Assessment of Key Factors Impacting Variability in AAV Vector Genome Titration by Digital PCR.

Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the rAAV genome titer is crucial for ensuring the safe and effective administration of clinical doses. The evolution of the rAAV genome titer assay from quantitative PCR (qPCR) to digital PCR (dPCR) has enhanced accuracy and precision, yet practical challenges persist. This study systematically investigated the impact of various operational factors on genome titration in a single-factor manner, aiming to address potential sources of variability in the quantitative determination process. Our findings revealed that a pretreatment procedure without genome extraction exhibits superior precision compared with titration with genome extraction. Additionally, notable variations in titration results across different brands of dPCR instruments were documented, with relative standard deviation (RSD) reaching 23.47% for AAV5 and 11.57% for AAV8. Notably, optimal operations about DNase I digestion were identified; we thought treatment time exceeding 30 min was necessary, and there was no need for thermal inactivation after digestion. And we highlighted that thermal capsid disruption before serial dilution substantially affected AAV genome titers, causing a greater than ten-fold decrease. Conversely, this study found that additive components of dilution buffer are not significant contributors to titration variations. Furthermore, we found that repeated freeze-thaw cycles significantly compromised AAV genome titers. In conclusion, a comprehensive dPCR titration protocol, incorporating insights from these impact factors, was proposed and successfully tested across multiple serotypes of AAV. The results demonstrate acceptable variations, with the RSD consistently below 5.00% for all tested AAV samples. This study provides valuable insights to reduce variability and improve the reproducibility of AAV genome titration using dPCR.

Distribution, source identification and health risk assessment of PFASs in groundwater from Jiangxi Province, China.

There is an urgent need to investigate on the distribution and fate of short-chain analogues and emerging per- and polyfluoroalkyl substances (PFASs) in groundwater, and little research on their source apportionment and health risks through the drinking water exposure pathway has been carried out. In present study, the concentration and source of 22 PFASs, including five alternatives: 6:2 fluorotelomer sulfonate (6:2 FTS), potassium 9-chlorohexadecafluoro-3-oxanonane-1-sulfonate (F-53B), hexafluoropropylene oxide trimer acid (HFPO-TA), hexafluoropropylene oxide dimer acid (HFPO-DA) and ammonium 4, 8-dioxa-3H-perfluorononanoate (ADONA), were analyzed in 88 groundwater samples from wells in Jiangxi Province, southeastern China. The total PFASs concentration (Σ18PFASs) in groundwater varied from 1.27 to 381.00 ng/L (mean 47.60 ng/L). Short-chain perfluorobutanoic acid (PFBA) and perfluoropentanoic acid (PFPeA) were the most abundant perfluorinated carboxylic acids (PFCAs), and short-chain perfluorobutanesulfonate (PFBS) was the most abundant perfluorinated sulfonic acids (PFSAs) in groundwater samples. The quantitative source apportionment by nonnegative matrix/tensor factorization coupled with k-means clustering (NMFk) model suggested that short-chain homologues and emerging alternatives have been used as substitutes for legacy PFOS and PFOA. Furthermore, the human risk assessment results showed that the estimated daily intakes (EDIs) for short-chain PFCAs were higher than that of PFOA, whereas the EDIs of PFBS, 6:2 FTS and F-53B were comparable to that of PFOS.

Distribution, source identification and health risk assessment of PFASs and two PFOS alternatives in groundwater from non-industrial areas.

Little research has been carried out for the per- and polyfluoroalkyl substances (PFASs) in groundwater from non-industrial areas, even though it has been proved that PFASs can transport for long distance. In this study, the concentration profiles and geographical distribution of 14 PFASs, including two alternatives of perfluorooctane sulfonate (PFOS), 6:2 fluorotelomer sulfonate (6:2 FTS) and potassium 9-chlorohexadecafluoro-3-oxanonane-1-sulfonate (F-53B), were analyzed in groundwater samples (n = 102) collected from water wells in non-industrial areas. The total concentrations of PFASs (Σ14PFASs) in groundwater samples ranged from 2.69 to 556 ng/L (mean 43.1 ng/L). The detection rates of shorter chain (C4-C9) PFASs were 62.75-100%, higher than those of long chain (> C10) PFASs with detection rates of less than 40%. The source identification using hierarchical cluster analysis and Spearman rank correlation analysis suggested that domestic sewage and atmospheric deposition may contribute significantly to the PFAS occurrence in groundwater in non-industrial areas, while the nearby industrial parks may contribute some, but not at a significant level. Furthermore, the human health risk assessment analysis shows that the health hazards associated with perfluorooctanoic acid (PFOA) and PFOS, two of the main PFAS constituents in groundwater from non-industrial areas, were one or two orders of magnitude higher than those in a previous study, but were unlikely to cause long-term harm to the residents via the drinking water exposure pathway alone.