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Updated: Aug 29, 2026
Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors
Published on: October 20, 2023
Enhanced beta2-adrenergic receptor (beta2AR) signaling by adeno-associated viral (AAV)-mediated gene transfer
Stacie M Jones1, F Charles Hiller, Sandie E Jacobi
1Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children's Hospital, Little Rock, Arkansas 72202, USA. JonesStacieM@uams.edu
Background:
Beta2-adrenergic receptors (beta2AR) play important regulatory roles in a variety of cells and organ systems and are important therapeutic targets in the treatment of airway and cardiovascular disease. Prolonged use of beta-agonists results in tolerance secondary to receptor down-regulation resulting in reduced therapeutic efficiency. The purpose of this work is to evaluate the signaling capabilities of the beta2AR expressed by a recombinant adeno-associated viral (AAV) vector that also included an enhanced green fluorescent protein (EGFP) gene (AAV-beta2AR/EGFP).
Results:
By epifluorescence microscopy, approximately 40% of infected HEK 293 cells demonstrated EGFP expression. beta2AR density measured with [3H]dihydroalprenolol ([3H]DHA) increased either 13- or 77-fold in infected cells compared to mock infected controls depending on the culture conditions used. The [3H]DHA binding was to a single receptor population with a dissociation constant of 0.42 nM, as would be expected for wild-type beta2AR. Agonist competition assays with [3H]DHA showed the following rank order of potency: isoproterenol>epinephrine> norepinephrine, consistent with beta2AR interaction. Isoproterenol-stimulated cyclic AMP levels were 5-fold higher in infected cells compared to controls (314 +/- 43 vs. 63.4 +/- 9.6 nmol/dish; n = 3). Receptor trafficking demonstrated surface expression of beta2AR with vehicle treatment and internalization following isoproterenol treatment.
Conclusions:
We conclude that HEK 293 cells infected with AAV-beta2AR/EGFP effectively express beta2AR and that increased expression of these receptors results in enhanced beta2AR signaling. This method of gene transfer may provide an important means to enhance function in in vivo systems.
Insights
Recombinant adeno-associated virus (AAV) vectors effectively express beta2-adrenergic receptors (beta2AR) in cells. This enhanced beta2AR expression leads to improved signaling, offering a potential gene transfer method for in vivo applications.
Area of Science:
- Molecular biology
- Cell biology
- Pharmacology
Background:
- Beta2-adrenergic receptors (beta2AR) are crucial for cellular regulation and are therapeutic targets for airway and cardiovascular diseases.
- Therapeutic efficacy of beta-agonists diminishes due to receptor downregulation, a phenomenon known as tolerance.
- Developing methods to enhance beta2AR expression and signaling is critical for overcoming treatment resistance.
Purpose of the Study:
- To evaluate the signaling capabilities of beta2AR expressed via a recombinant adeno-associated viral (AAV) vector.
- To assess the efficacy of AAV-beta2AR/EGFP in delivering and expressing functional beta2AR in HEK 293 cells.
Main Methods:
- HEK 293 cells were infected with AAV-beta2AR/EGFP, a vector co-expressing beta2AR and enhanced green fluorescent protein (EGFP).
- EGFP expression was monitored by epifluorescence microscopy.
- Beta2AR density was quantified using [3H]dihydroalprenolol ([3H]DHA) binding assays.
- Agonist potency and cyclic AMP (cAMP) levels were measured to assess receptor signaling.
- Receptor trafficking was investigated following agonist stimulation.
Main Results:
- Approximately 40% of infected cells showed EGFP expression, indicating successful transduction.
- Beta2AR density increased significantly (13- to 77-fold) in infected cells compared to controls.
- Binding assays confirmed the presence of functional beta2AR, with appropriate affinity and agonist rank order.
- Isoproterenol stimulation resulted in a 5-fold increase in cAMP levels in infected cells.
- Receptor internalization was observed upon agonist stimulation, consistent with normal beta2AR trafficking.
Conclusions:
- HEK 293 cells infected with AAV-beta2AR/EGFP efficiently express functional beta2AR.
- Increased beta2AR expression via this AAV vector enhances receptor-mediated signaling.
- This gene transfer approach holds promise for augmenting beta2AR function in in vivo models.
