We thank Anthony K. L. Leung of the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University for his technical help with designing the miR-181-sponge construct. We also thank Polina Sysa-Shah and Kathleen Gabrielson of the Department of Molecular and Comparative Pathobiology, Johns Hopkins Medical Institutions for their technical assistance by the in vivo imaging of the miRNA-Sponge delivery.
This work was supported by grants from the NIH, HL39752 (to Charles Steenbergen) and by a Scientist Development Grant from the American Heart Association 14SDG18890049 (to Samarjit Das). The rat cardio-specific promoter was generously provided by Jeffery D. Molkentin at the Cincinnati Children&#39;s Hospital.

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Johns Hopkins University.
1. Sponge Design
<ol>
	<li>MicroRNA binding 3&#8217;UTR 
	Note: A miRNA functions through specific base pairing interactions with partially complementary sites in the 3&#8217; untranslated region (UTR) of its target mRNAs (for a comprehensive review, see Bartel)15.
	<ol>
		<li>Design the inhibitors of miRNA expression as oligonucleotides that contain significant complementarity to the miRNA sequence. Sequester a miRNA in an oligonucleotide complex through extensive base pairing to block the miRNA function.</li>
	</ol>
	</li>
	<li>Design of a miRNA sponge
	<ol>
		<li>Design tandemly arrayed partially complementary miRNA sequences to include a bulge at the position normally cleaved by Argonaute 2. The bulge is positioned at nucleotides 9 - 12 of the mature miRNA sequence to prevent any RNA interference type cleavage and degradation of the sponge.</li>
		<li>If desired, design the decoy target sequence to bind to all miR-181 family members. Upon an examination and comparison of the miR-181 family miRNA sequences, find a common sequence at the 5&#8217;-side of the bulge containing 8 consecutive nucleotides, or find a sequence common to all 4 miR-181 family members on the 3&#8217;-side of the bulge of an additional 8 consecutive nucleotides. These 2 sequences represent the left and right half binding sites, respectively. 
		Note: The decoy sequence is a tandem array of the left and right half binding sites separated by a GGA spacer. Link together the 5&#8217;-end sequence and the 3&#8217;-side sequence (described in step 1.2.2) with a GGA spacer designed to create the bulge when annealed to a miRNA (as described in step 1.2.1). Repeat this 1 miRNA binding site 10x in the final miR-sponge construct.</li>
	</ol>
	</li>
	<li>Other miRNA sponge considerations 
	Note: In order to clone the sponge sequence into the recipient expression vector, restriction nuclease recognition sites must be included at each end of the sequence. An in silico digestion analysis should be used to confirm the non-specific cleavage of the sponge sequence by the chosen restriction enzymes and other restriction enzymes used in downstream cloning applications.
	<ol>
		<li>Add a double stop codon (TAA TAA) at the 5&#8217;-end of the synthetic 3&#8217;UTR, such that the miR-181-sponge sequence is not part of the coding sequence for an enhanced green fluorescent protein (EGFP).</li>
	</ol>
	</li>
	<li>Synthesis of the sponge DNA for cloning
	<ol>
		<li>Use a DNA synthesizer or employ a commercially available source to synthesize the final miRNA sponge sequence with restriction nuclease recognition sites in place for cloning. The sponge is shipped on a plasmid which can contain selectable markers for an amplification in bacteria. Further, the sponge can be amplified by a polymerase chain reaction (PCR) for cloning or simply dropped out the donor vector using the restriction sites described in step 1.3 (also, see step 3.1).</li>
	</ol>
	</li>
</ol>
2. Cloning the Recipient Vector to Include a Tissue-specific Promoter
Note: Use the pEGFP-C1 vector as the recipient vector for the expression cassette of the miRNA sponge. The pEGFP-C1 vector contains a reading frame for an EGFP driven by a cytomegalovirus (CMV) promoter. The CMV promoter is constitutively active in mammalian cells. If a tissue-specific promoter is required, the CMV promoter can be removed by digestion of AseI and NheI enzymes (see step 2.1). The plasmid contains an extensive multiple cloning site (MCS) as well as kanamycin (Kan) and neomycin (G418) markers for a selection in bacteria and a stable expression in mammalian cells, respectively.
<ol>
	<li>Removing the CMV promoter from pEGFP-C1
	<ol>
		<li>Make 50 &#956;L of digestion reaction containing 1x commercially available digest buffer suggested by the manufacture of the restriction enzymes used, 5 &#956;g of plasmid DNA (in water), and 5 &#956;L each of AseI and NheI enzymes. Add all components to a microcentrifuge tube and gently mix them by flicking it. Spin the tube for 10 s in a microcentrifuge to collect the reaction at the bottom. Incubate the reaction in a 37 &#176;C water bath for 15 min.</li>
		<li>Purify the digested linearized plasmid. Add 5x the reaction volume of column binding buffer (a proprietary mix of the appropriate binding buffer for the manufacture of the DNA purification columns used) and purify the reaction with a DNA purification column as described by the manufacturer. Elute with 25 &#956;L of elution buffer (water) added carefully to the center of the column.</li>
		<li>Gel purify the digested plasmid on 0.5% agarose gel as follows: add 5 &#956;L of loading buffer (50% glycerol-3x loading dye) to the eluted plasmid. Load the entire sample into 1 well on 0.5% agarose gel. Include a separate lane with 500 ng of uncut vector. Run it for 30 - 40 min until an adequate separation of cut and uncut plasmids is achieved.</li>
		<li>Purify the linearized plasmid as follows: visualize the DNA in the agarose gel on a UV light box. With a clean razor blade, carefully excise the band corresponding to the linear plasmid. 
		Note: The linear plasmid should run lower than the uncut plasmid and will appear as a single band at approximately 4.2 kb. The excised CMV promoter will appear as a light band at approximately 500 nucleotides (nt).</li>
		<li>Extract the DNA from the gel slice using a commercially available kit and elute the DNA from the column with 25 &#956;L of water.</li>
	</ol>
	</li>
	<li>Cloning a heart-specific promoter into pEGFP-C1 
	Note: The alpha-myosin heavy chain (&#945;-MHC) promoter has been previously characterized and demonstrated to direct robust levels of cardiac-restricted expression as described in the following Molkentin, Jobe, and Markham16. The next sections describe how to amplify the promoter of the vector for cloning.
	<ol>
		<li>Clone the &#945;-MHC promoter between elements +420 to -2934 relative to the transcription start site into the p40 plasmid16 by first designing PCR primers that flank this region and then the plasmid as a template for PCR. Amplify the promoter region using PCR primers previously described5. Forward and reverse PCR primers contain sequences of overlap to the digested ends of the pEGFP-C1 (step 2.1) to allow In-Fusion directed cloning. Consult the In-Fusion manual for a comprehensive explanation of the primer design for In-Fusion cloning. Primer sequences are found in Table 1.</li>
		<li>Prepare a 50 &#956;L PCR reaction, which contains 1 &#956;M of each primer and 10 ng of template DNA. Add an appropriate amount of PCR enzyme and dNTPs depending on the manufacture of the PCR reagents chosen. Perform PCR on a standard DNA cycling block. Perform a 3 min 98 &#176;C denaturing step, followed by 35 cycles of denaturing, annealing, and extension for 5, 30, and 120 s each, respectively. Include a final extension of 10 min at 72 &#176;C.</li>
		<li>Clean up the PCR and gel extraction. Upon completion of the PCR cycle, add 5x the reaction volume of the column binding buffer to the PCR reaction and purify it with a DNA purification column as described by the manufacturer. Elute the mixture with 25 &#956;L of elution buffer (water) added carefully to the center of the column.
		<ol>
			<li>Add 5 &#956;L of loading buffer [50% glycerol (3x) loading dye] to eluted plasmid. Load the entire sample into 1 well on 1% agarose gel. Run it for 30 - 40 min; visualize and excise the PCR product as described above (step 2.1.5).</li>
		</ol>
		</li>
		<li>Ligate the &#945;-MHC promoter with the pEGFP-C1 as follows: add the 10 &#956;L ligation reaction containing 1x commercially available ligation buffer, 2 &#956;L of purified PCR, and 1 &#956;L of linearized vector. Perform the ligation at 50 &#176;C for 15 min and then cool it on ice.</li>
		<li>Perform a bacterial transformation as follows: transfect 50 &#956;L of Competent Cells with 2.5 &#956;L of In-Fusion ligation mix by adding the components. Rest the Eppendorf tube on ice for 20 min, put it in a 42 &#176;C water bath for 40 s, and then place the tube on ice for 2 min.
		<ol>
			<li>Following the transformation, add 350 &#956;L of SOC media and grow cells at 37 &#176;C for 45 min. Plate 200 &#956;L of outgrowth solution on LB plates with Kan. Perform a colony PCR to identify Kan resistant cells which contain the &#945;-MHC promoter ligation. 
			Note: Screening primers were designed to PCR across the ligation junction.</li>
		</ol>
		</li>
		<li>Expand the PCR positive clones in LB-Kan broth and in plasmids purified using standard mini-prep plasmid purification protocols as described by the manufacturer.</li>
	</ol>
	</li>
</ol>
3. Cloning the Sponge
<ol>
	<li>Digest the recipient vector 
	<ol>
		<li>Make the &#945;-MHC-pEGFP plasmid linear for cloning with EcoRI and KpnI, and purify the gel as described above (step 2.1).</li>
	</ol>
	</li>
	<li>PCR amplify and digest the sponge DNA 
	<ol>
		<li>Use the miR-181-sponge and corresponding 284-nt-long scramble shipping vectors as the template for PCR. Amplify the sponge region using previously described PCR primers5. Note that forward and reverse PCR primers contain restriction enzyme sequences to permit ligation into &#945;-MHC-pEGFP-C1. Perform PCR as described above (step 2.2.2). Purify the PCR products prior to digestion as described (step 2.2.3) and eluted them with water for digestion. For primer sequences, see Table 1.</li>
		<li>Digest the sponge DNA for ligation. A 50 &#956;L digestion reaction contains 1x digest buffer, the gel-purified PCR reaction from step 3.2.1, and 5 &#956;L of both EcoRI and KpnI enzymes. Add all components to a microcentrifuge tube and gently mix them by flicking it. Spin the tube for 10 s in a microcentrifuge to collect the reaction at the bottom. Incubate the reaction in a 37 &#176;C water bath for 15 min.</li>
		<li>Purify the digested PCR sponge using a PCR clean-up column as described previously (step 2.2.1).</li>
	</ol>
	</li>
	<li>Ligate the miR-181-sponge into &#945;-MHC-pEGFP-C1 vector
	<ol>
		<li>Set up a 21 &#956;L ligation reaction which contains 1x ligation buffer, 3 &#956;L of digested and purified miR-181-sponge PCR, 1 &#956;L of linearized &#945;-MHC-pEGFP-C1 vector, 1x DNA buffer, and 1 &#956;L of DNA ligase. Perform the ligation at room temperature for 5 min and then place it on ice.</li>
		<li>Transform 50 &#956;L of XL1-blue competent cells with 2 &#956;L of ligation mix. Use the transformation and plating protocols as described above (step 2.2.5). Perform a colony PCR to identify Kan resistant bacteria which contain the correct &#945;-MHC-pEGFP-miR-181-sponge sequence. Design screening primers to the PCR across the ligation junction. See Table 1 for primer sequences.</li>
		<li>Amplify and sequence the vector. Expand the PCR positive clones in LB-Kan broth and in plasmids purified using standard mini-prep plasmid purification protocols. Perform a DNA sequencing to verify the plasmid expressing the desired DNA sequences.</li>
	</ol>
	</li>
</ol>
4. Generation of Stable H9c2 miR-181-sponge Expressing Cells
<ol>
	<li>Growth and maintenance of H9c2 cells
	<ol>
		<li>Culture the H9c2 cells in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) containing fetal bovine serum (FBS) to a final concentration of 10%. Sub-culture the cells using standard protocols and grow them at 37 &#176;C in 5% carbon dioxide.</li>
	</ol>
	</li>
	<li>Transfection and selection of H9c2 &#945;-MHC-pEGFP-C1-miR-181-sponge expressing cells
	<ol>
		<li>Perform an electroporation of H9c2 cells for the best results. Transfect rat myoblasts (H9c2) with either a scramble sponge (a 284-nt-long scramble sequence which replaces the miR-181-sponge-sequence) or the &#945;-MHC-pEGFP-miR-181-sponge construct with an electroporator.
		<ol>
			<li>Briefly, transfect 4x10-5 cells with 2 &#956;g of plasmid DNA (in 5 &#181;L of H2O) and 100 &#181;L of a solution compatible with the electroporation device. Use an electroporation program of DS-120 to transfect the H9c2 cells.</li>
			<li>Incubate the transfected cells in the cuvette for 10 min at room temperature. Add 500 &#181;L of DMEM directly to the cuvette. Gently transfer the total cuvette content to a well of a 6-well plate.</li>
		</ol>
		</li>
		<li>Select for GFP positive cells after 48 h post-transfection by FACs. Plate only the GFP-expressed cells into a 6-well plate with an addition of neomycin (G418) (400ng/mL) to the complete growth media. 
		Note: Prior to the G418 selection, a kill curve should be established to determine the amount of neomycin required for the selection. For H9c2 cells, 400 ng/mL of G418 resulted in an approximately 80% death rate of non-plasmid transfected H9c2 cells after 24 h and was considered to be the desired working concentration of G418 for these cells. The cell line was considered stable after growth was maintained in G418 for 16 days.</li>
		<li>Perform the FACs sorting of the G418 stable cells using the expression of GFP as a marker for a sponge positive selection. Seed 5 - 10 cells in each well of a 96-well plate by flow-sorting. Expand the monoclonal cells from the 96-well plate to 24-well plates over several passages. Use freezer stocks of cells from passage 3 (P3) cells as the starting point for subsequent experiments.</li>
		<li>Perform all cell-line-based experiments with low-passage cells between P5 and P10.</li>
	</ol>
	</li>
</ol>
5. Synthesis and Purification of Sponge-nanoparticles (miR-181-sponge Nanoparticles)
<ol>
	<li>Prepare the liposomal nanoparticles by dissolving cationic amphiphile (DOTAP) and co-lipids, cholesterol and DSPE-PEG-OMe, in a 5:5:0.1 mM ratio, respectively, in a mixture of chloroform and methanol in a glass vial.</li>
	<li>Remove the organic solvent at 44 - 45 &#176;C, under a vacuum by using a rotary evaporator, followed by a gentle flow of moisture-free nitrogen. Keep the remaining dried film of lipid under a high vacuum for 8 h.</li>
	<li>Prepare a mixture by adding 5% glucose to the vacuum-dried lipid film. Leave it overnight to allow this mixture to hydrate the film. Vortex the vial for 2 to 3 min at room temperature in order to produce multilamellar vesicles with an occasional shaking in a 45&#176; C water bath.
	<ol>
		<li>Sonicate the multilamellar vesicles to prepare small unilamellar vesicles in an ice bath for 3 - 4 min until clarity can be observed, at a 100% duty cycle and 25 W output power.</li>
	</ol>
	</li>
	<li>Mix either a scramble sequence or a miR-181-sponge sequence cloned into the pEGFP(&#945;-MHC) vectors and liposome on a 1:3 charge ratio basis to form the nanovector4. 
	Note: An electrostatic complex of positively-charged liposomal nanoparticles and negatively-charged plasmid DNA is known as a nanovector.</li>
</ol>
6. Systemic Delivery of Sponge-nanoparticles and Validation of In Vivo Effects
<ol>
	<li>Systemic delivery of sponge-nanoparticles in rats
	<ol>
		<li>Use Sprague-Dawley (SD) rats. Inject the rats intravenously with the miR-181-sponge nanovector or scramble nanovector through the tail vein. Use SD male rats, which are about 200 g in body weight.</li>
		<li>Perform the tail vein injection using a dose of 4 mg of nanovector/kg body weight and repeat this using a regimen of 6 tail vein injections over 2 weeks. Following the repeated nanovector delivery, allow a 1-week period (days 15 - 21) for the expression of the vector in vivo.</li>
		<li>Confirm the optimization by imaging the GFP signal before, during, and after the nanovector delivery. Analyze the GFP signal by tomographic imaging software, which generates semi-quantitative data.</li>
	</ol>
	</li>
	<li>Validation of in vivo miR-181 inhibition by the miR-181-sponge
	<ol>
		<li>Perform western blot in order to demonstrate the expression of a functional miR-181-sponge in the heart tissue. 
		Note: For this study, mt-COX1 expression was examined.</li>
	</ol>
	</li>
	<li>Functional consequences of in vivo miR-181-sponge expression in the heart 
	<ol>
		<li>Perform a two-dimensional, M-mode, and Doppler echocardiography during and after the nanovector delivery to analyze the cardiac function and morphological changes. For a better scanning capacity in the rodent hearts, use an ultrasound system equipped with a 15 MHz linear array transducer.</li>
		<li>Anesthesia may influence the cardiac contractility; therefore, perform the delivery of the miR-181-sponge nanovector or scramble nanovector to animals that are conscious and without any anesthetic reagent during the echocardiography process. Please see Das, et al.4 for further clarification.</li>
	</ol>
	</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+microRNA+with+antisense+oligonucleotides.">Inhibition of microRNA with antisense oligonucleotides.</a> Methods. 44 (1), 55-60 (2008).</li><li>Stenvang, J., Kauppinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+as+targets+for+antisense-based+therapeutics.">MicroRNAs as targets for antisense-based therapeutics.</a> Expert Opinion on Biological Therapy. 8 (1), 59-81 (2008).</li><li>Lennox, K. A., Behlke, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+comparison+of+anti-microRNA+oligonucleotide+potency.">A direct comparison of anti-microRNA oligonucleotide potency.</a> Pharmaceutical Research. 27 (9), 1788-1799 (2010).</li><li>Lennox, K. A., Behlke, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modification+and+design+of+anti-miRNA+oligonucleotides.">Chemical modification and design of anti-miRNA oligonucleotides.</a> Gene Therapy. 18 (12), 1111-1120 (2011).</li><li>van Rooij, E., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+therapeutics+for+cardiovascular+disease:+opportunities+and+obstacles.">MicroRNA therapeutics for cardiovascular disease: opportunities and obstacles.</a> Nature Reviews. Drug Discovery. 11 (11), 860-872 (2012).</li><li>Stenvang, J., Petri, A., Lindow, M., Obad, S., Kauppinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+microRNA+function+by+antimiR+oligonucleotides.">Inhibition of microRNA function by antimiR oligonucleotides.</a> Silence. 3 (1), 1 (2012).</li><li>Levin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+issues+in+the+pharmacokinetics+and+toxicology+of+phosphorothioate+antisense+oligonucleotides.">A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides.</a> Biochimica et Biophysica Acta. 1489 (1), 69-84 (1999).</li><li>Flynt, A. S., Li, N., Thatcher, E. J., Solnica-Krezel, L., Patton, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+miR-214+modulates+Hedgehog+signaling+to+specify+muscle+cell+fate.">Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate.</a> Nature Genetics. 39 (2), 259-263 (2007).</li><li>Kloosterman, W. P., Lagendijk, A. K., Ketting, R. F., Moulton, J. D., Plasterk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+inhibition+of+miRNA+maturation+with+morpholinos+reveals+a+role+for+miR-375+in+pancreatic+islet+development.">Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development.</a> PLoS Biology. 5 (8), e203 (2007).</li><li>Martello, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+control+of+Nodal+signalling.">MicroRNA control of Nodal signalling.</a> Nature. 449 (7159), 183-188 (2007).</li><li>Fabani, M. M., Gait, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-122+targeting+with+LNA/2'-O-methyl+oligonucleotide+mixmers,+peptide+nucleic+acids+(PNA),+and+PNA-peptide+conjugates.">miR-122 targeting with LNA/2'-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA-peptide conjugates.</a> RNA. 14 (2), 336-346 (2008).</li><li>Fabani, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+inhibition+of+miR-155+function+in+vivo+by+peptide+nucleic+acids.">Efficient inhibition of miR-155 function in vivo by peptide nucleic acids.</a> Nucleic Acids Research. 38 (13), 4466-4475 (2010).</li><li>Babar, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-based+therapy+in+an+in+vivo+microRNA-155+(miR-155)-dependent+mouse+model+of+lymphoma.">Nanoparticle-based therapy in an in vivo microRNA-155 (miR-155)-dependent mouse model of lymphoma.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (26), E1695-E1704 (2012).</li><li>Torres, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+structure+requirements+and+cellular+targeting+of+microRNA-122+by+peptide+nucleic+acids+anti-miRs.">Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs.</a> Nucleic Acids Research. 40 (5), 2152-2167 (2012).</li><li>Kent, O. A., McCall, M. N., Cornish, T. C., Halushka, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+miR-143/145:+the+importance+of+cell-type+localization+of+miRNAs.">Lessons from miR-143/145: the importance of cell-type localization of miRNAs.</a> Nucleic Acids Research. 42 (12), 7528-7538 (2014).</li></ol>

The authors have nothing to disclose.

This article described the design and synthesis of a miRNA-sponge and demonstrated how the tissue-specific expression of the sponge is a powerful tool to inhibit tissue-specific miRNA family expression.
We have demonstrated that a miR-181 family targeting sponge can be cloned into an expression plasmid with a cardiac-specific promoter. The plasmid can be efficiently packaged into a nanovector particle for delivery both in vitro and in vivo using electroporation or a tail vein...

Over the last two decades, there have been numerous studies that have pointed to the significant role of miRNAs in human disease. Findings from a large body of literature demonstrate the undeniable importance of miRNAs in the pathophysiology of diseases, such as cancer1 and cardiovascular disease2,3,4,5. For example, miR-21 is upregulated in many cancers, resulting in an increased cell cycle and cell proliferation6. In hepatitis C infections, miR-122 plays an important role in the replication of the virus7, and it has been shown that the inhibition of miR-122 decreases the viral load8. In cardiac hypertrophy, miR-212/132 is upregulated in the heart and is involved in the pathological phenotype9. The obvious importance of the downregulation or functional inhibition of an upregulated miRNA suggests opportunities for therapeutically exploiting the miRNA biology in almost all diseases.
The four miR-181 family members, miR-181a/b/c/d, are found in three genomic locations in the human genome. The intronic region of a non-coding RNA host gene (MIR181A1-HG) encodes the cluster of miR-181-a/b-1. The intronic region of the NR6A1 gene encodes the miR-181-a/b-2. The miR-181-c/d cluster is located in an uncharacterized transcript on chromosome 19. All the miR-181 family members share the same &#34;seed&#34; sequence and all four miR-181 family members can potentially regulate the same mRNA targets.
We3,4 and others10 have highlighted the importance of miR-181 family members during the end-stage heart failure. We have also recognized that a miR-181c upregulation occurs under pathological conditions associated with an increased risk of heart disease, such as type II diabetes, obesity, and aging3,4,5. It has been postulated that the overexpression of miR-181c causes oxidative stress which leads to a cardiac-dysfunction4.
Several groups have suggested that miRNA exist in mitochondria11,12,13,14, but we were the first to demonstrate that miR-181c is derived from the nuclear genome, processed, and subsequently translocated to the mitochondria in the RISC3. Furthermore, we have detected a low expression of miR-181a and miR-181b in the mitochondrial compartment of the heart5. Importantly, we have found that miR-181c represses the mt-COX1 mRNA expression, thereby demonstrating that miRNAs participate in the mitochondrial gene regulation and alter mitochondrial function3,4.
This article discusses the methodology required to design a miRNA-sponge to knock down the entire miR-181 family in cardiomyocytes. Moreover, we outline a protocol for the in vivo application of the miR-181-sponge.

<table><tbody><tr><td>pEGFP-C1 vector</td><td>Addgene</td><td>6084-1</td><td></td></tr><tr><td>In-fusion</td><td>Clontech</td><td>121416</td><td></td></tr><tr><td>QIAprep Miniprep</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>miR-181-sponge synthesis</td><td>Introgen GeneArt</td><td>custome made</td><td></td></tr><tr><td>PCR primers</td><td>Integrated DNA Technologies</td><td>custome</td><td></td></tr><tr><td>EcoRI enzymes</td><td>New Endland Biolabs</td><td>R0101S</td><td></td></tr><tr><td>KpnI enzymes</td><td>New Endland Biolabs</td><td>R0142S</td><td></td></tr><tr><td>Rapid DNA Ligation Kit</td><td>Sigma-Aldrich</td><td>11635379001</td><td></td></tr><tr><td>H9c2 cells</td><td>ATCC</td><td>CRL-1446</td><td></td></tr><tr><td>DMEM Media</td><td>Thermo Fisher Scientific</td><td>11965092</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>10082139</td><td></td></tr><tr><td>Nucleofector 2b Device</td><td>Lonza</td><td>AAB-1001</td><td></td></tr><tr><td>Nucleofector Kits for H9c2 (2-1)</td><td>Lonza</td><td>VCA-1005</td><td></td></tr><tr><td>G418, Geneticin</td><td>Thermo Fisher Scientific</td><td>11811023</td><td></td></tr><tr><td>FACSAria II Flow cytometer</td><td>BD Bioscience</td><td>644832</td><td></td></tr><tr><td>Branson 450 sonifier</td><td>Marshall Scientific</td><td>EDP 100-214-239</td><td></td></tr><tr><td>The Xenogen IVIS Spectrum optical imaging device</td><td>Caliper Life Sciences</td><td></td><td></td></tr><tr><td>Anti-MTCO1 antibody</td><td>Abcam</td><td>ab14705</td><td></td></tr><tr><td>&amp;alpha;-tubulin antibody</td><td>Abcam</td><td>ab7291</td><td></td></tr><tr><td>Sequoia C256 ultrasound system</td><td>Siemens</td><td></td><td></td></tr></tbody></table>

in vivo nanovector delivery of a heart-specific microrna-sponge

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and cardiovascular disease, have been shown to activate tissue and/or cell-specific miRNA expression associated with disease progression. The inhibition of miRNA expression offers the potential for a therapeutic intervention. However, traditional approaches to inhibit miRNAs, employing antagomir oligonucleotides, affect specific miRNA functions upon global delivery. Herein, we present a protocol for the in vivo cardio-specific inhibition of the miR-181 family in a rat model. A miRNA-sponge construct is designed to include 10 repeated anti-miR-181 binding sequences. The cardio-specific &#945;-MHC promoter is cloned into the pEGFP backbone to drive the cardio-specific miR-181 miRNA-sponge expression. To create a stable cell line expressing the miR-181-sponge, myoblast H9c2 cells are transfected with the &#945;-MHC-EGFP-miR-181-sponge construct and sorted by fluorescence-activated cell sorting (FACs) into GFP positive H9c2 cells which are cultured with neomycin (G418). Following stable growth in neomycin, monoclonal cell populations are established by additional FACs and single cell cloning. The resulting myoblast H9c2-miR-181-sponge-GFP cells exhibit a loss of function of miR-181 family members as assessed through the increased expression of miR-181 target proteins and compared to H9c2 cells expressing a scramble non-functional sponge. In addition, we develop a nanovector for the systemic delivery of the miR-181-sponge construct by complexing positively charged liposomal nanoparticles and negatively charged miR-181-sponge plasmids. In vivo imaging of GFP reveals that multiple tail vein injections of a nanovector over a three-week period are able to promote a significant expression of the miR-181-sponge in a cardio-specific manner. Importantly, a loss of miR-181 function is observed in the heart tissue but not in the kidney or the liver. The miRNA-sponge is a powerful method to inhibit tissue-specific miRNA expression. Driving the miRNA-sponge expression from a tissue-specific promoter provides specificity for the miRNA inhibition, which can be confined to a targeted organ or tissue. Furthermore, combining nanovector and miRNA-sponge technologies permits an effective delivery and tissue-specific miRNA inhibition in vivo.

In the stably transfected pEGFP-miR-181-sponge-expressing H9c2 cells (from step 4.2), the expression of the entire miR-181 family (miR-181a, miR-181b, miR-181c, and miR-181d) was moderately decreased relative to pEGFP-scrambled-expressing H9c2 cells. MiR-181-sponge serves as a competitive inhibitor of the entire miR-181 family, so we anticipated that the expression of the miR-181c mitochondrial target gene, mt-COX1, would increase. Western blot data suggest that the mt-COX1 expression was...

Watch this Scientific Journal Video about In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge at JoVE.com

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition.

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

in-vivo-nanovector-delivery-of-a-heart-specific-microrna-sponge

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

7.3K Views.  University of Toronto. Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition.

Video: In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising treatment option for heart disease, owing to its efficiency and exceptional therapeutic effects. In an effort to repair the damaged tissue post-MI, various studies have employed DNA-based or viral gene therapy but have faced considerable hurdles due to the poor and uncontrolled expression of the delivered genes, edema, arrhythmia, and cardiac hypertrophy. Synthetic modified mRNA (modRNA) presents a novel gene therapy approach that offers high, transient, safe, nonimmunogenic, and controlled mRNA delivery to the heart tissue without any risk of genomic integration. Due to these remarkable characteristics combined with its bell-shaped pharmacokinetics in the heart, modRNA has become an attractive approach for the treatment of heart disease. However, to increase its effectiveness in vivo, a consistent and reliable delivery method needs to be followed. Hence, to maximize modRNA delivery efficiency and yield consistency in modRNA use for in vivo applications, an optimized method of preparation and delivery of modRNA intracardiac injection in a mouse MI model is presented. This protocol will make modRNA delivery more accessible for basic and translational research.

This protocol presents a simple and coherent way to transiently upregulate a gene of interest using modRNA after myocardial infarction in mice.

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising ...

Genetics

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Developmental Biology

Nanodrug Targeting Oncogenic miRNA-10b for Treating Metastatic Breast Cancer

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

Cancer Research

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

Neuroscience

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Medicine

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Lentiviral Vector Preparation for In Vivo Gene Modulation 

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Author Spotlight: Exploring Macrophage Immunometabolism Through Lentiviral Vector&#45;Mediated Gene Manipulation

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...

Immunology and Infection

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing and inhibiting their translation in various kidney diseases. Therefore, alternation of miRNA expression by exogenous artificially synthesized miRNA mimics is a potentially useful treatment option for inhibiting the development of many kidney diseases. However, because serum RNAase immediately degrades systematically administered exogenous miRNA mimics in vivo, delivery of miRNA to the kidney remains a challenge. Therefore, vectors that can protect exogenous miRNA mimics from degradation by RNAase and significantly deliver them to the kidney are necessary. Many studies have used viral vectors to deliver exogenous miRNA mimics or inhibitors to the kidney. However, viral vectors may cause an interferon response and/or genetic instability. Therefore, the development of viral vectors is also a hurdle for the clinical use of exogenous miRNA mimics or inhibitors. To overcome these concerns regarding viral vectors, we developed a nonviral vector method to deliver miRNA mimics to the kidney using tail vein injection of polyethylenimine nanoparticles (PEI-NPs), which led to significant overexpression of target miRNAs in several mouse models of kidney disease.

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades")

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the development of numerous innovative applications. However, delivery of the Cas9 protein and single-guide RNA (sgRNA) into target cells can be technically challenge. Classical viral vectors, such as those derived from lentiviruses (LVs) or adeno-associated viruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9 protein and its associated sgRNA in many primary cells and in vivo. Nevertheless, these vectors can suffer from drawbacks such as integration of the transgene in the target cell genome, a limited cargo capacity, and long-term expression of the Cas9 protein and guide RNA in target cells.
To overcome some of these problems, a delivery vector based on the murine Leukemia virus (MLV) was developed to package the Cas9 protein and its associated guide RNA in the absence of any coding transgene. By fusing the Cas9 protein to the C-terminus of the structural protein Gag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA (named &#34;Nanoblades&#34;) were formed. Nanoblades can be collected from the culture medium of producer cells, purified, quantified, and used to transduce target cells and deliver the active Cas9/sgRNA complex. Nanoblades deliver their ribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary and immortalized cells and can be programmed for other applications, such as transient transcriptional activation of targeted genes, using modified Cas9 proteins. Nanoblades are capable of in vivo genome-editing in the liver of injected adult mice and in oocytes to generate transgenic animals. Finally, they can be complexed with donor DNA for &#34;transfection-free&#34; homology-directed repair. Nanoblade preparation is simple, relatively low-cost, and can be easily carried out in any cell biology laboratory.

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles "Nanoblades"

We have developed a simple and inexpensive protocol to load Cas9/single-guide RNA (sgRNA) ribonucleoprotein complexes within virus-like particles. These particles, called "Nanoblades", allow efficient delivery of the Cas9/sgRNA complex in immortalized and primary cells as well as in vivo.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the ...

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

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Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades")