Spleen route accelerates engraftment of human hematopoietic stem cells.

Intravenous injections of human hematopoietic stem cells (hHSCs) is routinely used in clinic and for modeling hematopoiesis in mice. However, unspecific dilution in vascular system and non-hematopoietic organs challenges engraftment efficiency. Although spleen is capable of extra medullar hematopoiesis, its ability to support human HSC transplantation has never been evaluated. We demonstrate that intra-splenic injection results in high and sustained engraftment of hHSCs into immune-deficient mice, with higher chimerisms than with intravenous or intra-femoral injections. Our results support that spleen microenvironment provides a niche for HSCs amplification and offers a new route for efficient HSC transplantation.

Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9.

The CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has led to gene editing in clinical applications. Comprehensive analysis of gene editing products at the targeted cut-site has revealed a complex spectrum of outcomes. ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and more sensitive detection methods. Here, we present two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable the detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH). These tools reveal rare complex chromosomal rearrangements caused by Cas9-nuclease and show that LOH frequency depends on cell division rate during editing and p53 status. Cell cycle arrest during editing suppresses the occurrence of LOH without compromising editing. These data are confirmed in human stem/progenitor cells, suggesting that clinical trials should consider p53 status and cell proliferation rate during editing to limit this risk by designing safer protocols.

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.

Lack of beta-catenin in early life induces abnormal glucose homeostasis in mice.

AIMS/HYPOTHESIS: Wingless and iNT-1 (WNT) pathway members are critical for pancreatic development and exocrine tissue formation. Recently, much attention has focused on delineating the roles of beta-catenin in pancreatic organogenesis. However, little is known about the involvement of beta-catenin in the endocrine or exocrine function of the mature pancreas. We report for the first time the impact of beta-catenin deletion in the pancreatic beta cells. METHODS: We targeted the deletion of the beta-catenin gene in pancreatic beta cells by crossing a floxed beta-catenin mouse strain with a RIP-Cre mouse strain. RESULTS: Surprisingly, the majority of the mutant mice died shortly after birth and had deregulated glucose and insulin levels. The newborn mutant pancreases demonstrated increased insulin content, reflecting a defect in insulin release confirmed in vitro. Moreover, there was a reduction in total endocrine tissue at birth, while cellularity in islets was greater, suggesting that lack of beta-catenin affects beta cell size. Some newborns survived beta-catenin deletion and showed a milder phenotype during adulthood. CONCLUSIONS/INTERPRETATION: The deletion of beta-catenin in the maturing beta cells negatively impacts on islet morphology and function. This work reveals that lack of beta-catenin in early life is related to severe deregulation of glucose homeostasis.

Congenital erythropoietic porphyria: mutation update and correlations between genotype and phenotype.

High quality genotype/phenotype analysis is a difficult issue in rare genetic diseases such as congenital erythropoietic porphyria (CEP) or Günther's disease, a heme biosynthesis defect due to uroporphyrinogen III synthase deficiency. The historical background and the main phenotypic features of the disease are depicted together with an update of published mutants and genotype/phenotype correlations. General rules concerning the prediction of disease severity are drawn as a guide for patient management and therapeutic choices. The phenotypic heterogeneity of the disease is presented in relation with a likely influence of modifying factors, either genetic or acquired.

Overexpression of the heat-shock protein 70 is associated to imatinib resistance in chronic myeloid leukemia.

Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the expression of the recombinant oncoprotein Bcr-Abl. In this investigation, we studied an imatinib-resistant cell line (K562-r) generated from the K562 cell line in which none of the previously described mechanisms of resistance had been detected. A threefold increase in the expression of the heat-shock protein 70 (Hsp70) was detected in these cells. This increase was not associated to heat-shock transcription factor-1 (HSF-1) overexpression or activation. RNA silencing of Hsp70 decreased dramatically its expression (90%), and was accompanied by a 34% reduction in cell viability. Overexpression of Hsp70 in the imatinib-sensitive K562 line induced resistance to imatinib as detected by a large reduction in cell death in the presence of 1 muM of imatinib. Hsp70 level was also increased in blast cells of CML patients resistant to imatinib, whereas the level remained low in responding patients. Taken together, the results demonstrate that overexpression of Hsp70 can lead to both in vitro and in vivo resistance to imatinib in CML cells. Moreover, the overexpression of Hsp70 detected in imatinib-resistant CML patients supports this mechanism and identifies potentially a marker and a therapeutic target of CML evolution.

Effect of tyrosine kinase inhibitors on stemness in normal and chronic myeloid leukemia cells.

Although tyrosine kinase inhibitors (TKIs) efficiently cure chronic myeloid leukemia (CML), they can fail to eradicate CML stem cells (CML-SCs). The mechanisms responsible for CML-SC survival need to be understood for designing therapies. Several previous studies suggest that TKIs could modulate CML-SC quiescence. Unfortunately, CML-SCs are insufficiently available. Induced pluripotent stem cells (iPSCs) offer a promising alternative. In this work, we used iPSCs derived from CML patients (Ph+). Ph+ iPSC clones expressed lower levels of stemness markers than normal iPSCs. BCR-ABL1 was found to be involved in stemness regulation and ERK1/2 to have a key role in the signaling pathway. TKIs unexpectedly promoted stemness marker expression in Ph+ iPSC clones. Imatinib also retained quiescence and induced stemness gene expression in CML-SCs. Our results suggest that TKIs might have a role in residual disease and confirm the need for a targeted therapy different from TKIs that could overcome the stemness-promoting effect caused by TKIs. Interestingly, a similar pro-stemness effect was observed in normal iPSCs and hematopoietic SCs. These findings could help to explain CML resistance mechanisms and the teratogenic side-effects of TKIs in embryonic cells.

Lentivirus-mediated gene transfer of uroporphyrinogen III synthase fully corrects the porphyric phenotype in human cells.

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.

Correction of the enzyme defect in cultured congenital erythropoietic porphyria disease cells by retrovirus-mediated gene transfer.

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.

Identification of two new mutations 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 (UROIIIS) activity. Six missense mutations in the UROIIIS gene, a deletion and an insertion have already been described in CEP. This work brings further evidence for the heterogeneity in the genetic defect found in CEP. Two new mutations are described, a point mutation (V99A) and a frame-shift mutation (633insA) in the same patient who had a mild to moderate form of Günther's disease. The mutation (V99A) had a detectable residual activity when expressed in Escherichia coli while the insertion (633insA), which introduced a premature stop, had no activity. In the patients studied in our laboratory, the mutation C73R, associated with a severe phenotype, remains the most frequently seen.

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.

Retroviral vector-mediated transfer of the interferon-alpha gene in chronic myeloid leukemia cells.

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.

[A model of congenital erythropoietic porphyria for gene transfer in hematopoietic cells]. / Modèle de la porphyrie érythropoïétique congénitale pour le transfert de gènes dans les cellules hématopoïétiques.

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.

[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.

[Congenital erythropoietic porphyria. Apropos of a fatal case in the neonatal period due to acute hemolysis with hepatic failure]. / Porphyrie érythropoïétique congénitale. A propos d'un cas fatal en période néonatale dû à une hémolyse aiguë avec insuffisance hépatique.

BACKGROUND: Congenital erythropoietic porphyria, an autosomal recessive disease, is characterized by deficiency of uroporphyrinogen III synthase. Clinical variability of the disease is related to the different mutations found in the patients. CASE REPORT: A newborn suffered one hour after birth from jaundice and polypnea with acute hemolysis. Severe cutaneous photosensitivity occurred after phototherapy. Congenital erythropoietic porphyria was suspected because of reddish-colored urine and confirmed by porphyrin analyses. The baby died one month later due to severe hemolytic anemia with hepatic failure. Uroporphyrinogen III synthase activity was decreased by 99% in bone marrow cells and established lymphoblastoid cells from the patient. Molecular biology studies demonstrated the presence of the Cys 73-->Arg substitution at the homozygous state in the patient. CONCLUSION: This mutation, the most frequently found in this disease, is responsible for a severe phenotype. Molecular characterization provides genotype/phenotype correlations in this porphyria and allows to clarify unusual cases of porphyrias.

The stem cell factor-c-KIT pathway must be inhibited to enable apoptosis induced by BCR-ABL inhibitors in chronic myelogenous leukemia cells.

Imatinib is an effective first-line therapy for chronic myelogenous leukemia (CML) that acts by targeting the tyrosine kinase activity of BCR-ABL. To overcome resistance, second-generation inhibitors of BCR-ABL have been developed. Among these, nilotinib is more potent against BCR-ABL than imatinib, and is effective against many imatinib-resistant BCR-ABL mutants. In this study, an in vitro flow cytometry assay to analyze imatinib- and nilotinib-induced apoptosis in CML cells has been developed. Both the drugs induced significant apoptosis in CD34+ cells from 36 CML bone marrow samples (P<10(-4)), whereas CD34+ cells from BCR-ABL negative samples were unaffected. When the experiments were carried out in the presence of a cocktail of cytokines, nilotinib- but not imatinib-induced apoptosis was inhibited. This differential inhibition was confirmed on K562 cells. A blocking anti-CD117 antibody alleviated the antiapoptotic effect of cytokines against nilotinib. Moreover, using short hairpin RNA against BCR-ABL, we showed that K562 cells were not dependent on BCR-ABL signaling as long as the stem cell factor (SCF) receptor pathway was activated. We conclude that the c-KIT pathway may substitute for BCR-ABL tyrosine kinase to activate survival signals, and that c-KIT must be inhibited besides Bcr-Abl to allow apoptosis of CML cells.

A knock-in mouse model of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is a recessive autosomal disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. The severity of the disease, the lack of specific treatment except for allogeneic bone marrow transplantation, and the knowledge of the molecular lesions are strong arguments for gene therapy. An animal model of CEP has been designed to evaluate the feasibility of retroviral gene transfer in hematopoietic stem cells. We have previously demonstrated that the knockout of the Uros gene is lethal in mice (Uros(del) model). This work describes the achievement of a knock-in model, which reproduces a mutation of the UROS gene responsible for a severe UROS deficiency in humans (P248Q missense mutant). Homozygous mice display erythrodontia, moderate photosensitivity, hepatosplenomegaly, and hemolytic anemia. Uroporphyrin (99% type I isomer) accumulates in urine. Total porphyrins are increased in erythrocytes and feces, while Uros enzymatic activity is below 1% of the normal level in the different tissues analyzed. These pathological findings closely mimic the CEP disease in humans and demonstrate that the Uros(mut248) mouse represents a suitable model of the human disease for pathophysiological, pharmaceutical, and therapeutic purposes.

Metabolic correction of congenital erythropoietic porphyria by retrovirus-mediated gene transfer into Epstein-Barr virus-transformed B-cell lines.

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.

Porphyrias: animal models and prospects for cellular and gene therapy.

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.

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.