Part:BBa_K2865009
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 671
Illegal NgoMIV site found at 818
Illegal NgoMIV site found at 1753 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 242
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