Mon. May 20th, 2024

Mouse muscle. (a) Transduction of tibialis anterior (TA) muscle tissues with AAV6 vectors carrying GFP reporters. Top, schematic of AAV proviruses containing enhanced GFP (eGFP) and hrGFP. Bottom, low energy photographs beneath fluorescent excitation showing common transduction of 1 1011 DNAse-resistant particle (DRP) eGFP and hrGFP AAV6 vectors, 1, 2, and four weeks right after injection with quantification of fluorescence units employing Bioquant software. Fluorescence intensity was drastically higher in eGFP mice, two and 4 weeks following injection (t-test, P 0.001; N = 4 legs per virus). (b) Low-power photomicrographs of hematoxylin and eosin stained mouse TA muscle tissues injected with 1 1011 DRP of indicated vectors, two weeks prior. Muscles expressing AAV6.CMV.hrGFP created widespread inflammatory lesions, whereas these containing comparable eGFP vectors didn’t. (c) Time course shows regeneration (as indicated by myofibers containing central nuclei) in mouse muscle tissues injected with 1 1011 DRP AAV6.CMV.hrGFP by four weeks. In contrast, muscle tissues expressing identical doses of AAV6.CMV.eGFP were relatively typical at 4 weeks, by comparison, even though focal inflammation, occasional central nuclei, and histological indications of myonecrosis were evident in some places (best suitable panel). (d) hrGFP persisted in some regenerated myofibers four weeks post-injection. White arrows indicate examples of GFP-negative, centrally nucleated myofibers; yellow arrows indicate examples of GFP-positive, centrally nucleated myofibers; asterisk centers a cluster of GFP-positive myofibers with nuclei at the normal peripheral location. AAV, adeno-associated virus; CMV, cytomegalovirus; ITR, inverted terminal repeat; PA, polyadenylation signal.Molecular Therapy ucleic AcidshrGFP Causes Dose-dependent Muscle Toxicity Wallace et al.table 1 AAV6.CMV.eGFP and AAV6.CMV.hrGFP vectors had baseline levels of endotoxin sample concentration dilution (eU/ml) 10 AAV6.Neurotrophin-3 Protein , Human (CHO) CMV. eGFP 50 0.050 0.048 0.026 0.022 0.048 0.045 0.011 0.011 0.325 0.404 0.063 0.083 0.345 0.345 0.081 0.092 concentration Imply (eU/ml) dilution sd 0.50 0.48 1.32 1.10 0.48 0.45 0.55 0.55 three.25 four.04 3.17 four.17 three.45 3.45 four.04 four.31 three.81 0.43 three 1010 three.66 0.52 eight 109 0.51 0.05 0.85 0.43 three a2 weeks4 weeks10 AAV6.CMV. hrGFPAAV6.CMV. eGFP, 5 EU spike controlAAV6.CMV. hrGFP, five EU spike controlAAV, adeno-associated virus; CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein; EU, endotoxin units; hrGFP, humanized Renilla reniformis GFP.beGFP showed only occasional focal inflammatory infiltrates and a few evidence of muscle regeneration (as indicated by presence of myofibers with centrally positioned nuclei). In comparison, by four weeks hrGFP-injected muscles had been virtually fully regenerated, as evidenced by myofibers with centrally positioned nuclei throughout the injected muscle.CHD-5 In Vivo Upon closer histological examination of transduced regions in 4-week cryosections, we identified hrGFP-positive myofibers with and without having central nuclei (Figure 1d; yellow and white arrows, respectively), and presumably undamaged hrGFP-positive myofibers containing only peripheral myonuclei (Figure 1d; asterisk indicates a cluster).PMID:23892407 This recommended that some hrGFP expression was tolerable, and we for that reason hypothesized that hrGFP-associated toxicity was dose-dependent. To test this, we determined the effect of decreasing AAV6.CMV.hrGFP vector dose on muscle toxicity. Muscle tissues injected with the lowest vector dosage (3 109 particles) have been histologically standard at two and 4 weeks (Figur.