David B. Schowalter

The generation of radiolabeled dna and rna probes with polymerase chain reaction

By including a radioactive triphosphate during polymerase chain reaction (PCR), probes of very high specific activity can be generated. The advantages of PCR labeling include (1) uniform labeling with a specific activity of 5 X 10(9) cpm/micrograms or higher (sensitivity of detection: 0.028 pg of target DNA per 24 h); (2) ease of regulation of both the specific activity and the amount of labeled probe produced; (3) efficient labeling of fragments less than 500 bp; (4) efficient incorporation over a wide range of input DNA template; (5) labeling with subnanogram amounts of input DNA; and (6) direct labeling of genomic DNA. The minimal amount of input DNA allows a virtually unlimited number of PCR labeling reactions to be performed on DNA generated by one amplification under the previously described nonlabeling conditions. This obviates the need for CsCl gradients or other large scale methods of DNA preparation. The above advantages except for the very high specific activity can also be achieved by transcript labeling after an amplification where one or both of PCR primers contain a phage promoter sequence.

Biochemical Correction of Very Long–chain Acyl-CoA Dehydrogenase Deficiency Following Adeno-associated Virus Gene Therapy

We report the development of a gene replacement strategy for very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. VLCAD is a mitochondrial enzyme involved in fatty acid beta-oxidation, a key step in energy production during times of fasting or stress. Deficiency of VLCAD classically presents as hepatic dysfunction, hypoglycemia, cardiomyopathy, rhabdomyolysis, and/or sudden death. While dietary therapy for VLCAD deficiency has proven beneficial in preventing some symptoms, a risk of metabolic catastrophic decompensation remains throughout life during times of increased energy demand. We designed a recombinant adeno-associated virus (AAV) expressing the human VLCAD gene (AAV8-hVLCAD). To demonstrate its in vivo activity, AAV8-hVLCAD was administered via the tail vein to VLCAD-knockout mice. A reduction in accumulated serum long-chain acylcarnitines and increased fasting tolerance judged on blood glucose concentrations were observed as of 11 days postinjections through >100 days. Western analysis of liver, skeletal muscle, and heart extracts using PEP1 anti-hVLCAD antibody revealed short-term hVLCAD expression in the liver and muscle and longer-term expression in heart. This demonstrates the ability of human VLCAD to correct the biochemical phenotype of VLCAD-deficient mice.

A method of sequencing without subcloning and its application to the identification of a novel ORF with a sequence suggestive of a transcriptional regulator in the water mold Achlya ambisexualis

Genomic amplification with transcript sequencing (GAWTS) is a method of direct sequencing that involves amplification with PCR using primers containing phage promoters, transcription of the amplified product, and sequencing with reverse transcriptase. GAWTS requires the generation of PCR primers that are specific for the sequences on both sides of a region. Here we describe promoter ligation and transcript sequencing (PLATS), a direct method for rapidly obtaining novel sequences that utilizes generic primers and only requires knowledge of the sequence on one side of a region. PLATS involves restriction digestion of the amplified vector insert, ligation with a phage promoter, and then GAWTS using phage promoter sequences as the PCR primers. The method is rapid and economical because it uses a limited set of oligonucleotides, and it is potentially amenable to automation because it does not require in vivo manipulations. PLATS facilitates the determination of a genomic sequence responsible for cross-hybridization in a Southern blot. Using PLATS, sequence has been obtained from a 1.1-kb segment in Achlya ambisexualis, which cross-hybridizes to the DNA-binding region of the chicken and Xenopus estrogen receptors. To our knowledge, this represents the first sequence reported from the Oomycetes, a large and widely distributed group of fungi. The sequence reveals a large, transcribed open reading frame that is markedly deficient in the dinucleotide TpA. A putative zinc finger containing three cysteines and one histidine (C-X2-C-X12-H-X3-C) and an acidic segment hint that this clone may be a member of a novel class of transcriptional regulators.

921. Steps toward Cardiomyopathy Gene Therapy: In Vivo Expression of Human VLCAD

Cardiomyopathy (CM) is an important cause of morbidity and mortality in children. Deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD), one of four nuclear encoded mitochondrial enzymes that catalyze the initial step in the beta-oxidation of straight-chain fatty acids, often presents with CM and/or sudden death. How VLCAD deficiency causes CM is unclear, but appears to be related to the accumulation of toxic long-chain acyl-carnitine species rather than a block in energy metabolism. While dietary therapy for VLCAD deficiency has been beneficial to some, many people do not respond, and intercurrent illness with diminished oral intake, exercise, or fasting all increase demand for fatty acid metabolism and can trigger catastrophic events even in those on diet therapy. While VLCAD deficiency is a rare metabolic deficiency, the development of a safe and durable gene therapy for VLCAD deficiency would be the first curative gene therapy for CM.

438. In Vitro Studies Towards the Development of a Recombinant Adeno-Associated Virus for the Treatment of Medium-Chain Acyl-CoA Dehydrogenase Deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly diagnosed inborn error of metabolism. Clinical presentation ranges from mild hypotonia and hypoglycemia in children to sudden unexpected death. Current therapies including avoidance of fasting, vitamin supplementation, and dietary management are effective, but may not prevent acute clinical decompensations associated with infectious illnesses. Together, this makes MCAD deficiency a good model disease for a safe and durable gene delivery system. The adeno-associated virus subtype 2 (AAV) is known to be a durable gene delivery system which can transduce non-dividing cells and has minimal toxicity in phase 1 human clinical trials. We report here the development of a recombinant AAV vector that expresses the human MCAD gene (AAV-hMCAD). Preliminary studies with the expression cassette demonstrated localization of the MCAD protein to the mitochondria, and correction of the metabolic block in fibroblasts isolated from MCAD deficient patients. Intact AAV-hMCAD particles were produced by triple plasmid transfection in 293 cells then characterized by electron microscopy and ELISA assay. Real-time PCR quantified viral genome containing particles. Human MCAD transcript was identified after AAV-hMCAD infection of MCAD deficient human and murine fibroblasts by RT-PCR. Acyl-carnitine analysis revealed functional correction of the metabolic block seen in MCAD deficient fibroblasts after infection with AAV-hMCAD. Together, these data provide important preliminary data for future in vivo studies in MCAD deficient mice.