Biomedical subjects

Cathryn Mah

Publications and source records attributed to Cathryn Mah.

6 recordsLinked to original sources

PubMed2006

Sustained FVIII expression and phenotypic correction of hemophilia A in neonatal mice using an endothelial-targeted sleeping beauty transposon.

Hemophilia A, deficiency of coagulation factor VIII (FVIII), is an attractive candidate for gene therapy as expression of modest amounts of FVIII can provide therapeutic benefit. Most gene transfer approaches for hemophilia have focused on the liver, as this is the major source of endogenous FVIII; however, increasing evidence suggests that endothelial cells are capable of synthesis and release of FVIII. Here the Sleeping Beauty (SB) transposon is employed to target long-term expression of the human B-domain-depleted FVIII gene (approved gene symbol F8) within lung endothelia of hemophilic mice. As the formation of inhibitory antibodies to FVIII has been a significant impediment toward achieving therapeutic plasma levels after gene or protein therapy, we chose to perform gene transfer in neonatal mice, which are more likely to be immune tolerant. Using this approach, low therapeutic levels of FVIII ( approximately 10%), as well as phenotypic correction of the bleeding disorder, were achieved in all animals that received the FVIII transposon and functional transposase throughout the duration of the study (24 weeks). Rechallenge of these animals with additional gene transfer did not result in significant increases in FVIII levels, due mainly to increases in inhibitory antibodies. These studies demonstrate the feasibility of using endothelial-targeted SB transposons for the treatment of hemophilia A.

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PubMed2005

Impact of humoral immune response on distribution and efficacy of recombinant adeno-associated virus-derived acid alpha-glucosidase in a model of glycogen storage disease type II.

Glycogen storage disease type II (GSDII) is a lysosomal storage disease caused by a deficiency in acid alpha-glucosidase (GAA), and leads to cardiorespiratory failure by the age of 2 years. In this study, we investigate the impact of anti-GAA antibody formation on cross-correction of the heart, diaphragm, and hind-limb muscles from liver-directed delivery of recombinant adeno-associated virus (rAAV)5- and rAAV8-GAA vectors. GAA(-/-) mice receiving 1 x 10(12) vector genomes of rAAV5- or rAAV8-DHBV-hGAA were analyzed for anti-GAA antibody response, GAA levels, glycogen reduction, and contractile function. We demonstrate that restoration of GAA to the affected muscles is dependent on the presence or absence of the antibody response. Immune-tolerant mice had significantly increased enzyme levels in the heart and skeletal muscles, whereas immune-responsive mice had background levels of GAA in all tissues except the diaphragm. The increased levels of activity in immune-tolerant mice correlated with reduced glycogen in the heart and diaphragm and, overall, contractile function of the soleus muscle was significantly improved. These findings highlight the importance of the immune response to rAAV-encoded GAA in correcting GSDII and provide additional understanding of the approach to treatment of GSDII.

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PubMed2004

A new method for recombinant adeno-associated virus vector delivery to murine diaphragm.

Genetically modified mice are important models for evaluation of potential gene therapies for human diseases. However, their small size often precludes the use of clinically feasible methods for vector delivery, therefore, alternative methods must be used. We have developed a gel-based method for delivery of recombinant adeno-associated virus vectors to the mouse diaphragm, an important target organ for many myopathic diseases. We hypothesized that delivery of vectors in a viscous solution would increase transduction by providing a longer exposure time to target cells. We demonstrate that gel-mediated delivery of rAAV serotypes 1, 2, and 5 results in higher transduction efficiencies than free vectors alone when administered in vivo to mouse diaphragms. We further establish greater tropism of rAAV1 vectors for the diaphragm compared to serotypes 2 and 5. This report describes a novel method for efficient delivery of rAAV vectors to the mouse diaphragm and is the first demonstration of gene transfer to the diaphragm using recombinant adeno-associated virus vectors.

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PubMed2003

Dual vectors expressing murine factor VIII result in sustained correction of hemophilia A mice.

Hemophilia A is a sex-linked disorder that results from a deficiency of functional factor VIII and is currently treated by protein replacement therapies. Within the past decade, gene therapy efforts have come to the forefront of novel therapeutics. In this work, a dual-vector approach was employed in which recombinant adeno-associated viral (rAAV) vectors expressing the heavy and light chains of the murine factor VIII gene were delivered either intramuscularly or intravenously to a mouse model of hemophilia A. From in vitro work, it was determined that coinfection with both vectors is required as heterodimerization of the heavy and light chains occurs intracellularly. In vivo, therapeutic levels of factor VIII expression were achieved throughout the duration of the study (22 weeks). Intravenous and intramuscular delivery resulted in a maximal average expression of 31.4 +/- 6.4 and 29 +/- 6.5% of normal murine factor VIII levels, respectively. Western blots of cryoprecipitate as well as immunostaining of injection sites with an anti-murine factor VIII light chain antibody also confirmed the expression of factor VIII. Because the murine form of the gene was used in the mouse model, less than 1 Bethesda unit of inhibitors was noted. This work demonstrates the feasibility of using rAAV vectors for the long-term treatment of hemophilia A.

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PubMed2002

Improved method of recombinant AAV2 delivery for systemic targeted gene therapy.

A major hurdle in most current gene therapy modalities is the ability to transduce target tissues at very high efficiencies that ultimately lead to therapeutic levels of transgene expression. We have developed a novel method of recombinant adeno-associated virus 2 (rAAV) delivery that results in increased vector transduction efficiencies using microspheres reversibly conjugated to rAAV vectors. We hypothesize that conjugation to microspheres should result in a higher effective concentration of vector as well as longer relative exposure time of vector to target cells as it moves through the tissue vasculature. In vitro experiments demonstrate that the same level of transduction seen with free vector can be achieved using 1% of vector when conjugated to microspheres. In addition, using magnetic microspheres, the region of infection can be targeted. In vivo, we demonstrate that microsphere-mediated delivery of rAAV vector results in higher transduction efficiencies than delivery with free vector alone when administered either intramuscularly or intravenously. Furthermore, we demonstrate targeting of transgene expression to specific tissues by retention of microsphere-bound vector in the capillary bed. These studies demonstrate a novel method to deliver rAAV vectors more effectively that could prove to be a successful alternative mode of virus-mediated human gene therapy.

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PubMed2002

Virus-based gene delivery systems.

Within the past decade, gene therapy strategies have come to the forefront of novel therapeutics. Tremendous advances in vector technology along with deeper understandings of vector biology and the molecular mechanisms of disease have significantly advanced the field of human gene therapy. This manuscript will discuss the viral-based subset of current gene transfer vectors. In particular, the most established viral vectors to date, including parvovirus, adenovirus, retrovirus, lentivirus, and herpesvirus-based vectors, are described, as well as the current innovative improvements being made to each. From past experience, it has become evident that in addition to optimising the vectors in terms of transgene expression, minimising vector-related immunology, and vector production, methods of vector delivery resulting in optimum vector transduction of target cells need to be established. This review will also illustrate several current improved physical delivery systems for optimal vector administration.

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