Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure

Abstract

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.

Unlocking the promise of mRNA therapeutics

“…Beyond the broad classes of potential applications discussed above, other innovative applications can be expected. For example, our group has used mRNA for in vivo expression of intracellular antibodies (intrabodies) in the prospective treatment of heart failure and as an in vitro disease-modeling tool to dissect key regulatory mechanisms 219,24 . Others have deployed mRNA to improve engraftment of human hematopoietic stem cells by transient overexpression of CXCR40 (ref.…”

Diagnostic and Therapeutic Aptamers

“… 101 , 102 , 103 Although promising, only 1 nanobody-based drug has been approved so far (caplacizumab [Sanofi] for targeting the vWF), whereas to the best of our knowledge, only 2 works describing the preclinical use of nAbs for defective calcium signaling components (RyR2 and PLN specific nanobodies) have been shown. 100 , 104 In fact, the main problem with regard to the use of this technology is the requirement of a safe, efficient, and cell-specific gene delivery method for their effective clinical translation. As a matter of fact, nAbs, like any other antibody, rely on conserved disulfide bonds, which in the reducing milieu of the cytosol strongly impair the formation of disulfide bonds.…”

Phospholamban R14del disease: The past, the present and the future

“…Collectively, exploitation of multiple approaches has provided promising prospects towards precision medicine strategies for therapeutic interventions in PLN-R14del patients ( Figure 2 ) ( 49 ). In addition, the discovery of new therapeutic tools, such as intracellular acting antibodies (intrabodies), may provide additional means that will help combat PLN-R14del pathogenesis and contribute towards the ultimate goal of providing a cure for PLN-R14del patients ( 87 , 88 ).…”