Frequent aneuploidy in primary human T cells after CRISPR-Cas9 cleavage.

Multiple clinical trials of allogeneic T cell therapy use site-specific nucleases to disrupt T cell receptor (TCR) and other genes1-6. In this study, using single-cell RNA sequencing, we investigated genome editing outcomes in primary human T cells transfected with CRISPR-Cas9 and guide RNAs targeting genes for TCR chains and programmed cell death protein 1. Four days after transfection, we found a loss of chromosome 14, harboring the TCRα locus, in up to 9% of the cells and a chromosome 14 gain in up to 1.4% of the cells. Chromosome 7, harboring the TCRß locus, was truncated in 9.9% of the cells. Aberrations were validated using fluorescence in situ hybridization and digital droplet PCR. Aneuploidy was associated with reduced proliferation, induced p53 activation and cell death. However, at 11 days after transfection, 0.9% of T cells still had a chromosome 14 loss. Aneuploidy and chromosomal truncations are, thus, frequent outcomes of CRISPR-Cas9 cleavage that should be monitored and minimized in clinical protocols.

In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice.

Transplantation of B cells engineered ex vivo to secrete broadly neutralizing antibodies (bNAbs) has shown efficacy in disease models. However, clinical translation of this approach would require specialized medical centers, technically demanding protocols and major histocompatibility complex compatibility of donor cells and recipients. Here we report in vivo B cell engineering using two adeno-associated viral vectors, with one coding for Staphylococcus aureus Cas9 (saCas9) and the other for 3BNC117, an anti-HIV bNAb. After intravenously injecting the vectors into mice, we observe successful editing of B cells leading to memory retention and bNAb secretion at neutralizing titers of up to 6.8 µg ml-1. We observed minimal clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 off-target cleavage as detected by unbiased CHANGE-sequencing analysis, whereas on-target cleavage in undesired tissues is reduced by expressing saCas9 from a B cell-specific promoter. In vivo B cell engineering to express therapeutic antibodies is a safe, potent and scalable method, which may be applicable not only to infectious diseases but also in the treatment of noncommunicable conditions, such as cancer and autoimmune disease.

Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion.

HIV viremia can be controlled by chronic antiretroviral therapy. As a potentially single-shot alternative, B cells engineered by CRISPR/Cas9 to express anti-HIV broadly neutralizing antibodies (bNAbs) are capable of secreting high antibody titers. Here, we show that, upon immunization of mice, adoptively transferred engineered B cells home to germinal centers (GC) where they predominate over the endogenous response and differentiate into memory and plasma cells while undergoing class switch recombination (CSR). Immunization with a high affinity antigen increases accumulation in GCs and CSR rates. Boost immunization increases the rate of engineered B cells in GCs and antibody secretion, indicating memory retention. Finally, antibody sequences of engineered B cells in the spleen show patterns of clonal selection. Therefore, B cells can be engineered into what could be a living and evolving drug.

Insulin-induced translocation of IR to the nucleus in insulin responsive cells requires a nuclear translocation sequence.

Insulin binding to its cell surface receptor (IR) activates a cascade of events leading to its biological effects. The Insulin-IR complex is rapidly internalized and then is either recycled back to the plasma membrane or sent to lysosomes for degradation. Although most of the receptor is recycled or degraded, a small amount may escape this pathway and migrate to the nucleus of the cell where it might be important in promulgation of receptor signals. In this study we explored the mechanism by which insulin induces IR translocation to the cell nucleus. Experiments were performed cultured L6 myoblasts, AML liver cells and 3T3-L1 adipocytes. Insulin treatment induced a rapid increase in nuclear IR protein levels within 2 to 5 min. Treatment with WGA, an inhibitor of nuclear import, reduced insulin-induced increases nuclear IR protein; IR was, however, translocated to a perinuclear location. Bioinformatics tools predicted a potential nuclear localization sequence (NLS) on IR. Immunofluorescence staining showed that a point mutation on the predicted NLS blocked insulin-induced IR nuclear translocation. In addition, blockade of nuclear IR activation in isolated nuclei by an IR blocking antibody abrogated insulin-induced increases in IR tyrosine phosphorylation and nuclear PKCδ levels. Furthermore, over expression of mutated IR reduced insulin-induced glucose uptake and PKB phosphorylation. When added to isolated nuclei, insulin induced IR phosphorylation but had no effect on nuclear IR protein levels. These results raise questions regarding the possible role of nuclear IR in IR signaling and insulin resistance.

Protein kinase Cδ but not PKCα is involved in insulin-induced glucose metabolism in hepatocytes.

The liver is a major insulin-responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin-induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin-induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin-induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML-12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin-induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin-induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin-induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin-induced glucose uptake and glycogenesis in hepatocytes.

Insulin stimulation of PKCδ triggers its rapid degradation via the ubiquitin-proteasome pathway.

Insulin rapidly upregulates protein levels of PKCδ in classical insulin target tissues skeletal muscle and liver. Insulin induces both a rapid increase in de novo synthesis of PKCδ protein. In this study we examined the possibility that insulin may also inhibit degradation of PKCδ. Experiments were performed on L6 skeletal muscle myoblasts or myotubes in culture. Phorbol ester (PMA)- and insulin-induced degradation of PKCδ were abrogated by proteasome inhibition. Both PMA and insulin induced ubiquitination of PKCδ, but not of that PKCα or PKCε and increased proteasome activity within 5 min. We examined the role of tyrosine phosphorylation of PKCδ in targeting PKCδ for degradation by the ubiquitin-proteasome pathway. Transfection of cells with PKCδY(311)F, which is not phosphorylated, resulted in abolition of insulin-induced ubiquitination of PKCδ and increase in proteasome activity. We conclude that insulin induces degradation of PKCδ via the ubiquitin-proteasome system, and that this effect requires phosphorylation on specific tyrosine residues for targeting PKCδ for degradation by the ubiquitin-proteasome pathway. These studies provide additional evidence for unique effects of insulin on regulation of PKCδ protein levels.

The regulatory domain of protein kinase C delta positively regulates insulin receptor signaling.

Protein kinase C delta (PKCdelta) is induced by insulin to rapidly associate with insulin receptor (IR) and upregulates insulin signaling. We utilized specific JM and CT receptor domains and chimeras of PKCalpha and PKCdelta regulatory and catalytic domains to elucidate which components of PKCdelta are responsible for positive regulatory effects of PKCdelta on IR signaling. Studies were performed on L6 and L8 skeletal muscle myoblasts and myotubes. PKCdelta was preferentially bound to the JM domain of IR, and insulin stimulation increased this binding. Both PKCdelta/alpha and PKCalpha/delta chimeras (regulatory/catalytic) were bound preferentially to the JM but not to the CT domain of IR. Although IR-PKCdelta binding was higher in cells expressing either the PKCdelta/alpha or PKCalpha/delta chimera than in control cells, upregulation of IR signaling was observed only in PKCdelta/alpha cells. Thus, in response to insulin increases in tyrosine phosphorylation of IR and insulin receptor substrate-1, downstream signaling to protein kinase B and glycogen synthase kinase 3 (GSK3) and glucose uptake were greater in cells overexpressing PKCdelta/alpha and the PKCdelta/delta domains than in cells expressing the PKCalpha/delta domains. Basal binding of Src to PKCdelta was higher in both PKCdelta/alpha- and PKCalpha/delta-expressing cells compared to control. Binding of Src to IR was decreased in PKCalpha/delta cells but remained elevated in the PKCdelta/alpha cells in response to insulin. Finally, insulin increased Src activity in PKCdelta/alpha-expressing cells but decreased it in PKCalpha/delta-expressing cells. Thus, the regulatory domain of PKCdelta via interaction with Src appears to determine the role of PKCdelta as a positive regulator of IR signaling in skeletal muscle.

Insulin increases nuclear protein kinase Cdelta in L6 skeletal muscle cells.

Protein kinase C (PKC) isoforms are involved in the transduction of a number of signals important for the regulation of cell growth, differentiation, apoptosis, and other cellular functions. PKC proteins reside in the cytoplasm in an inactive state translocate to various membranes to become fully activated in the presence of specific cofactors. Recent evidence indicates that PKC isoforms have an important role in the nucleus. We recently showed that insulin rapidly increases PKCdelta RNA and protein. In this study we initially found that insulin induces an increase in PKCdelta protein in the nuclear fraction. We therefore attempted to elucidate the mechanism of the insulin-induced increase in nuclear PKCdelta. Studies were performed on L6 skeletal myoblasts and myotubes. The increase in nuclear PKCdelta appeared to be unique to insulin because it was not induced by other growth factors or rosiglitazone. Inhibition of transcription or translation blocked the insulin-induced increase in nuclear PKCdelta, whereas inhibition of protein import did not. Inhibition of protein export from the nucleus reduced the insulin-induced increase in PKCdelta in the cytoplasm and increased it in the nucleus. The increase in nuclear PKCdelta induced by insulin was reduced but not abrogated by treatment of isolated nuclei by trypsin digestion. Finally, we showed that insulin induced incorporation of (35)S-methionine into nuclear PKCdelta protein; this effect was not blocked by inhibition of nuclear import. Thus, these results suggest that insulin may induce nuclear-associated, or possibly nuclear, translation of PKCdelta protein.

Involvement of PKCalpha in insulin-induced PKCdelta expression: Importance of SP-1 and NFkappaB transcription factors.

Protein kinase C delta (PKCdelta) is a key molecule in insulin signaling essential for insulin-induced glucose transport in skeletal muscle. Recent studies in our laboratory have shown that insulin rapidly stimulates PKCdelta activity and increases PKCdelta protein and RNA levels, and that the SP-1 transcription factor is involved in insulin-induced transcription of the PKCdelta gene. Activation of SP-1 involves serine phosphorylation and translocation to the nucleus. In this study we examined the possibility that PKCalpha might be involved in serine phosphorylation and activation of SP-1. We found that insulin rapidly phosphorylates and translocates SP-1. In the cytoplasm, SP-1 was constitutively associated with PKCalpha, and insulin stimulation caused these proteins to dissociate. In contrast, in the nucleus insulin induced an increase in association between PKCalpha and SP-1. PKCalpha inhibition blocked insulin-induced serine phosphorylation of SP-1 and its association with PKCalpha in the nucleus. Inhibition of PKCalpha also reduced the insulin-induced increase in PKCdelta RNA and protein in the cytoplasmic and nuclear fractions. We also attempted to determine if another transcription factor might be involved in regulation of PKCdelta expression. We earlier showed that insulin did not affect nuclear NFkappaB levels. Inhibition of NFkappaB, however, increased insulin-induced increase in PKCdelta RNA and protein in the cytoplasmic and nuclear fractions. Surprisingly, this inhibition reduced the insulin-induced increase in cytoplasmic and nuclear PKCalpha RNA and protein. Inhibition of PKCdelta reduced IkappaBalpha phosphorylation as well as NFkappaB activation. Thus, PKCalpha regulates insulin-induced PKCdelta expression levels and this regulation involves activation of SP-1 and NFkappaB.

Activation of the nuclear transcription factor SP-1 by insulin rapidly increases the expression of protein kinase C delta in skeletal muscle.

SP-1, a ubiquitous transcription factor involved in regulation of target genes participating in specific signaling pathways, is utilized by insulin for induction of gene transcription. Transcriptional activation generally occurs only after several (14-24) hours. A major element rapidly activated by insulin in skeletal muscle is PKCdelta, which plays a positive regulatory role in insulin signaling. We recently reported that insulin stimulation of skeletal muscle increases PKCdelta RNA expression and PKCdelta protein levels within 5 min. These effects were blocked by inhibitors of either translation or transcription. In this study, we investigated the possibility that SP-1 may participate in this unusually rapid effect. Studies were performed on myoblasts and myotubes of the L6 skeletal muscle cell line. Insulin rapidly increased SP-1 levels and stimulated SP-1 phosphorylation in the nuclear fraction of L6 myotubes. The increase in nuclear SP-1 was blocked by inhibition of nuclear import. Inhibition of SP-1, either pharmacologically or by suppression of SP-1 by RNAi, nearly completely abrogated insulin-induced increase in PKCdelta promoter activity. Insulin induced a rapid association of SP-1 with the PKCalpha promoter. In addition, SP-1 inhibition blocked insulin-induced increases in both PKCdelta RNA expression and PKCdelta protein levels. We conclude that insulin rapidly stimulates SP-1, which mediates the ability of this hormone to induce the rapid transcription of a major target gene utilized in the insulin signaling cascade.

Insulin rapidly upregulates protein kinase Cdelta gene expression in skeletal muscle.

Recent studies in our laboratories have shown that Protein Kinase C delta (PKCdelta) is essential for insulin-induced glucose transport in skeletal muscle, and that insulin rapidly stimulates PKCdelta activity skeletal muscle. The purpose of this study was to examine mechanisms of regulation of PKCdelta protein availability. Studies were done on several models of mammalian skeletal muscle and utilized whole cell lysates of differentiated myotubes. PKCdelta protein levels were determined by Western blotting techniques, and PKCdelta RNA levels were determined by Northern blotting, RT-PCR and Real-Time RT-PCR. Insulin stimulation increased PKCdelta protein levels in whole cell lysates. This effect was not due to an inhibition by insulin of the rate of PKCdelta protein degradation. Insulin also increased 35S-methionine incorporation into PKCdelta within 5-15 min. Pretreatment of cells with transcription or translation inhibitors abrogated the insulin-induced increase in PKCdelta protein levels. We also found that insulin rapidly increased the level of PKCdelta RNA, an effect abolished by inhibitors of transcription. The insulin-induced increase in PKCdelta expression was not reduced by inhibition of either PI3 Kinase or MAP kinase, indicating that these signaling mechanisms are not involved, consistent with insulin activation of PKCdelta. Studies on cells transfected with the PKCdelta promoter demonstrate that insulin activated the promoter within 5 min. This study indicates that the expression of PKCdelta may be regulated in a rapid manner during the course of insulin action in skeletal muscle and raise the possibility that PKCdelta may be an immediate early response gene activated by insulin.