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Background: Pancreatic ductal adenocarcinoma is a deadly malignancy resistant to current therapies. It is critical to test new strategies, including tumor-targeted delivery of therapeutic agents. This study tested the possibility to target the transfer of a suicide gene in tumor cells using an oncotropic lentiviral vector., Results: Three cell surface markers were evaluated to target the transduction of cells by lentiviruses pseudotyped with a modified glycoprotein from Sindbis virus. Only Mucin-4 and the Claudin-18 proteins were found efficient for targeted lentivirus transductions in vitro. In subcutaneous xenografts of human pancreatic cancer cells models, Claudin-18 failed to achieve efficient gene transfer but Mucin-4 was found very potent. Human pancreatic tumor cells were modified to express a fluorescent protein detectable in live animals by bioimaging, to perform a direct non invasive and costless follow up of the tumor growth. Targeted gene transfer of a bicistronic transgene bearing a luciferase gene and the herpes simplex virus thymidine kinase gene into orthotopic grafts was carried out with Mucin-4 oncotropic lentiviruses. By contrast to the broad tropism VSV-G carrying lentivirus, this oncotropic lentivirus was found to transduce specifically tumor cells, sparing normal pancreatic cells in vivo. Transduced cells disappeared after ganciclovir treatment while the orthotopic tumor growth was slowed down., Conclusion: This work considered for the first time three aspect of pancreatic adenocarcinoma targeted therapy. First, lentiviral transduction of human pancreatic tumor cells was possible when cells were grafted orthotopically. Second, we used a system targeting the tumor cells with cell surface antigens and sparing the normal cells. Finally, the TK/GCV anticancer system showed promising results in vivo. Importantly, the approach presented here appeared to be a safer, much more specific and an as efficient way to perform gene delivery in pancreatic tumors, in comparison with a broad tropism lentivirus. This study will be useful in future designing of targeted therapies for pancreatic cancer.

Congenital erythropoietic porphyria (CEP) is due to a deficiency in the enzymatic activity of uroporphyrinogen III synthase (UROS); such a deficiency leads to porphyrin accumulation and results in skin lesions and hemolytic anemia. CEP is a candidate for retrolentivirus-mediated gene therapy, but recent reports of insertional leukemogenesis underscore the need for safer methods. The discovery of induced pluripotent stem cells (iPSCs) has opened up new horizons in gene therapy because it might overcome the difficulty of obtaining sufficient amounts of autologous hematopoietic stem cells for transplantation and the risk of genotoxicity. In this study, we isolated keratinocytes from a CEP-affected individual and generated iPSCs with two excisable lentiviral vectors. Gene correction of CEP-derived iPSCs was obtained by lentiviral transduction of a therapeutic vector containing UROS cDNA under the control of an erythroid-specific promoter shielded by insulators. One iPSC clone, free of reprogramming genes, was obtained with a single proviral integration of the therapeutic vector in a genomic safe region. Metabolic correction of erythroblasts derived from iPSC clones was demonstrated by the disappearance of fluorocytes. This study reports the feasibility of porphyria gene therapy with the use of iPSCs., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Congenital erythropoietic porphyria (CEP) is a severe autosomal recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We recently demonstrated the definitive cure of a murine model of CEP by lentiviral vector-mediated hematopoietic stem cell (HSC) gene therapy. In the perspective of a gene therapy clinical trial, human cellular models are required to evaluate the therapeutic potential of lentiviral vectors in UROS-deficient cells. However, the rare incidence of the disease makes difficult the availability of HSCs derived from patients., Methods: RNA interference (RNAi) has been used to develop a new human model of the disease from normal cord blood HSCs. Lentivectors were developed for this purpose., Results: We were able to down-regulate the level of human UROS in human cell lines and primary hematopoietic cells. A 97% reduction of UROS activity led to spontaneous uroporphyrin accumulation in human erythroid bone marrow cells of transplanted immune-deficient mice, recapitulating the phenotype of cells derived from patients. A strong RNAi-induced UROS inhibition allowed us to test the efficiency of different lentiviral vectors with the aim of selecting a safer vector. Restoration of UROS activity in these small hairpin RNA-transduced CD34(+) cord blood cells by therapeutic lentivectors led to a partial correction of the phenotype in vivo., Conclusions: The RNAi strategy is an interesting new tool for preclinical gene therapy evaluation., (Copyright 2010 John Wiley & Sons, Ltd.)

The inherited porphyrias are inborn errors of haem biosynthesis, each resulting from the deficient activity of a specific enzyme of the haem biosynthetic pathway. Porphyrias are divided into erythropoietic and hepatic according to the predominant porphyrin-accumulating tissue. Three different erythropoietic porphyrias (EP) have been described: erythropoietic protoporphyria (EPP, MIM 177000) the most frequent, congenital erythropoietic porphyria (CEP, MIM 263700), and the very rare hepatoerythropoietic porphyria (HEP, MIM 176100). Bone marrow transplantation is considered as the only curative treatment for severe cases of erythropoietic porphyria (especially CEP), if donors are available. Some EPP patients who undergo liver failure may require hepatic transplantation. Murine models of EPP and CEP have been developed and mimic most of the human disease features. These models allow a better understanding of the pathophysiological mechanisms involved in EP as well as the development of new therapeutic strategies. The restoration of deficient enzymatic activity in the bone marrow compartment following gene therapy has been extensively studied. Murine oncoretroviral, and recently, lentiviral vectors have been successfully used to transduce hematopoietic stem cells, allowing full metabolic and phenotypic correction of both EPP and CEP mice. In CEP, a selective survival advantage of corrected cells was demonstrated in mice, reinforcing the arguments for a gene therapy approach in the human disease. These successful results form the basis for gene therapy clinical trials in severe forms of erythropoietic porphyrias.

Porphyrias are a group of disorders due to a genetic deficiency in one of the heme biosynthetic pathway enzymes. Congenital erythropoietic porphyria (CEP) is the most severe type characterized by a deficiency in uroporphyrinogen III synthase (UROS) activity. Bone marrow transplantation represents a curative treatment for patients, as long as human leucocyte antigen-compatible donor is available. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros(mut 248) mice resulted in a complete and long-term enzymatic, metabolic and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate for the first time that the cure of this mouse model of CEP at moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified HSCs.

Erythropoietic protoporphyria (EPP) is an inherited defect of the ferrochelatase (FECH) gene characterized by the accumulation of toxic protoporphyrin in the liver and bone marrow resulting in severe skin photosensitivity. We previously described successful gene therapy of an animal model of the disease with erythroid-specific lentiviral vectors in the absence of preselection of corrected cells. However, the high-level of gene transfer obtained in mice is not translatable to large animal models and humans if there is no selective advantage for genetically modified hematopoietic stem cells (HSCs) in vivo. We used bicistronic SIN-lentiviral vectors coexpressing EGFP or FECH and the G156A-mutated O6-methylguanine-DNA-methyltransferase (MGMT) gene, which allowed efficient in vivo selection of transduced HSCs after O6-benzylguanine and BCNU treatment. We demonstrate for the first time that the correction and in vivo expansion of deficient transduced HSC population can be obtained by this dual gene therapy, resulting in a progressive increase of normal RBCs in EPP mice and a complete correction of skin photosensitivity. Finally, we developed a novel bipromoter SIN-lentiviral vector with a constitutive expression of MGMT gene to allow the selection of HSCs and with an erythroid-specific expression of the FECH therapeutic gene.

Background: Erythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to the accumulation of toxic protoporphyrin in the liver, bone marrow and spleen. We have previously shown that a successful gene therapy of a murine model of the disease was possible with lentiviral vectors even in the absence of preselection of corrected cells, but lethal irradiation of the recipient was necessary to obtain an efficient bone marrow engraftment. To overcome a preconditioning regimen, a selective growth advantage has to be conferred to the corrected cells., Methods: We have developed a novel bicistronic lentiviral vector that contains the human alkylating drug resistance mutant O(6)-methylguanine DNA methyltransferase (MGMT G156A) and FECH cDNAs. We tested their capacity to protect hematopoietic cell lines efficiently from alkylating drug toxicity and correct enzymatic deficiency., Results: EPP lymphoblastoid (LB) cell lines, K562 and cord-blood-derived CD34(+) cells were transduced at a low multiplicity of infection (MOI) with the bicistronic constructs. Resistance to O(6)-benzylguanine (BG)/N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) was clearly shown in transduced cells, leading to the survival and expansion of provirus-containing cells. Corrected EPP LB cells were selectively amplified, leading to complete restoration of enzymatic activity and the absence of protoporphyrin accumulation., Conclusions: This study demonstrates that a lentiviral vector including therapeutic and G156A MGMT genes followed by BG/BCNU exposure can lead to a full metabolic correction of deficient cells. This vector might form the basis of new EPP mouse gene therapy protocols without a preconditioning regimen followed by in vivo selection of corrected hematopoietic stem cells., (Copyright 2003 John Wiley & Sons, Ltd.)

Congenital erythropoietic porphyria (CEP) is an inherited disease due to a deficiency in the uroporphyrinogen III synthase, the fourth enzyme of the heme biosynthesis pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood and other organs. The prognosis of CEP is poor, with death often occurring early in adult life. For severe transfusion-dependent cases, when allogeneic cell transplantation cannot be performed, the autografting of genetically modified primitive/stem cells may be the only alternative. In vitro gene transfer experiments have documented the feasibility of gene therapy via hematopoietic cells to treat this disease. In the present study lentiviral transduction of porphyric cell lines and primary CD34(+) cells with the therapeutic human uroporphyrinogen III synthase (UROS) cDNA resulted in both enzymatic and metabolic correction, as demonstrated by the increase in UROS activity and the suppression of porphyrin accumulation in transduced cells. Very high gene transfer efficiency (up to 90%) was achieved in both cell lines and CD34(+) cells without any selection. Expression of the transgene remained stable over long-term liquid culture. Furthermore, gene expression was maintained during in vitro erythroid differentiation of CD34(+) cells. Therefore the use of lentiviral vectors is promising for the future treatment of CEP patients by gene therapy.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell (HSC) disorder in which an acquired somatic mutation of the X-linked PIGA gene results in a deficiency in GPI-anchored surface proteins. Clinically, PNH is dominated by a chronic hemolytic anemia, often associated with recurrent nocturnal exacerbations, neutropenia, thrombocytopenia, and thrombotic tendency. Allogenic bone marrow transplantation is the only potentially curative treatment for severe forms of PNH but is associated with a high treatment-related morbidity and mortality. HSC gene therapy could provide a new therapeutic option, especially when an HLA-matched donor is not available. To develop an efficient gene transfer approach, we have designed a new SIN lentiviral vector (TEPW) that contains the PIGA cDNA driven by the human elongation factor 1 alpha promoter, the central DNA flap of HIV-1, and the WPRE cassette. TEPW transduction led to a complete surface expression of the GPI anchor and CD59 in PIGA-deficient cell lines without any selection procedure. Moreover, efficient gene transfer was achieved in bone marrow and mobilized peripheral blood CD34(+) cells derived from two patients with severe PNH disease. This expression was stable during erythroid, myeloid, and megakaryocytic liquid culture differentiation. CD59 surface cell expression was fully restored during 5 weeks of long-term culture.

Spatial and temporal control of transgene expression is one of the major prerequisites of efficient gene therapy. Recently, a noninvasive, physical approach has been presented based on local heat in combination with a heat-sensitive promoter. This strategy requires tight temperature control in vivo. Here, we use MRI-guided focused ultrasound (MRI-FUS) with real-time feedback control on a whole-body clinical MRI system for a completely automatic execution of a predefined temperature-time trajectory in the focal point. Feasibility studies on expression control were carried out on subcutaneously implanted rat tumors. A stable modified C6 glioma cell line was used carrying a fused gene coding for thymidine kinase (TK) and green fluorescent protein (GFP) under control of the human heat-shock protein 70 (HSP70) promoter. In vitro studies showed strong induction of the TK-GFP gene expression upon heat shock under various conditions and localization of the protein product in the nucleus. In vivo tumors were subjected to a 3-min temperature elevation using MRI-FUS with a constant temperature, and were analysed 24 hr after the heat shock with respect to GFP fluorescence. Preliminary results showed strong local induction in regions heated above 40 degrees C, and a good correspondence between temperature maps at the end of the heating period and elevated expression of TK-GFP.

Successful treatment of blood disorders by gene therapy has several complications, one of which is the frequent lack of selective advantage of genetically corrected cells. Erythropoietic protoporphyria (EPP), caused by a ferrochelatase deficiency, is a good model of hematological genetic disorders with a lack of spontaneous in vivo selection. This disease is characterized by accumulation of protoporphyrin in red blood cells, bone marrow, and other organs, resulting in severe skin photosensitivity. Here we develop a self-inactivating lentiviral vector containing human ferrochelatase cDNA driven by the human ankyrin-1/beta-globin HS-40 chimeric erythroid promoter/enhancer. We collected bone marrow cells from EPP male donor mice for lentiviral transduction and injected them into lethally irradiated female EPP recipient mice. We observed a high transduction efficiency of hematopoietic stem cells resulting in effective gene therapy of primary and secondary recipient EPP mice without any selectable system. Skin photosensitivity was corrected for all secondary engrafted mice and was associated with specific ferrochelatase expression in the erythroid lineage. An erythroid-specific expression was sufficient to reverse most of the clinical and biological manifestations of the disease. This improvement in the efficiency of gene transfer with lentiviruses may contribute to the development of successful clinical protocols for erythropoietic diseases.

Erythropoietic protoporphyria is characterized clinically by skin photosensitivity and biochemically by a ferrochelatase deficiency resulting in an excessive accumulation of photoreactive protoporphyrin in erythrocytes, plasma and other organs. The availability of the Fech(m1Pas)/Fech(m1Pas) murine model allowed us to test a gene therapy protocol to correct the porphyric phenotype. Gene therapy was performed by ex vivo transfer of human ferrochelatase cDNA with a retroviral vector to deficient hematopoietic cells, followed by re-injection of the transduced cells with or without selection in the porphyric mouse. Genetically corrected cells were separated by FACS from deficient ones by the absence of fluorescence when illuminated under ultraviolet light. Five months after transplantation, the number of fluorescent erythrocytes decreased from 61% (EPP mice) to 19% for EPP mice engrafted with low fluorescent selected BM cells. Absence of skin photosensitivity was observed in mice with less than 20% of fluorescent RBC. A partial phenotypic correction was found for animals with 20 to 40% of fluorescent RBC. In conclusion, a partial correction of bone marrow cells is sufficient to reverse the porphyric phenotype and restore normal hematopoiesis. This selection system represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.

Congenital erythropoietic porphyria (CEP) is an inherited disease due to a deficiency in the uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood, and other organs. The onset of most cases occurs in infancy and the main symptoms are cutaneous photosensitivity and hemolysis. For severe transfusion-dependent cases, when allogeneic cell transplantation cannot be performed, autografting of genetically modified primitive/stem cells is the only alternative. In the present study, efficient mobilization of peripheral blood primitive CD34(+) cells was performed on a young adult CEP patient. Retroviral transduction of this cell population with the therapeutic human UROS (hUS) gene resulted in both enzymatic and metabolic correction of CD34(+)-derived cells, as demonstrated by the increase in UROS activity and by a 53% drop in porphyrin accumulation. A 10-24% gene transfer efficiency was achieved in the most primitive cells, as demonstrated by the expression of enhanced green fluorescent protein (EGFP) in long-term culture-initiating cells (LTC-IC). Furthermore, gene expression remained stable during in vitro erythroid differentiation. Therefore, these results are promising for the future treatment of CEP patients by gene therapy.

Background: Congenital erythropoietic porphyria (CEP) is an inherited disease caused by a deficiency of uroporphyrinogen III synthase, the fourth enzyme of the haem biosynthesis pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood and other organs. The prognosis of CEP is poor with death occurring in early adult life and available treatments are only symptomatic and unsatisfactory. In vitro gene transfer experiments have documented the feasibility of gene therapy via haematopoietic stem cells to treat this disease. To facilitate future ex vivo gene therapy in humans, the design of efficient selection procedures to increase the frequency of genetically corrected cells prior to autologous transplantation is a critical step., Methods: An alternative selection procedure based upon expression of a transferred gene was performed on a lymphoblastoid (LB) cell line from a patient with congenital erythropoietic porphyria to obtain high frequencies of genetically modified cells. The presence of exogeneous delta-aminolevulinic acid (ALA), a haem precursor, induces an increase in porphyrin accumulation in LB deficient cells. Porphyrins exhibit a specific fluorescent emission and can be detected by cytofluorimetry under ultraviolet excitation., Results: In genetically modified cells, the restored metabolic flow from ALA to haem led to a lesser accumulation of porphyrins in the cells, which were easily separated from the deficient cells by flow cytometry cell sorting., Conclusion: This selection process represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.

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.

The transfer and expression of cytokine genes into tumor cells is reportedly a valuable approach to improve the antitumor activity of cytokines in various models. Interferon (IFN)-alpha may induce hematological remission in chronic myeloid leukemia (CML) patients, but only a small proportion of patients achieve a sustained, complete cytogenetic remission. We have investigated the possibility of transducing CML cells with the retroviral vector LIalpha2SN, which encodes the IFN-alpha2 gene. We first optimized the transduction efficiency using the CML-derived K562 cell line. A transduction efficiency of 50% and 85% after three and six infections, respectively, was obtained in K562 cells. We then expressed IFN-alpha2 in CML cells by transducing the latter with LIalpha2SN viral particles. The IFN-alpha secretion after three and six infections was 5,400 and 18,000 U/24 hours/10(6) cells for unselected K562 cells and 7,000 and 290 U/24 hours/10(6) cells for CML CD34+ cells at days 4 and 5. Moreover, the major histocompatibility complex class I antigens were overexpressed after infection with LIalpha2SN in both K562 and CML CD34+ cells. The proliferation (in liquid culture) and the cloning efficiency of these CML cells were significantly decreased after LIalpha2SN treatment. By contrast, the proliferation of cord blood CD34+ cells was not affected by transduction with LIalpha2SN. These results demonstrate the transduction efficiency of CML cells and suggest the possibility of CML cell immunotherapy with retroviral gene transfer of different cytokines such as IFN-alpha.

We have developed an efficient and rapid method to analyze transduction in human hematopoietic cells and to select them. We constructed two retroviral vectors using the recombinant humanized S65T green fluorescent protein (rHGFP) gene. Transduced cells appeared with specific green fluorescence on microscopy or fluorescence-activated cell sorting (FACS) analysis. The rHGFP gene was placed under the control of two different retroviral promotors (LTR) in the LGSN vector and in the SF-GFP vector. Amphotropic retroviruses were tested on NIH/3T3 fibroblasts or human hematopoietic (K562, TF-1) cell lines. Then CD34+ cells isolated from cord blood were infected three times after a 48-h prestimulation with IL-3, IL-6, SCF or with IL-3, IL-6, SCF, GM-CSF, Flt3-L and TPO. After 6 days of expansion, a similar number of total CD34(+)-derived cells, CD34+ cells and CFC was obtained in non-transduced and transduced cells, demonstrating the absence of toxicity of the GFP. A transduction up to 46% in total CD34(+)-derived cells and 21% of CD34+ cells was shown by FACS analysis. These results were confirmed by fluorescence of colonies in methyl-cellulose (up to 36% of CFU-GM and up to 25% of BFU-E). The FACS sorting of GFP cells led to 83-100% of GFP-positive colonies after 2 weeks of methyl-cellulose culture. Moreover, a mean gene transfer efficiency of 8% was also demonstrated in longterm culture initiating cells (LTC-IC). This rapid and efficient method represents a substantial improvement to monitor gene transfer and retroviral expression of various vectors in characterized human hematopoietic cells.

Congenital erythropoietic porphyria (CEP) is an inherited metabolic disorder characterized by an overproduction and accumulation of porphyrins in bone marrow. This autosomal recessive disease results from a deficiency of uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the haem biosynthetic pathway. It is phenotypically heterogeneous: patients with mild disease have cutaneous involvement, while more severely affected patients are transfusion dependent. The cloning of UROIIIS cDNA and genomic DNA has allowed the molecular characterization of the genetic defect in a number of families. To date, 22 different mutations have been characterized. Allogeneic bone marrow transplantation is the only curative treatment available for the severe, transfusion-dependent, cases. When bone marrow transplantation cannot be performed owing to the absence of a suitable donor, the autografting of genetically modified cells is an appealing alternative. The best approach to somatic gene therapy in this disease involves the use of recombinant retroviral vectors to transduce cells ex vivo, followed by autologous transplantation of the genetically modified cells. We investigated retroviral transfer in deficient human fibroblasts, immortalized lymphoblasts as well as bone marrow cells, and obtained a complete restoration of the enzymatic activity and full metabolic correction. Using K562 cells, an erythroleukaemic cell line, the expression of the transgene remained stable during 3 months and during erythroid differentiation of the cells. Finally, a 1.6- to 1.9-fold increase in enzyme activity compared to the endogenous level was found in normal CD34+ cells, a population of heterogeneous cells known to contain the progenitor/stem cells for long-term expression. The future availability of a mouse model of the disease will permit ex vivo gene therapy experiments on the entire animal.

CEP is a rare disease inherited as an autosomal recessive trait and characterized by an overproduction and accumulation of porphyrins in the bone-marrow. Because the predominant site of metabolic expression of the disease is the erythropoietic system, bone marrow transplantation represents a curative treatment for patients with severe phenotypes. This treatment can be considered in severe cases when the disease appears in the first few years of life. When bone marrow transplantation is not possible, gene therapy by transplantation of genetically modified hematopoietic cells is an attractive alternative for the future. In this report, we present the restoration of enzymatic activity and the metabolic correction of deficient cells in vitro after transduction with retroviral vectors. The future availability of a mouse model of the disease will permit ex vivo gene therapy experiments on the entire animal.

The rapid progress in the development of molecular technology has resulted in the identification of most of the genes of the heme biosynthesis pathway. Important problems in the pathogenesis and treatment of porphyrias now seem likely to be solved by the possibility of creating animal models and by the transfer of normal genes or cDNAs to target cells. Animal models of porphyrias naturally occur for erythropoietic protoporphyria and congenital erythropoietic porphyria, and different murine models have been or are being created for erythropoietic and hepatic porphyrias. The PBGD knock-out mouse will be useful for the understanding of nervous system dysfunction in acute porphyrias. Murine models of erythropoietic porphyrias are being used for bone-marrow transplantation experiments to study the features of erythropoietic and hepatic abnormalities. Gene transfer experiments have been started in vitro to look at the feasibility of somatic gene therapy in erythropoietic porphyrias. In particular, we have documented sufficient gene transfer rate and metabolic correction in different CEP disease cells to indicate that this porphyria is a good candidate for treatment by gene therapy in hematopoietic stem cells. With the rapid advancement of methods that may allow more precise and/or efficient gene targeting, gene therapy will become a new therapeutic option for porphyrias.

Congenital erythropoietic porphyria (CEP) is an inherited metabolic disorder resulting from the accumulation of porphyrins because of defective uroporphyrinogen III synthase (UROIIIS). This autosomal recessive disorder is phenotypically heterogeneous with respect to the age of onset and the severity of the symptoms. Different exonic point mutations in the UROIIIS gene have been identified, providing phenotype-genotype correlations in this disease. Severe cases may be treated by bone marrow transplantation and are potential candidates for somatic gene therapy. Epstein-Barr virus-transformed B-cell lines from patients with CEP provide a model system for the disease. We have used retrovirus-mediated expression of UROIIIS to restore enzymatic activity in a B-cell line from a patient. We have also demonstrated the metabolic correction of the disease, ie, porphyrin accumulation into the deficient transduced cells was reduced to the normal level. These data show the potential of gene therapy for this disease.

Congenital erythropoietic porphyria (CEP) is a genetic disease characterized by an overproduction and accumulation of porphyrins in bone marrow. The enzyme defect concerns uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the heme biosynthetic pathway. It is the most severe porphyria and the treatment is largely symptomatic: gene therapy would represent a great therapeutic improvement. As a step toward the development of an effective gene therapy, we have constructed two retroviral vectors, LUSN and pMFG-US (with and without the selectable marker Neo), containing a full-length human cDNA for UROIIIS. Recombinant retroviruses were obtained by transfection of the LUSN or pMFG-US plasmid into the amphotropic packaging cell line psi CRIP. For each construct, three different producing clones were selected for their high titer (LUSN) or for their ability to express the message at a high level (pMFG-US). In vitro amplification of genomic DNA from target tissue demonstrated the presence of vector sequences. Murine fibroblasts infected in vitro expressed the human enzyme efficiently, as indicated by RNA and enzymatic studies. Retroviral-mediated gene transfer was then used to introduce the UROIIIS cDNA into human deficient cells. Enzyme activity was increased from 2% (deficient fibroblasts) to 121-274% of the normal value for the different clones. Transduced cells selected with G418 presented an 18-fold increase in enzyme activity compared to the normal cells. Furthermore, high gene transfer rate into peripheral blood progenitor cells (PBPB) was documented by in vitro amplification (PCR). These results demonstrate the potential usefulness of somatic gene therapy for the treatment of CEP.