Helper-dependent adenovirus achieve more efficient and persistent liver transgene expression in non-human primates under immunosuppression.

Helper-dependent adenoviral (HDA) vectors constitute excellent gene therapy tools for metabolic liver diseases. We have previously shown that an HDA vector encoding human porphobilinogen deaminase (PBGD) corrects acute intermittent porphyria mice. Now, six non-human primates were injected in the left hepatic lobe with the PBGD-encoding HDA vector to study levels and persistence of transgene expression. Intrahepatic administration of 5 × 10(12) viral particles kg(-1) (10(10) infective units kg(-1)) of HDA only resulted in transient (≈14 weeks) transgene expression in one out of three individuals. In contrast, a more prolonged 90-day immunosuppressive regimen (tacrolimus, mycophenolate, rituximab and steroids) extended meaningful transgene expression for over 76 weeks in two out of two cases. Transgene expression under immunosuppression (IS) reached maximum levels 6 weeks after HDA administration and gradually declined reaching a stable plateau within the therapeutic range for acute porphyria. The non-injected liver lobes also expressed the transgene because of vector circulation. IS controlled anticapsid T-cell responses and decreased the induction of neutralizing antibodies. Re-administration of HDA-hPBGD at week +78 achieved therapeutically meaningful transgene expression only in those animals receiving IS again at the time of this second vector exposure. Overall, immunity against adenoviral capsids poses serious hurdles for long-term HDA-mediated liver transduction, which can be partially circumvented by pharmacological IS.

PET imaging of thymidine kinase gene expression in the liver of non-human primates following systemic delivery of an adenoviral vector.

Non-invasive in vivo imaging of transgene expression is currently providing very important means to optimize gene therapy regimes. Results in non-human primates are considered the most predictive models for the outcome in patients. In this study, we have documented that tumour and primary cell lines from human and non-human primates are comparably gene-transduced in vitro by serotype 5 adenovirus expressing HSV1-thymidine kinase. Transgene expression can be quantified in human and monkey cultured cells by positron emission tomography (PET) imaging when transduced cells are incubated with a fluoride-18 labelled penciclovir analogue. In our hands, PET images of cell cultures estimate the number of transduced cells rather than intensity of transgene expression once a threshold of TK per cell is reached. Interestingly, in vivo systemic administration of a clinical grade recombinant adenovirus expressing TK into macaques gives rise to an intense retention of the radiotracer in the liver parenchyma, providing an experimental system to visualize transgene expression that ought to be similar in human and macaques. Such imaging methodology might contribute to improve strategies based on adenoviral vectors.

Induction of hepatic aminolevulinate acid synthetase activity by isoflurane in a genetic model for erythropoietic protoporphyria.

Erythropoietic Protoporphyria (EPP) is an inherited deficiency of ferrochelatase, the last enzyme of the heme pathway. Under general anaesthesia, some patients develop neurological dysfunction suggesting upregulation in heme biosynthesis similar to that described for acute porphyrias after xenobiotic administration. Our aim has been to evaluate whether Isoflurane induces alterations in the heme pathway in a mouse model for EPP. Administration of Isoflurane (a single dose of 2 ml/kg, i.p) to wild-type (+/+), heterozygous (+/Fechm1Pas) and homozygous (Fechm1Pas/Fechm1Pas) mice, was evaluated by measuring the activity of delta-aminolevulinic acid synthetase (ALA-S) and Porphobilinogen-deaminase (PBG-D) in different tissues, as well as Heme oxygenase (HO), cytochrome P-450, CYP2E1 and glutathione levels in liver. Porphyrin precursors were measured in 24 h-urine samples. Fechm1Pas/Fechm1Pas mice receiving anaesthesia show enhanced ALA-S and CYP2E1 activities in the liver and increased urinary excretion of porphyrin precursors. No alterations were found in either PBG-D or HO activities. Diminished glutathione levels suggest that anaesthesia may produce oxidative stress in these animals. In conclusion, Isoflurane induces ALA-S activity and increased excretion of porphyrin precursors in EPP mice. These findings appear to confirm our previous hypothesis and indicate that Isoflurane may be an unsafe anaesthetic not only for patients with acute porphyrias but also for individuals with non acute porphyrias.

Identification and characterization of HMBS gene mutations in Spanish patients with acute intermittent porphyria.

Acute intermittent porphyria (AIP), the most common acute hepatic porphyria, is an autosomal dominant disorder with low penetrance that results from a partial deficiency of hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. The disease is clinically characterized by acute neurovisceral attacks that are precipitated by several factors including certain drugs, steroid hormones, alcohol and fasting. Early diagnosis and counselling are essential to prevent attacks, being mutation analysis the most reliable method to identify asymptomatic carriers in AIP families. In this study we have investigated the molecular defect in 15 unrelated Spanish AIP patients. Mutation analysis of the HMBS gene revealed a total of fourteen mutations including six novel ones, two of them were on the same allele in one patient. The novel mutations were three missense (R26L, R173G and D178H), two frameshift (c.749_765dup and c.874insC) and one intronic deletion (IVS12+3_+11delAGGGCCTGT). RT-PCR and sequencing demonstrated that the intronic mutation caused abnormal splicing and exon 12 skipping. Prokaryotic expression of the novel missense mutations showed that only D178H had significant residual activity. These findings will facilitate the accurate identification of presymptomatic AIP carriers in these families and they further emphasize the molecular heterogeneity of AIP in Spain.

Epidermal growth factor receptor ligands in murine models for erythropoietic protoporphyria: potential novel players in the progression of liver injury.

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.

Correlation between levels of free and protein-bound plasma porphyrin and urinary porphyrins in porphyria cutanea tarda.

Porphyria cutanea tarda (PCT) is a disorder of porphyrin metabolism that leads to massive overproduction and excretion of uroporphyrin. Most plasma porphyrins are bound to albumin and hemopexin. The aim of this work was to analyze the relationship between the concentrations of serum albumin and hemopexin, the levels of total, free and protein-bound plasma porphyrins and the urinary coproporphyrin and uroporphyrin excretion, in PCT patients at different stages of the disease. Urinary porphyrins showed a stronger correlation with total plasma porphyrin levels (r = 0.863) than with the free fraction of plasma porphyrins (r = 0.608). Patients considered in an active stage of PCT, have a higher mean level of total plasma porphyrins (8.80 micrograms/dl +/- 8.75) and a lower mean percentage of free plasma porphyrins (12.79% +/- 11.21) than those patients on remission (0.71 +/- 0.5 microgram/dl and 44.3 +/- 35.3%, respectively). 30% of patients showed hypohemopexinaemia, presumably due to hepatic damage. Despite the high affinity of this protein for porphyrins, no significant correlation was found between plasma porphyrin levels and hemopexin or albumin. It is concluded that (i) the kidney is not merely a passive filter for free plasma porphyrins and (ii) that the formation of hemopexin-porphyrin complex occurs when plasma porphyrins concentrations are increased (i.e. in those patients in an active stage of PCT).

A systematic analysis of the mutations of the uroporphyrinogen III synthase gene in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) or Günther's disease is an inborn error of heme biosynthesis, transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase activity (UROIIIS). The molecular defects observed in CEP are mainly heterogeneous, except for one missense mutation, C73R (Cys to Arg substitution at codon 73) which represents nearly 40% of the disease alleles. A convenient strategy was designed to establish a rapid diagnosis at the genetic level in samples from patients with CEP. As a first step, the most frequent mutation is searched for by restriction analysis from genomic. DNA amplified by PCR. Next, the nine coding exons and intron-exon boundaries are sequenced from genomic DNA. As an alternative, the mutation can be determined by sequencing the UROIIIS cDNA of the patient, using the RT-PCR technique on RNAs when a lymphoblastoid cell line can be established. Finally, for each new mutation in UROIIIS coding sequence, the corresponding mutant protein is expressed in Escherichia coli, in order to demonstrate the pathological significance of the mutation. This work describes the analysis of UROIIIS gene mutations in 10 new families with CEP and summarizes the data from 20 unrelated families studied in our laboratory. Three new missense mutations of UROIIIS coding sequence (H173Y, Q187P and P248Q) have been observed together with 8 known mutations. The significance of three intronic base changes (476 -31 T-->C; 562 -4 A-->T; 562 -23 A-->G) is discussed. In 6 alleles out of 40 (15%), the mutation remains undetermined.

A novel immunodeficient mouse model--RAG2 x common cytokine receptor gamma chain double mutants--requiring exogenous cytokine administration for human hematopoietic stem cell engraftment.

Gene transduction into immature human hematopoietic cells collected from umbilical cord blood, bone marrow, or mobilized peripheral blood cells could be useful for the treatment of genetic and acquired disorders of the hematopoietic system. Immunodeficient mouse models have been used frequently as recipients to assay the growth and differentiation of human hematopoietic stem/progenitor cells. Indeed, high levels of human cell engraftment were first reported in human/murine chimeras using NOD/SCID mice, which now are considered as the standard for these types of experiments. However, NOD/SCID mice have some clear disadvantages (including spontaneous tumor formation) that limit their general use. We have developed a new immunodeficient mouse model by combining recombinase activating gene-2 (RAG2) and common cytokine receptor gamma chain (gamma c) mutations. The RAG2-/-/gamma c- double mutant mice are completely alymphoid (T-, B-, NK-), show no spontaneous tumor formation, and exhibit normal hematopoietic parameters. Interestingly, human cord blood cell engraftment in RAG2-/-/gamma c- mice was greatly enhanced by the exogenous administration of human cytokines interleukin-(IL-3) granulocyte-macrophage colony-stimulating factor, (GM-CSF), and erythropoietin in contrast to the NOD/SCID model. This unique feature of the RAG2-/-/gamma c- mouse model should be particularly well suited for assessing the role of different cytokines in human lymphopoiesis and stem/progenitor cell function in vivo.

Salivary porphyrin concentration in porphyria cutanea tarda.

To assess the clinical utility of saliva samples, serum, urine and saliva porphyrin concentration were fluorometrically measured in 31 patients with porphyria cutanea tarda. In comparison with normal values, porphyrin mean levels were 20-fold increased in serum and urine but only 3-fold in saliva. Saliva porphyrin concentration exhibited significant but weak correlations with porphyrin levels in serum (r = 0.69) and urine (r = 0.67). However, saliva porphyrins were not more closely correlated with protein-unbound serum porphyrins (r = 0.57). This finding suggests that saliva porphyrin concentration does not simply correspond to the filtered free fraction of serum porphyrins. Nevertheless, measurement of saliva porphyrins could represent a valuable non-invasive alternative to serum porphyrins in the monitoring of patients with porphyria cutanea tarda.

[Prevalence of porphyria cutanea tarda in Madrid and relationship between urine porphyrin and ethanol intake in a multiple linear regression model]. / Prevalencia de porfiria cutánea tarda en Madrid y asociación entre la porfirinuria y el consumo de etanol en un modelo de regresión lineal múltiple.

BACKGROUND: The application of a simple fluorometric analytical method enabled us to quantify the urinary porphyrin excretion and to establish the prevalence of porphyria cutanea tarda (PCT) in the town of Madrid, Spain, in a cross-sectional study. PATIENTS AND METHODS: The study assessed 1,613 subjects from three districts in Madrid, in whom further variables potentially related to porphyrinuria such as ethanol intake or -in women-oral contraceptive use were measured and recorded. RESULTS: The estimated prevalence of the disease was 1.24 cases per 1,000 inhabitants (95% confidence interval 0.15-4.47 per thousand). After excluding from the study sample all cases with existent disease, an analysis was performed to ascertain an unilateral tolerance interval for urinary porphyrin concentration in the adult population; this level was established at 181.2 micrograms/l. The effect of ethanol intake on porphyrinuria was considered significant using a multiple linear regression model adjusted for the control variables gender, age and body mass index. In fertile women, contraceptive use did not attain statistical significance when that variable was included in a multiple regression model. CONCLUSIONS: A high prevalence has been estimated for PCT in the Madrid population. A significant association was further found between alcohol intake and porphyrinuria in non-porphyric adults.

[Prospects for gene therapy in hematology]. / Perspectives de thérapy génique en hématologie.

Gene transfer in hematopoietic cells is intended to treat patients with malignant disease and inherited monogenic (hematological, immunological, and metabolic) disorders. Hematopoietic progenitor or stem cells are a favoured target for gene therapy because these cells are easily withdrawn from the patient, expanded and genetically modified ex vivo and then reinjected into the organism. Retroviral vectors allow an efficient transfer of the genes of interest. Transduction of stem cells leads to a stable expression of the transgene for long periods of time. However, we are at the beginning of this new therapeutic application, the technique was being already successful in very few cases. Problems to be solved are mainly in the understanding of the physiology of the hematopoietic stem cell and in the improvement of technical qualities of the vectors for a targeted gene transfer in vivo.

[Laboratory approximation to the diagnosis and typification of porphyria]. / Aproximación de laboratorio al diagnóstico y tipificación de una porfiria.

The relationship between clinical features and specific alterations of heme metabolism allows the accurate diagnosis and classification of porphyrias. The acute porphyric attack is characterized by frequently confusing clinical pattern of abdominal-psiquic- and neurological symptoms. An increased urinary excretion of porphobilinogen, which can be quickly detected by the Hoesch test, confirms the diagnosis of this acute attack. Increased plasma porphyrin levels are associated with skin lesions, which are the characteristic features of the cutaneous porphyrias. Their presence is easily and rapidly detected by a fluorimetric scanning of PBS (phosphate buffer saline) diluted samples. Characterization of the molecular defects in genes coding for the enzymes involved in the heme synthetic pathway is complementary to the biochemical methods. Molecular analysis permits an accurate classification of those biochemically unclassified patients and allows prenatal diagnosis in those homozygotic cases where a severe prognosis is suspected.

Molecular heterogeneity of familial porphyria cutanea tarda in Spain: characterization of 10 novel mutations in the UROD gene.

BACKGROUND: Porphyria cutanea tarda (PCT) results from decreased hepatic uroporphyrinogen decarboxylase (UROD) activity. In the majority of patients, the disease is sporadic (S-PCT or type I) and the enzyme deficiency is limited to the liver. Familial PCT (F-PCT or type II) is observed in 20-30% of patients in whom mutations on one allele of the UROD gene reduce UROD activity by approximately 50% in all tissues. Another variant of PCT (type III) is characterized by family history of the disease although it is biochemically indistinguishable from S-PCT. OBJECTIVES: To investigate the molecular basis of PCT in Spain and to compare enzymatic and molecular analysis for the identification of patients with F-PCT. METHODS: Erythrocyte UROD activity measurement and mutation analysis of the UROD gene were carried out in a cohort of 61 unrelated Spanish patients with PCT and 50 control individuals. Furthermore, each novel missense mutation identified was characterized by prokaryotic expression studies. RESULTS: Of these 61 patients, 40 (66%) were classified as having S-PCT, 16 (26%) as having F-PCT and five (8%) as having type III PCT. Discordant results between enzymatic and molecular analysis were observed in two patients with F-PCT. In total, 14 distinct mutations were found, including 10 novel mutations: five missense, one nonsense, three deletions and an insertion. Prokaryotic expression of the novel missense mutations demonstrated that each results in decreased enzyme activity or stability. CONCLUSIONS: These results confirm the high degree of molecular heterogeneity of F-PCT in Spain and emphasize the usefulness of molecular genetic analysis to distinguish between F-PCT and S-PCT.

Reduced aminolevulinate dehydrase activity in rats with functional renal failure induced by cyclosporin A.

Patients with chronic failure evidence various abnormalities in heme metabolism, primarily erythrocyte aminolevulinate dehydrase hypoactivity and increased plasma and erythrocyte porphyrin levels. Such abnormalities have also been observed in animals with both acute and chronic experimental renal failure. The aim of this work was to study these parameters of porphyrin metabolism in an experimental model of functional renal failure. A group of 11 male Wistar rats received 13 doses (25 mg/kg body weight/day) of cyclosporin A. Serum creatinine did not vary, but the blood urea nitrogen levels increased and a significant decrease in the creatinine clearance was observed. The drug also caused a marked decrease in the erythrocyte aminolevulinate dehydrase activity, a slight reduction of the hematocrit value, and increased levels of blood porphyrins. The plasma of treated rats showed capacity to inhibit aminolevulinate dehydrase activity when incubated in vitro with erythrocytes from control rats. Porphyrin metabolism remained unchanged in the liver. The daily diuresis was significantly decreased in the cyclosporin as compared to the control group; however, the porphyrinuria showed no changes. The derangements in the erythrocyte heme biosynthesis pathway observed in patients with chronic renal failure are reproducible in an experimental model of cyclosporin A-induced functional renal failure.

Nonsynergic effect of ethanol and lead on heme metabolism in rats.

The heme biosynthetic pathway is a metabolic target of alcohol and lead poisoning. To analyze the interdependence of both xenobiotics on porphyrin metabolism, male Wistar rats (n=47) were divided into four groups and were fed Lieber-DeCarli semiliquid control or alcoholic diets containing or not containing lead acetate (160 mg/liter) for 8 weeks. After this period, hematocrit values and porphyrin concentration in liver and urine were similar in all groups, indicating that the goal of inducing only mild chronic intoxication was achieved. Compared with the control group, rats poisoned only with lead exhibited high levels of this metal in blood and liver, increased erythrocytic protoporphyrin, and hypoactivity of aminolevulinate dehydrase (ALA-D) in both blood and liver. Rats intoxicated only with alcohol exhibited mild hypoactivity of both hepatic and erythrocytic ALA-D, although such decreased enzymatic values did not achieve statistical significance. Rats receiving ethanol and lead simultaneously demonstrated abnormalities in heme biosynthesis similar to those in rats exposed to lead, although zinc hepatic levels decreased significantly only in animals exposed to both xenobiotics. Hepatic GSH and urinary ALA and porphyrin levels were maintained in a similar range in all groups.

Correction of uroporphyrinogen decarboxylase deficiency (hepatoerythropoietic porphyria) in Epstein-Barr virus-transformed B-cell lines by retrovirus-mediated gene transfer: fluorescence-based selection of transduced cells.

Hepatoerythropoietic porphyria (HEP) is an inherited metabolic disorder characterized by the accumulation of porphyrins resulting from a deficiency in uroporphyrinogen decarboxylase (UROD). This autosomal recessive disorder is severe, starting early in infancy with no specific treatment. Gene therapy would represent a great therapeutic improvement. Because hematopoietic cells are the target for somatic gene therapy in this porphyria, Epstein-Barr virus-transformed B-cell lines from patients with HEP provide a model system for the disease. Thus, retrovirus-mediated expression of UROD was used to restore enzymatic activity in B-cell lines from 3 HEP patients. The potential of gene therapy for the metabolic correction of the disease was demonstrated by a reduction of porphyrin accumulation to the normal level in deficient transduced cells. Mixed culture experiments demonstrated that there is no metabolic cross-correction of deficient cells by normal cells. However, the observation of cellular expansion in vitro and in vivo in immunodeficient mice suggested that genetically corrected cells have a competitive advantage. Finally, to facilitate future human gene therapy trials, we have developed a selection system based on the expression of the therapeutic gene. Genetically corrected cells are easily separated from deficient ones by the absence of fluorescence when illuminated under UV light.

Reference values of 5-aminolevulinate dehydrase and porphobilinogen deaminase in the Spanish population from Madrid.

5-Aminolevulinate dehydrase (ALA-D) and porphobilinogen deaminase (PBG-D) are cytosolic enzymes involved in heme biosynthesis. ALA-D activity is altered both genetically and by the action of various environmental factors, including exposure to lead. The activity of PBG-D is reduced in acute intermittent porphyria. The aim of this work is to establish the 95% reference range of the erythrocytic activity of ALA-D and PBG-D in a control population. ALA-D activity limits were 15.8 and 50.2 nmol of PBG/ml of red blood cells (RBCS) per minute. For the activity of ALA-D restored by addition of zinc and dithiothreitol ("restored ALA-D"), these limits were 44.1 and 86.5 units. It has been found that the "restored ALA-D"/ALA-D ratio is very useful for the evaluation of lead toxicity, and its 95% reference range was between 1.22 and 3.06. It has been demonstrated that the best method for measuring erythrocytic PBG-D is using PBG, but not ALA, as substrate; its 95% reference range was between 20.9 and 63.2 nmol of uroporphyrin/ml of RBCs per hour. Knowledge of these reference range values in a control population constitutes the basis for an accurate diagnosis of heavy metal intoxication and acute intermittent porphyria.

Efficiency of three different hemodialysis membranes for plasma porphyrin removal.

To assess the capability of three different membranes to remove porphyrins, plasma and dialysate porphyrin levels were fluorometrically measured in 10 patients with end-stage renal failure who were on hemodialysis. Three different hemodialysis membranes were used: cuprophan, polyacrylonitrile, and cellulose triacetate. Total plasma porphyrin concentrations decreased after dialysis, but to a lesser extent when using the cuprophan membrane (19%) than with the polyacrylonitrile (26%) or cellulose triacetate (30%) membranes (P < 0.01). However, since the free plasma porphyrin fraction remained unchanged, it can be assumed that the equilibrium between protein-bound and non-protein-bound (free) porphyrins is displaced toward the latter fraction. Dialysate porphyrin levels were lower (P < 0.01) when using the cuprophan membrane (10.1 micrograms/session) than when using polyacrylonitrile (17.8 micrograms/session) and cellulose triacetate (21.9 micrograms/session). Although most of the plasma porphyrins are protein bound, our results show that hemodialysis can remove significant amounts of non-protein-bound (free) porphyrins. The polyacrylonitrile and cellulose triacetate membranes had a greater capacity for porphyrin removal than cuprophan. Thus, two high-permeability membranes (polyacrylonitrile and cellulose triacetate) should be used whenever a reduction of plasma porphyrin levels is desired.

Clinical utility of fluorometric scanning of plasma porphyrins for the diagnosis and typing of porphyrias.

The fluorometric emission scanning (using excitation at 405 nm) of plasma samples, simply diluted five-fold in phosphate-buffered saline, allows the differentiation of three conditions according to their porphyrin content. The emission maximum at 626-628 nm is a specific finding in variegate porphyria, while in erythropoietic protoporphyria a characteristic peak is found at 636 nm. A fluorescence emission maximum at 618-622 nm corresponds to a third group that includes normal subjects, non-porphyria patients and patients suffering from acute intermittent porphyria, hereditary coproporphyria, congenital erythropoietic porphyria (Günther disease) and porphyria cutanea tarda. Therefore, this simple, quick and cheap screening test allows one to establish whether a patient with a photocutaneous syndrome has porphyria and whether this porphyria belongs to the types: variegate, protoporphyria or other cutaneous porphyrias.