Nucleotide metabolism in cancer cells fuels a UDP-driven macrophage cross-talk, promoting immunosuppression and immunotherapy resistance.

Many individuals with cancer are resistant to immunotherapies. Here, we identify the gene encoding the pyrimidine salvage pathway enzyme cytidine deaminase (CDA) among the top upregulated metabolic genes in several immunotherapy-resistant tumors. We show that CDA in cancer cells contributes to the uridine diphosphate (UDP) pool. Extracellular UDP hijacks immunosuppressive tumor-associated macrophages (TAMs) through its receptor P2Y6. Pharmacologic or genetic inhibition of CDA in cancer cells (or P2Y6 in TAMs) disrupts TAM-mediated immunosuppression, promoting cytotoxic T cell entry and susceptibility to anti-programmed cell death protein 1 (anti-PD-1) treatment in resistant pancreatic ductal adenocarcinoma (PDAC) and melanoma models. Conversely, CDA overexpression in CDA-depleted PDACs or anti-PD-1-responsive colorectal tumors or systemic UDP administration (re)establishes resistance. In individuals with PDAC, high CDA levels in cancer cells correlate with increased TAMs, lower cytotoxic T cells and possibly anti-PD-1 resistance. In a pan-cancer single-cell atlas, CDAhigh cancer cells match with T cell cytotoxicity dysfunction and P2RY6high TAMs. Overall, we suggest CDA and P2Y6 as potential targets for cancer immunotherapy.

A Metabolic Gene Survey Pinpoints Fucosylation as a Key Pathway Underlying the Suppressive Function of Regulatory T Cells in Cancer.

Forkhead box P3 (Foxp3)-expressing regulatory T cells (Treg) are the guardians of controlled immune reactions and prevent the development of autoimmune diseases. However, in the tumor context, their increased number suppresses antitumor immune responses, indicating the importance of understanding the mechanisms behind their function and stability. Metabolic reprogramming can affect Foxp3 regulation and, therefore, Treg suppressive function and fitness. Here, we performed a metabolic CRISPR/Cas9 screen and pinpointed novel candidate positive and negative metabolic regulators of Foxp3. Among the positive regulators, we revealed that targeting the GDP-fucose transporter Slc35c1, and more broadly fucosylation (Fuco), in Tregs compromises their proliferation and suppressive function both in vitro and in vivo, leading to alteration of the tumor microenvironment and impaired tumor progression and protumoral immune responses. Pharmacologic inhibition of Fuco dampened tumor immunosuppression mostly by targeting Tregs, thus resulting in reduced tumor growth. In order to substantiate these findings in humans, tumoral Tregs from patients with colorectal cancer were clustered on the basis of the expression of Fuco-related genes. FucoLOW Tregs were found to exhibit a more immunogenic profile compared with FucoHIGH Tregs. Furthermore, an enrichment of a FucoLOW signature, mainly derived from Tregs, correlated with better prognosis and response to immune checkpoint blockade in melanoma patients. In conclusion, Slc35c1-dependent Fuco is able to regulate the suppressive function of Tregs, and measuring its expression in Tregs might pave the way towards a useful biomarker model for patients with cancer. See related Spotlight by Silveria and DuPage, p. 1570.

Breast tumors interfere with endothelial TRAIL at the premetastatic niche to promote cancer cell seeding.

Endothelial cells (ECs) grant access of disseminated cancer cells to distant organs. However, the molecular players regulating the activation of quiescent ECs at the premetastatic niche (PMN) remain elusive. Here, we find that ECs at the PMN coexpress tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its cognate death receptor 5 (DR5). Unexpectedly, endothelial TRAIL interacts intracellularly with DR5 to prevent its signaling and preserve a quiescent vascular phenotype. In absence of endothelial TRAIL, DR5 activation induces EC death and nuclear factor κB/p38-dependent EC stickiness, compromising vascular integrity and promoting myeloid cell infiltration, breast cancer cell adhesion, and metastasis. Consistently, both down-regulation of endothelial TRAIL at the PMN by proangiogenic tumor-secreted factors and the presence of the endogenous TRAIL inhibitors decoy receptor 1 (DcR1) and DcR2 favor metastasis. This study discloses an intracrine mechanism whereby TRAIL blocks DR5 signaling in quiescent endothelia, acting as gatekeeper of the vascular barrier that is corrupted by the tumor during cancer cell dissemination.

Immunoediting instructs tumor metabolic reprogramming to support immune evasion.

Immunoediting sculpts immunogenicity and thwarts host anti-tumor responses in tumor cells during tumorigenesis; however, it remains unknown whether metabolic programming of tumor cells can be guided by immunosurveillance. Here, we report that T cell-mediated immunosurveillance in early-stage tumorigenesis instructs c-Myc upregulation and metabolic reprogramming in tumor cells. This previously unexplored tumor-immune interaction is controlled by non-canonical interferon gamma (IFNγ)-STAT3 signaling and supports tumor immune evasion. Our findings uncover that immunoediting instructs deregulated bioenergetic programs in tumor cells to empower them to disarm the T cell-mediated immunosurveillance by imposing metabolic tug-of-war between tumor and infiltrating T cells and forming the suppressive tumor microenvironment.

Targeting the bicarbonate transporter SLC4A4 overcomes immunosuppression and immunotherapy resistance in pancreatic cancer.

Solid tumors are generally characterized by an acidic tumor microenvironment (TME) that favors cancer progression, therapy resistance and immune evasion. By single-cell RNA-sequencing analysis in individuals with pancreatic ductal adenocarcinoma (PDAC), we reveal solute carrier family 4 member 4 (SLC4A4) as the most abundant bicarbonate transporter, predominantly expressed by epithelial ductal cells. Functionally, SLC4A4 inhibition in PDAC cancer cells mitigates the acidosis of the TME due to bicarbonate accumulation in the extracellular space and a decrease in lactate production by cancer cells as the result of reduced glycolysis. In PDAC-bearing mice, genetic or pharmacological SLC4A4 targeting improves T cell-mediated immune response and breaches macrophage-mediated immunosuppression, thus inhibiting tumor growth and metastases. In addition, Slc4a4 targeting in combination with immune checkpoint blockade is able to overcome immunotherapy resistance and prolong survival. Overall, our data propose SLC4A4 as a therapeutic target to unleash an antitumor immune response in PDAC.

Deletion of Lactate Dehydrogenase-A Impairs Oncogene-Induced Mouse Hepatocellular Carcinoma Development.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a multistep process whereby abnormally proliferating cancer cells undergo extensive metabolic reprogramming. Metabolic alterations in hepatocarcinogenesis depend on the activation of specific oncogenes, thus partially explaining HCC heterogeneity. c-Myc oncogene overexpression, frequently observed in human HCCs, leads to a metabolic rewiring toward a Warburg phenotype and production of lactate, resulting in the acidification of the extracellular space, favoring the emergence of an immune-permissive tumor microenvironment. Here, we investigated whether Ldha genetic ablation interferes with metabolic reprogramming and HCC development in the mouse. METHODS: We characterized the metabolic reprogramming in tumors induced in C57BL/6J mice hydrodynamically cotransfected with c-Myc and h-Ras. Using the same experimental model, we investigated the effect of Ldha inhibition-gained through the inducible and hepatocyte-specific Ldha knockout-on cancer cell metabolic reprogramming, number and size of HCC lesions, and tumor microenvironment alterations. RESULTS: c-Myc/h-Ras-driven tumors display a striking glycolytic metabolism, suggesting a switch to a Warburg phenotype. The tumors also exhibited enhanced pentose phosphate pathway activity, the switch of glutamine to sustain glutathione synthesis instead of the tricarboxylic acid cycle, and the impairment of oxidative phosphorylation. In addition, Ldha abrogation significantly hampered tumor number and size together with an evident inhibition of the Warburg-like metabolic feature and a remarkable increase of CD4+ lymphocytes compared with Ldha wild-type livers. CONCLUSIONS: These results demonstrate that Ldha deletion significantly impairs mouse HCC development and suggest lactate dehydrogenase as a potential target to enhance the efficacy of the current therapeutic options.

Plexin-A4 Mediates Cytotoxic T-cell Trafficking and Exclusion in Cancer.

Cytotoxic T cell (CTL) infiltration of the tumor carries the potential to limit cancer progression, but their exclusion by the immunosuppressive tumor microenvironment hampers the efficiency of immunotherapy. Here, we show that expression of the axon guidance molecule Plexin-A4 (Plxna4) in CTLs, especially in effector/memory CD8+ T cells, is induced upon T-cell activation, sustained in the circulation, but reduced when entering the tumor bed. Therefore, we deleted Plxna4 and observed that Plxna4-deficient CTLs acquired improved homing capacity to the lymph nodes and to the tumor, as well as increased proliferation, both achieved through enhanced Rac1 activation. Mice with stromal or hematopoietic Plxna4 deletion exhibited enhanced CTL infiltration and impaired tumor growth. In a melanoma model, adoptive transfer of CTLs lacking Plxna4 prolonged survival and improved therapeutic outcome, which was even stronger when combined with anti-programmed cell death protein 1 (PD-1) treatment. PLXNA4 abundance in circulating CTLs was augmented in melanoma patients versus healthy volunteers but decreased after the first cycle of anti-PD-1, alone or in combination with anti-cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), in those patients showing complete or partial response to the treatment. Altogether, our data suggest that Plxna4 acts as a "checkpoint," negatively regulating CTL migration and proliferation through cell-autonomous mechanisms independent of the interaction with host-derived Plxna4 ligands, semaphorins. These findings pave the way toward Plxna4-centric immunotherapies and propose Plxna4 detection in circulating CTLs as a potential way to monitor the response to immune checkpoint blockade in patients with metastatic melanoma.

Protein Phosphatase 2A Mediates YAP Activation in Endothelial Cells Upon VEGF Stimulation and Matrix Stiffness.

Angiogenesis is an essential process during development. Abnormal angiogenesis also contributes to many disease conditions such as tumor and retinal diseases. Previous studies have established the Hippo signaling pathway effector Yes-associated protein (YAP) as a crucial regulator of angiogenesis. In ECs, activated YAP promotes endothelial cell proliferation, migration and sprouting. YAP activity is regulated by vascular endothelial growth factor (VEGF) and mechanical cues such as extracellular matrix (ECM) stiffness. However, it is unclear how VEGF or ECM stiffness signal to YAP, especially how dephosphorylation of YAP occurs in response to VEGF stimulus or ECM stiffening. Here, we show that protein phosphatase 2A (PP2A) is required for this process. Blocking PP2A activity abolishes VEGF or ECM stiffening mediated YAP activation. Systemic administration of a PP2A inhibitor suppresses YAP activity in blood vessels in developmental and pathological angiogenesis mouse models. Consistently, PP2A inhibitor also inhibits sprouting angiogenesis. Mechanistically, PP2A directly interacts with YAP, and this interaction requires proper cytoskeleton dynamics. These findings identify PP2A as a crucial mediator of YAP activation in ECs and hence as an important regulator of angiogenesis.

Macrophage-derived glutamine boosts satellite cells and muscle regeneration.

Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells1-4. Macrophages and satellite cells communicate in different ways1-5, but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1-knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles.

Neutrophilic HGF-MET Signalling Exacerbates Intestinal Inflammation.

BACKGROUND AND AIMS: Ulcerative colitis [UC] is associated with excessive neutrophil infiltration and collateral tissue damage, but the link is not yet completely understood. Since c-MET receptor tyrosine kinase [MET] is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor [HGF], we aimed to identify the function of HGF-MET signalling in neutrophils in UC patients and in mice during intestinal inflammation. METHODS: Serum and colonic biopsies from healthy controls and UC patients with active [Mayo endoscopic subscore 2-3] and inactive [Mayo endoscopic subscore 0-1] disease were collected to assess the level of serum and colonic HGF. Disease progression and immune cell infiltration were assessed during dextran sodium sulphate [DSS] colitis in wild-type and MRP8-Cre MET-LoxP mice. RESULTS: Increased mucosal HGF expression was detected in patients with active UC, and in mice during the inflammatory phase of DSS colitis. Similarly, serum HGF was significantly increased in active UC patients and positively correlated with C-reactive protein and blood neutrophil counts. Flow cytometric analysis also demonstrated an upregulation of colonic MET+ neutrophils during DSS colitis. Genetic ablation of MET in neutrophils reduced the severity of DSS-induced colitis. Concomitantly, there was a decreased number of TH17 cells, which could be due to a decreased production of IL-1ß by MET-deficient neutrophils. CONCLUSIONS: These data highlight the central role of neutrophilic HGF-MET signalling in exacerbating damage during intestinal inflammation. Hence, selective blockade of this pathway in neutrophils could be considered as a novel therapeutic approach in UC.

Validation of miR-20a as a Tumor Suppressor Gene in Liver Carcinoma Using Hepatocyte-Specific Hyperactive piggyBac Transposons.

We established a semi-high-throughput in vivo screening platform using hyperactive piggyBac (hyPB) transposons (designated as PB-miR) to identify microRNAs (miRs) that inhibit hepatocellular carcinoma (HCC) development in vivo, following miR overexpression in hepatocytes. PB-miRs encoding six different miRs from the miR-17-92 cluster and nine miRs from outside this cluster were transfected into mouse livers that were chemically induced to develop HCC. In this slow-onset HCC model, miR-20a significantly inhibited HCC. Next, we developed a more aggressive HCC model by overexpression of oncogenic Harvey rat sarcoma viral oncogene homolog (HRASG12V) and c-MYC oncogenes that accelerated HCC development after only 6 weeks. The tumor suppressor effect of miR-20a could be demonstrated even in this rapid-onset HRASG12V/c-MYC HCC model, consistent with significantly prolonged survival and decreased HCC tumor burden. Comprehensive RNA expression profiling of 95 selected genes typically associated with HCC development revealed differentially expressed genes and functional pathways that were associated with miR-20a-mediated HCC suppression. To our knowledge, this is the first study establishing a direct causal relationship between miR-20a overexpression and liver cancer inhibition in vivo. Moreover, these results demonstrate that hepatocyte-specific hyPB transposons are an efficient platform to screen and identify miRs that affect overall survival and HCC tumor regression.

Podoplanin-Expressing Macrophages Promote Lymphangiogenesis and Lymphoinvasion in Breast Cancer.

Among mammary tumor-infiltrating immune cells, the highest expression of podoplanin (PDPN) is found in a subset of tumor-associated macrophages (TAMs). We hereby demonstrate that PDPN is involved in the attachment of this TAM subset to lymphatic endothelial cells (LECs). Mechanistically, the binding of PDPN to LEC-derived galectin 8 (GAL8) in a glycosylation-dependent manner promotes the activation of pro-migratory integrin ß1. When proximal to lymphatics, PDPN-expressing macrophages (PoEMs) stimulate local matrix remodeling and promote vessel growth and lymphoinvasion. Anti-integrin ß1 blockade, macrophage-specific Pdpn knockout, or GAL8 inhibition impairs TAM adhesion to LECs, restraining lymphangiogenesis and reducing lymphatic cancer spread. In breast cancer patients, association of PoEMs with tumor lymphatic vessels correlates with incidences of lymph node and distant organ metastasis.

The mTOR and PP2A Pathways Regulate PHD2 Phosphorylation to Fine-Tune HIF1α Levels and Colorectal Cancer Cell Survival under Hypoxia.

Oxygen-dependent HIF1α hydroxylation and degradation are strictly controlled by PHD2. In hypoxia, HIF1α partly escapes degradation because of low oxygen availability. Here, we show that PHD2 is phosphorylated on serine 125 (S125) by the mechanistic target of rapamycin (mTOR) downstream kinase P70S6K and that this phosphorylation increases its ability to degrade HIF1α. mTOR blockade in hypoxia by REDD1 restrains P70S6K and unleashes PP2A phosphatase activity. Through its regulatory subunit B55α, PP2A directly dephosphorylates PHD2 on S125, resulting in a further reduction of PHD2 activity that ultimately boosts HIF1α accumulation. These events promote autophagy-mediated cell survival in colorectal cancer (CRC) cells. B55α knockdown blocks neoplastic growth of CRC cells in vitro and in vivo in a PHD2-dependent manner. In patients, CRC tissue expresses higher levels of REDD1, B55α, and HIF1α but has lower phospho-S125 PHD2 compared with a healthy colon. Our data disclose a mechanism of PHD2 regulation that involves the mTOR and PP2A pathways and controls tumor growth.

Macrophage Metabolism Controls Tumor Blood Vessel Morphogenesis and Metastasis.

Hypoxic tumor-associated macrophages (TAMs) acquire angiogenic and immunosuppressive properties. Yet it remains unknown if metabolic changes influence these functions. Here, we argue that hypoxic TAMs strongly upregulate the expression of REDD1, a negative regulator of mTOR. REDD1-mediated mTOR inhibition hinders glycolysis in TAMs and curtails their excessive angiogenic response, with consequent formation of abnormal blood vessels. Accordingly, REDD1 deficiency in TAMs leads to the formation of smoothly aligned, pericyte-covered, functional vessels, which prevents vessel leakiness, hypoxia, and metastases. Mechanistically, highly glycolytic REDD1-deficient TAMs outcompete endothelial cells for glucose usage that thwarts vascular hyperactivation and promotes the formation of quiescent vascular junctions. Tuning down glycolysis in REDD1 knockout TAMs re-establishes abnormal angiogenesis and metastases. On this basis, we prove that the anti-tumor effect of mTOR inhibitors is partly countered by the deleterious outcome of these drugs on TAMs. Our data provide a functional link between TAM metabolism and tumor angiogenesis.

Tumour-educated circulating monocytes are powerful candidate biomarkers for diagnosis and disease follow-up of colorectal cancer.

OBJECTIVE: Cancer immunology is a growing field of research whose aim is to develop innovative therapies and diagnostic tests. Starting from the hypothesis that immune cells promptly respond to harmful stimuli, we used peripheral blood monocytes in order to characterise a distinct gene expression profile and to evaluate its potential as a candidate diagnostic biomarker in patients with colorectal cancer (CRC), a still unmet clinical need. DESIGN: We performed a case-control study including 360 peripheral blood monocyte samples from four European oncological centres and defined a gene expression profile specific to CRC. The robustness of the genetic profile and disease specificity were assessed in an independent setting. RESULTS: This screen returned 43 putative diagnostic markers, which we refined and validated in the confirmative multicentric analysis to 23 genes with outstanding diagnostic accuracy (area under the curve (AUC)=0.99 (0.99 to 1.00), Se=100.0% (100.0% to 100.0%), Sp=92.9% (78.6% to 100.0%) in multiple-gene receiver operating characteristic analysis). The diagnostic accuracy was robustly maintained in prospectively collected independent samples (AUC=0.95 (0.85 to 1.00), Se=92.6% (81.5% to 100.0%), Sp=92.3% (76.9% to 100.0%). This monocyte signature was expressed at early disease onset, remained robust over the course of disease progression, and was specific for the monocytic fraction of mononuclear cells. The gene modulation was induced specifically by soluble factors derived from transformed colon epithelium in comparison to normal colon or other cancer histotypes. Moreover, expression changes were plastic and reversible, as they were abrogated upon withdrawal of these tumour-released factors. Consistently, the modified set of genes reverted to normal expression upon curative treatment and was specific for CRC. CONCLUSIONS: Our study is the first to demonstrate monocyte plasticity in response to tumour-released soluble factors. The identified distinct signature in tumour-educated monocytes might be used as a candidate biomarker in CRC diagnosis and harbours the potential for disease follow-up and therapeutic monitoring.

piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.

MET is required for the recruitment of anti-tumoural neutrophils.

Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours, which rely on the constitutive engagement of this pathway for their growth and survival. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells, although its precise role in this compartment is not well characterized. Here we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF). Met deletion in mouse neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both the primary tumour and metastatic sites. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived tumour necrosis factor (TNF)-α or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and for inducible nitric oxide synthase production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. After systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect arising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential 'Achilles' heel' of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases.

Hyperactive piggyBac transposons for sustained and robust liver-targeted gene therapy.

The development of robust nonviral vectors could facilitate clinical gene therapy applications and may overcome some of the immune complications of viral vectors. Nevertheless, most nonviral gene deliver approaches typically yield only transient and/or low gene expression. To address these caveats, we have explored piggyBac transposons to correct hemophilia B by liver-directed factor IX (FIX) gene therapy in hemophilic mice. To achieve this, we combined the use of: (i) a hyperactive codon-optimized piggyBac transposase, (ii) a computationally enhanced liver-specific promoter, (iii) a hyperfunctional codon-optimized FIX transgene (FIX R338L Padua), and (iv) a modification of the transposon terminal repeats. This combination strategy resulted in a robust 400-fold improvement in vector performance in hepatocytes, yielding stable supraphysiologic human FIX activity (>1 year). Liver-specific expression resulted in the induction of FIX-specific immune tolerance. Remarkably, only very low transposon/transposase doses were required to cure the bleeding diathesis. Similarly, PB transposons could be used to express supraphysiologic factor VIII levels using low transposon/transposase doses. PB transposition did not induce tumors in a sensitive hepatocellular carcinoma-prone mouse model. These results underscore the potency and relative safety of the latest generation PB transposons, which constitutes a versatile platform for stable and robust secretion of therapeutic proteins.

Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and non-human primates.

The robustness and safety of liver-directed gene therapy can be substantially improved by enhancing expression of the therapeutic transgene in the liver. To achieve this, we developed a new approach of rational in silico vector design. This approach relies on a genome-wide bio-informatics strategy to identify cis-acting regulatory modules (CRMs) containing evolutionary conserved clusters of transcription factor binding site motifs that determine high tissue-specific gene expression. Incorporation of these CRMs into adeno-associated viral (AAV) and non-viral vectors enhanced gene expression in mice liver 10 to 100-fold, depending on the promoter used. Furthermore, these CRMs resulted in robust and sustained liver-specific expression of coagulation factor IX (FIX), validating their immediate therapeutic and translational relevance. Subsequent translational studies indicated that therapeutic FIX expression levels could be attained reaching 20-35% of normal levels after AAV-based liver-directed gene therapy in cynomolgus macaques. This study underscores the potential of rational vector design using computational approaches to improve their robustness and therefore allows for the use of lower and thus safer vector doses for gene therapy, while maximizing therapeutic efficacy.

Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice.

Gene therapy may provide a cure for hemophilia and overcome the limitations of protein replacement therapy. Increasing the potency of gene transfer vectors may allow improvement of their therapeutic index, as lower doses can be administered to achieve therapeutic benefit, reducing toxicity of in vivo administration. Here we generated codon-usage optimized and hyperfunctional factor IX (FIX) transgenes carrying an R338L amino acid substitution (FIX Padua), previously associated with clotting hyperactivity and thrombophilia. We delivered these transgenes to hemophilia B mice by hepatocyte-targeted integration-competent and -defective lentiviral vectors. The hyperfunctional FIX transgenes increased FIX activity reconstituted in the plasma without detectable adverse effects, allowing correction of the disease phenotype at lower vector doses and resulting in improved hemostasis in vivo. The combined effect of codon optimization with the hyperactivating FIX-R338L mutation resulted in a robust 15-fold gain in potency and therefore provides a promising strategy to improve the efficacy, feasibility, and safety of hemophilia gene therapy.