Cell fusion reprogramming leads to a specific hepatic expression pattern during mouse bone marrow derived hepatocyte formation in vivo.

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-ß1 (TGF-ß(1)) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation.

Rescue of pyruvate kinase deficiency in mice by gene therapy using the human isoenzyme.

Human erythrocyte R-type pyruvate kinase deficiency (PKD) is a disorder caused by mutations in the PKLR gene that produces chronic nonspherocytic hemolytic anemia. Besides periodic blood transfusion and splenectomy, severe cases require bone marrow (BM) transplant, which makes this disease a good candidate for gene therapy. Here, the normal human R-type pyruvate kinase (hRPK) complementary (cDNA) was expressed in hematopoietic stem cells (HSCs) derived from pklr deficient mice, using a retroviral vector system. These mice show a similar red blood cell phenotype to that observed in human PKD. Transduced HSCs were transplanted into myeloablated adult PKD mice or in utero injected into nonconditioned PKD fetuses. In the myeloablated recipients, the hematological manifestations of PKD were completely resolved and normal percentages of late erythroid progenitors, reticulocyte and erythrocyte counts, hemoglobin levels and erythrocyte biochemistry were restored. Corrected cells preserved their rescuing capacity after secondary and tertiary transplant. When corrected cells were in utero transplanted, partial correction of the erythrocyte disease was obtained, although a very low number of corrected cells became engrafted, suggesting a different efficiency of cell therapy applied in utero. Our data suggest that transduction of human RPK cDNA in PKLR mutated HSCs could be an effective strategy in severe cases of PKD.

In vivo proliferation advantage of genetically corrected hematopoietic stem cells in a mouse model of Fanconi anemia FA-D1.

Fanconi anemia (FA) is an inherited recessive DNA repair disorder mainly characterized by bone marrow failure and cancer predisposition. Studies in mosaic FA patients have shown that reversion of one inherited germ-line mutation resulting in a functional allele in one or a few hematopoietic stem cells (HSCs) can lead to the proliferation advantage of corrected cells, thus over time normalizing the hematologic status of the patient. In contrast to these observations, it is still unclear whether ex vivo genetic correction of FA HSCs also provides a similar proliferation advantage to FA HSCs. Using an FA mouse model with a marked hematopoietic phenotype, the FA-D1 (Brca2(Delta27/Delta27)) mice, we demonstrate that the lentivirus-mediated gene therapy of FA HSCs results in the progressive expansion of genetically corrected clones in mild-conditioned FA-D1 recipients. Consistent with these data, hematopoietic progenitors from FA recipients progressively became mitomycin C resistant and their chromosomal instability was reverted. No evidence of myelodysplasia, leukemias, or abnormal clonal repopulation was observed at multiple time points in primary or secondary recipients. Our results demonstrate that ectopic expression of BRCA2 confers a beneficial in vivo proliferation advantage to FA-D1 HSCs that enables the full hematopoietic repopulation of FA recipients with genetically corrected cells.

Characteristics of lentiviral vectors harboring the proximal promoter of the vav proto-oncogene: a weak and efficient promoter for gene therapy.

Recent published data have shown the efficacy of gene therapy treatments of certain monogenic diseases. Risks of insertional oncogenesis, however, indicate the necessity of developing new vectors with weaker or cell-restricted promoters to minimize the trans-activation activity of integrated proviruses. We have inserted the proximal promoter of the vav proto-oncogene into self-inactivating lentiviral vectors (vav-LVs) and investigated the expression pattern and therapeutic efficacy of these vectors. Compared with other LVs frequently used in gene therapy, vav-LVs mediated a weak, though homogeneous and stable, expression in in vitro-cultured cells. Transplantation experiments using transduced mouse bone marrow and human CD34(+) cells confirmed the stable activity of the promoter in vivo. To investigate whether the weak activity of this promoter was compatible with a therapeutic effect, a LV expressing the Fanconi anemia A (FANCA) gene was constructed (vav-FANCA LV). Although this vector induced a low expression of FANCA, compared to the expression induced by a LV harboring the spleen focus-forming virus (SFFV) promoter, the two vectors corrected the phenotype of cells from a patient with FA-A with the same efficacy. We propose that self-inactivating vectors harboring weak promoters, such as the vav promoter, will improve the safety of gene therapy and will be of particular interest for the treatment of diseases where a high expression of the transgene is not required.

Hematopoietic dysfunction in a mouse model for Fanconi anemia group D1.

We have investigated the hematopoietic phenotype of mice with a hypomorphic mutation in the Brca2/Fancd1 gene (Brca2(Delta27/Delta27) mutation). In contrast to observations made in other Fanconi anemia (FA) mouse models, low numbers of hematopoietic colony-forming cells (CFCs) were noted in Brca2(Delta27/Delta27) mice, either young or adult. Additionally, a high incidence of spontaneous chromosomal instability was observed in Brca2(Delta27/Delta27) bone marrow (BM) cells, but not in Brca2(+/Delta27) or Fanca(-/-) BM cells. Although Brca2(Delta27/Delta27) CFCs were not hypersensitive to ionizing radiation, a very severe hematopoietic syndrome was observed in irradiated Brca2(Delta27/Delta27) mice. Conventional BM competition experiments showed a marked repopulation defect in Brca2(Delta27/Delta27) hematopoietic stem cells (HSCs), compared to wild-type HSCs. Moreover, we have observed for the first time in a DNA repair disease model a very significant proliferation defect in Brca2(Delta27/Delta27) HSCs maintained in their natural physiological environment. The progressive repopulation of wild-type HSCs transplanted into unconditioned Brca2(Delta27/Delta27) recipients is reminiscent of the somatic mosaicism phenomenon observed in a number of genetic diseases, including FA. The hematopoietic phenotype associated with the Brca2(Delta27/Delta27) mutation suggests that this FA-D1 mouse model will constitute an important tool for the development of new therapies for FA, including gene therapy.

Cysticercosis in chronic psychiatric inpatients from a Venezuelan community.

Cysticercosis due to Taenia solium infection is endemic in developing countries of the Americas, Asia, and Africa. This study was designed to establish the prevalence of cysticercosis in 158 inpatients of a psychiatric institution in the state of Tachira (Venezuela) and in 127 healthy control subjects. Positive blood tests for cysticercosis by Western blotting were recorded in 18.35% of the patients and in 1.57% of the controls. Individuals with mental retardation were found to carry an increased risk of cysticercosis (RR: 2.92; 1.22 < 2.92 > 7.0; P < 0.05) compared with patients with other psychiatric disorders. Taeniasis by Taenia spp. was not demonstrated in the patient group, although a high incidence of infection by other helminths (95.1%) was detected. The high prevalence of cysticercosis in the psychiatric inpatient group, compared with healthy individuals, and the lack of a differential diagnosis of neurocysticercosis suggest cerebral cysticercosis in a large proportion of these patients. Cysticercosis could be the origin of the psychiatric disorders of these patients and may also be due to contact with the parasite in an environment with poor hygiene conditions and a deficient health care system.