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

Latest revision as of 01:19, 2 November 2017

PROJECT

Background

Obesity is a medical condition in which excess body fat has accumulated to the extent that it may have a negative effect on health. [1] People are generally considered obese when their body mass index (BMI), a measurement obtained by dividing a person's weight by the square of the person's height, is over 30 kg/m2, with the range 25–30 kg/m2 defined as overweight. [1]

We need to perceive this: obesity, or excess fat, is not simply an obstacle to many people pursuing beauty. It also stands in the way of health acquiring, sharing connection with various diseases, like cardiovascular diseases, obstructive sleep apnea, type 2 diabetes, depression, certain types of cancer and so on. [2][3] What’s worse, obesity is now one of the major evitable causes for death globally. [4][5] In general, obesity cut down life expectancy by six to seven years. As for the number of obese people, in 2016, more than 1.9 billion adults, aged 18 and above, were overweight. Of these over 650 million were obese. 39% of adults aged 18 years and over were overweight in 2016, and 13% were obese. [1]

Map of Obesity in Adult Males (% of adult population with en:BMI 30+) per country. Using data updated until December 2008.

In fact, given the severe circumstance, in 2013, the American medical association has classified obesity as a disease. [6]

While the prevalence of obesity continued to grow, people weren’t just sitting these years away and did nothing against obesity. To lose weight, some change their lifestyle. People trying to lose weight have to refuse high calorie food and switch to healthier food like fiber-rich vegetables, and try to exercise more. Besides, some people might get help from medications. The legal medications in the market usually work by inhibiting appetite (like Sibutramine), reducing the degradation and absorption of fat (like Orlistat), or regulating the metabolism of fat. [7]

We have seen the traditional methods to lose weight. Many of them are prevalent, and we cannot deny their efficacy. However, it’s not hard to find these traditional methods’ defects: changing lifestyle is usually unpleasant and perseverance-needed, and even if you get the positive feedback, the probability to regain weight is also quite high. [7] As for medications, they may bring out inevitable side effects, like oily stool. Some company had to retract their medications for safety concerns. For example, Sibutramine, an Australian drug, had to be recalled for harmful influence on heart diseases. [7]
Therefore, undoubtedly we need a better solution to address the obesity issue.

Left: fat mice treated by inducing adipocytes’ apoptosis

As we trace back to the source of obesity, we can see its relevance to adipocytes, which store fat derived from the diet and from liver metabolism. There are two types of adipose tissue, white adipose tissue (WAT) and brown adipose tissue (BAT), which are also known as white fat and brown fat, respectively, and comprise two types of fat cells.

When fat accumulates, the size of adipocytes increases. If the adipocytes in the body reach their maximum capacity of fat, they may replicate to allow additional fat storage.Since the number and the size of adipocytes affect human’s weight status, and it has been proved valid to decrease mice’s weight by targeted ablation of adipose tissue. [8]

Thus, we decided to use design a specific gene-targeted delivery system to help regulate the apoptosis of adipose tissues. To fulfil such a goal, a targeted gene, a gene-regulating tool, a targeting tool and a delivery vesicle have to be selected or created.

1) BCL-2 gene

BCL-2 gene encodes BCL-2 protein (B-cell lymphoma 2), the founding member of the Bcl-2 family of regulator proteins that regulate cell death (apoptosis), by either inducing (pro-apoptotic) or inhibiting (anti-apoptotic) apoptosis. [9][10] Since BCL-2 Can inhibit the activity of pro-apoptotic proteins in adipose tissues, we can induce adipocyte apoptosis by decrease BCL-2 gene’s expression.

2) RNAi

RNA interference (RNAi) is an endogenous regulatory pathway that is triggered by small interfering RNAs (siRNAs), and by the activated RNAi pathway, siRNA can silence the expression of any genes with high efficiency [11].

RNAi silencing is initiated when double-stranded RNA (dsRNA) is processed into siRNA between 19-26 base pairs in length by an RNase III enzyme called Dicer. Subsequently, siRNA unwinds, one of the strands is degraded, and the other strand is incorporated into RNA-induced silencing complexes (RISCs) and guides the complex to recognize messenger RNA (mRNA) sequences with perfect or nearly perfect complementarity, resulting in cleavage and degradation of the target, thereby interrupting protein synthesis of the target gene.

The RNAi process. dsRNA is processed into siRNA by the endonuclease Dicer. The siRNA is loaded into the RNA-induced silencing complex (RISC), followed by cleavage and release of the passenger strand. The guide strand then associates with a homologous mRNA strand by conventional base paring, and the mRNA strand is cleaved by RISC and released for further degradation within the cytoplasm.

RNAi is a promising applicable mechanism that has the potential to spark a revolution in medicine.
In the few years since biotechnologists have adopted RNAi, this technique has already earned a place among the major technology platforms. This powerful RNA-based mechanism by which cells can perform sequence-specific post-transcriptional gene silencing has great potential for therapeutic application in diseased cells. Initial studies were conducted in diseases requiring localized delivery. To date, locally delivered siRNAs have been used in clinical trials for diseases primarily involving the eye, such as age-related macular degeneration (AMD), diabetic macular edema (DME) and glaucoma, and a small number of other diseases involving respiratory syncytial virus (RSV) infections, pachyonychia congenita and pancreatic ductal adenocarcinoma. Later, with improvements in nanocarrier technology, systemically delivered siRNA-based therapeutics outnumbered locally delivered siRNA-based therapeutics, leading to a greater number of medical breakthroughs [12].

In the meantime, RNAi is already profitable. Although still in the proving stages as a therapeutic method, RNAi is providing a number of business opportunities. John Berriman, a director at the venture capital firm Abingworth Management Limited (London), estimates that such therapeutics could eventually capture as much as 10% of the drug market. He and others believe that this possibility could translate into billions of dollars in a market that sees revenues of hundreds of millions of dollars annually for some drugs [13]. Fascinatingly, nanomaterial delivery technologies motivate companies to invest. The global RNAi drug delivery market using nanomaterials is expected to grow to nearly $24 billion by 2015 at a 5 year compound annual growth rate of 27.9% [14].

Hopefully, in the long run, gene therapy using RNAi will become common practice in treating a range of diseases, particularly those associated with oncogenesis and infectious disease. And we chose this technique to regulate our target gene.

Global analysis on the RNAi market for 2014. (A) RNAi companies by continent. (B) RNAi market between 2004 and 2014.

3) tPep: CKGGRAKDC

tPep is a targeting peptide that actually serves as the ligand of the prohibitin, a multifunction membrane protein in white fat tissues. [8] We can take prohibitin as a marker of adipose tissues, which makes tPep a perfect targeting tool.

4) exosome

The biggest obstacle for successful clinical application of siRNA is to develop a safe and effective delivery system directed at the target tissues only. Current techniques for small RNA transfer use viruses or synthetic agents as delivery vehicles. The replacement of these delivery vehicles with a low toxicity and high target-specific approach is essential for making siRNA therapy feasible.

Recently, increasing attention has been given to the use of endogenous exosomes as a novel siRNA delivery system. Exosomes are natural, nano-sized intraluminal vesicles with a diameter ranging between 40 and 100 nm [15]. Released by most cell types, exosomes are present in almost all biological fluids and function as natural transporters of bioactive molecules (proteins, mRNAs, and small RNAs) between neighboring cells. The cargoes that exosomes carry can be gene expression-regulatory functionally in the recipient cells; thus, exosomes are known as the communicators of the microenvironment.

The detailed process of exosomal siRNA transfer is shown as follows. First, cytosolic siRNA is incorporated into the exosomes during invagination of the endosomal membrane. When the MVB fuses with the plasma membrane, exosomal siRNA is released into the circulation simultaneous with the release of exosomes. Subsequently, exosomes bind to the plasma membrane of a target cell, and then exosomes either fuse directly with the plasma membrane or are first endocytosed and then fuse with the delimiting membrane of an endocytic compartment. Both pathways result in the delivery of the exosomal siRNA to the cytosol of the target cell where it may associate with and silence corresponding mRNA [16] [17].

In conclusion, as exosomes have the intrinsic ability to traverse biological barriers and naturally transport functional small RNAs between cells with a character ——potentially better tolerated by the immune system because they are nano-carriers derived from endogenous cells, we picked exosomes to be our delivery vesicles.

Design

In general, the project design can be divided into three sessions: (1) RNAi module, (2) targeting module, (3) assembly.

RNAi module

RNA interference (RNAi) serves as a powerful tool that employs siRNAs to silence the expression of specific target genes. We designed specific BCL2 siRNAs to act as therapeutic agents to degrade BCL2 mRNA and block BCL2 expression and therefore allow higher apoptosis probabilities. Four siRNA sequences targeting different sites of the BCL2 mRNA open reading frame (ORF) were designed based on a free software accessible online. We conducted pre-experiments to find the best siRNA sequences on the targeted gene BCL2 to insure the maximum gene-specificity and silencing efficacy. This tool also designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. At least two of the four pre-designed shRNA plasmids are guaranteed to knock down expression of the targeted gene BCL2. The access to two effective sequences allows appropriate control on non-specific effects. When the shRNA plasmids of BCL2 are transfected into HEK293 cells, Dicer cleaves the shRNA into siRNA of BCL2. However, because siRNAs in vivo are vulnerable to degradation by plasma and tissue nucleases, nano-vesicles to facilitate siRNA uptake into the neurons need to be established.

Target module

Exosomes are natural nano-sized vesicles secreted by endogenous cells. Given their intrinsic role as natural carriers of bioactive molecules between cells, exosomes are reasonable siRNA carriers in terms of therapeutic use. Since exosomes can be genetically modified and are biocompatible with the immune system, they are capable of delivering siRNA to specific cellular environments without bringing cytotoxicity or triggering the immune response. To ensure that exosomes can transport siRNAs to targeted tissues, we redesigned natural exosomes through genetic modification.

We engineered our chassis, human embryonic kidney 293 (HEK293) cells, to express a fusion protein composed of the exosome membrane protein Lamp2b and a white fat tissue-targeted short peptide (tPep). Lamp2b (lysosomal-associated membrane protein 2b) is a protein ubiquitously expressed on the surface of exosomes. tPep is a specific ligand to the prohibitin that is abundantly located on the surface of adipocytes. By genetically linking the tPep to the outer membrane portion of Lamp2b, tPep can make its entrance on the surface of exosomes. Through these modifications, exosomes will be allowed to identify and target neuronal cells by binding exosome surface tPep to prohibitin on adipocytes.

We connected tPep to Lamp2b using a glycine-linker to construct our fusion protein and promoted its expression by the promoter Pcmv. Then, our site-specific exosomes could deliver siRNA to brain tissue.

Assembly

By expressing adipocyte-targeting peptide on the surface of exosomes, filling exosomes with BCL2 siRNA, we will harvest a large amount of exosomes targeted to the adipose tissues carrying BCL2 siRNA inside. When the modified exosomes are injected into the bloodstream, the exosomes will specifically identify adipocytes and fuse with them under the direction of the tPep. Once siRNAs get access into adipocytes, they will degrade BCL2 mRNA by base-pairing, resulting in a sharp decrease in BCL2s and thus an increase in apoptosis. Theoretically, the delivery of BCL2 siRNA to the targeted cells will be achieved, whereas non-specific uptake of BCL2 siRNA in other tissues will be avoided. Consequently, the increase of apoptosis will enable the treated animal (in our case, mice) to lose weight successfully.

Reference

[1]."Obesity and overweight Fact sheet N°311". WHO. January 2015. Retrieved 2 February 2016.
[2] .Haslam DW, James WP (2005). "Obesity". Lancet (Review). 366 (9492): 1197–209. PMID 16198769. doi:10.1016/S0140-6736(05)67483-1.
[3]. Luppino, FS; de Wit, LM; Bouvy, PF; Stijnen, T; Cuijpers, P; Penninx, BW; Zitman, FG (March 2010). "Overweight, obesity, and depression: a systematic review and meta-analysis of longitudinal studies.". Archives of General Psychiatry. 67 (3): 220–9. PMID 20194822. doi:10.1001/archgenpsychiatry.2010.2.
[4]Barness LA, Opitz JM, Gilbert-Barness E (December 2007). "Obesity: genetic, molecular, and environmental aspects". American Journal of Medical Genetics. 143A (24): 3016–34. PMID 18000969. doi:10.1002/ajmg.a.32035.
[5] Mokdad AH, Marks JS, Stroup DF, Gerberding JL (March 2004). "Actual causes of death in the United States, 2000" (PDF). JAMA. 291 (10): 1238–45. PMID 15010446. doi:10.1001/jama.291.10.1238.
[6] Pollack, Andrew (June 18, 2013). "A.M.A. Recognizes Obesity as a Disease". New York Times. Archived from the original on June 23, 2013.
[7] “Current and Future Medical Treatment of Obesity”, Gastrointestinal Endoscopy Clinics of North America, Author links open overlay panel DevikaUmashankerMD, MBALeon I.IgelMDRekha B.KumarMD, MSLouis J.AronneMD https: doi.org/10.1016/j.giec.2016.12.008
[8] Reversal of obesity by targeted ablation of adipose tissue Mikhail G Kolonin1, Pradip K Saha2, Lawrence Chan2, Renata Pasqualini1, 3 & Wadih Arap1, 3, Nature Medicine 10, 625 - 632 (2004) Published online: 9 May 2004; | doi:10.1038/nm1048
[9]Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM (Nov 1984). "Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation". Science. 226 (4678): 1097–9. Bibcode:1984Sci...226.1097T. PMID 6093263. doi:10.1126/science.6093263.
[10] Cleary ML, Smith SD, Sklar J (Oct 1986). "Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation". Cell. 47 (1): 19–28. PMID 2875799. doi:10.1016/0092- 8674(86)90362-4.
[11] Mihue Jang, Jong Hwan Kim, Hae Yun Nam, Ick Chan Kwon & Hyung Jun Ahn. Design of a platform technology for systemic delivery of siRNA to tumours using rolling circle transcription, Nature Communications
[12] Gulnihal Ozcan, Bulent Ozpolat, Robert L. Coleman, Anil K. Sood , Gabriel Lopez-Berestein , Preclinical and clinical development of siRNA-based therapeutics, Advanced Drug Delivery Reviews 87 (2015) 108–119
[13] Ken Howard, Unlocking the money-making potential of RNAi, Nature Biotechnology 21(2008)1441-1446.
[14] Joa ˜o Conde, Natalie Artzi, Are RNAi and miRNA therapeutics truly dead, Trends in Biotechnology 33(2015)141-144.
[15].Hagiwara K, Ochiya T, Kosaka N. A paradigm shift for extracellular vesicles as small RNA carriers: from cellular waste elimination to therapeutic applications[J]. Drug delivery and translational research. 2014,4(1):31-7.
[16] El Andaloussi S, Lakhal S, Mager I, Wood MJ. Exosomes for targeted siRNA delivery across biological barriers[J]. Advanced drug delivery reviews. 2013,65(3):391-7.
[17].Stoorvogel W. Functional transfer of microRNA by exosomes[J]. Blood. 2012,119(3):646-8.

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-                    <h1>PROJECT</h1>
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-         <h2>Description</h2>
-        <h3>Overview of cancer</h3>
-        <p>
-            Undeniably, no matter what we pursue, it is the health and wellness that people care about most. Everyone goes for a healthier and stronger body, but we are all clear about that there are numerous diseases around us, and it's still clueless for us to completely resist and cure a large quantity of them. People usually feel uncomfortable and scared by intractable and severe diseases. And cancer must be one of the nightmares.
-        <br />
-            Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. Once the spread, also known as metastasis, isn’t well controlled, it can result in death[1] . As one of the most frightening death threat, cancer can be aggressive and malignant. The harmful effects of cancer on individual, family and society are enormous and appalling.
-        </p>
-        <h3>Global</h3>
-        <p>
-            According to WHO and the latest global cancer statistics (2015), there were 14.1 million new cancer cases in 2012 worldwide and the corresponding estimates for total cancer deaths were 8.2 million (Fig. 1). Literally, about 22,000 cancer deaths happened a day. Besides, 1 in 7 deaths was related to cancer, and cancer caused more deaths than AIDS, tuberculosis and malaria combined. By 2030, the global burden is expected to grow to 21.7 million new cancer cases and 13 million cancer deaths on account of rapid growth and aging of population[1] . Due to the adoption of lifestyles that are known to increase cancer risk, such as smoking, poor diet, physical inactivity and reproductive changes (including lower parity and later age at first birth) in developing country, it’s reasonable to estimate that the actual figures will be considerably larger[2] .
-            <br />
-            Therefore, cancer is becoming one of the leading causes of death and the major public health problem around the world (Fig. 2).
-        </p>
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-    <div id="Design" style="background-color: #808080;color: #DCDCDC;">
-        <h2 style="color: #DCDCDC;">Design</h2>
-        <h3>RNAi & KRAS</h3>
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-             RNA interference (RNAi) is an emerging technology of gene silencing. It uses siRNA segments to destroy specific mRNA, thereby, shut down the expression of target genes. RNAi has been demonstrated as a novel treatment modality of cancer and we decided to utilize this technology to silence the expression of specific oncogene[1]. KRAS is one of the most commonly mutated oncogenes in lung cancer. The mutation rate of KRAS can be up to 25% in NSCLC (a main type of lung cancer)[2]. Thus, we picked KRAS as our target oncogene. Then we designed anti-KRAS siRNA as a therapeutic agent to degrade KRAS mRNA, therefore, repressing the expression and function of K-ras protein. We used a specialized software developed by team SYSU-software to find the ideal siRNA sequence. This tool also designed pairs of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. When the shRNA plasmids of KRAS are transfected into HEK293 cells, the dsRNA is cleaved into siRNA of KRAS by the enzyme Dicer, and then target KRAS mRNA. However, due to the short half-life period and poor cellular uptake of exogenous siRNA[3], an efficient delivery vehicle is needed to stabilize and enhance the naked small interference molecule's function.
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-                    <h2 style="color: white;float: left;font-size: 40px;padding: 175px 0 175px 100px;font-family: 'Microsoft YaHei';letter-spacing: 1.5px;">INTERLAB</h2>
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+                        Obesity is a medical condition in which excess body fat has accumulated to the extent that it may have a negative effect on health. [1] People are generally considered obese when their body mass index (BMI), a measurement obtained by dividing a person's weight by the square of the person's height, is over 30 kg/m2, with the range 25–30 kg/m2 defined as overweight. [1]
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+                        We need to perceive this: obesity, or excess fat, is not simply an obstacle to many people pursuing beauty. It also stands in the way of health acquiring, sharing connection with various diseases, like cardiovascular diseases, obstructive sleep apnea, type 2 diabetes, depression, certain types of cancer and so on. [2][3] What’s worse, obesity is now one of the major evitable causes for death globally. [4][5] In general, obesity cut down life expectancy by six to seven years. As for the number of obese people, in 2016, more than 1.9 billion adults, aged 18 and above, were overweight. Of these over 650 million were obese. 39% of adults aged 18 years and over were overweight in 2016, and 13% were obese. [1]
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+                    <i >Map of Obesity in Adult Males (% of adult population with en:BMI 30+) per country. Using data updated until December 2008.</i>
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+                    In fact, given the severe circumstance, in 2013, the American medical association has classified obesity as a disease. [6]
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+                    While the prevalence of obesity continued to grow, people weren’t just sitting these years away and did nothing against obesity. To lose weight, some change their lifestyle. People trying to lose weight have to refuse high calorie food and switch to healthier food like fiber-rich vegetables, and try to exercise more. Besides, some people might get help from medications. The legal medications in the market usually work by inhibiting appetite (like Sibutramine), reducing the degradation and absorption of fat (like Orlistat), or regulating the metabolism of fat. [7]
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+                    We have seen the traditional methods to lose weight. Many of them are prevalent, and we cannot deny their efficacy. However, it’s not hard to find these traditional methods’ defects: changing lifestyle is usually unpleasant and perseverance-needed, and even if you get the positive feedback, the probability to regain weight is also quite high. [7] As for medications, they may bring out inevitable side effects, like oily stool. Some company had to retract their medications for safety concerns. For example, Sibutramine, an Australian drug, had to be recalled for harmful influence on heart diseases. [7]
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+                    Therefore, undoubtedly we need a better solution to address the obesity issue.
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+                        <i style="margin-left: 10px;">Left: fat mice treated by inducing adipocytes’ apoptosis</i>
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+                        As we trace back to the source of obesity, we can see its relevance to adipocytes, which store fat derived from the diet and from liver metabolism. There are two types of adipose tissue, white adipose tissue (WAT) and brown adipose tissue (BAT), which are also known as white fat and brown fat, respectively, and comprise two types of fat cells.
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+                        When fat accumulates, the size of adipocytes increases. If the adipocytes in the body reach their maximum capacity of fat, they may replicate to allow additional fat storage.Since the number and the size of adipocytes affect human’s weight status, and it has been proved valid to decrease mice’s weight by targeted ablation of adipose tissue. [8]
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+                        Thus, we decided to use design a specific gene-targeted delivery system to help regulate the apoptosis of adipose tissues. To fulfil such a goal, a targeted gene, a gene-regulating tool, a targeting tool and a delivery vesicle have to be selected or created.
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+                <h3>1) BCL-2 gene</h3>
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+                    BCL-2 gene encodes BCL-2 protein (B-cell lymphoma 2), the founding member of the Bcl-2 family of regulator proteins that regulate cell death (apoptosis), by either inducing (pro-apoptotic) or inhibiting (anti-apoptotic) apoptosis. [9][10] Since BCL-2
+                    Can inhibit the activity of pro-apoptotic proteins in adipose tissues, we can induce adipocyte apoptosis by decrease BCL-2 gene’s expression.
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+                    <img src="https://static.igem.org/mediawiki/2017/c/c8/NJU_China_iGEM_2017_project_fig4.png" alt="" style="width: 70%; margin-left:150px;">
+                </div>
+                <h3>2) RNAi</h3>
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+                    RNA interference (RNAi) is an endogenous regulatory pathway that is triggered by small interfering RNAs (siRNAs), and by the activated RNAi pathway, siRNA can silence the expression of any genes with high efficiency [11].
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+                    RNAi silencing is initiated when double-stranded RNA (dsRNA) is processed into siRNA between 19-26 base pairs in length by an RNase III enzyme called Dicer. Subsequently, siRNA unwinds, one of the strands is degraded, and the other strand is incorporated into RNA-induced silencing complexes (RISCs) and guides the complex to recognize messenger RNA (mRNA) sequences with perfect or nearly perfect complementarity, resulting in cleavage and degradation of the target, thereby interrupting protein synthesis of the target gene.
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+                        The RNAi process. dsRNA is processed into siRNA by the endonuclease Dicer. The siRNA is loaded into the RNA-induced silencing complex (RISC), followed by cleavage and release of the passenger strand. The guide strand then associates with a homologous mRNA strand by conventional base paring, and the mRNA strand is cleaved by RISC and released for further degradation within the cytoplasm.
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+                    RNAi is a promising applicable mechanism that has the potential to spark a revolution in medicine.
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+                    In the few years since biotechnologists have adopted RNAi, this technique has already earned a place among the major technology platforms. This powerful RNA-based mechanism by which cells can perform sequence-specific post-transcriptional gene silencing has great potential for therapeutic application in diseased cells. Initial studies were conducted in diseases requiring localized delivery. To date, locally delivered siRNAs have been used in clinical trials for diseases primarily involving the eye, such as age-related macular degeneration (AMD), diabetic macular edema (DME) and glaucoma, and a small number of other diseases involving respiratory syncytial virus (RSV) infections, pachyonychia congenita and pancreatic ductal adenocarcinoma. Later, with improvements in nanocarrier technology, systemically delivered siRNA-based therapeutics outnumbered locally delivered siRNA-based therapeutics, leading to a greater number of medical breakthroughs [12].
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+                    In the meantime, RNAi is already profitable. Although still in the proving stages as a therapeutic method, RNAi is providing a number of business opportunities. John Berriman, a director at the venture capital firm Abingworth Management Limited (London), estimates that such therapeutics could eventually capture as much as 10% of the drug market. He and others believe that this possibility could translate into billions of dollars in a market that sees revenues of hundreds of millions of dollars annually for some drugs [13]. Fascinatingly, nanomaterial delivery technologies motivate companies to invest. The global RNAi drug delivery market using nanomaterials is expected to grow to nearly $24 billion by 2015 at a 5 year compound annual growth rate of 27.9% [14].
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+                    Hopefully, in the long run, gene therapy using RNAi will become common practice in treating a range of diseases, particularly those associated with oncogenesis and infectious disease. And we chose this technique to regulate our target gene.
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+                    <i>Global analysis on the RNAi market for 2014. (A) RNAi companies by continent. (B) RNAi market between 2004 and 2014.</i>
+                </div>
+                <h3>3) tPep: CKGGRAKDC</h3>
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+                    tPep is a targeting peptide that actually serves as the ligand of the prohibitin, a multifunction membrane protein in white fat tissues. [8] We can take prohibitin as a marker of adipose tissues, which makes tPep a perfect targeting tool.
+                </div>
+                <h3>4) exosome</h3>
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+                    The biggest obstacle for successful clinical application of siRNA is to develop a safe and effective delivery system directed at the target tissues only. Current techniques for small RNA transfer use viruses or synthetic agents as delivery vehicles. The replacement of these delivery vehicles with a low toxicity and high target-specific approach is essential for making siRNA therapy feasible.
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+                        Recently, increasing attention has been given to the use of endogenous exosomes as a novel siRNA delivery system. Exosomes are natural, nano-sized intraluminal vesicles with a diameter ranging between 40 and 100 nm [15]. Released by most cell types, exosomes are present in almost all biological fluids and function as natural transporters of bioactive molecules (proteins, mRNAs, and small RNAs) between neighboring cells. The cargoes that exosomes carry can be gene expression-regulatory functionally in the recipient cells; thus, exosomes are known as the communicators of the microenvironment.
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+                        The detailed process of exosomal siRNA transfer is shown as follows. First, cytosolic siRNA is incorporated into the exosomes during invagination of the endosomal membrane. When the MVB fuses with the plasma membrane, exosomal siRNA is released into the circulation simultaneous with the release of exosomes. Subsequently, exosomes bind to the plasma membrane of a target cell, and then exosomes either fuse directly with the plasma membrane or are first endocytosed and then fuse with the delimiting membrane of an endocytic compartment. Both pathways result in the delivery of the exosomal siRNA to the cytosol of the target cell where it may associate with and silence corresponding mRNA [16] [17].
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+                    In conclusion, as exosomes have the intrinsic ability to traverse biological barriers and naturally transport functional small RNAs between cells with a character ——potentially better tolerated by the immune system because they are nano-carriers derived from endogenous cells, we picked exosomes to be our delivery vesicles.
+                </div>
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+        <div id="Design" class="second" style="padding: 50px 0;">
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+            <h2>Design</h2>
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+                    <b>In general, the project design can be divided into three sessions: (1) RNAi module, (2) targeting module, (3) assembly.</b>
+                </div>
+                <h3>RNAi module</h3>
+                <div class="row">
+                    RNA interference (RNAi) serves as a powerful tool that employs siRNAs to silence the expression of specific target genes. We designed specific BCL2 siRNAs to act as   therapeutic agents to degrade BCL2 mRNA and block BCL2 expression and therefore allow higher apoptosis probabilities. Four siRNA sequences targeting different sites of the BCL2 mRNA open reading frame (ORF) were designed based on a free software accessible online. We conducted pre-experiments to find the best siRNA sequences on the targeted gene BCL2 to insure the maximum gene-specificity and silencing efficacy. This tool also designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. At least two of the four pre-designed shRNA plasmids are guaranteed to knock down expression of the targeted gene BCL2. The access to two effective sequences allows appropriate control on non-specific effects. When the shRNA plasmids of BCL2 are transfected into HEK293 cells, Dicer cleaves the shRNA into siRNA of BCL2. However, because siRNAs in vivo are vulnerable to degradation by plasma and tissue nucleases, nano-vesicles to facilitate siRNA uptake into the neurons need to be established.
+                </div>
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+                    <img src="https://static.igem.org/mediawiki/2017/7/7b/NJU_China_iGEM_2017_ProjectDesign_Fig1.png" alt="fig">
+                </div>
+                <h3>Target module</h3>
+                <div class="row">
+                    Exosomes are natural nano-sized vesicles secreted by endogenous cells. Given their intrinsic role as natural carriers of bioactive molecules between cells, exosomes are reasonable siRNA carriers in terms of therapeutic use. Since exosomes can be genetically modified and are biocompatible with the immune system, they are capable of delivering siRNA to specific cellular environments without bringing cytotoxicity or triggering the immune response. To ensure that exosomes can transport siRNAs to targeted tissues, we redesigned natural exosomes through genetic modification.
+                </div>
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+                        We engineered our chassis, human embryonic kidney 293 (HEK293) cells, to express a fusion protein composed of the exosome membrane protein Lamp2b and a white fat tissue-targeted short peptide (tPep). Lamp2b (lysosomal-associated membrane protein 2b) is a protein ubiquitously expressed on the surface of exosomes. tPep is a specific ligand to the prohibitin that is abundantly located on the surface of adipocytes. By genetically linking the tPep to the outer membrane portion of Lamp2b, tPep can make its entrance on the surface of exosomes. Through these modifications, exosomes will be allowed to identify and target neuronal cells by binding exosome surface tPep to prohibitin on adipocytes.
+                    </div>
+                    <div class="col-sm-5 col-md-5">
+                        <img src="https://static.igem.org/mediawiki/2017/e/e3/NJU_China_iGEM_2017_project_fig9.png" alt="">
+                    </div>
+                </div>
+                <div class="row" style="text-align: center;">
+                    <img src="https://static.igem.org/mediawiki/2017/c/cf/NJU_China_iGEM_2017_project_fig10.png" alt="">
+                    <br />
+                    We connected tPep to Lamp2b using a glycine-linker to construct our fusion protein and promoted its expression by the promoter Pcmv. Then, our site-specific exosomes could deliver siRNA to brain tissue.
+                </div>
+                <h3>Assembly</h3>
+                <div class="row">
+                    By expressing adipocyte-targeting peptide on the surface of exosomes, filling exosomes with BCL2 siRNA, we will harvest a large amount of exosomes targeted to the adipose tissues carrying BCL2 siRNA inside. When the modified exosomes are injected into the bloodstream, the exosomes will specifically identify adipocytes and fuse with them under the direction of the tPep. Once siRNAs get access into adipocytes, they will degrade BCL2 mRNA by base-pairing, resulting in a sharp decrease in BCL2s and thus an increase in apoptosis. Theoretically, the delivery of BCL2 siRNA to the targeted cells will be achieved, whereas non-specific uptake of BCL2 siRNA in other tissues will be avoided. Consequently, the increase of apoptosis will enable the treated animal (in our case, mice) to lose weight successfully.
+                </div>
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              </div>
+            <h2 > Reference </h2>
+            <div class="container">
+                <div class="row">
+            [1]."Obesity and overweight Fact sheet N°311". WHO. January 2015. Retrieved 2 February 2016.
+            <br />
+            [2] .Haslam DW, James WP (2005). "Obesity". Lancet (Review). 366 (9492): 1197–209.  PMID 16198769. doi:10.1016/S0140-6736(05)67483-1.
+            <br />
+            [3]. Luppino, FS; de Wit, LM; Bouvy, PF; Stijnen, T; Cuijpers, P; Penninx, BW; Zitman, FG (March  2010). "Overweight, obesity, and depression: a systematic review and meta-analysis of  longitudinal studies.". Archives of General Psychiatry. 67 (3): 220–9. PMID 20194822.  doi:10.1001/archgenpsychiatry.2010.2.
+            <br />
+            [4]Barness LA, Opitz JM, Gilbert-Barness E (December 2007). "Obesity: genetic,  molecular, and environmental aspects". American Journal of Medical Genetics. 143A  (24): 3016–34. PMID 18000969. doi:10.1002/ajmg.a.32035.
+            <br />
+            [5] Mokdad AH, Marks JS, Stroup DF, Gerberding JL (March 2004). "Actual causes  of death in the United States, 2000" (PDF). JAMA. 291 (10): 1238–45.  PMID 15010446. doi:10.1001/jama.291.10.1238.
+            <br />
+            [6] Pollack, Andrew (June 18, 2013). "A.M.A. Recognizes Obesity as a Disease". New  York Times. Archived from the original on June 23, 2013.
+            <br />
+            [7] “Current and Future Medical Treatment of Obesity”, Gastrointestinal Endoscopy Clinics of North America, Author links open overlay panel DevikaUmashankerMD, MBALeon I.IgelMDRekha B.KumarMD, MSLouis J.AronneMD https: doi.org/10.1016/j.giec.2016.12.008
+            <br />
+            [8] Reversal of obesity by targeted ablation of adipose tissue Mikhail G Kolonin1, Pradip K Saha2, Lawrence Chan2, Renata Pasqualini1, 3 & Wadih Arap1, 3, Nature Medicine  10, 625 - 632 (2004) Published online: 9 May 2004; | doi:10.1038/nm1048
+            <br />
+            [9]Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM (Nov 1984). "Cloning of the  chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome  translocation". Science. 226 (4678): 1097–9. Bibcode:1984Sci...226.1097T.  PMID 6093263. doi:10.1126/science.6093263.
+            <br />
+            [10] Cleary ML, Smith SD, Sklar J (Oct 1986). "Cloning and structural analysis of  cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the  t(14;18) translocation". Cell. 47 (1): 19–28. PMID 2875799. doi:10.1016/0092- 8674(86)90362-4.
+            <br />
+            [11] Mihue Jang, Jong Hwan Kim, Hae Yun Nam, Ick Chan Kwon & Hyung Jun Ahn. Design of a platform technology for systemic delivery of siRNA to tumours using rolling circle transcription, Nature Communications
+            <br />
+            [12] Gulnihal Ozcan, Bulent Ozpolat, Robert L. Coleman, Anil K. Sood , Gabriel Lopez-Berestein , Preclinical and clinical development of siRNA-based therapeutics, Advanced Drug Delivery Reviews 87 (2015) 108–119
+            <br />
+            [13] Ken Howard, Unlocking the money-making potential of RNAi, Nature Biotechnology 21(2008)1441-1446.
+            <br />
+            [14] Joa ˜o Conde, Natalie Artzi, Are RNAi and miRNA therapeutics truly dead, Trends in Biotechnology 33(2015)141-144.
+            <br />
+            [15].Hagiwara K, Ochiya T, Kosaka N. A paradigm shift for extracellular vesicles as small RNA carriers: from cellular waste elimination to therapeutic applications[J]. Drug delivery and translational research. 2014,4(1):31-7.
+            <br />
+            [16] El Andaloussi S, Lakhal S, Mager I, Wood MJ. Exosomes for targeted siRNA delivery across biological barriers[J]. Advanced drug delivery reviews. 2013,65(3):391-7.
+            <br />
+            [17].Stoorvogel W. Functional transfer of microRNA by exosomes[J]. Blood. 2012,119(3):646-8.
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+    <!-- /#wrapper -->
-        <script type="text/javascript" src="https://2017.igem.org/Team:NJU-China/Javascript:masterslider?action=raw&ctype=text/javascript"></script>
-        <script type="text/javascript" src="https://2017.igem.org/Team:NJU-China/Javascript:jquery-easing?action=raw&ctype=text/javascript"></script>
+    <script src="https://2017.igem.org/Team:NJU-China/Javascript:jquery-1110-min?action=raw&ctype=text/javascript" type="text/javascript"></script>
-        <script type="text/javascript" src="https://2017.igem.org/Team:NJU-China/Javascript:jquery-magnific-popup?action=raw&ctype=text/javascript"></script>
+    <script src="https://2017.igem.org/Team:NJU-China/Javascript:bootstrap-min-js?action=raw&ctype=text/javascript" type="text/javascript"></script>
-        <script type="text/javascript" src="https://2017.igem.org/Team:NJU-China/Javascript:owl-carousel?action=raw&ctype=text/javascript"></script>
+    <script type="text/javascript">
-        <script type="text/javascript" src="https://2017.igem.org/Team:NJU-China/Javascript:jquery-dlmenu?action=raw&ctype=text/javascript"></script>
+         $(document).ready(function () {
-        <script type="text/javascript" src="https://2017.igem.org/Team:NJU-China/Javascript:include?action=raw&ctype=text/javascript"></script>
+          var trigger = $('.hamburger'),
-        <script type="text/javascript" src="https://2017.igem.org/Team:NJU-China/Javascript:instagram-stream?action=raw&ctype=text/javascript"></script>
+              overlay = $('.overlay'),
-        <script type="text/javascript" src="https://2017.igem.org/Team:NJU-China/Javascript:TweenMax?action=raw&ctype=text/javascript"></script>
+             isClosed = false;
-         <script type="text/javascript" src="https://2017.igem.org/Team:NJU-China/Javascript:ScrollToPlugin?action=raw&ctype=text/javascript"></script>
-        <script type="text/javascript" src="https://2017.igem.org/Team:NJU-China/Javascript:jquery-jumpto?action=raw&ctype=text/javascript"></script>
-<script type="text/javascript" src="https://2017.igem.org/Team:NJU-China/Javascript:jquery-ba-bbq?action=raw&ctype=text/javascript"></script>
-<script type="text/javascript" src="https://2017.igem.org/Team:NJU-China/Javascript:jquery-smooth-scroll?action=raw&ctype=text/javascript"></script>
- <script>
-    jQuery(document).ready(function ($) {
-                'use strict';
-                // MASTER SLIDER START
-                var slider = new MasterSlider();
-                slider.setup('masterslider', {
-                    width: 1920, // slider standard width
-                    height: 1080, // slider standard height
-                    space: 0,
-                    layout: "fullwidth",
-                    speed: 50,
-                    centerControls: false,
-                    loop: false,
-                    autoplay: false,
-                    parallaxMode: "mouse"
-                            // more slider options goes here...
-                            // check slider options section in documentation for more options.
-                });
-                // adds Arrows navigation control to the slider.
-                MSScrollParallax.setup(slider, 50, 80, true);
-                //GALLERY LIGHTBOX
-                $('.triggerZoom').magnificPopup({
-                    type: 'image',
-                    gallery: {
-                        enabled: true
-                    }
-                });
-                // EVENTS CAROUSEL START
+            trigger.click(function () {
-                var owl = $("#events-carousel");
+              hamburger_cross();
+            });
-                owl.owlCarousel({
+            function hamburger_cross() {
-                    singleItem: true,
-                    pagination: true,
-                    autoPlay: true
-                });
-                 // INSTAGRAM STREAM START
+              if (isClosed == true) {
-                 $('.instagram-stream').instagramstream({
+                 overlay.hide();
-                    limit: 22, // number of images to fetch
+                 trigger.removeClass('is-open');
-                    username: 'NJU-CHINA', // your username
+                trigger.addClass('is-closed');
-                    overlay: true, // add overlay layer of hover effect
+                isClosed = false;
-                    textContainer: '.is-text', // default: '.is-text', pass jQuery object or selector
+              } else {
-                    textPosition: '10', // place that at this position
+                overlay.show();
-                    textSize: '4', // size of text e.g. 1 - has size like one image; 2 - has size of two images etc.
+                trigger.removeClass('is-closed');
-                    accessToken: '3315219258.4b0afec.8788057cf36948e98d27353bee5a77d9'
+                trigger.addClass('is-open');
-                });
+                isClosed = true;
+              }
-     });
+          }
-</script>
+          $('[data-toggle="offcanvas"]').click(function () {
+                $('#wrapper').toggleClass('toggled');
+          });
+        });
+     </script>
+<div class="loader"></div>
+        <script type="text/javascript">
+            $(window).load(function() {
+                $(".loader").fadeOut("slow");
+            })
+        </script>
 </body>
 </html>