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F D Ledley

Publications and source records attributed to F D Ledley.

At least 55 records · Page 3Linked to original sources
PubMed1993

Retrovirus-mediated gene transfer into canine thyroid using an ex vivo strategy.

We describe studies in a canine model aimed at establishing methods for ex vivo gene delivery to thyroid follicular cells. Canine follicular cells were harvested from tissue obtained by unilateral lobectomy, grown in thyrotropin-containing media, and transduced with amphotropic retroviral vectors carrying Escherichia coli beta-galactosidase or Tn7 neomycin-resistance genes. Up to 30% of cells were transduced with retroviral vectors containing the neomycin resistance gene, and transduced cells could be selected with G418. Significantly, transduced and selected cells exhibited the morphology of thyroid follicular cells and continued to express thyroglobulin. To assess the viability of cultivated and transduced cells for transplantation, cells were stained with the vital fluorescent dye DiI, recovered by trypsinization, and transplanted into the contralateral thyroid lobe of autologous animals. Engraftment was demonstrated by fluorescence microscopy and identification of proviral sequences 7-10 days after transplantation. Proviral transcripts were evident using coupled reverse transcription and the polymerase chain reaction using total RNA from transplanted glands. Thyroid follicular cells may represent an attractive target for gene therapy due to their proliferative potential, their large protein synthetic and secretory capacity, and their susceptibility to regulation. The thyroid might be a target for therapy of congenital or acquired thyroid diseases as well as disorders requiring regulated expression of proteins in the circulation. This work demonstrates the feasibility of ex vivo gene delivery to thyroid follicular cells that may be used in future investigations.

Animals↗
PubMed1993

Autologous, orthotopic thyroid follicular cell transplantation: a surgical component of ex vivo somatic gene therapy.

Ex vivo strategies for somatic gene therapy involve the harvest of primary cells from patients, the transfer of novel genetic material into these cells in cell culture, and reimplantation of the genetically engineered cells back into patients. In consideration of methods for targeting somatic gene therapy to the thyroid, we have studied the autologous, orthotopic transplantation of thyroid follicular cells in a canine model. Using the fluorescent dye Dil, we were able to stain follicular cells in vitro before transplantation and then follow the pattern of engraftment through histologic sectioning and microscopy up to 14 days after transplantation. The transplantations involved the direct injection of intact and disrupted follicles into a remaining thyroid lobe after cell harvest from the contralateral lobe. We also demonstrated engraftment of individual follicular cells recovered from primary monolayer cultures. Histologic studies revealed the presence of transplanted cells and follicles as well as focal regions of granulomatous reaction in close relation to the engrafted material. These studies demonstrate the feasibility of autologous, orthotopic transplantation of thyroid follicular cells. This method is an essential component of ex vivo strategies for targeting somatic gene therapy to the thyroid gland.

Animals↗
PubMed1993

Development of a clinical protocol for hepatic gene transfer: lessons learned in preclinical studies.

Strategies for hepatic gene therapy have been proposed that involve isolation of primary hepatocytes and introduction of recombinant genes into these cells in culture, followed by autologous hepatocellular transplantation (HCT). Consideration of clinical applications requires data suggesting that HCT can be performed safely in human subjects in addition to data indicating that recombinant gene expression can reverse a disease process. This report describes preclinical studies that underlie a clinical trial of HCT in which hepatocytes would be labeled with a marker gene to facilitate assessment of engraftment in the recipient. Human hepatocytes were harvested from liver segments preserved in Belzar's solution and transduced with an amphotropic retroviral vector carrying a recombinant marker gene (neomycin phosphotransferase II). Human hepatocytes were recovered from monolayer culture, stained with the fluorescent dye 1,1'-dioctadecyl-3,3,3,3'-tetra-methylindo-carbocyanine perchlorate (DiI) and transplanted into severe combined immunodeficient mice by splenic injection. Engrafted hepatocytes were identified in the liver and spleen of severe combined immunodeficient mice but not immunocompetent controls. Two large animal models of HCT are described. In a dog model, neomycin phosphotransferase II-containing hepatocytes were identified in the liver 7 wk after transplantation. In a baboon model, autologous HCT with DiI-stained cells demonstrated that transplanted cells assume a normal morphology and constitute up to 5% of hepatocytes. These data demonstrate transduction and transplantation of human hepatocytes and the feasibility of HCT in large animals. On the basis of these studies, the proposed clinical trial for gene transfer and transplantation in human subjects has been approved by the National Institutes of Health and the Food and Drug Administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

DNA- and viral-mediated gene transfer in follicular cells: progress toward gene therapy of the thyroid.

The authors investigate the in vitro component of an ex situ strategy for gene transfer into the thyroid gland using DNA complex and retroviral vectors. Canine follicular cells harvested by unilateral lobectomy and grown in low-serum media proliferated in culture and retained their differentiated state as evidenced by morphology and thyroglobulin expression. Transient and "stable" gene transfer in thyroid cells were evaluated by comparing DNA and retroviral transduction techniques. Effective gene transfer and expression was demonstrated by histochemical staining for the marker gene product beta-galactosidase. The efficiency of transduction was assessed using an amphotropic retroviral vector carrying the neomycin resistance gene and semiquantitative polymerase chain reaction (PCR) identification of integrated proviral sequences. This analysis demonstrated a proviral frequency in transduced cultures of 10% to 30%. Transduced cells showed no change in morphology or growth patterns and maintained differentiated function as assessed by antibody staining for thyroglobulin. The thyroid gland is an attractive target for somatic gene therapy because of its large protein-synthetic capacity, sensitivity to hormonal regulation, and proportionately high blood flow. Follicular cell gene therapy may be useful not only for treating congenital or acquired diseases of the thyroid, but also disorders of circulating proteins such as hypopituitarism, hemophilia, and diabetes.

Animals↗
PubMed1993

Academic careers: choice and activity of graduates of a pediatric residency program 1974-1986.

In summary, our data suggest that the playing field for academic medicine is changing. It is more patient care oriented, more multifaceted and supported more by clinical dollars than in the past. Greater flexibility in what constitutes "academic success" is necessary to assure a supportive environment in which tomorrow's academic faculty can develop and flourish. To accomplish these goals promotion systems that reward not only research but also teaching and clinical care accomplishments will be necessary. Clinicians will need to be compared with clinicians, teachers with teachers, clinical investigators with clinical investigators and basic investigators with basic investigators. Sources of support will need to be more clearly targeted along activity lines with clinical dollars supporting the clinician, medical education dollars supporting the teacher-educators and federal and foundation dollars supporting research. In our department, time and effort for research (45%) approximates dollar support for this activity (44%), while clinical dollars (43%) fund to a greater degree time and effort committed to clinical care (34%), and administration and teaching dollars (13%) under fund time and effort committed to these activities (21%). This suggests the need to identify increased funding to support teaching and education. Promotion expectations for women will need to be more flexible and adjusted to family responsibilities and demands. Most of all, however, we academic faculty must support enthusiastically the importance and joy of our work. We must be encouraging to our colleagues and our students and continue to recognize that for all of the difficulties and challenges, academic life is a rewarding and fulfilling enterprise.

Academic Medical Centers↗
PubMed1993

Prenatal application of somatic gene therapy.

Somatic gene therapy in the fetus or neonate represents an exciting new approach for the management of some congenital or inherited diseases. It is likely that clinical trials of these methods will take place in the future. In contrast, despite the potential for manipulation of embryos and inherited genetic material, the clinical applicability of these methods is uncertain.

Female↗
PubMed1993

Factors influencing the interests, career paths, and research activities of recent graduates from an academic, pediatric residency program.

OBJECTIVE: This study was undertaken to characterize the professional activity of a cohort of young pediatricians who completed an academically oriented residency program, to analyze factors which influenced their interests, career choices, and research activity, and to understand what steps might be effective in attracting more young physicians into academic and research career paths. METHOD: We studied by questionnaire a cohort of 150 pediatric residents from the Children's Hospital in Boston who completed residency training between 1976 and 1981. RESULTS: Questionnaires were returned by 117 (78%) of 150 individuals. Of these, 73% were in academic medicine and 37% were in clinical practice. Individuals were engaged in various activities with a mean of 47% effort in clinical care, 25% effort in administration and teaching, and 28% effort in basic or clinical research. The majority of respondents (71 [61%] of 117) reported at least 10% effort in all three activities. Only 9 (8%) of 117 reported no clinical activity, 8 (7%) of 117 reported no teaching or administrative activity, and 35 (30%) of 117 reported no research activity. Few individuals dedicated more than 50% of their time to research, and none reported a 100% commitment to research. Clinical sources provided 71% of all salary support, research grants 17%, government salaries 7%, and other sources 5%. For most respondents, interest in clinical practice predated medical school, whereas interest in research increased during preclinical training, particularly among those who engaged in research activities. Respondents identified previous research experience as the principle factor enhancing their decision and ability to do research. Factors that adversely influenced their decision or ability to do research included salary expectancy, funding, the perceived competitiveness of academic life, administrative duties, and family responsibilities. CONCLUSIONS: Efforts to encourage more young physicians to enter academic careers should focus on providing research experience during preclinical training and providing long-term support for research which enables physician scientists to maintain their interest and involvement in clinical medicine.

Adult↗
PubMed1993

Cloning of functional alpha propionyl CoA carboxylase and correction of enzyme deficiency in pccA fibroblasts.

Propionyl CoA carboxylase (PPC) is a heteromeric enzyme composed of alpha subunits (PCCA) and beta (PCCB) subunits. We describe cDNA clones expressing human PCCA and complementation of the genetic defect in pccA fibroblasts by DNA-mediated gene transfer. Two cDNA clones were constructed. The first corresponds to the previously reported, putatively full-length, open reading frame. The second encodes a chimera composed of the mitochondrial leader sequence of human methylmalonyl CoA mutase and the mature PCCA protein. Both clones reconstitute propionate flux to normal levels in fibroblasts from patients genetically deficient in PCCA (pccA). The maximal level of propionate flux approached, but never exceeded, the levels seen in control plates of normal cells. In contrast, the maximal level of PPC holoenzyme activity reached only 10%-20% that of normal controls, which corresponded roughly to the fraction of cells actually transformed with the recombinant gene. These data suggest that the level of PCCA expression in fibroblasts does not normally limit PCC holoenzyme activity or propionate flux. The fact that a small fraction of cells reconstitutes propionate flux to normal levels suggests that metabolic cooperation between cells is capable of increasing the metabolic capacity of recombinant enzyme in a subpopulation of cells. These factors may have important implications for the rational design of somatic gene therapy for PCCA deficiency.

Amino Acid Sequence↗
PubMed1993

Are contemporary methods for somatic gene therapy suitable for clinical applications?

Clinical trials involving the introduction of recombinant genes into human subjects began in 1989 after a decade of earnest debate concerning the technical, social, and ethical implications of somatic gene therapy. The initial trials involved the introduction of recombinant genes into peripheral blood lymphocytes to study the characteristics of tumor infiltrating lymphocytes, enhance immunotherapy for cancer, and treat severe combined immunodeficiency due to adenosine deaminase deficiency. Additional clinical trials involving the introduction of genes into bone marrow, hepatocytes, and tumor cells are underway. Are these clinical trials premature? This article reviews basic methods for somatic gene therapy and the clinical trials which have been proposed or performed to date. This clinical experience suggests that gene therapy can be performed in select clinical trials safely and with public acceptance. Clinical trials have provided essential data concerning the feasibility and safety of gene transfer in human subjects. These trials have also brought a clinical focus to the assessment of technologies currently being used in experimental models. While there will certainly be significant advances in somatic gene therapy in the future, existing methods may be employed fairly in clinical trials.

Clinical Trials as Topic↗
PubMed1992

Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors.

Experiments in animal models suggest that it is feasible to consider hepatic gene therapy using a strategy in which hepatocytes would be isolated by partial hepatectomy, transduced with recombinant retroviral vectors containing genes of therapeutic importance, and then transplanted back into the patient by autologous hepatocellular transplantation. The application of this strategy in clinical trials will require adapting these methods to human cells. We describe the transduction of primary human hepatocytes with two forms of retroviral vectors: amphotropic vectors, which have been used previously in clinical trials, and xenotropic vectors, which have a different host range. Human hepatocytes were harvested from organs preserved in Belzer's solution and were cultivated in a serum-free, tyrosine-free, hormonally defined medium. These cells proliferated for 3-5 days in culture, exhibited characteristic hepatocyte morphology, and expressed liver-specific functions, including phenylalanine hydroxylase, alpha 1-antitrypsin, and glutamine synthase. Transduction with an amphotropic LNL6 retroviral vector resulted in stable incorporation of the provirus into 1% of the cells as estimated by semiquantitative PCR. Consistently higher transduction efficiencies (as much as 10% of the cells) were observed with a xenotropic N2 vector. These data support the feasibility of using LNL6 as a marker gene in clinical trials of hepatocellular transplantation. These data also suggest that the efficiency of transducing hepatocytes with amphotropic vectors in animal models may not accurately reflect the utility of these vectors for human applications. Consideration should be given to the use of xenotropic vectors for optimizing the efficiency of transduction for human applications.

Animals↗
PubMed1992

Cobalamin deficiency associated with methylmalonic acidemia in a cat.

A 9-month-old sexually intact male longhair cat was examined because of lethargy, anorexia, cold intolerance, and failure to thrive since acquisition at an early age. Clinical signs of disease were less pronounced when the cat was fed a low-protein diet. Anemia, hypoglycemia, low total CO2 content, and hyperammonemia were detected. The cat was euthanatized. Urine obtained immediately before euthanasia contained a large amount of methylmalonic acid. Total serum cobalamin concentration was low. Hepatic methylmalonic-CoA mutase activity, with and without the addition of coenzyme adenosylcobalamin, was consistent with a cobalamin deficiency. Methylmalonic acidemia secondary to a putative defect in cobalamin absorption was diagnosed.

Absorption↗
PubMed1992

Phenotype of disease in three patients with identical mutations in methylmalonyl CoA mutase.

We have previously identified a mutation in the gene for methylmalonyl CoA mutase in a patient with the mut- phenotype of methylmalonic aciduria. This mutation (G717V) interferes with the binding of the deoxyadenosylcobalamin cofactor to the apoenzyme producing a mutant holoenzyme that is defective, but not completely inactive, in vitro. This report describes the clinical phenotype associated with this mutation in the original patient and two additional patients who are homozygous for this allele. All three patients presented in the first years of life with multiple episodes of life-threatening organic acidosis and hyperammonemia. None had evidence of disease in the perinatal period, and all three have low-normal intelligence. These three children exhibit a distinctive phenotype of disease that is intermediate between the fulminant and benign forms of methylmalonic aciduria. These data suggest that this phenotype is the specific consequence of the G717V mutation, and that the degree of residual enzyme activity associated with the G717V mutation is close to the threshold required in vivo for maintaining metabolic homeostasis.

Acidosis↗
PubMed1992

Propionate metabolism in cultured human cells after overexpression of recombinant methylmalonyl CoA mutase: implications for somatic gene therapy.

Strategies for somatic gene therapy must consider the metabolic consequences of expressing the recombinant gene product in addition to methods for gene transfer and expression. We describe studies of propionate metabolism in cultured cells transfected with methylmalonyl CoA mutase (MCM), the enzyme deficient in mut methylmalonic acidemia. Transfection of MCM into mut fibroblasts restores propionate metabolism to normal levels in a dose-dependent manner. Overexpression of MCM, or the addition of excess propionate, carnitine, or cobalamin, does not increase propionate metabolism in normal human fibroblasts, lymphoblasts, or hepatoma cells, although hepatic cells exhibit > 10-fold higher levels of propionate metabolism. Significantly, the restoration of propionate metabolism in mut fibroblasts is disproportionately greater than the efficiency of transfection, suggesting the presence of a cooperative phenomenon between cells. Intercellular participation in propionate metabolism is evident in cocultures of MCM-deficient and propionyl CoA carboxylase-deficient cells. We conclude that the liver is the preferred target for gene therapy of MCM deficiency because of its greater capacity for propionate metabolism and that cooperation between cells could enhance the biological effect of a subpopulation of cells transformed with recombinant MCM.

Amino Acid Metabolism, Inborn Errors↗
PubMed1992

Correction of methylmalonyl-CoA mutase deficiency in Mut0 fibroblasts and constitution of gene expression in primary human hepatocytes by retroviral-mediated gene transfer.

Methylmalonic acidemia is an often fatal inborn error of organic acid metabolism due to deficiency of methylmalonyl-CoA mutase. The cloning of genes encoding this enzyme and the advent of technologies for gene transfer have introduced the possibility of somatic gene therapy for this disorder. Gene therapy may require replacement of the defective enzyme in hepatocytes, which have a greater capacity for propionate metabolism than other somatic cells and represent the principle physiological site of propionate metabolism. We describe construction of an amphotropic retroviral vector containing the human methylmalonyl-CoA mutase cDNA. This vector is shown to transduce primary MCM-deficient fibroblasts and restore levels of [14C]propionate metabolism by cultures of nonselected cells to normal. This vector will transduce primary human hepatocytes and direct transcription of recombinant human MCM from the integrated provirus. This work demonstrates the feasibility of retroviral-mediated gene transfer of methylmalonyl-CoA mutase into primary human cells, including hepatocytes which represent a difficult, but potentially necessary, target for gene therapy of methylmalonic acidemia.

3T3 Cells↗
PubMed1992

The challenge of follow-up for clinical trials of somatic gene therapy.

Although extensive efforts have been made to optimize the safety of vectors for somatic gene transfer and somatic gene therapy, the safety of these agents must ultimately be assessed in clinical trials. A statistically significant assessment of safety will be complicated by the relatively small number of patients who will be enrolled in initial clinical trials, the need for long-term longitudinal follow-up of patients and perhaps their progeny, and the traditionally poor participation in long-term follow-up by many patients. This article reviews the potential risks of retroviral-mediated gene transfer, the statistical power required to assess the true incidence of potential complications, the number of patients who may participate in clinical trials involving retroviral vectors, and the factors that make thorough follow-up and uniform data ascertainment difficult. The role of the FDA in assessing the safety of retroviral vectors and the potential role of registries for patient tracking and data ascertainment are discussed.

Clinical Trials as Topic↗
PubMed1992

Somatic gene therapy in gastroenterology: approaches and applications.

Somatic gene therapy represents a new approach for treating a variety of genetic and acquired diseases and has many potential applications in gastroenterology. Somatic gene therapy is based on the ability to transfer recombinant genes efficiently into somatic tissues, such as the liver or intestine, and achieve expression of the recombinant gene product. Gene transfer could be used to replace genetically defective gene functions or prevent the progression of acquired or multifactorial diseases. The therapeutic application of gene transfer technologies requires development of methods for achieving efficient transfer and expression of recombinant gene products, research directed at developing strategies for altering the course of disease, and clinical research aimed at assessing both the technical feasibility and therapeutic efficacy of this approach.

Ethics, Medical↗