Difference between revisions of "Team:NJU-China/RNAi"

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<ul id="nav" class="dyn" style="height:100px;line-height:100px;margin-top:20%;margin-left:10%;list-style-type:none">
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</br>
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<li><a href="https://2015.igem.org/Team:NJU-China" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Home</a></li></br>
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Home</a></li>
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<li><a href="https://2015.igem.org/NJU-China-background.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Background</a></li></br>
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-background.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Background</a></li>
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<li><a href="https://2015.igem.org/NJU-China-project.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Project</a></li></br>
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<div style="line-height:250%;margin-left:10%" id="main1" onClick="document.all.child0.style.display=(document.all.child0.style.display =='none')?'':'none'" ><a href="#" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Project</a></div>
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<li><a href="#" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Model</a>
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<div id="child0" style="display:none">
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        <ul>  
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<ul>
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            <li><a href="https://2015.igem.org/Team:NJU-China/Modeling.html#deliverymodel" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Delivery model</a></li>  
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/Design" style="font-weight:bold;font-family:幼圆;color:black">design</a></li>
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            <li><a href="https://2015.igem.org/Team:NJU-China/Modeling#" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">RNAi model</a></li>  
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-project/result.html" style="font-weight:bold;font-family:幼圆;color:black">results</a></li>
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        </ul>  
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-project/conclusion.html" style="font-weight:bold;font-family:幼圆;color:black">conclusion</a></li>
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</li></br>
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-project/future work.html" style="font-weight:bold;font-family:幼圆;color:black">future work</a></li>
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<li style="line-height:250%"><a href="https://2015.igem.org/Team:NJU-China/Practices" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Human Practice</a></li></br>
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</ul>
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<li style="line-height:250%"><a href="https://2015.igem.org/NJU-China-parts.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Parts</a></li></br>
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</div>
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<li style="line-height:250%"><a href="https://2015.igem.org/NJU-China-team.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Team</a></li></br>
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<div style="line-height:250%;margin-left:10%" id="main1" onClick="document.all.child1.style.display=(document.all.child1.style.display =='none')?'':'none'" ><a href="#" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Model</a></div>
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<li style="line-height:250%"><a href="https://2015.igem.org/Team:NJU-China/Attributions" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Attribution</a></li></br>
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<div id="child1" style="display:none">
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<li style="line-height:250%"><a href="https://2015.igem.org/Team:NJU-China/Collaborations" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Colaborations</a></li></br>
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<ul>
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<li style="line-height:250%"><a href="https://2015.igem.org/NJU-China-safty.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Safety</a></li></br>
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-model.html" style="font-weight:bold;font-family:幼圆;color:black">Delivery model</a></li>
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<li style="line-height:250%"><a href="#" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Notebook</a>
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/RNAi" style="font-weight:bold;font-family:幼圆;color:black">RNAi model</a></li>
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        <ul>  
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/signaling" style="font-weight:bold;font-family:幼圆;color:black">Signaling</a></li>
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            <li><a href="https://2015.igem.org/NJU-China-notebook.html#methods" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Methods</a></li>  
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</ul>
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            <li><a href="https://2015.igem.org/NJU-China-notebook.html#protocal" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Protocal</a></li>  
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</div>
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            <li><a href="https://2015.igem.org/NJU-China-notebook.html#notebook" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Notebook</a></li>  
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/Practices" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Human Practice</a></li>
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        </ul>  
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-parts.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Parts</a></li>
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</li></br>
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-team.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Team</a></li>
 +
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-attribution.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Attribution</a></li>
 +
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/Collaborations" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Collaborations</a></li>
 +
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-safty.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Safety</a></li>
 +
<div style="line-height:250%;margin-left:10%" id="main2" onClick="document.all.child2.style.display=(document.all.child2.style.display =='none')?'':'none'" > <a href="#" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Notebook </div>
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<div id="child2" style="display:none">
 +
<ul>
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<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-notebook.html#methods" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Methods</a></li>
 +
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-notebook.html#protocal" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Protocal</a></li>
 +
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-notebook.html#notebook" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Notebook</a></li>
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</ul>
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你在火中涅槃，灵魂不死；你在花间长眠，记忆不消。绚烂的阳光是迎接你西去的光环，不知你独自一人去那方可会感到孤单？
+
<h1> 2. RNAi module </h1>
−
[一]魂去
+
 
−
      起初听到你的死，我是愕然的。毕竟听说你的病情好了不少，却不料那只是最后的回光返照。
+
<h2> 2.1 Introduction </h2>
−
      我没有流泪，甚至没有悲伤，脑子里只是混混沌沌的一团。死？那是我们所有人最后的归宿吧。或许于你更是能够减少痛苦，未尝不是一件好事。
+
 
−
      一切被我想得太简单，甚至太过理所当然了。理智，真是一个可怕的局。
+
 
−
[二]席间
+
RNA interference (RNAi) is a major tool for transiently suppressing the expression of
−
      小号、大鼓敲个不停，那种声音不得不说是一种噪音。良久声止，开始一个个念悼词。
+
 
−
      我这时才发现，原来我并没有那么冷漠，缘是城市的喧嚣麻木了我的心。想起那年你的温言软语、慈爱笑容，泪竟是就这样忍不住地从眼眶中溢出了。我撇过头，不愿让人看见，嘴角仍是挂着笑容。
+
genes. Many mathematical models have been constructed to elucidate the mechanism of RNA
−
      呵呵，我竟觉着在这葬礼上哭泣都是可耻的。望着周围人谈笑风生，我的心有点寒。到场的那么多人，究竟又有几个是真心的呢？可怜，还是可悲啊？
+
 
−
      原来人可以铁石心肠这种程度，可毕竟他们中很多人比我和你的关系更近啊！听着那个花钱雇来哭的人呜咽，看着那人热泪纵横，我终是忍不住苦笑，竟是一个毫不相识的外人哭得最伤心。这是讽刺，还是笑话？
+
interference and provide accurate predictions. Nevertheless, most of the current models
−
  [三]送葬
+
 
−
      你说，为什么他们还能那样淡然，可我偏偏忍不住泪流满面？为什么他们那个惊讶的表情让我竟是感到羞愧？好像自己做了什么惊天动地的事一样？
+
focus merely on RNAi and fail to consider the delivery process.
−
      执着一根香，撒下一碗酒，三拜三叩，泪融于酒，我如若痴人。
+
 
−
      照礼我是不必披麻戴孝的，只需戴着那顶白色的帽子即可。沿着幽僻的小路，我跟着吹着小号开路的队伍走在末尾。
+
<B>
−
      风景甚好，埋在那棵大树底下，你会不会成为那树的枝，树的叶？
+
We modeled the delivery process and the input variant in this module should be the
−
[四]感怀
+
 
−
      死生有命，无可更改。谋事在人，成事在天。任你生前浮华万千，到头来不过一抔尘土，一捧青灰。愿逝者安息，生者恬淡。
+
output result of the delivery module. </B>
−
      有时候我们看不到自己拥有的美好，直到失去才恍然叹息。逝者已矣，页已掀过，惜福、感恩，生活其实温暖如花。
+
 
−
      花开半夏，倾尽繁华，愿你回眸半刹。你可听到我在同你夜话？
+
<br><br>
 +
 
 +
 
 +
A challenge for the successful clinical application of RNAi-based drugs is determining
 +
 
 +
the dosing schedule required for efficacy. Patients may ask the following questions:
 +
 
 +
How soon will RNAi-based drugs take to exert efficacy after injection? How long will
 +
 
 +
the efficacy last? What is the dose I need to take, and will it be too costly? How soon
 +
 
 +
will the level of Mu opioid receptor (MOR) protein recover? Is RNAi therapy safe
 +
 
 +
enough? <B> Mathematical modeling using simple kinetic equations for each step in the
 +
 
 +
RNAi process can shed light on many of these questions. </B>
 +
 
 +
<br><br>
 +
 
 +
<h2> 2.2 Model methods </h2>
 +
 
 +
 
 +
This model is inspired by the paper written by Bartlett and Davis [1]. The system uses
 +
 
 +
the presence of the RISC complex, which is formed in exosomes and escaped from
 +
 
 +
endosome, as a stable source to provide silencing power. Then, the RISC units are
 +
 
 +
targeted to mRNA having the same sequence as the siRNA that triggers this process,
 +
 
 +
binding with mRNA to form an activated RISC-mRNA complex. Once bound to complementary
 +
 
 +
mRNA, activated RISC may induce the degradation of mRNA and further silence protein
 +
 
 +
expression.
 +
 
 +
<br><br>
 +
 +
<!-- 插入第五张图 --> <img src="https://static.igem.org/mediawiki/2015/5/55/NJU-China-
 +
 
 +
Model_Figure5.jpg" style="width:400px"> <br><br>
 +
 
 +
Figure 5. Schematic diagram of RNA interference pathway. Degradation of the RISC
 +
 
 +
complex, siRNA, mRNA and protein is not shown here for clear illustration. However,
 +
 
 +
these processes are included in the model equations.
 +
 
 +
<br><br>
 +
 
 +
Applying the usual mass action to the reaction network, we can easily obtain the
 +
 
 +
following model equations:
 +
 
 +
<br><br>
 +
 
 +
The RISC complex, derived from endosomal escape, may disassociate into free RISC and
 +
 
 +
siRNA, form an activated mRNA-RISC complex or be degraded, as represented by
 +
 
 +
<I>kdisRISC</I>, <I>kformRISCm</I> and <I>kdegRISC</I>, respectively. The free RISC and
 +
 
 +
siRNA may again form a RISC complex, which is represented by <I>kformRISC</I>. The
 +
 
 +
amount of free RISC proteins available for the formation of activated complex is
 +
 
 +
<I>rtot</I> (free RISC protein) – <I>R</I> – <I>C</I> – <I>kdisRISC</I>*<I>R</I>
 +
 
 +
(disassociated RISC protein derived from endosomes). Thus, the total numbers of siRNA-
 +
 
 +
RISC complexes can be modeled using the equations below.
 +
 
 +
<br><br>
 +
 
 +
<!-- 插入第一张公式 --> <img src="https://static.igem.org/mediawiki/2015/7/7a/NJU-China-
 +
 
 +
Equation_RNAi_1.jpg" style="width:450px"> <br><br>
 +
 
 +
&nbsp;&nbsp;&nbsp;
 +
 
 +
The number of free siRNA in the cytosol is governed by the equation below.
 +
 
 +
<br><br>
 +
 
 +
 
 +
<!-- 插入第二张公式 --> <img src="https://static.igem.org/mediawiki/2015/d/da/NJU-China-
 +
 
 +
Equation_RNAi_2.jpg" style="width:450px"> <br><br>
 +
 
 +
 
 +
Activated RISC complex bound to mRNA induces the cleavage of target mRNA
 +
 
 +
(<I>kcleavage</I>). Additionally, activated RISC complex may undergo degradation
 +
 
 +
(<I>kdegRISC</I>) or disassociation (<I>kdisRISCm</I>).
 +
 
 +
<br><br>
 +
 
 +
<!-- 插入第三张公式 --> <img src="https://static.igem.org/mediawiki/2015/a/a4/NJU-China-
 +
 
 +
Equation_RNAi_3.jpg" style="width:450px"> <br><br>
 +
 
 +
 
 +
The balance of formation (<I>kformmRNA</I>) and degradation (<I>kdegmRNA</I>) of mRNA
 +
 
 +
and protein (<I>kformprot</I> <I>kdegprot</I>) is interrupted by RISC-induced cleavage
 +
 
 +
of mRNA.
 +
 
 +
<br><br>
 +
 
 +
<!-- 插入第四张公式 --> <img src="https://static.igem.org/mediawiki/2015/1/1f/NJU-China-
 +
 
 +
Equation_RNAi_4.jpg" style="width:450px"> <br><br>
 +
 
 +
 
 +
The variables and parameters of this model can be accessed <a href="#var">here</a>. All the parameters we
 +
 
 +
used in this module are reported in the literature [1].
 +
 
 +
<br><br>
 +
 
 +
<h2> 2.3 Results </h2>
 +
 
 +
 
 +
The simulation results demonstrate the effect of siRNA on MOR knockdown in vivo.
 +
 
 +
<br><br>
 +
 
 +
<!-- 插入第六张图 --> <img src="https://static.igem.org/mediawiki/2015/2/26/NJU-China-
 +
 
 +
Model_Figure6.jpg"> <br><br>
 +
 
 +
Figure 6. Effect of siRNA on MOR mRNA (B) and protein (A) knockdown in vivo. The
 +
 
 +
quantity of total exosomes injected is 300 μg which contains 3 nmol siRNA.
 +
 
 +
<br><br>
 +
 
 +
 
 +
As depicted in the figure, the expression level of MOR protein exhibits a rapid
 +
 
 +
exponential decay and reaches lowest level 12 hours after exosome injection, following
 +
 
 +
a similar pattern observed with the relative level of MOR mRNA.
 +
 
 +
<br><br>
 +
 
 +
<!-- 插入第七张图 --> <img src="https://static.igem.org/mediawiki/2015/e/e9/NJU-China-
 +
 
 +
Model_Figure7.jpg" > <br><br>
 +
 
 +
Figure 7. Effect of dose on MOR mRNA (A) and protein (B) knockdown in vivo. The initial
 +
 
 +
quantity of total exosome injected was set at 50 μg, 100 μg, 200 μg, 400μg and 600
 +
 
 +
μg, containing 0.5 nmol, 1 nmol, 2 nmol, 4 nmol and 6 nmol siRNA, respectively.
 +
 
 +
<br><br>
 +
 
 +
 
 +
To explore the dose effect on MOR knockdown, we set different initial conditions and
 +
 
 +
ran simulations. The result shows that the concentrations of exosomes and siRNA have
 +
 
 +
significant impact on knockdown efficiency and recovery time. A high dosing schedule
 +
 
 +
leads to more complete knockdown of MOR protein and takes longer for MOR protein levels
 +
 
 +
to recover.
 +
 
 +
<br><br>
 +
 
 +
 
 +
To optimize the dosing schedule, the lasting times for efficiency and recovery needs to
 +
 
 +
be considered. Elongating the lasting time of drug efficiency while shortening the
 +
 
 +
recovery time seems paradoxical based on the simulation data. The literature has
 +
 
 +
reported that 3 nmol of siRNA is adequate for repressing reward effects after 7 days of
 +
 
 +
injection with the relative level of MOR protein reaching approxiamately 80%.
 +
 
 +
<B>
 +
If we assume that the threshold of relative level of MOR protein below which opioid
 +
 
 +
reward effects are repressed, is 80% [2], then injecting 400 μg exosome (4 nmol siRNA)
 +
 
 +
might be the best choice.
 +
</B>
 +
 
 +
The efficacy of the drug could last for about one week, and another week would be
 +
 
 +
required for MOR protein levels to absolutely recover. Increasing the frequency of
 +
 
 +
dosing may also help to lengthen the drug efficacy time.
 +
 
 +
<br><br>
 +
 
 +
 
 +
<h2 id="var"> 2.4 Model Variables </h2>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2015/8/85/NJU-China-model-rnai-vai.jpg" style="width:400px">
 +
 
 +
<br><br>
 +
 
 +
 
 +
<h2> 2.5 Model Parameters </h2>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2015/5/53/NJU-China-model-rnai-para.jpg" style="width:600px">
 +
 
 +
<br><br>
 +
 
 +
References: <br>
 +
1.Bartlett, D.W. and Davis, M.E. (2006) Insights into the kinetics of siRNA-mediated
 +
 
 +
gene silencing from live-cell and live-animal bioluminescent imaging. Nucleic Acids
 +
 
 +
Res, 34, 322-333. <br>
 +
2.Zhang, Y., Landthaler, M., Schlussman, S.D., Yuferov, V., Ho, A., Tuschl, T. and
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Kreek, M.J. (2009) Mu opioid receptor knockdown in the substantia nigra/ventral
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tegmental area by synthetic small interfering RNA blocks the rewarding and locomotor
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 +
effects of heroin. Neuroscience, 158, 474-483. <br>
 +
 
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 +
 
 +
 
 +
 
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 +
 
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</TD>
 
</TD>
 
     </TR>
 
     </TR>

Latest revision as of 18:53, 18 September 2015

model


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    2. RNAi module

    2.1 Introduction

    RNA interference (RNAi) is a major tool for transiently suppressing the expression of genes. Many mathematical models have been constructed to elucidate the mechanism of RNA interference and provide accurate predictions. Nevertheless, most of the current models focus merely on RNAi and fail to consider the delivery process. We modeled the delivery process and the input variant in this module should be the output result of the delivery module.

    A challenge for the successful clinical application of RNAi-based drugs is determining the dosing schedule required for efficacy. Patients may ask the following questions: How soon will RNAi-based drugs take to exert efficacy after injection? How long will the efficacy last? What is the dose I need to take, and will it be too costly? How soon will the level of Mu opioid receptor (MOR) protein recover? Is RNAi therapy safe enough? Mathematical modeling using simple kinetic equations for each step in the RNAi process can shed light on many of these questions.

    2.2 Model methods

    This model is inspired by the paper written by Bartlett and Davis [1]. The system uses the presence of the RISC complex, which is formed in exosomes and escaped from endosome, as a stable source to provide silencing power. Then, the RISC units are targeted to mRNA having the same sequence as the siRNA that triggers this process, binding with mRNA to form an activated RISC-mRNA complex. Once bound to complementary mRNA, activated RISC may induce the degradation of mRNA and further silence protein expression.



    Figure 5. Schematic diagram of RNA interference pathway. Degradation of the RISC complex, siRNA, mRNA and protein is not shown here for clear illustration. However, these processes are included in the model equations.

    Applying the usual mass action to the reaction network, we can easily obtain the following model equations:

    The RISC complex, derived from endosomal escape, may disassociate into free RISC and siRNA, form an activated mRNA-RISC complex or be degraded, as represented by kdisRISC, kformRISCm and kdegRISC, respectively. The free RISC and siRNA may again form a RISC complex, which is represented by kformRISC. The amount of free RISC proteins available for the formation of activated complex is rtot (free RISC protein) – R – C – kdisRISC*R (disassociated RISC protein derived from endosomes). Thus, the total numbers of siRNA- RISC complexes can be modeled using the equations below.



        The number of free siRNA in the cytosol is governed by the equation below.



    Activated RISC complex bound to mRNA induces the cleavage of target mRNA (kcleavage). Additionally, activated RISC complex may undergo degradation (kdegRISC) or disassociation (kdisRISCm).



    The balance of formation (kformmRNA) and degradation (kdegmRNA) of mRNA and protein (kformprot kdegprot) is interrupted by RISC-induced cleavage of mRNA.



    The variables and parameters of this model can be accessed here. All the parameters we used in this module are reported in the literature [1].

    2.3 Results

    The simulation results demonstrate the effect of siRNA on MOR knockdown in vivo.



    Figure 6. Effect of siRNA on MOR mRNA (B) and protein (A) knockdown in vivo. The quantity of total exosomes injected is 300 μg which contains 3 nmol siRNA.

    As depicted in the figure, the expression level of MOR protein exhibits a rapid exponential decay and reaches lowest level 12 hours after exosome injection, following a similar pattern observed with the relative level of MOR mRNA.



    Figure 7. Effect of dose on MOR mRNA (A) and protein (B) knockdown in vivo. The initial quantity of total exosome injected was set at 50 μg, 100 μg, 200 μg, 400μg and 600 μg, containing 0.5 nmol, 1 nmol, 2 nmol, 4 nmol and 6 nmol siRNA, respectively.

    To explore the dose effect on MOR knockdown, we set different initial conditions and ran simulations. The result shows that the concentrations of exosomes and siRNA have significant impact on knockdown efficiency and recovery time. A high dosing schedule leads to more complete knockdown of MOR protein and takes longer for MOR protein levels to recover.

    To optimize the dosing schedule, the lasting times for efficiency and recovery needs to be considered. Elongating the lasting time of drug efficiency while shortening the recovery time seems paradoxical based on the simulation data. The literature has reported that 3 nmol of siRNA is adequate for repressing reward effects after 7 days of injection with the relative level of MOR protein reaching approxiamately 80%. If we assume that the threshold of relative level of MOR protein below which opioid reward effects are repressed, is 80% [2], then injecting 400 μg exosome (4 nmol siRNA) might be the best choice. The efficacy of the drug could last for about one week, and another week would be required for MOR protein levels to absolutely recover. Increasing the frequency of dosing may also help to lengthen the drug efficacy time.

    2.4 Model Variables



    2.5 Model Parameters



    References:
    1.Bartlett, D.W. and Davis, M.E. (2006) Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging. Nucleic Acids Res, 34, 322-333.
    2.Zhang, Y., Landthaler, M., Schlussman, S.D., Yuferov, V., Ho, A., Tuschl, T. and Kreek, M.J. (2009) Mu opioid receptor knockdown in the substantia nigra/ventral tegmental area by synthetic small interfering RNA blocks the rewarding and locomotor effects of heroin. Neuroscience, 158, 474-483.