Unzu, C. (Carmen)
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- Decreased glucose-derivate uptake in primary somatosensorial cortex in the brain of female AIP mice. | International Porphyrins and Porphyria Meeting (10-13 April 2011. Cardiff, Wales, UK)(2011-04-13) Pozo, M.A. (Miguel Ángel); Delgado, M. (Mercedes); Benito, M. (Marina); Collantes, M. (María); Lanciego, J.L. (José Luis); Peñuelas-Sanchez, I. (Ivan); Desco, M. (Manuel); Prieto-Azcárate, E. (Elena); Molinet-Dronda, F. (Francisco); Unzu, C. (Carmen); Prieto, J. (Jesús); Enriquez-de-Salamanca, R. (Rafael); Fontanellas-Romá, A. (Antonio); Garcia-Garcia, L. (Luis); Rodriguez, I. (Ignacio)
- Renal failure affects the enzymatic activities of the three first steps in hepatic heme biosynthesis in the acute intermittent porphyria mouse(Public Library of Science, 2012) Unzu, C. (Carmen); Prieto, J. (Jesús); Enriquez-de-Salamanca, R. (Rafael); Fontanellas-Romá, A. (Antonio); Salido, E. (Eduardo); Sampedro, A. (Ana); Sardh, E. (Eliane); Harper, P. (Pauline); Mauleon, I. (Itsaso)Chronic kidney disease is a long-term complication in acute intermittent porphyria (AIP). The pathophysiological significance of hepatic overproduction of the porphyrin precursors aminolevulinate acid (ALA) and porphobilinogen (PBG) in chronic kidney disease is unclear. We have investigated the effect of repetitive acute attacks on renal function and the effect of total or five-sixth nephrectomy causing renal insufficiency on hepatic heme synthesis in the porphobilinogen deaminase (PBGD)-deficient (AIP) mouse. Phenobarbital challenge in the AIP-mice increased urinary porphyrin precursor excretion. Successive attacks throughout 14 weeks led to minor renal lesions with no impact on renal function. In the liver of wild type and AIP mice, 5/6 nephrectomy enhanced transcription of the first and rate-limiting ALA synthase. As a consequence, urinary PBG excretion increased in AIP mice. The PBG/ALA ratio increased from 1 in sham operated AIP animals to over 5 (males) and over 13 (females) in the 5/6 nephrectomized mice. Total nephrectomy caused a rapid decrease in PBGD activity without changes in enzyme protein level in the AIP mice but not in the wild type animals. In conclusion, high concentration of porphyrin precursors had little impact on renal function. However, progressive renal insufficiency aggravates porphyria attacks and increases the PBG/ALA ratio, which should be considered a warning sign for potentially life-threatening impairment in AIP patients with signs of renal failure.
- Innate functions of immunoglobulin M lessen liver gene transfer with helper-dependent adenovirus(2014) Dubrot, J. (Juan); Morales-Kastresana, A. (Aizea); Unzu, C. (Carmen); Serrano-Mendioroz, I. (Irantzu); Azpilicueta, A. (Arantza); Ochoa, M.C. (María Carmen); Melero, I. (Ignacio); Fontanellas-Romá, A. (Antonio); Sampedro, A. (Ana); Martinez-Anso, E. (Eduardo)The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA) vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors.
- Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in non-human primates(BioMed Central, 2012) Benito-Boilos, A. (Alberto); Mancheño, U. (Uxua); Alfaro, C. (Carlos); Unzu, C. (Carmen); Pety, H. (Harald); Hervas-Stubbs, S. (Sandra); Melero, I. (Ignacio); Beattie, S.G. (Stuart G.); Salamanca, E. (Enrique) de; Prieto, J. (Jesús); Fontanellas-Romá, A. (Antonio); Sampedro, A. (Ana); Mauleon, I. (Itsaso)BACKGROUND: Adeno-associated vectors (rAAV) have been used to attain long-term liver gene expression. In humans, the cellular immune response poses a serious obstacle for transgene persistence while neutralizing humoral immunity curtails re-administration. Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria) benefits from liver gene transfer in mouse models and clinical trials are about to begin. In this study, we sought to study in non-human primates the feasibility of repeated gene-transfer with intravenous administration of rAAV5 vectors under the effects of an intensive immunosuppressive regimen and to analyze its ability to circumvent T-cell immunity and thereby prolong transgene expression. METHODS: Three female Macaca fascicularis were intravenously injected with 1x1013 genome copies/kg of rAAV5 encoding the human PBGD. Mycophenolate mofetil (MMF), anti-thymocyte immunoglobulin, methylprednisolone, tacrolimus and rituximab were given in combination during 12 weeks to block T- and B-cell mediated adaptive immune responses in two macaques. Immunodeficient and immunocompetent mice were intravenously injected with 5x1012 genome copies/kg of rAAV5-encoding luciferase protein. Forty days later MMF, tacrolimus and rituximab were daily administrated to ascertain whether the immunosuppressants or their metabolites could interfere with transgene expression. RESULTS: Macaques given a rAAV5 vector encoding human PBGD developed cellular and humoral immunity against viral capsids but not towards the transgene. Anti-AAV humoral responses were attenuated during 12 weeks but intensely rebounded following cessation of the immunosuppressants. Accordingly, subsequent gene transfer with a rAAV5 vector encoding green fluorescent protein was impossible. One macaque showed enhanced PBGD expression 25 weeks after rAAV5-pbgd administration but overexpression had not been detected while the animal was under immunosuppression. As a potential explanation, MMF decreases transgene expression in mouse livers that had been successfully transduced by a rAAV5 several weeks before MMF onset. Such a silencing effect was independent of AAV complementary strand synthesis and requires an adaptive immune system. CONCLUSIONS: These results indicate that our transient and intensive pharmacological immunosuppression fails to improve AAV5-based liver gene transfer in non-human primates. The reasons include an incomplete restraint of humoral immune responses to viral capsids that interfere with repeated gene transfer in addition to an intriguing MMF-dependent drug-mediated interference with liver transgene expression.
- Intensive pharmacological immunosuppression allows for repetitive liver gene transfer with recombinant adenovirus in nonhuman primates(NATURE PUBLISHING GROUP, 2009-12-16) Dubrot, J. (Juan); Benito-Boilos, A. (Alberto); Mancheño, U. (Uxua); Alfaro, C. (Carlos); Collantes, M. (María); Peñuelas-Sanchez, I. (Ivan); Smerdou, C. (Cristian); Unzu, C. (Carmen); Hervas-Stubbs, S. (Sandra); Melero, I. (Ignacio); Palazon, A. (Asís); Prieto, J. (Jesús); Fontanellas-Romá, A. (Antonio); Sampedro, A. (Ana); Mauleon, I. (Itsaso)Repeated administration of gene therapies is hampered by host immunity toward vectors and transgenes. Attempts to circumvent antivector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permits repeated liver gene transfer to a limited number of nonhuman primates with recombinant adenovirus. Adenoviral vector–mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specific positron emission tomography (PET) technique, liver immunohistochemistry, and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell–mediated responses toward the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatment that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the first adenoviral exposure in a macaque, which has undergone a total of four treatments with the same adenoviral vector.
- Porphobilinogen deaminase over-expression in hepatocytes, but not in erythrocytes, prevents accumulation of toxic porphyrin precursors in a mouse model of acute intermittent porphyria(Elsevier, 2010) González-Aseguinolaza, G. (Gloria); Dubrot, J. (Juan); Unzu, C. (Carmen); Melero, I. (Ignacio); Prieto, J. (Jesús); Vanrell, L. (Lucía); Enriquez-de-Salamanca, R. (Rafael); Fontanellas-Romá, A. (Antonio); Sampedro, A. (Ana); Mauleon, I. (Itsaso)BACKGROUND & AIMS: Acute intermittent porphyria (AIP) is characterized by hepatic porphobilinogen deaminase (PBGD) deficiency resulting in a marked overproduction of presumably toxic porphyrin precursors. Our study aimed to assess the protective effects of bone marrow transplantation or PBGD gene transfer into the liver against phenotypic manifestations of acute porphyria attack induced in an AIP murine model. METHODS: Lethally irradiated AIP mice were intravenously injected with 5x10(6) nucleated bone marrow cells from wild type or AIP donor mice. To achieve liver gene transfer, AIP mice received via hydrodynamic injection plasmids expressing human PBGD or luciferase, driven by a liver-specific promoter. RESULTS: Erythrocyte PBGD activity increased 2.4-fold in AIP mice receiving bone marrow cells from normal animals. Nevertheless, phenobarbital administration in these mice reproduced key features of acute attacks, such as massively increased urinary porphyrin precursor excretion and decreased motor coordination. Hepatic PBGD activity increased 2.2-fold after hydrodynamic injection of therapeutic plasmid. Mice injected with the luciferase control plasmid showed a high excretion of porphyrin precursors after phenobarbital administration whereas just a small increase was observed in AIP mice injected with the PBGD plasmid. Furthermore, motor disturbance was almost completely abolished in AIP mice treated with the therapeutic plasmid. CONCLUSIONS: PBGD deficiency in erythroid tissue is not associated with phenotypic manifestations of acute porphyria. In contrast, PBGD over-expression in hepatocytes, albeit in a low proportion, reduced precursor accumulation, which is the hallmark of acute porphyric attacks. Liver-directed gene therapy might offer an alternative to liver transplantation applicable in patients with severe and recurrent manifestations.
- Epidermal growth factor receptor ligands in murine models for erythropoietic protoporphyria: potential novel players in the progression of liver injury(C.M.B. Association, 2009) Latasa, M.U. (María Ujué); Corrales, F.J. (Fernando José); Berasain, C. (Carmen); Matscheko, N. (N.); Avila, M.A. (Matías Antonio); Unzu, C. (Carmen); Goñi, S. (Saioa); Prieto, J. (Jesús); Enriquez-de-Salamanca, R. (Rafael); Fontanellas-Romá, A. (Antonio); Sampedro, A. (Ana); Garcia-Bravo, M. (M.); Mauleon, I. (Itsaso)Activation of the epidermal growth factor receptor (EGFR) plays an important role in liver regeneration and resistance to acute injury. However its chronic activation participates in the progression of liver disease, including fibrogenesis and malignant transformation. Hepatobiliary disease represents a constant feature in the clinically relevant Fechm1pas/Fechm1pas genetic model of erythropoietic protoporphyria (EPP). Similarly, chronic administration of griseofulvin to mice induces pathological changes similar to those found in patients with EPP-associated liver injury. We investigated the hepatic expression of the EGFR and its seven most relevant ligands in Fechm1pas/Fechm1pas mice bred in three different backgrounds, and in griseofulvin-induced protoporphyria. We observed that the expression of amphiregulin, betacellulin and epiregulin was significantly increased in young EPP mice when compared to aged-matched controls in all genetic backgrounds. The expression of these ligands was also tested in older (11 months) BALB/cJ EPP mice, and it was found to remain induced, while that of the EGFR was downregulated. Griseofulvin feeding also increased the expression of amphiregulin, betacellulin and epiregulin. Interestingly, protoporphyrin accumulation in cultured hepatic AML-12 cells readily elicited the expression of these three EGFR ligands. Our findings suggest that protoporphyrin could directly induce the hepatic expression of EGFR ligands, and that their chronic upregulation might participate in the pathogenesis of EPP-associated liver disease.
- Gene therapy for liver diseases — progress and challenges(Springer Nature, 2023) González-Aseguinolaza, G. (Gloria); Zabaleta-Lasarte, N. (Nerea); Weber, N.D. (Nicholas D.); Unzu, C. (Carmen)Gene therapy is poised to revolutionize modern medicine, with seemingly unlimited potential for treating and curing genetic disorders. For otherwise incurable indications, including most inherited metabolic liver disorders, gene therapy provides a realistic therapeutic option. In this Review, we discuss gene supplementation and gene editing involving the use of recombinant adeno-associated virus (rAAV) vectors for the treatment of inherited liver diseases, including updates on several ongoing clinical trials that are producing promising results. Clinical testing has been essential in highlighting many key translational challenges associated with this transformative therapy. In particular, the interaction of a patient's immune system with the vector raises issues of safety and the duration of treatment efficacy. Furthermore, several serious adverse events after the administration of high doses of rAAVs suggest greater involvement of innate immune responses and pre-existing hepatic conditions than initially anticipated. Finally, permanent modification of the host genome associated with rAAV genome integration and gene editing raises concerns about the risk of oncogenicity that require careful evaluation. We summarize the main progress, challenges and pathways forward for gene therapy for liver diseases.