Identification and quantification of novel per- and polyfluoroalkyl substances (PFAS) contamination in a Great Lakes urban-dominated watershed.

Per- and polyfluoroalkyl substances (PFAS) are a large group of synthetic organic fluoro-compounds that are oil-, water-, and flame-resistant, making them useful in a wide range of commercial and consumer products, as well as resistant to environmental degradation. To assess the impact of urbanization and wastewater treatment processes, surface water and sediment samples were collected at 27 sites within the Great Lakes in the Lake Huron to Lake Erie corridor (HEC), an international waterway including the highly urbanized Detroit and Rouge Rivers. Samples were analyzed for 92 PFAS via UHPLC-MS/MS. Our previous data in the HEC found the highest amount of PFAS contamination at the Rouge River mouth. In addition to evaluating the input of the Rouge River into the HEC, we evaluated the transport of PFAS into the HEC from other major tributaries. PFAS were detected in both surface water and sediment at all sites in this study, with a total of 10 congeners quantified in all surface water samples and 16 congeners quantified in all sediment samples, indicating ubiquitous contamination. Perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) were pervasive in the HEC as these two compounds were detected in all sites and matrices, often at concentrations above the US EPA's recommended lifetime interim updated health advisories. Surface water samples contained more perfluorohexanoic acid (PFHxA) than any other congener, with average aqueous PFHxA across all surface water samples exceeding the average concentration previously reported in the Great Lakes. Sediment samples were dominated by PFOS, but novel congeners, notably 3-Perfluoropentyl propanoic acid (FPePA), were also quantified in sediment. The Rouge River and other tributaries contribute significantly to the PFAS burden in the HEC including Lake Erie. Overall, our results indicate the need for expanding toxicological research and risk assessment focused on congeners such as PFHxA and PFAS mixtures, as well as regulation that is tighter at the onset of production and encompasses PFAS as a group at a national level.

Efficient long-term multilineage engraftment of CD33-edited hematopoietic stem/progenitor cells in nonhuman primates.

Current immunotherapeutic targets are often shared between neoplastic and normal hematopoietic stem and progenitor cells (HSPCs), leading to unwanted on-target, off-tumor toxicities. Deletion or modification of such targets to protect normal HSPCs is, therefore, of great interest. Although HSPC modifications commonly aim to mimic naturally occurring phenotypes, the long-term persistence and safety of gene-edited cells need to be evaluated. Here, we deleted the V-set domain of CD33, the immune-dominant domain targeted by most anti-CD33 antibodies used to treat CD33-positive malignancies, including acute myeloid leukemia, in the HSPCs of two rhesus macaques, performed autologous transplantation after myeloablative conditioning, and followed the animals for up to 3 years. CD33-edited HSPCs engrafted without any delay in recovery of neutrophils, the primary cell type expressing CD33. No impact on the blood composition, reconstitution of the bone marrow stem cell compartment, or myeloid differentiation potential was observed. Up to 20% long-term gene editing in HSPCs and blood cell lineages was seen with robust loss of CD33 detection on myeloid lineages. In conclusion, deletion of the V-set domain of CD33 on HSPCs, progenitors, and myeloid lineages did not show any adverse effects on their homing and engraftment potential or the differentiation and functionality of myeloid progenitors and lineages.

Multiplex CRISPR/Cas9 genome editing in hematopoietic stem cells for fetal hemoglobin reinduction generates chromosomal translocations.

Sickle cell disease and ß-thalassemia are common monogenic disorders that cause significant morbidity and mortality globally. The only curative treatment currently is allogeneic hematopoietic stem cell transplantation, which is unavailable to many patients due to a lack of matched donors and carries risks including graft-versus-host disease. Genome editing therapies targeting either the BCL11A erythroid enhancer or the HBG promoter are already demonstrating success in reinducing fetal hemoglobin. However, where a single locus is targeted, reliably achieving levels high enough to deliver an effective cure remains a challenge. We investigated the application of a CRISPR/Cas9 multiplex genome editing approach, in which both the BCL11A erythroid enhancer and HBG promoter are disrupted within human hematopoietic stem cells. We demonstrate superior fetal hemoglobin reinduction with this dual-editing approach without compromising engraftment or lineage differentiation potential of edited cells post-xenotransplantation. However, multiplex editing consistently resulted in the generation of chromosomal rearrangement events that persisted in vivo following transplantation into immunodeficient mice. The risk of oncogenic events resulting from such translocations therefore currently prohibits its clinical translation, but it is anticipated that, in the future, alternative editing platforms will help alleviate this risk.