Rafael Enríquez de Salamanca

Porphyria Cutanea Tarda and Hepatocellular Carcinoma: Frequency of Occurrence and Related Factors

In order to assess the incidence of hepatocellular carcinoma (HCC) in porphyria cutanea tarda (PCT), 83 patients (77 males, 6 females, mean age 57.4 years) were studied. Thirteen patients (15.7%) had HCC, all of whom were male and cirrhotics with a mean age of 58.5 years. HCC patients showed a statistically significant (P less than 0.0005) longer evolution time (23 years since onset of the cutaneous disease) than patients without HCC (9.4 years), while the age of onset was similar in both groups. Differences in alcohol intake and hepatitis B virus (HBV) markers were non-significant, although high prevalence (54%) of past HBV infection was found in both groups. In HCC development, attributable risks of 100% were found for cirrhosis (P less than 0.001), male sex (P = NS) and for age over 51 (P less than 0.025). Therefore, PCT harbours a high incidence of HCC; evolution time, cirrhosis and age over 51 appear to be the most important contributing factors.

Phase I open label liver-directed gene therapy clinical trial for acute intermittent porphyria

BACKGROUND & AIMS Acute intermittent porphyria (AIP) results from porphobilinogen deaminase (PBGD) haploinsufficiency, which leads to hepatic over-production of the neurotoxic heme precursors porphobilinogen (PBG) and delta-aminolevulinic acid (ALA) and the occurrence of neurovisceral attacks. Severe AIP is a devastating disease that can only be corrected by liver transplantation. Gene therapy represents a promising curative option. The objective of this study was to investigate the safety of a recombinant adeno-associated vector expressing PBGD (rAAV2/5-PBGD) administered for the first time in humans for the treatment of AIP. METHODS In this phase I, open label, dose-escalation, multicenter clinical trial, four cohorts of 2 patients each received a single intravenous injection of the vector ranging from 5×10(11) to 1.8×10(13) genome copies/kg. Adverse events and changes in urinary PBG and ALA and in the clinical course of the disease were periodically evaluated prior and after treatment. Viral shedding, immune response against the vector and vector persistence in the liver were investigated. RESULTS Treatment was safe in all cases. All patients developed anti-AAV5 neutralizing antibodies but no cellular responses against AAV5 or PBGD were observed. There was a trend towards a reduction of hospitalizations and heme treatments, although ALA and PBG levels remained unchanged. Vector genomes and transgene expression could be detected in the liver one year after therapy. CONCLUSIONS rAAV2/5-PBGD administration is safe but AIP metabolic correction was not achieved at the doses tested in this trial. Notwithstanding, the treatment had a positive impact in clinical outcomes in most patients. LAY SUMMARY Studies in an acute intermittent porphyria (AIP) animal model have shown that gene delivery of PBGD to hepatocytes using an adeno-associated virus vector (rAAV2/5-PBG) prevent mice from suffering porphyria acute attacks. In this phase I, open label, dose-escalation, multicenter clinical trial we show that the administration of rAAV2/5-PBGD to patients with severe AIP is safe but metabolic correction was not achieved at the doses tested; the treatment, however, had a positive but heterogeneous impact on clinical outcomes among treated patients and 2 out of 8 patients have stopped hematin treatment. CLINICAL TRIAL NUMBER The observational phase was registered at Clinicaltrial.gov as NCT 02076763. The interventional phase study was registered at EudraCT as n° 2011-005590-23 and at Clinicaltrial.gov as NCT02082860.

Cerebrospinal fluid levels of alpha-tocopherol in patients with multiple sclerosis

We compared cerebrospinal fluid (CSF) and serum levels, and the CSF/serum ratio of alpha-tocopherol (vitamin E), measured by HPLC, in 36 patients with multiple sclerosis (MS) and 32 matched controls. The mean CSF vitamin E levels and the CSF/serum vitamin E ratio did not differ significantly between the two study groups. The serum levels of vitamin E and the serum vitamin E/cholesterol ratio were significantly lower in MS patients when compared with controls (P < 0.05 and P < 0.01, respectively). These values were not correlated with age, age at onset and duration of the disease in the patients group. These results suggest that CSF vitamin E concentrations are not a marker of activity of MS activity.

Sustained Enzymatic Correction by rAAV-Mediated Liver Gene Therapy Protects Against Induced Motor Neuropathy in Acute Porphyria Mice

Acute intermittent porphyria (AIP) is characterized by a hereditary deficiency of hepatic porphobilinogen deaminase (PBGD) activity. Clinical features are acute neurovisceral attacks accompanied by overproduction of porphyrin precursors in the liver. Recurrent life-threatening attacks can be cured only by liver transplantation. We developed recombinant adeno-associated virus (rAAV) vectors expressing human PBGD protein driven by a liver-specific promoter to provide sustained protection against induced attacks in a predictive model for AIP. Phenobarbital injections in AIP mice induced porphyrin precursor accumulation, functional block of nerve conduction, and progressive loss of large-caliber axons in the sciatic nerve. Hepatocyte transduction showed no gender variation after rAAV2/8 injection, while rAAV2/5 showed lower transduction efficiency in females than males. Full protection against induced phenobarbital-attacks was achieved in animals showing over 10% of hepatocytes expressing high amounts of PBGD. More importantly, sustained hepatic expression of hPBGD protected against loss of large-caliber axons in the sciatic nerve and disturbances in nerve conduction velocity as induced by recurrent phenobarbital administrations. These data show for the first time that porphyrin precursors generated in the liver interfere with motor function. rAAV2/5-hPBGD vector can be produced in sufficient quantity for an intended gene therapy trial in patients with recurrent life-threatening porphyria attacks.

Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in non-human primates

BackgroundAdeno-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 work, 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.MethodsThree 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.ResultsMacaques 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.ConclusionsThese 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.

MECHANISTIC STUDIES ON UROPORPHYRIN I-INDUCED PHOTOINACTIVATION OF SOME HEME-ENZYMES

Aerobic and anaerobic studies have demonstrated that uroporphyrin I-induced inactivation of delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase was dependent on oxygen and mediated by reactive oxygen species. The mechanism of photoinactivation of those heme-enzymes from human erythrocytes by uroporphyrin I by u.v. light was investigated. Enzymes of the heme pathway were preincubated in the presence of specific scavengers for several reactive oxygen species and then exposed to uroporphyrin I and u.v. light. Upon exposure of the enzymes to the porphyrin under u.v. light, and in an aerobic atmosphere, the percentage of enzyme activities with respect to the corresponding controls were 50.2 +/- 5.1 (SD, n = 6), 25.3 +/- 3.0 (SD, n = 6), 25.9 +/- 2.8 (SD, n = 6) and 49.7 +/- 7.5 (SD, n = 8) for delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase, respectively. The presence of sodium azide, histidine or superoxide dismutase did not protect the enzymes against the effects of uroporphyrin I. However, both cysteine and potassium ferrycyanide prevented the enzyme photoinactivation induced by uroporphyrin I. In the presence of either catalase or GSH, the enzyme photoinactivation was lower. Ethanol, glucose and dimethylsulfoxide had no effect on enzyme activity, while ion chelators had variable effects. This study shows that the type II mechanism is not the predominant reaction mediating the uroporphyrin I effect and enzyme photoinactivation would involve an electron transfer. Hydrogen peroxide and hydroxyl radicals could possibly mediate the uroporphyrin I-induced enzyme photoinactivation.

Photodynamic inactivation of red cell uroporphyrinogen decarboxylase by porphyrins

The effects of light and porphyrins on the activity of red cell uroporphyrinogen decarboxylase were studied. Photoinactivation of uroporphyrinogen decarboxylase was dependent on uroporphyrin concentration, irradiation time and temperature. Using 40 W/m2 of UV light intensity, 40-45% decreased activity was produced with 200 microM uroporphyrin I, at 37 degrees C and after 2 hr of illumination. It has been demonstrated that porphyrins photoinactivate uroporphyrinogen decarboxylase and a mechanism for this action in relation to skin lesions is proposed.

Porphobilinogen deaminase over-expression in hepatocytes, but not in erythrocytes, prevents accumulation of toxic porphyrin precursors in a mouse model of acute intermittent porphyria

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.

Administration of the anesthetic isoflurane to mice: A model for acute intermittent porphyria?

The effects of isoflurane, a commonly used volatile anesthetic, on the activity of some haem enzymes in liver, kidney, and blood, and glucose content in liver and blood were studied. Mice were injected with different doses of the drug (0.5-6 mL/kg) and killed at varying intervals after injection (5-240 min). Within this dose range, optimal effects on alteration of haem metabolism were obtained at 2 mL/kg. The time-response profile for each enzyme was different. Blood porphobilinogenase (PBGase) and deaminase showed lower activities 20 min after anesthesia. This diminution coupled with the induction of delta-aminolevulinate synthetase activity observed soon after anesthesia (5 min) would fit well with the expected biochemical changes occurring in acute intermittent porphyria, indicating that this may be a suitable animal model for this disease.

Helper-dependent adenoviral liver gene therapy protects against induced attacks and corrects protein folding stress in acute intermittent porphyria mice

Acute intermittent porphyria (AIP) is a hepatic metabolic disease that results from haplo-insufficient activity of porphobilinogen deaminase (PBGD). The dominant clinical feature is acute intermittent attacks when hepatic heme synthesis is activated by endocrine or exogenous factors. Gene therapy vectors over-expressing PBGD protein in the liver offers potential as a cure for AIP. Here, we developed a helper-dependent adenovirus (HDA) encoding human PBGD (hPBGD) and assessed its therapeutic efficacy in a murine model of AIP. Intravenous or intrahepatic administration of HDA-hPBGD to AIP mice resulted in a sustained hepatic hPBGD expression in a dose-dependent manner. Intrahepatic administration conveyed full protection against induced porphyria attacks at a significantly lower viral dose than intravenous injection. Transgenic hPBGD accumulated only in the cytosol of hepatocytes as the endogenous protein. Characterization of PBGD-deficient mouse strains revealed that a strong PBGD deficiency causes the chronic disturbance of cytosolic and endoplasmic reticulum folding machineries. This disturbance was completely restored over time by the over-expression of hPBGD. HDA-hPBGD is a promising vector that protects against porphyria attacks and resolves the chronic folding stress associated with low levels of PBGD activity.