Difference between revisions of "Part:BBa K2865001"

Revision as of 15:09, 7 October 2018

AR185-T2A-EGFP, nanobody inhibiting RyR2 phosphorylation

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.

Usage and Biology

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).

Therefore,in this study, we hypothesis 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 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.

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.

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.

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.

2.

Intrabody AR185 rescues cardiac function and reverses remodeling in failing rat myocardium in vivo 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).

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.

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. 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).

Sequence and Features