Why commercialization of gene therapy stalled; examining the life cycles of gene therapy technologies.

This report examines the commercialization of gene therapy in the context of innovation theories that posit a relationship between the maturation of a technology through its life cycle and prospects for successful product development. We show that the field of gene therapy has matured steadily since the 1980s, with the congruent accumulation of >35 000 papers, >16 000 US patents, >1800 clinical trials and >$4.3 billion in capital investment in gene therapy companies. Gene therapy technologies comprise a series of dissimilar approaches for gene delivery, each of which has introduced a distinct product architecture. Using bibliometric methods, we quantify the maturation of each technology through a characteristic life cycle S-curve, from a Nascent stage, through a Growing stage of exponential advance, toward an Established stage and projected limit. Capital investment in gene therapy is shown to have occurred predominantly in Nascent stage technologies and to be negatively correlated with maturity. Gene therapy technologies are now achieving the level of maturity that innovation research and biotechnology experience suggest may be requisite for efficient product development. Asynchrony between the maturation of gene therapy technologies and capital investment in development-focused business models may have stalled the commercialization of gene therapy.

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 mut(o) 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 mut(o) 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.

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 x 10(6) 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.

[A molecular study of methylmalonic aciduria: structure-function correlations]. / Etude moléculaire de l'acidurie méthylmalonique: corrélations structure-fonction.

Cobalamin (Vitamin B12) non-responsive methylmalonic acidemia is caused by mutations in the MUT locus on chromosome 6p21 encoding the enzyme methylmalonyl CoA mutase (EC 5.4.99.2). This disorder has been extensively studied by biochemical, somatic cell genetic and molecular techniques. Mutations have been identified which cause classic mut(o) phenotypes in which there is no detectable enzymatic activity, as well as mut- phenotypes in which there is residual cobalamin-dependent activity. Mutations which exhibit interallelic complementation have been identified within both of these groups. 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. The homology of the cobalamin binding region has allowed mutations of the mutase to be mapped onto the x-ray structure of methionine synthase (EC 2.1.1.13).

Molecular basis for dysfunction of some mutant forms of methylmalonyl-CoA mutase: deductions from the structure of methionine synthase.

Inherited defects in the gene for methylmalonyl-CoA mutase (EC 5.4.99.2) result in the mut forms of methylmalonic aciduria. mut- mutations lead to the absence of detectable mutase activity and are not corrected by excess cobalamin, whereas mut- mutations exhibit residual activity when exposed to excess cobalamin. Many of the mutations that cause methylmalonic aciduria in humans affect residues in the C-terminal region of the methylmalonyl-CoA mutase. This portion of the methylmalonyl-CoA mutase sequence can be aligned with regions in other B12 (cobalamin)-dependent enzymes, including the C-terminal portion of the cobalamin-binding region of methionine synthase. The alignments allow the mutations of human methylmalonyl-CoA mutase to be mapped onto the structure of the cobalamin-binding fragment of methionine synthase from Escherichia coli (EC 2.1.1.13), which has recently been determined by x-ray crystallography. In this structure, the dimethylbenzimidazole ligand to the cobalt in free cobalamin has been displaced by a histidine ligand, and the dimethylbenzimidazole nucleotide "tail" is thrust into a deep hydrophobic pocket in the protein. Previously identified mut0 and mut- mutations (Gly-623 --> Arg, Gly-626 --> Cys, and Gly-648 --> Asp) of the mutase are predicted to interfere with the structure and/or stability of the loop that carries His-627, the presumed lower axial ligand to the cobalt of adenosylcobalamin. Two mutants that lead to severe impairment (mut0) are Gly-630 --> Glu and Gly-703 --> Arg, which map to the binding site for the dimethylbenzimidazole nucleotide substituent of adenosylcobalamin. The substitution of larger residues for glycine is predicted to block the binding of adenosylcobalamin.

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.

Nonviral gene therapy: the promise of genes as pharmaceutical products.

Although most research on gene therapy has focused on the use of recombinant viruses to deliver genes to cells in vivo, progress also has been made toward developing nonviral, pharmaceutical formulations of genes for in vivo human therapy. Various methods for nonviral gene therapy have been proposed. Some approaches are aimed at developing "artificial viruses" that attempt to mimic the process of viral infection using synthetic materials. Others apply the theory and methods of advanced, particulate drug delivery to deliver DNA to select somatic targets. These approaches employ DNA complexes containing lipid, protein, peptide, or polymeric carriers as well as ligands capable of targeting the DNA complex to cell-surface receptors on the target cell and ligands for directing the intracellular trafficking of DNA to the nucleus. Nonviral systems have been used to deliver genes to the lung, liver, endothelium, epithelium, and tumor cells and have been shown to be generally safe. More than a dozen clinical trials are currently underway using nonviral systems for disease indications including cystic fibrosis and cancer. Future advances in nonviral systems will be based on an emerging appreciation of the biological constraints on the fate and function of DNA within the body and within the cell.

Retroviral gene transfer into the intestinal epithelium.

The epithelial cells of the gastrointestinal tract may be attractive targets for somatic gene therapy. In these studies, we have used rats and mice to explore the feasibility of gene transfer into the small intestinal epithelium using retroviral vectors. The first series of experiments was conducted in mature Sprague-Dawley rats using an ecotropic retroviral vector that has bacterial beta-galactosidase (beta-Gal) as the reporter gene. The vector was introduced into the lumen of ligated segments of terminal ileum. After a 4-hr exposure period, the ligatures were removed. Sham-operated animals were subjected to the same ligation procedure but received only tissue culture medium in the ligated segment. All animals were sacrificed 6 days later, and tissue from both the experimental segment and an upstream control segment was assessed for cytoplasmic beta-Gal activity using X-Gal histochemistry. Expression of the reporter gene was observed in the crypt epithelium of tissue exposed to the vector. In the villus epithelium, high background staining precluded accurate assessment of reporter gene expression. To obviate the latter problem, we sought an alternative reporter gene for which there would be no background staining in control animals. We repeated the experiments with beta-glucuronidase as the reporter gene in MPS VII mutant mice, which are devoid of this enzyme. In these studies, ileal segments exposed to the vector demonstrated expression of the reporter gene in both the crypt and villus epithelium 4 days after exposure. These results indicate that genes can be transferred into the intestinal epithelium using retroviral vectors introduced luminally.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene transfer to synovial cells by intra-articular administration of plasmid DNA.

We studied reporter gene expression in synovial tissue after intra-articular administration of an expression plasmid into the knees of rabbits and rats. In both species, administration of a plasmid encoding beta-galactosidase led to gene expression in the synovial cells lining the joint. Expression correlated with the presence of plasmid DNA in synovial tissue extracts. Studies with a plasmid encoding chloramphenicol acetyltransferase demonstrated that gene expression persists for 2-5 days after administration. Southern blotting demonstrated that the administered plasmid was taken up rapidly by synovial tissue and degraded. By 24 hr after administration, no intact plasmid could be detected by Southern blotting, although small amounts of plasmid could be amplified by PCR up to 7 days. Administration of a plasmid encoding human growth hormone demonstrated that this product could be expressed from synovial cells and secreted into the synovial fluid. The histological distribution of gene expression in synovium resembles the known distribution of particulate materials injected into the joint and suggests that plasmid DNA is taken up by nonspecific endocytosis like other particulate materials during the remodeling of synovial fluid.

Infection by retroviral vectors outside of their host range in the presence of replication-defective adenovirus.

Retrovirus infection is normally limited to cells within a specific host range which express a cognate receptor that is recognized by the product of the env gene. We describe retrovirus infection of cells outside of their normal host range when the infection is performed in the presence of a replication-defective adenovirus (dl312). In the presence of adenovirus, several different ecotropic vectors are shown to infect human cell lines (HeLa and PLC/PRF), and a xenotropic vector is shown to infect murine cells (NIH 3T3). Infectivity is demonstrated by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, selection with G418 for neomycin resistance, and PCR identification of the provirus in infected cells. Infectivity is quantitatively dependent upon both the concentration of adenovirus (10(6) to 10(8) PFU/ml) and the concentration of retrovirus. Infection requires the simultaneous presence of adenovirus in the retrovirus infection medium and is not stimulated by preincubation and removal of adenovirus from the cells before retrovirus infection. The presence of adenovirus is shown to enhance the uptake of fluorescently labeled retrovirus particles into cells outside of their normal host range, demonstrating that the adenovirus enhances viral entry into cells in the absence of the recognized cognate receptor. This observation suggests new opportunities for developing safe retroviral vectors for gene therapy and new mechanisms for the pathogenesis of retroviral disease.