Regulated control of gene therapies by drug-induced splicing.

So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote Xon, does not require the co-expression of any regulatory proteins. Using Xon, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of Xon to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.

Regulation of immune cells by local-tissue oxygen tension: HIF1 alpha and adenosine receptors.

Immune cells are often exposed to low oxygen tensions, which markedly affect cellular metabolism. We describe how activated T cells adapt to the changing energy supplies in hypoxic areas of inflamed tissues by using hypoxia-inducible factor 1 (HIF1) to switch to glycolysis as the main source of energy and by signalling through extracellular-adenosine receptors. This hypoxic regulation might alter the balance between T helper 1 cells and T helper 2 cells and might alter the activities of cells of the innate immune system, thereby qualitatively and quantitatively affecting immune responses. This regulatory mechanism should be taken into account in the design and interpretation of in vitro and in vivo studies of immune-cell effector functions.

Genetic deletion of the HIF-1α isoform I.1 in T cells enhances antibacterial immunity and improves survival in a murine peritonitis model.

Hypoxia-adenosinergic suppression and redirection of the immune response has been implicated in the regulation of antipathogen and antitumor immunity, with hypoxia-inducible factor 1α (HIF-1α) playing a major role. In this study, we investigated the role of isoform I.1, a quantitatively minor alternative isoform of HIF-1α, in antibacterial immunity and sepsis survival. By using the cecal ligation and puncture model of bacterial peritonitis, we studied the function of I.1 isoform in T cells using mice with total I.1 isoform deficiency and mice with T-cell-targeted I.1 knockdown. We found that genetic deletion of the I.1 isoform resulted in enhanced resistance to septic lethality, significantly reduced bacterial load in peripheral blood, increased M1 macrophage polarization, augmented levels of proinflammatory cytokines in serum, and significantly decreased levels of the anti-inflammatory cytokine IL-10. Our data suggest a previously unrecognized immunosuppressive role for the I.1 isoform in T cells during bacterial sepsis. We interpret these data as indicative that the activation-inducible isoform I.1 hinders the contribution of T cells to the antibacterial response by affecting M1/M2 macrophage polarization and microbicidal function.

A2B adenosine receptor blockade enhances macrophage-mediated bacterial phagocytosis and improves polymicrobial sepsis survival in mice.

Antimicrobial treatment strategies must improve to reduce the high mortality rates in septic patients. In noninfectious models of acute inflammation, activation of A2B adenosine receptors (A2BR) in extracellular adenosine-rich microenvironments causes immunosuppression. We examined A2BR in antibacterial responses in the cecal ligation and puncture (CLP) model of sepsis. Antagonism of A2BR significantly increased survival, enhanced bacterial phagocytosis, and decreased IL-6 and MIP-2 (a CXC chemokine) levels after CLP in outbred (ICR/CD-1) mice. During the CLP-induced septic response in A2BR knockout mice, hemodynamic parameters were improved compared with wild-type mice in addition to better survival and decreased plasma IL-6 levels. A2BR deficiency resulted in a dramatic 4-log reduction in peritoneal bacteria. The mechanism of these improvements was due to enhanced macrophage phagocytic activity without augmenting neutrophil phagocytosis of bacteria. Following ex vivo LPS stimulation, septic macrophages from A2BR knockout mice had increased IL-6 and TNF-α secretion compared with wild-type mice. A therapeutic intervention with A2BR blockade was studied by using a plasma biomarker to direct therapy to those mice predicted to die. Pharmacological blockade of A2BR even 32 h after the onset of sepsis increased survival by 65% in those mice predicted to die. Thus, even the late treatment with an A2BR antagonist significantly improved survival of mice (ICR/CD-1) that were otherwise determined to die according to plasma IL-6 levels. Our findings of enhanced bacterial clearance and host survival suggest that antagonism of A2BRs offers a therapeutic target to improve macrophage function in a late treatment protocol that improves sepsis survival.

An orally available, brain penetrant, small molecule lowers huntingtin levels by enhancing pseudoexon inclusion.

Huntington's Disease (HD) is a progressive neurodegenerative disorder caused by CAG trinucleotide repeat expansions in exon 1 of the huntingtin (HTT) gene. The mutant HTT (mHTT) protein causes neuronal dysfunction, causing progressive motor, cognitive and behavioral abnormalities. Current treatments for HD only alleviate symptoms, but cerebral spinal fluid (CSF) or central nervous system (CNS) delivery of antisense oligonucleotides (ASOs) or virus vectors expressing RNA-induced silencing (RNAi) moieties designed to induce mHTT mRNA lowering have progressed to clinical trials. Here, we present an alternative disease modifying therapy the orally available, brain penetrant small molecule branaplam. By promoting inclusion of a pseudoexon in the primary transcript, branaplam lowers mHTT protein levels in HD patient cells, in an HD mouse model and in blood samples from Spinal Muscular Atrophy (SMA) Type I patients dosed orally for SMA (NCT02268552). Our work paves the way for evaluating branaplam's utility as an  HD therapy, leveraging small molecule splicing modulators to reduce expression of dominant disease genes by driving pseudoexon inclusion.

Hypoxia-adenosinergic immunosuppression: tumor protection by T regulatory cells and cancerous tissue hypoxia.

Cancerous tissue protection from tumor-recognizing CD8(+) and CD4(+) T cells (antitumor T cells) limits the therapeutic potential of immunotherapies. We propose that tumor protection is to a large extent due to (a) inhibition of antitumor T cells by hypoxia-driven accumulation of extracellular adenosine in local tumor microenvironment and due to (b) T regulatory cell-produced extracellular adenosine. The adenosine triggers the immunosuppressive signaling via intracellular cyclic AMP-elevating A2A adenosine receptors (A2AR) on antitumor T cells. In addition, the activated antitumor T cells in hypoxic tumor microenvironment could be inhibited by elevated levels of immunosuppressive hypoxia-inducible factor-1alpha. Complete rejection or tumor growth retardation was observed when A2AR has been genetically eliminated or antagonized with synthetic drug or with natural A2AR antagonist 1,3,7-trimethylxanthine (caffeine). The promising strategy may be in combining the anti-hypoxia-adenosinergic treatment that prevents inhibition of antitumor T cells by tumor-produced and T regulatory cell-produced adenosine with targeting of other negative regulators, such as CTL antigen-4 blockade. Observations of tumor rejection in mice and massive prospective epidemiologic studies support the feasibility of anti-hypoxia-adenosinergic combined immunotherapy.

Preferential expression of the novel alternative isoform I.3 of hypoxia-inducible factor 1alpha in activated human T lymphocytes.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is critical not only in the regulation of oxygen homeostasis but also in the regulation of innate and adaptive immune systems. We previously reported that T-cell receptor-mediated activation of T cells in mice leads to the expression of an alternative isoform of HIF-1alpha that inhibits activated T cells in a delayed negative feed-back manner. In this paper, we describe a novel mRNA isoform of human HIF-1alpha that is upregulated in peripheral T lymphocytes after T-cell receptor stimulation. This activation- inducible isoform is expressed using the alternative first exon I.3, and it encodes a protein that is 24 amino acids longer than the ubiquitous HIF-1alpha isoform. This mRNA isoform I.3 of HIF-1alpha is expressed in a tissue-specific manner with the highest expression found in peripheral blood leukocytes and the thymus.

TSGA10 prevents nuclear localization of the hypoxia-inducible factor (HIF)-1alpha.

The hypoxia-inducible factor (HIF)-1 is a transcriptional regulator of genes involved in oxygen homeostasis. We previously described testis-specific isoforms of HIF-1alpha (mHIF-1alphaI.1 and hHIF-1alphaTe). Using mHIF-1alpha exon I.1 knock-out mice we confirmed the specific expression of mHIF-1alphaI.1 in the sperm tail. A protein-protein interaction between HIF-1alpha and the testis specific gene antigen 10 (TSGA10) was identified by yeast two-hybrid screening. TSGA10 is expressed in testis but also in other organs and malignant tissues. Immunofluorescence analysis indicated that the C-terminal part of TSGA10 accumulates in the midpiece of spermatozoa, where it co-localizes with HIF-1alpha. HIF-1alpha nuclear localization and HIF-1 transcriptional activity were significantly affected by overexpressed TSGA10.

Immunological mechanisms of the antitumor effects of supplemental oxygenation.

Antitumor T cells either avoid or are inhibited in hypoxic and extracellular adenosine-rich tumor microenvironments (TMEs) by A2A adenosine receptors. This may limit further advances in cancer immunotherapy. There is a need for readily available and safe treatments that weaken the hypoxia-A2-adenosinergic immunosuppression in the TME. Recently, we reported that respiratory hyperoxia decreases intratumoral hypoxia and concentrations of extracellular adenosine. We show that it also reverses the hypoxia-adenosinergic immunosuppression in the TME. This, in turn, stimulates (i) enhanced intratumoral infiltration and reduced inhibition of endogenously developed or adoptively transfered tumor-reactive CD8 T cells, (ii) increased proinflammatory cytokines and decreased immunosuppressive molecules, such as transforming growth factor-ß (TGF-ß), (iii) weakened immunosuppression by regulatory T cells, and (iv) improved lung tumor regression and long-term survival in mice. Respiratory hyperoxia also promoted the regression of spontaneous metastasis from orthotopically grown breast tumors. These effects are entirely T cell- and natural killer cell-dependent, thereby justifying the testing of supplemental oxygen as an immunological coadjuvant to combine with existing immunotherapies for cancer.

Targeting hypoxia--A(2A) adenosine receptor-mediated mechanisms of tissue protection.

Despite inflammation having beneficial effects, the action of toxic proinflammatory molecules can result in excessive tissue damage that subsequently contributes to the pathogenesis of many major diseases. The development of novel drugs and therapeutic strategies for the treatment of inflammation requires an improved understanding of the molecular mechanisms that terminate inflammation. The physiological hypothesis proposes that excessive levels of inflammatory tissue damage result in local hypoxia and accumulation of extracellular adenosine. The A(2A) adenosine receptor and hypoxia-inducible factor play important roles in the attenuation of proinflammatory processes in a delayed, negative-feedback manner and thus protect organs from excessive damage. Targeting individual stages of the hypoxia-A(2A) receptor signaling pathway represents an attractive strategy for the modulation of inflammation.

Analysis of A2a receptor-deficient mice reveals no significant compensatory increases in the expression of A2b, A1, and A3 adenosine receptors in lymphoid organs.

Although recent genetic and pharmacologic in vivo studies of acute inflammation models in mice demonstrated that the cyclic AMP-elevating A2a receptor plays a non-redundant role in protection from excessive acute inflammatory tissue damage and in the down-regulation of proinflammatory cytokine production, it remained to be established whether genetic deficiency of the A2a receptor is accompanied by a compensatory up-regulation of the cAMP-elevating A2b receptor and/or other adenosine receptors. Here, we show that most of the cAMP response to adenosine is abolished in lymphoid tissues of A2a receptor-deficient mice, although some response remains in splenocytes. No significant changes were observed in A2b, A1, and A3 mRNA levels in the thymus or lymph nodes of A2a receptor-deficient mice, but small increases in mRNA expression of these receptors were detected in the spleen. These data suggest that regulation of the expression of A2b, A1, and A3 receptors is not affected significantly by the absence of A2a receptors and may provide further explanation of earlier in vivo observations of increased tissue damage and of longer persistence of proinflammatory cytokines in animals with inactivated A2a receptors.

Novel RNA interference-based therapies for sepsis.

INTRODUCTION: Sepsis is an extremely fast-paced disease, initiated by an infection that can progress to multiple organ dysfunction and death. The complexity associated with sepsis makes the therapies difficult to develop. Moreover, the 'one-fits-all' kind of therapy is far from being realistic. AREAS COVERED: This review provides a conspectus of the current results of sepsis therapies and their benefits, focusing on the development of small interfering RNA (siRNA) therapeutics for targeting immune cells and sepsis pathways. EXPERT OPINION: The question, 'When will an effective therapy for sepsis be available for patients?' remains unanswered. New RNA interference-mediated therapies are emerging as novel approaches for the treatment of sepsis by downregulating key inflammatory cytokine expression. Strategies that exploit multimodal gene silencing using siRNA and targeted delivery systems are discussed in this review. Some of these strategies have shown positive results in preclinical model of sepsis.

Hostile, hypoxia-A2-adenosinergic tumor biology as the next barrier to overcome for tumor immunologists.

Hypoxia-driven, A2A adenosine receptor (A2AR)-mediated (hypoxia-A2-adenosinergic), T-cell-autonomous immunosuppression was first recognized as critical and nonredundant in protecting normal tissues from inflammatory damage and autoimmunity. However, this immunosuppressive mechanism can be highjacked by bacteria and tumors to provide misguided protection for pathogens and cancerous tissues. Inhibitors of the hypoxia-A2-adenosinergic pathway represent a conceptually novel type of immunologic coadjuvants that could be combined with cancer vaccines, adoptive cell transfer, and/or blockade of negative immunologic regulators to further prolong patient survival and to minimize treatment-related side effects. In support of this approach are preclinical studies and findings that some human cancers are resistant to chemotherapies and immunotherapies due to the tumor-generated extracellular adenosine and A2AR on antitumor T and natural killer (NK) cells. Among the coadjuvants are (i) antagonists of A2AR, (ii) extracellular adenosine-degrading drugs, (iii) inhibitors of adenosine generation by CD39/CD73 ectoenzymes, and (iv) inhibitors of hypoxia-HIF-1α signaling. Combining these coadjuvants with CTLA-4 and/or PD-1 blockade is expected to have additive or even synergistic effects of targeting two different antitumor protective mechanisms. It is expected that even after multicombinatorial blockade of negative immunologic regulators, the antitumor T and NK cells would still be vulnerable to inhibition by hypoxia and A2AR. Yet to be tested is the potential capacity of coadjuvants to minimize the side effects of CTLA-4 and/or PD-1 blockade by decreasing the dose of blocking antibodies or by eliminating the need for dual blockade.

Systemic oxygenation weakens the hypoxia and hypoxia inducible factor 1α-dependent and extracellular adenosine-mediated tumor protection.

UNLABELLED: Intratumoral hypoxia and hypoxia inducible factor-1α (HIF-1-α)-dependent CD39/CD73 ectoenzymes may govern the accumulation of tumor-protecting extracellular adenosine and signaling through A2A adenosine receptors (A2AR) in tumor microenvironments (TME). Here, we explored the conceptually novel motivation to use supplemental oxygen as a treatment to inhibit the hypoxia/HIF-1α-CD39/CD73-driven accumulation of extracellular adenosine in the TME in order to weaken the tumor protection. We report that hyperoxic breathing (60 % O2) decreased the TME hypoxia, as well as levels of HIF-1α and downstream target proteins of HIF-1α in the TME according to proteomic studies in mice. Importantly, oxygenation also downregulated the expression of adenosine-generating ectoenzymes and significantly lowered levels of tumor-protecting extracellular adenosine in the TME. Using supplemental oxygen as a tool in studies of the TME, we also identified FHL-1 as a potentially useful marker for the conversion of hypoxic into normoxic TME. Hyperoxic breathing resulted in the upregulation of antigen-presenting MHC class I molecules on tumor cells and in the better recognition and increased susceptibility to killing by tumor-reactive cytotoxic T cells. Therapeutic breathing of 60 % oxygen resulted in the significant inhibition of growth of established B16.F10 melanoma tumors and prolonged survival of mice. Taken together, the data presented here provide proof-of principle for the therapeutic potential of systemic oxygenation to convert the hypoxic, adenosine-rich and tumor-protecting TME into a normoxic and extracellular adenosine-poor TME that, in turn, may facilitate tumor regression. We propose to explore the combination of supplemental oxygen with existing immunotherapies of cancer. KEY MESSAGES: Oxygenation decreases levels of tumor protecting hypoxia. Oxygenation decreases levels of tumor protecting extracellular adenosine. Oxygenation decreases expression of HIF-1alpha dependent tumor-protecting proteins. Oxygenation increases MHC class I expression and enables tumor regression.

In vivo hypoxic preconditioning protects from warm liver ischemia-reperfusion injury through the adenosine A2B receptor.

BACKGROUND: Liver ischemia-reperfusion injury (IRI) is a known risk factor for the postoperative outcome of patients undergoing liver surgery/transplantation. Attempts to protect from organ damage require multidisciplinary strategies and are of emerging interest in view of patients with higher age and American Society of Anesthesiology status. Ischemic preconditioning has been successfully applied to prevent from IRI during liver resection/transplantation. Because even short periods of ischemia during preconditioning inevitably lead to hypoxia and formation of anti-inflammatory/cytoprotective acting adenosine, we reasoned that short nonischemic hypoxia also protects against hepatic IRI. METHODS: Mice underwent hypoxic preconditioning (HPC) by breathing 10% oxygen for 10 min followed by 10 min of 21% oxygen before left liver lobe ischemia (45 min) and reperfusion (4 hr). The interactions of hypoxia→adenosine→adenosine receptors were tested by pharmacologic antagonism at adenosine receptor (AR) sites in wild-type mice and in mice with genetic deletions at the A1, A2A, A2B, and A3 ARs. Hepatocellular damage, inflammation, and metabolic effects were quantified by enzyme activities, cytokines, liver myeloperoxidase, blood adenosine, and tissue AMP, respectively. RESULTS: Hepatoprotection by HPC was significant in wild-type and A1, A2A, and A3 AR knockout mice as quantified by lower alanine aminotransferase serum activities, cytokine levels, histologic damage scores, tissue myeloperoxidase concentrations, and preserved AMP concentrations. Protection by HPC was blunted in mice pretreated with the A2B AR antagonist MRS1754 or in A2B AR knockout mice. CONCLUSIONS: Because liver protective effects of HPC are negated when the A2B receptor is nonfunctional, the hypoxia→adenosine→A2B receptor pathway plays a critical role in the prevention of warm IRI in vivo. Hypoxic activation of this pathway warrants use of selective A2B AR agonists or even intermittent hypoxia (e.g., in deceased organ donors) to protect from liver IRI.

Equilibrative nucleoside transporter 1 (ENT1) regulates postischemic blood flow during acute kidney injury in mice.

A complex biologic network regulates kidney perfusion under physiologic conditions. This system is profoundly perturbed following renal ischemia, a leading cause of acute kidney injury (AKI) - a life-threatening condition that frequently complicates the care of hospitalized patients. Therapeutic approaches to prevent and treat AKI are extremely limited. Better understanding of the molecular pathways promoting postischemic reflow could provide new candidate targets for AKI therapeutics. Due to its role in adapting tissues to hypoxia, we hypothesized that extracellular adenosine has a regulatory function in the postischemic control of renal perfusion. Consistent with the notion that equilibrative nucleoside transporters (ENTs) terminate adenosine signaling, we observed that pharmacologic ENT inhibition in mice elevated renal adenosine levels and dampened AKI. Deletion of the ENTs resulted in selective protection in Ent1-/- mice. Comprehensive examination of adenosine receptor-knockout mice exposed to AKI demonstrated that renal protection by ENT inhibitors involves the A2B adenosine receptor. Indeed, crosstalk between renal Ent1 and Adora2b expressed on vascular endothelia effectively prevented a postischemic no-reflow phenomenon. These studies identify ENT1 and adenosine receptors as key to the process of reestablishing renal perfusion following ischemic AKI. If translatable from mice to humans, these data have important therapeutic implications.

Toll-like receptor 3 regulates angiogenesis and apoptosis in prostate cancer cell lines through hypoxia-inducible factor 1 alpha.

Toll-like receptors (TLRs) recognize microbial/viral-derived components that trigger innate immune response and conflicting data implicate TLR agonists in cancer, either as protumor or antitumor agents. We previously demonstrated that TLR3 activation mediated by its agonist poly(I:C) induces antitumor signaling, leading to apoptosis of prostate cancer cells LNCaP and PC3 with much more efficiency in the former than in the second more aggressive line. The transcription factor hypoxia-inducible factor 1 (HIF-1) regulates several cellular processes, including apoptosis, in response to hypoxia and to other stimuli also in normoxic conditions. Here we describe a novel protumor machinery triggered by TLR3 activation in PC3 cells consisting of increased expression of the specific I.3 isoform of HIF-1 alpha and nuclear accumulation of HIF-1 complex in normoxia, resulting in reduced apoptosis and in secretion of functional vascular endothelial growth factor (VEGF). Moreover, we report that, in the less aggressive LNCaP cells, TLR3 activation fails to induce nuclear accumulation of HIF-1 alpha. However, the transfection of I.3 isoform of hif-1 alpha in LNCaP cells allows poly(I:C)-induced HIF-1 activation, resulting in apoptosis protection and VEGF secretion. Altogether, our findings demonstrate that differences in the basal level of HIF-1 alpha expression in different prostate cancer cell lines underlie their differential response to TLR3 activation, suggesting a correlation between different stages of malignancy, hypoxic gene expression, and beneficial responsiveness to TLR agonists.

The antihypoxia-adenosinergic pathogenesis as a result of collateral damage by overactive immune cells.

Here, we attract attention to the possibility of iatrogenic exacerbation of immune-mediated tissue damage as a result of the unintended weakening of the tissue-protecting, hypoxia-adenosinergic pathway. These immunosuppressive, anti-inflammatory pathways play a critical and nonredundant role in the protection of normal tissues from collateral damage during an inflammatory response. We believe that it is the tissue hypoxia associated with inflammatory damage that leads to local inhibition of overactive immune cells by activating A2AR and A2BR and stabilizing HIF-1alpha. We show in an animal model of acute lung injury that oxygenation (i.e., inspiring supplemental oxygen) reverses tissue hypoxia and exacerbates ongoing inflammatory lung tissue damage. However, little has been done to carefully investigate and prevent this in a clinical setting. Similarly, the consumption of caffeine antagonizes A2ARs, resulting in exacerbation of ongoing acute inflammation. It is suggested that although the elimination of hypoxia-adenosinergic immunosuppression is desirable to improve vaccines, it is important to take into account the unintentional effects of supplemental oxygen and caffeine, which may increase collateral, inflammatory tissue damage.