Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy

Abstract

Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 1012 vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD−/− mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD−/− mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD−/− mice maintained euglycemia, whereas untreated VLCAD−/− mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

Reprogramming Adipose Tissue-Derived Mesenchymal Stem Cells into Pluripotent Stem Cells by a Mutant Adeno-Associated Viral Vector

“…The vector-specific primers used in PCR to detect rAAV DNA were CMV-F (5¢-CGTGTACGGTGGGAGGTCTATATAA-3¢) and CMV-R (5¢-GGATCGGTCCCGGTGTCT-3¢). For the quantification of rAAV vector DNA in rAAV-iPSC clones, quantitative real-time PCR (qPCR) was performed with vector-specific primers and a probe for CMV promoter sequences: forward primer, 5¢-CGTGTACGGTGGGAGGTC TATATAA-3¢; reverse primer, 5¢-GGATCGGTCCCGGTG TCT-3¢; and probe, 5¢-6FAM-ACGCCATCCACGCTGTTTT GACCT-TAMRA-3¢ (Keeler et al, 2012). In RT-PCR analysis, total RNA was purified with TRIzol reagent (Invitrogen).…”

Cell and Gene Therapy for Genetic Diseases: Inherited Disorders Affecting the Lung and Those Mimicking Sudden Infant Death Syndrome

“…In this study long-term expression of VLCAD protein, 147-182 days post injection, was observed in the liver and skeletal and cardiac muscle as well as brown fat (Keeler et al, 2012). Systemic correction was seen in reduction of blood acylcarnitine accumulation, but tissue-specific accumulation was measured in the liver and heart and muscle ex vivo and in the liver and muscle in vivo by MRS. Also for the first time disease-specific phenotypes of cold intolerance and coldinduced hypoglycemia and hypotonia were observed after correction, and animals receiving AAV9-expressing VLCAD behaved like wild-type animals (Keeler et al, 2012) (Fig. 3).…”

Increased mean aliphatic lipid chain length in left ventricular hypertrophy secondary to arterial hypertension

“…[34] Moreover, blood profiles from animals and patients with VLCAD deficiency are characterized by large accumulations of long chain acyl carnitines. [35,36] …”