Small Nppa and Myl2 Promoters Are Sufficient to Maintain Chamber‐specific Expression on an AAV9 Platform
Authors: Bilal Alina S. , Blackwood Erik A. , Thuerauf Donna J. , Glembotski Christopher C. 
Published: April 2020

Objective

To generate an AAV9 vector with small promoters that selectively express transgenes in a chamber‐specific manner.

Introduction

The adeno‐associated virus serotype 9 (AAV9) vector exhibits high transduction efficiency and cardiotropism which has allowed for the elucidation of tissue‐specific functions of genes implicated in cardiac diseases. However, cardiac diseases such as atrial fibrillation, require a more selective strategy to allow for the study of gene function in a chamber‐specific manner. AAV9 vectors carrying chamber‐specific promoters, such as Nppa and SLN for atrial‐specific expression, have been published; however, these vectors contain 632 and 1000 base pairs (bp), respectively, limiting the carrying capacity for transgenes.

Hypothesis

Less than 500 bp of the Nppa and Myl2 promoters is sufficient to confer potent AAV9‐mediated atrial and ventricular cardiomyocyte specific expression, respectively.

Methods

Fragments of the Nppa promoter, consisting of about 600 and 400 bp, and of the Myl2 promoter, consisting of about 700 and 300 bp, were inserted into a Green Fluorescent Protein (GFP) expressing vector. These vectors were electroporated into primary neonatal rat atrial and ventricular cardiomyocytes (NRAMS and NRVMs). Because pathological hypertrophy is known to upregulate expression of Nppa in ventricular cardiomyocytes, NRAMs and NRVMs were treated with 50 uM of phenylephrine (PE), a chemical inducer of hypertrophy, for 48 hours, to assess the faithfulness of our promoters to maintain chamber‐specific GFP expression. Promoter activity was analyzed by immunoblot (IB) for GFP and fluorescent microscopy. The promoters which expressed GFP robustly and retained chamber‐specificity with PE treatment were selected for testing in mice in an AAV9 vector. The mouse model used was a two‐color fluorescent Cre Recombinase (Cre) reporter, consisting of membrane targeted Tomato (mT) and GFP (mG). In this model, mT fluorescence is exclusively expressed; upon introduction of Cre, mT is excised, allowing mG to express. We generated AAV9‐Cre driven by the Nppa or Myl2 promoters, injected mice via tail‐vein with 1×1011 viral particles, performed TAC for 4 weeks, and determined chamber‐specific expression of Cre by IB detection of GFP from atrial and ventricular tissue.

Results

In NRAMs and NRVMs, IB and fluorescent microscopy confirmed that promoters as small as 400 bp and 300 bp for Nppa and Myl2, respectively, elicited robust atrial and ventricular specific GFP expression, respectively. Interestingly, 600 bp of the Nppa promoter expressed GFP in NRVMs with PE treatment, while 400 bp did not. In an AAV9‐Cre vector, 400 and 300 bp of the Nppa and Myl2 promoter, respectively, were sufficient to mediate chamber‐specific excision of mT, as determined by IB for GFP.

Conclusion

Small promoter regions of Nppa and Myl2, consisting of about 400 and 300 bp, respectively, can be utilized in an AAV9 vector to selectively express transgenes in atrial and ventricular cardiomyocytes, respectively. Importantly, the smaller size of the Nppa and Myl2 promoters leaves room for larger transgenes, alleviating the limitations imposed upon the small carrying capacity of AAV9 vectors.

Support or Funding Information

American Heart Association

National Institute of Health