Parkerwrenn8869

The present work provides a benchmark for applications of XFELs for soft x-ray absorption studies of carrier-specific dynamics in semiconductors, and future opportunities enabled by this method are discussed.Regulatory T cells (Tregs) are emerging as a new cell-based therapy in solid organ transplantation. Adoptive transfer of Tregs has been shown preclinically to protect from graft rejection, and the safety of Treg therapy has been demonstrated in clinical trials. Despite these successes, the in vivo distribution and persistence of adoptively transferred Tregs remained elusive, which hampers clinical translation. Here we isolated human Tregs using a GMP-compatible protocol and lentivirally transduced them with the human sodium iodide symporter to render them traceable in vivo by radionuclide imaging. Engineered human Tregs were characterized for phenotype, survival, suppressive capacity, and reporter function. To study their trafficking behavior, they were subsequently administered to humanized mice with human skin transplants. Traceable Tregs were quantified in skin grafts by non-invasive nano-single-photon emission computed tomography (nanoSPECT)/computed tomography (CT) for up to 40 days, and the results were validated ex vivo. Using this approach, we demonstrated that Treg trafficking to skin grafts was regulated by the presence of recipient Gr-1+ innate immune cells. We demonstrated the utility of radionuclide reporter gene-afforded quantitative Treg in vivo tracking, addressing a fundamental need in Treg therapy development and offering a clinically compatible methodology for future Treg therapy imaging in humans.Gaucher disease type 1 (GD1) is an inherited lysosomal disorder with multisystemic effects in patients. Hallmark symptoms include hepatosplenomegaly, cytopenias, and bone disease with varying degrees of severity. Mutations in a single gene, glucosidase beta acid 1 (GBA1), are the underlying cause for the disorder, resulting in insufficient activity of the enzyme glucocerebrosidase, which in turn leads to a progressive accumulation of the lipid component glucocerebroside. In this study, we treat mice with signs consistent with GD1, with hematopoietic stem/progenitor cells transduced with a lentiviral vector containing an RNA transcript that, after reverse transcription, results in codon-optimized cDNA that, upon its integration into the genome encodes for functional human glucocerebrosidase. Five months after gene transfer, a highly significant reduction in glucocerebroside accumulation with subsequent reversal of hepatosplenomegaly, restoration of blood parameters, and a tendency of increased bone mass and density was evident in vector-treated mice compared to non-treated controls. Furthermore, histopathology revealed a prominent reduction of Gaucher cell infiltration after gene therapy. The vector displayed an oligoclonal distribution pattern but with no sign of vector-induced clonal dominance and a typical lentiviral vector integration profile. Cumulatively, our findings support the initiation of the first clinical trial for GD1 using the lentiviral vector described here.Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete silencing of the disease locus, leading to pathogenic misexpression of DUX4 in skeletal muscle. Previously, we showed that CRISPR inhibition could successfully target and repress DUX4 in FSHD myocytes. However, an effective therapy will require both efficient delivery of therapeutic components to skeletal muscles and long-term repression of the disease locus. Thus, we re-engineered our platform to allow in vivo delivery of more potent epigenetic repressors. We designed an FSHD-optimized regulatory cassette to drive skeletal muscle-specific expression of dCas9 from Staphylococcus aureus fused to HP1α, HP1γ, the MeCP2 transcriptional repression domain, or the SUV39H1 SET domain. Targeting each regulator to the DUX4 promoter/exon 1 increased chromatin repression at the locus, specifically suppressing DUX4 and its target genes in FSHD myocytes and in a mouse model of the disease. Importantly, minimizing the regulatory cassette and using the smaller Cas9 ortholog allowed our therapeutic cassettes to be effectively packaged into adeno-associated virus (AAV) vectors for in vivo delivery. By engineering a muscle-specific epigenetic CRISPR platform compatible with AAV vectors for gene therapy, we have laid the groundwork for clinical use of dCas9-based chromatin effectors in skeletal muscle disorders.A clinical trial using adeno-associated virus serotype 8 (AAV8)-human uridine diphosphate glucuronosyltransferase 1A1 (hUGT1A1) to treat inherited severe unconjugated hyperbilirubinemia (Crigler-Najjar syndrome) is ongoing, but preclinical data suggest that long-term efficacy in children is impaired due to loss of transgene expression upon hepatocyte proliferation in a growing liver. This study aims to determine at what age long-term efficacy can be obtained in the relevant animal model and whether immune modulation allows re-treatment using the same AAV vector. Neonatal, suckling, and juvenile Ugt1a1-deficient rats received a clinically relevant dose of AAV8-hUGT1A1, and serum bilirubin levels and anti-AAV8 neutralizing antibodies (NAbs) in serum were monitored. The possibility of preventing the immune response toward the vector was investigated using a rapamycin-based regimen with daily intraperitoneal (i.p.) injections starting 2 days before and ending 21 days after vector administration. Pidnarulex In rats treated at postnatal day 1 (P1) or P14, the correction was (partially) lost after 12 weeks, whereas the correction was stable in rats injected at P28. Combining initial vector administration with the immune-suppressive regimen prevented induction of NAbs in female rats, allowing at least partially effective re-administration. Induction of NAbs upon re-injection could not be prevented, suggesting that this strategy will be ineffective in patients with low levels of preexisting anti-AAV NAbs.Machine learning (ML) can aid in novel discoveries in the field of viral gene therapy. Specifically, big data gathered through next-generation sequencing (NGS) of complex capsid libraries is an especially prominent source of lost potential in data analysis and prediction. Furthermore, adeno-associated virus (AAV)-based capsid libraries are becoming increasingly popular as a tool to select candidates for gene therapy vectors. These higher complexity AAV capsid libraries have previously been created and selected in vivo; however, in silico analysis using ML computer algorithms may augment smarter and more robust libraries for selection. In this study, data of AAV capsid libraries gathered before and after viral assembly are used to train ML algorithms. We found that two ML computer algorithms, artificial neural networks (ANNs), and support vector machines (SVMs), can be trained to predict whether unknown capsid variants may assemble into viable virus-like structures. Using the most accurate models constructed, hypothetical mutation patterns in library construction were simulated to suggest the importance of N495, G546, and I554 in AAV2-derived capsids.