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Data validation and comparative analysis of EGFP fluorescence from NE-TR125-AFP-EGFP, Linear open-end (L) AFP-EGFP, and circular Plasmid pAFP-EGFP. Two techniques were used at Day 3 to determine the best quantifiable methodology. (A) Quantified fluorescence imaging data using fluorescence microscopy and ImageJ analysis of EGFP transgene expression at 3 days post-transfection. (B) Quantified fluorescence as measured by spectrophotometry as artificial fluorescent units (AFU). Fluorescence imaging data were collected at day 28 (C) post-transfection and quantified by ImageJ. Two-tailed unequal variance t-test utilized to generate p-values. Y-axis of each bar graph are adjusted to the respective data. *p < 0.05. Bar graphs represent the mean fluorescence of the triplicates with ±1SD error bars.
Source publication
Recombinant AAV serotype vectors and their variants have been or are currently being used for gene therapy for hemophilia in several phase I/II/III clinical trials in humans. However, none of these trials have included children with hemophilia since the traditional liver-directed AAV gene therapy will not work in these patients because of the follo...
Citations
... All of the slides were stored in À80 C until imaging, at which time slides were stained with DAPI and subjected to fluorescence microscopy. 51 Vector genome copy numbers were determined by qPCR using total genomic DNA isolated from all of the tissues. The same tissue samples (20 mg each) were used for the isolation of total genomic DNA and RNA using the QIAmp DNA Mini Kit (catalog no. ...
The first generation of adeno-associated virus (AAV) vectors composed of the naturally occurring capsids and genomes, although effective in some instances, are unlikely to be optimal for gene therapy in humans. The use of the first generation of two different AAV serotype vectors (AAV9 and AAVrh74) in four separate clinical trials failed to be effective in patients with Duchenne muscular dystrophy, although some efficacy was observed in a subset of patients with AAVrh74 vectors leading to US Food and Drug Administration approval (Elevidys). In two trials with the first generation of AAV9 vectors, several serious adverse events were observed, including the death of a patient in one trial, and more recently, in the death of a second patient in an N-of-1 clinical trial. In a fourth trial with the first generation of AAVrh74 vectors, myositis and myocarditis were also observed. Here, we report that capsid- and genome-modified optimized AAVrh74 vectors are significantly more efficient in transducing primary human skeletal muscle cells in vitro and in all major muscle tissues in vivo following systemic administration in a murine model. The availability of optimized AAVrh74 vectors promises to be safe and effective in the potential gene therapy of muscle diseases in humans.
