Part:BBa_K2865013
Left ITR-CMV-AR185-T2A-EGFP-Poly(A)-Right ITR
This device is a shuttle plasmid of the AAV helper-free system. It consists of a CMV promoter, nanobody AR185-T2A-EGFP (BBa_K2865001), SV40 polyA signal and flanked by two inverted terminal repeats (BBa_K2865002 and BBa_K2865003).
The use of this part is
1) to produce AAV9 particles
2) to express our novel intrabody AR185 linked with reporter EGFP in failing heart
3) to test its efficacy for heart failure
Usage and Biology
Introduction to AAV-9
1)We used an AAV helper-free system to produce AAV-9 viruses and introduce our gene of interest into cardiac muscle cells. This system is composed of three plasmids: shuttle plasmid, RC9 plasmid and helper plasmid. The AAV Helper-Free System allows the production of infectious recombinant human adeno-associated virus-9 (AAV-9) virions without the use of a helper virus, while traditional methods required co-infection with a helper adenovirus or herpes virus for productive infection.
Among the three plasmids mentioned above the shuttle plasmid plays a leading role, for it is this plasmid that carries our gene of interest. The left and right inverted terminal repeats (ITRs) on both sides of the plasmid contain all the cis-acting elements necessary for replication and packaging.
Recombinant AAV has proven especially valuable for long-term gene expression. This advantage can be attributed to the ability of AAV to replicate epichromosomally under replication permissive conditions. Since cardiomyocyte is a kind of non-dividing cells, it can maintain AAV genome over time, and gene expression will be stable while the AAV-9 genome is maintained.[1]
Until now, several AAV serotypes have been discovered and each of them has diverse tissue specificity. Among these serotypes, AAV-9 has a high cardiomyocyte specificity, thus has become a new and promising vector for gene therapy of heart diseases.
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.
Characterization
We have previously isolated the nanobody AR185 and a negative control AR117 via phage display screening and ELISA analysis. We also performed co-immunoprecipitation experiments to examine their antigen binding ability. Results revealed that AR185 has high affinity to RyR2 and can effectively inhibit its phosphorylation in vitro. Details of these experiments are available in part page of AR185 (BBa_K2865001).
Based on the previous work, we next set out to access this device’s ability in producing AAV virons and curative effect of heart failure.
AAV9 Transfection Efficiency and Specificity in Different Organs
This shuttle plasmid was co-transfected into HEK-293 cells with helper plasmid (carrying adenovirus-derived genes), and RC9 plasmid (carrying AAV9 replication and capsid genes), which together supply all of the trans-acting factors required for AAV replication and packaging in the host cells. Recombinant AAV9 viral particles were prepared from infected HEK-293 cells and transmission electron microscope was used to access the AAV9 particles (Figure.3 A). Next, we evaluated the efficiency of gene expression delivered by AAV in vivo. To assess the expression of AR185, we attached a reporter EGFP to AR185 thus the amount of expression could be evaluated by detecting the fluorescent intensity. A dosage of AAV9-AR185 particles was delivered to each rat at 1×1012 genome containing particles (gcp). After four weeks, organs from the sacrificed rats were removed, weighed, treated for measuring 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 (Figure.3 B and Figure.4). The results shows that AAV-9 has a high cardiomyocyte specificity, thus is a promising vector for gene therapy of heart diseases.
Intrabody AR185 rescues cardiac function and reverses remodeling in failing rat myocardium in vivo
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 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, left ventricular (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 heart failure(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 (Figure. 5A). To determine whether AAV9-AR185 treatment prevented adverse remodeling of the heart after myocardial infarction (MI), Masson trichrome staining of cardiac sections was performed to measure cardiac fibrosis (Figure. 5B). 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（Figure. 5C）. 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 (Figure. 5D). 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.
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.
Table 1 Contractile properties of cardiomyocytes from groups
| Sham | HF | AAV-AR117 | AAV-AR185 | |
| Fractional shortening (%) | 10.35±0.53** | 7.03±1.00 | 7.12±1.13 | 9.49±0.49* |
| +dl/dt (μm/s) | 3.37±0.13** | 2.30±0.16 | 2.26±0.18 | 2.96±0.18* |
| –dl/dt (μm/s) | 3.02±0.23** | 2.01±0.15 | 2.04±0.21 | 2.78±0.15* |
| TPS50% | 64.75±2.36** | 75.06±5.22 | 74.25±5.58 | 69.90±3.88* |
| TR50% | 155.70±9.56# | 196.37±10.47 | 186.67±15.22 | 162.18±8.91* |
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 Figure. 6A, 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 Figure. 6B 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 (Figure. 6C). AAV9-AR185 treatment increased peak of amplitude of evoked Ca2+ release and rate of Ca2+ rise during Ca2+ release.
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.
Table 2 Ca2+ transient in cardiomyocytes from groups
| Sham | HF | AAV-AR117 | AAV-AR185 | |
| Peak Ca2+ amplitude | 45.73±3.32 | 13.06±1.39 | 12.25±1.28 | 23.11±2.12 |
| Rate of Ca2+ rise (/sec) | 687.12±73.95 | 87.19±9.35 | 83.90±6.83 | 334.28±59.11 |
Table 3 Spontaneous Ca2+ release in cardiomyocytes from groups
| Sham | HF | AAV-AR117 | AAV-AR185 | |
| Frequency of Ca2+ sparks (per 50um/sec) | 0.7±0.34 | 12.6±1.38 | 12.3±1.25 | 1.1±0.34 |
| Duration of Ca2+ sparks (msec) | 44.7±3.1 | 48.1±4.2 | 49.4±3.7 | 46.9±4.0 |
| Peak Ca2+ amplitude | 37.5±4.3 | 18.4±2.9 | 18.6±2.1 | 32.3±3.6 |
| Rate of Ca2+ rise (/msec) | 31.32±4.05 | 4.67±0.54 | 4.58±0.36 | 27.15±3.59 |
| T50 decay (msec) | 7.75±0.15 | 10.85±0.57 | 10.57±0.70 | 8.46±0.19 |
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. (Figure. 7).
References
[1] Russell, D. W., Alexander, I. E. and Miller, A. D. (1995) Proc Natl Acad Sci U S A 92(12):5719-23.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2360
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1000
Illegal NgoMIV site found at 1147
Illegal NgoMIV site found at 2082 - 1000COMPATIBLE WITH RFC[1000]
| None |