Difference between revisions of "Part:BBa K2865001"

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	<partinfo>BBa_K2865001 short</partinfo>		<partinfo>BBa_K2865001 short</partinfo>
−		+	<p class="MsoNormal"><span lang="EN-US">AR185 is an RyR2-specific VHH antibody which is designed to treat heart failure by inhibiting RyR2 phosphorylation. To access expression of AR185, we added a “self-cleaving” T2A peptide to co-express an EGFP reporter downstream of the VHH. This part (BBa K2865001) is a combination of nanobody AR185 and EGFP by self-cleaving peptide T2A.</span></p>
−	AR185 is a camel single-domain antibody that have the ability to inhibit PKA dependent RyR2 phosphorylation. This part (BBa_K2865001) is a combination of nanobody AR185 and reporter eGFP by self-cleaving peptide T2A.	+
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	<!-- Add more about the biology of this part here -->		<!-- Add more about the biology of this part here -->
	===Usage and Biology===		===Usage and Biology===
−		+	<p class="MsoNormal"><span lang="EN-US">
−	Heart failure is the end stage of most heart diseases.Chronic PKA phosphorylation of RyR2 has been shown to increased diastolic SR "calcium leakage" which is considered to be an important pathological mechanism for myocardial injury and heart failure development. (8, 9).	+	The cardiac ryanodine receptor type 2 (RyR2) controls the release of Ca2+ from the sarcoplasmic reticulum (SR) during each excitation–contraction cycle. Chronic PKA phosphorylation of RyR2 has been shown to increased diastolic SR "calcium leakage" which is considered to be an important pathological mechanism for myocardial injury and heart failure development. So far three phosphorylation sites have been identified. They are separately S2808, S2814, and S2030. Understanding the regulation of RyR phosphorylation will promote the development of strategies to heart failure.[[#References|[1]]] </span></p>
−		+	<p class="MsoNormal"><span lang="EN-US">
−	Therefore,in this study, we hypothesis that targeting RyR2 using anti-phosphorylation agents may improve treatment efficacy.	+	The earliest article demonstrating changes in RyR2 phosphorylation in patients with heart failure was published by Marx et al. in 2000. Marx et al., Wehrens et al. and Shan et al. proposed a hypothesis: sympathetic excitation in heart failure, activation of PKA-mediated RyR2 S2808 Hyperphosphorylation, while inhibiting the binding of FKBP12.6 to RyR2, increases the probability of RyR2 opening. Wehrens et al. believe that RyR2 S2808 phosphorylation plays a key role in the experimental myocardial ischemia-induced heart impaired process. They established a model of heart failure induced by myocardial ischemia in RyR2 S2808 knockout mice and wild-type mice. Heart function tests were performed 4 weeks after ischemia. The results showed that RyR2 S2808 knockout mice showed significant improvement in cardiac function such as ejection fraction, shortening fraction, maximal rate of left ventricular pressure. At the same time, they also found that RyR2 of RyR2 S2808 knockout mice could not be phosphorylated by PKA, and PKA lost regulation of FKBP12.6, resulting in FKBP12.6 not being able to dissociate from RyR2.Here, we hypothesize that targeting RyR2 using anti-phosphorylation agents may improve treatment efficacy.   </span></p>
−	Nanobodies are single domain antibodies consisting of the heavy chain variable domain (VHH) in the camelid family which lacks the light chain. Currently, a variety of nanobodies have entered the clinical research stage. Compared with traditional antibodies, nanobodies have the advantages of low molecular weight, high affinity, high stability, low immunogenicity and strong penetrability. Based on the characteristics of nanobodies and VHH, the use of adeno-associated virus vectors to mediate nanobody treatment of heart failure has great potential.	+	<p class="MsoNormal"><span lang="EN-US">
−		+	Nanobodies are single domain antibodies consisting of the heavy chain variable domain (VHH) in the camelid family which lacks the light chain. Currently, a variety of nanobodies have entered the clinical research stage. Compared with traditional antibodies, nanobodies have the advantages of low molecular weight, high affinity, high stability, low immunogenicity and strong penetrability. Based on the characteristics of nanobodies and VHH, the use of adeno-associated virus vectors to mediate nanobody treatment of heart failure has great potential. </span></p>
−	We successfully expressed AR185 nanobodies which specifically bind to RyR2 in rat cardiomyocytes and have the ability to inhibit PKA dependent S2808 phosphorylation in vitro. To evaluate its potential use 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.	+	<p class="MsoNormal"><span lang="EN-US">
−		+	We successfully identified a camel single-domain antibody termed AR185 that have the ability to specifically bind to RyR2 in rat cardiomyocytes and have the ability to inhibit PKA dependent S2808 phosphorylation in vitro. To evaluate its potential use 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>
−	The construct of the intracellular antibody fragment, AR185, was engineered to express an upstream EGFP reporter separated from the nanobody sequence by a T2A sequence. The sequence encoding the whole fragments construct was subcloned into the pAAV vector resulting in AAV.AR185. For the production of AAV9 pseudotyped vectors, the plasmids were used for cotransfection in 293T cells together with pAAV-RC9, encoding the AAV-9 cap sequence, and rAAV2-retro helper vector, containing the AAV-2 rep gene as well as adenoviral helper sequences. AAV vectors were produced, purified, and titrated using standard procedures.	+
−	Our experimental data demonstrate that intracellular antibody treatment is effective in heart disease rats and does not present a significant safety risk.	+
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	===Characterzation===		===Characterzation===
−	1. To examine the ability of VHH-AR185 to interact with RyR2 when they are expressed in the cytoplasm of eukaryotic cells, co-immunoprecipitation experiments were carried out on lysates of transfected neonatal cardiomyocytes cells. As shown in fig. S1, anti-his antibody efficiently co-precipitated RyR2 from cells expressing VHH-AR185-HIS, but not from cells expressing VHH-AR117-HIS. Conversely, anti-RyR2 antibody was able to co-precipitate VHH-AR185-HIS, but not VHH-AR185-HIS, with RyR2, indicating that the VHH-AR185 could correctly fold as a soluble protein in the reducing environment of the cytoplasm and retain its antigen binding ability.	+	<p class="MsoNormal"><span lang="EN-US"><B> </B> </span></p>
−		+	<p class="MsoNormal"><span lang="EN-US"><B>Co-immunoprecipitation</B></span></p>
−	Figure 2. Delivery of a cardiac-specific intracellular nanobody. (A) Schematic diagram of constructs expressing nanobody targeting RyR2 along with EGFP using the T2A ribosomal skipping sequence. (B) Transmission electron micrographs of AAV9 AR185. The samples were negatively stained with uranyl acetate. Scale bars = 100 nm. (C) The distribution of AAV9.AR185. SD Rat were intravenously injected with AAV9.AR185 3 days after injection, the animals were sacrificed and different tissue samples were collected and assayed for fluorescence intensity; right (FI). Data are presented as % FI/g of tissue and values are the mean ± SD.	+	<p class="MsoNormal"><span lang="EN-US">The interaction of VHH-AR185 to RyR2 in the cytoplasm of eukaryotic cells was examined by co-immunoprecipitation experiments. VHH-AR185 and RyR2 were expressed in neonatal cardiomyocytes cells and the lysates of transfected cells were detected. As the result in figure. 3, anti-his antibody was able to efficiently co-precipitate RyR2 from the cells that expressed VHH-AR185-HIS, but could not co-precipitate RyR2 from cells expressing VHH-AR117-HIS. Conversely, anti-RyR2 antibody was able to co-precipitate VHH-AR185-HIS with RyR2, but not VHH-AR185-HIS. This result indicated that the VHH-AR185 could maintain its antigen binding ability in the cytoplasm and fold as a soluble protein.
−		+	</span></p>
−	2. 然后都是动物实验	+	[[File:T--SMMU-China--AR185 figure 3.png|200px|thumb|center|Fig.3]]
−		+	<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p> To identify the epitopes recognized by AR185, phage clones were isolated by panning the PhD.-7 phage display peptide library with AR185. Three rounds of selection were performed, and, at each round, the library was pre-cleared with a control AR177 nanobody. After the third round of panning, the binding of the isolated phage clones to AR185 was determined by ELISA. Sequence analysis of AR185-positive phage clones identified five and six distinct amino acid sequences, respectively (figure.4). Alignment of these sequences revealed the consensus motifs DKLAC, which could be aligned with the (2725) DKLAN (2729) sequence located at P2 Domain of RyR2.
−	Intrabody AR185 rescues cardiac function and reverses remodeling in failing rat myocardium in vivo	+	[[File:T--SMMU-China--AR185 figure 4.png|400px|thumb|center|Fig.4]]
−	We constructed an AAV9 vector containing a VHH-AR185 expression cassette flanked by two AAV2 inverted terminal repeats and is pseudotyped with an AAV9 capsid, termed as AAV9.AR185. VHH-AR117 were also constructed as a negative control, termed as AAV9.AR117. To access cardiac expression of VHH, we add a downstream GFP reporter coexpressed with VHHs using the “self-cleaving” T2A peptide (Fig. 2A). We used the HEK-293 cells expression of different AAV9 particles in vitro and Transmission electron microscope was used to access the AAV9 particles (Fig. 2B). Next, we evaluated AAV mediated gene expression in vivo. The AAV9.AR185 particles was delivered at a dosage of 1× 1012 genome containing particles (gcp) per rat, whereas AAV9 empty vector was given to the control group (n = 6). Four weeks later, all organs of rat were then removed, weighed, and assayed for fluorescence intensity. The AAV9 mediated gene expression and targeting were evaluated by the percent of fluorescence intensity per gram of tissue in rat (Fig. 2C and D).	+	<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p><B></B>
−		+	<table class="MsoTableGrid" style="border: medium none ; width: 491pt; border-collapse: collapse;" border="0" cellpadding="0" cellspacing="0" width="655">
−	To explore the therapeutic potential of VHH, coronary ligation was chosen as a model of heart failure to model ischemic cardiomyopathy. Following the myocardial-infarcted surgery, rats were randomized to receive control virus (AAV9.AR117,) or AAV9.AR185 treatment. Moreover, sham-operated animals (sham-OP) and myocardial-infarction induced heart failure (HF) animals (n=6-8) were used as controls. All the AAV9 particles were delivered at a dosage of 1× 1012 genome containing particles (gcp) per animal. Nine weeks after surgery and injection of AAV particles, Cardiac echo was used to measure LV dimensions in the short-axis view and to calculate Ejection Fraction and fractional shortening. Our data shows that rats of HF group and AAV9.AR117 group exhibited progressive cardiac dysfunction and LV enlargement, while AAV9.AR185-treated animals showed significant improvement. Moreover, Ejection Fraction and fractional shortening was markedly improved in AAV9.AR185 group compared with HF group and AAV9.AR117 group (Fig. 3A). To determine whether AAV9.AR185 treatment prevented adverse remodeling of the heart after MI, Masson trichrome staining of cardiac sections was performed to measure cardiac fibrosis (Fig. 3B). Whereas there was a significant increase in the development of cardiac fibrosis in Rats of HF group and AAV9.AR117 group after HF, whereas the amount of fibrosis was significant reduced in AAV9.AR185-treated animals. Additionally, HF rat and AAV9.AR117 treated rat had development of a significant increase of heart weight-to-body weight ratios (HW/BW) after MI compared with sham-operated rat, which is indicative of cardiac remodeling in the context of congestive HF（Fig. 3C）. In contrast, there was no significant increase in HW/BW ratio after MI in AAV9.AR185-treated rat compared with sham-operated rat  Ultrastructures including sarcomeres and mitochondria of myocardial cells in the left ventricle were observed under TEM (Fig. 3D). Complete sarcomeres, neatly arranged myofilaments and clear Z lines were present in the AAV9.AR185 treated and Sham groups, and these groups showed better performance However, in the HF and AAV9.AR117 groups, some sarcomeres were incomplete, and a disordered arrangement of myofilaments appeared frequently with some vacuoles. In both HF and AAV9.AR117 groups, the electron density of some mitochondria increased, and the mitochondrial cristae were separated. The Sham-OP group mitochondria were well shaped, and the cristae of the mitochondria were arranged regularly. There were analogous observations in the AAV9.AR185 treated group. Taken together with the alteration of cardiac function and changes in microstructure in different groups, the TEM images further support a therapeutic role of VHH-AR185 in treating heart failure.	+	===Reference===
−		+	<p class="MsoNormal"><span lang="EN-US">
−	We next accessed the contractile kinetics of isolated LV cardiomyocytes（Table1）. When cells were stimulated at a frequency of 0.5 Hz, velocities of cell shortening and relengthening were significantly slower in HF and AAV9.AR117 treated myocytes than in AAV9.AR185 treated myocytes. Fractional shortening also was less in myocytes isolated from HF and AAV9.AR117 treated animals, and times to 50% peak myocyte shortening (TPS50%) and relengthening (TR50%) were longer. AAV9.AR185 treatment blunted the reduction in contractile kinetics induced by MI. However, only the index of TR50% in myocytes from AAV9.AR185 treated animals returned to a level similar to those of sham operated animals.	+	[1] Huke, S. and D.M. Bers, Ryanodine receptor phosphorylation at Serine 2030, 2808 and 2814 in rat cardiomyocytes. Biochem Biophys Res Commun, 2008. 376(1): p. 80-5.</span></p>
−	AR185 gene therapy restores cardiomyocyte and SR calcium handling in failing myocardium	+
−		+
−	We next measured the sarcoplasmic reticulum Ca2+ content of cardiomyocytes isolated from different groups of rats using the fluorescent dye Fluo-5N/AM by laser scanning confocal microscopy. As shown in Fig. 4A, basal sarcoplasmic reticulum Ca2+ levels in HF and AAV9.AR117 treated animals were lower than in AAV9.AR185 treated and sham-operated animals.	+
−	Additionally, we measured cytoplasmic Ca2+ with Fluo-4/AM by the method of caffeine perfusion. The colorful images in Fig. 4B show representative line-scan images of evoked Ca2+ transients from Shams, HFs, AAV9.AR117s and AAV9.AR185s. When challenged with 20 mM caffeine, myocytes from AAV9.AR117 group released less Ca2+ from the SR compared with myocytes from AAV9.AR185 treated rats. The results showed that the amplitude of Ca2+ transients was also significantly reduced in the HFs and AR117s compared to that of AR185s. The decrease in SR Ca2+ load may therefore be related to the decrease in Ca2+ transient amplitude. Rate of Ca2+ rise also was significantly slower in HF and AAV9.AR117 myocytes than in AAV9.AR185 treated myocytes (Fig 4C). AAV9.AR185 increased the amplitudes of evoked Ca2+ releases, increased the rate and amplitude of caffeine-releasable Ca2+.	+
−		+
−	Table 2 shows representative line-scan images of spontaneous Ca2+ release in myocytes from Sham (A), HF (B), AAV9.AR117 (C), and AAV9.AR185 animals (D). The data showed that a significantly higher frequency of Ca2+ sparks was observed in the HF and AAV9.AR117 group compared with the AAV9.AR185 group. The duration of Ca2+ sparks in HF and AR117 myocytes were similar to those in Sham and AR185 myocytes, but rate of Ca2+ rise was slower, peak Ca2+ amplitude was less and T50 decay was longer.	+
−	VHH-AR185 inhibits phosphorylation of RyR2 S2808 in failing hearts	+
−		+
−	To examine whether AAV9.AR185 treatment results in dephosphorylation of RyR2 and in vivo, cardiomyocyte lysates were further subjected to ELISA analysis, our data shows treatment with AAV9.AR185significantly reduced the level of pRyR2 (S2808) in the cardiomyocytes compared with HF group and AAV9.AR117 treatment (p = 0.0003, Dunnett’s test). Moreover, immunohistochemical analysis of the heart tissues in different treatment group also revealed that an increased accumulation of RyR2 phosphorylation was also observed in the AAV9.AR117 treated group, AAV9.AR185 treatment decreased the level of pRyR2 stain of cells in the myocardium, which indicated that VHH185 has blockage effect of RyR2 phosphorylation. Together, these data demonstrate that AAV9.AR185 treatment leads to inhibition of the RyR2 phosphorylation in vivo. (Fig. 3b1, b2).	+
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	<span class='h3bb'>Sequence and Features</span>		<span class='h3bb'>Sequence and Features</span>
	<partinfo>BBa_K2865001 SequenceAndFeatures</partinfo>		<partinfo>BBa_K2865001 SequenceAndFeatures</partinfo>
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	<!-- Uncomment this to enable Functional Parameter display		<!-- Uncomment this to enable Functional Parameter display

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Latest revision as of 02:06, 13 October 2018

AR185-T2A-EGFP, nanobody inhibiting RyR2 phosphorylation

AR185 is an RyR2-specific VHH antibody which is designed to treat heart failure by inhibiting RyR2 phosphorylation. To access expression of AR185, we added a “self-cleaving” T2A peptide to co-express an EGFP reporter downstream of the VHH. This part (BBa K2865001) is a combination of nanobody AR185 and EGFP by self-cleaving peptide T2A.

Usage and Biology

The cardiac ryanodine receptor type 2 (RyR2) controls the release of Ca2+ from the sarcoplasmic reticulum (SR) during each excitation–contraction cycle. Chronic PKA phosphorylation of RyR2 has been shown to increased diastolic SR "calcium leakage" which is considered to be an important pathological mechanism for myocardial injury and heart failure development. So far three phosphorylation sites have been identified. They are separately S2808, S2814, and S2030. Understanding the regulation of RyR phosphorylation will promote the development of strategies to heart failure.[1]

The earliest article demonstrating changes in RyR2 phosphorylation in patients with heart failure was published by Marx et al. in 2000. Marx et al., Wehrens et al. and Shan et al. proposed a hypothesis: sympathetic excitation in heart failure, activation of PKA-mediated RyR2 S2808 Hyperphosphorylation, while inhibiting the binding of FKBP12.6 to RyR2, increases the probability of RyR2 opening. Wehrens et al. believe that RyR2 S2808 phosphorylation plays a key role in the experimental myocardial ischemia-induced heart impaired process. They established a model of heart failure induced by myocardial ischemia in RyR2 S2808 knockout mice and wild-type mice. Heart function tests were performed 4 weeks after ischemia. The results showed that RyR2 S2808 knockout mice showed significant improvement in cardiac function such as ejection fraction, shortening fraction, maximal rate of left ventricular pressure. At the same time, they also found that RyR2 of RyR2 S2808 knockout mice could not be phosphorylated by PKA, and PKA lost regulation of FKBP12.6, resulting in FKBP12.6 not being able to dissociate from RyR2.Here, we hypothesize that targeting RyR2 using anti-phosphorylation agents may improve treatment efficacy.

Nanobodies are single domain antibodies consisting of the heavy chain variable domain (VHH) in the camelid family which lacks the light chain. Currently, a variety of nanobodies have entered the clinical research stage. Compared with traditional antibodies, nanobodies have the advantages of low molecular weight, high affinity, high stability, low immunogenicity and strong penetrability. Based on the characteristics of nanobodies and VHH, the use of adeno-associated virus vectors to mediate nanobody treatment of heart failure has great potential.

We successfully identified a camel single-domain antibody termed AR185 that have the ability to specifically bind to RyR2 in rat cardiomyocytes and have the ability to inhibit PKA dependent S2808 phosphorylation in vitro. To evaluate its potential use 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.

Characterzation

Co-immunoprecipitation

The interaction of VHH-AR185 to RyR2 in the cytoplasm of eukaryotic cells was examined by co-immunoprecipitation experiments. VHH-AR185 and RyR2 were expressed in neonatal cardiomyocytes cells and the lysates of transfected cells were detected. As the result in figure. 3, anti-his antibody was able to efficiently co-precipitate RyR2 from the cells that expressed VHH-AR185-HIS, but could not co-precipitate RyR2 from cells expressing VHH-AR117-HIS. Conversely, anti-RyR2 antibody was able to co-precipitate VHH-AR185-HIS with RyR2, but not VHH-AR185-HIS. This result indicated that the VHH-AR185 could maintain its antigen binding ability in the cytoplasm and fold as a soluble protein.

 

To identify the epitopes recognized by AR185, phage clones were isolated by panning the PhD.-7 phage display peptide library with AR185. Three rounds of selection were performed, and, at each round, the library was pre-cleared with a control AR177 nanobody. After the third round of panning, the binding of the isolated phage clones to AR185 was determined by ELISA. Sequence analysis of AR185-positive phage clones identified five and six distinct amino acid sequences, respectively (figure.4). Alignment of these sequences revealed the consensus motifs DKLAC, which could be aligned with the (2725) DKLAN (2729) sequence located at P2 Domain of RyR2.

 

Reference

[1] Huke, S. and D.M. Bers, Ryanodine receptor phosphorylation at Serine 2030, 2808 and 2814 in rat cardiomyocytes. Biochem Biophys Res Commun, 2008. 376(1): p. 80-5.

Sequence and Features