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ABSTRACT: Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by the half-normal activity of hydroxymethylbilane synthase (HMB-synthase). Affected individuals can experience episodic, life-threatening, acute neurological attacks that are precipitated by various drugs, dieting, and hormonal changes. Intravenous hematin is used to treat the attacks, but a more effective, preventive therapy is needed, especially for patients with frequent attacks. Since the disease is a hepatic encephalopathy, efforts were focused towards evaluating four different combinations of liver-specific enhancers and promoters for maximal hepatic HMB-synthase expression.
Four different mammalian expression vectors, each carrying a unique combination of liver-specific enhancers and promoters driving murine HMB-synthase cDNA expression, were transiently transfected into HepG2 cells. The vectors included: HMBS-1; human alpha1-microglobulin enhancer/alpha1-antityrpsin promoter (alpha1Me/alpha1ATp), HMBS-2; alpha1Me/human serum albumin promoter (alpha1Me/SAp), HMBS-3; human prothrombin enhancer/SAp (PTe/SAp), and HMBS-4; (PTe/alpha1ATp). Each HMB-synthase construct and a luciferase reporter construct were hydrodynamically coinjected into mice with HMB-synthase deficiency and evaluated for hepatic expression 24 h post-injection, the time-point of peak hepatic HMB-synthase expression.
Following transient transfection into HepG2 cells, HMBS-1 (alpha1Me/alpha1ATp) had the highest HMB-synthase expression level, with an approximately 8-fold increase over endogenous cellular activities. Construct HMBS-1 also had the highest hepatic HMB-synthase activity following hydrodynamic delivery into HMB-synthase deficient mice, with a approximately 6-fold increase over saline-treated mice.
These studies support the use of a gene therapy vector containing the alpha1Me/alpha1ATp combination for preclinical studies of the efficacy and safety of liver-targeted gene therapy for AIP.
The Journal of Gene Medicine 10/2007; 9(9):806-11. · 2.48 Impact Factor
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ABSTRACT: The advent of novel adeno-associated virus (AAV) serotype vectors with higher transduction activity has encouraged a re-evaluation of the merits of this delivery platform for a variety of diseases. We report here that administration of a recombinant AAV8-based serotype vector encoding human alpha-galactosidase A into Fabry mice facilitated more rapid and significantly higher levels of production of the enzyme than an AAV2 vector. This translated into improved clearance of globotriaosylceramide, the glycosphingolipid that accumulates in the lysosomes of affected Fabry cells, and to correction of the peripheral neuropathy shown associated with this disease. The higher levels of alpha-galactosidase A expression also allowed for a more rapid induction of immunotolerance to the enzyme. Recombinant AAV8 vectors that facilitated hepatic-restricted expression of high levels of alpha-galactosidase A conferred immunotolerance to the expressed enzyme as early as 30 days post-treatment. Animals expressing lower levels of the hydrolase, such as those treated with an AAV2-based vector or with lower doses of the AAV8-based vector, were also able to develop immunotolerance, but only after a more extended time period. Adoptive transfer of T cells isolated from the spleens of immunotolerized mice suppressed the formation of antibodies in naïve recipient animals, suggesting the possible role of regulatory T cells in effecting this state.
Molecular Therapy 04/2007; 15(3):492-500. · 6.87 Impact Factor
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ABSTRACT: Gaucher disease is the most common of the lysosomal storage disorders. The primary manifestation is the accumulation of glucosylceramide (GL-1) in the macrophages of liver and spleen (Gaucher cells), due to a deficiency in the lysosomal hydrolase glucocerebrosidase (GC). A Gaucher mouse model (D409V/null) exhibiting reduced GC activity and accumulation of GL-1 was used to evaluate adeno-associated viral (AAV)-mediated gene therapy.
A recombinant AAV8 serotype vector bearing human GC (hGC) was administered intravenously to the mice. The levels of hGC in blood and tissues were determined, as were the effects of gene transfer on the levels of GL-1. Histopathological evaluation was performed on liver, spleen and lungs.
Vector administration to pre-symptomatic Gaucher mice resulted in sustained hepatic secretion of hGC at levels that prevented GL-1 accumulation and the appearance of Gaucher cells in the liver, spleen and lungs. AAV administration to older mice with established disease resulted in normalization of GL-1 levels in the spleen and liver and partially reduced that in the lung. Analysis of the bronchoalveolar lavage fluid (BALF) from treated mice showed significant correction of the abnormal cellularity and cell differentials. No antibodies to the expressed hGC were detected following a challenge with recombinant enzyme suggesting the animals were tolerized to human enzyme.
These data demonstrate the effectiveness of AAV-mediated gene therapy at preventing and correcting the biochemical and pathological abnormalities in a Gaucher mouse model, and thus support the continued consideration of this vector as an alternative approach to treating Gaucher disease.
The Journal of Gene Medicine 07/2006; 8(6):719-29. · 2.48 Impact Factor
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ABSTRACT: Acid sphingomyelinase deficiency is a lysosomal storage disorder in which the defective lysosomal hydrolase fails to degrade sphingomyelin. The resulting accumulation of substrate in the lysosomes of histiocytic cells leads to hepatosplenomegaly and severe pulmonary inflammation. Administration of a recombinant AAV1 vector encoding human acid sphingomyelinase to acid sphingomyelinase knockout (ASMKO) mice effectively reduced the accumulated substrate in all of the affected visceral organs. However, more complete and rapid clearance of sphingomyelin was observed when an AAV8-based serotype vector was used in lieu of AAV1. Importantly, AAV8-mediated hepatic expression of higher and sustained levels of the enzyme also corrected the abnormal cellularity, cell differentials, and levels of the chemokine MIP-1alpha in the bronchoalveolar lavage fluids of the ASMKO mice. Treatment also reversed the morphological aberrations associated with the alveolar macrophages of ASMKO mice and restored their phagocytic activity. No antibodies to the expressed enzyme were detected when the viral vectors were used in conjunction with a transcription cassette harboring a liver-restricted enhancer/promoter. Together, these data support the continued development of AAV8-mediated hepatic gene transfer as an approach to treat the visceral manifestations observed in individuals with acid sphingomyelinase deficiency.
Molecular Therapy 10/2005; 12(3):431-40. · 6.87 Impact Factor
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ABSTRACT: Molecular Therapy (2004) 9, S324–S325; doi: 10.1016/j.ymthe.2004.06.762
854. Feasibility of AAV-Mediated Gene Therapy Examined Using a New Murine Model (D409V/null) of Gaucher Disease
John Marshall1, Kerry McEachern1, Jennifer B. Nietupski1, Julie A. Cavanagh Kyros1, Donna Armentano1, Gregory A. Grabowski2 and Seng H. Cheng11Genzyme Corporation, Framingham, MA2Divisions of Human Genetics and Pathology, Cincinnati Children's Hospital Research Foundation, Cincinnati, OH
Molecular Therapy 04/2004; · 6.87 Impact Factor
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Robin J Ziegler,
Scott M Lonning, Donna Armentano,
Chester Li,
David W Souza,
Maribeth Cherry,
Christine Ford,
Christine M Barbon,
Robert J Desnick,
Guangping Gao,
James M Wilson,
Richard Peluso,
Simon Godwin,
Barrie J Carter,
Richard J Gregory,
Samuel C Wadsworth,
Seng H Cheng
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ABSTRACT: The successful application of gene therapy for the treatment of genetic diseases such as Fabry is reliant on the development of vectors that are safe and that facilitate sustained expression of therapeutic levels of the transgene product. Here, we report that intravenous administration of a recombinant AAV2 vector encoding human alpha-galactosidase A under the transcriptional control of a liver-restricted enhancer/promoter (AAV2/DC190-alphagal) generated significantly higher levels of expression in BALB/c and Fabry mice than could be realized using the ubiquitous CMV promoter (AAV2/CMVHI-alphagal). Moreover, AAV2/DC190-alphagal-mediated hepatic expression of alpha-galactosidase A was sustained for 12 months in BALB/c mice and was associated with a significantly reduced immune response to the expressed enzyme. Subsequent challenge of the AAV2/DC190-alphagal-treated animals with recombinant human alpha-galactosidase A at 6 months failed to elicit the production of anti-alpha-galactosidase A antibodies, suggesting the induction of immune tolerance in these animals. The levels of expression attained with AAV2/DC190-alphagal in the Fabry mice were sufficient to reduce the abnormal accumulation of globotriaosylceramide in the liver, spleen, and heart to basal levels and in the kidney by approximately 40% at 8 weeks. Together, these results demonstrate that AAV2-mediated gene transfer that limits the expression of alpha-galactosidase A to the liver may be a viable strategy for treating Fabry disease.
Molecular Therapy 03/2004; 9(2):231-40. · 6.87 Impact Factor
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Robin J. Ziegler,
Scott M. Lonning, Donna Armentano,
Chester Li,
David W. Souza,
Maribeth Cherry,
Christine Ford,
Christine M. Barbon,
Robert J. Desnick,
Guangping Gao,
James M. Wilson,
Richard Peluso,
Simon Godwin,
Barrie J. Carter,
Richard J. Gregory,
Samuel C. Wadsworth,
Seng H. Cheng
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ABSTRACT: The successful application of gene therapy for the treatment of genetic diseases such as Fabry is reliant on the development of vectors that are safe and that facilitate sustained expression of therapeutic levels of the transgene product. Here, we report that intravenous administration of a recombinant AAV2 vector encoding human -galactosidase A under the transcriptional control of a liver-restricted enhancer/promoter (AAV2/DC190-gal) generated significantly higher levels of expression in BALB/c and Fabry mice than could be realized using the ubiquitous CMV promoter (AAV2/CMVHI-gal). Moreover, AAV2/DC190-gal-mediated hepatic expression of -galactosidase A was sustained for 12 months in BALB/c mice and was associated with a significantly reduced immune response to the expressed enzyme. Subsequent challenge of the AAV2/DC190-gal-treated animals with recombinant human -galactosidase A at 6 months failed to elicit the production of anti--galactosidase A antibodies, suggesting the induction of immune tolerance in these animals. The levels of expression attained with AAV2/DC190-gal in the Fabry mice were sufficient to reduce the abnormal accumulation of globotriaosylceramide in the liver, spleen, and heart to basal levels and in the kidney by approximately 40% at 8 weeks. Together, these results demonstrate that AAV2-mediated gene transfer that limits the expression of -galactosidase A to the liver may be a viable strategy for treating Fabry disease.Keywords: AAV2 vector, gene therapy, liver-specific promoters, Fabry disease, lysosomal storage diseases, glycosphingolipids, immune tolerance
Molecular Therapy 01/2004; 9(2):231-240. · 6.87 Impact Factor
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ABSTRACT: Group C adenovirus-mediated gene transfer to central nervous system cells is inefficient. We found that wild-type group D viruses, or recombinant adenovirus type 2 (Ad2) (group C) modified to contain Ad17 (group D) fiber, were more efficient in infecting primary cultures of neurons. Together with studies on primary vascular endothelial cells and tissue culture cell lines, our results indicate that there is not a universally applicable adenovirus serotype for use as a gene transfer vector.
