Part:BBa_K2865001
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.
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