AAV Vectors for Efficient Gene Delivery to Rodent Hearts

Abstract

Currently, gene therapy is one of the most promising fields in biomedicine, with great therapeutic potential for an array of inherited and acquired diseases. Adeno-associated viral (AAV) vectors have emerged as promising tools to deliver selectively a therapeutic payload to target organs, including the heart. In this chapter, we describe the production and quality control of recombinant AAV (rAAV) vectors of the serotype 9, the most cardiotropic AAV serotype when delivered systemically in rodents. We also describe the systemic administration of rAAV vectors and the local delivery of rAAV vectors by direct intramyocardial injection. Taken together, the methods described in this chapter will allow the reader to deliver efficiently therapeutic genes to the rodent heart, both globally and regionally.

Viral expression of a SERCA2a-activating PLB mutant improves calcium cycling and synchronicity in dilated cardiomyopathic hiPSC-CMs

“…AAV viruses were purified according to the two-plasmid method with iodixanol gradient as described. 32…”

iTRAQ‑based quantitative proteomics analysis of the potential application of secretoneurin gene therapy for cardiac hypertrophy induced by DL‑isoproterenol hydrochloride in mice

“…After loss of the righting reflex, the mice were ventilated by intratracheal intubation with a rodent ventilator (Harvard Apparatus). The chest of each mouse was opened to expose the heart and 2x10 10 particles of the Ad-con or Ad-SN (injection volume, 50 µl) were injected into the myocardium at five different sites, as previously described (26,27). Subsequently, the mice received subcutaneous injection of either ISO (5 mg/kg/day) or saline into their back for seven days (28).…”

The specific inhibition of the cardiac electrogenic sodium/bicarbonate cotransporter leads to cardiac hypertrophy

“…Recombinant AAV9-shNBCe1 or AAV9-shControl was produced essentially as previously described (Orlowski et al, 2021 and Lopez-Gordo et al, 2019) 21,22 . Brie y, HEK293T/17 (ATCC) cells were cultured with DMEM (Thermo Fisher Scienti c) supplemented with 10% fetal bovine serum (Natocor) and penicillin/streptomycin (Thermo Fisher Scienti c) at 37°C and 5% CO2.…”

“…The gradients were centrifuged at 350,333 × g (avg) for 75 min at 18°C in a Beckman type 70 Ti xed angle rotor. Fractions were obtained from the bottom of the tube and the presence of the virus were detected by alkaline gel 22 . Positive fractions were buffer exchange lactated Ringer's solution (Baxter International), ltered through a 0.22 µm pore lter (Merck Millipore), and stored at −80°C.…”