit Sinha Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00252 • Publication Date (Web): 14 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

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Bioconjugate Chemistry

Graphical Abstract:

ACS Paragon Plus Environment


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†

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, Jayanta Kundu†

,¶

moiety has been identified as the responsible molecular feature for cellular delivery, the other features such as backbone rigidity, static presentation of guanidinium 12 13 groups and amphiphilic characters of delivery moiety are also important to increase cellular uptake properties.

INTRODUCTION Phosphorodiamidate morpholino oligonucleotides 1,2 (PMOs, Figure 1a) are chemically modified DNA analogues which are routinely used for gene silencing following “steric blocking” mechanisim at ribosome binding 3-5 site. Their enzymatic stability, backbone neutrality, water solubility, low toxicity and predictive antisense properties make them promising therapeutics for cancer and other 5 genetic diseases. However poor cell penetration property limits their therapeutic applications and perhaps the reason there is no FDA-approved PMO-based drug till date. Due to the neutral backbone of PMO, complexation with cationic lipids or transfection reagents is not the choice of PMO delivery. For in vivo delivery, PPMO or Vivo-PMO (Figure 1b) were developed by conjugating PMO either with 6 arginine-rich cell penetrating peptides (CPPs), or terminal 3 guanidinium functionalized dendritic cellular transporters. Though such modified PMOs can penetrate a wide variety of cell types, the correlation between transfection efficiency and cytotoxicity of such transporters has been unsatisfactory because of too many flexibly attached guanidinium groups or CPPs prone to in vivo hydrolysis by peptidases. Recent application of vivo-morpholinos in mice model showed that it causes clotting of blood due to increased cationic charges associated with the dendritic 7 scaffold. Hence, a less toxic or ideally non-toxic efficient cellular transporter is highly desirable that can transport PMO for clinical applications which has become a challenge 8 for PMO delivery. Since the discovery of CPPs, originally conceptualized from HIV-Tat, several types of peptide and nonpeptide-based per-guanidinylated scaffolds have been 9-11 developed for cellular delivery. Though guanidinium

Figure 1. Chemical structures of a) PMO b) PPMO and VivoPMO; c) IGT-PMO.

As a part of our research efforts to develop cellpenetrating PMO, we intended to device a new class of delivery agent that will have intrinsic rigid structures with static orientation of the guanidiniums, while posing enough hydrophobic characters. We presumed that such kind of features could effectively be obtained by an internally substituted guanidinium-linked oligomer that can broadly 14 resemble rigid polyproline-like structures. To the best of our knowledge, in the literature, with the exception of the 15 Girlat’s bicyclic guanidinium oligomers and our guanidinium-linked morpholino oligomers (GMOs) (which may have its own advantage in at-a-stretch GMO-PMO, 16 chimera synthesis), no cell-penetration data is available for non-peptidic and disubstituted internal guanidinium transporters. Here we report a scalable approach to develop structurally simple, rigid non-peptidic internal oligoguanidinium transporters (IGTs) and demonstrate the applicability to deliver antisense PMOs as IGT-PMO (Figure 1c) conjugates for Gli1 silencing in sonic hedgehog cell lines (Shh-Light 2) and the temporal regulation of no tail (Ntl) gene in zebrafish (Danio rerio) embryos. Comparative studies of genes silencing and toxicities with commercially available Vivo-PMO have also been evaluated.


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RESULTS AND DISCUSSION Devised oligo-guanidines are of two types: type 1 – guanidinium linked 4-aminopiperidines with piperazine (7); type 2 – guanidinium linked 2-aminomethyl morpholines (9). Type 1 represents the most rigid structure, as one of the “nitrogens” of the guanidinium group is directly attached to the piperidine ring whereas in type 2 the nitrogen is attached with the conformationally flexible aminomethyl group of morpholine ring. These alicyclic amines are chosen because they are amphipathic in nature, known to improve 17 pharmacokinetics of many drugs as a derivatives by increasing the stability against Cytochrome P450 and can adopt stable chair like conformation for rigidity. IGTs were synthesized (Scheme 1) using Fmoc-protected active 18 monomer 2 with the modification of Bruice’s protocol. Our initial attempt using Bz (benzoyl) protected active monomer failed as the deprotection of Bz was not successful in the final step (Schemes S1, S2). IGT 9 (Figure 2) was synthesized using thioactivated Tr-protected-morpholino methylamine monomer (Scheme S3).

of sodium laurate (Figure S2). Transfection efficieny of 5a having one guanidinium group less was reduced, however, 16 5a was better than our previously reported TTTT-GMO (Figure S3). FACS analysis of cellular uptake of 7b in a wide variety of cell lines MCF7, A549, PC3 and Shh Light 2 indicated that it was dependent on time and cell line (Figure S4). Cellular internalization of 7b was observed in a wide variety of fixed cell lines PC-3, A549, MCF7 including CHO, and also in suspension live monocyte cells (Figure S5). The cellular uptake of 7b in comparision to other devised IGT implied that both number and spatial orientation of guanidinium groups have role in cellular transfection. The role of “Boc” group in 7 in transfection was verified as the Boc-deprotected product showed very poor cellular transfection in FACS analysis at the same 500 nM conc (Figure 3c). The transfection of 7b was energy dependent 19 and followed endocytosis pathway as the uptake was significantly reduced at 4 °C (Figure 3d) and confocal microscopy images showed reduced fluorescence intensity in the presence of endocytosis inhibitors sucrose and chloropromazine (Figure S6). MTT assay of 7a in CHO-K1 cells showed 90% viability at 100 µM concentration (Figure S7).

Figure 3. Cellular uptake measurement of IGTs using FACS studies in CHO-K1 cells in 10% serum-containing medium with varying concentrations of IGTs after 4 h incubation (a) IGT 7b at 37oC; (b) IGT 9b at 37oC; (c) Boc-deprotected 7b at 37oC; (d) 7b at 4oC. Scheme 1. Synthesis of internal oligoguanidinium molecular transporter type 1

Figure 2. Type 2: Morpholine ring oligoguanidinium molecular transporter.

containing

internal

Cellular transfection efficiency of devised IGTs were measured using fluorescence activated cell sorting (FACS) studies in CHO-k1 cells. FACS analysis data revealed that transfection efficiency of 7b (99.9%) was better than 9b (83.0%) at 500 nM concentration (Figure 3a, b) and also confirmed by live cell imaging in CHO-k1 cells (Figure S1). IGT 7b is more lipophilic than 9b which was confirmed by octanol/water partitioning of respective IGTs after addition

As the cellular uptake property of BODIPY-conjugated IGT 7b is better than 9b, we became interested to study the presence of secondary structures of IGT 7a and 9a. CD spectra were then recorded at 3 mM concentration in water. The CD patterns of both the compounds were observed to be different (Figures 4a, b). A characteristic band shape in CD spectrum of 7a was oserved perhaps due to a repetitive spatial arrangement of the gunidine (chromophore) resulting 20 in molecular chirality from achiral unit. The different CD spectra of 7a and 9a clearly indicated that these are struc16 turally different from our previously reported GMO. For further structural elucidation of 7a, atomic force microscopy images were recorded. IGT 7a was dropcasted on different surfaces such as glass, mica and gold at 10 mM concentration in water. Interestingly on glass surface it formed aggregation looking like pairs of closely spaced right circular cylinder of height ~ 400 nm for a scanned space of 7.9µm × 7.9µm on glass surface (Figures 4c-e). This kind of aggregation disappeared when the surface shifted from



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rough to smooth surface (Figure S8). No such aggregation was found for 9a in AFM images on glass surface as well as on other surface at the same 10 mM concentration. Taking into account these results, we propose that 7a adopts helical conformation with an organized structure and is involved in transfection in both monomeric and aggregation form like 12,21,22 CPPs.

Figure 4. CD spectra of (a) 7a and (b) 9a in water at 3 mM concentration at 25oC. (c) AFM images of 7a (concentration: 10 mM in water, 25oC) on glass surface.

After having encouraging results of the cell-penetration properties of 7b, as a proof of principle, we became interested to evaluate the ability of 7a to deliver PMO for antisense applications targeting Gli1, a transcription factor 23-25 required for hedgehog signaling and no tail (Ntl) gene of zebrafish. For this purpose regular Gli1 PMO of sequence 7′-GTC ATT GGA TTG AAC ATG GCG TCT C-4′ and Ntl PMO of sequence 7′-GAC TTG AGG CAG ACA TAT TTC CGA T-4′ were procured from Gene Tools and functionalized to acetylinic-PMO and conjugated with azido 7a using Copper-catalyzed Azide-Alkyne click reaction to obtain the IGT-Gli1 PMO (8a) and IGT-Ntl PMO (8b) conjugates, respectively (Scheme 2). 8a and 8b were purified by HPLC using C-18 column, solvent A: 0.1% TFA in water, solvent B: Acetonitrile, Gradient: 0 – 10% B in A, 25 min, flow rate 1 ml/min. 8a and 8b were eluted at 5.121 and 5.855 mins, respectively. Fractions collected and lyophilised for biological applications.

Scheme 2. Synthesis of IGT-PMO conjugates 8a and 8b using click conjugation. 23

For in-vitro applicaton we have used Shh-Light 2 cells, derived from NIH3T3 (mouse embryo fibroblast) cell line, stably transfected with Gli-dependent firefly luciferase and constitutive renilla luciferase reporters, which was stimulated with ShhN – conditioned media. To ensure cellular uptake and antisense property of 8a, luciferase assay was performed which showed a dose-dependent inhibition of luciferase expression. The immunoblot analysis

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of Gli1 protein using IGT-Gli1 PMO 8a and Vivo-Gli PMO ( procured from Gene Tools) at 5 µM concentration implied that 8a has better antisense effects than Vivo-PMO. Again MTT assay showed almost no toxicity of 8a upto 50 μM concentration whereas survivability of Vivo-Gli1 PMO treated cells was 58% only at the same concentration and toxicity started from 20 μM concentration and cells looked round and yellowish in color (Figure 5).

Figure 5. (a) Inhibition of luciferase expression with IGT-Gli1 in Shh Light 2 cells by stimulating the pathway with ShhN conditioned media; (b) Immunoblot analysis in Shh Light 2 cells. IGT-Gli inhibited Gli1 expression at 5 µM concentration and no inhibition by Vivo-Gli. Lysate was prepared after 40 h of treatment of respective compounds. For densitometric analysis of relative expression level of Gli1, the band intensity of Gli1 was normalized by the corresponding band intensity of GAPDH; (c) MTT assay of IGT-Gli1 PMO and Vivo-Gli1 PMO in Shh Light 2 cells. For Vivo-Gli1 toxicity started from 20 µM concentration.

To understand the ability of 7b to transfect in-vivo Zebrafish embryos, we injected 7b (0.1 ng/nL, 3-4 nL, injected embryos - 30) into the yolk and also into the chorion (injected embryos - 30) of zebrafish embryos at 64cell stage (2 hpf). A wide spread fluorescence was observed throughout the hatched embryos. Fluorescence intensity was comparatively more for the yolk injected embryo than chorion (Figure 6a) and fluorescence intensity gradually reduced with time. 7b neither interfered in embryonic development nor showed any visible side effects and distribution of 7b was observed throughout the cells within 30 min after the delivery of 7b into the yolk at 2-cell stage (Figure S9). As PMOs function through an RNase-H independent mechanism, it is suitable for gene knockdown in zebrafish 26 model developmental biology. However, due to poor cellular transfection properties of PMOs, gene knockdown in zebrafish embryos can only be achieved by microinjection in 27,28 early developmental stages (between 1 to 4 -cell stage). After that cytoplasmic bridges doesn’t allow to rapid diffusion for ubiquitous delivery. So, spatiotemporal regulation of gene expression is almost impossible and after 4-cell stage, caged morpholinos are the only tools used for 29-33 spatiotemporal regulation of gene in zebrafish embryos.


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Bioconjugate Chemistry 8b injected zebrafish embryos in comparison with water injected control embryos. Embryos were microinjected at sphere stage (4 hpf) (injected embryos - 60) and fixed at 90% epiboly stage (9 hpf), (i) Control and (i‘) 8b; bud-stage (10 hpf), (ii) control and (ii‘) 8b; 3somite stage (11 hpf), (iii) control and (iii‘) 8b; 10-somite stage (14 hpf), (iv) control (iv‘) 8b and at 24 hpf, (v) control and (v‘) 8b; (d) Western blot of Ntl protein for different phenotypes. Embryos microinjected (at each stage injected embryos - 60) with IGT-Ntl at 64 cell stage (2 hpf), high cell stage (3.3 hpf) and at 50% epiboly stage (5.3 hpf) were cultured at 28.5oC temperature in E3 medium for 36 h. Then phenotypes were catagorized as severe, medium and mild for immunobloting of no tail protein. 50 µg of total protein was loaded per lane as determined by Bradford assay.

So, we became interested to use our IGT-Ntl PMO 8b for temporal regulation of ntl gene in zebrafish embryos and 8b was microinjected into the embryos at different developmental stages. Ntl gene was chosen becasue it is well characterized and expressed from early to late developmental stage. Initially, 8b (9 ng/ nL) was injected into zebrafish embryos at 2-cell stage (0.75 hpf, injected 34 embryos - 45) following Ekker’s Protocol and observed ntl 35,36 mutants phenotypes as reported earlier (Figure S10). Phenotype includes a complete loss of vacuolated notochord cells, posterior structures and U-shaped somites rather than normal V-shaped due to the lack of notochordderived hedgehog protein expression. Next we injected 8b into the embryos at different developmental stages including 16 cell (1.5 hpf), 64 cell (2 hpf), 128 cell (2.25 hph), 512 cell (2.75 hpf), 1K cell (3 hpf), sphere (4 hpf), dome (4.3 hpf), 30% epiboly (4.7 hpf), 50% epiboly (5.3 hpf), shield (6 hpf), 70% epiboly (8 hpf), 90% epiboly (9 hpf), bud (10 hpf), 3somite (11 hpf) and 10 somite (14 hpf) stages (injected embryos – 45 at each stage). To our delight, predictable ntldependent phenotypes were observed in all the cases (Figure 6b, Figure S11) with very good survivability rate (70%–90%) and categorized into severe (16 cell to 1K cell stage injected embryos), medium upto 90 % epiboly stage injected embryos and mild phenotypes from bud to 10somite stage injected embryos (Figures 6c and S12). Highest expression of the Ntl protein was reported in the period of 50% epiboly (5.3 hpf) to 90% epiboly (9 hpf) 36 stages. Embryos injected upto 1K cell stage led to major severe phenotypes as it can be distributed ubiquitously before Ntl protein expression has been started. After high cell stage (3.3 hpf) 8b injected embryos led to medium or mild phenotypes attributed to the fact that it only knocked down the residual protein expression. Whole mount immunostaining of Ntl protein expression of 8b injected embryos showed that the intensity of targeted protein diminished significantly as compared to control (Figure 6d). The arrow indicated the expression of no tail protein which is discontinued for 8b injected embryos, and at the bud stage (ii‘) it is not detectable. At 24 hpf, no tail is restricted to the notochord and the tail bud (v) which is partially detectable in the case of 8b injected embryos (v‘). The loss of Ntl protein expression of severe, medium, and mild phenotypic embryos was confirmed by western blot analysis (Figure 6e). Figure 6. (a) IGT-7b injected embryos, (injected embryos - 30, fluoresence images of larvae at 2 dpf are shown; (b) 8b injected embryos at different developmental stages Number of injected embryos in each stage: 45; (c) Percentage of phenotypic distribution and survivabilty graph for 8b injected zebrafish embryos at different developmental stages in comparision with water and IGT 7a injected embryos as control (d) Whole mount immunostaining of

Although Vivo-Ntl is commercially available, however, to the best of our knowledge there is no literature report on the use of Vivo-Ntl PMO for embryological study except few 7 applications in adult zebrafish. So for comparison, we injected Gene Tools Vivo-Ntl at different developmental stages into zebrafish embryos in a similar fashion of 8b injection. Though severe, medium and mild ntl phenotypic embryos were obtained, however, severe phenotypes were



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not identical with either ntl-mutant phenotypes or regular ntlPMO injected embryos and embryos contained some black patches, edema formation, missing of yolk sac with high mortality rate, possibly due to the high level of toxicity of Vivo-PMO (Figures S13, S14). In the context of current clinical trial of PMO for the treatment of Duchenne Muscular 8,37-40 Dystrophy (DMD) IGT-PMO could be an improved delivery technology for the successful treatment of DMD. CONCLUSION In summary, we have developed a pharmacologically compatible, nonpeptide-based, and highly water soluble inernal oligo guanidinium containing cellular transporter (IGT) with the composition of 4-amino piperidine and piperazine or morpholine units. It has successfully delivered antisense PMO both in in-vitro and in-vivo and showed better antisense efficacy than Vivo-PMO. It is worth to mention here that this conjugated PMO could be useful for temporal regulation of gene without having any photocaging technology. It’s simple and high yielding synthetic protocol promises for large scale synthesis. It can be further useful to deliver any other important non-penetrable biomolecules or drugs and work is in progress.

1. Summerton, J., and Weller, D. (1997) Morpholino antisense oligomers: Design, preparation, and properties, Antisense Nucleic Acid Drug Dev. 7, 187–195. 2. Summerton, J., and Weller, D. (1993) Uncharged morpholino-based polymers having phosphorous-containing chiral intersubunit linkages, US Patent. 5, 185, 444. 3. Moulton, J. D., and Jiang, S. (2009) Gene knockdowns in adult animals: PPMOs and Vivo-morpholinos. Molecules 14, 1304–1323. 4. Wada, T., Hara, M., Taneda, T., Qingfu, C., Takata, R., Moro, K., Takeda, K., Kishimoto, T., and Handa, H. (2012) Antisense morpholino

Page 6 of 7

targeting just upstream from a poly (A) tail junction of maternal mRNA removes the tail and inhibits translation, Nucl. Acid Res. 40, 1–10. 5. Amantana, A., Iversen, P. L. (2005) Pharmacokinetics and biodistribution of phosphorodiamidate morpholino antisense oligomers, Curr. Opin. Pharmacol. 5, 550–555. 6. Hassane, F. S., Saleh, A. F., Abes, R., Gait, M. J., and Lebleu, B. (2010) Cell penetrating peptides overview and applications to the delivery of oligonucleotides, Cell. Mol. Life Sci. 2010, 67, 715–726. 7. Ferguson, D. P., Dangott, L. J., Lightfoot, J. T. (2014) Lessons learned from vivo-morpholinos: How to avoid vivo-morpholino toxicity, Biotechniques 56, 251– 256. 8. Li Y. F. (2014) To discover, develop and deliver a right drug: A showcase for antisense technology, J. Drug Discov. Develop. and Deliv. 1, 1–3. 9. Stanzl, E. G., Trantow, B. M., Vargas, J. R., and Wender, P. A. (2013) Fifteen years of cell-penetrating, guanidinium-rich molecular transporters: Basic science, research tools, and clinical applications, Acc. Chem. Res. 46, 2944–2954. 10. Wexselblatt, E., Esko, J. D., and Tor, Y. (2014) On guanidinium and cellular uptake, J. Org. Chem. 79, 6766−6774. 11. McKinlay, C. J., Waymouth, R. M., and Wender, P. A. (2016) CellPenetrating, Guanidinium-Rich Oligophosphoesters: Effective and Versatile Molecular Transporters for Drug and Probe Delivery, J. Am. Chem. Soc. 138, 3510–3517. 12. Lattig-Tunnermann, G., Prinz, M., Hoffmenn, D., Behlke, J., PalmApergi, C., Morano, I., Herce, H. D., and Cardoso, M. C. (2011) Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides, Nature Commun. 2, 1–6. 13. Geihe, E. I., Cooley, C. B., Simon, J. R., Kiesewetter, M. K., Edward, J. A., Hickerson, R. P., Kaspar, R. L., Hedrick, J. L., Waymouth, R. M., and Wender, P. A. (2012) Designed guanidinium-rich amphipathic oligocarbonate molecular transporters complex, deliver and release siRNA in cells, Proc. Natl. Acad. Sci. USA. 109, 13171–13176. 14. Fillon, Y. A., Anderson, J. P., and Chmielewski, J. (2005) Cell penetrating agents based on a polyproline helix scaffold, J. Am. Chem. Soc. 127, 11798–11803. 15. Fernandez-Carneado, J., Gool, M. V., Martos, V., Castel, S., Prados, P., Mendoza, J. D., and Giralt, E. (2005) Highly efficient nonpeptidic oligoguanidinium vectors that selectively internalize into mitochondria, J. Am. Chem. Soc. 127, 869–874. 16. Pattanayak, S., Khatra, H., Saha, S., and Sinha, S. (2014) Cationic morpholino antisense oligomer conjugate: Synthesis, cellular uptake and inhibition of Gli1 in hedgehog signalling pathway, RSC Adv. 4, 1951– 1954. 17. Sun, H., and Scott, D. O. (2011) Metabolism of 4-aminopiperidine drugs by cytochrome P450s: Molecular and quantum mechanical insights into drug design, ACS Med. Chem. Lett. 2, 638–643. 18. Jain, M. L., Bruice, P. Y., Szabo, I. E., and Bruice, T. C. (2012) Incorporation of positively charged linkages into DNA and RNA backbones: A novel strategy for antigene and antisense agents, Chem. Rev. 112, 1284–1309. 19. Richard, J. P., Melikov, K., Vives, E., Ramos, C., Verbeure, B., Gait, M. J., Chernomordik, L. V., and Lebleu, B. (2003) Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake, J. Biol. Chem. 278, 585–590. 20. Cho, J., Tanaka, M., Sato, S., Kinbara, K., and Aida, T. (2010) Oligo(4-aminopiperidine-4-carboxylic acid): An Unusual Basic Oligopeptide with an Acid-Induced Helical Conformation, J. Am. Chem. Soc. 132, 13176–13178. 21. Pujals, S., Fernandex-Carneado, J., Lopez-Iglesias, C., Kogan, M. J., and Giralt, E. (2006) Mechanistic aspects of CPP-mediated intracellular drug delivery: Relevance of CPP self-assembly, Biochim. Biophys. Acta, 1758, 264–279. 22. Ezzat, K., Aoki, Y., Koo, T., McClorey, G., Benner, L., CoenenStass, A., O’Donovan, L., Lehto, T., Garcia-Guerra, A., Nordin, J., et. al (2015) Self-Assembly into nanoparticles is essential for receptor mediated uptake of therapeutic antisense oligonucleotides, Nano Lett. 15, 4364– 4373. 23. Taipale, J., Chen, J. K., Cooper, M. K.,Wang, B., Mann, R. K., Milenkovic, L., Scott, M. P., and Beachy, P. A. (2000) Effects of oncogenic mutations in smoothened and patched can be reversed by cyclopamine, Nature 406, 1005–1009.


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24. Ingham, P. W., and McMahon, A. P. (2001) Hedgehog signaling in animal development: paradigms and principles, Genes Dev. 15, 3059– 3087. 25. Hooper, J. E., Scott, M. P. (2005) Communicating with hedgehogs, Nat. Rev. Mol. Cell Biol. 6, 306–317. 26. Westerfield, M. (2000) The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Daniorerio) (Univ. of Oregon Press, Eugene). 27. Essner, J. L., Amack, J. D., Nyholm, M. K., Harris, E. B., Yost, H. J. (2005) Kupffer’s vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut, Development 132, 1247–1260. 28. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., and Schilling, T. F. (1995) Stages of Embryonic Development of the Zebrafish, Dev Dyn. 203, 253–310. 29. Griepenburg, J. C., Rapp, T. L., Carroll, P. J., Eberwine, J., and Dmochowski, I. J. (2015) Ruthenium-caged antisense morpholinos for regulating gene expression in zebrafish embryos, Chem. Sci. 6, 2342– 2346. 30. Shestopalov, I. A., Sinha, S., and Chen, J. K. (2007) Lightcontrolled gene silencing in, zebrafish embryos, Nat. Chem. Biol. 3, 650– 651. 31. Wu, L., Wang, Y., Wu, J., Lv, C., Wang, J., and Tang, X. (2013) Caged circular antisense oligonucleotides for photomodulation of RNA digestion and gene expression in cells, Nucl. Acid Res. 41, 677–686. 32. Yamazoe, S., Liu, Q., McQuade, L. E., Deiters, A., and Chen, J. K. (2014) Sequential gene silencing using wavelength-selective caged morpholinos, Angew. Chem. Int. Ed. 53, 10114–10118. 33. Deiters, A., Garner, R. A., Lusic, H., Govan, J. M., Dush, M., Nascone-Yoder, and N. M., Yoder, J. A. (2010) Photocaged morpholino oligomers for the light-regulation of gene function in zebrafish and xenopus embryos, J. Am. Chem. Soc. 2010, 132, 15644–15650. 34. Nasevicius, A., and Ekker, S. C. (2000) Effective targeted gene knockdown in zebrafish, Nat. Genet. 26, 216–220. 35. Schulte-Merker, S. (1995) The zebrafish no tail gene, Dev. Bio. 6, 411–416. 36. Schulte-Merker, S., Ho, R. K., Herrmann, B. G., and NussleinVolhard, C. (1992) The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo, Development 116, 1021–1032. 37. Cirak, S., Arechavala-Gomeza, V., Guglieri, M., Feng, L., Torelli, S., Anthony, K., Abbs, S., Garralda, M. E., Bourke, J., Wells, D. J., et al (2011) Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study, Lancet 378, 595–605. 38. O’ Donovan, L., Okamoto, I., Arzumanov, A. A., Williams, D. L., Deuss, P., and Gait, M. J. (2015) Parallel synthesis of cell-penetrating peptide conjugates of PMO toward exon skipping enhancement in Duchenne muscular dystrophy, Nucl. Acids Ther. 25, 1 – 10. 39. Popplewell, L. J., Graham, I. R., Malerba, A., and Dickson, G. (2011) Bioinformatic and functional optimization of antisense phosphorodiamidate morpholino oligomers (PMOs) for therapeutic modulation of RNA splicing in muscle, Methods in Mol. Biol. 709, 153 – 78. 40. Nowak, K. J., and Davies, K. E. (2004) Duchenne muscular dystrophy and dystrophin; pathogenesis and opportunities for treatment, EMBO Reports 5, 872 – 876.


Internal Oligoguanidinium-Based Cellular Transporter Enhances

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