Coding

Part:BBa_K2865001

Designed by: Chuqi Wang   Group: iGEM18_SMMU-China   (2018-09-29)
Revision as of 02:54, 4 October 2018 by William Wang (Talk | contribs)


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. Despite the use of drugs, implanted cardiac assist devices and surgical treatments, many patients' conditions will still irreversibly deteriorate, eventually being difficult to control and rescue. 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.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 155
    Illegal NgoMIV site found at 302
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None