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Intestinal delivery to zebrafish from fish water

Progatzky F, Sangha NJ, Yoshida N, McBrien M, Cheung J, Shia A, Scott J, Marchesi JR, Lamb JR, Bugeon L, Dallman MJ. Dietary cholesterol directly induces acute inflammasome-dependent intestinal inflammation. Nat Commun. 2014 Dec 23;5:5864. doi: 10.1038/ncomms6864.

This paper claims delivery of Morpholino to the intestinal epithelium of zebrafish by putting Morpholinos in the fish water around feeding time.

"As the knockdown efficiency of MO injected into single-cell stage embryos diminishes after 5 days post fertilization (dpf) ... we devised a novel delivery method by simply adding the MO to the fish water 24-h before and during the 6-h feeding period."

"First, we validated this new method by imaging MO uptake in the intestine ..."

"Second, analysis by flow cytometry following treatment with the FITC-conjugated ​ASC splice-blocking MO revealed an increase in FITC fluorescence in Cytokeratin+ epithelial cells but not in mCherry+ macrophages in Tg(​fms:mCherry), or in dsRed+ neutrophils in Tg(​lyz:dsRed) larvae ..."

"Finally, we confirmed the ability of this MO to significantly knock down ​ASC mRNA by performing reverse-transcription PCR (RT-PCR) on injected embryos (Supplementary Fig. 10) and quantitative RT-PCR (qRT-PCR) on sorted FITC+ cells"

"...we used a GFP MO (not tagged with FITC) to target GFP in Tg(ubi:EGFP) fish35 and found significantly decreased fluorescence intensity in intestinal cells (Supplementary Fig. 11)."

Protocol, figure 1 legend:
"Fig. 1. Schematic representation of the protocol used for experiments. Zebrafish larvae at 6 days post-fertilisation (dpf) were pre-treated with indicated drugs for 30 minutes (min) followed by feeding with ZM control diet or high cholesterol diet (HCD) for 6 hours (h) in the presence of drugs (top panel). Zebrafish larvae (6 dpf) were pre-treated with indicated morpholino oligonucleotides for 24 hours and followed by feeding with ZM control diet or HCD for 6 hours in the presence of morpholinos (bottom panel). The quantification of total cell numbers of indicated immune cells in the intestine were performed at 18 hours post feeding."

Rescues with wild-type or mutant mRNA

Here is an interesting strategy for nailing down the relationship between a phenotype and a particular mutation. Using a zebrafish pnpla6 morphant, co-injection of wild-type human pnpla6 mRNA rescued the morphant phenotype while mutant human pnpla6 mRNA failed to rescue.

Hufnagel RB, Arno G, Hein ND, Hersheson J, Prasad M, Anderson Y, Krueger LA, Gregory LC, Stoetzel C, Jaworek TJ, Hull S, Li A, Plagnol V, Willen CM, Morgan TM, Prows CA, Hegde RS, Riazuddin S, Grabowski GA, Richardson RJ, Dieterich K, Huang T, Revesz T, Martinez-Barbera JP, Sisk RA, Jefferies C, Houlden H, Dattani MT, Fink JK, Dollfus H, Moore AT, Ahmed ZM. Neuropathy target esterase impairments cause Oliver-McFarlane and Laurence-Moon syndromes. J Med Genet. 2014 Dec 5. pii: jmedgenet-2014-102856. doi: 10.1136/jmedgenet-2014-102856. [Epub ahead of print]

Of Mice and Morpholinos, 2014

These are some papers using Morpholinos in mouse, published in 2014.


WT1 controls antagonistic FGF and BMP-pSMAD pathways in early renal progenitors . Motamedi FJ, Badro DA, Clarkson M, Rita Lecca M, Bradford ST, Buske FA, Saar K, Hübner N, Brändli AW, Schedl A. Nat Commun. 2014 Jul 17;5:4444. doi: 10.1038/ncomms5444.
Vivo-Morpholino in kidney organ culture.

Effect of Combined Systemic and Local Morpholino Treatment on the Spinal Muscular Atrophy Δ7 Mouse Model Phenotype. Nizzardo N, Simone C, Salani S, Ruepp M-D, Rizzo F, Ruggieri M, Zanetta C, Brajkovic S, Moulton HM, Müehlemann O, Bresolin N, Comi GP, Corti S. Clin Therap. 2014. 36(3):340-56. doi:10.1016/j.clinthera.2014.02.004.
Vivo-Morpholino in SMA model.

miR-142-3p balances proliferation and differentiation of mesenchymal cells during lung development. Carraro G, Shrestha A, Rostkovius J, Contreras A, Chao CM, El Agha E, Mackenzie B, Dilai S, Guidolin D, Taketo MM, Günther A, Kumar ME, Seeger W, De Langhe S, Barreto G, Bellusci S. Development. 2014 Feb 19. [Epub ahead of print].
Vivo-Morpholino in lung explant culture.

Secretin Stimulates Biliary Cell Proliferation by Regulating Expression of MicroRNA 125b and MicroRNA let7a in Mice. Glaser S, Meng F, Han Y, Onori P, Chow BK, Francis H, Venter J, McDaniel K, Marzioni M, Invernizzi P, Ueno Y, Lai JM, Huang L, Standeford H, Alvaro D, Gaudio E, Franchitto A, Alpini G. Gastroenterology. 2014 Feb 25. pii: S0016-5085(14)00241-8. doi: 10.1053/j.gastro.2014.02.030. [Epub ahead of print].

Differential Skeletal Muscle Proteome of High and Low Active Mice. Ferguson DP, Dangott LJ, Schmitt EE, Vellers HL, Lightfoot JT. Differential J Appl Physiol. 2014 Feb 6. [Epub ahead of print].

A sensitive assay system to test antisense oligonucleotides for splice suppression therapy in the mouse liver. Gallego-Villar L, Viecelli HM, Pérez B, Harding CO, Ugarte M, Thöny B, Desviat LR. Mol Ther Nucleic Acids. 2014 Sep 16;3:e193. doi: 10.1038/mtna.2014.44.

Regulation of the extrinsic apoptotic pathway by microRNA-21 in alcoholic liver injury. Francis H, McDaniel K, Han Y, Liu X, Kennedy L, Yang F, McCarra J, Zhou T, Glaser S, Venter J, Huang L, Levine P, Lai J-M, Liu C-G, Alpini G, Meng F. J Biol Chem. 2014;[Epub ahead of print] doi:10.1074/jbc.M114.602383.

Antisense-mediated Exon Skipping Decreases Tau Protein Expression: A Potential Therapy For Tauopathies. Sud R, Geller ET, Schellenberg GD. Mol Ther Nucleic Acids. 2014 Jul 29;3:e180. doi: 10.1038/mtna.2014.30.

Gli1 Activation and Protection Against Hepatic Encephalopathy is Suppressed by Circulating Transforming Growth Factor β1 in Mice. McMillin M, Galindo C, Pae HY, Frampton G, Di Patre PL, Quinn M, Whittington E, DeMorrow S. J Hepatol. 2014;[Epub ahead of print] doi:10.1016/j.jhep.2014.07.015.

Genetic Targets of Hydrogen Sulfide in Ventilator-Induced Lung Injury - A Microarray Study. Spassov S, Pfeifer D, Strosing K, Ryter S, Hummel M, Faller S, Hoetzel A. PLoS One. 2014 Jul 15;9(7):e102401. doi: 10.1371/journal.pone.0102401. eCollection 2014.

TLR7 is a key regulator of innate immunity against Japanese Encephalitis Virus infection. Nazmi A, Mukherjee S, Kundu K, Dutta K, Mahadevan A, Shankar SK, Basu A. Neurobiol Dis. 2014 Jun 5. pii: S0969-9961(14)00154-5. doi: 10.1016/j.nbd.2014.05.036. [Epub ahead of print].

Lessons learned from vivo-morpholinos: How to avoid vivo-morpholino toxicity. Ferguson DP, Dangott JJ, Timothy Lightfoot JT. BioTechniques. 2014;56(5):251-6.

Dynamic Expression of Chymotrypsin-Like Elastase 1 Over the Course of Murine Lung Development. Liu S, Young SM, Varisco BM. Am J Physiol Lung Cell Mol Physiol. 2014 May 2. [Epub ahead of print].

De novo mutation in ATP6V1B2 impairs lysosome acidification and causes dominant deafness-onychodystrophy syndrome. Yuan Y, Zhang J, Chang Q, Zeng J, Xin F, Wang J, Zhu Q, Wu J, Lu J, Guo W, Yan X, Jiang H, Zhou B, Li Q, Gao X, Yuan H, Yang S, Han D, Mao Z, Chen P, Lin X, Dai P. Cell Res. 2014 Jun 10. doi: 10.1038/cr.2014.77. [Epub ahead of print].
Intracochlear injection of Vivo-Morpholino.

Peptide-linked Morpholino

Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease. Clayton NP, Nelson CA, Weeden T, Taylor KM, Moreland RJ, Scheule RK, Phillips L, Leger AJ, Cheng SH, Wentworth BM. Mol Ther Nucleic Acids. 2014 Oct 28;3:e206. doi: 10.1038/mtna.2014.57.
Injection & Electroporation or Peptide-linked Morpholino.

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model. Bestas B, Moreno PM, Blomberg KE, Mohammad DK, Saleh AF, Sutlu T, Nordin JZ, Guterstam P, Gustafsson MO, Kharazi S, Piątosa B, Roberts TC, Behlke MA, Wood MJ, Gait MJ, Lundin KE, El Andaloussi S, Månsson R, Berglöf A, Wengel J, Smith CI. J Clin Invest. 2014 Aug 8. pii: 76175. doi: 10.1172/JCI76175. [Epub ahead of print].
Peptide-linked Morpholino.

Assessment of RT-qPCR Normalization Strategies for Accurate Quantification of Extracellular microRNAs in Murine Serum. Roberts TC, Coenen-Stass AML, Wood MJA. PLoS ONE. 2014;9(2):e89237. doi:10.1371/journal.pone.0089237.
Peptide-linked Morpholino.

Exon skipping restores dystrophin expression, but fails to prevent disease progression in later stage dystrophic dko mice. Wu C, Cloer C, Lu P, Milazi S, Shaban M, Shah SN, Marston-Poe L, Moulton HM, Lu QL. Gene Ther. 2014;[Epub ahead of print] doi:10.1038/gt.2014.53.
Peptide-linked Morpholino.

Unanchored K48-Linked Polyubiquitin Synthesized by the E3-Ubiquitin Ligase TRIM6 Stimulates the Interferon-IKKε Kinase-Mediated Antiviral Response. Rajsbaum R, Versteeg GA, Schmid S, Maestre AM, Belicha-Villanueva A, Martínez-Romero C, Patel JR, Morrison J, Pisanelli G, Miorin L, Laurent-Rolle M, Moulton HM, Stein DA, Fernandez-Sesma A, tenOever BR, García-Sastre A. Immunity. 2014;[Epub ahead of print] doi:10.1016/j.immuni.2014.04.018.
Peptide-linked Morpholino.

Induced IL-10 Splice Altering Approach to Antiviral Drug Discovery. Panchal RG, Mourich DV, Bradfute S, Hauck LL, Warfield KL, Iversen PL, Bavari S. Nucleic Acid Ther. 2014;24(3):179-185. doi:10.1089/nat.2013.0457.
Peptide-linked Morpholino.

Effective dystrophin restoration by a novel muscle-homing peptide-morpholino conjugate in dystrophin-deficient mdx mice. Gao X, Zhao J, Han G, Zhang Y, Dong X, Cao L, Wang Q, Moulton HM, Yin H. Mol Ther. 2014 Apr 15. doi: 10.1038/mt.2014.63. [Epub ahead of print].
Peptide-linked Morpholino.

Oocyte/Embryo Injection

Hira-Mediated H3.3 Incorporation Is Required for DNA Replication and Ribosomal RNA Transcription in the Mouse Zygote. Lin C-J, Koh FM, Wong P, Conti M, Ramalho-Santos M. Dev Cell. 2014;[Epub ahead of print] doi:10.1016/j.devcel.2014.06.022.
Oocyte microinjection.

WASH complex regulates Arp2/3 complex for actin-based polar body extrusion in mouse oocytes. Wang F, Zhang L, Zhang GL, Wang ZB, Cui XS, Kim NH, Sun SC. Sci Rep. 2014 Jul 7;4:5596. doi: 10.1038/srep05596.
Oocyte microinjection.

Possible Role of p38 MAPK-MNK1-EMI2 Cascade in Metaphase-II Arrest of Mouse Oocytes. Miyagaki Y, Kanemori Y, Tanaka F, Baba T. Biol Reprod. 2014;[Epub ahead of print] doi:10.1095/biolreprod.113.116962.
Oocyte microinjection.

Knockdown of RBBP7 unveils a requirement of histone deacetylation for CPC function in mouse oocytes. Balboula AZ, Stein P, Schultz RM, Schindler K. Cell Cycle. 2014;[epub ahead of print]13:0 - -1; PMID: 24317350.
Oocyte microinjection.

Comparison of Epigenetic Mediator Expression and Function in Mouse and Human Embryonic Blastomeres. Chavez SL, McElroy SL, Bossert NL, De Jonge CJ, Rodriguez MV, Leong DE, Behr B, Westphal LM, Pera RAR. Hum Mol Genet. 2014;[Epub ahead of print] doi:10.1093/hmg/ddu212.
Zygote microinjection.

Intracerebroventricular Injection of Bare Morpholino

Morpholino Antisense Oligonucleotides Targeting Intronic Repressor Element1 Improve Phenotype in SMA Mouse Models. Osman EY, Miller MR, Robbins KL, Lombardi AM, Atkinson AK, Brehm AJ, Lorson CL. Hum Mol Genet. 2014;[Epub ahead of print] doi:10.1093/hmg/ddu198.
SMNΔ7 ICV injection.

Electrophysiological Biomarkers in Spinal Muscular Atrophy: Proof of Concept. Arnold WD, Porensky PN, McGovern VL, Iyer CC, Duque S, Li X, Meyer K, Schmelzer L, Kaspar BK, Kolb SJ, Kissel JT, Burghes AH. Ann Clin Transl Neurol. 2014 Jan 1;1(1):34-44.
ICV injection.

Pharmacology of a central nervous system delivered 2'-o-methoxyethyl-modified survival of motor neuron splicing oligonucleotide in mice and nonhuman primates. Rigo F, Chun SJ, Norris DA, Hung G, Lee S, Matson J, Fey RA, Gaus H, Hua Y, Grundy JS, Krainer AR, Henry SP, Bennett CF. J Pharmacol Exp Ther. 2014 Jul;350(1):46-55. doi: 10.1124/jpet.113.212407. Epub 2014 Apr 30.
ICV injection.

RIG-I knockdown impedes neurogenesis in a murine model of Japanese encephalitis. Mukherjee S, Ghosh S, Nazmi A, Basu A. Cell Biol Int. 2014 Jul 31. doi: 10.1002/cbin.10354. [Epub ahead of print].
Intracerebral injection; murine neurospheres.

Other Delivery Strategies

HSPA5 is an essential host factor for Ebola virus infection. Patrick Reid S, Shurtleff AC, Costantino JA, Tritsch SR, Retterer C, Spurgers KB, Bavari S. Antiviral Res. 2014 Sep;109:171-4. doi: 10.1016/j.antiviral.2014.07.004. Epub 2014 Jul 11.
IP injection.

Serum-Free Culture of Mid-gestation Mouse Embryos: A Tool for the Study of Endoderm-Derived Organs. Gordon J, Moore BA, Blackburn CC, Manley NR. Methods Mol Biol 2014;[Epub ahead of print] doi: 10.1007/978-1-60327-292-6_12.
Electroporation into mouse embryo.

MiR-153 targets the nuclear factor-1 family and protects against teratogenic effects of ethanol exposure in fetal neural stem cells. Tsai P-C, Bake S, Balaraman S, Rawlings J, Holgate RR, Dubois D, Miranda RC. Biology Open. 2014;[Epub]doi:10.1242/bio.20147765.
Electroporation into single-cell suspensions from murine neurospheres.

Near-Infrared-Light-Based Nano-Platform Boosts Endosomal Escape and Controls Gene Knockdown in Vivo. Jayakumar MK, Bansal A, Huang K, Yao R, Li BN, Zhang Y. ACS Nano. 2014 Apr 14. [Epub ahead of print].

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine? Ljubimova JY, Ding H, Portilla-Arias J, Patil R, Gangalum PR, Chesnokova A, Inoue S, Rekechenetskiy A, Nassoura T, Black KL, Holler E. J Vis Exp. 2014;(88):e50668. doi:10.3791/50668.

Blockade of CD47 mediated CTSS-PAR2 signaling provides a therapeutic target for hepatocellular carcinoma. Lee TKW, Cheung VCH, Lu P, Lau EYT, Ma S, Tang KH, Tong M, Lo J, Ng IOL. Hepatology. 2014;[Epub ahead of print] doi:10.1002/hep.27070.
Intratumoral injection into human xenograft in mice.

Bubble Liposomes and Ultrasound Exposure Improve Localized Morpholino Oligomer Delivery into the Skeletal Muscles of Dystrophic mdx Mice. Negishi Y, Ishii Y, Shiono H, Akiyama S, Sekine S, Kojima T, Mayama S, Kikuchi T, Hamano N, Endo-Takahashi Y, Suzuki R, Maruyama K, Aramaki Y. Molec Pharmaceutics. 2014;[epub ahead of print] doi:10.1021/mp4004755.
Bubble liposomes & ultrasound delivery.

Evaluation of Tris[2-(acryloyloxy)ethyl]isocyanurate Cross-linked Polyethylenimine as Antisense Morpholino Oligomer Delivery Vehicle in Cell Culture and Dystrophic mdx Mice. Wang M. Hum Gene Ther. 2014 Jan 9. [Epub ahead of print].
Covalent PEI.

Splicing-Directed Therapy in a New Mouse Model of Human Accelerated Aging. Osorio FG, Navarro CL, Cadiñanos J, López-Mejía IC, Quirós PM, Bartoli C, Rivera J, Tazi J, Guzmán G, Varela I, Depetris D, de Carlos F, Cobo J, Andrés V, Sandre-Giovannoli AD, Freije JM, Lévy N, López-Otín C. Sci Transl Med. 2011 Oct 26;3(106):106ra107. doi: 10.1126/scitranslmed.3002847.

Phosphorodiamidate Morpholino Oligomers (PMOs) suppress mutant huntingtin expression and attenuate neurotoxicity. Sun X, Marque LO, Cordner Z, Pruitt JL, Bhat M, Li P, Kannan G, Ladenheim EE, Moran TH, Margolis RL, Rudnicki DD. Hum Mol Genet. 2014;[Epub ahead of print] doi:10.1093/hmg/ddu349.

Impaired functional communication between the L-type calcium channel and mitochondria contributes to metabolic inhibition in the mdx heart. Viola HM, Adams AM, Davies SM, Fletcher S, Filipovska A, Hool LC. Proc Natl Acad Sci U S A. 2014 Jun 26. pii: 201402544. [Epub ahead of print].

Cardiac CD47 Drives Left Ventricular Heart Failure Through Ca2+‐CaMKII‐Regulated Induction of HDAC3arifi‐Sanjani M, Shoushtari AH, Quiroz M, Baust J, Sestito SF, Mosher M, Ross M, McTiernan CF, St. Croix CM, Bilonick RA, Champion HC, Isenberg JS. J Am Heart Assoc. 2014;3:e000670, originally published June 10, 2014, doi:10.1161/JAHA.113.000670.

Lomofungin and dilomofungin: inhibitors of MBNL1-CUG RNA binding with distinct cellular effects. Hoskins JW, Ofori LO, Chen CZ, Kumar A, Sobczak K, Nakamori M, Southall N, Patnaik S, Marugan JJ, Zheng W, Austin CP, Disney MD, Miller BL, Thornton CA. Nucleic Acids Res. 2014 May 5. [Epub ahead of print].
Electroporation into cultured mouse C2C12 myogenic cells.

Some Morpholino Citations for Hematology: 2014

Representative Recent Morpholino Citations for Hematology: 2014
Last edit: 4 Dec 2014

Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.
Nuzzo F, Bulato C, Nielsen BI, Lee K, Wielders SJ, Simioni P, Key NS, Castoldi E. Haemophilia. 2014 Dec 3. doi: 10.1111/hae.12554. [Epub ahead of print].

Loss of function mutations in RPL27 and RPS27 identified by whole-exome sequencing in Diamond-Blackfan anaemia. Wang R, Yoshida K, Toki T, Sawada T, Uechi T, Okuno Y, Sato-Otsubo A, Kudo K, Kamimaki I, Kanezaki R, Shiraishi Y, Chiba K, Tanaka H, Terui K, Sato T, Iribe Y, Ohga S, Kuramitsu M, Hamaguchi I, Ohara A, Hara J, Goi K, Matsubara K, Koike K, Ishiguro A, Okamoto Y, Watanabe K, Kanno H, Kojima S, Miyano S, Kenmochi N, Ogawa S, Ito E. Br J Haematol. 2014 Nov 25. doi: 10.1111/bjh.13229. [Epub ahead of print]

Live Imaging and Gene Expression Analysis in Zebrafish Identifies a Link between Neutrophils and Epithelial to Mesenchymal Transition. Freisinger CM, Huttenlocher A. PLoS One. 2014 Nov 5;9(11):e112183. doi: 10.1371/journal.pone.0112183. eCollection 2014.

Nkx2.5 is involved in myeloid cell differentiation at anterior ventral blood islands in the Xenopus embryo. Sakata H, Maéno M. Dev Growth Differ. 2014 Oct 3. doi: 10.1111/dgd.12155. [Epub ahead of print]

Proinflammatory Signaling Regulates Hematopoietic Stem Cell Emergence. Espín-Palazón R, Stachura DL, Campbell CA, García-Moreno D, Del Cid N, Kim AD, Candel S, Meseguer J, Mulero V, Traver D. Cell. 2014;[Epub ahead of print] doi:10.1016/j.cell.2014.10.031

Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production. Li Y, Esain V, Teng L, Xu J, Kwan W, Frost IM, Yzaguirre AD, Cai X, Cortes M, Maijenburg MW, Tober J, Dzierzak E, Orkin SH, Tan K, North TE, Speck NA. Genes Dev. 2014;[Epub ahead of print] doi:10.1101/gad.253302.114

Identification of Cdca7 as a novel Notch transcriptional target involved in hematopoietic stem cell emergence. Guiu J, Bergen DJM, De Pater E, Islam ABMMK, Ayllón V, Gama-Norton L, Ruiz-Herguido C, González J, López-Bigas N, Menendez P, Dzierzak E, Espinosa L, Bigas A. J Exp Med. 2014;[Epub ahead of print] doi:10.1084/jem.20131857

ETV6 (TEL1) regulates embryonic hematopoiesis in zebrafish. Rasighaemi P, Onnebo SM, Liongue C, Ward AC. Haematologica. 2014 Oct 3. pii: haematol.2014.104091. [Epub ahead of print].

Discrete Notch signaling requirements in the specification of hematopoietic stem cells. Kim AD, Melick CH, Clements WK, Stachura DL, Distel M, Panáková D, MacRae C, Mork LA, Crump JG, Traver D. EMBO J. 2014 Oct 16;33(20):2363-73. doi: 10.15252/embj.201488784. Epub 2014 Sep 17.

Systematic transcriptome analysis of the zebrafish model of diamond-blackfan anemia induced by RPS24 deficiency. Song B, Zhang Q, Zhang Z, Wan Y, Jia Q, Wang X, Zhu X, Leung A Y-H, Cheng T, Fang X, Yuan W, Jia H. BMC Genomics. 2014;15:759. doi:10.1186/1471-2164-15-759.

miR-142-3p acts as an essential modulator of neutrophil development in zebrafish. Fan HB, Liu YJ, Wang L, Du TT, Dong M, Gao L, Meng ZZ, Jin Y, Chen Y, Deng M, Yang HT, Jing Q, Gu AH, Liu TX, Zhou Y. Blood. 2014 Aug 21;124(8):1320-30. doi: 10.1182/blood-2013-12-545012. Epub 2014 Jul 2.

The DPY30 subunit in SET1/MLL complexes regulates the proliferation and differentiation of hematopoietic progenitor cells. Yang Z, Augustin J, Chang C, Hu J, Shah K, Chang C-W, Townes T, Jiang H. Blood. 2014 Sep 25;124(13):2025-33. doi: 10.1182/blood-2014-01-549220. Epub 2014 Aug 18.

Drl.3 governs primitive hematopoiesis in zebrafish. Pimtong W, Datta M, Ulrich AM, Rhodes J. Sci Rep. 2014 Sep 4;15(3):376-91. doi: 10.1016/j.stem.2014.06.005. Epub 2014 Jul 17.

A Systems Biology Approach for Defining the Molecular Framework of the Hematopoietic Stem Cell Niche. Charbord P, Pouget C, Binder H, Dumont F, Stik G, Levy P, Allain F, Marchal1 C, Richter J, Uzan B, Pflumio F, Letourneur F, Wirth H, Dzierzak E, Traver D, Jaffredo T, Charles Durand C. Cell Stem Cell. 2014;[Epub ahead of print] doi:10.1016/j.stem.2014.06.005.

A Loss of Function Screen of Identified Genome-Wide Association Study Loci Reveals New Genes Controlling Hematopoiesis. Bielczyk-Maczyńska E, Serbanovic-Canic J, Ferreira L, Soranzo N, Stemple DL, Ouwehand WH, Cvejic A. PLoS Genet. 2014;10(7):e1004450. doi:10.1371/journal.pgen.1004450.

Ncor2 is required for hematopoietic stem cell emergence by inhibiting Fos signaling in zebrafish. Wei Y, Ma D, Gao Y, Zhang C, Wang L, Liu F. Blood. 2014 Sep 4;124(10):1578-85. doi: 10.1182/blood-2013-11-541391. Epub 2014 Jul 8.

Gcsf-Chr19 Promotes Neutrophil Migration to Damaged Tissue through Blood Vessels in Zebrafish. Galdames JA, Zuñiga-Traslaviña C, Reyes AE, Feijóo CG. J Immunol. 2014 Jul 1;193(1):372-8. doi: 10.4049/jimmunol.1303220. Epub 2014 Jun 2.

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia. Danilova N, Bibikova E, Covey TM, Nathanson D, Dimitrova E, Konto Y, Lindgren A, Glader B, Radu CG, Sakamoto KM, Lin S. Dis Model Mech. 2014 Jul;7(7):895-905. doi: 10.1242/dmm.015495. Epub 2014 May 8.

Estrogen defines the dorsal-ventral limit of VEGF regulation to specify the location of the hemogenic endothelial niche. Carroll KJ, Esain V, Garnaas MK, Cortes M, Dovey MC, Nissim S, Frechette GM, Liu SY, Kwan W, Cutting CC, Harris JM, Gorelick DA, Halpern ME, Lawson ND, Goessling W, North TE. Dev Cell. 2014 May 27;29(4):437-53. doi: 10.1016/j.devcel.2014.04.012.

Lysophosphatidic acid acts as a nutrient-derived developmental cue to regulate early hematopoiesis. Li H, Yue R, Wei B, Gao G, Du J, Pei G. 2014 Jun 17;33(12):1383-96. doi: 10.15252/embj.201387594. Epub 2014 May 14.

Homeobox transcription factor VentX regulates differentiation and maturation of human dendritic cells. Wu X, Gao H, Bleday R, Zhu Z. J Biol Chem. 2014 May 23;289(21):14633-43. doi: 10.1074/jbc.M113.509158. Epub 2014 Apr 4.

Protein tyrosine phosphatase PTPN9 regulates erythroid cell development through STAT3 dephosphorylation in zebrafish. Bu Y, Su F, Wang X, Gao H, Lei L, Chang N, Wu Q, Hu K, Zhu X, Chang Z, Meng K, Xiong J-W. J Cell Sci. 2014 Jun 15;127(Pt 12):2761-70. doi: 10.1242/jcs.145367. Epub 2014 Apr 11.

Functions of FMS-like tyrosine kinase 3 (flt3) in zebrafish hematopoiesis and its relevance to human acute myeloid leukemia. He B-L, Shi X, Man CH, Ma ACH, Ekker SC, Chow HCH, So CWE, Choi WWL, Zhang W, Zhang Y, Leung AYH. Blood. 2014 Apr 17;123(16):2518-29. doi: 10.1182/blood-2013-02-486688. Epub 2014 Mar 3.

Zebrafish eaf1 Suppresses foxo3b Expression to Modulate Transcriptional Activity of gata1 and spi1 in Primitive Hematopoiesis. Hu B, Zhang W, Feng X, Ji W, Xie X, Xiao W. Dev Biol. 2014 Apr 1;388(1):81-93. doi: 10.1016/j.ydbio.2014.01.005. Epub 2014 Jan 17.

Elavl1a regulates zebrafish erythropoiesis via post-transcriptional control of gata1. Li X, Lu YC, Dai K, Torregroza I, Hla T, Evans T. Blood. 2014 Feb 27;123(9):1384-92. doi: 10.1182/blood-2013-09-526962. Epub 2014 Jan 14.

TET2 plays an essential role in erythropoiesis by regulating lineage-specific genes via DNA oxidative demethylation in a zebrafish model. Ge L, Zhang RP, Wan F, Guo DY, Wang P, Xiang LX, Shao JZ. Mol Cell Biol. 2014 Mar;34(6):989-1002. doi: 10.1128/MCB.01061-13. Epub 2014 Jan 6.

Therapeutic antagonists of microRNAs deplete leukemia-initiating cell activity. Velu CS, Chaubey A, Phelan JD, Horman SR, Wunderlich M, Guzman ML, Jegga AG, Zeleznik-Le NJ, Chen J, Mulloy JC, Cancelas JA, Jordan CT, Aronow BJ, Marcucci G, Bhat B, Gebelein B, Grimes HL. J Clin Invest. 2014 Jan 2;124(1):222-36. doi: 10.1172/JCI66005. Epub 2013 Dec 16.

Citations for Morpholino work with Ebola or Marburg filovirus

Following are citations of Ebola or Marburg filovirus drug development using Morpholino antisense oligos.


Enterlein S, Warfield KL, Swenson DL, Stein DA, Smith JL, Gamble CS, Kroeker AD, Iversen PL, Bavari S, Muhlberger E. VP35 Knockdown Inhibits Ebola Virus Amplification and Protects against Lethal Infection in Mice. Antimicrob Agents Chemother. 2006 Mar;50(3):984-93.

Phosphorodiamidate morpholino oligomers (PMO) are a class of uncharged single-stranded DNA analogs modified such that each subunit includes a phosphorodiamidate linkage and morpholine ring. PMO antisense agents have been reported to effectively interfere with the replication of several positive-strand RNA viruses in cell culture. The filoviruses, Marburg virus and Ebola virus (EBOV), are negative-strand RNA viruses that cause up to 90% lethality in human outbreaks. There is currently no commercially available vaccine or efficacious therapeutic for any filovirus. In this study, PMO conjugated to arginine-rich cell-penetrating peptide (P-PMO) and nonconjugated PMO were assayed for the ability to inhibit EBOV infection in cell culture and in a mouse model of lethal EBOV infection. A 22-mer P-PMO designed to base pair with the translation start site region of EBOV VP35 positive-sense RNA generated sequence-specific and time- and dose-dependent inhibition of EBOV amplification in cell culture. The same oligomer provided complete protection to mice when administered before or after an otherwise lethal infection of EBOV. A corresponding nonconjugated PMO, as well as nonconjugated truncated versions of 16 and 19 base residues, provided length-dependent protection to mice when administered prophylactically. Together, these data suggest that antisense PMO and P-PMO have the potential to control EBOV infection and are promising therapeutic candidates.

mice and cell culture


Warfield KL, Swenson DL, Olinger GG, Nichols DK, Pratt WD, Blouch R, Stein DA, Aman MJ, Iversen PL, Bavari S. Gene-Specific Countermeasures against Ebola Virus Based on Antisense Phosphorodiamidate Morpholino Oligomers. PLoS Pathog. 2006 Jan;2(1):e1. Epub 2006 Jan 13.

The filoviruses Marburg virus and Ebola virus (EBOV) quickly outpace host immune responses and cause hemorrhagic fever, resulting in case fatality rates as high as 90% in humans and nearly 100% in nonhuman primates. The development of an effective therapeutic for EBOV is a daunting public health challenge and is hampered by a paucity of knowledge regarding filovirus pathogenesis. This report describes a successful strategy for interfering with EBOV infection using antisense phosphorodiamidate morpholino oligomers (PMOs). A combination of EBOV-specific PMOs targeting sequences of viral mRNAs for the viral proteins (VPs) VP24, VP35, and RNA polymerase L protected rodents in both pre- and post-exposure therapeutic regimens. In a prophylactic proof-of-principal trial, the PMOs also protected 75% of rhesus macaques from lethal EBOV infection. The work described here may contribute to development of designer, \"druggable\" countermeasures for filoviruses and other microbial pathogens.

Rhesus macaque, mouse, guinea pig, Vero cells


Vander-Linden CL. Gene-Specific Ebola Therapies Protect Nonhuman Primates from Lethal Diseases. Chem-Bio Defense Quarterly. 2006 3(2):8-9.

Also this press release from USAMRIID:


Silvestri LS, Ruthel G, Kallstrom G, Warfield KL, Swenson DL, Nelle T, Iversen PL, Bavari S, Aman MJ. Involvement of Vacuolar Protein Sorting Pathway in Ebola Virus Release Independent of TSG101 Interaction. J Infect Dis. 2007 Nov 15;196 Suppl 2:S264-70.

Budding of Ebola virus (EBOV) particles from the plasma membrane of infected cells requires viral and host proteins. EBOV virus matrix protein VP40 recruits TSG101, an ESCRT-1 (host cell endosomal sorting complex required for transport-1) complex protein in the vacuolar protein sorting (vps) pathway, to the plasma membrane during budding. Involvement of other vps proteins in EBOV budding has not been established. Therefore, we used VP40 deletion analysis, virus-like particle-release assays, and confocal microscopy to investigate the potential role of ESCRT-1 proteins VPS4, VPS28, and VPS37B in EBOV budding. We found that VP40 could redirect each protein from endosomes to the cell surface independently of TSG101 interaction. A lack of VPS4 adenosine triphosphatase activity reduced budding by up to 80%. Inhibition of VPS4 gene expression by use of phosphorodiamidite morpholino antisense oligonucleotides protected mice from lethal EBOV infection. These data show that EBOV can use vps proteins independently of TSG101 for budding and reveal VPS4 as a potential target for filovirus therapeutics.


Swenson DL, Warfield KL, Warren TK, Lovejoy C, Hassinger JN, Ruthel G, Blouch RE, Moulton HM, Weller DD, Iversen PL, Bavari S. Chemical modifications of antisense morpholino oligomers enhance their efficacy against ebolavirus infection. Antimicrob Agents Chemother. 2009 Feb 17. [Epub ahead of print].

Phosphorodiamidate morpholino oligomers (PMO) are uncharged nucleic acid-like molecules designed to inactivate specific gene expression via antisense-based steric hindrance of mRNA translation. PMOs have been successful in knockdown of viral gene expression and replication in the case of acute viral infections in animal models and are well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents to combat filoviral infections. Previously we have shown that mice treated with a PMO complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebolavirus challenge. In the present study, we report on the ability of two additional VP24-specific PMOs to reduce cell-free translation of a VP24 reporter, to inhibit in vitro replication of Ebolavirus, and to protect mice against lethal challenge when delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of variable length and composition, positioning of conjugated peptides to either the 5' or 3' terminus, and charge modifications conferred by addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and in mice during lethal Ebolavirus challenge.

mice, cell cultures


Iversen PL, Warren TK, Wells JB, Garza NL, Mourich DV, Welch LS, Panchal RG, Bavari S. Discovery and Early Development of AVI-7537 and AVI-7288 for the Treatment of Ebola Virus and Marburg Virus Infections. Viruses. 2012;4(11):2806-2830. doi:10.3390/v4112806.

There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates.


Heald AE, Iversen PL, Saoud JB, Sazani P, Charleston JS, Axtelle T, Wong M, Smith WB, Vutikullird A, Kaye E. Safety and Pharmacokinetic Profiles of Phosphorodiamidate Morpholino Oligomers with Activity against Ebola Virus and Marburg Virus: Results of Two Single Ascending Dose Studies. Antimicrob Agents Chemother. 2014 Aug 25. pii: AAC.03442-14. [Epub ahead of print]

Two identical single ascending dose (SAD) studies evaluated the safety and pharmacokinetics (PK) of AVI-6002 and AVI-6003, two experimental combinations of phosphorodiamidate morpholino oligomers with positive charges (PMOplus®) that target viral messenger RNA (mRNA) encoding Ebola virus and Marburg virus proteins, respectively. Both AVI-6002 and AVI-6003 were found to suppress disease in virus-infected nonhuman primates (NHPs) in previous studies. AVI-6002 (a combination of AVI-7537 and AVI-7539) or AVI-6003 (a combination of AVI-7287 and AVI-7288) were administered as sequential intravenous (IV) infusions of a 1:1 fixed dose ratio of the two subcomponents. In each study, 30 healthy male and female subjects between 18 and 50 years of age were enrolled in 6 dose escalation cohorts of 5 subjects each and received a single IV infusion of active study drug (0.005, 0.05, 0.5, 1.5, 3, and 4.5 mg/kg per component) or placebo in a 4:1 ratio. Both AVI-6002 and AVI-6003 were safe and well tolerated at the doses studied. A maximum tolerated dose (MTD) was not observed in either study. The four chemically similar PMOplus components exhibited generally similar PK profiles. The mean peak plasma concentration (Cmax) and area under the concentration curve (AUC) values of the four components exhibited dose-proportional PK. The estimated plasma half-life of all four components was 2 to 5 hours. The safety of the two combinations and the PK of the four components were similar, regardless of the target RNA sequence.

human iv infusion


Patrick Reid S, Shurtleff AC, Costantino JA, Tritsch SR, Retterer C, Spurgers KB, Bavari S. HSPA5 is an essential host factor for Ebola virus infection. Antiviral Res. 2014 Sep;109:171-4. doi: 10.1016/j.antiviral.2014.07.004. Epub 2014 Jul 11.

Development of novel strategies targeting the highly virulent ebolaviruses is urgently required. A proteomic study identified the ER chaperone HSPA5 as an ebolavirus-associated host protein. Here, we show using the HSPA5 inhibitor (-)- epigallocatechin gallate (EGCG) that the chaperone is essential for virus infection, thereby demonstrating a functional significance for the association. Furthermore, in vitro and in vivo gene targeting impaired viral replication and protected animals in a lethal infection model. These findings demonstrate that HSPA5 is vital for replication and can serve as a viable target for the design of host-based countermeasures.


Clinical trials

APC Is an RNA-Binding Protein, and Its Interactome Provides a Link to Neural Development and Microtubule Assembly

Preitner N, Quan J, Nowakowski DW, Hancock ML, Shi J, Tcherkezian J, Young-Pearse TL, Flanagan JG. APC Is an RNA-Binding Protein, and Its Interactome Provides a Link to Neural Development and Microtubule Assembly. Cell. 2014 Jul 17;158(2):368-82. doi: 10.1016/j.cell.2014.05.042.

This paper reports rat brain in utero injection then electroporation of Morpohlinos as well as mouse experiments with peptide nucleic acids.

Oligos targeted to trigger nonsense-mediated decay (NMD)

This paper provides some experimental support for targeting steric-blocking oligos to alter splicing and trigger nonsense-mediated decay. The paper compared uniform 2'-methoxyethyl phosphorothioate oligos with 2'-MOE phosphorothioate gapmers (steric blocking versus RNase-H competent, respectively).

Nonsense-mediated decay as a terminating mechanism for antisense oligonucleotides.
Ward AJ, Norrbom M, Chun S, Bennett CF, Rigo F.
Nucleic Acids Res. 2014 May 1;42(9):5871-9. doi: 10.1093/nar/gku184. Epub 2014 Mar 3.

Intranasal administration in mice of peptide-conjugated Morpholinos

Peptide-conjugated Morpholinos were administered intranasally to mice to alter the immune response to subsequent intranasal influenza doses. "BALB/c mice were anesthetized with ketamine, followed by i.n. administration with 100 mg (approximately 5 mg/kg) PPMO (in 40 ml of PBS)." Repeat dosing at 24 hours, influenza inoculation at 48 hours.

"TRIM6 expression was silenced in the lungs of mice via peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO). In line with our results from hDCs and cell lines, the designed TRIM6 PPMOs efficiently and specifically downregulated TRIM6 protein levels in MEFs, resulting in a reduction of ISG54 protein expression upon IFN-b treatement."

"Treatment with PPMOs resulted in 50% reduction in TRIM6 mRNA and protein compared to nontargeting PPMO control in the lungs of infected and noninfected mice."

Rajsbaum R, Versteeg GA, Schmid S, Maestre AM, Belicha-Villanueva A, Martínez-Romero C, Patel JR, Morrison J, Pisanelli G, Miorin L, Laurent-Rolle M, Moulton HM, Stein DA, Fernandez-Sesma A, tenOever BR, García-Sastre A. Unanchored K48-Linked Polyubiquitin Synthesized by the E3-Ubiquitin Ligase TRIM6 Stimulates the Interferon-IKKε Kinase-Mediated Antiviral Response. Immunity. 2014;[Epub ahead of print] doi:10.1016/j.immuni.2014.04.018

Knockdowns in mosquito guts

Vivo-Morpholinos were fed to mosquitos. The oligos decreased the amount of MAPK protein in the midgut and the number of Plasmodium oocysts in the mudgut.

"The feeding protocol described in the present study provides several advantages over current methods for gene knockdown in insects. The method is easy to apply, requires no special skills for delivery, and is highly target specific. ... This protocol should therefore be considered as an efficient alternative in studies requiring target specific protein knockdown in mosquitoes and other arthropods."

Pietri JE, Cheung KW, Luckhart S. Knockdown of mitogen-activated protein kinase (MAPK) signalling in the midgut of Anopheles stephensi mosquitoes using antisense morpholinos. Insect Mol Biol. 2014 May 28. doi: 10.1111/imb.12103. [Epub ahead of print]

Morpholino electroporated into human T-cells induces alternative splicing without interferon response

Michel M, Wilhelmi I, Schultz A-S, Preussner M, Heyd F. Activation-induced Tumor Necrosis Factor Receptor-associated Factor 3 (Traf3) Alternative Splicing Controls the Noncanonical Nuclear Factor κB Pathway and Chemokine Expression in Human T Cells. J Biol Chem. 2014;289:13651-60. doi:10.1074/jbc.M113.526269

"Furthermore, the interferon response, which can be regulated by Traf3 (35), was not altered in Traf3E8 MO-treated cells (data not shown)" supports that the Morpholino did not induce an innate immune response.


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