Jon-D's blog

From the paper:
Lambert JD, Johnson AB, Hudson CN, Chan A. Dpp/BMP2-4 Mediates Signaling from the D-Quadrant Organizer in a Spiralian Embryo. Curr Biol. 2016;[Epub ahead of print] doi:10.1016/j.cub.2016.05.059
http://www.cell.com/current-biology/abstract/S0960-9822(16)30556-5

From Supplemental Information: Supplemental Experimental Procedures: Injection and knockdown with antisense morpholino oligos:
"We have made some improvements to our previously reported injection protocol [S2,
S4]. First, we find that most MOs we use, including IoDppMO3 and 4, will lose activity
2-3 months after they are resuspended, but can be reactivated by autoclaving.
Similarly, we have stopped heating the morpholinos and fluorescent dye before
injection, because this can hasten evaporation and higher concentrations of either MO
stock or the fluorescent dye can cause the MO to lose activity when injected. Especially
with these improvements, we continue to find MOs to be highly reproducible and
invaluable tools for probing gene function in Ilyanassa."

There's a Morpholino special issue of the Austin Journal of Drug Discovery, Development and Delivery.
You may have to click “Antisense Drug Research and Development” to open the list of the papers.

Antisense antimicrobial therapeutics.

Sully EK, Geller BL.

Curr Opin Microbiol. 2016 Jun 29;33:47-55. doi: 10.1016/j.mib.2016.05.017. [Epub ahead of print] Review.

http://www.sciencedirect.com/science/article/pii/S1369527416300807

(open-access)

A Morpholino user asked me about his oligo design, which I had marked with a Tm of 82.5°C. He wondered about efficacy and specificity of such an oligo. Here's my response.

I think you can get pretty good activity from an oligo with a Tm of 80°C. I mark oligos with Tm of 80°C to 85°C as "fairly low" or "moderately low" RNA affinity so that when deciding between two good oligo designs, you might take the one without a fairly/moderately low Tm note. If I didn't mark it, there would be no way to tell between a very good oligo and a pretty good oligo. I don't start worrying until the Tm is down in the 70s, and those get a "very low" or "quite low" Tm note (at that point I am trying to warn people away). I won't send out oligo designs in the 60s or less. Once an oligo Tm is much over 100°C, I'll include a note saying that this might be too high an RNA affinity for good target specificity.

Since your oligo has a Tm at the lower end of the good range, that is good for the oligo specificity. More bases will need to pair to a target RNA before the oligo has any activity.

Here's a review I'd like to keep track of.

Splice-switching antisense oligonucleotides as therapeutic drugs.

Havens MA, Hastings ML.

Nucleic Acids Res. 2016 Jun 10. pii: gkw533. [Epub ahead of print]

http://nar.oxfordjournals.org/content/early/2016/06/10/nar.gkw533.long

This was written to a researcher in response to his question about timing of assays in a translation-blocking experiment.

-----------------------------------------------

Since you are using a translation blocker, the optimum delay between treatment and analysis will be strongly affected by the turnover time of the protein. It will also be affected by the delivery technique used to get the Morpholino into the cytosol. Rapid techniques like electroporation or microinjection will get the Morpholino in quickly and start the knockdown immediately, while endocytosis-mediated techniques like Endo-Porter or Vivo-Morpholinos might take a day to achieve sufficient cytosolic Morpholino concentrations for a good knockdown. Once translation is suppressed, the preexisting protein is degrading without replacement. However, some proteins are very stable and might have half-lives of days. Rapid-turnover proteins like most transcription factors will disappear quickly, perhaps in hours.

If you are targeting cells in culture using an unmodified Morpholino with Endo-Porter as an endosomal escape agent, and you are targeting a protein with a one-day half-life, I'd try about three days between oligo delivery and measurement of the protein. That gives a day for delivery and two half-lives for protein degradation.

If the protein is especially stable, it might take another delivery of Morpholino on day four to keep translation suppressed long enough for most of the protein to degrade.

I think you can tell by my tone that all of these estimates are pretty rough. Still, if you consider delivery and half-life I think you can find an effective time course within a few experiments.

I'm headed for the American Association for Immunologists conference (AAI 2016). I assembled a citation list of many of the immunology papers published over the last couple of years. Here it is.

Some Morpholino Citations for Immunology (AAI 2016)

Furukawa R, Tamaki K, Kaneko H. Two macrophage migration inhibitory factors regulate starfish larval immune cell chemotaxis. Immunol Cell Biol. 2016 Apr;94(4):315-21. doi: 10.1038/icb.2016.6. Epub 2016 Feb 2. Patiria pectinifera (sea star)

Salewsky B, Hildebrand G, Rothe S, Parplys AC, Radszewski J, Kieslich M, Wessendorf P, Krenzlin H, Borgmann K, Nussenzweig A, Sperling K, Digweed M. Directed Alternative Splicing in Nijmegen Breakage Syndrome: Proof of Principle Concerning Its Therapeutical Application. Mol Ther. 2016 Feb;24(1):117-24. doi: 10.1038/mt.2015.144. Epub 2015 Aug 12. Mouse Vivo-Morpholino

Dewaele M, Tabaglio T, Willekens K, Bezzi M, Teo SX, Low DHP, Koh CM, Rambow F, Fiers M, Rogiers A, Radaelli E, Al-Haddawi M, Tan SY, Hermans E, Amant F, Yan H, Lakshmanan M, Koumar RC, Lim ST, Derheimer FA, Campbell RM, Bonday Z, Tergaonkar V, Shackleton M, Blattner C, Marine J-C, Guccione E. Antisense oligonucleotide-mediated MDM4 exon 6 skipping impairs tumor growth. J Clin Invest. 2016;126(1):68-84. doi:10.1172/JCI82534. Cell culture MDM4-expressing melanoma cell lines, diffuse, large B cell lymphoma DLBCL cells, mice

-----------------------------------2015-----------------------------------

Martinez NM, Agosto L, Qiu J, Mallory MJ, Gazzara MR, Barash Y, Fu XD, Lynch KW. Widespread JNK-dependent alternative splicing induces a positive feedback loop through CELF2-mediated regulation of MKK7 during T-cell activation. Genes Dev. 2015 Oct 1;29(19):2054-66. doi: 10.1101/gad.267245.115. cell culture: Jurkat T cells.

Hartley JM, Chu T-W, Peterson EM, Zhang R, Yang J, Harris J, Kopecek J. Super-Resolution Imaging and Quantitative Analysis of Membrane Protein/Lipid Raft Clustering Mediated by Cell Surface Self-Assembly of Hybrid Nanoconjugates. ChemBioChem. 2015 Aug 17;16(12):1725-9. doi: 10.1002/cbic.201500278. Epub 2015 Jul 2. Cell culture: Human Burkitt’s B-cell non-Hodgkin’s lymphoma Raji cell line

Ridnour LA, Cheng RY, Weiss JM, Soto-Pantoja DR, Basudhar D, Heinecke JL, Stewart A, DeGraff WG, Sowers A, Thetford A, Kesarwala AH, Roberts DD, Young HA, Mitchell JB, Trinchieri G, Wiltrout RH, Wink DA. Nitric Oxide Synthase Inhibition Modulates Immune Polarization and Improves Radiation-Induced Tumor Growth Delay. Cancer Res. 2015 Jul 15;75(14):2788-99. doi: 10.1158/0008-5472.CAN-14-3011. Epub 2015 May 19. cell culture: Raw 267.4, mouse

Curtis AM, Fagundes CT, Yang G, Palsson-McDermott EM, Wochal P, McGettrick AF, Foley NH, Early JO, Chen L, Zhang H, Xue C, Geiger SS, Hokamp K, Reilly MP, Coogan AN, Vigorito E, FitzGerald GA, O'Neill LA. Circadian control of innate immunity in macrophages by miR-155 targeting Bmal1. Proc Natl Acad Sci U S A. 2015 Jun 9;112(23):7231-6. doi: 10.1073/pnas.1501327112. Epub 2015 May 20. cell culture: bone-marrow-derived macrophage (BMDM); Endo-Porter

Mino T, Murakawa Y, Fukao A, Vandenbon A, Wessels HH, Ori D, Uehata T, Tartey S, Akira S, Suzuki Y, Vinuesa CG, Ohler U, Standley DM, Landthaler M, Fujiwara T, Takeuchi O. Regnase-1 and Roquin Regulate a Common Element in Inflammatory mRNAs by Spatiotemporally Distinct Mechanisms. Cell. 2015 May 21;161(5):1058-73. doi: 10.1016/j.cell.2015.04.029. cell culture: HeLa, murine bone marrow macrophages; Endo-Porter

Shi X, He BL, Ma AC, Guo Y, Chi Y, Man CH, Zhang W, Zhang Y, Wen Z, Cheng T, Leung AY. Functions of idh1 and its mutation in the regulation of developmental hematopoiesis in zebrafish. Blood. 2015 May 7;125(19):2974-84. doi: 10.1182/blood-2014-09-601187. Epub 2015 Mar 16. Zebrafish

Ruiz-González I, Minten M, Wang X, Dunlap K, Bazer FW. Involvement of TLR7 and TLR8 in Conceptus Development and Establishment of Pregnancy in Sheep. Reproduction. 2015 Apr;149(4):305-16. doi: 10.1530/REP-14-0537. Epub 2015 Jan 19. Ovis aries (sheep)

Chu TW, Zhang R, Yang J, Chao MP, Shami PJ, Kopeček J. A Two-Step Pretargeted Nanotherapy for CD20 Crosslinking May Achieve Superior Anti-Lymphoma Efficacy to Rituximab. Theranostics. 2015;5(8):834-46. doi:10.7150/thno.12040. cell culture: patient mantle cell lymphoma

Rolig AS, Parthasarathy R, Burns AR, Bohannan BJM, Guillemin K. Individual Members of the Microbiota Disproportionately Modulate Host Innate Immune Responses. Cell Host Microbe. 2015;18(5):613-20. doi:10.1016/j.chom.2015.10.009. Zebrafish

Yang S, Marín-Juez R, Meijer AH, Spaink HP. Common and specific downstream signaling targets controlled by Tlr2 and Tlr5 innate immune signaling in zebrafish. BMC Genomics. 2015;16:547 doi:10.1186/s12864-015-1740-9. Zebrafish

Cator LJ, Pietri JE, Murdock CC, Ohm JR, Lewis EE, Read AF, Luckhart S, Thomas MB. Immune response and insulin signalling alter mosquito feeding behaviour to enhance malaria transmission potential. Sci Rep. 2015;5:11947
doi:10.1038/srep11947. Anopheles stephensi (mosquito), Vivo-Morpholinos in feed

Benard EL, Racz PI, Rougeot J, Nezhinsky AE, Verbeek FJ, Spaink HP, Meijer AH. Macrophage-Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti-Bacterial Function in Zebrafish. J Innate Immun. 2015;7(2):136-52. doi: 10.1159/000366103. Epub 2014 Sep 19. Zebrafish

-----------------------------------2014-----------------------------------

Chu TW, Kosak KM, Shami PJ, Kopeček J. Drug-Free Macromolecular Therapeutics Induce Apoptosis of Patient Chronic Lymphocytic Leukemia Cells. Drug Deliv Transl Res. 2014 Dec;4(5-6):389-394. cell culture: human chronic lymphocytic leukemia cells

Soto-Pantoja DR, Terabe M, Ghosh A, Ridnour LA, DeGraff WG, Wink DA, Berzofsky JA, Roberts DD. CD47 in the Tumor Microenvironment Limits Cooperation between Antitumor T-cell Immunity and Radiotherapy. Cancer Res. 2014 Dec 1;74(23):6771-83. doi: 10.1158/0008-5472.CAN-14-0037-T. Epub 2014 Oct 8. Mouse

Varela M, Romero A, Dios S, van der Vaart M, Figueras A, Meijer AH, Novoa B. Cellular visualization of macrophage pyroptosis and interleukin-1β release in a viral hemorrhagic infection in zebrafish larvae. J Virol. 2014 Oct;88(20):12026-40. doi: 10.1128/JVI.02056-14. Epub 2014 Aug 6. Zebrafish

Knox BP, Deng Q, Rood M, Eickhoff JC, Keller NP, Huttenlocher A. Distinct Innate Immune Phagocyte Responses to Aspergillus fumigatus Conidia and Hyphae in Zebrafish Larvae. Eukaryot Cell. 2014 Oct;13(10):1266-77. doi: 10.1128/EC.00080-14. Epub 2014 May 30. Zebrafish

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

Nazmi A, Mukherjee S, Kundu K, Dutta K, Mahadevan A, Shankar SK, Basu A. TLR7 is a key regulator of innate immunity against Japanese Encephalitis Virus infection. Neurobiol Dis. 2014 Sep;69:235-47. doi: 10.1016/j.nbd.2014.05.036. Epub 2014 Jun 5. Mouse

Ouyang C, Nie L, Gu M, Wu A, Han X, Wang X, Shao J, Xia Z. TGF-β-activated kinase 1 (TAK1) Activation Requires Phosphorylation of Serine 412 by Protein Kinase A Catalytic Subunit α (PKACα) and Protein Kinase X (PRKX). J Biol Chem. 2014 Aug 29;289(35):24226-37. doi: 10.1074/jbc.M114.559963. Epub 2014 Jul 15. Zebrafish

Marín-Juez R, Jong-Raadsen S, Yang S, Spaink HP. Hyperinsulinemia induces insulin resistance and immune suppression via Ptpn6/Shp1 in zebrafish. J Endocrinol. 2014 Aug;222(2):229-41. doi: 10.1530/JOE-14-0178. Epub 2014 Jun 5. 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. miR-142-3p acts as an essential modulator of neutrophil development in zebrafish. Blood. 2014 Aug 21;124(8):1320-30. doi: 10.1182/blood-2013-12-545012. Epub 2014 Jul 2. Zebrafish

Elks PM, van der Vaart M, van Hensbergen V, Schutz E, Redd MJ, Murayama E, Spaink HP, Meijer AH. Mycobacteria counteract a TLR-mediated nitrosative defense mechanism in a zebrafish infection model. PLoS One. 2014 Jun 26;9(6):e100928. doi: 10.1371/journal.pone.0100928. eCollection 2014. Zebrafish

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

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

Fink IR, Benard EL, Hermsen T, Meijer AH, Forlenza M, Wiegertjes GF. Molecular and functional characterization of the scavenger receptor CD36 in zebrafish and common carp. Mol Immunol. 2015 Feb;63(2):381-93. doi: 10.1016/j.molimm.2014.09.010. Epub 2014 Oct 11. Zebrafish

Cambier CJ, Takaki KK, Larson RP, Hernandez RE, Tobin DM, Urdahl KB, Cosma CL, Ramakrishnan L. Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids. Nature. 2014 Jan 9;505(7482):218-222. doi: 10.1038/nature12799. Epub 2013 Dec 15. Zebrafish

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. Therapeutic antagonists of microRNAs deplete leukemia-initiating cell activity. J Clin Invest. 2014 Jan 2;124(1):222-36. doi: 10.1172/JCI66005. Epub 2013 Dec 16. cell culture: c-Kit+ CD45.1+ CD45.2+ MLL-AF9-initiated leukemia cells; Vivo-Morpholino

Hepburn L, Prajsnar TK, Klapholz C, Moreno P, Loynes CA, Ogryzko NV, Brown K, Schiebler M, Hegyi K, Antrobus R, Hammond KL, Connolly J, Ochoa B, Bryant C, Otto M, Surewaard B, Seneviratne SL, Grogono DM, Cachat J, Ny T, Kaser A, Török ME, Peacock SJ, Holden M, Blundell T, Wang L, Ligoxygakis P, Minichiello L, Woods CG, Foster SJ, Renshaw SA, Floto RA. A Spaetzle-like role for nerve growth factor β in vertebrate immunity to Staphylococcus aureus. Science. 2014;346(6209):641-6. doi:10.1126/science.1258705. Zebrafish

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. Cell culture: jsl1 t-cells

van der Vaart M, Korbee CJ, Lamers GEM, Tengeler AC, Hosseini R, Haks MC, Ottenhoff THM, Spaink HP, Meijeremail AH. The DNA Damage-Regulated Autophagy Modulator DRAM1 Links Mycobacterial Recognition via TLP-MYD88 to Authophagic Defense. Cell Host Microbe. 2014;(15)6:753-67. doi:10.1016/j.chom.2014.05.005. Zebrafish

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

Yang F, Xiong J, Jia X-E, Gu Z-H, Shi J-Y, Zhao Y, Li J-M, Chen S-J, Zhao W-L. GSTT1 Deletion Is Related to Polycyclic Aromatic Hydrocarbons-Induced DNA Damage and Lymphoma Progression. PLoS ONE. 2014;9(2):e89302. doi:10.1371/journal.pone.0089302. Zebrafish

My open-access review paper was just published:
Moulton JD. Guide for Morpholino Users: Toward Therapeutics. Austin Journal of Drug Discovery, Development and Delivery. 2016;3(2):1023.
http://austinpublishinggroup.com/drug-development/download.php?file=full...

Review
Advances in the Study of Heart Development and Disease Using Zebrafish

Daniel R. Brown, Leigh Ann Samsa, Li Qian, Jiandong Liu.
J. Cardiovasc. Dev. Dis. 2016, 3(2), 13; doi: 10.3390/jcdd3020013

http://www.mdpi.com/2308-3425/3/2/13/htm

Keyword search "Morpholino" or go to section "3.1.2. Morpholinos" for a brief discussion of Morpholino efficacy, specificity and controls.

There are a few points in this discussion (3.1.2. Morpholinos) where I disagree, others where I would like to expand on their suggestions.

First, the authors write that Morpholinos are designed to bind to translation-initiation sites of mRNA. We have made many successful oligos targeting upstream of the translation initiation AUG; Morpholinos targeting the 5'-UTR block the scanning complex from completing its journey from the 5'-cap to the start codon.

The authors claim that Morpholinos are slowly degraded by cellular processes. However, when this was explicitly tested no degradation was found [1,2]. Morpholino activity does decrease with time; we hypothesize that when a Morpholino binds an RNA and the RNA is degraded by nucleases, a footprint of RNA is protected bound to the Morpholino. The slow degradation of the footprint and release of single-stranded Morpholino activity explains the persistent low-level splice-modifying activity observed months after Morpholino dosing [3].

In Box 2, the Specificity section mentions several control Morpholinos. In section 4, they list the standard control, the 5-base pair mismatch, and the p53 oligo. I've comments about each.

Standard control: This is an oligo targeting an intronic site in human beta-globin that is mutated in some cases of beta-thalaslemia. It has been extensively used as a negative control for developmental biology, so extensively that some experienced Morpholino users have suggested there is no reason to ever inject it again. I agree, but results from your reviewers may vary.

Five base pair mismatch: This is an old-style specificity control oligo, but I think a better control for specificity is to use a second non-overlapping Morpholino to phenocopy the first oligo's results. Sometimes there is phenotypic bleed-through with the five-mispair oligo. On the whole, I'd be happier if everyone used the second targeting oligo approach.

p53 oligo: this is a very important control, used to determine whether the p53-mediated apoptotic cascade is triggered by loss of a protein [4]. The Morpholino results in a zebrafish paper are stronger if, along with reporting the outcome on injecting a targeting oligo, the results of a p53 oligo co-injected with the targeting oligo are also reported.

[1] Hudziak RM, Barofsky E, Barofsky DF, Weller DL, Huang SB, Weller DD. Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acid Drug Dev 1996 Winter;6(4):267-72.

[2] Youngblood DS, Hatlevig SA, Hassinger JN, Iversen PL, Moulton HM. Stability of cell-penetrating Peptide-morpholino oligomer conjugates in human serum and in cells. Bioconjug Chem. 2007 Jan-Feb;18(1):50-60.

[3] Wells DJ. Gene doping: the hype and the reality. Br J Pharmacol. 2008 Jun;154(3):623-31. Epub 2008 Apr 21.

[4] Robu ME, Larson JD, Nasevicius A, Beiraghi S, Brenner C, Farber SA, Ekker SC. p53 activation by knockdown technologies. PLoS Genet. 2007 May 25;3(5):e78. Epub 2007 Apr 10.

Makino S, Mishima Y, Inoue K, Inada T. Roles of mRNA fate modulators Dhh1 and Pat1 in TNRC6-dependent gene silencing recapitulated in yeast. J Biol Chem. 2015 Mar 27;290(13):8331-47. doi: 10.1074/jbc.M114.615088. Epub 2015 Feb 5.
http://www.jbc.org/content/290/13/8331.long

"To address this question, we injected GFP reporter mRNA into zebrafish embryos and then blocked its translation using a morpholino oligomer (ATG MO) that specifically masks the start codon of the GFP ORF."