Difference between revisions of "Part:BBa K2865012"

Revision as of 15:43, 16 October 2018

Left ITR-BNP-AR185-T2A-eGFP-Poly(A)- Right ITR

This device is a shuttle plasmid in an AAV-helper-free assembly system and is the center of our heart failure gene therapy project. It is composed of 5 basic parts: the HF inducible BNP promoter (BBa_K2865000), our novel nanobody AR185-T2A-EGFP (BBa_K2865001), SV40 polyA signal and two inverted terminal repeats (BBa_K2865002 and BBa_K2865003), as is shown in figure.1. The use of this device is 1) to produce Adeno-associated virus 9 (AAV9) particles, 2) to initiate expression of therapeutic genes driven by BNP promoter, and 3) to rescue heart failure by inhibiting RyR2 phosphorylation.

Figure 1. Schematic diagram of constructs of Left ITR-BNP-AR185-T2A-EGFP- Poly(A)-Right ITR

Usage and Biology

We used an AAV-helper free system to prepare AAV9 virons, which contained three plasmids: shuttle plasmid, RC9 plasmid (carrying AAV9 replication and capsid genes) and helper plasmid (carrying adenovirus-derived genes). These three plasmids were co-transfected into HEK-293cells when producing the viruses (Figure. 2). The viruses prepared in the previous step were then used to infect mammalian cells and introduce gene of interest into targeting organs.

Figure 2. The process of transfection

The usage and biology of our intrabody AR185 (BBa_K2865001) and BNP promoter (BBa_K2865000) has been separately elaborated in their part pages. Here, we combine this two parts together, aiming to develop an approach that could specifically function in failing cardiomyocytes and is safe in normal cells simultaneously. There are two aspects designed to ensure the biosafety in this system. They are AAV9 and BNP promoter, since AAV9 has high selectivity for heart tissues and BNP promoter has been reported as HF inducible in a lot of studies.[1][2]These data demonstrate that AAV9-AR185 treatment leads to inhibition of the RyR2 phosphorylation in vivo.

The part of BNP promotor is a regulator which can be used to control gene expression in heart failure gene therapy. Because BNP, known as B-type natriuretic peptide, has been regarded as an important biomarker in the diagnosis of heart failure (HF), whose level remains low in normal hearts and elevates dramatically in the blood of patients with HF. What’s more, BNP promoter activity is related to the severity of HF. Therefore, our team wants to utilize the ability that BNP promoter can be activited in the HF environment in order to regulate the related sequences expression for heart failure.

The subsequent part is AR185 which is a coding and can be translated for one of the RyR2-specific nanobodies isolated from a phage display library of variable domains of camellidae heavy chain-only antibodies (VHH).With the feature of inhibiting PKA dependent S2808 phosphorylation on RyR2 in an vitro assay, it was chosen for further investigation on the treatment of heart failure. 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.

Figure 3. Design and Function of part of Left ITR-BNP-AR185-Poly(A)-Right ITR

References

[1]. Sergeeva, I.A. and V.M. Christoffels, Regulation of expression of atrial and brain natriuretic peptide, biomarkers for heart development and disease. Biochim Biophys Acta, 2013. 1832(12): p. 2403-13. [2]. Carlson, C., et al., Phenotypic screening with human iPS cell-derived cardiomyocytes: HTS-compatible assays for interrogating cardiac hypertrophy. J Biomol Screen, 2013. 18(10): p. 1203-11. Sequence and Features