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AAV-MEDIATED GENE TRANSFER TO MODELS OF MUSCULAR DYSTROPHY: INSIGHTS INTO ASSEMBLY OF MULTI-SUBUNIT MEMBRANE PROTEINS.

Dressman, Devin Charles (2002) AAV-MEDIATED GENE TRANSFER TO MODELS OF MUSCULAR DYSTROPHY: INSIGHTS INTO ASSEMBLY OF MULTI-SUBUNIT MEMBRANE PROTEINS. Doctoral Dissertation, University of Pittsburgh.

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    Abstract

    The sarcoglycanopathies are a subset of the limb girdle muscular dystrophies (LGMD) caused by mutations in the sarcoglycan genes (alpha, beta, gamma and delta). In collaborative studies, delta-sarcoglycan was delivered to deficient hamsters using a recombinant adeno-associated virus (AAV), which rescued muscle biochemically, histologically, and functionally. Murine knockouts for the other sarcoglycans permitted us to pursue AAV-mediated gene delivery. AAV-mediated gene delivery of beta-sarcoglycan to deficient mice provided long-term biochemical and histological rescue. AAV-mediated gene delivery of alpha-sarcoglycan to deficient mice showed initial rescue of biochemical and histological defects, although expression was not persistent. Severe Combined Immune-Deficient (SCID) mouse studies indicated that alpha-sarcoglycan over-expression leads to cytotoxicity. The apparent cytotoxicity can be interpreted with emerging models of sarcoglycan complex assembly. These studies show that AAV-mediated delivery of even closely related proteins can lead to different outcomes, and aspects of protein biochemistry can alter efficacy of gene delivery.Inherited muscle disorders typically have defined primary biochemical defects. However, there are likely secondary responses that mitigate gene delivery success. To dissect such variables, we studied the immunostimulatory properties of dystrophic muscle. We hypothesized that immune cell infiltrate accompanying degeneration/regeneration could be immunostimulatory, which could elicit an immune response to delivered transgenes, hampering the success of gene delivery. To study this, we tested antibody response to and persistence of, beta-galactosidase in normal and dystrophic muscle. Consistent with our hypothesis, dystrophic muscle showed increased immune surveillance and recognition of beta-galactosidase, evidenced by antibody titers and clearance of transduced cells. Furthermore, biochemical rescue of the dystrophy quenched the immune response. This indicated that dystrophic muscle is more prone to immune responses and that aspects of tissue pathology influence the persistence and efficacy of gene delivery. Our results suggest that full biochemical rescue will attenuate immunostimulatory effects. We also address a hurdle facing AAV-mediated gene therapy; namely, delivery methods. We developed an injection manifold, which was used to safely, accurately, and consistently deliver genes to 20 mm2 regions of muscle. Taken together, these results more clearly define barriers to gene delivery. Future research will finely tune regulation of transgenes and enable full rescue of biochemical defects.


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    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmailORCID
    Committee ChairHoffman, Ericehoffman@cnmcresearch.org
    Committee MemberStewart, Alexstewartaf@msx.upmc.edu
    Committee MemberWatchko, Jonjwatchko@mail.magee.edu
    Committee MemberRobbins, Paulprobb@pitt.edu
    Committee MemberClemens, Paulapclemens@pitt.edu
    Committee MemberWatkins, Simonswatkins@pitt.edu
    Title: AAV-MEDIATED GENE TRANSFER TO MODELS OF MUSCULAR DYSTROPHY: INSIGHTS INTO ASSEMBLY OF MULTI-SUBUNIT MEMBRANE PROTEINS.
    Status: Unpublished
    Abstract: The sarcoglycanopathies are a subset of the limb girdle muscular dystrophies (LGMD) caused by mutations in the sarcoglycan genes (alpha, beta, gamma and delta). In collaborative studies, delta-sarcoglycan was delivered to deficient hamsters using a recombinant adeno-associated virus (AAV), which rescued muscle biochemically, histologically, and functionally. Murine knockouts for the other sarcoglycans permitted us to pursue AAV-mediated gene delivery. AAV-mediated gene delivery of beta-sarcoglycan to deficient mice provided long-term biochemical and histological rescue. AAV-mediated gene delivery of alpha-sarcoglycan to deficient mice showed initial rescue of biochemical and histological defects, although expression was not persistent. Severe Combined Immune-Deficient (SCID) mouse studies indicated that alpha-sarcoglycan over-expression leads to cytotoxicity. The apparent cytotoxicity can be interpreted with emerging models of sarcoglycan complex assembly. These studies show that AAV-mediated delivery of even closely related proteins can lead to different outcomes, and aspects of protein biochemistry can alter efficacy of gene delivery.Inherited muscle disorders typically have defined primary biochemical defects. However, there are likely secondary responses that mitigate gene delivery success. To dissect such variables, we studied the immunostimulatory properties of dystrophic muscle. We hypothesized that immune cell infiltrate accompanying degeneration/regeneration could be immunostimulatory, which could elicit an immune response to delivered transgenes, hampering the success of gene delivery. To study this, we tested antibody response to and persistence of, beta-galactosidase in normal and dystrophic muscle. Consistent with our hypothesis, dystrophic muscle showed increased immune surveillance and recognition of beta-galactosidase, evidenced by antibody titers and clearance of transduced cells. Furthermore, biochemical rescue of the dystrophy quenched the immune response. This indicated that dystrophic muscle is more prone to immune responses and that aspects of tissue pathology influence the persistence and efficacy of gene delivery. Our results suggest that full biochemical rescue will attenuate immunostimulatory effects. We also address a hurdle facing AAV-mediated gene therapy; namely, delivery methods. We developed an injection manifold, which was used to safely, accurately, and consistently deliver genes to 20 mm2 regions of muscle. Taken together, these results more clearly define barriers to gene delivery. Future research will finely tune regulation of transgenes and enable full rescue of biochemical defects.
    Date: 25 April 2002
    Date Type: Completion
    Defense Date: 15 December 2001
    Approval Date: 25 April 2002
    Submission Date: 22 April 2002
    Access Restriction: No restriction; The work is available for access worldwide immediately.
    Patent pending: No
    Institution: University of Pittsburgh
    Thesis Type: Doctoral Dissertation
    Refereed: Yes
    Degree: PhD - Doctor of Philosophy
    URN: etd-04222002-213643
    Uncontrolled Keywords: gene therapy; muscular dystropy
    Schools and Programs: School of Medicine > Biochemistry and Molecular Genetics
    Date Deposited: 10 Nov 2011 14:40
    Last Modified: 29 May 2012 16:29
    Other ID: http://etd.library.pitt.edu:80/ETD/available/etd-04222002-213643/, etd-04222002-213643

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