Ex vivo CRISPR gene editing in hematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation, sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci, cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. Engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to nontreated cells, with no differences in differentiation. Here we demonstrate nontoxic delivery of the entire CRISPR payload into primary human blood progenitors. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Parameters of humoral and cellular immunity were measured in thirty-five patients with atopic eczema. The mean serum IgE level was raised but levels of the other major immunoglobulin classes were normal. Ten per cent of patients failed to respond to tetanus immunization. All patients responded to S. typhi H antigen. Fourteen per cent of patients failed to mount delayed hypersensitivity reactions to a battery of three intradermal antigens. The phytohaemagglutinin-stimulated uptake of 3H thymidine by lymphocytes was normal in the presence of autologous or of fetal calf serum, as was the spontaneous lymphocyte uptake. T and B lymphocyte numbers in the peripheral blood were normal. These results are similar to those found in asthmatic patients and support the hypothesis that, in some patients, atopic eczema is associated with an immunodeficiency state.
Surface tension at liquid–air interfaces is a major barrier that needs to be surmounted by a wide range of organisms; surfactant and interfacially active proteins have evolved for this purpose. Although these proteins are essential for a variety of biological processes, our understanding of how they elicit their function has been limited. However, with the recent determination of high-resolution 3D structures of several examples, we have gained insight into the distinct shapes and mechanisms that have evolved to confer interfacial activity. It is now a matter of harnessing this information, and these systems, for biotechnological purposes.
The skin at the site of HSV-2 reactivation is enriched for HSV-2 specific T cells. To evaluate whether an immunotherapeutic vaccine could elicit skin-based memory T cells we studied skin biopsies and HSV-2-reactive CD4+ T cells from peripheral blood mononuclear cells (PBMCs) by T-cell receptor (TCR) sequencing before and after vaccination with a replication-incompetent whole virus HSV-2 vaccine candidate (HSV529). The representation of HSV-2-reactive CD4+ T cell sequences from PBMCs increased from a median of 0.03% (range 0-0.09%) to 0.6% (range 0-1.3%) of the total skin TCR repertoire after the first vaccine dose. We found sustained expansion after vaccination in unique, skin-based T-cell clonotypes that were not detected in HSV-2-reactive CD4+ T cells isolated from PBMCs. While detection of skin clonotypes in the blood was related to abundance in the skin it was not related to expansion after vaccination. In one participant a switch in immunodominance was observed after vaccination with the emergence of a newly dominant TCRa/b pair in skin that was not detected in blood. We confirmed that the newly dominant clonotype was derived from an HSV-specific CD4+ T cell by creation of a synthetic TCR in a Jurkat-based cell line with a NR4A1-mNeonGreen reporter system. Our data indicate that the skin in areas of HSV-2 reactivation possesses an oligoclonal TCR repertoire that is distinct from the circulation with prominent clonotypes infrequently detected in the circulation by standard methods. Defining the influence of therapeutic vaccination on the HSV-2-specific TCR repertoire requires tissue-based evaluation.
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