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Biomedical subjects

Emanuela Bruscia

Publications and source records attributed to Emanuela Bruscia.

7 recordsLinked to original sources
PubMed2005

In vitro restoration of functional SMN protein in human trophoblast cells affected by spinal muscular atrophy by small fragment homologous replacement.

The majority of patients affected by spinal muscular atrophy (SMA) have deletion of the survival of motor neuron 1 (SMN1) gene, but they retain a "nonfunctional" copy of the duplicate gene (SMN2) in their genome. SMN2 produces defective SMN protein because of a C --> T transition in exon 7, which causes the skipping of exon 7 during SMN mRNA maturation. Many attempts have been made to correct altered SMN gene expression and to increase the level of normal SMN protein, but to date an effective treatment for this disease has not been established. Small Fragment Homologous Replacement (SFHR) is a site-specific gene modification approach that has the potential to maintain the genomic organization necessary for expression. The target modification in the genome is mediated by small DNA fragments (SDFs) 400-800 bp in length. In this study we used SFHR to induce a T --> C transition at codon 280 in exon 7 of the SMN2 gene in order to produce an increase in functional SMN protein. SDFs were transfected in vitro into cells obtained from five human fetal chorionic villi of embryos, homozygous for the SMN1 deletion, by either electroporation or microinjection. Transfected SMA cells showed an increase of up to 53% in full-length SMN mRNA compared with untransfected controls, as detected by real-time polymerase chain reaction. Consistent with the RNA data, immunocytochemistry and immunoblotting revealed a significant 2-fold increase in wild-type SMN protein. Furthermore, genotype and phenotype of transfected cells remained stable after several in vitro passages, demonstrating the stability of the correction over time.

Cells, Cultured↗
PubMed2005

Engraftment of bone marrow-derived epithelial cells.

The long-held concept that transplanted bone marrow (BM)-derived cells contribute only to cells of the hematopoietic system was challenged by data from our laboratory showing that a single male BM-derived cell could not only reconstitute the hematopoietic system of an irradiated female recipient, but could also lead to the generation of mature BM-derived epithelial cells in the liver, lung, skin, and gastrointestinal tract. Careful costaining and single-cell analyses have been used to rule out false positive cells due to inadequate detection techniques in microscopy or cell overlay. Since this initial discovery, we have sought to understand the mechanisms underlying the formation of BM-derived epithelial cells, and to evaluate their therapeutic use for gene therapy and/or tissue regeneration. Several reports have shown that donor BM-derived cells, possibly macrophages, can fuse with existing host epithelial cells to form heterokaryons that express both donor and tissue-specific markers. While this is certainly true for murine tyrosinemia models, we have used a Cre-lox system to demonstrate that fusion is not a requirement for the generation of BM-derived epithelial cells and is likely not responsible for the BM-derived epithelial cells generated after standard BM transplantation. In a proof of principal experiment for potential gene therapy applications, we have shown that autologous BM-derived cells transfected with a transgene prior to BM transplantation are able to develop into mature type-II pneumocytes that express the transgene. We also discuss future research directions in the field and the therapeutic potential of BM-derived epithelia, including ongoing work to test whether combined cell and gene therapy can be used therapeutically in preclinical mouse models of human disease.

Bone Marrow Cells↗
PubMed2004

Plasticity of bone marrow-derived stem cells.

Stem cell plasticity refers to the ability of adult stem cells to acquire mature phenotypes that are different from their tissue of origin. Adult bone marrow cells (BMCs) include two populations of bone marrow stem cells (BMCs): hematopoietic stem cells (HSCs), which give rise to all mature lineages of blood, and mesenchymal stem cells (MSCs), which can differentiate into bone, cartilage, and fat. In this article, we review the literature that lends credibility to the theory that highly plastic BMCs have a role in maintenance and repair of nonhematopoietic tissue. We discuss the possible mechanisms by which this may occur. Also reviewed is the possibility that adult BMCs can change their gene expression profile after fusion with a mature cell, which has brought into question whether this stem cell plasticity is real.

Animals↗
PubMed2003

Sequence-specific modification of genomic DNA by small DNA fragments.

Small DNA fragments have been used to modify endogenous genomic DNA in both human and mouse cells. This strategy for sequence-specific modification or genomic editing, known as small-fragment homologous replacement (SFHR), has yet to be characterized in terms of its underlying mechanisms. Genotypic and phenotypic analyses following SFHR have shown specific modification of disease-causing genetic loci associated with cystic fibrosis, beta-thalassemia, and Duchenne muscular dystrophy, suggesting that SFHR has potential as a therapeutic modality for the treatment of monogenic inherited disease.

Anemia, Sickle Cell↗
PubMed2002

In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique.

BACKGROUND: SFHR (small fragment homologous replacement)-mediated targeting is a process that has been used to correct specific mutations in mammalian cells. This process involves both chemical and cellular factors that are not yet defined. To evaluate potential of this technique for gene therapy it is necessary to characterize gene transfer efficacy in terms of the transfection vehicle, the genetic target, and the cellular processing of the DNA and DNA-vehicle complex. METHODS: In this study, small fragments of genomic cystic fibrosis (CF) transmembrane conductance regulator (CFTR) DNA, that comprise the wild-type and DeltaF508 sequences, were transfected into immortalized CF and normal airway epithelial cells, respectively. Homologous replacement was evaluated using PCR and sequence-based analyses of cellular DNA and RNA. Individual stages of cationic lipid-facilitated SFHR in cultured cell lines were also examined using transmission electron microscopy (TEM). RESULTS: We demonstrated that the lipid/DNA (+/-) ratio influences the mode of entry into the cell and therefore affects the efficacy of SFHR-mediated gene targeting. Lipid/DNA complexes with more negative ratios entered the cell via a plasma membrane fusion pathway. Transfer of the DNA that relies on an endocytic pathway appeared more effective at mediating SFHR. In addition, it was also clear that there is a correlation between the specific cell line transfected and the optimal lipid/DNA ratio. CONCLUSIONS: These studies provide new insights into factors that underlie SFHR-mediated gene targeting efficacy and into the parameters that can be modulated for its optimization.

Journal Article↗
PubMed2002

Towards the pharmacogenomics of cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal recessive genetic disease affecting children in Europe and the US. CF is a multiorgan disease and may present a variety of clinical symptoms, like chronic obstructive lung disease, exocrine pancreatic insufficiency (PI) and elevated sweat chloride concentration. CF mutations have also been found in other related clinical diseases such as congenital bilateral absence of the vas deferens (CBAVD), disseminated bronchiectasis and chronic pancreatitis. These clinical overlaps pose etiopathogenetic, diagnostic and therapeutic questions. Despite stunning advances in genomic technologies and drug discovery, drug therapy often improves disease symptoms but does not cure the disease. One of the main causes of this failure in CF cure may be attributable to genetic variability and to the scarce knowledge of CF biochemistry. Therefore, knowing the genotype of a patient might help improve drug efficacy, reduce toxicity and suggests innovative genomic-based therapy approaches.

Cystic Fibrosis↗