Despite the proven safety and efficacy of adeno-associated virus serotype 2 (AAV2) vectors in gene therapy, their clinical application is often limited by dose-dependent immune responses, particularly CD8+ T cell activation against the capsid proteins. This immune reaction becomes prominent at high vector doses—commonly used in systemic delivery for liver-targeted therapies—leading to transgene silencing and potential loss of therapeutic benefit. To address this challenge, we developed a novel class of bioengineered AAV2 vectors through targeted mutagenesis of key serine, threonine, and lysine residues on the capsid surface.
We focused on residues within phosphodegron regions—structural motifs that serve as recognition sites for host kinases and ubiquitin ligases. Phosphorylation of these residues triggers ubiquitination and subsequent proteasomal degradation of incoming viral particles, reducing their transduction efficiency. We hypothesized that mutating these residues would not only enhance intracellular stability but also alter antigenic epitopes, thereby reducing immunogenicity. Using structural modeling and evolutionary conservation analysis, we identified 15 candidate residues across three phosphodegrons: S489, S498, S662, S668, T251, T454, T503, T671, T701, T713, T716, K39, K137, K143, K161, K490, K507, K527, K532, and K544. Single and double mutants were generated using site-directed mutagenesis, with serine/threonine substituted by alanine (S/A, T/A) and lysine replaced by arginine (K/R).
All mutant vectors were packaged into self-complementary EGFP-expressing constructs and purified via iodixanol gradient ultracentrifugation and column chromatography. Physical titers ranged from 1 × 10¹² to 3.Doxycycline hyclate supplier 2 × 10¹² VG/ml, confirming successful packaging with minimal disruption to capsid integrity.HKUST-1 In Vitro In vitro testing in HeLa and HEK-293 cells showed that 11 S/T/A and 7 K/R mutants significantly outperformed AAV2-WT, achieving up to 90% higher transduction efficiency at a multiplicity of infection of 2 × 10³ VG/cell. The S489A mutant consistently ranked among the top performers, with nearly 8-fold greater EGFP expression compared to wild-type.
In vivo evaluation was conducted in C57BL/6 mice receiving tail vein injections of 5 × 10¹⁰ VG per animal. Four weeks post-injection, hepatic tissues were analyzed for transgene expression. Fluorescence microscopy revealed strong EGFP signal in livers from animals treated with S/T/K mutants, particularly S489A, S498A, K532R, and K544R. Quantitative PCR confirmed up to 4.9-fold higher vector genome copy numbers per diploid genome in mutant-treated groups versus AAV2-WT controls. Real-time RT-PCR further demonstrated a 14-fold increase in EGFP transcript levels, indicating robust and sustained expression.
Crucially, immune profiling revealed a dramatic reduction in neutralizing antibody formation. Serial dilution assays showed that the S489A mutant induced approximately 8-fold lower neutralizing antibody titers than AAV2-WT. This suggests that the mutation alters capsid epitopes recognized by B cells and antibodies, potentially reducing cross-reactivity with wild-type vectors. Notably, despite its enhanced transduction, the S489A vector elicited no significant inflammatory response, with histological examination showing normal liver architecture and median inflammation scores of 0–1 across all groups.PMID:35061220
Further mechanistic studies using an in vitro ubiquitination assay demonstrated that the K532R mutant exhibited markedly reduced ubiquitin conjugation compared to both AAV2-WT and AAV5-WT. This supports the idea that lysine mutations directly interfere with the ubiquitin ligase machinery, leading to prolonged cytoplasmic survival and improved nuclear entry of viral genomes.
These findings highlight a dual advantage of engineered AAV2 vectors: enhanced transduction efficiency due to evasion of proteasomal degradation and reduced immunogenicity due to altered capsid antigenicity. By combining these benefits, the modified vectors enable effective gene delivery at significantly lower doses, minimizing the risk of immune activation while maximizing therapeutic output. This strategy represents a major step forward in developing safer, more effective AAV-based therapies for inherited liver diseases such as hemophilia B, where durable expression and repeat dosing are critical for long-term success.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com