Difference between revisions of "Part:BBa K2865009"

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	__NOTOC__		__NOTOC__
	<partinfo>BBa_K2865009 short</partinfo>		<partinfo>BBa_K2865009 short</partinfo>
−		+	The composite part is the combination of BNP Promotor(<partinfo>BBa_K2865000</partinfo>) and AR185-T2A-EGFP (<partinfo>BBa_K2865001</partinfo>).The use of this part is to express our novel intrabody AR185 linked with reporter EGFP in failing heart under control of BNP Promotor.
−	AR185 linked with eGFP by Tr2 is attached to the PloyA under control of BNP Promotor(BB...).The use of this part is to express AR185 under control of BNP Promotor induced by some factors. And to certify its potential efficacy on heart failure gene therapy.	+	<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>[[File:T--SMMU-China--B185.png|450px|thumb|center|Figure 1. Schematic diagram of constructs of BNP-AR185-Poly(A)]]
	<!-- Add more about the biology of this part here -->		<!-- Add more about the biology of this part here -->
	<h2>Usage and Biology</h2>		<h2>Usage and Biology</h2>
	<h3>Respective induction of parts </h3>		<h3>Respective induction of parts </h3>
		+	<p class="MsoNormal"><span lang="EN-US"><B>BNP Promotor</B></span></p>
		+	<p class="MsoNormal"><span lang="EN-US">Brain natriuretic peptide (BNP), also known as B-type natriuretic peptide, is a hormone secreted by cardiomyocytes in the heart ventricles. The testing of BNP to standard clinical assessment has been considered as an accurate and efficient diagnosis and prognostication of HF, and the use of BNP may be a potential targeting to improve clinical outcomes. [[#References|[1][2][3]]]</span></p>
		+	<p class="MsoNormal"><span lang="EN-US">Circulating levels of BNP are normally very low in healthy individuals. And BNP level correlate well with ventricular wall stress and severity of HF, that is to say, BNP promoter activity is related to the severity of HF[[#References|[4]]]. Based on the qualities mentioned above, it is natural to think that we can utilize BNP promoter to control gene expression in HF gene therapy. However, despite decades of research, the mechanisms regulating the BNP genes during development and disease are not well understood. According to previous studies, BNP promoter can be activated by a number of factors associated with HF, including endothelin (ET), angiotensin II (Ang II), mechanical strain, and hypoxia, among others. These regulating activities could be attributed to the combination of a large variety of transcriptional factors and cis-acting elements at the upstream of the promoter.</span></p>
		+	<p class="MsoNormal"><span lang="EN-US"><B>Nanobody AR185</B></span></p>
		+	<p class="MsoNormal"><span lang="EN-US">Here, we designed a novel nanobody AR185 (BBa_K2865001) to treat heart failure by targeting cardiac ryanodine receptor type 2 (RyR2) to restore Ca<sup>2+</sup> cycling.</span></p>
		+	<p class="MsoNormal"><span lang="EN-US">Recent years, proteins relevant to the Ca<sup>2+</sup> cycling in myocardium have emerged as a potential target for the treatment of severe heart failure. Chronic PKA phosphorylation of RyR2 has been shown to increase diastolic SR "calcium leakage" which is considered to be an important pathological mechanism for myocardial injury and heart failure development. This nanobody, AR185, was designed to restore heart function and ameliorate heart failure by inhibiting hyperphosphorylation of RyR2.</span></p>
		+	<p class="MsoNormal"><span lang="EN-US">Nanobody, also called VHH, corresponds to the variable region of a heavy chain of a camelid antibody and has a very small size of around 15 kDa (Figure.2). Compared with human immunoglobulin which is around 150 kDa, VHH has bigger probability to function in cells and bind to hidden epitopes not accessible to whole antibodies. Therefore, it is a qualified candidate for intracellular antibody (Intrabody).</span></p>
		+	<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>[[File:T--SMMU-China--nanobody.png|450px|thumb|center|Figure.2 Human IgG, camelid heavy-chain-only antibody and its derivative VHH]]
		+	<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>More details of the nanobody’s functions, mechanisms, and how we obtained it can be found in the part page of <partinfo>BBa_K2865001</partinfo>.
		+	<h3>Experimental Setup</h3>
		+	<p class="MsoNormal"><span lang="EN-US">This part is meant to express the AR185 gene under control of BNP promoter.AR185 is a protein monomer which can conjuge with s2808 site on RyR2 receptor to inhibit its phosphorylation. This protein is transported as an intracellular antibody. To characterize AR185 in vivo, we used ET-1, one stimulus factor, to induce BNP promoter expression. Then, the later sequence of the part, AR185, also can be expressed. We created two E. coli strains. One carried the AR185 gene that codes for AR185 under control of the BNP promoter (pSB1C3 plasmid <partinfo>BBa_K2865009</partinfo>), whereas a second strain carried an additional second plasmid with the CMV promoter (pSB1C3 plasmid;<partinfo>BBa_K2865010</partinfo>). </span></p>
		+	<p class="MsoNormal"><span lang="EN-US">To evaluate the potential use of anti-phosphorylation agents targeting RyR2 for the treatment of heart failure, an adeno-associated virus (AAV) based intracellular antibody delivery strategy were adopt to achieve cardiac-specific gene-therapy and demonstrated therapeutic effect both in cell-based assays and in vivo models.</span></p>
−	Brain natriuretic peptide (BNP), also known as B-type natriuretic peptide, is a hormone secreted by cardiomyocytes in the heart ventricles. The testing of BNP to standard clinical assessment has been considered as an accurate and efficient diagnosis and prognostication of HF, and the use of BNP may be a potential targeting to improve clinical outcomes.[1-3]	+	<h2>References</h2>
−	Circulating levels of BNP are normally very low in healthy individuals. And BNP level correlate well with ventricular wall stress and severity of HF, that is to say, BNP promoter activity is related to the severity of HF [4]. Based on the qualities mentioned above, it is natural to think that we can utilize BNP promoter to control gene expression in HF gene therapy. However, despite decades of research, the mechanisms regulating the BNP genes during development and disease are not well understood. According to previous studies, BNP promoter can be activated by a number of factors associated with HF, including endothelin (ET), angiotensin II (Ang II), mechanical strain, and hypoxia, among others. These regulating activities could be attributed to the combination of a large variety of transcriptional factors and cis-acting elements at the upstream of the promoter.	+	1. Maisel AS, Krishnaswamy P, Nowak RM, McCord J, Hollander JE, Duc P, Omland T, Storrow AB, Abraham WT, Wu AH, Clopton P, Steg PG, Westheim A, Knudsen CW, Perez A, Kazanegra R, Herrmann HC, McCullough PA. Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure. N Engl J Med. 2002;347:161–167.
−	AR185 nanobodies which specifically bind to RyR2 in rat cardiomyocytes and have the ability to inhibit PKA dependent S2808 phosphorylation in vitro (detailed showing in BBa_K2865001).	+
−	<h3>Experimental Setup</h3>	+	2. Januzzi JL Jr, Peacock WF, Maisel AS, Chae CU, Jesse RL, Baggish AL, O’Donoghue M, Sakhuja R, Chen AA, van Kimmenade RR, Lewandrowski KB, Lloyd-Jones DM, Wu AH. Measurement of the interleukin family member ST2 in patients with acute dyspnea: resultsfromthePRIDE(Pro-BrainNatriuretic Peptide Investigation of Dyspnea in the Emergency Department) study. J Am Coll Cardiol. 2007;50:607–613.
−	This part is meant to express the AR185 gene under control of BNP promoter.AR185 is a protein monomer which can conjuge with s2808 site on RyR2 receptor to inhibit its phosphorylation. This protein is transported as an intracellular antibody. To characterize AR185 in vivo, we used ET-1, one stimulus factor, to induce BNP promoter expression. Then, the later sequence of the part, AR185, also can be expressed. We created two E. coli strains. One carried the AR185 gene that codes for AR185 under control of the BNP promoter (,,,,,plasmid BBa_I712008), whereas a second strain carried an additional second plasmid with the CMV promoter (,,,,,,,,,plasmid;BBa_I712006).	+
−	To evaluate the potential use of anti-phosphorylation agents targeting RyR2 for the treatment of heart failure, an adeno-associated virus (AAV) based intracellular antibody delivery strategy were adopt to achieve cardiac-specific gene-therapy and demonstrated therapeutic effect both in cell-based assays and in vivo models.	+	3. Felker GM, Hasselblad V, Hernandez AF, O’Connor CM. Biomarker-guided therapy in chronic heart failure: a meta-analysis of randomized controlled trials. Am Heart J. 2009; 158:422–430.
−	<h2>Cloning</h2>	+
−	<h3>PCR Amplification</h3>	+	4.  Januzzi JL Jr, Camargo CA, Anwaruddin S, Baggish AL, Chen AA, Krauser DG, Tung R, Cameron R, Nagurney JT, Chae CU, Lloyd-Jones DM, Brown DF, ForanMelanson S, Sluss PM, Lee-Lewandrowski E, Lewandrowski KB. The N-terminal Pro-BNP Investigation of Dyspnea in the Emergency department (PRIDE) study. Am J Cardiol. 2005;95:948–954.
−	The concentration of each of the 6 parts was increased using PCR amplification.	+
−	<h3>Gel Extraction</h3>	+
−	After PCR amplification, gel extraction was carried out to ensure that the parts were correct, and to separate them.	+
−	<h3>Transformation</h3>	+
−	Transformation was carried out according to standard protocol using competent E. Coli Top 10 cells.	+
	<!-- -->		<!-- -->
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		+
	<span class='h3bb'>Sequence and Features</span>		<span class='h3bb'>Sequence and Features</span>
	<partinfo>BBa_K2865009 SequenceAndFeatures</partinfo>		<partinfo>BBa_K2865009 SequenceAndFeatures</partinfo>

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Latest revision as of 14:57, 16 October 2018

BNP-AR185-Poly(A) The composite part is the combination of BNP Promotor(BBa_K2865000) and AR185-T2A-EGFP (BBa_K2865001).The use of this part is to express our novel intrabody AR185 linked with reporter EGFP in failing heart under control of BNP Promotor.

 

Usage and Biology

Respective induction of parts

BNP Promotor

Brain natriuretic peptide (BNP), also known as B-type natriuretic peptide, is a hormone secreted by cardiomyocytes in the heart ventricles. The testing of BNP to standard clinical assessment has been considered as an accurate and efficient diagnosis and prognostication of HF, and the use of BNP may be a potential targeting to improve clinical outcomes. [1][2][3]

Circulating levels of BNP are normally very low in healthy individuals. And BNP level correlate well with ventricular wall stress and severity of HF, that is to say, BNP promoter activity is related to the severity of HF[4]. Based on the qualities mentioned above, it is natural to think that we can utilize BNP promoter to control gene expression in HF gene therapy. However, despite decades of research, the mechanisms regulating the BNP genes during development and disease are not well understood. According to previous studies, BNP promoter can be activated by a number of factors associated with HF, including endothelin (ET), angiotensin II (Ang II), mechanical strain, and hypoxia, among others. These regulating activities could be attributed to the combination of a large variety of transcriptional factors and cis-acting elements at the upstream of the promoter.

Nanobody AR185

Here, we designed a novel nanobody AR185 (BBa_K2865001) to treat heart failure by targeting cardiac ryanodine receptor type 2 (RyR2) to restore Ca²⁺ cycling.

Recent years, proteins relevant to the Ca²⁺ cycling in myocardium have emerged as a potential target for the treatment of severe heart failure. Chronic PKA phosphorylation of RyR2 has been shown to increase diastolic SR "calcium leakage" which is considered to be an important pathological mechanism for myocardial injury and heart failure development. This nanobody, AR185, was designed to restore heart function and ameliorate heart failure by inhibiting hyperphosphorylation of RyR2.

Nanobody, also called VHH, corresponds to the variable region of a heavy chain of a camelid antibody and has a very small size of around 15 kDa (Figure.2). Compared with human immunoglobulin which is around 150 kDa, VHH has bigger probability to function in cells and bind to hidden epitopes not accessible to whole antibodies. Therefore, it is a qualified candidate for intracellular antibody (Intrabody).

 

 

More details of the nanobody’s functions, mechanisms, and how we obtained it can be found in the part page of BBa_K2865001.

Experimental Setup

This part is meant to express the AR185 gene under control of BNP promoter.AR185 is a protein monomer which can conjuge with s2808 site on RyR2 receptor to inhibit its phosphorylation. This protein is transported as an intracellular antibody. To characterize AR185 in vivo, we used ET-1, one stimulus factor, to induce BNP promoter expression. Then, the later sequence of the part, AR185, also can be expressed. We created two E. coli strains. One carried the AR185 gene that codes for AR185 under control of the BNP promoter (pSB1C3 plasmid BBa_K2865009), whereas a second strain carried an additional second plasmid with the CMV promoter (pSB1C3 plasmid;BBa_K2865010).

To evaluate the potential use of anti-phosphorylation agents targeting RyR2 for the treatment of heart failure, an adeno-associated virus (AAV) based intracellular antibody delivery strategy were adopt to achieve cardiac-specific gene-therapy and demonstrated therapeutic effect both in cell-based assays and in vivo models.

References

1. Maisel AS, Krishnaswamy P, Nowak RM, McCord J, Hollander JE, Duc P, Omland T, Storrow AB, Abraham WT, Wu AH, Clopton P, Steg PG, Westheim A, Knudsen CW, Perez A, Kazanegra R, Herrmann HC, McCullough PA. Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure. N Engl J Med. 2002;347:161–167.

2. Januzzi JL Jr, Peacock WF, Maisel AS, Chae CU, Jesse RL, Baggish AL, O’Donoghue M, Sakhuja R, Chen AA, van Kimmenade RR, Lewandrowski KB, Lloyd-Jones DM, Wu AH. Measurement of the interleukin family member ST2 in patients with acute dyspnea: resultsfromthePRIDE(Pro-BrainNatriuretic Peptide Investigation of Dyspnea in the Emergency Department) study. J Am Coll Cardiol. 2007;50:607–613.

3. Felker GM, Hasselblad V, Hernandez AF, O’Connor CM. Biomarker-guided therapy in chronic heart failure: a meta-analysis of randomized controlled trials. Am Heart J. 2009; 158:422–430.

4. Januzzi JL Jr, Camargo CA, Anwaruddin S, Baggish AL, Chen AA, Krauser DG, Tung R, Cameron R, Nagurney JT, Chae CU, Lloyd-Jones DM, Brown DF, ForanMelanson S, Sluss PM, Lee-Lewandrowski E, Lewandrowski KB. The N-terminal Pro-BNP Investigation of Dyspnea in the Emergency department (PRIDE) study. Am J Cardiol. 2005;95:948–954.

Sequence and Features