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PRODUCTION AND PURIFICATION OF HSV-1 VECTORS AND ITS USE FOR GENE TRANSFER TO HUMAN CD34⁺ CELLS

Wechuck, James Brian (2002) PRODUCTION AND PURIFICATION OF HSV-1 VECTORS AND ITS USE FOR GENE TRANSFER TO HUMAN CD34⁺ CELLS. Doctoral Dissertation, University of Pittsburgh.

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    Abstract

    The delivery of therapeutic DNA to patients with genetic or acquired disorders has evolved into a realistic alternate treatment to surgery, drug delivery, or as an adjuvant to therapies. A specific area of this research is to transduce CD34⁺ cells with therapeutic DNA containing vectors developed from viruses. Explanted CD34⁺ progenitor cells are exposed to these vectors and then are transplanted to a host. Despite the positive advances, major limitations exist in the ability to efficiently transduce cells with current vector systems. In addition, if vectors are proven effective at ameliorating disease, the expense of manufacturing sufficient quantities required to treat large populations of patients may prohibit their widespread applications. Our lab utilizes replication-defective herpes simplex virus type-1 (HSV-1) as a gene delivery vector. HSV-1 possesses the capability to deliver large or multiple genes and infects a wide variety of cell types. In order to optimize transduction of human umbilical cord blood CD34⁺ cells, I examined this population for presence and functionality of the three different HSV receptors required for virus binding and entry. Optimal transduction efficiencies of 75% were obtained by systematically varying vector concentration and adsorption time. Additionally, several vector constructs were developed in order to examine the effects of various promoter systems and additional gene deletions on gene expression and vector-associated toxicity. Throughout these studies we were confronted with the difficulty of obtaining sufficient amounts of high quality vector needed for testing the efficacy of HSV-1 vectors in various applications. To further improve the vector yield, I examined the effect of temperature and media conditions on the stability of an HSV-1 vector. Applying a temperature shift to production conditions resulted in increased vector yields that remained stable at peak levels in cultures incubated at 33°C rather than 37°C. To enhance vector recovery, alternative purification strategies were examined. A two-stage tangential flow filtration system coupled to a chromatography step was developed to isolate vector from large debris such as cells and smaller molecules such as protein and DNA. This new system for vector concentration and purification proved superior to the standard protocols currently in use.


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    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmail
    Committee ChairAtaai, Mohammad M.Ataai@engrng.pitt.edu
    Committee MemberGlorioso, Joseph C.
    Committee MemberFederspiel, William
    Committee MemberWagner, William
    Title: PRODUCTION AND PURIFICATION OF HSV-1 VECTORS AND ITS USE FOR GENE TRANSFER TO HUMAN CD34⁺ CELLS
    Status: Unpublished
    Abstract: The delivery of therapeutic DNA to patients with genetic or acquired disorders has evolved into a realistic alternate treatment to surgery, drug delivery, or as an adjuvant to therapies. A specific area of this research is to transduce CD34⁺ cells with therapeutic DNA containing vectors developed from viruses. Explanted CD34⁺ progenitor cells are exposed to these vectors and then are transplanted to a host. Despite the positive advances, major limitations exist in the ability to efficiently transduce cells with current vector systems. In addition, if vectors are proven effective at ameliorating disease, the expense of manufacturing sufficient quantities required to treat large populations of patients may prohibit their widespread applications. Our lab utilizes replication-defective herpes simplex virus type-1 (HSV-1) as a gene delivery vector. HSV-1 possesses the capability to deliver large or multiple genes and infects a wide variety of cell types. In order to optimize transduction of human umbilical cord blood CD34⁺ cells, I examined this population for presence and functionality of the three different HSV receptors required for virus binding and entry. Optimal transduction efficiencies of 75% were obtained by systematically varying vector concentration and adsorption time. Additionally, several vector constructs were developed in order to examine the effects of various promoter systems and additional gene deletions on gene expression and vector-associated toxicity. Throughout these studies we were confronted with the difficulty of obtaining sufficient amounts of high quality vector needed for testing the efficacy of HSV-1 vectors in various applications. To further improve the vector yield, I examined the effect of temperature and media conditions on the stability of an HSV-1 vector. Applying a temperature shift to production conditions resulted in increased vector yields that remained stable at peak levels in cultures incubated at 33°C rather than 37°C. To enhance vector recovery, alternative purification strategies were examined. A two-stage tangential flow filtration system coupled to a chromatography step was developed to isolate vector from large debris such as cells and smaller molecules such as protein and DNA. This new system for vector concentration and purification proved superior to the standard protocols currently in use.
    Date: 15 March 2002
    Date Type: Completion
    Defense Date: 29 January 2002
    Approval Date: 15 March 2002
    Submission Date: 18 February 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-02182002-135311
    Uncontrolled Keywords: CD34⁺ Cells; Cell Culture; Gene Therapy; Heparin Chromatography; Herpes Simplex Virus; Tangential-Flow Filtration; Vector Production; Vector Purification; Viral Vector
    Schools and Programs: Swanson School of Engineering > Chemical Engineering
    Date Deposited: 10 Nov 2011 14:31
    Last Modified: 22 Feb 2012 13:11
    Other ID: http://etd.library.pitt.edu/ETD/available/etd-02182002-135311/, etd-02182002-135311

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