Oleuropein-driven reprogramming of the myeloid cell compartment to sensitise tumours to PD-1/PD-L1 blockade strategies.

BACKGROUND: Previous studies have shown that functional systemic immunity is required for the efficacy of PD-1/PD-L1 blockade immunotherapies in cancer. Hence, systemic reprogramming of immunosuppressive dysfunctional myeloid cells could overcome resistance to cancer immunotherapy. METHODS: Reprogramming of tumour-associated myeloid cells with oleuropein was studied by quantitative differential proteomics, phenotypic and functional assays in mice and lung cancer patients. Combinations of oleuropein and two different delivery methods of anti-PD-1 antibodies were tested in colorectal cancer tumour models and in immunotherapy-resistant lung cancer models. RESULTS: Oleuropein treatment reprogrammed monocytic and granulocytic myeloid-derived suppressor cells, and tumour-associated macrophages towards differentiation of immunostimulatory subsets. Oleuropein regulated major differentiation programmes associated to immune modulation in myeloid cells, which potentiated T cell responses and PD-1 blockade. PD-1 antibodies were delivered by two different strategies, either systemically or expressed within tumours using a self-amplifying RNA vector. Combination anti-PD-1 therapies with oleuropein increased tumour infiltration by immunostimulatory dendritic cells in draining lymph nodes, leading to systemic antitumour T cell responses. Potent therapeutic activities were achieved in colon cancer and lung cancer models resistant to immunotherapies, even leading to complete tumour regression. DISCUSSION: Oleuropein significantly improves the outcome of PD-1/PD-L1 blockade immunotherapy strategies by reprogramming myeloid cells.

Engineering U1-Based Tetracycline-Inducible Riboswitches to Control Gene Expression in Mammals.

Synthetic riboswitches are promising regulatory devices due to their small size, lack of immunogenicity, and ability to fine-tune gene expression in the absence of exogenous trans-acting factors. Based on a gene inhibitory system developed at our lab, termed U1snRNP interference (U1i), we developed tetracycline (TC)-inducible riboswitches that modulate mRNA polyadenylation through selective U1 snRNP recruitment. First, we engineered different TC-U1i riboswitches, which repress gene expression unless TC is added, leading to inductions of gene expression of 3-to-4-fold. Second, we developed a technique called Systematic Evolution of Riboswitches by Exponential Enrichment (SEREX), to isolate riboswitches with enhanced U1 snRNP binding capacity and activity, achieving inducibilities of up to 8-fold. Interestingly, by multiplexing riboswitches we increased inductions up to 37-fold. Finally, we demonstrated that U1i-based riboswitches are dose-dependent and reversible and can regulate the expression of reporter and endogenous genes in culture cells and mouse models, resulting in attractive systems for gene therapy applications. Our work probes SEREX as a much-needed technology for the in vitro identification of riboswitches capable of regulating gene expression in vivo.

ISR8/IRF1-AS1 Is Relevant for IFNα and NF-κB Responses.

The study of the interferon (IFN) α-induced cell transcriptome has shown altered expression of several long non-coding RNAs (lncRNAs). ISR8/IRF1-AS1 (IFN stimulated RNA 8), located close to IFN regulatory factor 1 (IRF1) coding gene, transcribes a lncRNA induced at early times after IFNα treatment or IRF1 or NF-κB activation. Depletion or overexpression of ISR8 RNA does not lead to detected deregulation of the IFN response. Surprisingly, disruption of ISR8 locus with CRISPR-Cas9 genome editing results in cells that fail to induce several key ISGs and pro-inflammatory cytokines after a trigger with IFNα or overexpression of IRF1 or the NF-κB subunit RELA. This suggests that the ISR8 locus may play a relevant role in IFNα and NF-κB pathways. Interestingly, IFNα, IRFs and NF-κB-responding luciferase reporters are normally induced in ISR8-disrupted cells when expressed from a plasmid but not when integrated into the genome. Therefore, IFNα and NF-κB pathways are functional to induce the expression of exogenous episomic transcripts but fail to activate transcription from genomic promoters. Transcription from these promoters is not restored with silencing inhibitors, by decreasing the levels of several negative regulators or by overexpression of inducers. Transcriptome analyses indicate that ISR8-disrupted cells have a drastic increase in the levels of negative regulators such as XIST and Zinc finger proteins. Our results agree with ISR8 loci being an enhancer region that is fundamental for proper antiviral and proinflammatory responses. These results are relevant because several SNPs located in the ISR8 region are associated with chronic inflammatory and autoimmune diseases including Crohn's disease, inflammatory bowel disease, ulcerative colitis or asthma.

U1A is a positive regulator of the expression of heterologous and cellular genes involved in cell proliferation and migration.

Here, we show that direct recruitment of U1A to target transcripts can increase gene expression. This is a new regulatory role, in addition to previous knowledge showing that U1A decreases the levels of U1A mRNA and other specific targets. In fact, genome-wide, U1A more often increases rather than represses gene expression and many U1A-upregulated transcripts are directly bound by U1A according to individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) studies. Interestingly, U1A-mediated positive regulation can be transferred to a heterologous system for biotechnological purposes. Finally, U1A-bound genes are enriched for those involved in cell cycle and adhesion. In agreement with this, higher U1A mRNA expression associates with lower disease-free survival and overall survival in many cancer types, and U1A mRNA levels positively correlate with those of some oncogenes involved in cell proliferation. Accordingly, U1A depletion leads to decreased expression of these genes and the migration-related gene CCN2/CTGF, which shows the strongest regulation by U1A. A decrease in U1A causes a strong drop in CCN2 expression and CTGF secretion and defects in the expression of CTGF EMT targets, cell migration, and proliferation. These results support U1A as a putative therapeutic target for cancer treatment. In addition, U1A-binding sequences should be considered in biotechnological applications.

Long Noncoding RNA NIHCOLE Promotes Ligation Efficiency of DNA Double-Strand Breaks in Hepatocellular Carcinoma.

Long noncoding RNAs (lncRNA) are emerging as key players in cancer as parts of poorly understood molecular mechanisms. Here, we investigated lncRNAs that play a role in hepatocellular carcinoma (HCC) and identified NIHCOLE, a novel lncRNA induced in HCC with oncogenic potential and a role in the ligation efficiency of DNA double-stranded breaks (DSB). NIHCOLE expression was associated with poor prognosis and survival of HCC patients. Depletion of NIHCOLE from HCC cells led to impaired proliferation and increased apoptosis. NIHCOLE deficiency led to accumulation of DNA damage due to a specific decrease in the activity of the nonhomologous end-joining (NHEJ) pathway of DSB repair. DNA damage induction in NIHCOLE-depleted cells further decreased HCC cell growth. NIHCOLE was associated with DSB markers and recruited several molecules of the Ku70/Ku80 heterodimer. Further, NIHCOLE putative structural domains supported stable multimeric complexes formed by several NHEJ factors including Ku70/80, APLF, XRCC4, and DNA ligase IV. NHEJ reconstitution assays showed that NIHCOLE promoted the ligation efficiency of blunt-ended DSBs. Collectively, these data show that NIHCOLE serves as a scaffold and facilitator of NHEJ machinery and confers an advantage to HCC cells, which could be exploited as a targetable vulnerability. SIGNIFICANCE: This study characterizes the role of lncRNA NIHCOLE in DNA repair and cellular fitness in HCC, thus implicating it as a therapeutic target.See related commentary by Barcena-Varela and Lujambio, p. 4899.

Long Noncoding RNA EGOT Responds to Stress Signals to Regulate Cell Inflammation and Growth.

The cell has several mechanisms to sense and neutralize stress. Stress-related stimuli activate pathways that counteract danger, support cell survival, and activate the inflammatory response. We use human cells to show that these processes are modulated by EGOT, a long noncoding RNA highly induced by viral infection, whose inhibition results in increased levels of antiviral IFN-stimulated genes (ISGs) and decreased viral replication. We now show that EGOT is induced in response to cell stress, viral replication, or the presence of pathogen-associated molecular patterns via the PI3K/AKT, MAPKs, and NF-κB pathways, which lead to cell survival and inflammation. Transcriptome analysis and validation experiments show that EGOT modulates PI3K/AKT and NF-κB responses. On the one hand, EGOT inhibition decreases expression of PI3K/AKT-induced cellular receptors and cell proliferation. In fact, EGOT levels are increased in several tumors. On the other hand, EGOT inhibition results in decreased levels of key NF-κB target genes, including those required for inflammation and ISGs in those cells that build an antiviral response. Mechanistically, EGOT depletion decreases the levels of the key coactivator TBLR1, essential for transcription by NF-κB. In summary, EGOT is induced in response to stress and may function as a switch that represses ISG transcription until a proper antiviral or stress response is initiated. EGOT then helps PI3K/AKT, MAPKs, and NF-κB pathways to activate the antiviral response, cell inflammation, and growth. We believe that modulation of EGOT levels could be used as a therapy for the treatment of certain viral infections, immune diseases, and cancer.

The Mir181ab1 cluster promotes KRAS-driven oncogenesis and progression in lung and pancreas.

Few therapies are currently available for patients with KRAS-driven cancers, highlighting the need to identify new molecular targets that modulate central downstream effector pathways. Here we found that the microRNA (miRNA) cluster including miR181ab1 is a key modulator of KRAS-driven oncogenesis. Ablation of Mir181ab1 in genetically engineered mouse models of Kras-driven lung and pancreatic cancer was deleterious to tumor initiation and progression. Expression of both resident miRNAs in the Mir181ab1 cluster, miR181a1 and miR181b1, was necessary to rescue the Mir181ab1-loss phenotype, underscoring their nonredundant role. In human cancer cells, depletion of miR181ab1 impaired proliferation and 3D growth, whereas overexpression provided a proliferative advantage. Lastly, we unveiled miR181ab1-regulated genes responsible for this phenotype. These studies identified what we believe to be a previously unknown role for miR181ab1 as a potential therapeutic target in 2 highly aggressive and difficult to treat KRAS-mutated cancers.

The cirrhotic liver is depleted of docosahexaenoic acid (DHA), a key modulator of NF-κB and TGFß pathways in hepatic stellate cells.

Liver cirrhosis results from chronic hepatic damage and is characterized by derangement of the organ architecture with increased liver fibrogenesis and defective hepatocellular function. It frequently evolves into progressive hepatic insufficiency associated with high mortality unless liver transplantation is performed. We have hypothesized that the deficiency of critical nutrients such as essential omega-3 fatty acids might play a role in the progression of liver cirrhosis. Here we evaluated by LC-MS/MS the liver content of omega-3 docosahexaenoic fatty acid (DHA) in cirrhotic patients and investigated the effect of DHA in a murine model of liver injury and in the response of hepatic stellate cells (HSCs) (the main producers of collagen in the liver) to pro-fibrogenic stimuli. We found that cirrhotic livers exhibit a marked depletion of DHA and that this alteration correlates with the progression of the disease. Administration of DHA exerts potent anti-fibrogenic effects in an acute model of liver damage. Studies with HSCs show that DHA inhibits fibrogenesis more intensely than other omega-3 fatty acids. Data from expression arrays revealed that DHA blocks TGFß and NF-κB pathways. Mechanistically, DHA decreases late, but not early, SMAD3 nuclear accumulation and inhibits p65/RelA-S536 phosphorylation, which is required for HSC survival. Notably, DHA increases ADRP expression, leading to the formation of typical quiescence-associated perinuclear lipid droplets. In conclusion, a marked depletion of DHA is present in the liver of patients with advanced cirrhosis. DHA displays anti-fibrogenic activities on HSCs targeting NF-κB and TGFß pathways and inducing ADPR expression and quiescence in these cells.

Deregulation of linc-PINT in acute lymphoblastic leukemia is implicated in abnormal proliferation of leukemic cells.

Long Non-Coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides in length. Several lncRNAs are involved in cell proliferation and are deregulated in several human tumors. Few lncRNAs have been described to play a role in Acute Lymphoblastic Leukemia (ALL). In this study, we carried out a genome wide lncRNA expression profiling in ALL samples and peripheral blood samples obtained from healthy donors. We detected 43 lncRNAs that were aberrantly expressed in ALL. Interestingly, among them, linc-PINT showed a significant downregulation in T and B-ALL. Re-expression of linc-PINT in ALL cells induced inhibition of leukemic cell growth that was associated with apoptosis induction and cell cycle arrest in G2/M phase. linc-PINT induced the transcription of HMOX1 which reduced the viability of ALL cells. Intriguingly, we observed that treatment with anti-tumoral epigenetic drugs like LBH-589 (Panobinostat) and Curcumin induced the expression of linc-PINT and HMOX1 in ALL. These results indicate that the downregulation of linc-PINT plays a relevant role in the pathogenesis of ALL, and linc-PINT re-expression may be one of the mechanisms exerted by epigenetic drugs to reduce cell proliferation in ALL.

Transient depletion of specific immune cell populations to improve adenovirus-mediated transgene expression in the liver.

BACKGROUND & AIMS: Adenoviral (Ad) vectors are currently one of the most efficient tools for in vivo gene transfer to the liver. However, anti-Ad immune responses limit the safety and efficacy of these vectors. The initial inflammatory reaction is a concern in terms of toxicity, and it favours the development of cellular and humoral responses leading to short transgene persistence and inefficient vector re-administrations. Therefore, safe and simple ways to interfere with these processes are needed. Study ways to deplete specific immune cell populations and their impact on liver-directed gene transfer. METHODS: First-generation Ad vectors encoding reporter genes (luciferase or ß-galactosidase) were injected intravenously into Balb/c mice. Kupffer cells and splenic macrophages were depleted by intravenous administration of clodronate liposomes. B lymphocytes, CD4(+) , CD8(+) T lymphocytes or NK cells were depleted by intraperitoneal injection of anti-M plus anti-D, anti-CD4, anti-CD8 or anti-asialo-GM1 antibodies respectively. Long-term evolution of luciferase expression in the liver was monitored by bioluminescence imaging. RESULTS: The anti-CD4 monoclonal antibody impaired cellular and humoral immune responses, leading to efficient vector re-administration. Clodronate liposomes had no impact on humoral responses but caused a 100-1000 fold increase in liver transduction, stabilized transgene expression, reduced the concentration of inflammatory cytokines, and inhibited lymphocyte activation. CONCLUSIONS: Transient CD4(+) T-cell depletion using antibodies is a clinically feasible procedure that allows efficient Ad redosing. Systemic administration of clodronate liposomes may further increase the safety and efficacy of vectors.

Increased in vivo inhibition of gene expression by combining RNA interference and U1 inhibition.

Inhibition of gene expression can be achieved with RNA interference (RNAi) or U1 small nuclear RNA-snRNA-interference (U1i). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to inhibit polyadenylation of a target pre-mRNA. In culture, we have shown that the combination of RNAi and U1i results in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. We have now used these techniques to inhibit gene expression in mice. We show that U1ins can induce strong inhibition of the expression of target genes in vivo. Furthermore, combining U1i and RNAi results in synergistic inhibitions also in mice. This is shown for the inhibition of hepatitis B virus (HBV) sequences or endogenous Notch1. Surprisingly, inhibition obtained by combining a U1in and a RNAi mediator is higher than that obtained by combining two U1ins or two RNAi mediators. Our results suggest that RNAi and U1i cooperate by unknown mechanisms to result in synergistic inhibitions. Analysis of toxicity and specificity indicates that expression of U1i inhibitors is safe. Therefore, we believe that the combination of RNAi and U1i will be a good option to block damaging endogenous genes, HBV and other infectious agents in vivo.

Insulin-like growth factor I gene transfer to cirrhotic liver induces fibrolysis and reduces fibrogenesis leading to cirrhosis reversion in rats.

UNLABELLED: We investigated whether gene transfer of insulin-like growth factor I (IGF-I) to the hepatic tissue was able to improve liver histology and function in established liver cirrhosis. Rats with liver cirrhosis induced by carbon tetrachloride (CCl(4)) given orally for 8 weeks were injected through the hepatic artery with saline or with Simian virus 40 vectors encoding IGF-I (SVIGF-I), or luciferase (SVLuc). Animals were sacrificed 8 weeks after vector injection. In cirrhotic rats we observed that, whereas IGF-I was synthesized by hepatocytes, IGF-I receptor was predominantly expressed by nonparenchymal cells, mainly in fibrous septa surrounding hepatic nodules. Rats treated with SVIGF-I showed increased hepatic levels of IGF-I, improved liver function tests, and reduced fibrosis in association with diminished alpha-smooth muscle actin expression, up-regulation of matrix metalloproteases (MMPs) and decreased expression of the tissue inhibitors of MMPs TIM-1 and TIM-2. SVIGF-I therapy induced down-regulation of the profibrogenic molecules transforming growth factor beta (TGFbeta), amphiregulin, platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), and vascular endothelium growth factor (VEGF) and induction of the antifibrogenic and cytoprotective hepatocyte growth factor (HGF). Furthermore, SVIGF-I-treated animals showed decreased expression of Wilms tumor-1 (WT-1; a nuclear factor involved in hepatocyte dedifferentiation) and up-regulation of hepatocyte nuclear factor 4 alpha (HNF4alpha) (which stimulates hepatocellular differentiation). The therapeutic potential of SVIGF-I was also tested in rats with thioacetamide-induced liver cirrhosis. Also in this model, SVIGF-I improved liver function and reduced liver fibrosis in association with up-regulation of HGF and MMPs and down-regulation of tissue inhibitor of metalloproteinase 1 (TIMP-1). CONCLUSION: IGF-I gene transfer to cirrhotic livers induces MMPs and hepatoprotective factors leading to reversion of fibrosis and improvement of liver function. IGF-I gene therapy may be a useful alternative therapy for patients with advanced cirrhosis without timely access to liver transplantation.

Adenovirus VA RNA-derived miRNAs target cellular genes involved in cell growth, gene expression and DNA repair.

Adenovirus virus-associated (VA) RNAs are processed to functional viral miRNAs or mivaRNAs. mivaRNAs are important for virus production, suggesting that they may target cellular or viral genes that affect the virus cell cycle. To look for cellular targets of mivaRNAs, we first identified genes downregulated in the presence of VA RNAs by microarray analysis. These genes were then screened for mivaRNA target sites using several bioinformatic tools. The combination of microarray analysis and bioinformatics allowed us to select the splicing and translation regulator TIA-1 as a putative mivaRNA target. We show that TIA-1 is downregulated at mRNA and protein levels in infected cells expressing functional mivaRNAs and in transfected cells that express mivaRNAI-138, one of the most abundant adenoviral miRNAs. Also, reporter assays show that TIA-1 is downregulated directly by mivaRNAI-138. To determine whether mivaRNAs could target other cellular genes we analyzed 50 additional putative targets. Thirty of them were downregulated in infected or transfected cells expressing mivaRNAs. Some of these genes are important for cell growth, transcription, RNA metabolism and DNA repair. We believe that a mivaRNA-mediated fine tune of the expression of some of these genes could be important in adenovirus cell cycle.

Development of a new noncytopathic Semliki Forest virus vector providing high expression levels and stability.

Alphavirus vectors express high levels of recombinant proteins in mammalian cells, but their cytopathic nature makes this expression transient. In order to generate a Semliki Forest virus (SFV) noncytopathic vector we introduced mutations previously described to turn Sindbis virus noncytopathic into a conserved position in an SFV vector expressing LacZ. Interestingly, mutant P718T in replicase nsp2 subunit was able to replicate in only a small percentage of BHK cells, producing beta-gal-expressing colonies without selection. Puromycin N-acetyl-transferase (pac) gene was used to replace LacZ in this mutant allowing selection of an SFV noncytopathic replicon containing a second mutation in nsp2 nuclear localization signal (R649H). This latter mutation did not confer a noncytopathic phenotype by itself and did not alter nsp2 nuclear translocation. Replicase synthesis was diminished in the SFV double mutant, leading to genomic and subgenomic RNA levels that were 125-fold and 66-fold lower than in wild-type vector, respectively. Interestingly, this mutant expressed beta-gal levels similar to parental vector. By coexpressing pac and LacZ from independent subgenomic promoters this vector was able to generate stable cell lines maintaining high expression levels during at least 10 passages, indicating that it could be used as a powerful system for protein production in mammalian cells.

Effect of adenovirus-mediated RNA interference on endogenous microRNAs in a mouse model of multidrug resistance protein 2 gene silencing.

RNA interference with viral vectors that express short hairpin RNAs (shRNAs) has emerged as a powerful tool for functional genomics and therapeutic purposes. However, little is known about shRNA in vivo processing, accumulation, functional kinetics, and side effects related to shRNA saturation of the cellular gene silencing machinery. Therefore, we constructed first-generation recombinant adenoviruses encoding different shRNAs against murine ATP-binding cassette multidrug resistance protein 2 (Abcc2), which is involved in liver transport of bilirubin to bile, and analyzed Abcc2 silencing kinetics. C57/BL6 mice injected with these viruses showed significant impairment of Abcc2 function for up to 3 weeks, as reflected by increased serum bilirubin levels. The lack of Abcc2 function correlated with a specific reduction of Abcc2 mRNA and with high levels of processed shRNAs targeting Abcc2. Inhibition was lost at longer times postinfection, correlating with a decrease in the accumulation of processed shRNAs. This finding suggests that a minimal amount of processed shRNAs is required for efficient silencing in vivo. This system was also used to evaluate the effect of shRNA expression on the saturation of silencing factors. Saturation of the cellular silencing processing machinery alters the accumulation and functionality of endogenous microRNAs (miRNAs) and pre-miRNAs. However, expression of functional exogenous shRNAs did not change the levels of endogenous miRNAs or their precursors. In summary, this work shows that adenoviral vectors can deliver sufficient shRNAs to mediate inhibition of gene expression without saturating the silencing machinery.

Adenovirus virus-associated RNA is processed to functional interfering RNAs involved in virus production.

Posttranscriptional gene silencing allows sequence-specific control of gene expression. Specificity is guaranteed by small antisense RNAs such as microRNAs (miRNAs) or small interfering RNAs (siRNAs). Functional miRNAs derive from longer double-stranded RNA (dsRNA) molecules that are cleaved to pre-miRNAs in the nucleus and are transported by exportin 5 (Exp 5) to the cytoplasm. Adenovirus-infected cells express virus-associated (VA) RNAs, which are dsRNA molecules similar in structure to pre-miRNAs. VA RNAs are also transported by Exp 5 to the cytoplasm, where they accumulate. Here we show that small RNAs derived from VA RNAs (svaRNAs), similar to miRNAs, can be found in adenovirus-infected cells. VA RNA processing to svaRNAs requires neither viral replication nor viral protein expression, as evidenced by the fact that svaRNA accumulation can be detected in cells transfected with VA sequences. svaRNAs are efficiently bound by Argonaute 2, the endonuclease of the RNA-induced silencing complex, and behave as functional siRNAs, in that they inhibit the expression of reporter genes with complementary sequences. Blocking svaRNA-mediated inhibition affects efficient adenovirus production, indicating that svaRNAs are required for virus viability. Thus, svaRNA-mediated silencing could represent a novel mechanism used by adenoviruses to control cellular or viral gene expression.

Intratumoral injection of dendritic cells transduced by an SV40-based vector expressing interleukin-15 induces curative immunity mediated by CD8+ T lymphocytes and NK cells.

Cancer immunotherapy has been extensively attempted by gene transfer of cytokines with viral vectors. In this work, we compared the therapeutic effects of interleukin 12 and 15 (IL-12 and IL-15) genes transferred to tumor cells or to dendritic cells (DCs), which were subsequently injected into established tumors. For this purpose, we used viral vectors based on simian virus 40 (rSV40). Importantly, we observed that nonmatured DCs infected with rSV40 vectors remained phenotypically immature. Infection of CT-26 tumor cells with rSV40 expressing IL-12 (rSVIL-12) or IL-15 (rSVIL-15) failed to inhibit tumor development. In contrast, the intratumoral administration of syngeneic DCs transduced with rSVIL-12 or rSVIL-15 was associated with a strong antitumor response; up to 40% tumor remissions were achieved with DCs transduced by rSVIL-12 and 73% with DCs expressing IL-15. This antitumor effect correlated with the in vivo priming of tumor-specific CD8+ T lymphocytes. Depletion studies showed that rSVIL-15-mediated antitumor efficacy was mediated mainly by CD8+ T lymphocytes and NK cells. We conclude that (i) SV40-derived vectors are an advantageous alternative to transduce genes into DCs and (ii) DCs transferred with IL-15 have an enhanced capability to induce curative antitumor immunity when injected into malignant lesions.

In vitro and in vivo comparative study of chimeric liver-specific promoters.

Targeting therapeutic genes to the liver is essential to improve gene therapy protocols of hepatic diseases and of some hereditary disorders. Transcriptional targeting can be achieved using liver-specific promoters. In this study we have made chimeric constructs combining promoter and enhancer regions of the albumin, alpha 1-antitrypsin, hepatitis B virus core protein, and hemopexin genes. Tissue specificity, activity, and length of gene expression driven from these chimeric regulatory sequences have been analyzed in cultured cells from hepatic and nonhepatic origin as well as in mice livers and other organs. We have identified a collection of liver-specific promoters whose activities range from twofold to less than 1% of the CMV promoter in human hepatoma cells. We found that the best liver specificity was attained when both enhancer and promoter sequences of hepatic genes were combined. In vivo studies were performed to analyze promoter function during a period of 50 days after gene transfer to the mouse liver. We found that among the various chimeric constructs tested in this work, the alpha1-antitrypsin promoter alone or linked to the albumin or hepatitis B enhancers is the most potent in directing stable gene expression in liver cells.