1 Summary

This year, we subscribed altogether 13 parts, including 5 basic parts and 8 composite parts to the registry. The purpose of these parts is to construct an AAV9-BNP-AR185 system and implement heart failure targeting gene therapy. Experiments were carried out to test these parts’ performance alone or in combination. The results showed that AR185 has good curative effect by inhibiting RyR2. Besides, the shuttle plasmid devices worked well in packaging AAV9 viruses and the viruses had high cardiac specificity. To our dismay, BNP promoter didn’t response to stimuls in H9C2 cells cultured in vitro as we expected, and its function remains to be investigated in the future.

2 Results of AR185

Generation of Anti-RyR2 nanobodies that specifically inhibits the Phosphorylation of RyR2 S2808

We first obtained and purified RyR2 from rat heart by using GST-fused FKBP12 as the published strategies described. To construct the camel VHH library, blood samples of 30 non-immunized, four-year-old male Bactrian camel were collected. B lymphocyte cDNA encoding VHHs was used to construct a phage display VHH library that consisted of approximately 3×10⁸ individual colonies. VHH gene corresponded to the size of insert of over 98% colonies. For confirming the heterogeneity of the individual clones from the library, we sequenced fifty randomly selected clones, and each clone showed a distinct VHH sequence.

In order to select nanobodies with specific ability to bind RyR2, bio-panning was performed with immobilized RyR2 protein. After the third round of panning, the result showed an obvious enrichment of phage particles that carried RyR2-specific VHH (Fig. 1A). Phage clones exhibited increased binding to RyR2 after the second round of panning. During four rounds of panning there was no phage clone that was found binding to BSA (Fig. 1B). VHH fragments of 300 individual colonies that were randomly chosen were expressed in an ELISA for screening colonies which bound to RyR2. Among these clones, 276 antibody fragments specifically bound to RyR2. One antibody fragment which did not bind to RyR2 was choose as a negative control, termed as VHH-AR117. To obtain antibodies that functionally inhibit of RyR2 phosphorylation, each of the antibody fragments was tested for its effect in an ELSA based RyR2 phosphorylation assay. 4 antibody fragments were potent inhibitors of RyR2 phosphorylation. The complementary determining regions (CDRs) were confirmed by sequence analysis and the result revealed that there was only one unique clone in this panel of antibody fragments, termed as VHH-AR185. To investigating the basis of dephosphorylation of RyR2 by VHH-AR185, the binding affinity of VHH-AR185 to RyR2 was measured by surface plasmon resonance. VHHs were purified for these experiments by expressing and secreting from the E. coli cytosol. As shown in Fig. 1D, the affinity (KD) of VHH-AR185 to RyR2 was estimated to be 1.93 nM. The result of affinity studies likely explained the inhibition of RyR2 phosphorylation due to the extremely slow dissociation rate of VHH-AR185 from RyR2.

Figure 1. Isolation of RyR2-specific nanobody by phage display.
(A) Phage-displayed nanobody fragments were selected against RyR2 by four rounds of panning. A gradual increase in phage titers was detected after each round of panning. (B) Polyclonal phage ELISA from the output phage of each round of panning. Control group used BSA as the irrelevant antigen. (C) Heat map generated from ELISA data of purified RyR2 channels which were phosphorylated in the presence of the PKA. (D) Kinetic analysis of AR185 binding to RyR2 was performed by SPR.

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 fig. S1, 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.

Figure S1

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 (fig. S2). 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.

Figure S2

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

We constructed an AAV9 vector containing a VHH-AR185 expression between the two AAV2 inverted terminal repeats and the vector was pseudotyped with a capsid of AAV serotype 9, termed as AAV9.AR185. VHH-AR117 were also constructed as a negative control, termed as AAV9.AR117. To access cardiac expression of VHH, we used the method of adding “self-cleaving” T2A peptide to co-expressed a GFP reporter downstream of VHHs (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 the efficiency of gene expression delivered by AAV in vivo. A dosage of AAV9.AR185 particles was delivered to each rat at 1×10¹² genome containing particles (gcp), whereas AAV9.AR117 vector was given to the control treated group (n = 5) at the same dosage. After four weeks, we removed all organs from the sacrificed rat, weighed, and assayed treated tissue for fluorescence intensity. Efficiency of gene expression and ability of targeting were evaluated by the ratio of fluorescence intensity to mass of tissue under fluorescence microscope (Fig. 2C and D).

Figure 2. Delivery of a cardiac-specific intracellular nanobody.
(A) Schematic diagram of constructs that have the function of expressing nanobody targeting RyR2 along with EGFP by using the T2A sequence. (B) Transmission electron micrographs of AAV9.AR185 particles. The specimens were prepared with negative staining by uranyl acetate. Scale bars = 100 nm. (C) The distribution of AAV9.AR185 expressing in vivo. 4 weeks after intravenous injection of AAV.AR185, SD rats were sacrificed, samples from different tissue were collected and assayed for fluorescence intensity. Data are presented as % FI/g of tissue and displayed as the mean ± SD. (D) Representative fluorescent image of heart that was infected by AAV9.AR185.

To explore the therapeutic potential of VHH, we chose the mode of ischemic heart failure induced by coronary artery ligation for this study. Following the ligation operation, rats were divided into different groups as described in Methods and received control virus (AAV9.AR117), AAV9.AR185 treatment or saline (HF) (n=7-8). The sham-operated animals (Sham) were used as healthy controls. Nine weeks after ligation operation and injection of AAV particles, LV dimensions in the short-axis view was measured by cardiac echo and we also calculated and analyzed the value of 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. Sarcomeres and mitochondria were the most important index for analysis of ultra-structures of cardiomyocytes from left ventricle that were observed by transmission electron microscopy (Fig. 3D). In the AAV9.AR185 treated and Sham groups, myofilaments were neatly arranged, sarcomeres were intact and Z lines were clear. Conversely, in the HF and AAV9.AR117 groups, MI leaded to disordered arrangement of sarcomeres, dissolution of myofilaments, and frequent vacuoles. In both HF and AAV9.AR117 groups, a lot of mitochondria were swollen and even ruptured, and the separated mitochondrial cristae frequently appeared. The mitochondria in Sham group were well shaped, and the cristae of the mitochondria were obvious and tightly packed. The observations of mitochondria were improved in the AAV9.AR185 treated group compared with AAV9.AR117 treated group. Comprehensively considering the alteration of cardiac function and changes in structure of different groups, the TEM images further support that VHH-AR185 had therapeutic effect in treating heart failure.

Figure 3. AAV9.AR185 gene therapy rescues cardiac function and reverses remodeling in failing rat myocardium in vivo.
(A) Representative wall motion showed by echo cardiograms in different treatment group. Arrows point to septum in all echoes and reduced wall motion appeared in the HF and AAV.AR117 group. In addition, rats in HF and AAV9.AR117 group showed significant reduction of EF and FS (%) compared with Sham and AAV.AR185. (B) Representative microstructure of transverse heart sections from four different groups was observed after Masson’s trichrome staining. (C) There were significant rises of heart weight to body weight ratio in HF (n =3) and AAV.AR117 treated animals compared with Sham or AAV9.AR185 treated animals. (D) The ultrastructure of myocardium acquired by TEM in different treatment groups.

We next accessed the contractile kinetics of isolated LV cardiomyocytes（Table1）. When cardiomyocytes were field-stimulated at a frequency of 1 Hz, HF and AAV9.AR117 treated myocytes had significantly slower velocities of shortening and relengthening in than AAV9.AR185 treated myocytes. Fractional shortening of myocytes that were isolated from HF and AAV9.AR117 treated animals also decreased, and time to 50% peak shortening (TPS50%) and time to 50% relengthening (TR50%) became longer. AAV9.AR185 treatment protected cardiomyocytes contractility reserve from the impairment 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 used laser scanning confocal microscopy recorded the fluorescence intensity to measure the sarcoplasmic reticulum Ca²⁺ content of cardiomyocytes from different groups by incubation in the fluorescent dye Fluo-5N/AM. As shown in Fig. 4A, basal sarcoplasmic reticulum Ca²⁺ contents in HF and AAV9.AR117 treated animals were lower than in AAV9.AR185 treated and sham-operated animals.

Additionally, we measured amplitude of calcium transient by incubation in Fluo-4/AM and caffeine perfusion. The representative colorful images in Fig. 4B and Table 2 show line-scan results of evoked Ca²⁺ transients from Shams, HFs, AAV9.AR117s and AAV9.AR185s. When challenged with 20 mM caffeine, less Ca²⁺ was released from the SR of myocytes from AAV9.AR117 group compared with myocytes from AAV9.AR185 treated rats. The results also showed there was significantly reduction of the amplitude of Ca²⁺ transients in the HFs and AR117s compared to that in AR185s. Therefore, the decrease in Ca²⁺ transient amplitude may be the causative factor of the impairment in SR Ca²⁺ load. Rate of Ca²⁺ rise also was significantly slower in HF and AAV9.AR117 myocytes than in AAV9.AR185 treated myocytes (Fig. 4C). AAV9.AR185 treatment increased peak of amplitude of evoked Ca²⁺ release and rate of Ca²⁺ rise during Ca²⁺ release.

Figure 4. AAV9.AR185 gene therapy restores cardiomyocyte and SR calcium handling in failing myocardium.
(A) For cardiomyocytes from different treatment group, the fluorescence intensity that reflected sarcoplasmic reticulum Ca²⁺ content was detected by incubating with Fluo-5N/AM. (B) Processed fluorescent images of cardiomyocytes that recorded by line scanning model showed the amplitude of caffeine-induced Ca²⁺ transients in different groups. (C) Chart shows Ca²⁺ transient characteristics of (B). (D) Spontaneous Ca²⁺ sparks appeared during diastole in cardiomyocytes from different treatment group and were showed by the representative processed line-scan images (respectively, n=30 cardiomyocytes from 3 or 4 different hearts were studied for three experiments)

Table 3 shows representative line-scan images of Ca²⁺ release during the resting stage of cardiomyocytes from Sham (A), HF (B), AAV9.AR117 (C), and AAV9.AR185 animals (D). The data showed that the frequency of Ca²⁺ release was significantly higher and Ca²⁺ sparks occurred frequently 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 the Ca²⁺ rise rate of sparks was slower, fluorescence intensity of Ca²⁺ sparks was decreased 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.AR185 significantly 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. 5).

Figure 5. AAV9.AR185 gene therapy inhibits phosphorylation of RyR2 S2808 in failing hearts.
(A) Myocardial tissue was harvested, homogenized, and analyzed for pRyR2 (Ser2808) and total RyR2by using ELISA. The statistical significance was determined by using Dunnett’s test. (B) Histologic evaluation of myocardial tissue in different treatment groups. Scale bars = 100 mm. (C) Quantification is expressed as means ± SEM; n = 3. Statistical analysis was done by one-way ANOVA followed by Tukey post-test.

3 BNP promoter

To characterize the BNP promoter, it was connected with EGFP and transfected into H9c2 cell line via liposome transfection. To stimulate heart failure condition, we added two widely reported reagents, AngⅡ and ET-1 into the medium. The cells were observed under fluorescent microscope 48 h after drug administration, contrary to our expectation, there was no evident fluorescence.

Figure 6. Expression of BNP-EGFP 48 h after the administration of ET-1 and AngⅡ.
(A) and (B) were cells treated with 10-7 mol/L ET-1.(A) was taken under fluorescent microscope and (B) light microscope. (C) and (D) were cells treated with 10-7mol/L AngⅡ.

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