PubMed Health⌕ Search

Biomedical subjects

Francesca Ficara

Publications and source records attributed to Francesca Ficara.

8 recordsLinked to original sources
PubMed2004

IL-3 or IL-7 increases ex vivo gene transfer efficiency in ADA-SCID BM CD34+ cells while maintaining in vivo lymphoid potential.

To improve maintenance and gene transfer of human lymphoid progenitors for clinical use in gene therapy of adenosine deaminase (ADA)-deficient SCID we investigated several gene transfer protocols using various stem cell-enriched sources. The lymphoid differentiation potential was measured by an in vitro clonal assay for B/NK cells and in the in vivo SCID-hu mouse model. Ex vivo culture with the cytokines TPO, FLT3-ligand, and SCF (T/F/S) plus IL-3 or IL-7 substantially increased the yield of transduced bone marrow (BM) CD34(+) cells purified from ADA-SCID patients or healthy donors, compared to T/F/S alone. Moreover, the use of IL-3 or IL-7 significantly improved the maintenance of in vitro B cell progenitors from ADA-SCID BM cells and allowed the efficient transduction of B and NK cell progenitors. Under these optimized conditions transduced CD34(+) cells were efficiently engrafted into SCID-hu mice and gave rise to B and T cell progeny, demonstrating the maintenance of in vivo lymphoid reconstitution capacity. The protocol based on the T/F/S + IL-3 combination was included in a gene therapy clinical trial for ADA-SCID, resulting in long-term engraftment of stem/progenitor cells. Remarkably, gene-corrected BM CD34(+) cells obtained from one patient 4 and 11 months after gene therapy were capable of repopulating the lymphoid compartment of SCID-hu hosts.

Adenosine Deaminase↗
PubMed2004

Mobilized blood CD34+ cells transduced and selected with a clinically applicable protocol reconstitute lymphopoiesis in SCID-Hu mice.

We developed a clinically applicable gene transfer procedure into mobilized peripheral blood (MPB) CD34(+) hematopoietic progenitor cells, based on single viral exposure and selection of engineered cells. CD34(+) cells were transduced with a retroviral vector carrying the truncated form of the nerve growth factor receptor (Delta NGFR) marker gene, and immunoselected for Delta NGFR expression. Optimal time and procedure for viral exposure, length of culture, and transgene expression of MPB CD34(+) cells were determined using in vitro assays. The multipotent capacity of MPB CD34(+)-transduced cells was demonstrated in the SCID-hu bone/liver/thymus mouse model. Transduced Delta NGFR(+) cells retained 50% of long-term culture-colony forming cells (LTC-CFC) compared to unmanipulated CD34(+) cells. In SCID-hu mice, 52% of CD45(+) cells, 27% of CD34(+) cells, 49% of B cells, and more than 50% of T cells were derived from transplanted CD34(+)/Delta NGFR(+) cells. Furthermore, transplantation of purified transduced cells greatly reduced the competition with untransduced progenitors occurring in unselected grafts. These data demonstrate that MPB CD34(+) cells, transduced with a single viral exposure and selected by transgene expression, retain multilineage reconstitution capacity and remarkable transgene expression.

Animals↗
PubMed2004

Efficient gene transfer into primitive hematopoietic progenitors using a bone marrow microenvironment cell line engineered to produce retroviral vectors.

BACKGROUND AND OBJECTIVES: Effective gene transfer into human hematopoietic stem/progenitor cells is a compromise between achieving high transduction efficiency and maintaining the desired biological characteristics of the target cell. The aim of our work was to exploit the stromal microenvironment to increase gene transfer and maintenance of hematopoietic progenitors. DESIGN AND METHODS: The murine bone marrow stromal cell line MS-5, known to support primitive human progenitors, was modified into an amphotropic packaging cell, by the stable introduction of DNA coding for retroviral structural proteins, and a viral vector encoding a marker gene. The gene transfer efficiency of the recombinant virus was evaluated by flow cytometry, in vitro assays for committed (CFC) and primitive (LTC-CFC) progenitors, as well as a clonal assay for B and NK lymphoid progenitors. RESULTS: The new packaging cell line (NEXUS) produced equivalent levels of virus as did the established GP+Am12 system, also under serum-free conditions. On average 30% of human mobilized peripheral blood CD34(+) cells were transduced by a single exposure to NEXUS supernatant, representing a three-fold increase over GP+Am12-based technology. Gene transfer into both committed and primitive progenitors increased on average two-fold using NEXUS retroviral supernatant. Furthermore, CD34(+) CD38(low) early progenitor cells purified from umbilical cord blood were efficiently transduced with NEXUS retroviral vector and gave rise to a high frequency of marked B and NK lymphocytes. INTERPRETATION AND CONCLUSIONS: Our data show that that an established bone marrow stromal cell can be engineered to enhance the genetic modification of primitive hematopoietic and lymphoid progenitors using a clinically relevant method.

Animals↗
PubMed2003

A T-cell epitope encoded by a subset of HLA-DPB1 alleles determines nonpermissive mismatches for hematologic stem cell transplantation.

The importance of HLA-DPB1 matching for the outcome of allogeneic hematologic stem cell (HSC) transplantation is controversial. We have previously identified HLA-DPB1*0901 as a target of cytotoxic T cells mediating in vivo rejection of an HSC allograft. Here we show that HLA-DPB1*0901 encodes a T-cell epitope shared by a subset of DPB1 alleles that determines nonpermissive mismatches for HSC transplantation. Several T-cell clones obtained from the patient at the time of rejection showed HLA-DP restricted recognition of allogeneic targets expressing HLA-DPB1*0901, *1001, *1701, *0301, *1401, and *4501, but not other alleles. Based on these findings, we developed an algorithm for prediction of nonpermissive HLA-DPB1 mismatches. Retrospective evaluation of 118 transplantations showed that the presence of nonpermissive HLA-DPB1 mismatches was correlated with significantly increased hazards of acute grade II to IV graft-versus-host disease (HR = 1.87, P =.046) and transplantation-related mortality (HR = 2.69, P =.027) but not relapse (HR = 0.98, P =.939), as compared with the permissive group. There was also a marked but statistically not significant increase in the hazards of overall mortality (HR = 1.64, P =.1). These data suggest that biologic characterization of in vivo alloreactivity can be a tool for definition of clinically relevant nonpermissive HLA mismatches for unrelated HSC transplantation.

Algorithms↗
PubMed2003

Gene therapy for adenosine deaminase deficiency.

PURPOSE OF REVIEW: Gene therapy for severe combined immunodeficiency due to adenosine deaminase deficiency has moved from the early trials of safety and feasibility to recent studies demonstrating efficacy and clinical benefit. This review describes the latest advances in gene therapy trials for this condition using peripheral blood lymphocytes or hematopoietic progenitors. RECENT FINDINGS: In the first patients with severe combined immunodeficiency due to adenosine deaminase deficiency treated with peripheral blood lymphocytes, transduced T cells have been shown to persist for over 10 years, expressing transgenic adenosine deaminase, but the therapeutic effect of gene therapy remained difficult to assess because of the concomitant treatment with bovine adenosine deaminase conjugated to polyethylene-glycol (PEG-ADA). A recent report showed that discontinuation of PEG-ADA resulted in a strong selective advantage of gene corrected T cells associated with restoration of T cell functions and antibody responses to neoantigen, but incomplete correction of the metabolic defect. Follow-up studies in patients treated with engineered hematopoietic progenitors in the early trials revealed low marking levels of long-term living progenitors and limited clinical effect. Recently, an improved gene transfer protocol in bone marrow CD34+ cells combined with low-dose busulfan resulted in multilineage, stable engraftment of transduced progenitors at substantial levels, restoration of immune functions, correction of the adenosine deaminase metabolic defect, and proven clinical benefit, in the absence of PEG-ADA. Overall, no adverse effect or toxicity has been observed in patients treated with adenosine deaminase gene transfer in mature lymphocytes or hematopoietic progenitors. SUMMARY: Gene transfer in hematopoietic stem cells combined with nonmyeloablative conditioning is efficacious and might be extended to the treatment of other inherited and acquired disorders of the hematopoietic system.

Adenosine Deaminase↗
PubMed2002

Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.

Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID.

Adenosine Deaminase↗
PubMed2002

Developmental expression of the T-box transcription factor T-bet/Tbx21 during mouse embryogenesis.

A novel type of DNA-binding domain, the 'T-box' domain, characterizes an increasingly large family of transcription factors (Trends Genet. 15 (1999) 154). We have identified and characterized the expression pattern of a new member of the Tbr1 subfamily of T-box genes; this gene has been recently named T-bet/Tbx21 (Genomics 70 (2000) 41; Cell 17 (2000) 655; Science 292 (2001) 1907; Science 295 (2002) 338). The sequence and expression of Tbr1 and eomesodermin/Tbr2 are closely related to T-bet/Tbx21. The expression of Tbr1 (Neuron 15 (1995) 63) and Tbr2 (Mech Dev 84 (1999) 133) have virtually identical onset, at around E10.5, and expression domains in the mouse telencephalon. While Tbr1 is expressed in postmitotic neurons, Tbr2 (which is also expressed during gastrulation is also expressed in neural progenitors. We have used in situ hybridization to determine the temporal and spatial distribution of T-bet/Tbx21 expression during mouse development. T-bet/Tbx21 expression is exclusively restricted to the olfactory bulb and the thymus. To assess the distribution of T-BET/TBX21 expression in the haematopoietic compartment we used reverse transcriptase-polymerase chain reaction and found its expression in several human blood cell lineages, including progenitors/stem cells, immature B cells and peripheral T cells.

Amino Acid Sequence↗