Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A.

BACKGROUND: We tested the safety of a nonviral somatic-cell gene-therapy system in patients with severe hemophilia A. METHODS: An open-label, phase 1 trial was conducted in six patients with severe hemophilia A. Dermal fibroblasts obtained from each patient by skin biopsy were grown in culture and transfected with a plasmid containing sequences of the gene that encodes factor VIII. Cells that produced factor VIII were selected, cloned, and propagated in vitro. The cloned cells were then harvested and administered to the patients by laparoscopic injection into the omentum. The patients were followed for 12 months after the implantation of the genetically altered cells. An interim analysis was performed. RESULTS: There were no serious adverse events related to the use of factor VIII-producing fibroblasts or the implantation procedure. No long-term complications developed, and no inhibitors of factor VIII were detected. In four of the six patients, plasma levels of factor VIII activity rose above the levels observed before the procedure. The increase in factor VIII activity coincided with a decrease in bleeding, a reduction in the use of exogenous factor VIII, or both. In the patient with the highest level of factor VIII activity, the clinical changes lasted approximately 10 months. CONCLUSIONS: Implantation of genetically altered fibroblasts that produce factor VIII is safe and well tolerated. This form of gene therapy is feasible in patients with severe hemophilia A.

Infusion of alpha-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease.

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb(3); also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified alpha-gal A, 10 patients with Fabry disease received a single i.v. infusion of one of five escalating dose levels of the enzyme. The objectives of this study were: (i) to evaluate the safety of administered alpha-gal A, (ii) to assess the pharmacokinetics of i.v.-administered alpha-gal A in plasma and liver, and (iii) to determine the effect of this replacement enzyme on hepatic, urine sediment and plasma concentrations of Gb(3). alpha-Gal A infusions were well tolerated in all patients. Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified alpha-gal A in several cell types, including sinusoidal endothelial cells, Kupffer cells, and hepatocytes, suggesting diffuse uptake via the mannose 6-phosphate receptor. The tissue half-life in the liver was greater than 24 hr. After the single dose of alpha-gal A, nine of the 10 patients had significantly reduced Gb(3) levels both in the liver and shed renal tubular epithelial cells in the urine sediment. These data demonstrate that single infusions of alpha-gal A prepared from transfected human fibroblasts are both safe and biochemically active in patients with Fabry disease. The degree of substrate reduction seen in the study is potentially clinically significant in view of the fact that Gb(3) burden in Fabry patients increases gradually over decades. Taken together, these results suggest that enzyme replacement is likely to be an effective therapy for patients with this metabolic disorder.

Identification of common cystic fibrosis mutations in African-Americans with cystic fibrosis increases the detection rate to 75%.

Cystic fibrosis (CF)--an autosomal recessive disorder caused by mutations in CF transmembrane conductance regulator (CFTR) and characterized by abnormal chloride conduction across epithelial membranes, leading to chronic lung and exocrine pancreatic disease--is less common in African-Americans than in Caucasians. No large-scale studies of mutation identification and screening in African-American CF patients have been reported, to date. In this study, the entire coding and flanking intronic sequence of the CFTR gene was analyzed by denaturing gradient-gel electrophoresis and sequencing in an index group of 82 African-American CF chromosomes to identify mutations. One novel mutation, 3120+1G-->A, occurred with a frequency of 12.3% and was also detected in a native African patient. To establish frequencies, an additional group of 66 African-American CF chromosomes were screened for mutations identified in two or more African-American patients. Screening for 16 "common Caucasian" mutations identified 52% of CF alleles in African-Americans, while screening for 8 "common African" mutations accounted for an additional 23%. The combined detection rate of 75% was comparable to the sensitivity of mutation analysis in Caucasian CF patients. These results indicate that African-Americans have their own set of "common" CF mutations that originate from the native African population. Inclusion of these "common" mutations substantially improves CF mutation detection rates in African-Americans.

Non-viral gene therapy.

Gene therapy is a medical/surgical intervention currently being developed, in which genes are introduced into cells in order to treat or cure a wide variety of human diseases. The field has evolved over the past four decades, with most experimental gene-therapy studies based on the use of viruses to deliver the genes of therapeutic interest. More recently, a large number of non-viral approaches to gene therapy have emerged, yielding promising pre-clinical results, and which are currently being evaluated in early stage clinical trials.

Long-term production and delivery of human growth hormone in vivo.

The application of somatic cell gene therapy to large patient populations will require the development of safe and practical approaches to the generation and characterization of genetically manipulated cells. Transkaryotic implantation is a gene therapy system based on the production of clonal strains of engineered primary and secondary cells, using non-viral methods. We demonstrate here that, on implantation, these clonal cell strains stably and reproducibly deliver pharmacologic quantities of protein for the lifetime of the experimental animals.

Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases.

The human growth hormone transgene: expression in hemizygous and homozygous mice.

Female transgenic mice carrying the mouse metallothionein-I/human growth hormone (hGH) fusion gene are sterile. Transmission of the transgene has been limited to the male germ line, resulting in the production of hemizygous (He) progeny containing only a single (paternal) copy of the gene. Using ovary transfer, we have developed procedures for producing homozygous (Ho) TG mice, viz., male TG mice were mated with control (non-TG) females carrying ovaries donated by female TG mice. In both He and Ho TG animals, serum levels of hGH were higher (1.5-fold) in males than in females, tended to decrease with age of the animal, and were increased (about 5-fold) by zinc induction. However, in comparison to He animals of the same sex, the Ho TG mice attained a greater body weight and had more than 2-fold higher levels of liver hGH-mRNA and serum hGH, both under basal conditions and in response to zinc induction. That is, the expression of the transgene was qualitatively similar in He and Ho TG mice, but the level of transgene activity was greater in the Ho animals. We interpret this to indicate that both copies (maternal and paternal) of the transgene were active and expressed additively (or cooperatively) in the Ho TG animal.

Tolerance induced by physiological levels of secreted proteins in transgenic mice expressing human insulin.

We have used transgenic mice to study immune tolerance to autologous, non-MHC encoded proteins that are expressed at physiological levels in the circulation. The transgenic mice used in these studies express the human preproinsulin gene and synthesize human proinsulin. Human and mouse insulin are secreted from the pancreatic islets of transgenic mice in response to normal physiological stimuli, such as glucose. Our data demonstrate that the transgenic mice have acquired tolerance to human insulin. The repertoire of T cells specific for exogenous antigens is shaped by the acquired tolerance to autologous proteins since pork but not beef or sheep insulin is also nonimmunogenic in the transgenic mice. We also found that the transgenic mice were tolerant to human proinsulin, the intracellular precursor of insulin. Unresponsiveness to human proinsulin most likely results from tolerance of insulin-specific and proinsulin-specific T cells that recognize the secreted enzymatic cleavage products of proinsulin, insulin and C-peptide.

Glucocorticoid regulation of human growth hormone expression in transgenic mice and transiently transfected cells.

A mouse metallothionein-I/human growth hormone fusion gene was microinjected into fertilized mouse eggs, the embryos were implanted into pseudopregnant foster mothers, and the offspring analysed. Five of twenty-six mice born after one series of injections contained from one to eight copies of the fusion gene stably integrated into their genomes and had human growth hormone in their serum. When several of these transgenic mice and transgenic offspring were treated with glucocorticoids, serum growth hormone levels were elevated from 1.5- to 6.3-fold. A fourfold induction in fusion gene mRNA in the liver of one of the five mice was also observed after treatment with glucocorticoids. When the fusion gene was transiently transfected into mouse L cells, dexamethasone caused a three- to fourfold induction of fusion gene mRNA and secreted human growth hormone. A deletion analysis of regulatory elements required for inducibility in L cells shows that DNA sequences responsible for the observed inductions are located within the transcribed region of the human growth hormone gene. However, a previously described glucocorticoid receptor binding site in the first intron of the gene is not required for response to the hormone.

Expression of the human growth hormone variant gene in cultured fibroblasts and transgenic mice.

The nucleotide sequence of the human growth hormone variant gene, one of the five members of the growth hormone gene family, predicts that it encodes a growth hormone-like protein. As a first step in determining whether this gene is functional in humans, we have expressed a mouse metallothionein I/human growth hormone variant fusion gene in mouse L cells and in transgenic mice. The growth hormone variant protein expressed in transiently transfected L cells is distinct from growth hormone itself with respect to reactivity with anti-growth hormone monoclonal antibodies, behavior during column chromatography, and isoelectric point. Transgenic mice expressing the growth hormone variant protein are 1.4- to 1.9-fold larger than nontransgenic controls, suggesting that the protein has growth-promoting properties.

Implantation of genetically engineered fibroblasts into mice: implications for gene therapy.

In a variety of human genetic diseases, replacement of the absent or defective protein provides significant therapeutic benefits. As a model for a somatic cell gene therapy system, cultured murine fibroblasts were transfected with a human growth hormone (hGH) fusion gene and cells from one of the resulting clonal lines were subsequently implanted into various locations in mice. Such implants synthesized and secreted hGH, which was detectable in the serum. The function of the implants depended on their location and size, and on the histocompatibility of the donor cells with their recipients. The expression of hGH could be modified by addition of regulatory effectors, and, with appropriate immunosuppression, the implants survived for more than 3 months. This approach to gene therapy, here termed "transkaryotic implantation," is potentially applicable to many genetic diseases in that the transfected cell line can be extensively characterized prior to implantation, several anatomical sites are suitable for implantation, and regulated expression of the gene of therapeutic interest can be obtained.

Talc poudrage in the treatment of spontaneous pneumothoraces in patients with cystic fibrosis.

As patients with cystic fibrosis live longer, spontaneous pneumothoraces are seen with increasing frequency. Severe underlying pulmonary disease in these patients makes them particularly susceptible to life-threatening respiratory distress. Several modalities, including chemical sclerosis and open thoracotomy with pleurectomy, have been used to treat pneumothoraces in these patients. In the past 4 years, pneumothoraces in five patients (ages 9-22 years) with cystic fibrosis have been treated with thoracoscopy and talc poudrage. All procedures were performed under either regional or general anesthesia, depending on the age of the patient. Thoracoscopy was performed with a rod lens system and a 5.5-mm trocar, using biopsy forceps to lyse pleural adhesions, all of which ensures access to the entire pleural surface. United States Pharmacopeia-certified talc was insufflated to cover the entire pleural surface. There were no complications, and the patients had minimal pleural pain. Follow-up ranged from 6 months to 4 years. No patient has had a recurrent pneumothorax on the treated side. Thoracoscopy with talc poudrage is a preferable alternative to chemical sclerosis or thoracotomy for treating pneumothoraces in patients with cystic fibrosis. The procedure may be performed under regional anesthesia and allows rapid and complete sclerosis of the pleural cavity.

Human growth hormone as a reporter gene in regulation studies employing transient gene expression.

The human growth hormone (hGH) transient assay system described here is based on the expression of hGH directed by cells transfected with hGH fusion genes. Levels of secreted hGH in the medium, measured by a simple radioimmunoassay, are proportional to both levels of cytoplasmic hGH mRNA and the amount of transfected DNA. The system is extremely sensitive, easy to perform, and is qualitatively different from other transient expression systems in that the medium is assayed and the cells themselves are not destroyed. The hGH transient assay system is appropriate for analyses of regulation of gene expression and was utilized here to investigate the effect of the simian virus 40 enhancer on the herpes simplex virus thymidine kinase promoter and the effect of zinc on the mouse metallothionein-I promoter. The expression of hGH can also be used as an internal control to monitor transfection efficiency along with any other transient expression system. All cell types tested thus far (including AtT-20, CV-1, GC, GH4, JEG, L, and primary pituitary cells) were able to secrete hGH into the medium.

Regulation of human insulin gene expression in transgenic mice.

Insulin is a polypeptide hormone of major physiological importance in the regulation of fuel homeostasis in animals (reviewed in refs 1,2). It is synthesized by the beta-cells of pancreatic islets, and circulating insulin levels are regulated by several small molecules, notably glucose, amino acids, fatty acids and certain pharmacological agents. Insulin consists of two polypeptide chains (A and B, linked by disulphide bonds) that are derived from the proteolytic cleavage of proinsulin, generating equimolar amounts of the mature insulin and a connecting peptide (C-peptide). Humans, like most vertebrates, contain one proinsulin gene, although several species, including mice and rats, have two highly homologous insulin genes. We have studied the regulation of serum insulin levels and of insulin gene expression by generating a series of transgenic mice containing the human insulin gene. We report here that the human insulin gene is expressed in a tissue-specific manner in the islets of these transgenic mice, and that serum human insulin levels are properly regulated by glucose, amino acids and tolbutamide, an oral hypoglycaemic agent.

Cefsulodin sodium therapy in cystic fibrosis patients.

Cefsulodin sodium is a narrow-spectrum cephalosporin with marked in vitro activity against clinical isolates of Pseudomonas aeruginosa. We have studied the antibiotic in a clinical trial in 10 patients admitted to the Pediatric Ward of the University of Virginia Medical Center with cystic fibrosis and recurrent acute lower respiratory tract infections with P. aeruginosa isolated from their sputa. The patients received 500 to 1,500 mg of cefsulodin every 6 hours by intravenous infusion for 10 to 22 days. Mean peak drug levels in plasma after 500, 1,000, and 1,500 mg were 46, 71, and 90 micrograms/ml, respectively, and the mean minimal inhibitory concentration of all organisms was 7.5 micrograms/ml. Detectable levels of cefsulodin in sputa were found in approximately half of the random samples and ranged from 2 to 5 micrograms/ml. The clinical response was satisfactory in nine (90%) of the patients. One patient gained weight and had improved pulmonary function tests but showed no reduction in sputum production and no improvement in arterial blood gas values. In pulmonary function tests, four of five patients tested showed an average 43% increase in forced vital capacity after initiation of therapy and five of five had an average 51% increase in forced expired volume in 1 s. No adverse effects were observed.

Transfer RNA genes ofZea mays chloroplast DNA.

A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA2 (Ile) and tRNA(Ala) map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAs(Leu) and the three for tRNAs(Ser), as well as the two isoaccepting species for tRNA(Asn), tRNA(Gly), tRNAs(Ile), tRNAs(Met), tRNAs(Thr), are shown to be encoded at different loci.Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were(32)P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases.

Otitis media and the immotile cilia syndrome.

The immotile cilia syndrome appears to be a congenital defect in the ultrastructure of cilia that renders them incapable of movement. Respiratory tract cilia and sperm are predominantly affected. Bronchiectasis, sinusitis and male sterility are the main clinical findings. Situs inversus may be found. To these findings can be added otitis media. The defect appears to be a complete or partial absence of dynein arms which are believed to be essential for generating movement of cilia or sperm tails. Six patients suspected of having immotile cilia were compared to six patients in a control group. In affected patients, no cilia movement in the middle ear or nasopharynx was observed using the operating microscope. Electron microscopy of cilia from the mucosa of the middle ear and nasopharynx appeared to confirm the ultrastructural defect in two of six patients suspected of having the syndrome.

Avasucular necrosis of pheochromocytoma followed by spontaneous remission.

Following an acute spontaneous hypertensive crisis and shock a patient with pheochromocytoma was found to have an exceedingly high catecholamine excretion rate. After this episode, the patient remained normotensive and urinary excretion of catecholamines returned to normal. During surgery, a large pheochromocytoma was found and removed that showed avascualr necrosis. In phenochromocytoma, a sudden and exceedingly high rate of catecholamine release may cause intense vasoconstriction both generally and within the tumor itself. In this patient, avascular tumor necrosis led to a spontaneous remission of clinical symptoms.