Name: predicting gene silencing through the spatiotemporal control of sirna release from photo-responsive polymeric nanocarriers
Uploaded: 2017-07-21T15:00:00
Description: Watch this Scientific Journal Video about Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers at JoVE.com

The authors thank the National Institute of General Medical Sciences of the National Institutes of Health (NIH) for financial support through an Institutional Development Award (IDeA) under grant number P20GM103541 as well as grant number P20GM10344615. The statements herein do not reflect the views of the NIH. We also acknowledge the Delaware Biotechnology Institute (DBI) and Delaware Economic Development Office (DEDO) for financial support through the Bioscience Center for Advanced Technology (Bioscience CAT) award (12A00448).

1. Formulation of siRNA Nanocarriers
<ol>
	<li>Prepare separate solutions of siRNA and mPEG-b-P(APNBMA) with equal volumes diluted in 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer at pH 6.0.
	<ol>
		<li>Add siRNA at a concentration of 32 &#181;g/mL to 20 mM HEPES solution. 
		NOTE: The siRNA was a non-targeted, universal negative control sequence; however, the siRNA can be designed to target any gene of interest.</li>
		<li>Dissolve mPEG-b-P(APNBMA) poly...

<ol>
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There are a few steps in the method that are particularly critical. When formulating the nanocarriers, the order of component addition and mixing speed are two important parameters influencing efficacy39. This protocol requires that the cationic component, mPEG-b-P(APNBMA), is added to the anionic component, siRNA, in a dropwise fashion while vortexing. Depending on the total formulation volume, this mixing process takes 3-6 s. To test if the nanocarriers were formed properly, measure the...

Small interfering RNAs (siRNAs) mediate post-transcriptional gene silencing through a catalytic RNAi pathway that is highly specific, potent, and tailorable to virtually any target gene1. These promising characteristics have enabled siRNA therapeutics to advance in human clinical trials for the treatment of numerous diseases, including metastatic melanoma and hemophilia2,3. However, significant delivery issues persist that have hindered translation4. In particular, delivery vehicles must remain stable and protect siRNAs from extracellular degradation, yet also release the payload into the cytoplasm5. Furthermore, many RNAi applications require improved methods to regulate gene silencing in space and time6, which will reduce side effects in siRNA therapeutics7 and enable transformative advances in applications ranging from cell microarrays for drug discovery8 to modulation of cell responses in regenerative scaffolds9. These challenges highlight the need for new materials and methods to better control binding vs. release in siRNA nanocarriers.
One of the most promising strategies for controlling siRNA release and enhancing spatiotemporal regulation is the use of stimuli-responsive materials10. For example, a wide variety of biomaterials have been engineered with changeable nucleic acid binding affinity in response to altered redox potential or pH, or applied magnetic fields, ultrasound, or light11. Although many of these systems demonstrate improved control over nucleic acid activity, the use of light as a trigger is particularly advantageous due to its instantaneous temporal response, precise spatial resolution, and ease of tunability12. Moreover, the potential of photo-sensitive technologies for regulating gene expression has been demonstrated by state-of-the-art inducible promoter and optogenetic regulator systems; however, these systems suffer from numerous challenges including limited capacities to regulate endogenous genes, safety concerns such as immunogenicity, and difficulties in delivering multi-component assemblies13,14,15. Photo-responsive siRNA nanocarriers are ideally suited to overcome these drawbacks and provide a simpler and more robust approach to spatiotemporally modulate gene expression16,17,18. Unfortunately, methods to accurately predict the resulting protein knockdown response remain elusive.
A key challenge is that quantitative evaluations of siRNA release are rare19,20, and even when these evaluations are performed, they have not been coupled to analyses of siRNA/protein turnover dynamics. Both the amount of siRNA released and its persistence/lifetime are important determinants of the resulting gene silencing dynamics; hence, a lack of such information is a major disconnect that precludes accurate prediction of dose-response in RNAi21. Addressing this challenge would expedite the formulation of the appropriate structure-function relationships in nanocarriers and better inform biomaterial designs22. Furthermore, such approaches would enable development of more effective siRNA dosing protocols. In an attempt to understand the dynamic silencing response, several groups have investigated mathematical models of RNAi23,24,25. These frameworks were successful in providing insights into siRNA-mediated changes in gene expression and identifying rate-limiting steps26. However, these models have been applied only to commercial gene delivery systems (e.g., Lipofectamine and polyethylenimine (PEI)) that are not capable of controlled siRNA release, and the complexity of the models has severely limited their utility27. These shortcomings highlight an unmet need for new materials capable of precisely tunable siRNA release combined with streamlined and easy-to-use predictive kinetic models.
Our method addresses all of these challenges through the integration of a light-sensitive nanocarrier platform with coupled methods to quantify free siRNA and model RNAi dynamics. In particular, our platform&#39;s precisely controlled siRNA release28 is monitored by two complementary methods for accurately quantifying encapsulated vs. unbound siRNA. The experimental data from these assays are entered into a simple kinetic model to predict gene silencing efficiencies a priori29. Finally, the on/off nature of the nanocarriers is easily exploited to generate cell patterns in gene expression with spatial control on the cellular length scale. Thus, this method provides an easily adaptable method to control and predict gene silencing in a variety of applications that would benefit from spatiotemporal regulation of cell behavior.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">siRNA</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:145px;">SIC001</td>
			<td style="width:195px;">non-targeted, universal negative control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">mPEG-b-P(APNBMA)</td>
			<td style="width:157px;">synthesized in our lab</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">photo-responsive polymer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">HEPES</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">BP310-100</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">sodium dodecyl sulfate&#160;</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:145px;">436143</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">rubber gasket</td>
			<td style="width:157px;">McMaster-Carr</td>
			<td style="width:145px;">3788T21</td>
			<td style="width:195px;">0.5 mL thick</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">UV laser&#160;</td>
			<td style="width:157px;">Excelitas Technologies</td>
			<td style="width:145px;">Omnicure S2000</td>
			<td style="width:195px;">collimating lens and 365 nm filter used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">agarose</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">BP160-100</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">ethidium bromide</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">BP1302-10</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">siRNA labelled with Dy547</td>
			<td style="width:157px;">GE Healthcare Dharmacon, Inc.</td>
			<td style="width:145px;">custom order</td>
			<td style="width:195px;">fluorophore conjugated to 5&#8217; end of sense strand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">microscope slide</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">12-550-A3</td>
			<td style="width:195px;">pre-cleaned glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Secure-Seal Spacer</td>
			<td style="width:157px;">Life Technologies</td>
			<td style="width:145px;">S24735</td>
			<td style="width:195px;">double-sided adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">LSM 780&#160;</td>
			<td style="width:157px;">Carl Zeiss</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">confocal microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">ZEN 2010</td>
			<td style="width:157px;">Carl Zeiss</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">FCS analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">MATLAB</td>
			<td style="width:157px;">MathWorks</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">programming language</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">NIH/3T3 cells&#160;</td>
			<td style="width:157px;">ATCC</td>
			<td style="width:145px;">ATCC CRL-1658</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">DMEM</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">10-013-CV</td>
			<td style="width:195px;">growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">fetal bovine serum</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">35-011-CV</td>
			<td style="width:195px;">heat-inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">penicillin-streptomycin</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">&#160;30-002-CI</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">6-well plates</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">08-772-1B</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Opti-MEM</td>
			<td style="width:157px;">Life Technologies</td>
			<td style="width:145px;">11058021</td>
			<td style="width:195px;">transfection media</td>
		</tr>
	</tbody>
</table>

predicting gene silencing through the spatiotemporal control of sirna release from photo-responsive polymeric nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

Following the formulation of the nanocarriers, siRNA release assays were conducted to inform the irradiation conditions to be used in the in vitro transfections. Various dosages of light were applied to determine the percent of siRNA that was released. The first assay used gel electrophoresis to separate the free siRNA molecules from the siRNA molecules still complexed/associated with the polymer. Nanocarriers that were not treated with light remained stable and did not release a...

Watch this Scientific Journal Video about Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers at JoVE.com

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Research

JoVE Journal

Bioengineering

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