Fred D. Ledley

Pharmaceutical Approach to Somatic Gene Therapy

The pharmaceutical approach to somatic gene therapy is based on consideration of a gene as a chemical entity with specific physical, chemical and colloidal properties. The genes that are required for gene therapy are large molecules (>1 × 106 Daltons, >100 nm diameter) with a net negative charge that prevents diffusion through biological barriers such as an intact endothelium, the plasma membrane or the nuclear membrane. New methods for gene therapy are based on increasing knowledge of the pathways by which DNA may be internalized into cells and traffic to the nucleus, pharmaceutical experience with particulate drug delivery systems, and the ability to control gene expression with recombined genetic elements. This article reviews two themes in the development of gene therapies: first, the current approaches involving the administration of cells, viruses and plasmid DNA; second, the emerging pharmaceutical approach to gene therapy based on the pharmaceutical characteristics of DNA itself and methods for advanced drug delivery.

Benign Methylmalonic Aciduria

Methylmalonic aciduria due to methylmalonyl-CoA mutase deficiency is usually considered to be a serious, often life-threatening disease. However, through routine screening of urine in neonates or screening of siblings of clinically affected neonates, we have identified eight children who have a benign clinical variant of this disorder. Their urinary methylmalonic acid levels have ranged from 1.0 to 3.4 mg per milligram of creatinine, with serum values ranging from an undetectable level to 1.7 mg per deciliter (130 nmol per liter). The children have not received dietary or vitamin therapy, have had normal growth and development (age range, 18 months to 13 years), and have performed as well as their unaffected siblings on psychometric testing. These children have no evidence of a deficiency of vitamin B12, which acts as a cofactor with methylmalonyl-CoA mutase, and they did not respond to the administration of vitamin B12. Two siblings were found by complementation analysis to have a defect in the methylmalonyl-CoA mutase apoenzyme; complementation analysis was not performed on the other patients. We conclude that the clinical spectrum of methylmalonyl-CoA mutase deficiency is wider than indicated by previously reported cases.

Sequence similarity between cholera toxin and glycoprotein hormones: Implications for structure activity relationship and mechanism of action

Abstract The B chain of cholera toxin and the β subunits of thyrotropin, luteinizing hormone, human chorionic gonadotropin, and follicle-stimulating hormone are shown to have a region of sequence analogy believed to correlate with their ability to bind to receptors on cell membranes. A possible sequence analogy is also defined in the α subunits of these glycoprotein hormones and a region of the cholera toxin A 1 chain believed to be responsible for adenylate cyclase activation.

Effective cryopreservation and long-term storage of primary human hepatocytes with recovery of viability, differentiation, and replicative potential.

Despite reports of successful cryopreservation of primary human hepatocytes, existing methods do not produce sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocellular transplantation. We now describe a protocol for efficient cryopreservation of primary human hepatocytes using University of Wisconsin (UW) solution, fetal bovine serum, and dimethyl sulfoxide (DMSO). This method provides > 90% viability of differentiated, primary human hepatocytes 8 mo after cryopreservation as measured by trypan blue exclusion, preserves hepatocyte morphology, liver-specific gene expression (alpha 1 antitrypsin), and replication. The effectiveness of UW solution as a cryopreservative agent suggests that metabolic as well as ultrastructural factors may be important in the effective cryopreservation of primary human hepatocytes. The present method represents an effective protocol for cryopreserving differentiated primary human hepatocytes for research. This method may allow characterization and banking of human hepatocytes for clinical applications, including hepatocellular transplantation and hepatic assist devices.

Mutations in mut methylmalonic acidemia: Clinical and enzymatic correlations

Mut methylmalonic acidemia is caused by mutations in the MUT locus encoding the enzyme methylmalonyl CoA mutase. Genotypic and phenotypic variability in this disease has been studied extensively by biochemical and somatic cell genetic techniques, by molecular cloning, and by gene transfer. Mutations have been identified that cause classic muto phenotypes in which there is no detectable enzymatic activity, mut− phenotypes in which there is residual cobalamin‐dependent activity, as well as a subset within both muto and mut− phenotypes that exhibit interallelic complementation. These mutations illustrate the position, structure, and function of critical domains within this cobalamin‐binding enzyme and provide new insights into the biochemical and clinical consequences of enzyme deficiency.

Correlation between polymorphic DNA haplotypes at phenylalanine hydroxylase locus and clinical phenotypes of phenylketonuria

Eight polymorphic sites for seven restriction endonucleases have been reported at the human phenylalanine hydroxylase locus. The composite profile of the presence or absence for each of the eight polymorphic sites within an allele defines the haplotype of the corresponding allele. Twelve such haplotypes associated with normal and mutant phenylalanine hydroxylase alleles have been identified in 33 Danish families with children with phenylketonuria. Of the 66 mutant alleles analyzed, 59 (89%) were associated with only four haplotypes. The identification of individual phenylalanine hydroxylase alleles by haplotype analysis enables correlation of the hyperphenylalaninemic phenotypes of the patients with their genotypes. Patients who were either homozygous or heterozygous for the mutant alleles of haplotypes 2 and 3 had a severe clinical course. Patients who had a mutant allele of either haplotype 1 or 4 usually had a less severe clinical phenotype. The recent demonstration that the mutation responsible for classic phenylketonuria associated with haplotype 3 is not present in mutant alleles of other haplotypes provides unambiguous evidence that there are multiple mutations in the phenylalanine hydroxylase gene and supports the hypothesis that different combinations of mutant alleles may be responsible for the clinical diversity of phenylketonuria.

Molecular analysis of the inheritance of phenylketonuria and mild hyperphenylalaninemia in families with both disorders

Clinical phenylketonuria and mild hyperphenylalaninemia represent two different phenotypes of phenylalanine hydroxylase deficiency. To determine the genetic relation between these two phenotypes, we studied two families in which one member had phenylketonuria and other members had mild hyperphenylalaninemia. We identified restriction-fragment-length polymorphisms that differentiated the four phenylalanine hydroxylase alleles in each family. Phenylketonuria and mild hyperphenylalaninemia were found to be allelic; certain pairs of alleles induced the more severe phenylketonuria phenotype, and other pairs induced the less severe hyperphenylalaninemia phenotype. Several of the alleles that were identified can contribute to either phenylketonuria or mild hyperphenylalaninemia. These results demonstrate that there are multiple and distinct mutations in the phenylalanine hydroxylase gene, with different levels of severity, and that various combinations of the mutant alleles can result in different phenotypes of the metabolic disorders of hyperphenylalaninemia.

Thyrotropin interactions with human fat cell membrane preparations and the finding of a soluble thyrotropin binding component

Summary Both human and guinea pig adipocyte membranes are shown to have specific thyrotropin receptor sites, and the thyrotropin binding to human retro-orbital tissue adipocyte membranes is shown to be enhanced by gamma globulin from patients with exophthalmos. In addition, supernatant solutions from crude human and guinea pig membrane preparations are shown to contain a thermolabile, non-dialyzable inhibitor of thyrotropin binding which acts by forming a thyrotropin-inhibitor addut rather than by directly interacting with the membrane receptors.

Production of discrete high specific activity DNA probes using the polymerase chain reaction

Conditions are described for the synthesis of discrete DNA probes with high specific activity using the polymerase chain reaction. This method enables the production of DNA probes between any two oligonucleotide sequences from cloned or uncloned templates. These probes are uniform in length and their specific activity (1 x 10(9) cpm/microgram) is comparable to probes produced by other methods.

Cloning and expression of a mutant methylmalonyl coenzyme A mutase with altered cobalamin affinity that causes mut- methylmalonic aciduria.

Distinct genotypic and phenotypic forms of methylmalonyl CoA mutase (MCM) apoenzyme deficiency can be delineated by biochemical analysis of mutant fibroblasts. One form, designated mut-, expresses a phenotype in which residual enzyme activity is evident in cultured cells exposed to high concentrations of hydroxycobalamin. We describe cloning of an MCM cDNA from cells exhibiting a mut- phenotype and characterization of the mutant gene product overexpressed in primary muto human fibroblasts and Saccharomyces cerevisiae. Three novel base changes were observed. Recombinant clones containing one of these base changes (G717V) express four characteristics of the mut- phenotype: failure to constitute [14C]propionate incorporation activity in fibroblasts assayed under basal cell culture conditions, constitution of [14C]propionate incorporation activity in fibroblasts stimulated with 0.1-1.0 micrograms/ml hydroxycobalamin, interallelic complementation with alleles bearing an R93H mutation, and an apparent Km (adenosylcobalamin) 1,000-fold higher than normal. These results demonstrate that the G717V mutation produces the mut- phenotype and localizes determinants for adenosylcobalamin binding near the carboxyl terminus of MCM.