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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.

http://nar.oxfordjournals.org/content/42/9/5871.long

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]

http://onlinelibrary.wiley.com/doi/10.1111/imb.12103/abstract

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.

Vivo-Morpholino in rat brain

Shen H-w, Scofield MD, Boger H, Hensley M, Kalivas PW. Synaptic Glutamate Spillover Due to Impaired Glutamate Uptake Mediates Heroin Relapse. J Neurosci. 2014;34(16):5649-57 doi:10.1523/JNEUROSCI.4564-13.2014

http://www.jneurosci.org/content/34/16/5649.abstract

Vivo-Morpholinos targeting the glial excitatory amino acid transporter 2 (GLT-1) were administered to adult male Sprague Dawley rats by infusion through microdialysis guide cannulas (26 ga) implanted bilaterally in their nucleus accumbens (position details in paper). Vivo-Morpholino stocks (0.5 mM) were diluted 50-fold in PBS. 1 ul was infused at 0.5 ul/min though 33ga microinjectors extending 2mm below the cannula, with the injectors left in place for 60 seconds after infusion was completed. Injections were done once per day for three days and GLT-1 assessed from tissue taken seven days later. Their protocol was shown to reduce GLT-1 expression by 56% (+/- 10%) in the nucleus accumbens seven days after the last infusion.

Nanoparticle activated by near-IR delivers and cleaves Photo-Morpholino

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

http://www.cell.com/current-biology/abstract/S0960-9822%2812%2901327-9

Nanoparticles of about 30 nm diameter were constructed based on β-NaYF4 crystals doped with Yb3+ and Tm3+ inside and Yb3+ and Er3+ in the shell. When illuminated with a 980nm IR laser, the particles emitted in the visible and UV bands. The particles were coated with mesoporous silica and loaded with TPPS2a (mesotetraphenylporphine with two sulfonate groups on adjacent phenyl rings) and Photo-Morpholino duplexes. The TPPS2a is a photosensitizer for photochemical internalization and the Photo-Morpholino duplexes consisted of antisense Morpholino targeting STAT3 and a complementary sense Photo-Morpholino strand to cage the antisense activity.

In B16F0 cells, which express STAT3, the STAT3 expression decreased to about 50% after incubation with nanoparticles and irradiation with the IR laser. Uptake was temperature dependent.

To set up a melanoma tumor model in mice, B16F0 cells were injected subcutaneously on day 0. Nanoparticles were injected day 5 and day 8 and tumors were irradiated with the IR laser. The treatment reduced the tumor volume relative to controls by about eightfold. The mice maintained their weight through the treatments.

I've skipped describing many controls and characterizations of materials. The bottom line is that their system delivered antisense activity to the IR-illuminated tumors. For more detail, see their fascinating paper.

Using a GFP reporter to test a knockdown

Romaker D, Kumar V, Cerqueira DM, Cox RM, Wessely O. MicroRNAs are critical regulators of tuberous sclerosis complex and mTORC1 activity in the size control of the Xenopus kidney. Proc Natl Acad Sci U S A. 2014 Apr 14. [Epub ahead of print]

The authors report several test constructs in which their upstream Morpholino target is ligated to a GFP coding sequence and then tested for its response to a Morpholino translation blocker. See figure S6 in the supporting online information.

http://www.pnas.org/content/early/2014/04/09/1320577111.abstract

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