Stephanie K. Dougan

Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining

Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator–like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by non-homologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing using Cas9 in mammalian cell lines and in mice for all four genes examined up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator like effector nucleases, and to non-mammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.

XBP‐1 regulates signal transduction, transcription factors and bone marrow colonization in B cells

XBP‐1, a transcription factor that drives the unfolded protein response (UPR), is activated in B cells when they differentiate to plasma cells. Here, we show that in the B cells, whose capacity to secrete IgM has been eliminated, XBP‐1 is induced normally on induction of differentiation, suggesting that activation of XBP‐1 in B cells is a differentiation‐dependent event, but not the result of a UPR caused by the abundant synthesis of secreted IgM. Without XBP‐1, B cells fail to signal effectively through the B‐cell receptor. The signalling defects lead to aberrant expression of the plasma cell transcription factors IRF4 and Blimp‐1, and altered levels of activation‐induced cytidine deaminase and sphingosine‐1‐phosphate receptor. Using XBP‐1‐deficient/Blimp‐1‐GFP transgenic mice, we find that XBP‐1‐deficient B cells form antibody‐secreting plasmablasts in response to initial immunization; however, these plasmablasts respond ineffectively to CXCL12. They fail to colonize the bone marrow and do not sustain antibody production. These findings define the role of XBP‐1 in normal plasma cell development and have implications for management of B‐cell malignancies.

Sortase-catalyzed transformations that improve the properties of cytokines

Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortase-mediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety of proteins, provided that their termini are not involved in receptor binding or function.

CD1 expression on antigen-presenting cells.

CD1 proteins present self and microbial glycolipids to CD 1-restricted T cells, or in the case of CD1d, to NKT cells. The CD1 family in humans consists of group I proteins CDla, CDlb, CDlc, and CDle and the group II protein CDld. Rodents express only CDld, but as CD1d is broadly expressed and traffics to all endosomal compartments, this single CD1 family member is thereby able to acquire antigens in many subcellular compartments. A complete understanding of the CD 1 family requires an appreciation of which cells express CD1 and how CD1 contributes to the unique function of each cell type. While group I CD 1 expression is limited to thymocytes and professional APCs, CD1d has a wider tissue distribution and can be found on many nonhematopoietic cells. The expression and regulation of CD1 are presented here with particular emphasis on the function of CD1 in thymocytes, B cells, monocytes and macrophages, dendritic cells (DCs), and intestinal epithelial cells (IECs). Altered expression of CD 1 in cancer, autoimmunity, and infectious disease is well documented, and the implication of CD 1 expression in these diseases is discussed.

In vivo discovery of immunotherapy targets in the tumour microenvironment

Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments.

Preparation of unnatural N-to-N and C-to-C protein fusions

Standard genetic approaches allow the production of protein composites by fusion of polypeptides in head-to-tail fashion. Some applications would benefit from constructions that are genetically impossible, such as the site-specific linkage of proteins via their N or C termini, when a remaining free terminus is required for biological activity. We developed a method for the production of N-to-N and C-to-C dimers, with full retention of the biological activity of both fusion partners and without inflicting chemical damage on the proteins to be joined. We use sortase A to install on the N or C terminus of proteins of interest the requisite modifications to execute a strain-promoted copper-free cycloaddition and show that the ensuing ligation proceeds efficiently. Applied here to protein–protein fusions, the method reported can be extended to connecting proteins with any entity of interest.

Microsomal triglyceride transfer protein in plasma and cellular lipid metabolism.

Purpose of review This review summarizes recent advances about the role of microsomal triglyceride transfer protein in plasma and tissue lipid homeostasis. Recent findings Microsomal triglyceride transfer protein emerged as a phospholipid transfer protein and acquired triacylglycerol transfer activity during evolution from invertebrates to vertebrates. These activities are proposed to participate in ‘nucleation’ and ‘desorption’ steps during the biosynthesis of primordial apoB-containing lipoproteins. Microsomal triglyceride transfer protein also transfers phospholipids to the glycolipid antigen presentation molecule CD1d. Under physiologic conditions, plasma apoB-containing lipoproteins and microsomal triglyceride transfer protein expression exhibit diurnal variations synchronized by food and light. Microsomal triglyceride transfer protein is regulated at the transcriptional level. HNF4α is critical for its transcription. Other transcription factors along with coactivators and corepressors modulate microsomal triglyceride transfer protein expression. Reductions in microsomal triglyceride transfer protein mRNA and activity are related to steatosis in HCV-3 infected patients. CCl4 induces steatosis by enhancing proteasomal degradation of microsomal triglyceride transfer protein and can be partially avoided by inhibiting this degradation. Chemical antagonists cause hepatosteatosis, but this was not seen in the absence of fatty acid binding protein. Summary Microsomal triglyceride transfer protein is a target to lower plasma lipids and to reduce inflammation in certain immune disorders. More knowledge is required, however, regarding its regulation and its role in the biosynthesis of apoB-containing lipoproteins and CD1d.

Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes

Significance Engineered human RBCs are attractive carriers for the introduction of novel therapeutics, immunomodulatory agents, and diagnostic imaging probes into the human body. Normal murine and human RBCs can be produced in tissue culture from progenitors; we have introduced genes into these cells encoding surface proteins that can be covalently and site-specifically modified on the cell surface. The engineering and labeling processes do not damage the cells or affect their survival in vivo. Most importantly, the engineered RBCs can be labeled with a wide array of functional probes, including small molecules, peptides, and proteins, and thus have the potential to be carriers of a variety of therapeutic substances into the bloodstream. We developed modified RBCs to serve as carriers for systemic delivery of a wide array of payloads. These RBCs contain modified proteins on their plasma membrane, which can be labeled in a sortase-catalyzed reaction under native conditions without inflicting damage to the target membrane or cell. Sortase accommodates a wide range of natural and synthetic payloads that allow modification of RBCs with substituents that cannot be encoded genetically. As proof of principle, we demonstrate site-specific conjugation of biotin to in vitro-differentiated mouse erythroblasts as well as to mature mouse RBCs. Thus modified, RBCs remain in the bloodstream for up to 28 d. A single domain antibody attached enzymatically to RBCs enables them to bind specifically to target cells that express the antibody target. We extend these experiments to human RBCs and demonstrate efficient sortase-mediated labeling of in vitro-differentiated human reticulocytes.

IAP inhibitors enhance co-stimulation to promote tumor immunity

The inhibitor of apoptosis proteins (IAPs) have recently been shown to modulate nuclear factor κB (NF-κB) signaling downstream of tumor necrosis factor (TNF) family receptors, positioning them as essential survival factors in several cancer cell lines, as indicated by the cytotoxic activity of several novel small molecule IAP antagonists. In addition to roles in cancer, increasing evidence suggests that IAPs have an important function in immunity; however, the impact of IAP antagonists on antitumor immune responses is unknown. In this study, we examine the consequences of IAP antagonism on T cell function in vitro and in the context of a tumor vaccine in vivo. We find that IAP antagonists can augment human and mouse T cell responses to physiologically relevant stimuli. The activity of IAP antagonists depends on the activation of NF-κB2 signaling, a mechanism paralleling that responsible for the cytotoxic activity in cancer cells. We further show that IAP antagonists can augment both prophylactic and therapeutic antitumor vaccines in vivo. These findings indicate an important role for the IAPs in regulating T cell–dependent responses and suggest that targeting IAPs using small molecule antagonists may be a strategy for developing novel immunomodulating therapies against cancer.

Primary deficiency of microsomal triglyceride transfer protein in human abetalipoproteinemia is associated with loss of CD1 function

Abetalipoproteinemia (ABL) is a rare Mendelian disorder of lipid metabolism due to genetic deficiency in microsomal triglyceride transfer protein (MTP). It is associated with defects in MTP-mediated lipid transfer onto apolipoprotein B (APOB) and impaired secretion of APOB-containing lipoproteins. Recently, MTP was shown to regulate the CD1 family of lipid antigen-presenting molecules, but little is known about immune function in ABL patients. Here, we have shown that ABL is characterized by immune defects affecting presentation of self and microbial lipid antigens by group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 molecules. In dendritic cells isolated from ABL patients, MTP deficiency was associated with increased proteasomal degradation of group 1 CD1 molecules. Although CD1d escaped degradation, it was unable to load antigens and exhibited functional defects similar to those affecting the group 1 CD1 molecules. The reduction in CD1 function resulted in impaired activation of CD1-restricted T and invariant natural killer T (iNKT) cells and reduced numbers and phenotypic alterations of iNKT cells consistent with central and peripheral CD1 defects in vivo. These data highlight MTP as a unique regulator of human metabolic and immune pathways and reveal that ABL is not only a disorder of lipid metabolism but also an immune disease involving CD1.